Publications
2024
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(2024) Life Science Alliance. 7, 10, Abstract
Ineffective endometrial matrix remodeling, a key factor in infertility, impedes embryo implantation in the uterine wall. Our study reveals the cellular and molecular impact of human collagenase-1 administration in mouse uteri, demonstrating enhanced embryo implantation rates. Collagenase-1 promotes remodeling of the endometrial ECM, degrading collagen fibers and proteoglycans. This process releases matrix-bound bioactive factors (e.g., VEGF, decorin), facilitating vascular permeability and angiogenesis. Collagenase-1 elevates embryo implantation regulators, including NK cell infiltration and the key cytokine LIF. Remarkably, uterine tissue maintains structural integrity despite reduced endometrial collagen fiber tension. In-utero collagenase-1 application rescues implantation in heat stress and embryo transfer models, known for low implantation rates. Importantly, ex vivo exposure of human uterine tissue to collagenase-1 induces collagen de-tensioning and VEGF release, mirroring remodeling observed in mice. Our research highlights the potential of collagenases to induce and orchestrate cellular and molecular processes enhancing uterine receptivity for effective embryo implantation. This innovative approach underscores ECM remodeling mechanisms critical for embryo implantation.
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(2024) Nanoscale Advances. 6, 14, p. 3655-3667 Abstract
Fibrillar collagen accumulation emerges as a promising biomarker in several diseases, such as desmoplastic tumors and unstable atherosclerotic plaque. Gold nanorods (GNRs) hold great potential as contrast agents in high-resolution, biomedically safe, and non-invasive photoacoustic imaging (PAI). This study presents the design and characterization of a specialized imaging tool which exploits GNR assisted targeted photoacoustic imaging that is tailored for the identification of fibrillar collagen. In addition to the photoacoustic characterization of collagen in the NIR 1 and 2 regions, we demonstrate the detailed steps of conjugating a decoy to GNRs. This study serves as a proof of concept, that demonstrates that conjugated collagenase-1 (MMP-1) generates a distinct and collagen-specific photoacoustic signal, facilitating real-time visualization in the wavelength range of 700-970 nm (NIR I). As most of the reported studies utilized the endogenous contrast of collagen in the NIR II wavelength that has major limitations to perform in vivo deep tissue imaging, the approach that we are proposing is unique and it highlights the promise of MMP-1 decoy-functionalized GNRs as novel contrast agents for photoacoustic imaging of collagen in the NIR 1 region. To our knowledge this is the first time functionalized GNRs are optimized for the detection of fibrillar collagen and utilized in the field of non-invasive photoacoustic imaging that can facilitate a better prognosis of desmoplastic tumors and broken atherosclerotic plaques.
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(2024) Nature Medicine. 30, 8, p. 2170-2180 Abstract
Metastasis occurs frequently after resection of pancreatic cancer (PaC). In this study, we hypothesized that multi-parametric analysis of pre-metastatic liver biopsies would classify patients according to their metastatic risk, timing and organ site. Liver biopsies obtained during pancreatectomy from 49 patients with localized PaC and 19 control patients with non-cancerous pancreatic lesions were analyzed, combining metabolomic, tissue and single-cell transcriptomics and multiplex imaging approaches. Patients were followed prospectively (median 3 years) and classified into four recurrence groups; early (6 months after resection) liver metastasis (LiM); extrahepatic metastasis (EHM); and disease-free survivors (no evidence of disease (NED)). Overall, PaC livers exhibited signs of augmented inflammation compared to controls. Enrichment of neutrophil extracellular traps (NETs), Ki-67 upregulation and decreased liver creatine significantly distinguished those with future metastasis from NED. Patients with future LiM were characterized by scant T cell lobular infiltration, less steatosis and higher levels of citrullinated H3 compared to patients who developed EHM, who had overexpression of interferon target genes (MX1 and NR1D1) and an increase of CD11B+ natural killer (NK) cells. Upregulation of sortilin-1 and prominent NETs, together with the lack of T cells and a reduction in CD11B+ NK cells, differentiated patients with early-onset LiM from those with late-onset LiM. Liver profiles of NED closely resembled those of controls. Using the above parameters, a machine-learning-based model was developed that successfully predicted the metastatic outcome at the time of surgery with 78% accuracy. Therefore, multi-parametric profiling of liver biopsies at the time of PaC diagnosis may determine metastatic risk and organotropism and guide clinical stratification for optimal treatment selection.
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(2024) EMBO Journal. 43, 17, p. 3553-3586 Abstract
Extracellular vesicles (EVs) are important mediators of communication between cells. Here, we reveal a new mode of intercellular communication by melanosomes, large EVs secreted by melanocytes for melanin transport. Unlike small EVs, which are disintegrated within the receiver cell, melanosomes stay intact within them, gain a unique protein signature, and can then be further transferred to another cell as \u201csecond-hand\u201d EVs. We show that melanoma-secreted melanosomes passaged through epidermal keratinocytes or dermal fibroblasts can be further engulfed by resident macrophages. This process leads to macrophage polarization into pro-tumor or pro-immune cell infiltration phenotypes. Melanosomes that are transferred through fibroblasts can carry AKT1, which induces VEGF secretion from macrophages in an mTOR-dependent manner, promoting angiogenesis and metastasis in vivo. In melanoma patients, macrophages that are co-localized with AKT1 are correlated with disease aggressiveness, and immunotherapy non-responders are enriched in macrophages containing melanosome markers. Our findings suggest that interactions mediated by second-hand extracellular vesicles contribute to the formation of the metastatic niche, and that blocking the melanosome cues of macrophage diversification could be helpful in halting melanoma progression.
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(2024) Journal of Proteome Research. 23, 4, p. 1420-1432 Abstract
Colitis has a multifactorial pathogenesis with a strong cross-talk among microbiota, hypoxia, and tissue metabolism. Here, we aimed to characterize the molecular signature of the disease in symptomatic and presymptomatic stages of the inflammatory process at the tissue and fecal level. The study is based on two different murine models for colitis, and HR-MAS NMR on \u201cintact\u201d colon tissues and LC-MS/MS on colon tissue extracts were used to derive untargeted metabolomics and proteomics information, respectively. Solution NMR was used to derive metabolomic profiles of the fecal extracts. By combining metabolomic and proteomic analyses of the tissues, we found increased anaerobic glycolysis, accompanied by an altered citric acid cycle and oxidative phosphorylation in inflamed colons; these changes associate with inflammation-induced hypoxia taking place in colon tissues. Different colitis states were also characterized by significantly different metabolomic profiles of fecal extracts, attributable to both the dysbiosis characteristic of colitis as well as the dysregulated tissue metabolism. Strong and distinctive tissue and fecal metabolomic signatures can be detected before the onset of symptoms. Therefore, untargeted metabolomics of tissues and fecal extracts provides a comprehensive picture of the changes accompanying the disease onset already at preclinical stages, highlighting the diagnostic potential of global metabolomics for inflammatory diseases.
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(2024) Biomedicines. 12, 2, 434. Abstract
Proteolysis of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) plays a crucial role in the immune response to bacterial infections. Here we report the secretion of MMPs associated with proteolytic extracellular vesicles (EVs) released by macrophages in response to Salmonella enterica serovar Typhimurium infection. Specifically, we used global proteomics, in vitro, and in vivo approaches to investigate the composition and function of these proteolytic EVs. Using a model of S. Typhimurium infection in murine macrophages, we isolated and characterized a population of small EVs. Bulk proteomics analysis revealed significant changes in protein cargo of naïve and S. Typhimurium-infected macrophage-derived EVs, including the upregulation of MMP-9. The increased levels of MMP-9 observed in immune cells exposed to S. Typhimurium were found to be regulated by the toll-like receptor 4 (TLR-4)-mediated response to bacterial lipopolysaccharide. Macrophage-derived EV-associated MMP-9 enhanced the macrophage invasion through Matrigel as selective inhibition of MMP-9 reduced macrophage invasion. Systemic administration of fluorescently labeled EVs into immunocompromised mice demonstrated that EV-associated MMP activity facilitated increased accumulation of EVs in spleen and liver tissues. This study suggests that macrophages secrete proteolytic EVs to enhance invasion and ECM remodeling during bacterial infections, shedding light on an essential aspect of the immune response.
2023
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(2023) Trends in Molecular Medicine. Abstract
Proteolytic processes on cell surfaces and extracellular matrix (ECM) sustain cell behavior and tissue integrity in health and disease. Matrix metalloproteases (MMPs) and a disintegrin and metalloproteases (ADAMs) remodel cell microenvironments through irreversible proteolysis of ECM proteins and cell surface bioactive molecules. Pan-MMP inhibitors in inflammation and cancer clinical trials have encountered challenges due to promiscuous activities of MMPs. Systems biology advances revealed that MMPs initiate multifactorial proteolytic cascades, creating new substrates, activating or suppressing other MMPs, and generating signaling molecules. This review highlights the intricate network that underscores the role of MMPs beyond individual substrateenzyme activities. Gaining insight into MMP function and tissue specificity is crucial for developing effective drug discovery strategies and novel therapeutics. This requires considering the dynamic cellular processes and consequences of network proteolysis.
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(2023) Nature Biomedical Engineering. 7, p. 1343-1345 Abstract
Advanced glycation end products mediate the extensive crosslinking of the extracellular matrix in cirrhotic liver tissue.
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(2023) Neurophotonics. 10, 1, 015008. Abstract
Significance: Perineuronal nets (PNNs) are extracellular matrix structures implicated in learning, memory, information processing, synaptic plasticity, and neuroprotection. However, our understanding of mechanisms governing the evidently important contribution of PNNs to central nervous system function is lacking. A primary cause for this gap of knowledge is the absence of direct experimental tools to study their role in vivo. Aim: We introduce a robust approach for quantitative longitudinal imaging of PNNs in brains of awake mice at subcellular resolution. Approach: We label PNNs in vivo with commercially available compounds and monitor their dynamics with two-photon imaging. Results: Using our approach, we show that it is possible to longitudinally follow the same PNNs in vivo while monitoring degradation and reconstitution of PNNs. We demonstrate the compatibility of our method to simultaneously monitor neuronal calcium dynamics in vivo and compare the activity of neurons with and without PNNs. Conclusion: Our approach is tailored for studying the intricate role of PNNs in vivo, while paving the road for elucidating their role in different neuropathological conditions.
2022
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(2022) Proceedings of the National Academy of Sciences. 119, 42, e221374411. Abstract
Acute and chronic pancreatitis, the latter associated with fibrosis, are multifactorial inflammatory disorders and leading causes of gastrointestinal disease-related hospitalization. Despite the global health burden of pancreatitis, currently, there are no effective therapeutic agents. In this regard, the protease A Disintegrin And Metalloproteinase 17 (ADAM17) mediates inflammatory responses through shedding of bioactive inflammatory cytokines and mediators, including tumor necrosis factor α (TNFα) and the soluble interleukin (IL)-6 receptor (sIL-6R), the latter of which drives proinflammatory IL-6 trans-signaling. However, the role of ADAM17 in pancreatitis is unclear. To address this, Adam17ex/ex mice-which are homozygous for the hypomorphic Adam17ex allele resulting in marked reduction in ADAM17 expression-and their wild-type (WT) littermates were exposed to the cerulein-induced acute pancreatitis model, and acute (1-wk) and chronic (20-wk) pancreatitis models induced by the cigarette smoke carcinogen nicotine-derived nitrosamine ketone (NNK). Our data reveal that ADAM17 expression was up-regulated in pancreatic tissues of animal models of pancreatitis. Moreover, the genetic (Adam17ex/ex mice) and therapeutic (ADAM17 prodomain inhibitor [A17pro]) targeting of ADAM17 ameliorated experimental pancreatitis, which was associated with a reduction in the IL-6 trans-signaling/STAT3 axis. This led to reduced inflammatory cell infiltration, including T cells and neutrophils, as well as necrosis and fibrosis in the pancreas. Furthermore, up-regulation of the ADAM17/IL-6 trans-signaling/STAT3 axis was a feature of pancreatitis patients. Collectively, our findings indicate that the ADAM17 protease plays a pivotal role in the pathogenesis of pancreatitis, which could pave the way for devising novel therapeutic options to be deployed against this disease.
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(2022) NPJ Parkinson's Disease. 8, 1, 103. Abstract
Several mutations that cause Parkinsons disease (PD) have been identified over the past decade. These account for 1525% of PD cases; the rest of the cases are considered sporadic. Currently, it is accepted that PD is not a single monolithic disease but rather a constellation of diseases with some common phenotypes. While rodent models exist for some of the PD-causing mutations, research on the sporadic forms of PD is lagging due to a lack of cellular models. In our study, we differentiated PD patient-derived dopaminergic (DA) neurons from the induced pluripotent stem cells (iPSCs) of several PD-causing mutations as well as from sporadic PD patients. Strikingly, we observed a common neurophysiological phenotype: neurons derived from PD patients had a severe reduction in the rate of synaptic currents compared to those derived from healthy controls. While the relationship between mutations in genes such as the SNCA and LRRK2 and a reduction in synaptic transmission has been investigated before, here we show evidence that the pathogenesis of the synapses in neurons is a general phenotype in PD. Analysis of RNA sequencing results displayed changes in gene expression in different synaptic mechanisms as well as other affected pathways such as extracellular matrix-related pathways. Some of these dysregulated pathways are common to all PD patients (monogenic or idiopathic). Our data, therefore, show changes that are central and convergent to PD and suggest a strong involvement of the tetra-partite synapse in PD pathophysiology.
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(2022) Viruses. 14, 8, 1627. Abstract
The COVID-19 pandemic caused by the SARS-CoV-2 infection induced lung inflammation characterized by cytokine storm and fulminant immune response of both resident and migrated immune cells, accelerating alveolar damage. In this work we identified members of the matrix metalloprotease (MMPs) family associated with lung extra-cellular matrix (ECM) destruction using K18-hACE2-transgenic mice (K18-hACE2) infected intranasally with SARS-CoV-2. Five days post infection, the lungs exhibited overall alveolar damage of epithelial cells and massive leukocytes infiltration. A substantial pulmonary increase in MMP8, MMP9, and MMP14 in the lungs post SARS-CoV-2 infection was associated with degradation of ECM components including collagen, laminin, and proteoglycans. The process of tissue damage and ECM degradation during SARS-CoV-2 lung infection is suggested to be associated with activity of members of the MMPs family, which in turn may be used as a therapeutic intervention.
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(2022) Cell. 185, 7, p. 1208-1222 Abstract
The tumor microenvironment hosts antibody-secreting cells (ASCs) associated with a favorable prognosis in several types of cancer. Patient-derived antibodies have diagnostic and therapeutic potential; yet, it remains unclear how antibodies gain autoreactivity and target tumors. Here, we found that somatic hypermutations (SHMs) promote antibody antitumor reactivity against surface autoantigens in high-grade serous ovarian carcinoma (HGSOC). Patient-derived tumor cells were frequently coated with IgGs. Intratumoral ASCs in HGSOC were both mutated and clonally expanded and produced tumor-reactive antibodies that targeted MMP14, which is abundantly expressed on the tumor cell surface. The reversion of monoclonal antibodies to their germline configuration revealed two types of classes: one dependent on SHMs for tumor binding and a second with germline-encoded autoreactivity. Thus, tumor-reactive autoantibodies are either naturally occurring or evolve through an antigen-driven selection process. These findings highlight the origin and potential applicability of autoantibodies directed at surface antigens for tumor targeting in cancer patients.
2021
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(2021) Journal of Experimental Medicine. 219, 1, e20201039. Abstract
Metastasis is the major cause of death in cancer patients. Circulating tumor cells need to migrate through the endothelial layer of blood vessels to escape the hostile circulation and establish metastases at distant organ sites. Here, we identified the membrane-bound metalloprotease ADAM17 on endothelial cells as a key driver of metastasis. We show that TNFR1-dependent tumor cell-induced endothelial cell death, tumor cell extravasation, and subsequent metastatic seeding is dependent on the activity of endothelial ADAM17. Moreover, we reveal that ADAM17-mediated TNFR1 ectodomain shedding and subsequent processing by the gamma-secretase complex is required for the induction of TNF-induced necroptosis. Consequently, genetic ablation of ADAM17 in endothelial cells as well as short-term pharmacological inhibition of ADAM17 prevents long-term metastases formation in the lung. Thus, our data identified ADAM17 as a novel essential regulator of necroptosis and as a new promising target for antimetastatic and advanced-stage cancer therapies.Metastasis is the leading cause of death in cancer patients. We discovered that proteolytic processing of endothelial TNFR1 by the metalloprotease ADAM17 is essential for metastases formation. Based on our findings, we present a novel therapeutic approach to target metastasis.
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(2021) Frontiers in Genetics. 12, 676182. Abstract
The Hippo signaling pathway has been shown to be involved in regulating cellular identity, cell/tissue size maintenance and mechanotransduction. The Hippo pathway consists of a kinase cascade which determines the nucleo-cytoplasmic localization of YAP in the cell. YAP is the effector protein in the Hippo pathway, which acts as a transcriptional cofactor for TEAD. Phosphorylation of YAP upon activation of the Hippo pathway prevents it from entering the nucleus and abrogates its function in the transcription of the target genes. In Cnidaria, the information on the regulatory roles of the Hippo pathway is virtually lacking. Here, we report the existence of a complete set of Hippo pathway core components in Hydra for the first time. By studying their phylogeny and domain organization, we report evolutionary conservation of the components of the Hippo pathway. Protein modelling suggested the conservation of YAP-TEAD interaction in Hydra. Further, we characterized the expression pattern of the homologs of yap, hippo, mob and sav in Hydra using whole-mount RNA in situ hybridization and report their possible role in stem cell maintenance. Immunofluorescence assay revealed that Hvul_YAP expressing cells occur in clusters in the body column and are excluded in the terminally differentiated regions. Actively proliferating cells marked by Ki67 exhibit YAP colocalization in their nuclei. Strikingly, a subset of these colocalized cells is actively recruited to the newly developing bud. Disruption of the YAP-TEAD interaction increased the budding rate indicating a critical role of YAP in regulating cell proliferation in Hydra. Collectively, we posit that the Hippo pathway is an essential signaling system in Hydra; its components are ubiquitously expressed in the Hydra body column and play a crucial role in Hydra tissue homeostasis.
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(2021) JACS Au. 1, 7, p. 1076-1085 Abstract
Although it is well-known that limited local mutations of enzymes, such as matrix metalloproteinases (MMPs), may change enzyme activity by orders of magnitude as well as its stability, the completely rational design of proteins is still challenging. These local changes alter the electrostatic potential and thus local electrostatic fields, which impacts the dynamics of water molecules close the protein surface. Here we show by a combined computational design, experimental, and molecular dynamics (MD) study that local mutations have not only a local but also a global effect on the solvent: In the specific case of the matrix metalloprotease MMP14, we found that the nature of local mutations, coupled with surface morphology, have the ability to influence large patches of the water hydrogen-bonding network at the protein surface, which is correlated with stability. The solvent contribution can be experimentally probed via terahertz (THz) spectroscopy, thus opening the door to the exciting perspective of rational protein design in which a systematic tuning of hydration water properties allows manipulation of protein stability and enzymatic activity.
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(2021) Cancers. 13, 7, 1679. Abstract
Matrix metalloproteases (MMPs) undergo post-translational modifications including pro-domain shedding. The activated forms of these enzymes are effective drug targets, but generating potent biological inhibitors against them remains challenging. We report the generation of anti-MMP-7 inhibitory monoclonal antibody (GSM-192), using an alternating immunization strategy with an active site mimicry antigen and the activated enzyme. Our protocol yielded highly selective anti-MMP-7 monoclonal antibody, which specifically inhibits MMP-7 s enzyme activity with high affinity (IC50 = 132 ± 10 nM). The atomic model of the MMP-7-GSM-192 Fab complex exhibited antibody binding to unique epitopes at the rim of the enzyme active site, sterically preventing entry of substrates into the catalytic cleft. In human PDAC biopsies, tissue staining with GSM-192 showed characteristic spatial distribution of activated MMP-7. Treatment with GSM-192 in vitro induced apoptosis via stabilization of cell surface Fas ligand and retarded cell migration. Co-treatment with GSM-192 and chemotherapeutics, gemcitabine and oxaliplatin elicited a synergistic effect. Our data illustrate the advantage of precisely targeting catalytic MMP-7 mediated disease specific activity.
