Publications

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells by binding to the angiotensin-converting enzyme 2 (hACE2) receptor. This process is aided by the transmembrane protease serine 2 (TMPRSS2), which enhances entry efficiency and infectiousness by cleaving the SARS-CoV-2 surface glycoprotein (Spike). The cleavage primes the Spike protein, promoting membrane fusion instead of receptor-mediated endocytosis. Despite the pivotal role played by TMPRSS2, our understanding of its non-protease distinct domains remains limited. In this report, we present evidence indicating the potential phosphorylation of a minimum of six tyrosine residues within the cytosolic tail (CT) of TMPRSS2. Via the use of TMPRSS2 CT phospho-mimetic mutants, we observed a reduction in TMPRSS2 protease activity, accompanied by a decrease in SARS-CoV-2 pseudovirus transduction, which was found to occur mainly via the endosomal pathway. We expanded our investigation beyond TMPRSS2 CT and discovered the involvement of other non-protease domains in regulating infection. Our co-immunoprecipitation experiments demonstrated a strong interaction between TMPRSS2 and Spike. We revealed a 21 amino acid long TMPRSS2-Spike-binding region (TSBR) within the TMPRSS2 scavenger receptor cysteine-rich (SRCR) domain that contributes to this interaction. Our study sheds light on novel functionalities associated with TMPRSS2s cytosolic tail and SRCR region. Both of these regions have the capability to regulate SARS-CoV-2 entry pathways. These findings contribute to a deeper understanding of the complex interplay between viral entry and host factors, opening new avenues for potential therapeutic interventions.

Increasing evidence suggests that ribosome composition and modifications contribute to translation control. Whether direct mRNA binding by ribosomal proteins regulates the translation of specific mRNA and contributes to ribosome specialization has been poorly investigated. Here, we used CRISPR-Cas9 to mutate the RPS26 C-terminus (RPS26dC) predicted to bind AUG upstream nucleotides at the exit channel. RPS26 binding to positions -10 to -16 of short 5' untranslated region (5'UTR) mRNAs exerts positive and negative effects on translation directed by Kozak and Translation Initiator of Short 5'UTR (TISU), respectively. Consistent with that, shortening the 5'UTR from 16 to 10 nt diminished Kozak and enhanced TISU-driven translation. As TISU is resistant and Kozak is sensitive to energy stress, we examined stress responses and found that the RPS26dC mutation confers resistance to glucose starvation and mTOR inhibition. Furthermore, the basal mTOR activity is reduced while AMP-activated protein kinase is activated in RPS26dC cells, mirroring energy-deprived wild-type (WT) cells. Likewise, the translatome of RPS26dC cells is correlated to glucose-starved WT cells. Our findings uncover the central roles of RPS26 C-terminal RNA binding in energy metabolism, in the translation of mRNAs bearing specific features and in the translation tolerance of TISU genes to energy stress.

The COVID-19 pandemic resulted from the global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since its first appearance in 2019, new SARS-CoV-2 variants of concern (VOCs) have emerged frequently, changing the infections dynamic. SARS-CoV-2 infects cells via two distinct entry routes; receptor-mediated endocytosis or membrane fusion, depending on the absence or presence of transmembrane serine protease 2 (TMPRSS2), respectively. In laboratory conditions, the Omicron SARS-CoV-2 strain inefficiently infects cells predominantly via endocytosis and is phenotypically characterized by decreased syncytia formation compared to the earlier Delta variant. Thus, it is important to characterize Omicrons unique mutations and their phenotypic manifestations. Here, by utilizing SARS-CoV-2 pseudovirions, we report that the specific Omicron Spike F375 residue decreases infectivity, and its conversion to the Delta S375 sequence significantly increases Omicron infectivity. Further, we identified that residue Y655 decreases Omicrons TMPRSS2 dependency and entry via membrane fusion. The Y655H, K764N, K856N and K969N Omicron revertant mutations, bearing the Delta variant sequence, increased the cytopathic effect of cellcell fusion, suggesting these Omicron-specific residues reduced the severity of SARS-CoV-2. This study of the correlation of the mutational profile with the phenotypic outcome should sensitize our alertness towards emerging VOCs.

