Publications

Cancer exploits different mechanisms to escape T-cell immunosurveillance, including overexpression of checkpoint ligands, secretion of immunosuppressive molecules, and aberrant glycosylation. Herein, we report that IFNγ, a potent immunomodulator secreted in the tumor microenvironment, can induce α2,6 hypersialylation in cancer cell lines derived from various histologies. We focused on Siglec-9, a receptor for sialic acid moieties, and demonstrated that the Siglec-9⁺ T-cell population displayed reduced effector function. We speculated that Siglec-9 in primary human T cells can act as a checkpoint molecule and demonstrated that knocking out Siglec-9 using a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system enhanced the functionality of primary human T cells. Finally, we aimed to augment cancer-specific T-cell activity by taking advantage of tumor hypersialylation. Thus, we designed several Siglec-9based chimeric switch receptors (CSR), which included an intracellular moiety derived from costimulatory molecules (CD28/41BB) and different hinge regions. In an antigen-specific context, T cells transduced with Siglec-9 CSRs demonstrated increased cytokine secretions and upregulation of activation markers. Moreover, T cells equipped with specific Siglec-9 CSRs mediated robust antitumor activity in a xenograft model of human tumors. Overall, this work sheds light on tumor evasion mechanisms mediated by sialylated residues and exemplifies an approach to improve engineered T cellbased cancer treatment.

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.

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.

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.

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.

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.

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.

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 ANTXR2^KO 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 ų Da^-1; the solvent content is estimated to be 57.6%.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 PEG_nNa⁺ 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.

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.