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(2021) Journal of Dental Research. 100, 2, p. 179-186 Abstract
The periodontal ligament (PDL) plays a critical role in providing immediate response to abrupt high loads during mastication while also facilitating slow remodeling of the alveolar bone. The PDL exceptional functionality is permitted by the unique nonuniform structure of the tissue. Two distinct areas that are critical to PDL function were previously identified: the furcation and the dense collar. Despite their hypothesized functions in tooth movement and maintenance, these 2 regions have not yet been compared within the context of their native environment. Therefore, the objective of this study is to elucidate the extracellular matrix (ECM) structure, composition, and biomechanical function of the furcation and the collar regions while maintaining the 3-dimensional (3D) structure in the murine PDL. We identify significant difference between the collar and furcation regions in both structure and mechanical properties. Specifically, we observed unique longitudinal structures in the dense collar that correlate with type VI collagen and LOX, both of which are associated with increased type I collagen density and tissue stiffness and are therefore proposed to function as scaffolds for tooth stabilization. We also found that the collar region is stiffer than the furcation region and therefore suggest that the dense collar acts as a suspense structure of the tooth within the bone during physiological loading. The furcation region of the PDL contained more proteins associated with reduced stiffness and higher tissue remodeling, as well as a dual mechanical behavior, suggesting a critical function in loads transfer and remodeling of the alveolar bone. In summary, this work unravels the nonuniform nature of the PDL within the 3D structural context and establishes understanding of regional PDL function, which opens new avenues for future studies of remodeling, regeneration, and disease.
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(2021) Matrix Biology. 96, p. 47-68 Abstract
Identification of early processes leading to complex tissue pathologies, such as inflammatory bowel diseases, \u200eposes a major scientific and clinical challenge that is imperative for improved diagnosis and treatment. Most studies of inflammation onset focus on cellular processes and signaling molecules, while overlooking the environment in which they take place, the continuously remodeled extracellular matrix. In this study, we used colitis models for investigating extracellularmatrix dynamics during disease onset, while treating the matrix as a complete and defined entity. Through the analysis of matrix structure, stiffness and composition, we unexpectedly revealed that even prior to the first clinical symptoms, the colon displays its own unique extracellularmatrix signature and found specific markers of clinical potential, which were also validated in human subjects. We also show that the emergence of this pre-symptomatic matrix is mediated by subclinical infiltration of immune cells bearing remodeling enzymes. Remarkably, whether the inflammation is chronic or acute, its matrix signature converges at pre-symptomatic states. We suggest that the existence of a pre-symptomatic extracellularmatrix is general and relevant to a wide range of diseases.
2020
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(2020) Scientific Reports. 10, 1, 19116. Abstract
Various respiratory viral infections in general and seasonal influenza in particular may increase the susceptibility to bacterial infections. Plague caused by Yersinia pestis endangers large populations during outbreaks or bioterrorism attacks. Recommended antibiotic countermeasures include well-established protocols based on animal studies and corroborated by effective treatment of human cases. Until now, prior exposure to viral respiratory infections was not taken into consideration when selecting the appropriate treatment for plague. Here, we show that as late as 25 days after exposure to influenza virus, convalescent mice still exhibited an increased susceptibility to sublethal doses of Y. pestis, presented with aberrant cytokine expression, and impaired neutrophil infiltration in the lungs. Increased levels of M2 alveolar macrophages and type II epithelial cells, as well as induction in metalloproteases expression and collagen and laminin degradation, suggested that the previous viral infection was under resolution, correlating with enhanced susceptibility to plague. Surprisingly, postexposure prophylaxis treatment with the recommended drugs revealed that ciprofloxacin was superior to doxycycline in mice recovering from influenza infection. These results suggest that after an influenza infection, the consequences, such as impaired immunity and lung tissue remodeling and damage, should be considered when treating subsequent Y. pestis exposure.
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(2020) International Journal of Molecular Sciences. 21, 21, p. 1-16 8200. Abstract
Hyaline fibromatosis syndrome (HFS), resulting from ANTXR2 mutations, is an ultra-rare disease that causes intestinal lymphangiectasia and protein-losing enteropathy (PLE). The mechanisms leading to the gastrointestinal phenotype in these patients are not well defined. We present two patients with congenital diarrhea, severe PLE and unique clinical features resulting from deleterious ANTXR2 mutations. Intestinal organoids were generated from one of the patients, along with CRISPR-Cas9 ANTXR2 knockout, and compared with organoids from two healthy controls. The ANTXR2-deficient organoids displayed normal growth and polarity, compared to controls. Using an anthrax-toxin assay we showed that the c.155C>T mutation causes loss-of-function of ANTXR2 protein. An intrinsic defect of monolayer formation in patient-derived or ANTXR2KO organoids was not apparent, suggesting normal epithelial function. However, electron microscopy and second harmonic generation imaging showed abnormal collagen deposition in duodenal samples of these patients. Specifically, collagen VI, which is known to bind ANTXR2, was highly expressed in the duodenum of these patients. In conclusion, despite resistance to anthrax-toxin, epithelial cell function, and specifically monolayer formation, is intact in patients with HFS. Nevertheless, loss of ANTXR2-mediated signaling leads to collagen VI accumulation in the duodenum and abnormal extracellular matrix composition, which likely plays a role in development of PLE.
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(2020) Journal of Experimental Biology. 223, 20, jeb232702. Abstract
The bell-shaped members of the Cnidaria typically move around by swimming, whereas the Hydra polyp can perform locomotion on solid substrates in an aquatic environment. To address the biomechanics of locomotion on rigid substrates, we studied the 'somersaulting' locomotion in Hydra. We applied atomic force microscopy to measure the local mechanical properties of Hydra's body column and identified the existence of differential Young's modulus between the shoulder region versus rest of the body column at 3:1 ratio. We show that somersaulting primarily depends on differential tissue stiffness of the body column and is explained by computational models that accurately recapitulate the mechanics involved in this process. We demonstrate that perturbation of the observed stiffness variation in the body column by modulating the extracellular matrix polymerization impairs the 'somersault' movement. These results provide a mechanistic basis for the evolutionary significance of differential extracellular matrix properties and tissue stiffness.
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(2020) ACS Chemical Neuroscience. 11, 15, p. 2243-2255 Abstract
Oligomers of amyloid β-protein (Aβ) are thought to be the proximal toxic agents initiating the neuropathologic process in Alzheimer's disease (AD). Therefore, targeting the self-assembly and oligomerization of Aβ has been an important strategy for designing AD therapeutics. In parallel, research into the metallobiology of AD has shown that Zn2+ can strongly modulate the aggregation of Aβ in vitro and both promote and inhibit the neurotoxicity of Aβ, depending on the experimental conditions. Thus, successful inhibitors of Aβ self-assembly may have to inhibit the toxicity not only of Aβ oligomers themselves, but also of Aβ-Zn2+ complexes. However, there has been relatively little research investigating the effects of Aβ self-assembly and toxicity inhibitors in the presence of Zn2+. Our group has characterized previously a series of Aβ42 C-terminal fragments (CTFs), some of which have been shown to inhibit Aβ oligomerization and neurotoxicity. Here, we asked whether three CTFs shown to be potent inhibitors of Aβ42 toxicity maintained their activity in the presence of Zn2+. Biophysical analysis showed that the CTFs had different effects on oligomer, β-sheet, and fibril formation by Aβ42-Zn2+ complexes. However, cell viability experiments in differentiated PC-12 cells incubated with Aβ42-Zn2+ complexes in the absence or presence of these CTFs showed that the CTFs completely lost their inhibitory activity in the presence of Zn2+ even when applied at 10-fold excess relative to Aβ42. In light of these results, we tested another inhibitor, the molecular tweezer CLR01, which coincidentally had been shown to have a high affinity for Zn2+, suggesting that it could disrupt both Aβ42 oligomerization and Aβ42-Zn2+ complexation. Indeed, we found that CLR01 effectively inhibited the toxicity of Aβ42-Zn2+ complexes. Moreover, it did so at a lower concentration than needed for inhibiting the toxicity of Aβ42 alone. In agreement with these results, CLR01 inhibited β-sheet and fibril formation in Aβ42-Zn2+ complexes. Our data suggest that for development of efficient therapeutic agents, inhibitors of Aβ self-assembly and toxicity should be examined in the presence of relevant metal ions, and that molecular tweezers may be particularly attractive candidates for therapy development.
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(2020) Cell. 182, 4, p. 1044-1061.e18 Abstract
There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.
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(2020) The FEBS journal. 287, 13, p. 2636-2646 Abstract
The extracellular matrix (ECM) is a key non-cellular component in all organs and tissues. It is composed of a large number of proteins including collagens, glycoproteins (GP), and ECM-associated proteins, which show diversity of biochemical and biophysical functions. The ECM is dynamic both in normal physiology of tissues and under pathological conditions. One cellular phenomenon associated with changes in both ECM components expression and in ECM remodeling enzymes secretion is cellular senescence. It represents a stable state form of cell cycle arrest induced in proliferating cells by various forms of stress. Short-term induction of senescence is essential for tumor suppression and tissue repair. However, long-term presence of senescent cells in tissues may have a detrimental role in promoting tissue damage and aging. Up to date, there is insufficient knowledge about the interplay between the ECM and senescence cells. Since changes in the ECM occur in many physiological and pathological conditions in which senescent cells are present, a better understanding of ECM-senescence interactions is necessary. Here, we will review the functions of the different ECM components and will discuss the current knowledge about their regulation in senescent cells and their influence on the senescence state.
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(2020) Seminars in Cancer Biology. 62, p. 192-200 Abstract
Extracellular matrix (ECM) plays a central and dynamic role in the creation of tumor microenvironment. Herein we discuss the emerging biophysical and biochemical aspects of ECM buildup and proteolysis in cancer niche formation. Dysregulated ECM remodeling by cancer cells facilitate irreversible proteolysis and crosslinking, which in turn influence cell signaling, micro environmental cues, angiogenesis and tissue biomechanics. Further, we introduce the emerging roles of cancer microbiome in aberrant tumor ECM remodeling and membrane bound nano-sized vesicles called exosomes in creation of distant pre-metastatic niches. A detailed molecular and biophysical understanding of the ECM morphologies and its components such as key enzymes, structural and signaling molecules are critical in identifying the next generation of therapeutic and diagnostic targets in cancer.
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(2020) Nature Communications. 11, 1, 1975. Abstract
Treatment paradigms for patients with upper tract urothelial carcinoma (UTUC) are typically extrapolated from studies of bladder cancer despite their distinct clinical and molecular characteristics. The advancement of UTUC research is hampered by the lack of disease-specific models. Here, we report the establishment of patient derived xenograft (PDX) and cell line models that reflect the genomic and biological heterogeneity of the human disease. Models demonstrate high genomic concordance with the corresponding patient tumors, with invasive tumors more likely to successfully engraft. Treatment of PDX models with chemotherapy recapitulates responses observed in patients. Analysis of a HER2 S310F-mutant PDX suggests that an antibody drug conjugate targeting HER2 would have superior efficacy versus selective HER2 kinase inhibitors. In sum, the biological and phenotypic concordance between patient and PDXs suggest that these models could facilitate studies of intrinsic and acquired resistance and the development of personalized medicine strategies for UTUC patients.
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(2020) PLoS ONE. 15, 4, e0231202. Abstract
OBJECTIVE: Monoclonal antibody derivatives are promising drugs for the treatment of various diseases due to their high matrix metalloproteinases (MMP) active site specificity. We studied the effects of a novel antibody, SDS3, which specifically recognizes the mature active site of MMP9/2 during ventricular remodeling progression in a mouse model of chronic volume overload (VO).
METHODS: VO was induced by creating an aortocaval fistula (ACF) in 10- to 12-week-old C57BL male mice. The VO-induced mice were treated with either vehicle control (PBS) or with SDS3 twice weekly by intraperitoneal (ip) injection. The relative changes in cardiac parameters between baseline (day 1) and end-point (day 30), were evaluated by echocardiography. The effects of SDS3 treatment on cardiac fibrosis, cardiomyocyte volume, and cardiac inflammation were tested by cardiac staining with Masson's trichrome, wheat Germ Agglutinin (WGA), and CD45, respectively. Serum levels of TNFα and IL-6 with and without SDS3 treatment were tested by ELISA.
RESULTS: SDS3 significantly reduced cardiac dilatation, left ventricular (LV) mass, and cardiomyocyte hypertrophy compared to the vehicle treated animals. The antibody also reduced the heart-to-body weight ratio of the ACF animals to values comparable to those of the controls. Interestingly, the SDS3 group underwent significant reduction of cardiac inflammation and pro-inflammatory cytokine production, indicating a regulatory role for MMP9/2 in tissue remodeling, possibly by tumor necrosis factor alpha (TNFα) activation. In addition, significant changes in the expression of proteins related to mitochondrial function were observed in ACF animals, these changes were reversed following treatment with SDS3.
CONCLUSION: The data suggest that MMP9/2 blockage with SDS3 attenuates myocardial remodeling associated with chronic VO by three potential pathways: downregulating the extracellular matrix proteolytic cleavage, reducing the cardiac inflammatory responses, and preserving the cardiac mitochondrial structure and function. -
(2020) International Journal of Cancer. 146, 8, p. 2209-2217 Abstract
Pancreatic cancers, both adenocarcinomas and endocrine tumors are characterized by varying levels of aberrant angiogenesis and fibrotic microenvironment. The difficulty to deliver drugs and treat the disease has been attributed in part to the vascular architecture and tissue/ECM density. Here we present longitudinal three-dimensional intravital imaging of vascular and tumor microenvironment remodeling in spontaneous transgenic tumors (RIP1-Tag2 insulinomas) and orthotopically injected tumors (KPC adenocarcinomas). Analysis of the data acquired in insulinomas revealed major differences in tumor blood vessel branching, fraction volume, number of branch points segments, vessel straightness and length compared to the normal tissue. The aggressive adenocarcinoma presented widespread peritumoral vascular remodeling and heterogeneous vascular distribution. Longitudinal imaging was used to acquire sequential vascular remodeling data during tumor progression. This work demonstrates the potential for using a pancreatic intravital imaging window for direct visualization of the tumor heterogenic microenvironments during tumor progression. This article is protected by copyright. All rights reserved.
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(2020) Frontiers in Immunology. 11, 11, p. 480-480 480. Abstract
Liver fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) proteins and enzymes, especially fibrillary collagens, and represents a major cause of morbidity and mortality worldwide. Lysyl oxidases (LOXs) drive covalent crosslinking of collagen fibers, thereby promoting stabilization and accumulation of liver fibrosis while limiting its resolution. Here we show in a carbon tetrachloride (CCl4)-induced liver fibrosis murine model that treatment with a novel anti-lysyl oxidase like 2 (LOXL2) neutralizing antibody, which targets extracellular LOXL2, significantly improves fibrosis resolution. LOXL2 inhibition following the onset of fibrosis accelerated and augmented collagen degradation. This was accompanied by increased localization of reparative monocyte-derived macrophages (MoMFs) in the proximity of fibrotic fibers and their representation in the liver. These cells secreted collagenolytic matrix metalloproteinases (MMPs) and, in particular, the membrane-bound MT1-MMP (MMP-14) collagenase. Inducible and selective ablation of infiltrating MoMFs negated the increased \u201con-fiber\u201d accumulation of MMP-14-expressing MoMFs and the accelerated collagenolytic activity observed in the anti-LOXL2-treated mice. Many studies of liver fibrosis focus on preventing the progression of the fibrotic process. In contrast, the therapeutic mechanism of LOXL2 inhibition presented herein aims at reversing existing fibrosis and facilitating endogenous liver regeneration by paving the way for collagenolytic macrophages.
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(2020) BioRxiv. Abstract
Two murine models for colitis were used to study multi-level changes and derive molecular signatures of colitis onset and development. By combining metabolomics data on tissues and fecal extracts with proteomics data on tissues, we provide a comprehensive picture of the metabolic profile of acute and chronic states of the disease, and most importantly, of two early pre-symptomatic states. We show that, increased anaerobic glycolysis, accompanied by altered TCA cycle and oxidative phosphorylation, associates with inflammation-induced hypoxia taking place in colon tissues. We also demonstrate significant changes in the metabolomic profiles of fecal extracts in different colitis states, most likely associated with the dysbiosis characteristic of colitis, as well as the dysregulated tissue metabolism. Most remarkably, strong and distinctive tissue and fecal metabolomic signatures can be detected before onset of symptoms. These results highlight the diagnostic potential of global metabolomics for inflammatory diseases.
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(2020) Nucleic Acids Research. 29, 24, e122. Abstract
Nucleic acid fragmentation (footprinting) by ·OH radicals is used often as a tool to probe nucleic acid structure and nucleic acid-protein interactions. This method has proven valuable because it provides structural information with single base pair resolution. Recent developments in the field introduced the 'synchrotron X-ray footprinting' method, which uses a high-flux X-ray source to produce single base pair fragmentation of nucleic acid in tens of milliseconds. We developed a complementary method that utilizes X-rays generated from a conventional rotating anode machine in which nucleic acid footprints can be generated by X-ray exposures as short as 100-300ms. Our theoretical and experimental studies indicate that efficient cleavage of nucleic acids by X-rays depends upon sample preparation, energy of the X-ray source and the beam intensity. In addition, using this experimental set up, we demonstrated the feasibility of conducting X-ray footprinting to produce protein-DNA protection portraits at subsecond timescales.
2019
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MMP9 modulates the metastatic cascade and immune landscape for breast cancer anti-metastatic therapy(2019) Life Science Alliance. 2, 6, e201800226. Abstract
Metastasis, the main cause of cancer-related death, has traditionally been viewed as a late-occurring process during cancer progression. Using the MMTV-PyMT luminal B breast cancer model, we demonstrate that the lung metastatic niche is established early during tumorigenesis. We found that matrix metalloproteinase 9 (MMP9) is an important component of the metastatic niche early in tumorigenesis and promotes circulating tumor cells to colonize the lungs. Blocking active MMP9, using a monoclonal antibody specific to the active form of gelatinases, inhibited endogenous and experimental lung metastases in the MMTV-PyMT model. Mechanistically, inhibiting MMP9 attenuated migration, invasion, and colony formation and promoted CD8+ T cell infiltration and activation. Interestingly, primary tumor burden was unaffected, suggesting that inhibiting active MMP9 is primarily effective during the early metastatic cascade. These findings suggest that the early metastatic circuit can be disrupted by inhibiting active MMP9 and warrant further studies of MMP9-targeted anti-metastatic breast cancer therapy.
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(2019) FASEB Journal. 33, 11, p. 11925-11940 Abstract
Meprin β is a membrane-bound metalloprotease involved in extracellular matrix assembly and inflammatory processes in health and disease. A disintegrin and metalloproteinase (ADAM)10 and ADAM17 are physiologic relevant sheddases of inactive promeprin β, which influences its substrate repertoire and subsequent biologic functions. Proteomic analysis also revealed several ADAMs as putative meprin β substrates. Here, we demonstrate specific N-terminal processing of ADAM9, 10, and 17 by meprin β and identify cleavage sites within their prodomains. Because ADAM prodomains can act as specific inhibitors, we postulate a role for meprin β in the regulation of ADAM activities. Indeed, prodomain cleavage by meprin β caused increased ADAM protease activities, as observed by peptide-based cleavage assays and demonstrated by increased ectodomain shedding activity. Direct interaction of meprin β and ADAM proteases could be shown by immunofluorescence microscopy and immunoprecipitation experiments. As demonstrated by a bacterial activator of meprin β and additional measurement of TNF-α shedding on bone marrow-derived macrophages, meprin β/ADAM protease interactions likely influence inflammatory conditions. Thus, we identified a novel proteolytic pathway of meprin β with ADAM proteases to control protease activities at the cell surface as part of the protease web.-Wichert, R., Scharfenberg, F., Colmorgen, C., Koudelka, T., Schwarz, J., Wetzel, S., Potempa, B., Potempa, J., Bartsch, J. W., Sagi, I., Tholey, A., Saftig, P., Rose-John, S., Becker-Pauly, C. Meprin β induces activities of A disintegrin and metalloproteinases 9, 10, and 17 by specific prodomain cleavage.
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(2019) Chemistry-A European Journal. 64, p. 14679-14687 Abstract
Chemiluminescence is being considered an effective imaging modality as it offers low background and high sensitivity. Recent discovery by our group has led to development of new phenoxy-dioxetane chemiluminescence luminophores, which are highly bright under physiological conditions. However, the current scope of probes based on these luminophores is limited, as they can only be turned on by phenol protecting group removal. Here we present a new chemiluminescence resonance energy transfer (CRET) system, Glow-CRET, in which light emission is triggered by proteolytic cleavage of a peptide substrate that links a dioxetane luminophore and a quencher. In order to compose such system, a new phenoxy-dioxetane luminophore, 7-HC-CL, was developed. This luminophore exhibits intense and persistent glow chemiluminescence; it undergoes very slow chemiexcitation, and it has the highest chemiluminescence quantum yield ever reported under physiological conditions. Based on 7-HC-CL, a Glow-CRET probe for matrix metalloproteinases, MMP-CL, was synthesized. Incubation of MMP-CL with its cognate protease resulted in 160-fold increase in chemiluminescence signal. MMP-CL was also able to detect matrix metalloproteinase activity in cancer cells with significantly higher signal-to-background ratio than an analogous fluorescence resonance energy transfer (FRET)-based probe. This work is expected to open new horizons in chemiluminescence imaging, as it enables to use the dioxetanes in ways that had not been possible. We anticipate that 7-HC-CL and future derivatives will be utilized not only for the construction of further Glow-CRET probes, but also for other applications, such as chemiluminescence tagging of proteins.