CRISPR technology affords a simple and robust way to edit the genomes of cells, providing powerful tools for basic research and medicine. While using Cas9 to target a genomic site is very efficient, making a specific mutation at that site is much less so, as it depends on the endogenous DNA repair machinery. Various strategies have been developed to increase the efficiency of knock-in mutagenesis, but often the desired cells remain a small percentage of the total population. To improve efficiency, strategies to select edited cells have been developed. In some applications, a selectable foreign gene is linked directly to the gene of interest (GOI). Alternatively, co-editing, where the GOI is edited along with a selectable gene, enriches the desired cells since the cells that successfully edited the selectable gene are likely to have also edited the GOI. To minimize perturbations of the host genome, "scarless" selection strategies have been developed, where the modified cells are mutated solely in the GOI. In this review, we will discuss strategies employed to improve specific genome editing in mammalian cells, focusing on ways to select successfully edited cells.

Silent information regulator 2-related enzyme 1 (SIRT1) is an NAD⁺-dependent class III deacetylase and a key component of the cellular metabolic sensing pathway. The requirement of NAD⁺ for SIRT1 activity led us to assume that NQO1, an NADH oxidoreductase producing NAD⁺, regulates SIRT1 activity. We show here that SIRT1 is capable of increasing NQO1 (NAD(P)H Dehydrogenase Quinone 1) transcription and protein levels. NQO1 physically interacts with SIRT1 but not with an enzymatically dead SIRT1 H363Y mutant. The interaction of NQO1 with SIRT1 is markedly increased under mitochondrial inhibition. Interestingly, under this condition the nuclear pool of NQO1 is elevated. Depletion of NQO1 compromises the role of SIRT1 in inducing transcription of several target genes and eliminates the protective role of SIRT1 following mitochondrial inhibition. Our results suggest that SIRT1 and NQO1 form a regulatory loop where SIRT1 regulates NQO1 expression and NQO1 binds and mediates the protective role of SIRT1 during mitochondrial stress. The interplay between an NADH oxidoreductase enzyme and an NAD⁺ dependent deacetylase may act as a rheostat in sensing mitochondrial stress.

RFX proteins are a family of conserved DNA binding proteins involved in various, essential cellular and developmental processes. RFX1 is a ubiquitously expressed, dual-activity transcription factor capable of both activation and repression of target genes.The exact mechanism by which RFX1 regulates its target is not known yet. In this work, we show that the C-terminal repression domain of RFX1 interacts with the Serine/Threonine protein phosphatase PP1c, and that interaction with RFX1 can target PP1c to specific sites in the genome. Given that PP1c was shown to de-phosphorylate several transcription factors, as well as the regulatory C-terminal domain of RNA Polymerase II the recruitment of PP1c to promoters may be a mechanism by which RFX1 regulates the target genes. (C) 2019 Elsevier Inc. All rights reserved.

The polyomavirus middle T antigen (PyMT) is an oncogene that activates the non-receptor tyrosine kinase, c-Src, and physically interacts with Taz (WWTR1). Taz is a pro-oncogenic transcription coactivator of the Tead transcription factors. The Hippo tumor suppressor pathway activates the kinase Lats, which phosphorylates Taz, leading to its nuclear exclusion and blunting Tead coactivation. We found that Taz was required for transformation by PyMT, but counter-intuitively, Taz was exclusively cytoplasmic in the presence of PyMT. We demonstrate that in the presence of PyMT, wild-type Taz was phosphorylated by Lats, in a Src-dependent manner. Consistently, a Lats refractory Taz mutant did not undergo cytoplasmic retention by PyMT. We show that Yap, the Taz paralog, and Shp2 phosphatase were nuclear excluded as well. Our findings describe a noncanonical activation of Lats, and an unprecedented Tead-independent role for Taz and Yap in viral-mediated oncogenesis.

YES-associated protein (YAP) is a central transcription coactivator that functions as an oncogene in a number of experimental systems. However, under DNA damage, YAP activates pro-apoptotic genes in conjunction with p73. This program switching is mediated by c-Abl (Abelson murine leukemia viral oncogene) via phosphorylation of YAP at the Y357 residue (pY357). YAP as an oncogene coactivates the TEAD (transcriptional enhancer activator domain) family transcription factors. Here we asked whether c-Abl regulates the YAP-TEAD functional module. We found that DNA damage, through c-Abl activation, specifically depressed YAP-TEAD-induced transcription. Remarkably, c-Abl counteracts YAP-induced transformation by interfering with the YAP-TEAD transcriptional program. c-Abl induced TEAD1 phosphorylation, but the YAP-TEAD complex remained unaffected. In contrast, TEAD coactivation was compromised by phosphomimetic YAP Y357E mutation but not Y357F, as demonstrated at the level of reporter genes and endogenous TEAD target genes. Furthermore, YAP Y357E also severely compromised the role of YAP in cell transformation, migration, anchorage-independent growth, and epithelial-to-mesenchymal transition (EMT) in human mammary MCF10A cells. These results suggest that YAP pY357 lost TEAD transcription activation function. Our results demonstrate that YAP pY357 inactivates YAP oncogenic function and establish a role for YAP Y357 phosphorylation in cell-fate decision.

The p53 family of proteins has an important role in determining cell fate in response to different types of stress, such as DNA damage, hypoxia, or oncogenic stress. In recent years, p53 has also been shown to respond to metabolic stress, and to be induced by the AMP-activated protein kinase (AMPK), a central cellular energy sensor. A bioinformatic analysis revealed three putative AMPK phopshorylation sites in p73, a p53 tumor suppressor paralog. In vitro and in vivo assays confirmed that AMPK phosphorylates p73 on a novel residue, S426. Following specific pharmacologic stimulation of AMPK in cells, p73 protein half-life was prolonged leading to p73 accumulation in the nucleus. We show that p73 escaped the E3 ligase Itch resulting in reduced p73 ubiquitination and proteasomal degradation. Furthermore, chronic activation of AMPK led to apoptosis that was p73 dependent, but only in p53-expressing cells. Surprisingly, we found that p73 was required for p53 stabilization and accumulation under AMPK activation, but was dispensable under DNA damage. Our findings couple p73 with p53 in determining cell fate under AMPK-induced metabolic stress.

PGC-1α is a key transcription coactivator regulating energy metabolism in a tissue-specific manner. PGC-1α expression is tightly regulated, it is a highly labile protein, and it interacts with various proteins-the known attributes of intrinsically disordered proteins (IDPs). In this study, we characterize PGC-1α as an IDP and demonstrate that it is susceptible to 20S proteasomal degradation by default. We further demonstrate that PGC-1α degradation is inhibited by NQO1, a 20S gatekeeper protein. NQO1 binds and protects PGC-1α from degradation in an NADH-dependent manner. Using different cellular physiological settings, we also demonstrate that NQO1-mediated PGC-1α protection plays an important role in controlling both basal and physiologically induced PGC-1α protein level and activity. Our findings link NQO1, a cellular redox sensor, to the metabolite-sensing network that tunes PGC-1α expression and activity in regulating energy metabolism.