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(2019) Cancer and Metastasis Reviews. 38, 3, p. 455-468 Abstract
Rapidly increasing scientific reports of exosomes and their biological effects have improved our understanding of their cellular sources and their cell-to-cell communication. These nano-sized vesicles act as potent carriers of regulatory bio-macromolecules and can induce regulatory functions by delivering them from its source to recipient cells. The details of their communication network are less understood. Recent studies have shown that apart from delivering its cargo to the cells, it can directly act on extracellular matrix (ECM) proteins and growth factors and can induce various remodeling events. More importantly, exosomes carry many surface-bound proteases, which can cleave different ECM proteins and carbohydrates and can shed cell surface receptors. These local extracellular events can modulate signaling cascades, which consequently influences the whole tissue and organ. This review aims to highlight the critical roles of exosomal proteases and their mechanistic insights within the cellular and extracellular environment.
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(2019) Cellular and Molecular Life Sciences. 76, 16, p. 3229-3248 Abstract
The extracellular matrix (ECM) plays diverse roles in several physiological and pathological conditions. In the brain, the ECM is unique both in its composition and in functions. Furthermore, almost all the cells in the central nervous system contribute to different aspects of this intricate structure. Brain ECM, enriched with proteoglycans and other small proteins, aggregate into distinct structures around neurons and oligodendrocytes. These special structures have cardinal functions in the normal functioning of the brain, such as learning, memory, and synapse regulation. In this review, we have compiled the current knowledge about the structure and function of important ECM molecules in the brain and their proteolytic remodeling by matrix metalloproteinases and other enzymes, highlighting the special structures they form. In particular, the proteoglycans in brain ECM, which are essential for several vital functions, are emphasized in detail.
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(2019) Carcinogenesis. 41, 4, p. 527-538 Abstract
Lung cancer is the leading cause of cancer-related mortality, with most cases attributed to tobacco smoking, in which nicotine-derived nitrosamine ketone (NNK) is the most potent lung carcinogen. The ADAM17 protease is responsible for the ectodomain shedding of many pro-tumourigenic cytokines, growth factors and receptors, and therefore is an attractive target in cancer. However, the role of ADAM17 in promoting tobacco smoke carcinogen-induced lung carcinogenesis is unknown. The hypomorphic Adam17ex/ex mice - characterized by reduced global ADAM17 expression - were backcrossed onto the NNK-sensitive pseudo-A/J background. CRISPR-driven and inhibitor-based (GW280264X, and ADAM17 prodomain) ADAM17 targeting was employed in the human lung adenocarcinoma cell lines A549 and NCI-H23. Human lung cancer biopsies were also used for analyses. The Adam17ex/ex mice displayed marked protection against NNK-induced lung adenocarcinoma. Specifically, the number and size of lung lesions in NNK-treated pseudo-A/J Adam17ex/ex mice were significantly reduced compared to wild-type littermate controls. This was associated with lower proliferative index throughout the lung epithelium. ADAM17 targeting in A549 and NCI-H23 cells led to reduced proliferative and colony-forming capacities. Notably, among select ADAM17 substrates, ADAM17 deficiency abrogated shedding of the soluble IL-6 receptor (sIL-6R), which coincided with the blockade of sIL-6R-mediated trans-signaling via ERK MAPK cascade. Furthermore, NNK upregulated phosphorylation of p38 MAPK, whose pharmacological inhibition suppressed ADAM17 threonine phosphorylation. Importantly, ADAM17 threonine phosphorylation was significantly upregulated in human lung adenocarcinoma with smoking history compared to their cancer-free controls. Our study identifies the ADAM17/sIL-6R/ERK MAPK axis as a candidate therapeutic strategy against tobacco smoke associated lung carcinogenesis.
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(2019) EMBO Molecular Medicine. 11, 4, 9976. Abstract
Oncogenic KRAS mutations are major drivers of lung adenocarcinoma (LAC), yet the direct therapeutic targeting of KRAS has been problematic. Here, we reveal an obligate requirement by oncogenic KRAS for the ADAM17 protease in LAC. In genetically engineered and xenograft (human cell line and patient-derived) Kras
G12D -driven LAC models, the specific blockade of ADAM17, including with a non-toxic prodomain inhibitor, suppressed tumor burden by reducing cellular proliferation. The pro-tumorigenic activity of ADAM17 was dependent upon its threonine phosphorylation by p38 MAPK, along with the preferential shedding of the ADAM17 substrate, IL-6R, to release soluble IL-6R that drives IL-6 trans-signaling via the ERK1/2 MAPK pathway. The requirement for ADAM17 in Kras
G12D -driven LAC was independent of bone marrow-derived immune cells. Furthermore, in KRAS mutant human LAC, there was a significant positive correlation between augmented phospho-ADAM17 levels, observed primarily in epithelial rather than immune cells, and activation of ERK and p38 MAPK pathways. Collectively, these findings identify ADAM17 as a druggable target for oncogenic KRAS-driven LAC and provide the rationale to employ ADAM17-based therapeutic strategies for targeting KRAS mutant cancers. -
(2019) Collagen. Sagi I. & Afratis N..(eds.). p. 129-133 (trueMethods in Molecular Biology). Abstract
Scanning electron microscopy is a useful tool for high-resolution morphological characterization of the extracellular matrix. In certain tissues and surfaces, imaging of the extracellular matrix requires cell removal before sample preparation. In this protocol, we will describe a method for preparing extracellular matrices derived from murine colon for imaging under a scanning electron microscope.
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(2019) New York, NY: . Vol. 1944. (trueMethods in Molecular Biology). Abstract
This detailed volume compiles state-of-the-art protocols that will serve as recipes for scientists researching collagen, an abundant protein with great importance to health and disease, as well as in applications like food, cosmetics, pharmaceuticals, cosmetic surgery, artificial skin, and glue. Beginning with a section on in vitro models for the characterization of collagen formation, the book continues by highlighting large-scale analysis of collagen with mass spectrometry in order to elucidate the proteomics, degradomics, interactomes, and cross-linking of collagen, high resolution imaging approaches for collagen by the use of scanning electron microscopy and multiphoton imaging, as well as the role of collagen during physiological and pathological conditions. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Collagen: Methods and Protocols is an ideal guide to high quality and repeatable protocols in this vital field of study.
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(2019) The Extracellular Matrix. Theocharis A. D. & Vigetti D.(eds.). Vol. 1952. p. 261-275 (trueMethods in Molecular Biology). Abstract
Extracellular matrix (ECM) macromolecules, apart from structural role for the surrounding tissue, have also been defined as crucial mediators in several cell mechanisms. The proteolytic and cross-linking cascades of ECM have fundamental importance in health and disease, which is increasingly becoming acknowledged. However, formidable challenges remain to identify the diverse and novel role of ECM molecules, especially with regard to their distinct biophysical, biochemical, and structural properties. Considering the heterogeneous, dynamic, and hierarchical nature of ECM, the characterization of 3D functional molecular view of ECM in atomic detail will be very useful for further ECM-related studies. Nowadays, the creation of a pioneer ECM multidisciplinary integrated platform in order to decipher ECM homeostasis is more possible than ever. The access to cutting-edge technologies, such as optical imaging and electron and atomic force microscopies, along with diffraction and X-ray-based spectroscopic methods can integrate spanning wide ranges of spatial and time resolutions. Subsequently, ECM image-guided site-directed proteomics can reveal molecular compositions in defined native and reconstituted ECM microenvironments. In addition, the use of highly selective ECM enzyme inhibitors enables the comparative molecular analyses within pre-classified remodeled ECM microenvironments. Mechanistic information which will be derived can be used to develop novel protein-based inhibitors for effective diagnostic and/or therapeutic modalities targeting ECM reactions within tissue microenvironment.
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(2019) American Journal Of Physiology-Lung Cellular And Molecular Physiology. 316, 1, p. L255-L268 Abstract
Irrespective of its diverse etiologies, acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) leads to increased permeability of the alveolar-capillary barrier, which in turn promotes edema formation and respiratory failure. We investigated the mechanism of ALI/ARDS lung hyperpermeability triggered by pulmonary exposure of mice to the highly toxic plant-derived toxin ricin. One prominent hallmark of ricin-mediated pulmonary intoxication is the rapid and massive influx of neutrophils to the lungs, where they contribute to the developing inflammation yet may also cause tissue damage, thereby promoting ricin-mediated morbidity. Here we show that pulmonary exposure of mice to ricin results in the rapid diminution of the junction proteins VE-cadherin, claudin 5, and connexin 43, belonging, respectively, to the adherens, tight, and gap junction protein families. Depletion of neutrophils in ricin-intoxicated mice attenuated the damage caused to these junction proteins, alleviated pulmonary edema, and significantly postponed the time to death of the intoxicated mice. Inhibition of matrix metalloproteinase (MMP) activity recapitulated the response to neutrophil depletion observed in ricin-intoxicated mice and was associated with decreased insult to the junction proteins and alveolar-capillary barrier. However, neutrophil-mediated MMP activity was not the sole mechanism responsible for pulmonary hyperpermeability, as exemplified by the ricin-mediated disruption of claudin 18, via a neutrophil-independent mechanism involving tyrosine phosphorylation. This in-depth study of the early stage mechanisms governing pulmonary tissue integrity during ALI/ARDS is expected to facilitate the tailoring of novel therapeutic approaches for the treatment of these diseases.
2018
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(2018) Cell Stem Cell. 23, 4, p. 572-585 Abstract
Hematopoietic stem and progenitor cells (HSPCs) tightly couple maintenance of the bone marrow (BM) reservoir, including undifferentiated long-term repopulating hematopoietic stem cells (LT-HSCs), with intensive daily production of mature leukocytes and blood replenishment. We found two daily peaks of BM HSPC activity that are initiated by onset of light and darkness providing this coupling. Both peaks follow transient elevation of BM norepinephrine and TNF secretion, which temporarily increase HSPC reactive oxygen species (ROS) levels. Light-induced norepinephrine and TNF secretion augments HSPC differentiation and increases vascular permeability to replenish the blood. In contrast, darkness-induced TNF increases melatonin secretion to drive renewal of HSPCs and LT-HSC potential through modulating surface CD150 and c-Kit expression, increasing COX-2/αSMA
+ macrophages, diminishing vascular permeability, and reducing HSPC ROS levels. These findings reveal that light- and darkness-induced daily bursts of norepinephrine, TNF, and melatonin within the BM are essential for synchronized mature blood cell production and HSPC pool repopulation. Golan et al. report that daily onsets of light and dark induce NE and TNF bursts that induce two different peaks of BM HSPC activity. Light-induced NE promotes HSPC differentiation and egress, replenishing mature blood cells. Dark-induced TNF promotes melatonin-dependent renewal of CD150
+ HSCs and their long-term repopulation potential. -
(2018) Matrix Biology. 68-69, p. 167-179 Abstract
Fibrosis is the extensive accumulation and buildup of extracellular matrix components, especially fibrillar collagens, during wound healing in response to tissue injury. During all individual stages of fibrosis ECM proteases, mainly matrix metalloproteinases, have diverse roles. The functional role of MMPs and their endogenous inhibitors are differentiated among their family members, and according to the different stages of fibrosis. MMPs levels are elevated in several inflammatory and non-inflammatory fibrotic tissues contributing to the development, progression or resolution of the disease, whereas in other tissues their expression levels can be diminished or be stable to the baseline. The biological roles of MMPs during fibrosis are not fully resolved, but they seem to differ according the specific member of the family, the affected tissue and the stage of the fibrotic response. Remarkably, some members of the family exhibit profibrotic actions while other function as antifibrotic molecules. Diverse animal models indicate that MMPs are contributing in processes related to immunity, tissue repair and ECM turnover, providing significant impact on mechanisms related to fibrosis. For that purpose, these proteases are considered as pharmacological targets and new biological drugs have been developed in order to treat fibrosis.
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(2018) Advanced Drug Delivery Reviews. 129, p. 4-15 Abstract
Progressive loss of organ function in most organs is associated with fibrosis, a tissue state associated with abnormal matrix buildup. If highly progressive, the fibrotic process eventually leads to organ failure and death. Fibrosis is a basic connective tissue lesion defined by the increase in the amount of fibrillar extracellular matrix (ECM) components in a tissue or organ. In addition, intrinsic changes in important structural cells can induce the fibrotic response by regulating the differentiation, recruitment, proliferation and activation of extracellular matrix-producing myofibroblasts. ECM enzymes belonging to the family of matrix metalloproteinases (MMPs) and lysyl oxidases (LOXs) play a crucial role in ECM remodeling and regeneration. MMPs have a catalytic role in degradation of ECM, whereas LOX/LOXLs mediate ECM, especially collagen, cross-linking and stiffening. Importantly, enzymes from both families are elevated during the fibrotic response to tissue injury and its resolution. Yet, the apparent molecular competition or antagonistic activities of these enzyme families during the various stages of fibrosis is often overlooked. In this review, we discuss the diverse roles of MMPs and LOX/LOXL2 in chronic organ fibrosis. Finally, we review contemporary therapeutic strategies for fibrosis treatment, based on neutralization of MMP and LOX activity, as well as the development of novel drug delivery approaches.
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(2018) The FEBS journal. 285, 4, p. 734-751 Abstract
Phagocytes, such as tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs), are abundant in the stroma of experimental and human tumors and are locally educated to mediate important biological functions that profoundly affect tumor initiation, growth, and dissemination. Of considerable importance is the noncellular component of the tumor microenvironment, namely-the extracellular matrix (ECM). This milieu is often overlooked due to its complexity and vast heterogeneity. Biophysical and biomechanical cues provided by the dynamically evolving tumorigenic ECM fundamentally modulate every behavioral facet of the cancer cells and of associated stromal cells. In this review, we discuss the intricate interplay between phagocytes and ECM that are lined up to support tumor progression. TAMs and TANs shape the tumorigenic ECM by providing key matrix-remodeling enzymes and structural proteins and in turn, the altered tumor ECM modulates their migration and function. A better mechanistic comprehension of this reciprocal dependence has exciting implications for the development of new therapeutic options for cancer.
2017
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(2017) Physical Chemistry Chemical Physics. 19, 45, p. 30316-30331 Abstract
Matrix metalloproteinases (MMP) are an important family of proteases which catalyze the degradation of extracellular matrix components. While the mechanism of peptide cleavage is well established, the process of enzyme regeneration, which represents the rate limiting step of the catalytic cycle, remains unresolved. This step involves the loss of the newly formed N-terminus (amine) and C-terminus (carboxylate) protein fragments from the site of catalysis coupled with the inclusion of one or more solvent waters. Here we report a novel crystal structure of membrane type I MMP (MT1-MMP or MMP-14), which includes a small peptide bound at the catalytic Zn site via its C-terminus. This structure models the initial product state formed immediately after peptide cleavage but before the final proton transfer to the bound amine; the amine is not present in our system and as such proton transfer cannot occur. Modeling of the protein, including earlier structural data of Bertini and coworkers [I. Bertini, et al., Angew. Chem., Int. Ed., 2006, 45, 7952-7955], suggests that the C-terminus of the peptide is positioned to form an H-bond network to the amine site, which is mediated by a single oxygen of the functionally important Glu240 residue, facilitating efficient proton transfer. Additional quantum chemical calculations complemented with magneto-optical and magnetic resonance spectroscopies clarify the role of two additional, non-catalytic first coordination sphere waters identified in the crystal structure. One of these auxiliary waters acts to stabilize key intermediates of the reaction, while the second is proposed to facilitate C-fragment release, triggered by protonation of the amine. Together these results complete the enzymatic cycle of MMPs and provide new design criteria for inhibitors with improved efficacy.
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(2017) Biochimica et Biophysica Acta - Molecular Cell Research. 1864, 11, p. 2220-2227 Abstract
Cutaneous wound healing is a complex mechanism with multiple processes orchestrating harmoniously for structural and functional restoration of the damaged tissue. Chronic non-healing wounds plagued with infection create a major healthcare burden and is one of the most frustrating clinical problems. Chronic wounds are manifested by prolonged inflammation, defective re-epithelialization and haphazard remodeling. Matrix metalloproteinases (MMPs) are zinc dependent enzymes that play cardinal functions in wound healing. Understanding the pathological events mediated by MMPs during wound healing may pave way in identifying novel drug targets for chronic wounds. Here, we discuss the functions and skewed regulation of different MMPs during infection and chronic tissue repair. This review also points out the potential of MMPs and their inhibitors as therapeutic agents in treating chronic wounds during distinct phases of the wound healing. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.
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(2017) Biochimica et Biophysica Acta - Molecular Cell Research. 1864, 11, p. 1927-1939 Abstract
Enzymatic proteolysis of cell surface proteins and extracellular matrix (ECM) is critical for tissue homeostasis and cell signaling. These proteolytic activities are mediated predominantly by a family of proteases termed matrix metalloproteinases (MMPs). The growing evidence in recent years that ECM and non-ECM bioactive molecules (e.g., growth factors, cytokines, chemokines, on top of matrikines and matricryptins) have versatile functions redefines our view on the roles matrix remodeling enzymes play in many physiological and pathological processes, and underscores the notion that ECM proteolytic reaction mechanisms represent master switches in the regulation of critical biological processes and govern cell behavior. Accordingly, MMPs are not only responsible for direct degradation of ECM molecules but are also key modulators of cardinal bioactive factors. Many attempts were made to manipulate ECM degradation by targeting MMPs using small peptidic and organic inhibitors. However, due to the high structural homology shared by these enzymes, the majority of the developed compounds are broad-spectrum inhibitors affecting the proteolytic activity of various MMPs and other zinc-related proteases. These inhibitors, in many cases, failed as therapeutic agents, mainly due to the bilateral role of MMPs in pathological conditions such as cancer, in which MMPs have both pro- and anti-tumorigenic effects. Despite the important role of MMPs in many human diseases, none of the broad-range synthetic MMP inhibitors that were designed have successfully passed clinical trials. It appears that, designing highly selective MMP inhibitors that are also effective in vivo, is not trivial. The challenges related to designing selective and effective metalloprotease inhibitors, are associated in part with the aforesaid high structural homology and the dynamic nature of their protein scaffolds. Great progress was achieved in the last decade in understanding the biochemistry and biology of MMPs activity. This knowledge, combined with lessons from the past has drawn new \u201cboundaries\u201d for the development of the next-generation MMP inhibitors. These novel agents are currently designed to be highly specific, capable to discriminate between the homologous MMPs and ideally administered as a short-term topical treatment. In this review we discuss the latest progress in the fields of MMP inhibitors in terms of structure, function and their specific activity. The development of novel highly specific inhibitors targeting MMPs paves the path to study complex biological processes associated with ECM proteolysis in health and disease. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.
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(2017) Oncogene. 36, 40, p. 5639-5647 Abstract
Our increasing knowledge of the mechanisms behind the progression of pancreatic cancer (PC) has not yet translated into effective treatments. Many promising drugs have failed in the clinic, highlighting the need for better preclinical models to assess drug efficacy and characterize mechanisms of resistance. Using different experimental models, including patient-derived xenografts (PDXs), we gauged the efficacy of therapies aimed at two hallmark lesions of PCs: Activation of signaling pathways by oncogenic KRAS and inactivation of tumor-suppressor genes. Although the drug targeting inactivation of tumor suppressors by DNA methylation had little effect, the inhibition of Mek, a K-Ras effector, in combination with the standard of care (chemotherapy consisting of gemcitabine/Nab-paclitaxel), reduced the growth of three out of five PC-PDXs and impaired metastasis. The two least responding PC-PDXs were composed of genetically diverse cells, which displayed sensitivities to the Mek inhibitor differing by >10-fold. Unexpectedly, our analysis of this genetic diversity unveiled different KRAS mutations. As mutation in KRAS occurs early during progression, this heterogeneity may reflect the simultaneous appearance of different malignant cellular clones or, alternatively, that cells containing two mutations of KRAS are selected during tumor evolution. In vitro and in vivo analyses indicated that the intratumoral heterogeneity, along with the selective pressure imposed by the Mek inhibitor, resulted in rapid selection of resistant cells. Together with the gemcitabine/Nab-paclitaxel backbone, Mek inhibition could be effective in treatment of PC. However, resistance because of intratumoral heterogeneity is likely to develop frequently, pointing to the necessity of identifying the factors and mechanisms of resistance to further develop this therapy.
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(2017) Proceedings Of The National Academy Of Sciences Of The United States Of America-Physical Sciences. 114, 42, p. 11139-11144 Abstract
Metal ions play essential roles in many aspects of biological chemistry. Detecting their presence and location in proteins and cells is important for understanding biological function. Conventional structural methods such as X-ray crystallography and cryo-transmission electron microscopy can identify metal atoms on protein only if the protein structure is solved to atomic resolution. We demonstrate here the detection of isolated atoms of Zn and Fe on ferritin, using cryogenic annular dark-field scanning transmission electron microscopy (cryo-STEM) coupled with single-particle 3D reconstructions. Zn atoms are found in a pattern that matches precisely their location at the ferroxidase sites determined earlier by X-ray crystallography. By contrast, the Fe distribution is smeared along an arc corresponding to the proposed path from the ferroxidase sites to the mineral nucleation sites along the twofold axes. In this case the single-particle reconstruction is interpreted as a probability distribution function based on the average of individual locations. These results establish conditions for detection of isolated metal atoms in the broader context of electron cryo-microscopy and tomography.