Intrinsically unstructured proteins (IUPs) like the structured proteins are subjected to proteasomal degradation. However, unlike the structured ones, there is no crucial need of protein unfolding step to access the IUPs to the 20S catalytic subunit of the proteasome. This distinctive behavior set the stage for operational definition of the IUPs based on their susceptibility to the 20S degradation in a cell free system. Numerous studies revealed that this is the case in the cells as well, although no comprehensive analysis was performed to date. IUPs are degraded by the 20S proteasome subunit by default, without being polyubiquitinated or undergoing any other modifications. IUPs escape the process of degradation by default by a number of mechanisms, of which a more general one is interaction with a partner named nanny. Based on these attributes one can define IUP by conducting a set of cell free and cell culture experiments as outlined in this chapter.

Intrinsically disordered proteins (IDPs), also known as intrinsically unstructured proteins (IUPs), lack a well-defined 3D structure in vitro and, in some cases, also in vivo. Here, we discuss the question of proteolytic sensitivity of IDPs, with a view to better explaining their in vivo characteristics. After an initial assessment of the status of IDPs in vivo, we briefly survey the intracellular proteolytic systems. Subsequently, we discuss the evidence for IDPs being inherently sensitive to proteolysis. Such sensitivity would not, however, result in enhanced degradation if the protease-sensitive sites were sequestered. Accordingly, IDP access to and degradation by the proteasome, the major proteolytic complex within eukaryotic cells, are discussed in detail. The emerging picture appears to be that IDPs are inherently sensitive to proteasomal degradation along the lines of the "degradation by default" model. However, available data sets of intracellular protein half-lives suggest that intrinsic disorder does not imply a significantly shorter half-life. We assess the power of available systemic half-life measurements, but also discuss possible mechanisms that could protect IDPs from intracellular degradation. Finally, we discuss the relevance of the proteolytic sensitivity of IDPs to their function and evolution.

BIM-extra long (BIM_EL), a pro-apoptotic BH3-only protein and part of the BCL-2 family, is degraded by the proteasome following activation of the ERK1/2 signalling pathway. Although studies have demonstrated poly-ubiquitylation of BIM_EL in cells, the nature of the ubiquitin chain linkage has not been defined. Using ubiquitin-binding domains (UBDs) specific for defined ubiquitin chain linkages, we show that BIM_EL undergoes K48-linked poly-ubiquitylation at either of two lysine residues. Surprisingly, BIM_ELΔKK, which lacks both lysine residues, was not poly-ubiquitylated but still underwent ERK1/2-driven, proteasome-dependent turnover. BIM has been proposed to be an intrinsically disordered protein (IDP) and some IDPs can be degraded by uncapped 20S proteasomes in the absence of poly-ubiquitylation. We show that BIM_EL is degraded by isolated 20S proteasomes but that this is prevented when BIM_EL is bound to its pro-survival target protein MCL-1. Furthermore, knockdown of the proteasome cap component Rpn2 does not prevent BIM_EL turnover in cells, and inhibition of the E3 ubiquitin ligase β-TrCP, which catalyses poly-Ub of BIM_EL, causes Cdc25A accumulation but does not inhibit BIM _EL turnover. These results provide new insights into the regulation of BIM_EL by defining a novel ubiquitin-independent pathway for the proteasome-dependent destruction of this highly toxic protein.

The short-lived proto-oncoprotein c-Fos is a component of the activator protein 1 (AP-1) transcription factor. A large region of c-Fos is intrinsically unstructured and susceptible to a recently characterized proteasomal ubiquitin-independent degradation (UID) pathway. UID is active by a default mechanism that is inhibited by NAD(P)H:quinone oxidoreductase 1 (NQO1), a 20S proteasome gatekeeper. Here, we show that NQO1 binds and induces robust c-Fos accumulation by blocking the UID pathway. c-Jun, a partner of c-Fos, also protects c-Fos from proteasomal degradation by default. Our findings suggest that NQO1 protects monomeric c-Fos from proteasomal UID, a function that is fulfilled later by c-Jun. We show that this process regulates c-Fos homeostasis (proteostasis) in response to serum stimulation, phosphorylation, nuclear translocation, and transcription activity. In addition, we show that NQO1 is important to ensure immediate c-Fos accumulation in response to serum, since a delayed response was observed under low NQO1 expression. These data suggest that in vivo, protein unstructured regions determine the kinetics and the homeostasis of regulatory proteins. Our data provide evidence for another layer of regulation of key regulatory proteins that functions at the level of protein degradation and is designed to ensure optimal formation of functional complexes such as AP-1.