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(2017) Frontiers in Molecular Biosciences. 4, AUG, 61. Abstract
Inflammatory bowel disease (IBD) is a multifactorial disease characterized by the dysregulated activity of many pro-inflammatory factors. Thus, bi-specific inhibitors for the simultaneous inhibition of two pro-inflammatory factors can exhibit high therapeutic potential. Here, we developed a novel bi-specific inhibitor targeting the TL1A cytokine and ADAM17/TACE metalloprotease. Biochemical analysis of the bi-specific inhibitor revealed high TL1A binding and TACE inhibition that is similar to the two respective mono-specific inhibitors. Interestingly, cell based assays for TL1A inhibition revealed strong synergism between the inhibitory domains showing an up to 80-fold increase in potency of the bi-specific inhibitor. The dramatic increase in potency is associated with binding to cell membranes through the TACE inhibitory domain leading to increased concentration of the inhibitor on the cell surface. Our study highlights the high potential of the simultaneous targeting of cell surface metalloprotease (TACE) and soluble pro-inflammatory cytokine (TL1A) as a potential therapeutic approach in IBD.
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(2017) Nature. 547, 7662, p. 179-184 Abstract
The adult mammalian heart is non-regenerative owing to the post-mitotic nature of cardiomyocytes. The neonatal mouse heart can regenerate, but only during the first week of life. Here we show that changes in the composition of the extracellular matrix during this week can affect cardiomyocyte growth and differentiation in mice. We identify agrin, a component of neonatal extracellular matrix, as required for the full regenerative capacity of neonatal mouse hearts. In vitro, recombinant agrin promotes the division of cardiomyocytes that are derived from mouse and human induced pluripotent stem cells through a mechanism that involves the disassembly of the dystrophin-glycoprotein complex, and Yap- and ERK-mediated signalling. In vivo, a single administration of agrin promotes cardiac regeneration in adult mice after myocardial infarction, although the degree of cardiomyocyte proliferation observed in this model suggests that there are additional therapeutic mechanisms. Together, our results uncover a new inducer of mammalian heart regeneration and highlight fundamental roles of the extracellular matrix in cardiac repair.
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(2017) Cell Reports. 19, 4, p. 774-784 Abstract
Surgery remains the most successful curative treatment for cancer. However, some patients with early-stage disease who undergo surgery eventually succumb to distant metastasis. Here, we show that in response to surgery, the lungs become more vulnerable to metastasis due to extracellular matrix remodeling. Mice that undergo surgery or that are preconditioned with plasma from donor mice that underwent surgery succumb to lung metastases earlier than controls. Increased lysyl oxidase (LOX) activity and expression, fibrillary collagen crosslinking, and focal adhesion signaling contribute to this effect, with the hypoxic surgical site serving as the source of LOX. Furthermore, the lungs of recipient mice injected with plasma from post-surgical colorectal cancer patients are more prone to metastatic seeding than mice injected with baseline plasma. Downregulation of LOX activity or levels reduces lung metastasis after surgery and increases survival, highlighting the potential of LOX inhibition in reducing the risk of metastasis following surgery.
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(2017) Journal of Biological Chemistry. 292, 8, p. 3481-3495 Abstract
Degradation of the extracellular matrices in the human body is controlled by matrix metalloproteinases (MMPs), a family of more than 20 homologous enzymes. Imbalance in MMP activity can result in many diseases, such as arthritis, cardiovascular diseases, neurological disorders, fibrosis, and cancers. Thus, MMPs present attractive targets for drug design and have been a focus for inhibitor design for as long as 3 decades. Yet, to date, all MMP inhibitors have failed in clinical trials because of their broad activity against numerous MMP family members and the serious side effects of the proposed treatment. In this study, we integrated a computational method and a yeast surface display technique to obtain highly specific inhibitors of MMP-14 by modifying the natural non-specific broad MMP inhibitor protein N-TIMP2 to interact optimally with MMP-14. We identified an N-TIMP2 mutant, with five mutations in its interface, that has an MMP-14 inhibition constant (K-i) of 0.9 pm, the strongest MMP-14 inhibitor reported so far. Compared with wild-type N-TIMP2, this variant displays approximate to 900-fold improved affinity toward MMP-14 and up to 16,000-fold greater specificity toward MMP-14 relative to other MMPs. In an in vitro and cell-based model of MMP-dependent breast cancer cellular invasiveness, this N-TIMP2 mutant acted as a functional inhibitor. Thus, our study demonstrates the enormous potential of a combined computational/directed evolution approach to protein engineering. Furthermore, it offers fundamental clues into the molecular basis of MMP regulation by N-TIMP2 and identifies a promising MMP-14 inhibitor as a starting point for the development of protein-based anticancer therapeutics.
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(2017) Journal of Physical Chemistry B. 121, 21, p. 5340-5346 Abstract
The dielectric relaxation of hydrated collagen powders was studied over a wide temperature and frequency range. We revealed two mechanisms of dielectric relaxation in hydration water that are driven by the migration of ionic and orientation defects. At high water fractions in powders (h > 0.2), the hydration shell around the collagen triple helixes presents a spatial H-bonded network consisting of structural water bridges and cleft water channels. These two water phases provide the long-range paths for proton hopping and orientation defect migration. At low water fractions (h
2016
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(2016) Scientific Reports. 6, 35598. Abstract
Dysregulated activity of A Disintegrin And Metalloproteinase 17 (ADAM17)/TNF alpha Converting Enzyme (TACE) is associated with inflammatory disorders and cancer progression by releasing regulatory membrane-tethered proteins like TNF alpha, IL6R and EGFR ligands. Although specific inhibition of TACE is thought to be a viable strategy for inflammatory disorders and for malignancies treatment, the generation of effective inhibitors in vivo has been proven to be challenging. Here we report on the development of a protein inhibitor that leverages the endogenous modulator of TACE. We have generated a stable form of the auto-inhibitory TACE prodomain (TPD), which specifically inhibits in vitro and cell-surface TACE, but not the related ADAM10, and effectively modulated TNF alpha secretion in cells. TPD significantly attenuated TACE-mediated disease models of sepsis, rheumatoid arthritis (RA) and inflammatory bowel disease (IBD), and reduced TNFa in synovial fluids from RA patients. Our results demonstrate that intervening with endogenous ADAM sheddase modulatory mechanisms holds potential as a general strategy for the design of ADAM inhibitors.
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(2016) Journal of Experimental Medicine. 213, 11, p. 2315-2331 Abstract
Tumor-associated macrophages (TAMs) promote tumor development, invasion, and dissemination by various mechanisms. In this study, using an orthotopic colorectal cancer (CRC) model, we found that monocyte-derived TAMs advance tumor development by the remodeling of its extracellular matrix (ECM) composition and structure. Unbiased transcriptomic and proteomic analyses of (a) TAM-abundant and -deficient tumor tissues and (b) sorted tumor-associated and -resident colonic macrophage subpopulations defined a distinct TAM-induced ECM molecular signature composed of an ensemble of matricellular proteins and remodeling enzymes they provide to the tumor microenvironment. Remarkably, many of these ECM proteins are specifically increased in human CRC versus healthy colon. Specifically, we demonstrate that although differentiating into TAMs, monocytes up-regulate matrix-remodeling programs associated with the synthesis and assembly of collagenous ECM, specifically collagen types I, VI, and XIV. This finding was further established by advanced imaging showing that TAMs instruct the deposition, cross-linking, and linearization of collagen fibers during tumor development, especially at areas of tumor invasiveness. Finally, we show that cancer-associated fibroblasts are significantly outnumbered by TAMs in this model and that their expression of collagen XIV and I is reduced by TAM deficiency. Here, we outline a novel TAM protumoral function associated with building of the collagenous ECM niche.
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(2016) Cell Host & Microbe. 20, 4, p. 458-470 Abstract
Mounting an effective immune response, while also protecting tissue integrity, is critical for host survival. We used a combined genomic and proteomic approach to investigate the role of extracellular matrix (ECM) proteolysis in achieving this balance in the lung during influenza virus infection. We identified the membrane-tethered matrix metalloprotease MT1-MMP as a prominent host-ECM-remodeling collagenase in influenza infection. Selective inhibition of MT1-MMP protected the tissue from infection-related structural and compositional tissue damage. MT1-MMP inhibition did not significantly alter the immune response or cytokine expression. The available flu therapeutic Oseltamivir did not prevent lung ECM damage and was less effective than anti-MT1-MMP in influenza virus Streptococcus pneumoniae coinfection paradigms. Combination therapy of Oseltamivir with anti-MT1-MMP showed a strong synergistic effect and resulted in complete recovery of infected mice. This study highlights the importance of tissue resilience in surviving infection and the potential of such host-pathogen therapy combinations for respiratory infections.
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(2016) Proceedings of the National Academy of Sciences of the United States of America. 113, 39, p. 10884-10889 Abstract
It is well established that the expression profiles of multiple and possibly redundant matrix-remodeling proteases (e.g., collagenases) differ strongly in health, disease, and development. Although enzymatic redundancy might be inferred from their close similarity in structure, their in vivo activity can lead to extremely diverse tissueremodeling outcomes. We observed that proteolysis of collagen-rich natural extracellular matrix (ECM), performed uniquely by individual homologous proteases, leads to distinct events that eventually affect overall ECM morphology, viscoelastic properties, and molecular composition. We revealed striking differences in the motility and signaling patterns, morphology, and gene-expression profiles of cells interacting with natural collagen-rich ECM degraded by different collagenases. Thus, in contrast to previous notions, matrix-remodeling systems are not redundant and give rise to precise ECM-cell crosstalk. Because ECM proteolysis is an abundant biochemical process that is critical for tissue homoeostasis, these results improve our fundamental understanding its complexity and its impact on cell behavior.
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(2016) Africa Insight. 1, 13, e87023. Abstract
Kidney fibrosis following kidney injury is an unresolved health problem and causes significant morbidity and mortality worldwide. In a study into its molecular mechanism, we identified essential causative features. Acute or chronic kidney injury causes sustained elevation of a disintegrin and metalloprotease 17 (ADAM17); of its cleavage-activated proligand substrates, in particular of pro-TNFα and the EGFR ligand amphiregulin (pro-AREG); and of the substrates' receptors. As a consequence, EGFR is persistently activated and triggers the synthesis and release of proinflammatory and profibrotic factors, resulting in macrophage/neutrophil ingress and fibrosis. ADAM17 hypomorphic mice, specific ADAM17 inhibitor-treated WT mice, or mice with inducible KO of ADAM17 in proximal tubule (Slc34a1-Cre) were significantly protected against these effects. In vitro, in proximal tubule cells, we show that AREG has unique profibrotic actions that are potentiated by TNFα-induced AREG cleavage. In vivo, in acute kidney injury (AKI) and chronic kidney disease (CKD, fibrosis) patients, soluble AREG is indeed highly upregulated in human urine, and both ADAM17 and AREG expression show strong positive correlation with fibrosis markers in related kidney biopsies. Our results indicate that targeting of the ADAM17 pathway represents a therapeutic target for human kidney fibrosis.
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(2016) Cancer Res.. 76, 14, 052206 . Abstract
Abnormal architectures of collagen fibers in the extracellular matrix (ECM) are hallmarks of many invasive diseases, including cancer. Targeting specific stages of collagen assembly in vivo presents a great challenge due to the involvement of various crosslinking enzymes in the multistep, hierarchical process of ECM build-up. Using advanced microscopic tools, we monitored stages of fibrillary collagen assembly in a native fibroblast-derived 3D matrix system and identified anti-lysyl oxidase-like 2 (LOXL2) antibodies that alter the natural alignment and width of endogenic fibrillary collagens without affecting ECM composition. The disrupted collagen morphologies interfered with the adhesion and invasion properties of human breast cancer cells. Treatment of mice bearing breast cancer xenografts with the inhibitory antibodies resulted in disruption of the tumorigenic collagen superstructure and in reduction of primary tumor growth. Our approach could serve as a general methodology to identify novel therapeutics targeting fibrillary protein organization to treat ECMassociated pathologies.
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(2016) Metalloproteinases In Medicine. 3, p. 31-47 Abstract
The development of therapeutic matrix metalloproteinase (MMP) inhibitors has evolved from broad-spectrum peptidomimetic inhibitors with deleterious side effects, to highly selective agents. These range from small molecules to antibodies, antisense inhibitors, and engineered N-terminal tissue inhibitors of metalloproteinase domain. The advances in inhibitor design along with promising new global molecular insights into MMP structures, the protease web, and the role of extracellular matrix in diseases have contributed toward a renewed interest in using MMPs as valid drug targets. This review aims to address the advances and challenges concerning the design, development, and current status of anti-MMP agents in this new era of post-broad-spectrum MMP inhibitors. Highly selective inhibitors of MMPs promise to usher in an era of specific targeting of diseased tissue proteolysis networks, with markedly reduced negative repercussions, and to uncover the molecular and mechanistic roles of MMP isoforms in cancer, inflammation, and infection.
2015
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(2015) Nature Medicine. 21, 11, p. 1307-1317 Abstract
Retention of long-term repopulating hematopoietic stem cells (LT-HSCs) in the bone marrow is essential for hematopoiesis and for protection from myelotoxic injury. We report that signaling cascades that are traditionally viewed as coagulation related also control retention of endothelial protein C receptor-positive (EPCR +) LT-HSCs in the bone marrow and their recruitment to the blood via two pathways mediated by protease activated receptor 1 (PAR1). Thrombin-PAR1 signaling induces nitric oxide (NO) production, leading to EPCR shedding mediated by tumor necrosis factor-α-converting enzyme (TACE), enhanced CXCL12-CXCR4-induced motility and rapid stem and progenitor cell mobilization. Conversely, bone marrow blood vessels provide a microenvironment enriched with activated protein C (aPC) that retains EPCR + LT-HSCs by limiting NO generation, reducing Cdc42 activity and enhancing integrin VLA4 affinity and adhesion. Inhibition of NO production by aPC-EPCR-PAR1 signaling reduces progenitor cell egress from the bone marrow, increases retention of bone marrow NO low EPCR + LT-HSCs and protects mice from chemotherapy-induced hematological failure and death. Our study reveals new roles for PAR1 and EPCR in controlling NO production to balance maintenance and recruitment of bone marrow EPCR + LT-HSCs, with potential clinical relevance for stem cell transplantation.
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(2015) Nature Immunology. 16, 7, p. 737-745 Abstract
Aire is a transcriptional regulator that induces the promiscuous expression of thousands of tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs), a step critical for the induction of immunological self-tolerance. Studies have offered molecular insights into how Aire operates, but more comprehensive understanding of this process still remains elusive. Here we found abundant expression of the protein deacetylase Sirtuin-1 (Sirt1) in mature Aire + mTECs, wherein it was required for the expression of Aire-dependent TRA-encoding genes and the subsequent induction of immunological self-tolerance. Our study elucidates a previously unknown molecular mechanism for Aire-mediated transcriptional regulation and identifies a unique function for Sirt1 in preventing organ-specific autoimmunity.
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(2015) Matrix Biology. 44-46, p. 191-199 Abstract
The matrix metalloproteinases (MMPs) play a crucial role in irreversible remodeling of the extracellular matrix (ECM) in normal homeostasis and pathological states. Accumulating data from various studies strongly suggest that MMPs are tightly regulated, starting from the level of gene expression all the way to zymogen activation and endogenous inhibition, with each level controlled by multiple factors. Recent in vivo findings indicate that cell-ECM and cell-cell interactions, as well as ECM bio-active products, contribute an additional layer of regulation at all levels, indicating that individual MMP expression and activity in vivo are highly coordinated and tissue specific processes.
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(2015) Abstract
Discussing recent advances in the field of matrix metalloproteinase (MMP) research from a multidisciplinary perspective, Matrix Metalloproteinase Biologyis a collection of chapters written by leaders in the field of MMPs. The book focuses on the challenges of understanding the mechanisms substrate degradation by MMPs, as well as how these enzymes are able to degrade large, highly ordered substrates such as collagen. All topics addressed are considered in relation to disease progression including roles in cancer metastasis, rheumatoid arthritis and other inflammatory diseases. The text first provides an overview of MMPs, focusing on the history, the development and failures of small molecule inhibitors in clinical trials, and work with TIMPS, the endogenous inhibitors of MMPs. These introductory chapters establish the foundation for later discussion of the recent progress on the design of different types of inhibitors, including novel antibody based therapeutics. The following section emphasizes research using novel methods to further the study of the MMPs. The third and final section focuses on in vivo research, particularly with respect to cancer models, degradation of the extracellular matrix, and MMP involvement in other disease states.
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(2015) The journal of Biological chemistry. 290, 19, p. 12135-12146 Abstract
Proenzyme maturation is a general mechanism to control the activation of enzymes. Catalytically active members of the A Disintegrin And Metalloprotease (ADAM) family of membrane-anchored metalloproteases are synthesized as proenzymes, in which the latency is maintained by their autoinhibitory pro-domains. A proteolytic processing then transforms the proenzyme into a catalytically active form. The removal of the pro-domain of ADAMs is currently thought to depend on processing at a canonical consensus site for the proprotein convertase Furin (RXXR) between the pro- and the catalytic domain. Here, we demonstrate that this previously described canonical site is a secondary cleavage site to a prerequisite cleavage in a newly characterized upstream PC site embedded within the pro-domain sequence. The novel upstream regulatory site is important for the maturation of several ADAM proenzymes. Mutations in the upstream regulatory site of ADAM17, ADAM10, and ADAM9 do not prevent pro-domain processing between the pro- and metalloprotease domain, but nevertheless, cause significantly reduced catalytic activity. Thus, our results have uncovered a novel functionally relevant PC processing site in the N-terminal part of the pro-domain that is important for the activation of these ADAMs. These results suggest that the novel PC site is part of a general mechanism underlying proenzyme maturation of ADAMs that is independent of processing at the previously identified canonical Furin cleavage site.
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(2015) Biochemical Journal. 465, p. 259-270 Abstract
Gelatinase B/matrix metalloproteinase-9 (MMP-9) (EC 3.4.24.35) cleaves many substrates and is produced by most cell types as a zymogen, proMMP-9, in complex with the tissue inhibitor of metalloproteinases-1 (TIMP-1). Natural proMMP-9 occurs as monomers, homomultimers and heterocomplexes, but our knowledge about the overall structure of proMMP-9 monomers and multimers is limited. We investigated biochemical, biophysical and functional characteristics of zymogen and activated forms of MMP-9 monomers and multimers. In contrast with a conventional notion of a dimeric nature of MMP-9 homomultimers, we demonstrate that these are reduction-sensitive trimers. Based on the information from electrophoresis, AFM and TEM, we generated a 3D structure model of the proMMP-9 trimer. Remarkably, the proMMP-9 trimers possessed a 50-fold higher affinity for TIMP-1 than the monomers. In vivo, this finding was reflected in a higher extent of TIMP-1 inhibition of angiogenesis induced by trimers compared with monomers. Our results show that proMMP-9 trimers constitute a novel structural and functional entity that is differentially regulated by TIMP-1.
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(2015) Oncotarget. 6, 42, p. 44151-44160 Abstract
Genetic deficiencies provide insights into gene function in humans. Here we describe a patient with a very rare genetic deficiency of ADAM17. We show that the patient's PBMCs had impaired cytokine secretion in response to LPS stimulation, correlating with the clinical picture of severe bacteremia from which the patient suffered. ADAM17 was shown to cleave CD16, a major NK killer receptor. Functional analysis of patient's NK cells demonstrated that his NK cells express normal levels of activating receptors and maintain high surface levels of CD16 following mAb stimulation. Activation of individual NK cell receptors showed that the patient's NK cells are more potent when activated directly by CD16, albeit no difference was observed in Antibody Depedent Cytotoxicity (ADCC) assays. Our data suggest that ADAM17 inhibitors currently considered for clinical use to boost CD16 activity should be cautiously applied, as they might have severe side effects resulting from impaired cytokine secretion.
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(2015) Development (Cambridge). 142, 5, p. 983-993 Abstract
Muscle is an integrated tissue composed of distinct cell types and extracellular matrix. While much emphasis has been placed on the factors required for the specification of the cells that comprise muscle, little is known about the crosstalk between them that enables the development of a patterned and functional tissue.We find in mice that deletion of lysyl oxidase (Lox), an extracellular enzyme regulating collagen maturation and organization, uncouples the balance between the amount of myofibers and that of muscle connective tissue (MCT). We show that Lox secreted from the myofibers attenuates TGFβ signaling, an inhibitor of myofiber differentiation and promoter of MCT development. We further demonstrate that a TGFβ-Lox feedback loop between the MCT and myofibers maintains the dynamic developmental homeostasis between muscle components while also regulating MCT organization. Our results allow a better understanding of diseases such as Duchenne muscular dystrophy, in which LOX and TGFβ signaling have been implicated and the balance between muscle constituents is disturbed.
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(2015) Structure. 23, 1, p. 104-115 Abstract
Membrane type 1 metalloprotease (MT1-MMP) is a membrane-anchored, zinc-dependent protease. MT1-MMP is an important mediator of cell migration and invasion, and overexpression of this enzyme has been correlated with the malignancy of various tumor types. Therefore, modulators of MT1-MMP activity are proposed to possess therapeutic potential in numerous invasive diseases. Here we report the inhibition mode of MT1-MMP by LEM-2/15 antibody, which targets a surface epitope of MT1-MMP. Specifically, the crystal structures of Fab LEM-2/15 in complex with the MT1-MMP surface antigen suggest that conformational swiveling of the enzyme surface loop is required for effective binding and consequent inhibition of MT1-MMP activity on the cell membrane. This inhibition mechanism appears to be effective in controlling active MT1-MMP in endothelial cells and at the leading edge of migratory cancer cells.
2014
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(2014) Proceedings of the National Academy of Sciences of the United States of America. 111, 50, p. 17857-17862 Abstract
The main focus of enzymology is on the enzyme rates, substrate structures, and reactivity, whereas the role of solvent dynamics in mediating the biological reaction is often left aside owing to its complex molecular behavior. We used integrated X-ray- and terahertz-based time-resolved spectroscopic tools to study protein-water dynamics during proteolysis of collagen-like substrates by a matrix metalloproteinase. We show equilibration of structural kinetic transitions in the millisecond timescale during degradation of the two model substrates collagen and gelatin, which have different supersecondary structure and flexibility. Unexpectedly, the detected changes in collective enzyme-substrate-water-coupled motions persisted well beyond steady state for both substrates while displaying substrate-specific behaviors. Molecular dynamics simulations further showed that a hydration funnel (i.e., a gradient in retardation of hydrogen bond (HB) dynamics toward the active site) is substrate-dependent, exhibiting a steeper gradient for the more complex enzyme-collagen system. The long-lasting changes in protein-water dynamics reflect a collection of local energetic equilibrium states specifically formed during substrate conversion. Thus, the observed long-lasting water dynamics contribute to the net enzyme reactivity, impacting substrate binding, positional catalysis, and product release.
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(2014) Chemical Communications. 50, 4, p. 421-423 Abstract
Bioinspired silica coprecipitates of enzymes find a wide range of applications for analysis and catalysis in industrial and academic research. Here we used SSNMR as a proxy for the 3D structure of enzymes trapped in bioinspired silica. We show that it is easy to assess whether the enzymes retain their native conformation in atomic detail. We thus propose SSNMR as a rapid and reliable tool to analyze this kind of samples at high resolution.
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(2014) Scientific Reports. 4, 5987. Abstract
A complete fingerprint of a tissue sample requires a detailed description of its cellular and extracellular components while minimizing artifacts. We introduce the application of a novel scanning electron microscope (airSEM™) in conjunction with light microscopy for functional analysis of tissue preparations at nanometric resolution (
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(2014) Molecular Endocrinology. 28, 7, p. 1039-1054 Abstract
Ovulation and inflammation share common attributes, including immune cell invasion into the ovary. The present study aims at deciphering the role of dendritic cells (DCs) in ovulation and corpus luteum formation. Using a CD11c-EYFP transgenic mouse model, ovarian transplantation experiments, and fluorescence-activated cell sorting analyses, we demonstrate that CD11c-positive, F4/80-negative cells, representing DCs, are recruited to the ovary under gonadotropin regulation. By conditional ablation of these cells in CD11c-DTR transgenic mice, we revealed that they are essential for expansion of the cumulus-oocyte complex, release of the ovum from the ovarian follicle, formation of a functional corpus luteum, and enhanced lymphangiogenesis. These experiments were complemented by allogeneic DC transplantation after conditional ablation of CD11cpositive cells that rescued ovulation. The pro-ovulatory effects of these cells were mediated by up-regulation of ovulation-essential genes. Interestingly, we detected a remarkable anti-inflammatory capacity of ovarian DCs, which seemingly serves to restrict the ovulatory-associated inflammation. In addition to discovering the role of DCs in ovulation, this study implies the extended capabilities of these cells, beyond their classic immunologic role, which is relevant also to other biological systems.
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(2014) Nature Communications. 5, 4058. Abstract
During embryonic development, axons can gain and lose sensitivity to guidance cues, and this flexibility is essential for the correct wiring of the nervous system. Yet, the underlying molecular mechanisms are largely unknown. Here we show that receptor cleavage by ADAM (A Disintegrin And Metalloprotease) metalloproteases promotes murine sensory axons loss of responsiveness to the chemorepellant Sema3A. Genetic ablation of ADAM10 and ADAM17 disrupts the developmental downregulation of Neuropilin-1 (Nrp1), the receptor for Sema3A, in sensory axons. Moreover, this is correlated with gain of repulsive response to Sema3A. Overexpression of Nrp1 in neurons reverses axonal desensitization to Sema3A, but this is hampered in a mutant Nrp1 with high susceptibility to cleavage. Lastly, we detect guidance errors of proprioceptive axons in ADAM knockouts that are consistent with enhanced response to Sema3A. Our results provide the first evidence for involvement of ADAMs in regulating developmental switch in responsiveness to axonal guidance cues.
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(2014) PLoS ONE. 9, 4, e93712. Abstract
Multispecific proteins play a major role in controlling various functions such as signaling, regulation of transcription/translation, and immune response. Hence, a thorough understanding of the atomic-level principles governing multispecific interactions is important not only for the advancement of basic science but also for applied research such as drug design. Here, we study evolution of an exemplary multispecific protein, a Tissue Inhibitor of Matrix Metalloproteinases 2 (TIMP2) that binds with comparable affinities to more than twenty-six members of the Matrix Metalloproteinase (MMP) and the related ADAMs families. We postulate that due to its multispecific nature, TIMP2 is not optimized to bind to any individual MMP type, but rather embodies a compromise required for interactions with all MMPs. To explore this hypothesis, we perform computational saturation mutagenesis of the TIMP2 binding interface and predict changes in free energy of binding to eight MMP targets. Computational results reveal the non-optimality of the TIMP2 binding interface for all studied proteins, identifying many affinity-enhancing mutations at multiple positions. Several TIMP2 point mutants predicted to enhance binding affinity and/or binding specificity towards MMP14 were selected for experimental verification. Experimental results show high abundance of affinity-enhancing mutations in TIMP2, with some point mutations producing more than ten-fold improvement in affinity to MMP14. Our computational and experimental results collaboratively demonstrate that the TIMP2 sequence lies far from the fitness maximum when interacting with its target enzymes. This non-optimality of the binding interface and high potential for improvement might characterize all proteins evolved for binding to multiple targets.
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(2014) Oncogene. 33, 8, p. 1006-1016 Abstract
Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD5+ B lymphocytes in peripheral blood, lymphoid organs and bone marrow. The main feature of the disease is accumulation of the malignant cells due to decreased apoptosis. CD84 belongs to the signaling lymphocyte activating molecule family of immunoreceptors, and has an unknown function in CLL cells. Here, we show that the expression of CD84 is significantly elevated from the early stages of the disease, and is regulated by macrophage migration inhibitory factor and its receptor, CD74. Activation of cell surface CD84 initiates a signaling cascade that enhances CLL cell survival. Both downmodulation of CD84 expression and its immune-mediated blockade induce cell death in vitro and in vivo. In addition, analysis of samples derived from an on-going clinical trial, in which human subjects were treated with humanized anti-CD74 (milatuzumab), shows a decrease in CD84 messenger RNA and protein levels in milatuzumab-treated cells. This downregulation was correlated with reduction of Bcl-2 and Mcl-1 expression. Thus, our data show that overexpression of CD84 in CLL is an important survival mechanism that appears to be an early event in the pathogenesis of the disease. These findings suggest novel therapeutic strategies based on the blockade of this CD84-dependent survival pathway.
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(2014) Acta Crystallographica Section F-Structural Biology And Crystallization Communications. 70, 2, p. 232-235 Abstract
Membrane type 1 matrix metalloproteinase (MT1-MMP) belongs to the large family of zinc-dependent endopeptidases termed MMPs that are located in the extracellular matrix. MT1-MMP was crystallized at 277 K using the vapour-diffusion method with PEG as a precipitating agent. Data sets for MT1-MMP were collected to 2.24 Å resolution at 100 K. The crystals belonged to space group P43212, with unit-cell parameters a = 62.99, c = 122.60 Å. The crystal contained one molecule per asymmetric unit, with a Matthews coefficient (V M) of 2.90 Å3 Da-1; the solvent content is estimated to be 57.6%.
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(2014) International Conference of Computational Methods in Sciences and Engineering (ICCMSE 2014). Monovasilis T., Simos TE. & Kalogiratou Z.(eds.). p. 121-124 (trueAIP Conference Proceedings). Abstract
Neurotoxic assemblies of amyloid beta-protein (A beta) are widely believed to be the cause for Alzheimer's disease (AD). Therefore, understanding the factors and mechanisms that control, modulate, and inhibit formation of these assemblies is crucial for the development of therapeutic intervention of AD. This information also can contribute significantly to our understanding of the mechanisms of other amyloidosis diseases, such as Parkinson's disease, Huntington's disease, type 2 diabetes, amyotrophic lateral sclerosis (Lou Gehrig's disease) and prion diseases (e.g. Mad Cow disease). We have developed a multidisciplinary experimental strategy to study structural and dynamic mechanistic aspects that underlie the A beta assembly process. Utilizing this strategy, we explored the molecular basis leading to the perturbation of the A beta assembly process by divalent metal ions, mainly Zn2+ ions. Using Zn2+ as reaction physiological relevant probes, it was demonstrated that Zn2+ rapidly (milliseconds) induce self-assembly of A beta aggregates and stabilize them in a manner that prevents formation of A beta fibrils. Importantly, the early-formed intermediates are substantially more neurotoxic than fibrils. Our results suggest that relevant A beta modulators should be targeted against the rapidly evolved intermediate states of A beta assembly. The design of such modulators is challenging, as they have to compete with different natural mediators (such as Zn2+) of A beta aggregation, which diverse A beta assemblies in both specific and nonspecific manners.
2013
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(2013) Journal of Molecular Biology. 425, 13, p. 2330-2346 Abstract
Monitoring enzymatic activity in vivo of individual homologous enzymes such as the matrix metalloproteinases (MMPs) by antagonist molecules is highly desired for defining physiological and pathophysiological pathways. However, the rational design of antagonists targeting enzyme catalytic moieties specific to one of the homologous enzymes often appears to be an extremely difficult task. This is mainly due to the high structural homology at the enzyme active sites shared by members of the protein family. Accordingly, controlling enzymatic activity via alternative allosteric sites has become an attractive proposition for drug design targeting individual homologous enzymes. Yet, the challenge remains to identify such regulatory alternative sites that are often hidden and scattered over different locations on the protein's surface. We have designed branched amphiphilic molecules exhibiting specific inhibitory activity towards individual members of the MMP family. These amphiphilic isomers share the same chemical nature, providing versatile nonspecific binding reactivity that allows to probe hidden regulatory residues on a given protein surface. Using the advantage provided by amphiphilic ligands, here we explore a new approach for determining hidden regulatory sites. This approach includes diverse experimental analysis, such as structural spectroscopic analyses, NMR, and protein crystallography combined with computational prediction of effector binding sites. We demonstrate how our approach works by analyzing members of the MMP family that possess a unique set of such sites. Our work provides a proof of principle for using ligand effectors to unravel hidden regulatory sites specific to members of the structurally homologous MMP family. This approach may be exploited for the design of novel molecular effectors and therapeutic agents affecting protein catalytic function via interactions with structure-specific regulatory sites.
2012
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(2012) Oncogene. 31, 31, p. 3569-3583 Abstract
The HER2/neu oncogene encodes a receptor-like tyrosine kinase whose overexpression in breast cancer predicts poor prognosis and resistance to conventional therapies. However, the mechanisms underlying aggressiveness of HER2 (human epidermal growth factor receptor 2)-overexpressing tumors remain incompletely understood. Because it assists epidermal growth factor (EGF) and neuregulin receptors, we overexpressed HER2 in MCF10A mammary cells and applied growth factors. HER2-overexpressing cells grown in extracellular matrix formed filled spheroids, which protruded outgrowths upon growth factor stimulation. Our transcriptome analyses imply a two-hit model for invasive growth: HER2-induced proliferation and evasion from anoikis generate filled structures, which are morphologically and transcriptionally analogous to preinvasive patients lesions. In the second hit, EGF escalates signaling and transcriptional responses leading to invasive growth. Consistent with clinical relevance, a gene expression signature based on the HER2/EGF-activated transcriptional program can predict poorer prognosis of a subgroup of HER2-overexpressing patients. In conclusion, the integration of a three-dimensional cellular model and clinical data attributes progression of HER2-overexpressing lesions to EGF-like growth factors acting in the context of the tumor's microenvironment.
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(2012) Journal of Biological Chemistry. 287, 24, p. 20555-20564 Abstract
The roles of metal ions in promoting amyloid β-protein (Aβ) oligomerization associated with Alzheimer disease are increasingly recognized. However, the detailed structures dictating toxicity remain elusive for Aβ oligomers stabilized by metal ions. Here, we show that small Zn 2+-bound Aβ1-40 (Zn2+- Aβ40) oligomers formed in cell culture medium exhibit quasispherical structures similar to native amylospheroids isolated recently from Alzheimer disease patients. These quasi-spherical Zn2+-Aβ40 oligomers irreversibly inhibit spontaneous neuronal activity and cause massive cell death in primary hippocampal neurons. Spectroscopic and x-ray diffraction structural analyses indicate that despite their non-fibrillar morphology, the metastable Zn 2+-Aβ40 oligomers are rich in β-sheet and cross-β structures. Thus, Zn2+ promotes Aβ40 neurotoxicity by structural organization mechanisms mediated by coordination chemistry.
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Application of Stopped-Flow and Time-Resolved X-Ray Absorption Spectroscopy to the Study of Metalloproteins Molecular Mechanisms(2012) X-Ray Spectroscopy. Sharma S. K.(eds.). p. 245-264 Abstract
Revealing detailed molecular mechanisms associated with metalloproteins is highly desired for the advancement of both basic and clinical research. Metalloproteins represent one of the most diverse classes of proteins, containing intrinsic metal ions which provide catalytic, regulatory, or structural roles critical to protein function. The metal ion is usually coordinated by nitrogen, oxygen, or sulfur atoms belonging to amino acids in a polypeptide chain and/or in a macrocyclic ligand incorporated into the protein. The presence of a metal ion rich in electrons allows metalloproteins to perform functions such as redox reactions, phosphorylation, electron transfer, and acid-base catalytic reactions that cannot easily be performed by the limited set of functional groups found in amino acids. Approximately onethird of all proteins possess a bound metal1, and almost half of all enzymes require the presence of a metal atom to function2. The most abundant metal ions in vivo are Mg and Zn, while Fe, Ca, Co, Mn, and Ni are also frequently observed. Metalloproteins play important roles in key biological processes such as photosynthesis, signaling, metabolism, proliferation, and immune response 3-5. For instance, the principal oxygen carrier protein in human, hemoglobin, uses iron (II) ions coordinated to porphyrin rings to bind the dioxygen molecules; calmodulin is a calcium-binding protein mediating a large variety of signal transduction processes in response to calcium binding; cytochromes mediate electrontransport and facilitate a variety of redox reactions using iron, which interconverts between Fe2+ (reduced) and Fe3+ (oxidized) states; and many proteolytic enzymes contain zinc ions in their active sites that is highly crucial for peptide bond hydrolysis reaction. [first paragraph]
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(2012) Nature Medicine. 18, 1, p. 143-147 Abstract
Endogenous tissue inhibitors of metalloproteinases (TIMPs) have key roles in regulating physiological and pathological cellular processes. Imitating the inhibitory molecular mechanisms of TIMPs while increasing selectivity has been a challenging but desired approach for antibody-based therapy. TIMPs use hybrid protein-protein interactions to form an energetic bond with the catalytic metal ion, as well as with enzyme surface residues. We used an innovative immunization strategy that exploits aspects of molecular mimicry to produce inhibitory antibodies that show TIMP-like binding mechanisms toward the activated forms of gelatinases (matrix metalloproteinases 2 and 9). Specifically, we immunized mice with a synthetic molecule that mimics the conserved structure of the metalloenzyme catalytic zinc-histidine complex residing within the enzyme active site. This immunization procedure yielded selective function-blocking monoclonal antibodies directed against the catalytic zinc-protein complex and enzyme surface conformational epitopes of endogenous gelatinases. The therapeutic potential of these antibodies has been demonstrated with relevant mouse models of inflammatory bowel disease. Here we propose a general experimental strategy for generating inhibitory antibodies that effectively target the in vivo activity of dysregulated metalloproteinases by mimicking the mechanism employed by TIMPs.
2011
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(2011) PLoS ONE. 6, 12, e27969. Abstract
The inflammatory response in the injured spinal cord, an immune privileged site, has been mainly associated with the poor prognosis. However, recent data demonstrated that, in fact, some leukocytes, namely monocytes, are pivotal for repair due to their alternative anti-inflammatory phenotype. Given the pro-inflammatory milieu within the traumatized spinal cord, known to skew monocytes towards a classical phenotype, a pertinent question is how parenchymal-invading monocytes acquire resolving properties essential for healing, under such unfavorable conditions. In light of the spatial association between resolving (interleukin (IL)-10 producing) monocytes and the glial scar matrix chondroitin sulfate proteoglycan (CSPG), in this study we examined the mutual relationship between these two components. By inhibiting the de novo production of CSPG following spinal cord injury, we demonstrated that this extracellular matrix, mainly known for its ability to inhibit axonal growth, serves as a critical template skewing the entering monocytes towards the resolving phenotype. In vitro cell culture studies demonstrated that this matrix alone is sufficient to induce such monocyte polarization. Reciprocal conditional ablation of the monocyte-derived macrophages concentrated at the lesion margins, using diphtheria toxin, revealed that these cells have scar matrix-resolving properties. Replenishment of monocytic cell populations to the ablated mice demonstrated that this extracellular remodeling ability of the infiltrating monocytes requires their expression of the matrix-degrading enzyme, matrix metalloproteinase 13 (MMP-13), a property that was found here to be crucial for functional recovery. Altogether, this study demonstrates that the glial scar-matrix, a known obstacle to regeneration, is a critical component skewing the encountering monocytes towards a resolving phenotype. In an apparent feedback loop, monocytes were found to regulate scar resolution. This cross-regulation between the glial scar and monocytes primes the resolution of this interim phase of spinal cord repair, thereby providing a fundamental platform for the dynamic healing response.
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(2011) Current Opinion in Structural Biology. 21, 5, p. 678-685 Abstract
A network of dynamic protein interactions with their protein partners, substrates, and ligands is known to be crucial for biological function. Revealing molecular and structural-based mechanisms at atomic resolution and in real-time is fundamental for achieving a basic understanding of cellular processes. These technically challenging goals may be achieved by combining time-resolved spectroscopic and structural-kinetic tools, thus providing broad insights into specific molecular events over a wide range of timescales. Here we review representative studies utilizing such an integrated real-time structural approach designed to reveal molecular mechanisms underlying protein interactions at atomic resolution.
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(2011) Journal of the American Chemical Society. 133, 39, p. 15514-15523 Abstract
The RNA helicase DbpA promotes RNA remodeling coupled to ATP hydrolysis. It is unique because of its specificity to hairpin 92 of 23S rRNA (HP92). Although DbpA kinetic pathways leading to ATP hydrolysis and RNA unwinding have been recently elucidated, the molecular (atomic) basis for the coupling of ATP hydrolysis to RNA remodeling remains unclear. This is, in part, due to the lack of detailed structural information on the ATPase site in the presence and absence of RNA in solution. We used high-field pulse ENDOR (electron-nuclear double resonance) spectroscopy to detect and analyze fine conformational changes in the proteins ATPase site in solution. Specifically, we substituted the essential Mg 2+ cofactor in the ATPase active site for paramagnetic Mn 2+ and determined its close environment with different nucleotides (ADP, ATP, and the ATP analogues ATPγS and AMPPnP) in complex with single- and double-stranded RNA. We monitored the Mn 2+ interactions with the nucleotide phosphates through the 31P hyperfine couplings and the coordination by protein residues through 13C hyperfine coupling from 13C-enriched DbpA. We observed that the nucleotide binding site of DbpA adopts different conformational states upon binding of different nucleotides. The ENDOR spectra revealed a clear distinction between hydrolyzable and nonhydrolyzable nucleotides prior to RNA binding. Furthermore, both the 13C and the 31P ENDOR spectra were found to be highly sensitive to changes in the local environment of the Mn 2+ ion induced by the hydrolysis. More specifically, ATPγS was efficiently hydrolyzed upon binding of RNA, similar to ATP. Importantly, the Mn 2+ cofactor remains bound to a single protein side chain and to one or two nucleotide phosphates in all complexes, whereas the remaining metal coordination positions are occupied by water. The conformational changes in the proteins ATPase active site associated with the different DbpA states occur in remote coordination shells of the Mn 2+ ion. Finally, a competitive Mn 2+ binding site was found for single-stranded RNA construct.
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(2011) Expert Opinion on Drug Discovery. 6, 5, p. 527-542 Abstract
Introduction: MMPs (matrix metalloproteinases) and ADAMs (a disintegrin and metalloproteinases) are endopeptidases central to the degradation and remodeling of the extracellular matrix. These proteases also exhibit regulatory activity in cell signaling pathways and thus tissue homeostasis under normal conditions and in many diseases. Consequently, individual members of the MMP and ADAM protein families were identified as important therapeutic targets. However, designing effective inhibitors in vivo for this class of enzymes appears to be extremely challenging. This is attributed to the broad structural similarity of their active sites and to the dynamic functional interconnectivity of MMPs with other proteases, their inhibitors, and substrates (the so-called degradome) in healthy and disease tissues. Areas covered: The article covers the progress in designing metalloproteinase inhibitors, based on recent advancements in our understanding of enzyme structures and their function as master regulators. It also discusses the potential of utilizing structure-based drug design strategies in conjunction with systems biology experimental approaches for designing potent and therapeutically effective metalloproteinase inhibitors. Expert opinion: We highlight the use of protein-based drug design strategies, for example, antibodies and protein scaffolds, targeting extracatalytic domains, which are central to proteolytic and non-proteolytic enzyme functions. Such rationally designed function-blocking inhibitors may create new opportunities in disease management and in emerging therapies that require control of dysregulatedMMP activity without causing severe side effects. Importantly, the lessons learned from studying these protein-based inhibitors can be implemented to design new and effective small or medium sized synthetic antagonists.
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(2011) Connective Tissue Research. 52, 1, p. 18-24 Abstract
Fibrillar collagens form the structural basis of organs and tissues including the vasculature, bone, and tendon. They are also dynamic, organizational scaffolds that present binding and recognition sites for ligands, cells, and platelets. We interpret recently published X-ray diffraction findings and use atomic force microscopy data to illustrate the significance of new insights into the functional organization of the collagen fibril. These data indicate that collagen's most crucial functional domains localize primarily to the overlap region, comprising a constellation of sites we call the "master control region." Moreover, the collagen's most exposed aspect contains its most stable partthe C-terminal region that controls collagen assembly, cross-linking, and blood clotting. Hidden beneath the fibril surface exists a constellation of "cryptic" sequences poised to promote hemostasis and cellcollagen interactions in tissue injury and regeneration. These findings begin to address several important, and previously unresolved, questions: How functional domains are organized in the fibril, which domains are accessible, and which require proteolysis or structural trauma to become exposed Here we speculate as to how collagen fibrillar organization impacts molecular processes relating to tissue growth, development, and repair.
2010
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(2010) Nature Structural & Molecular Biology. 18, 10, p. 1102-1108 Abstract
Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme-inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site.
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(2010) Biochemistry. 49, 29, p. 6184-6192 Abstract
Protein flexibility is thought to play key roles in numerous biological processes, including antibody affinity maturation, signal transduction, and enzyme catalysis, yet only limited information is available regarding the molecular details linking protein dynamics with function. A single point mutation at the distal site of the endogenous tissue inhibitor of metalloproteinase 1 (TIMP-1) enables this clinical target protein to tightly bind and inhibit membrane type 1 matrix metalloproteinase (MT1-MMP) by increasing only the association constant. The high-resolution X-ray structure of this complex determined at 2 could not explain the mechanism of enhanced binding and pointed to a role for protein conformational dynamics. Molecular dynamics (MD) simulations reveal that the high-affinity TIMP-1 mutants exhibit significantly reduced binding interface flexibility and more stable hydrogen bond networks. This was accompanied by a redistribution of the ensemble of substrates to favorable binding conformations that fit the enzyme catalytic site. Apparently, the decrease in backbone flexibility led to a lower entropy cost upon formation of the complex. This work quantifies the effect of a single point mutation on the protein conformational dynamics and function of TIMP-1. Here we argue that controlling the intrinsic protein dynamics of MMP endogenous inhibitors may be utilized for rationalizing the design of selective novel protein inhibitors for this class of enzymes.
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(2010) Journal of the American Chemical Society. 132, 24, p. 8232-8233 Abstract
Derived from the extensive work in the area of small molecule zinc(II) ion sensors, chelating fragment libraries of quinoline- and benzimidazole- sulfonamides have been prepared and screened against several different zinc(II)-dependent matrix metalloproteinases (MMPs). The fragments show impressive inhibition of these metalloenzymes and preferences for different MMPs based on the nature of the chelating group. The findings show that focused chelator libraries are a powerful strategy for the discovery of lead fragments for metalloprotein inhibition.
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(2010) Chemistry of Materials. 22, 1, p. 161-166 Abstract
Magnesium is a key component used by many organisms in biomineralization. One role for magnesium is in stabilizing an otherwise unstable amorphous calcium carbonate (ACC) phase. The way in which this stabilization is achieved is unknown. Here, we address this question by studying the chemical environment around magnesium in biogenic and synthetic ACCs using Mg K-edge X-ray absorption spectroscopy (XAS). We show that although the short-range structure around the Mg ion is different in the various minerals studied, they all involve a shortening of the Mg-O bond length compared to crystalline anhydrous MgCO 3 minerals. We propose that the compact structure around magnesium introduces distortion in the CaCO3 host mineral, thus inhibiting its crystallization. This study also shows that despite technical challenges in the soft X-ray energy regime, Mg K-edge XAS is a valuable tool for structural analysis of Mg containing amorphous materials, in biology and materials science.
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(2010) PLoS ONE. 5, 6, e11043. Abstract
Enzymatic processing of extracellular matrix (ECM) macromolecules by matrix metalloproteases (MMPs) is crucial in mediating physiological and pathological cell processes. However, the molecular mechanisms leading to effective physiological enzyme-ECM interactions remain elusive. Only scant information is available on the mode by which matrix proteases degrade ECM substrates. An example is the enzymatic degradation of triple helical collagen II fragments, generated by the collagenase MMP-8 cleavage, during the course of acute inflammatory conditions by gelatinase B/MMP-9. As is the case for many other matrix proteases, it is not clear how MMP-9 recognizes, binds and digests collagen in this important physiological process. We used single molecule imaging to directly visualize this protease during its interaction with collagen fragments. We show that the initial binding is mediated by the diffusion of the protease along the ordered helix on the collagen 3/4 fragment, with preferential binding of the collagen tail. As the reaction progressed and prior to collagen degradation, gelatin-like morphologies resulting from the denaturation of the triple helical collagen were observed. Remarkably, this activity was independent of enzyme proteolysis and was accompanied by significant conformational changes of the working protease. Here we provide the first direct visualization of highly complex mechanisms of macromolecular interactions governing the enzymatic processing of ECM substrates by physiological protease.
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(2010) Biochimica et Biophysica Acta - Molecular Cell Research. 1803, 1, p. 29-38 Abstract
The zinc-dependent matrix metalloproteinases (MMPs) belong to a large family of structurally homologous enzymes. These enzymes are involved in a wide variety of biological processes ranging from physiological cell proliferation and differentiation to pathological states associated with tumor metastasis, inflammation, tissue degeneration, and cell death. Controlling the enzymatic activity of specific individual MMPs by antagonist molecules is highly desirable, first, for studying their individual roles, and second as potential therapeutic agents. However, blocking the enzymatic activity with synthetic small inhibitors appears to be an extremely difficult task. Thus, this is an unmet need presumably due to the high structural homology between MMP catalytic domains. Recent reports have recognized a potential role for exosite or allosteric protein regions, distinct from the extended catalytic pocket, in mediating MMP activation and substrate hydrolysis. This raises the possibility that MMP enzymatic and non-enzymatic activities may be modified via antagonist molecules targeted to such allosteric sites or to alternative enzyme domains. In this review, we discuss the structural and functional bases for potential allosteric control of MMPs and highlight potential alternative enzyme domains as targets for designing highly selective MMP inhibitors.
2009
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(2009) Journal of the American Chemical Society. 131, 47, p. 17270-17276 Abstract
Natural RNAs, unlike many proteins, have never been reported to form extended nanostructures, despite their wide variety of cellular functions. This is all the more striking, as synthetic DNA and RNA forming large nanostructures have long been successfully designed. Here, we show that DsrA, a 87-nt noncoding RNA of Escherishia coli, self-assembles into a hierarchy of nanostructures through antisense interactions of three contiguous self-complementary regions. Yet, the extended nanostructures, observed using atomic force microscopy (AFM) and fluorescence microscopy, are easily disrupted into >100 nm long helical bundles of DsrA filaments, including hundreds of DsrA monomers, and are surprisingly resistant to heat and urea denaturation. Molecular modeling demonstrates that this structural switch of DsrA nanostructures into filament bundles results from the relaxation of stored torsional constraints and suggests possible implications for DsrA regulatory function.
2008
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(2008) Proceedings of the National Academy of Sciences of the United States of America. 105, 50, p. 20045 Abstract
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(2008) Proceedings of the National Academy of Sciences of the United States of America. 105, 45, p. 17362-17366 Abstract
Sea urchin larval spicules transform amorphous calcium carbonate (ACC) into calcite single crystals. The mechanism of transformation is enigmatic: the transforming spicule displays both amorphous and crystalline properties, with no defined crystallization front. Here, we use X-ray photoelectron emission spectromicroscopy with probing size of 40-200 nm. We resolve 3 distinct mineral phases: An initial short-lived, presumably hydrated ACC phase, followed by an intermediate transient form of ACC, and finally the biogenic crystalline calcite phase. The amorphous and crystalline phases are juxtaposed, often appearing in adjacent sites at a scale of tens of nanometers. We propose that the amorphous-crystal transformation propagates in a tortuous path through preexisting 40- to 100-nm amorphous units, via a secondary nucleation mechanism.
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(2008) Phytochemistry. 69, 14, p. 2586-2592 Abstract
Pectin methyl esterases (PMEs) and their endogenous inhibitors are involved in the regulation of many processes in plant physiology, ranging from tissue growth and fruit ripening to parasitic plant haustorial formation and host invasion. Thus, control of PME activity is critical for enhancing our understanding of plant physiological processes and regulation. Here, we report on the identification of epigallocatechin gallate (EGCG), a green tea component, as a natural inhibitor for pectin methyl esterases. In a gel assay for PME activity, EGCG blocked esterase activity of pure PME as well as PME extracts from citrus and from parasitic plants. Fluorometric tests were used to determine the IC50 for a synthetic substrate. Molecular docking analysis of PME and EGCG suggests close interaction of EGCG with the catalytic cleft of PME. Inhibition of PME by the green tea compound, EGCG, provides the means to study the diverse roles of PMEs in cell wall metabolism and plant development. In addition, this study introduces the use of EGCG as natural product to be used in the food industry and agriculture.
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(2008) Journal of the American Chemical Society. 130, 4, p. 1376-1383 Abstract
The role of zinc, an essential element for normal brain function, in the pathology of Alzheimer's disease (AD) is poorly understood. On one hand, physiological and genetic evidence from transgenic mouse models supports its pathogenic role in promoting the deposition of the amyloid β-protein (Aβ) in senile plaques. On the other hand, levels of extracellular ("free") zinc in the brain, as inferred by the levels of zinc in cerebrospinal fluid, were found to be too low for inducing Aβ aggregation. Remarkably, the release of transient high local concentrations of zinc during rapid synaptic events was reported. The role of such free zinc pulses in promoting Aβ aggregation has never been established. Using a range of time-resolved structural and spectroscopic techniques, we found that zinc, when introduced in millisecond pulses of micromolar concentrations, immediately interacts with Aβ 1-40 and promotes its aggregation. These interactions specifically stabilize non-fibrillar pathogenic related aggregate forms and prevent the formation of Aβ fibrils (more benign species) presumably by interfering with the self-assembly process of Aβ. These in vitro results strongly suggest a significant role for zinc pulses in Aβ pathology. We further propose that by interfering with Aβ self-assembly, which leads to insoluble, non-pathological fibrillar forms, zinc stabilizes transient, harmful amyloid forms. This report argues that zinc represents a class of molecular pathogens that effectively perturb the self-assembly of benign Aβ fibrils, and stabilize harmful non-fibrillar forms.
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2007
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(2007) Journal of the American Chemical Society. 129, 44, p. 13566-13574 Abstract
Activation of matrix metalloproteinase zymogen (pro-MMP) is a vital homeostatic process, yet its molecular basis remains unresolved. Using stopped-flow X-ray spectroscopy of the active site zinc ion, we determined the temporal sequence of pro-MMP-9 activation catalyzed by tissue kallikrein protease in milliseconds to several minutes. The identity of three intermediates seen by X-ray spectroscopy was corroborated by molecular dynamics simulations and quantum mechanics/molecular mechanics calculations. The cysteine-zinc interaction that maintains enzyme latency is disrupted via active-site proton transfers that mediate transient metal-protein coordination events and eventual binding of water. Unexpectedly, these events ensue as a direct result of complexation of pro-MMP-9 and kallikrein and occur before proteolysis and eventual dissociation of the pro-peptide from the catalytic site. Here we demonstrate the synergism among long-range protein conformational transitions, local structural rearrangements, and fine atomic events in the process of zymogen activation.
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(2007) Structure. 15, 10, p. 1227-1236 Abstract
The multidomain zinc endopeptidase matrix metalloproteinase-9 (MMP-9) is a recognized therapeutic target in autoimmune diseases, vascular pathologies, and cancer. Despite its importance, structural characterization of full-length pro-MMP-9 is incomplete. Here, we report the structural model of full-length pro-MMP-9 and, in particular, the molecular character of its unique proline-rich and heavily O-glycosylated (OG) domain. Using a powerful combination of small-angle X-ray scattering and single-molecule imaging, we demonstrate that pro-MMP-9 possesses an elongated structure with two terminal globular domains connected by an unstructured OG domain. Image analysis highlights the flexibility of the OG domain, implicating its role in the varied enzyme conformations and in facilitating independent movements of the terminal domains. This may endorse recognition, binding, and processing of substrates, ligands, as well as receptors and marks this domain as an additional target for the design of selective regulators.
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(2007) Proceedings of the National Academy of Sciences of the United States of America. 104, 12, p. 4931-4936 Abstract
Despite their key roles in many normal and pathological processes, the molecular details by which zinc-dependent proteases hydrolyze their physiological substrates remain elusive. Advanced theoretical analyses have suggested reaction models for which there is limited and controversial experimental evidence. Here we report the structure, chemistry and lifetime of transient metal-protein reaction intermediates evolving during the substrate turnover reaction of a metalloproteinase, the tumor necrosis factor-α converting enzyme (TACE). TACE controls multiple signal transduction pathways through the proteolytic release of the extracellular domain of a host of membrane-bound factors and receptors. Using stopped-flow x-ray spectroscopy methods together with transient kinetic analyses, we demonstrate that TACE's catalytic zinc ion undergoes dynamic charge transitions before substrate binding to the metal ion. This indicates previously undescribed communication pathways taking place between distal protein sites and the enzyme catalytic core. The observed charge transitions are synchronized with distinct phases in the reaction kinetics and changes in metal coordination chemistry mediated by the binding of the peptide substrate to the catalytic metal ion and product release. Here we report key local charge transitions critical for proteolysis as well as long sought evidence for the proposed reaction model of peptide hydrolysis. This study provides a general approach for gaining critical insights into the molecular basis of substrate recognition and turnover by zinc metalloproteinases that may be used for drug design.
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(2007) Structure And Biophysics - New Technologies For Current Challenges In Biology Beyond. 231, p. 111-120 Abstract
Conformational flexibility and molecular dynamics play an important role in protein function. Quantification and structural characterization of protein flexibility is essential for the assignment of both the physiological and molecular mechanisms of biological macromolecules. Structural and spectroscopic tools have emerged that allow the study of local and global changes in protein structure and dynamics involved in ligand binding, protein activation, and protein-protein interactions. We have recently introduced the use of time-resolved freeze-quench X-ray absorption spectroscopy (XAS) to study detailed reaction mechanisms of metalloenzymes. Combining such measurements with molecular dynamics simulations provides novel insights into the reaction mechanism. Here we discuss the utilization of this structural dynamic approach to the design of novel inhibitors.
2006
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(2006) Advanced Functional Materials. 16, 10, p. 1289-1298 Abstract
Sea urchin embryos form their calcitic spicular skeletons via a transient precursor phase composed of amorphous calcium carbonate (ACC). Transition of ACC to calcite in whole larvae and isolated spicules during development has been monitored using X-ray absorption spectroscopy (XAS). Remarkably, the changing nature of the mineral phase can clearly be monitored in the whole embryo samples. More detailed analyses of isolated spicules at different stages of development using both XAS and infrared spectroscopy demonstrate that the short-range order of the transient ACC phase resembles calcite, even though infrared spectra show that the spicules are mostly composed of an amorphous mineral phase. The coordination sphere is at first distorted but soon adopts the octahedral symmetry typical of calcite. Long-range lattice rearrangement follows to form the calcite single crystal of the mature spicule. These studies demonstrate the feasibility of real-time monitoring of mineralized-tissue development using XAS, including the structural characterization of transient amorphous phases at the atomic level.
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(2006) Journal of Molecular Biology. 355, 4, p. 697-707 Abstract
The Escherichia coli DEAD-box protein A (DbpA) belongs to the highly conserved superfamily-II of nucleic acid helicases that play key roles in RNA metabolism. A central question regarding helicase activity is whether the process of coupling ATP hydrolysis to nucleic acid unwinding requires an oligomeric form of the enzyme. We have investigated the structural and functional properties of DbpA by multi-angle laser light-scattering, size-exclusion chromatography, analytical ultracentrifugation, chemical cross-linking and hydrodynamic modeling. DbpA is monomeric in solution up to a concentration of 25 μM and over the temperature range of 4°C to 22°C. Binding of neither nucleotide (ATP or ADP) nor peptidyl transferase center (PTC) RNA, the presumed physiological RNA substrate, favor oligomerization. The hydrodynamic parameters were used together with hydrodynamic bead modeling and structural homology in conjunction with ab initio structure prediction methods to define plausible shapes of DbpA. Collectively, the results favor models where DbpA functions as an active monomer that possesses two distinct RNA binding sites, one in the helicase core domain and the other in the carboxyl-terminal domain that recognizes 23 S rRNA and interacts specifically with hairpin 92 of the PTC.
2005
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(2005) Journal of Synchrotron Radiation. 12, 4, p. 392-401 Abstract
Many inorganic species are now recognized as being essential for life, including many forms of sulfur, phosphate and numerous classes of metal ions. For example, recent progress in the fields of biochemistry and biology has pointed out the critical importance of sulfur in the biosynthesis of vital cofactors and active sites in proteins, and in the complex reaction mechanisms often involved. Special attention has also been drawn to the diverse roles of alkaline (Na-, K-) and alkaline earth (Mg2+, Ca2+) metal ions in mediating the activity of RNA, proteins and many processes in living cells. While the general effect of these ions in biology is mostly understood, information on their detailed role is deficient. Here the application of softer X-ray absorption spectroscopy (XAS) to probe the local structural and electronic environment of such ions within their biological complexes and during physiological reactions is discussed. In addition, the required experimental set-up and the difficulties associated with conducting softer XAS experiments on biological samples are presented.
2004
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(2004) Journal of Inorganic Biochemistry. 98, 11, p. 1750-1756 Abstract
Irradiation of plasmid DNA in the presence of Ru(II)-2, a modified tris(2,2-bipyridyl)Ru(II) complex, in which two hydroxamic acid groups are attached to one of the three bipyridyl ligands, results in total fragmentation of the DNA. The photo-chemical reaction products were analyzed by gel electrophoresis, which revealed complete fragmentation. Further evidence for the complete degradation of the DNA was obtained by imaging the pre- and post-treated plasmid DNA using atomic force microscopy (AFM). A mechanism for the reaction is proposed. It initially involves the photo-chemical generation of Ru(III) ions and superoxide radicals, as corroborated by absorbance difference spectroscopy and electron paramagnetic resonance (EPR). Consequently, Ru(III) preferentially oxidizes guanine, liberating superoxide radicals that yield OH radicals. The OH radicals were identified by observing the spectral change at 532 nm of a 5-dAdG substrate forming a colored adduct with thiobarbituric acid. These radicals are associated with the major non-specific damage exerted to DNA.
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(2004) Journal of Biological Chemistry. 279, 30, p. 31638-31645 Abstract
Tumor necrosis factor-α-converting enzyme (TACE) is a disintegrin metalloproteinase that processes tumor necrosis factor and a host of other ectodomains. TACE is biosynthesized as a zymogen, and activation requires the removal of an inhibitory pro domain. Little is known about how the pro domain exerts inhibition for this class of enzymes. To study the inhibitory properties of the pro domain of TACE, we have expressed it in isolation from the rest of the protease. Here we show that the TACE pro domain (TACE Pro) is a stably folded protein that is able to inhibit this enzyme. TACE Pro inhibited the catalytic domain of TACE with an IC50. of 70 nM. In contrast, this inhibitory potency decreased over 30-fold against a TACE form containing the catalytic plus disintegrin/cysteine-rich domains (IC50 greater that 2 μM). The disintegrin/cysteine-rich region in isolation also decreases the interaction of TACE Pro with the catalytic domain. Surprisingly, we found that the cysteine switch motif located in TACE Pro was not essential for inhibition of the enzymatic activity of TACE; the pro domain variant C184A showed the same inhibitory potency against both TACE forms as wild type TACE Pro. X-ray absorption spectroscopy experiments indicate that binding of TACE Pro to the catalytic domain does include ligation of the catalytic zinc ion via the sulfur atom of its conserved Cys184 residue. Moreover, the binding of TACE Pro to the catalytic zinc ion partially oxidizes the catalytic zinc ion of the enzyme. Despite this, the nature of the interaction between the pro and catalytic domains of TACE is not consistent with a simple competitive model of inhibition based on cysteine switch ligation of the zinc ion within the active site of TACE.
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(2004) Journal of Biological Chemistry. 279, 30, p. 31646-31654 Abstract
The metalloproteinase tumor necrosis factor-α-converting enzyme (TACE) is involved in the regulation of several key physiological and pathological processes. Therefore, potent and selective synthetic inhibitors are highly sought for the study of the physiological roles of TACE as well as for therapeutic purposes. Because of the high structural similarities between the active site of TACE and those of other related zinc endopeptidases such as disintegrin (ADAMs) and matrix metalloproteinases (MMPs), the design of such tailor-made inhibitors is not trivial. To obtain new insights into this problem, we have used a selective MMP inhibitor as a probe to examine the structural and kinetic effects occurring at the active site of TACE upon inhibition. Specifically, we used the selective MMP mechanism-based inhibitor SB-3CT to characterize the fine structural and electronic differences between the catalytic zinc ions within the active sites of TACE and MMP-2. We show that SB-3CT directly binds the metal ion of TACE as observed before with MMP-2. However, in contrast to MMP-2, the binding mode of SB-3CT to the catalytic zinc ion of TACE is different in the length of the Zn-S(SB-3CT) bond distance and the total effective charge of the catalytic zinc ion. In addition, SB-3CT inhibits TACE in a non-competitive fashion by inducing significant conformational changes in the structure. For MMP-2, SB-3CT behaved as a competitive inhibitor and no significant conformational changes were observed. An examination of the second shell amino acids surrounding the catalytic zinc ion of these enzymes indicated that the active site of TACE is more polar than that of MMP-2 and of other MMPs. On the basis of these results, we propose that although there is a seemingly high structural similarity between TACE and MMP-2, these enzymes are significantly diverse in the electronic and chemical properties within their active sites.
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(2004) Biochemistry. 43, 22, p. 7151-7161 Abstract
Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) is a zinc-dependent NADP+/H-linked class enzyme that reversibly catalyzes the oxidation of secondary alcohols to their corresponding ketones. Cobalt substitution studies of other members of the alcohol dehydrogenase (ADH) family showed that the cobalt-containing ADHs have a similar active site structure but slightly decreased activity compared to wild-type zinc ADHs. In contrast, the cobalt-substituted TbADH (Co-TbADH) exhibits an increase in specific activity compared to the native enzyme [Bogin, O., Peretz, M., and Burstein, Y. (1997) Protein Sci. 6, 450-458]. However, the structural basis underlying this behavior is not yet clear. To shed more light on this issue, we studied the local structure and electronics at the catalytic metal site in Co-TbADH by combining X-ray absorption (XAS) and quantum chemical calculations. Importantly, we show that the first metal-ligand coordination shell of Co-TbADH is distorted compared to its native tetrahedral coordination shell and forms an octahedral structure. This is mediated presumably by the addition of two water molecules and results in more positively charged catalytic metal ions. Recently, we have shown that the metal-ligand coordination number of the zinc ion in TbADH changes dynamically during substrate turnover. These structural changes are associated with a higher coordination number of the native catalytic zinc ion and the consequent buildup of a positive charge. Here we propose that the accumulation of a higher coordination number and positive charge at the catalytic metal ion in TbADH stabilizes the structure of the catalytic transition state and hence lowers the barrier for enzyme catalysis.
2003
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(2003) International Journal of Mass Spectrometry. 229, 1-2, p. 55-60 Abstract
We describe a new mass spectrometric technique that is based on the use of a linear electrostatic ion trap and a newly discovered self-bunching phenomenon. Ions are stored in the trap and their oscillation frequencies are determined by Fourier transform of their oscillation times. Using this system, we demonstrate that it is possible to simultaneously trap several masses and obtain their mass spectra with high resolution. The instrument is compared to time-of-flight mass, as well as to ion cyclotron resonance mass spectrometers.
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(2003) Journal of Biological Chemistry. 278, 29, p. 27009-27015 Abstract
The zinc-dependent gelatinases belong to the family of matrix metalloproteinases (MMPs), enzymes that have been shown to play a key role in angiogenesis and tumor metastasis. These enzymes are capable of hydrolyzing extracellular matrix (ECM) components under physiological conditions. Specific and selective inhibitors aimed at blocking their activity are highly sought for use as potential therapeutic agents. We report herein on a novel mode of inhibition of gelatinase A (MMP-2) by the recently characterized inhibitors 4-(4-phenoxphenylsulfonyl)butane-1,2-dithiol (inhibitor 1) and 5-(4-phenoxphenylsulfonyl) pentane-1,2-dithiol (inhibitor 2). These synthetic inhibitors are selective for MMP-2 and MMP-9. We show that the dithiolate moiety of these inhibitors chelates the catalytic zinc ion of MMP-2 via two sulfur atoms. This mode of binding results in alternation of the coordination number of the metal ion and the induction of conformational changes at the microenvironment of the catalytic zinc ion; a set of events that is likely to be at the root of the potent slow binding inhibition behavior exhibited by these inhibitors. This study demonstrates a distinct approach for the understanding of the structural mechanism governing the molecular interactions between potent inhibitors and catalytic sites of MMPs, which may aid in the design of effective inhibitors.
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(2003) IEEE Transactions on Nanobioscience. 2, 2, p. 70-74 Abstract
RNA molecules play essential roles in many biological processes, including the storage and transfer of information in the cell. These events are mediated via RNA-protein interactions or by catalytic RNA molecules. It is now recognized that unique RNA folds are associated with biological functions. Therefore, to study the intrinsic structural changes and dynamics which regulate the various functions of RNA, it is necessary to probe its three-dimensional structure in solution. In this respect, using single-molecule methodologies may allow study of native RNA molecules independent of their size and in real time. However, this may require the immobilization of RNA on a surface. Here, we report a novel approach to immobilize RNA on a glass. The procedures involve both chemical and enzymatic modifications of long RNA molecules. In addition, we demonstrate the application of an optical tweezers apparatus to measure the length and, hence, the dynamics of immobilized intact ribosomal RNA molecules as a function of different solution conditions.
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(2003) Protein Science. 12, 3, p. 468-479 Abstract
Glu-60 of the zinc-dependent Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) is a strictly conserved residue in all members of the alcohol dehydrogenase (ADH) family. Unlike most other ADHs, the crystal structures of TbADH and its analogs, ADH from Clostridium beijerinckii (CbADH), exhibit a unique zinc coordination environment in which this conserved residue is directly coordinated to the catalytic zinc ion in the native form of the enzymes. To explore the role of Glu-60 in TbADH catalysis, we have replaced it by alanine (E60A-TbADH) and aspartate (E60D-TbADH). Steady-state kinetic measurements show that the catalytic efficiency of these mutants is only four- and eightfold, respectively, lower than that of wild-type TbADH. We applied X-ray absorption fine-structure (EXAFS) and near-UV circular dichroism to characterize the local environment around the catalytic zinc ion in the variant enzymes in their native, cofactor-bound, and inhibited forms. We show that the catalytic zinc site in the studied complexes of the variant enzymes exhibits minor changes relative to the analogous complexes of wild-type TbADH. These moderate changes in the kinetic parameters and in the zinc ion environment imply that the Glu-60 in TbADH does not remain bound to the catalytic zinc ion during catalysis. Furthermore, our results suggest that a water molecule replaces this residue during substrate turnover.
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(2003) Nature Structural Biology. 10, 2, p. 98-103 Abstract
Zinc-dependent enzymes play important roles in many cellular processes. Assignment of their reaction mechanisms is often a subject of debate because the zinc ion is silent in several spectroscopic techniques. We have combined time-resolved X-ray absorption spectroscopy, pre-steady state kinetics and computational quantum chemistry to study the active site zinc ion of bacterial alcohol dehydrogenase during single substrate turnover. We detect a series of alternations in the coordination number and structure of the catalytic zinc ion with concomitant changes in metal-ligand bond distances. These structural changes are reflected in the effective charge of the metal ion. The present work emphasizes the flexibility of catalytic zinc sites during catalysis and provides novel mechanistic insights into alcohol dehydrogenase catalysis.
2002
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(2002) Matrix Biology. 21, 7, p. 567-577 Abstract
Matrix Metalloproteinases (MMPs) are cell-secreted soluble and membrane-tethered enzymes that degrade extracellular matrix (ECM) proteins. These proteases play a key role in diverse physiological and pathological processes, including embryonic development, wound repair, inflammatory diseases and cancer. Yet, there is insufficient knowledge on the mode by which cell-produced MMPs conduct their action on the ECM. Specifically, the localization and the mode of the degradation within the pericellular space are of great interest. To provide new insights to these questions we utilized Fourier transform infrared (FTIR) micro-spectroscopy to follow proteolytic processes, induced by invasive cancer cells, on insoluble collagen-based matrices. Here we show that FTIR micro-spectroscopy have a great potential for monitoring degradation events near cells. Using this tool we demonstrate that the net proteolysis is unevenly distributed around the cell boundary. The degradation patterns show different levels of proteolytic activity by MMPs within the pericellular space. In addition, our spectral analysis suggests that the enzymatic proteolysis of the collagen-based matrices induces unwinding of the triple helical structures of the macromolecules within the collagen network.
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(2002) Journal of Biological Chemistry. 277, 48, p. 46559-46565 Abstract
The motor enzymes that belong to the family of RNA helicases catalyze the strand separation of duplex RNA via ATP hydrolysis. Among these enzymes, Escherichia coli DbpA is a unique RNA helicase because it possesses ATPase-specific activity toward the peptidyl transferase center in 23 S ribosomal RNA. For this reason, it has been the subject of numerous biochemical and structure-function studies. The ATP-stimulated unwinding activity of DbpA toward specific and nonspecific RNA duplexes has been demonstrated. However, the underlying molecular and structural basis, which facilitates its helicase activities, is presently not known. We combined time-dependent limited proteolysis digestion, fluorescence spectroscopy, and three-dimensional structural homology modeling techniques to study the structural conformations of DbpA with respect to its binding to stoichiometric ratios of RNA and cofactors. We show that the conformational state of DbpA is markedly different in the ADP-bound state than in any other state (ATP- or RNA-bound). These results, together with structural homology studies, suggest that a hinge region located in the core domain of DbpA mediates such conformational changes.
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(2002) Journal of Chemical Physics. 116, 21, p. 9449-9456 Abstract
Phase speciation by extended x-ray absorption fine structure (EXAFS) spectroscopy was discussed. Two approaches for the EXAFS data analysis of mixtures: principal component analysis (PCA) and residual phase analysis (RPA) were developed. The number of chemically distinct species in the heterogeneous samples model can be obtained with PCA by analyzing the samples' EXAFS spectra. The RPA is a better technique for the identification of samples. The reliability and limitations of the multiple dataset (MDS), PCA, and RPA were compared by studying a test case of a heterogeneous mixture of two organometallic Co compounds.
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(2002) Journal Of Physical Chemistry B. 106, 17, p. 4552-4559 Abstract
Six zinc complexes, Zn(imidazole)3X (where X = water, CI3S-, hydroxamate, formylhydroxylamine, hypophosphite, and acetate), have been calculated with the density functional theory (DFT) method B3LYP/ 6-31G* to probe the interaction between the exogenous ligands and the catalytic zinc ion in matrix metalloproteinases (MMPs). According to our calculation, their interaction modes can be divided into three classes. The structural features of these complexes are in agreement with the X-ray data in the Cambridge Structural Database and the Protein Data Bank. The atomic charge analyses proved that the catalytic water molecule in the active site of MMPs can be activated after coordinating with the zinc ion and thus acts as a good nucleophile to attack and degrade the substrate of MMPs. The geometric parameters of these complexes, the charge transfers from the exogenous ligands to the Zn(II), and the calculated relative binding free energy characterize well the binding ability of these ligands with the zinc ion. The inhibitory activities of the MMP inhibitors, which contain the same substituents and different zinc binding groups (ZBGs), correlate well with the binding free energies predicted by the DFT calculation with correlation coefficients of 0.969 for matrilysin and 0.939 for human fibroblast collagenase (HFC). This gives a possible good strategy for predicting the inhibitory activity for the newly designed inhibitors of MMPs; those potential inhibitors that would exhibit strong binding ability of ZBGs to Zn(II) using this paradigm would therefore be expected to have greater inhibitory activity.
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(2002) Advanced Functional Materials. 12, 1, p. 43-48 Abstract
We compare the organization of the first coordination shells around the calcium ion in biogenic ACC phases from three different sources. The results show that although the three biogenic samples have the same chemical composition, which is referred to collectively under the name "amorphous calcium carbonate", they are structurally different from one another. These differences may be attributed to the diverse modes of formation of such biogenic materials and may account for their known variations in stability.
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(2002) Physical review letters. 89, 28, Abstract
We demonstrate that the synchronization effect observed [Pedersen et al., Phys. Rev. Lett. 87, 055001 (2001)], when a bunch of ions oscillates between two mirrors in an electrostatic ion beam trap, can be explained as a negative mass instability. We derive simple necessary conditions for the existence of a regime in which this dispersionless behavior occurs and demonstrate that in this regime, the ion trap can be used as a high resolution mass spectrometer.
2001
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Ion motion synchronization in an ion-trap resonator(2001) Physical review letters. 87, 5, p. 055001/1-055001/4 055001. Abstract
The synchronized oscillations of a large number of ions forming a bunch in an electrostatic ion trap are described. It is shown that the coupling mechanism of these ions is the repulsive Coulomb interaction between them. The absence of dispersion, despite the finite energy spread of the ions, are exploited to transform the trap into a mass spectrometer with extremely high resolution.
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(2001) Journal of Biological Chemistry. 276, 20, p. 17125-17131 Abstract
Malignant tumors express high levels of zinc-dependent endopeptidases called matrix metalloproteinases (MMPs), which are thought to facilitate tumor metastasis and angiogenesis by hydrolyzing components of the extracellular matrix. Of these enzymes, gelatinases A (MMP-2) and B (MMP-9), have especially been implicated in malignant processes, and thus, they have been a target for drugs designed to block their activity. Therefore, understanding their molecular structure is key for a rational approach to inhibitor design. Here, we have conducted x-ray absorption spectroscopy of the full-length human MMP-2 in its latent, active, and inhibited states and report the structural changes at the zinc ion site upon enzyme activation and inhibition. We have also examined the molecular structure of NMP-2 in complex with SB-3CT, a recently reported novel mechanism-based synthetic inhibitor that was designed to be highly selective in gelatinases (1). It is shown that SB-3CT directly binds the catalytic zinc ion of MMP-2. Interestingly, the novel mode of binding of the inhibitor to the catalytic zinc reconstructs the conformational environment around the active site metal ion back to that of the proenzyme.
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(2001) Proceedings of the National Academy of Sciences of the United States of America. 98, 9, p. 5007-5012 Abstract
The Escherichia coli protein DbpA is unique in its subclass of DEAD box RNA helicases, because it possesses ATPase-specific activity toward the peptidyl transferase center in 23S rRNA. Although its remarkable ATPase activity had been well defined toward various substrates, its RNA helicase activity remained to be characterized. Herein, we show by using biochemical assays and atomic force microscopy that DbpA exhibits ATP-stimulated unwinding activity of RNA duplex regardless of its primary sequence. This work presents an attempt to investigate the action of DEAD box proteins by a single-molecule visualization methodology. Our atomic force microscopy images enabled us to observe directly the unwinding reaction of a DEAD box helicase on long stretches of double-stranded RNA. Specifically, we could differentiate between the binding of DbpA to RNA in the absence of ATP and the formation of a Y-shaped intermediate after its progression through double-stranded RNA in the presence of ATP. Recent studies have questioned the designation of DbpA, in particular, and DEAD box proteins in general as RNA helicases. However, accumulated evidence and the results reported herein suggest that these proteins are indeed helicases that resemble in many aspects the DNA helicases.
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(2001) Journal of Synchrotron Radiation. 8, 2, p. 978-980 Abstract
The understanding of structure-function relationships in proteins has been significantly advanced with the advent of the biotechnological revolution. A goal yet to be realized for many metalloenzyme systems is to characterize the dynamic changes in structure that bridge the static endpoints provided by crystallography. We present here a series of edge and EXAFS spectra of the metalloenzyme alcohol dehydrogenase from Thermoanaerobacter brockii (TbADH) complexed with its substrate. The enzyme-substrate complexes were trapped by fast freezing at various times, following their enzyme activity. Our edge and EXAFS analyses both reveal the time-dependent changes in the structure of the active site of TbADH.
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Simulations for x-ray synchrotron beams using the EGS4 code system in medical applications(2001) Advanced Monte Carlo For Radiation Physics, Particle Transport Simulation And Applications. p. 93-98 Abstract
X-ray synchrotron beams are commonly used in biological and medical research. The availability of intense, polarized low-energy photons from the synchrotron beams provides a high dose transfer to biological materials. The EGS4 code system, which includes the photoelectron angular distribution, electron motion inside a magnetic field, and the LSCAT package, found to be the appropriate Monte Carlo code for synchrotron-produced x-ray simulations. The LSCAT package was developed in 1995 for the EGS4 code to contain the routines to simulate the linear polarization, the bound Compton, and the incoherent scattering functions. Three medical applications were demonstrated using the EGS4 Monte Carlo code as a proficient simulation code system for the synchrotron low-energy x-ray source.
2000
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(2000) Journal of Biological Chemistry. 275, 44, p. 34335-34343 Abstract
Matrix metalloproteinases are endopeptidases that have a leading role in the catabolism of the macromolecular components of the extracellular matrix in a variety of normal and pathological processes. Human gelatinase B is a zinc-dependent proteinase and a member of the matrix metalloproteinase family that is involved in inflammation, tissue remodeling, and cancer. We have conducted x-ray absorption spectroscopy, atomic emission, and quantum mechanics studies of natural and activated human gelatinase B. Our results show that the natural enzyme contains one catalytic zinc ion that is central to catalysis. In addition, upon enzyme activation, the catalytic zinc site exhibits a conformation change that results in the expansion of the bond distances around the zinc ion and the replacement of one sulfur with oxygen. Interestingly, quantum mechanics calculations show that oxygen ligation at the catalytic zinc ion exhibits a greater affinity to the binding of an oxygen from an amino acid residue rather than from an external water molecule. These results suggest that the catalytic zinc ion plays a key role in both substrate binding and catalysis.
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(2000) Analytical Chemistry. 72, 17, p. 4041-4046 Abstract
We report on the application of an electrostatic ion beam trap as a mass spectrometer. The instrument is analogous to an optical resonator; ions are trapped between focusing mirrors. The storage time is limited by the residual gas pressure and reaches up to several seconds, resulting in long ion flight paths. The oscillation of ion bunches between the mirrors is monitored by nondestructive image charge detection in a field-free region and mass spectra are obtained via Fourier transform. The principle of operation is demonstrated by measuring the mass spectrum of trapped Ar+ and Xe+ particles, produced by a standard electron impact ion source. Also, mass spectra of heavier PEGnNa+ and bradykinin ions from a pulsed MALDI ion source were obtained. The long ion flight path, combined with mass-independent charge detection, makes this system particularly interesting for the investigation of large molecules.
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(2000) Biochemistry. 39, 26, p. 7702-7711 Abstract
Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) catalyzes the reversible oxidation of secondary alcohols to the corresponding ketones using NADP+ as the cofactor. The active site of the enzyme contains a zinc ion that is tetrahedrally coordinated by four protein residues. The enzymatic reaction leads to the formation of a ternary enzyme-cofactor-substrate complex; and catalytic hydride ion transfer is believed to take place directly between the substrate and cofactor at the ternary complex. Although crystallographic data of TbADH and other alcohol dehydrogenases as well as their complexes are available, their mode of action remains to be determined. It is firmly established that the zinc ion is essential for catalysis. However, there is no clear agreement about the coordination environment of the metal ion and the competent reaction intermediates during catalysis. We used a combination of X-ray absorption, circular dichroism (CD), and fluorescence spectroscopy, together with structural analysis and modeling studies, to investigate the ternary complexes of TbADH that are bound to a transition-state analogue inhibitor. Our structural and spectroscopic studies indicated that the coordination sphere of the catalytic zinc site in TbADH undergoes conformational changes when it binds the inhibitor and forms a pentacoordinated complex at the zinc ion. These studies provide the first active site structure of bacterial ADH bound to a substrate analogue. Here, we suggest the active site structure of the central intermediate complex and, more specifically, propose the substrate-binding site in TbADH.
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(2000) Journal Of The Chemical Society-Dalton Transactions. 2000, 21, p. 3977-3982 Abstract
Amorphous calcium carbonate is formed by a surprisingly large number of organisms, bearing in mind how unstable it is. Organisms use amorphous calcium carbonate as temporary storage sites for ions, as precursor phases that transform into more stable crystalline calcium carbonate polymorphs, or in a stabilized form for mechanical purposes. Here one example is examined that fulfils the latter function; the so-called antler shaped spicules formed by the ascidian Pyura pachydermatina. These spicules are composed of amorphous calcium carbonate containing 16% (w/w) water and 14 mole% phosphate. A detailed X-ray absorption spectroscopy (XAS) study of the calcium K-edge in the spicules shows a first co-ordination shell with seven/eight oxygen atoms, and a second co-ordination shell with four/five carbon atoms. The best fit was obtained using monohydrocalcite as model, and is consistent with a slightly expanded hydrated structure. It is noteworthy that the XAS spectrum of these spicules is quite different from that reported previously for the amorphous calcium carbonate of plant cystoliths. This raises the intriguing possibility that the biogenic amorphous phases differ structurally from each other, and that the differences can account for their diverse modes of formation and function.
1999
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(1999) Journal of Synchrotron Radiation. 6, 3, p. 409-410 Abstract
The structure of vanadate, a phosphate analogue which functions in the presence of tightly bound ADP and divalent cations as a transition state inhibitor of CF1-ATPase, was investigated by X-ray absorption spectroscopy. There was a decrease in the intensity of the pre-edge transition and a change in the shape and energy of the K-edge upon binding of ADP-vanadate-Mg(II) to CF1. The changes were due to alterations in the structure of vanadium from tetrahedral to five-coordinated trigonal bipyramidal geometry. Simulation of the edge shape and energies and EXAFS analysis confirmed the presence of pentacoordinated vanadium bound to the enzyme. This structure was analogous to the proposed transition state of the phosphate during the synthesis and the hydrolysis of ATP by CF1.
1998
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(1998) Photosynthesis Research. 57, 3, p. 275-285 Abstract
The structure of vanadate, a phosphate analogue which was suggested to function in the presence of tightly bound ADP and divalent cations as a transition state inhibitor of CF1-ATPase, was investigated by X-ray absorption spectroscopy. Analysis of the vanadium K-edge was used for determination of the structure of vanadate bound to a single site in CF1-ATPase containing a single tightly bound ADP. There was a decrease in the intensity of the 1s-3d pre-edge transition and a change in the shape of two other shoulders at the edge region upon binding of vanadate to CF1 in the presence of Mg2+ ions. The changes are due to alteration in the structure of vanadium from tetrahedral to a five-coordinated trigonal bipyramidal geometry. Comparison of the pre-edge peak intensity of ADP-vanadate complex, and model compound resolved by crystallography support the proposed structure of CF1-bound vanadate. 51V NMR measurements were used to verify the pentacoordinated structure of ADP-vanadate complex used as a model in the X-ray absorption studies. The inhibition of a single and multiple site activity by vanadate and by MgADP was measured. Vanadate inhibition of CF1-ATPase activity decreased more than 90 fold in the presence of MgADP. A differential specificity of the inhibition in single and multiple mode of activity was observed. It is suggested that ADP-vanadate binds to the active sites of the enzyme as a pentacoordinated vanadium having approximate trigonal bipyramidal geometry. This structure is analogous to the proposed transition state of the phosphate during the synthesis and the hydrolysis of ATP by CF1.
1997
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(1997) Acta Physica Polonica A. 91, 5, p. 871-875 Abstract
X-ray absorption spectra of photosystem I core protein containing Fx, the low potential iron-sulfur cluster was measured. X-ray absorption of the iron K-edge was obtained in the reduced and oxidized forms of Fx. An edge shift of 0.5 eV to lower energy and a change in the X-ray absorption near edge structure was observed. Some of the alterations in edge shape of Fx are interpreted to indicate structural changes of the iron-sulfur cluster in Fx upon reduction.
1996
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(1996) Journal of Crystal Growth. 168, 1, p. 308-323 Abstract
Preliminary phases were determined by the application of the isomorphous replacement method at low and intermediate resolution for structure factor amplitudes collected from crystals of large and small ribosomal subunits from halophilic and thermophilic bacteria. Derivatization was performed with dense heavy atom clusters, either by soaking or by specific covalent binding prior to the crystallization. The resulting initial electron density maps contain features comparable in size to those expected for the corresponding particles. The packing arrangements of these maps have been compared with motifs observed by electron microscopy in positively stained thin sections of embedded three-dimensional crystals, as well as with phase sets obtained by ab-initio computations. Aimed at higher resolution phasing, procedures are being developed for multi-site binding of relatively small dense metal clusters at selected locations. Potential sites are being inserted either by mutagenesis or by chemical modifications to facilitate cluster binding to the large halophilic and the small thermophilic ribosomal subunits which yield crystals diffracting to the highest resolution obtained so far for ribosomes, 2.9 and 7.3 Angstrom, respectively. For this purpose the surfaces of these ribosomal particles have been characterized and conditions for quantitative reversible detachment of selected ribosomal proteins have been found. The corresponding genes are being cloned, sequenced, mutated to introduce the reactive side-groups (mainly cysteines) and overexpressed. To assist the interpretation of the anticipated electron density maps, sub-ribosomal stable complexes were isolated from H50S. One of these complexes is composed of two proteins and the other is made of a stretch of the rRNA and a protein. For exploiting the exposed parts of the surface of these complexes for heavy atom binding and for attempting the models reconstructed from tilt series of crystalline arrays of ribosomal partic
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The combination of functional, genetics, biochemical, microscopical and crystallographic studies led to initial phasing of data collected from ribosomal crystals at intermediate resolution(1996) Biological structure and dynamics. p. 25-41 Abstract
1995
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(1995) Biochemistry And Cell Biology-Biochimie Et Biologie Cellulaire. 73, 11-12, p. 739-749 Abstract
Preliminary electron density maps of the large and the small ribosomal particles from halophilic and thermophilic sources, phased by the isomorphous replacement method, have been constructed at intermediate resolution. These maps contain features comparable in size with what is expected for the corresponding particles, and their packing arrangements are in accord with the schemes obtained by ab-initio procedures as well as with the motifs observed in thin sections of the crystals by electron microscopy. To phase higher resolution data, procedures are being developed for derivatization by specific labeling of the ribosomal particles at selected locations with rather small and dense clusters. Potential binding sites are being inserted either by site directed mutagenesis or by chemical modifications to facilitate duster binding on the surface of the halophilic large and the thermophilic small ribosomal particles, which yield the crystals diffracting to highest resolution (2.9 and 7.3 Angstrom (1 Angstrom = 0.1 nm), respectively). For this purpose, the surface of these ribosomal particles is being characterized and procedures are being developed for quantitative detachment of selected ribosomal proteins and for their incorporation into core particles. The genes of these proteins are being cloned, sequenced, mutated to introduce reactive side groups, mainly cysteines, and overexpressed. In parallel, two in situ small and stable complexes were isolated from the halophilic ribosome. Procedures for their crystal production in large quantities are currently being developed. Models, reconstructed at low resolution from crystalline arrays of ribosomes and their large subunits, are being used for initial low-resolution phasing of the X-ray amplitudes. The interpretation of these models stimulated the design and the crystallization of complexes mimicking defined functional states of a higher quality than those obtained for isolated ribosomes. These models also inspired modell
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(1995) Biophysical Chemistry. 55, 1-2, p. 31-41 Abstract
Crystals of various ribosomal particles, diffracting best to 2.9 Å resolution were grown. Crystallographic data were collected from shock frozen crystals with intense synchrotron radiation at cryo temperature. For obtaining phase information, monofunctional reagents were prepared from an undecagold and a tetrairidium cluster, by attaching to them chemically reactive handles, specific for sulfhydryl moieties. Heavy-atom derivatives were prepared by a specific and quantitative binding of the undecagold cluster to an exposed sulfhydryl prior to the crystallization. To create potential binding sites on the halophilic and thermophilic ribosomal particles, which yield our best and most interesting crystals, exposed reactive moieties were inserted, using genetic and chemical procedures. In order to choose the appropriate locations for these insertions, the surfaces of the ribosomal particles were mapped by direct chemical determination of exposed amino and sulfhydryl groups.
1994
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(1994) Biochemistry. 33, 20, p. 6310-6315 Abstract
Sulfur-containing cobalamins are thought to have a special role in the intracellular conversion of cyanocobalamin to its coenzyme forms through a Co(I) intermediate. Glutathionylcobalamin is especially interesting as a possible precursor of cobalamin coenzymes [Wagner et al. (1969) Ann. N.Y. Acad. Sci. 112, 580; Pezacka et al. (1990) Biochem. Biophys. Res. Commun. 169, 443]. Recent NMR data [Brown et al. (1993) Biochemistry 32, 8421] strongly support the hypothesis that glutathione coordinates to the cobalt through the sulfur atom in glutathionylcobalamin. In this study threesulfur containing cobalamin derivatives (glutathionylcobalamin, sulfitocobalamin, and cysteinylcobalamin) have been characterized by X-ray absorption spectroscopy. We give evidence for the sulfur coordination in these compounds and present the corresponding structural information. The Co-Neq distances in the sulfur-containing cobalamins are very close to one another (1.90 ± 0.01 Å). The Co-S and Co-Nax distances are also similar (Co-2.282.35 Å and Co-Nax: 2.13-2.16 Å) and in the expected range. The Xray edge positions for the sulfur derivatives shift to lower energies with respect to cyanocobalamin. This indicates strong electron donation from the sulfur to the cobalt and suggests that the effective charge on the cobalt ion in sulfur cobalamins is largely reduced from +3.
1993
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(1993) Systematic and Applied Microbiology. 16, 4, p. 697-705 Abstract
Ribosomal particles from Haloarcula marismortui were crystallized. The best crystals, diffracting to 2.9 Å resolution and yielding crystallographic data of reasonable quality were grown from the large ribosomal subunits. Attempts at crystallization of functional ribosomal complexes are in progress, benefiting from experience gained from crystals of ribosomes of an extreme thermophilic bacterium, Thermus thermophilus. For obtaining phase information, a monofunctional reagent was prepared from an undecagold cluster, by attaching to it a chemically reactive handle, specific for sulfhydryl moieties. Heavy atom derivatives were prepared by binding this cluster to exposed sulfhydryls prior to the crystallization. Cores of halophilic ribosomal particles, lacking four ribosomal proteins, were prepared using dioxane. All detached proteins could be fully reconstituted. However, blocking the -SH group of one of them (HmaL11), prevented its incorporation into the core particles. The so obtained depleted 50S subunits crystallize under the same conditions as native ones and show apparent isomorphism with them. Most of the genes of several r-proteins were cloned. These are being used for sequencing as well as for providing new locations for binding heavy atom clusters by genetic insertion of cysteines on the ribosomal surface, according to accessibility data, obtained either chemically or by limited proteolysis. A nucleoprotein complex of protein HmaL1 and a fragment of the 23S rRNA was isolated from ribosomes. Chimeric complexes were reconstituted with E. coli ribosomal components, indicating rather high homolgy, despite the evolution distance.
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(1993) The Translational Apparatus: Structure, Function, Regulation, Evolution. Subramanian A. R., Wittmann-Liebold B., Franceschi F., Nierhaus K. H. & Erdmann V. A.(eds.). Boston, MA: . p. 397-410 Abstract
The studies reported here were initiated and inspired by the late Prof. H.G. Wittmann. From the early stages of this project, when it was widely believed that even the initial steps in determining the molecular structure of ribosomes are impossible, until his last days, Prof. Wittmann was actively involved in the experimental design and in the actual studies. We have no doubt that without his motivation, optimism, guidance and support, this project would not have reached its current stage.
1992
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Extended x-ray absorption fine structure studies of a retrovirus: equine infectious anemia virus cysteine arrays are coordinated to zinc.: Equine infectious anemia virus cysteine arrays are coordinated to zinc(1992) Proceedings Of The National Academy Of Sciences Of The United States Of America-Biological Sciences. 89, 21, p. 10041-10045 Abstract
Zinc finger arrays have been established as a critical structural feature of proteins involved in DNA recognition. Retroviral nucleocapsid proteins, which are involved in the binding of viral RNA, contain conserved cysteine-rich arrays that have been suggested to coordinate zinc. We provide metalloprotein structural data from an intact virus preparation that validate this hypothesis. Extended x-ray absorption fine structure (EXAFS) spectroscopy of well-characterized and active preparations of equine infectious anemia virus, compared with a peptide with known coordination and in combination with available biochemical and genetic data, defines a Cys3His1 coordination environment for zinc. The average of the Zn-S distances is 2.30(1) A and that of the Zn-N distance (to histidine) is 2.01(3) A.
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(1992) Journal of the American Chemical Society. 114, 21, p. 8061-8066 Abstract
Although a number of (nonalky1)cobalamins are considered essential to human nutrition, providing starting materials in the synthesis of alkylcobalamin coenzymes, their structures are either not available or are very old and unreliable.'*2 We present cobalt-ligand distance information in solution for methylcobalamin, cyanocobalamin, and aquocobalamin using extendedX-ray absorption fine Structure (EXAFS) spectroscopy. Methylcobalamin was examined as a control to verify our ability to accurately assign cobalt-ligand distances, since this compound has a known X-ray ~tructure.~ The structures of cyanocobalamin and aquocobalamin show a remarkable and unexpectedly long cobalt-ligand bond distance of 2.14-2.15 * 0.03 A to the 5,6-dimethylbenzimidazole (DMB) ligand, while the cobalt-ligand distances to CN and H20 fall in the expected range of 1.90 0.03 A. The structural trans effects of CN and H20 are reasonably considered to be weak; therefore, the relativelylong Co-N(DMB) bond must be the result of steric repulsions between the DMB ligand and the corrin equatorial ligand.
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(1992) Protein Science. 1, 5, p. 563-574 Abstract
Abstract All retroviral nucleocapsid (NC) proteins contain one or two copies of an invariant 3Cys?1His array (CCHC = C?X2?C?X4?H?X4?C; C = Cys, H = His, X = variable amino acid) that are essential for RNA genome packaging and infectivity and have been proposed to function as zinc?binding domains. Although the arrays are capable of binding zinc in vitro, the physiological relevance of zinc coordination has not been firmly established. We have obtained zinc?edge extended X?ray absorption fine structure (EXAFS) spectra for intact retroviruses in order to determine if virus?bound zinc, which is present in quantities nearly stoichiometric with the CCHC arrays (Bess, J.W., Jr., Powell, P.J., Issaq, H.J., Schumack, L.J., Grimes, M.K., Henderson, L.E., & Arthur, L.O., 1992, J. Virol. 66, 840?847), exists in a unique coordination environment. The viral EXAFS spectra obtained are remarkably similar to the spectrum of a model CCHC zinc finger peptide with known 3Cys?1His zinc coordination structure. This finding, combined with other biochemical results, indicates that the majority of the viral zinc is coordinated to the NC CCHC arrays in mature retroviruses. Based on these findings, we have extended our NMR studies of the HIV?1 NC protein and have determined its three?dimensional solution?state structure. The CCHC arrays of HIV?1 NC exist as independently folded, noninteracting domains on a flexible polypeptide chain, with conservatively substituted aromatic residues forming hydrophobic patches on the zinc finger surfaces. These residues are essential for RNA genome recognition, and fluorescence measurements indicate that at least one residue (Trp37) participates directly in binding to nucleic acids in vitro. The NC is only the third HIV?1 protein to be structurally characterized, and the combined EXAFS, structural, and nucleic acid?binding results provide a basis for the rational design of new NC?targeted antiviral agents and vaccines for the control of AIDS.
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(1992) Biophysical Journal. 63, 2, p. 412-417 Abstract
X-ray edge and extended x-ray absorption fine structure (EXAFS) techniques provide powerful tools for analysis of local molecular structure of complexes in solution. We present EXAFS results for Co(I) B12 that demonstrate a four-coordinate (distorted) square-planar configuration. Comparison of EXAFS solutions for Co(I) and Co(II) B12 (collected previously; Sagi et al. 1990. J. Am. Chem. Soc. 112:8639-8644) suggest that modulation of the Co-N bond to the axial 5,6-dimethylbenzimidazole (DMB), in the absence of changes in Co-N (equatorial) bond distances, may be a key mechanism in promoting homolytic versus heterolytic cleavage. As Co-C bond homolysis occurs, the Co-N (DMB) bond becomes stronger. However, for heterolytic cleavage to occur, earlier electrochemical studies (D. Lexa and J. M. Saveant. 1976. J. Am. Chem. Soc. 98:2652-2658) and recent studies of methylcobalamin-dependent Clostridium thermoaceticum (Ragsdale et al. 1987. J. Biol. Chem. 262:14289-14297) suggest that removal of the DMB ligand (before Co-C bond cleavage) favors formation of the four-coordinate square-planar Co(I) species while inhibiting formation of the five-coordinate Co(II) B12 complex. This paper presents the first direct evidence that formation of the Co(I) B12 intermediate must involve breaking of the Co-N (DMB) bond.
1991
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(1991) Journal of the American Chemical Society. 113, 14, p. 5299-5304 Abstract
Geometric conformations for the cobalt(1) and cobalt(I1) BI2 intermediates in solution are examined by X-ray edge spectroscopy. Integration of 1s-3d and 1s-4p + SD (shakedown) transitions of edge spectra facilitates a direct comparisonof relative preedge intensities of the lower valence BI2 intermediates to model compounds of known structure. Our results indicate that the structure of the cobalt(1) and cobalt(I1) forms of B12 in solution are distorted square-planar and distorted square-pyramidal, respectively. Base-off Co(II1) adenosylcobalamin, generated by acidification, is shown to be a distorted octahedron where the axial 5,6-dimethylbenzimidazole (DMB) ligand is replaced by a water molecule at low temperature.The demonstration that Co(1) BI2 has a detached DMB axial ligand indicates that cleavage of this bond is required for enzyme reactions like those catalyzed by methionine synthetase (Frasca et al. Biochemistry 1988, 27, 8458). Such a mechanism must be distinct from those of enzymes involved in homolytic cleavage, where EXAFS studies show a strengthening of the bond from cobalt to DMB for the Co(I1) BI2 species (Sagi et al. J. Am. Chem. Soc. 1990, 112, 8639). Shifts in the cobalt absorption threshold (main peak of the first derivative spectrum) are shown to be characteristic of the effective charge on the cobalt ion.Edge shifts to lower energy for the reduction of Co(II1) to Co(I1) to Co(1) cobalamins and cobaloximes follow a linear relationship between the absorption threshold position and the effective charge. The u donor effects of alkyl ligands bonded to cobalt are also evidenced by shifts in the threshold. A reduced positive charge for these compounds may be important in lowering thebarrier to formation of lower oxidation states upon cleavage of the cobalt-carbon bond.
1990
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(1990) Journal of the American Chemical Society. 112, 24, p. 8639-8644 Abstract
The prevalence and importance of BI,-dependent enzyme reactions is well-known; however, the structures of the catalytic intermediates are not well-understood. Homolytic cleavage of the cobalt-carbon bond occurs for a number of BI2 enzyme reactions, so that the reduced, Co(II), form of BI2 is well-established as an important intermediate. Using EXAFS spectroscopy, we have examined the structure of Co(1I) B,2 in solution prepared by reduction of cyanocobalamin and by photolysis of adenosylcobalamin. The two EXAFS structures are very similar, with the average of the cobalt-nitrogen distances to the corrin ring at 1.88 f 0.02 A, and a cobalt-nitrogen (Co-N,) distance to the dimethylbenzimidazole ligand of 1.99 f 0.03 A. In addition, X-ray edge measurements indicate that the Co(I1) BIZ complex is five coordinate. Our results suggest that when the cobalt-carbon bond breaks, the Co-Nd bond becomes much stronger, while the Co-N equatorial distances show minimal changes.