Hepatitis B virus (HBV) infects the liver and uses its cell host for gene expression and propagation. Therefore, targeting host factors essential for HBV gene expression is a potential anti-viral strategy. Here we show that treating HBV expressing cells with the natural phenolic compound curcumin inhibits HBV gene expression and replication. This inhibition is mediated via down-regulation of PGC-1 alpha, a starvation-induced protein that initiates the gluconeogenesis cascade and that has been shown to robustly coactivate HBV transcription. We suggest curcumin as a host targeted therapy for HBV infection that may complement current virus-specific therapies.

The degradation of the majority of cellular proteins is mediated by the proteasomes. Ubiquitin - dependent proteasomal protein degradation is executed by a number of enzymes that interact to modify the substrates prior to their engagement with the 26S proteasomes. The 26S proteasome is made of two complexes, the 20S and the 19S. The role of 19S is to unfold the proteins to gain entry into the 20S particle, where the protein is cleaved into short peptides. Thus, some of the functions of the 19S complex are expected to be dispensable for degradation of intrinsically disordered proteins, or IDPs. Indeed, in cell - free systems, at least some of the IDPs are digested by 20S particles in the absence of the 19S. In fact, it appears that susceptibility to the 20S proteasome may represent a hallmark of IDPs. Recent evidence suggests that IDPs are susceptible to degradation in vivo by the 20S proteasome as well. The process is ubiquitin independent and takes place by default. However, the process of default degradation can be regulated by different strategies. The described process of IDP degradation provokes new predictions and explanations in the field of protein regulation and functionality.

Researchers from around the world met for two days in April this year in Rome, Italy, to discuss progress in the rapidly developing field of Hippo signaling, which is relevant to cancer and the control of organ size. Most of the participants presented data related to previously uncharacterized proteins that physically and functionally interact with known components of the Hippo pathway and regulate its biological output.

The N-terminal transcription activation domain of p53 is intrinsically unstructured.Weshow in vitro and in vivo that this domain initiates p53 degradation by the 20 S proteasome in a ubiquitin-independent fashion. The decay of metabolically labeled p53 follows biphasic kinetics with an immediate fast phase that is ubiquitin-independent and a second slower phase that is ubiquitin-dependent. The 20 S proteasome executes the first phase by default, whereas the second phase requires the 26 S proteasome. p53 N-terminal binding proteins, such as Hdmx, can selectively block the first phase of degradation. Remarkably, γ-irradiation inhibits both p53 decay phases, whereas UV selectively negates the second phase, giving rise to discrete levels of p53 accumulation. Our data of a single protein experiencing double mode degradation mechanisms each with unique kinetics provide the mechanistic basis for programmable protein homeostasis (proteostasis).

Rsf-1 interacts with human sucrose nonfermenting protein 2 homologue (hSNF2H) to form a chromatin remodeling complex that participates in several biological processes. We have previously shown that Rsf-1 gene amplification was associated with the most aggressive type of ovarian cancer and cancer cells with Rsf-1 overexpression depended on Rsf-1 to survive. In this report, we determine if formation of the Rsf-1/hSNF2H complex could be one of the mechanisms contributing to tumor cell survival and growth in ovarian carcinomas. Based on immunohistochemistry, we found that Rsf-1 and hSNF2H were co-upregulated in ovarian cancer tissues. Ectopic expression of Rsf-1 in SKOV3 ovarian cancer cells with undetectable endogenous Rsf-1 expression enhanced hSNF2H protein levels and promoted SKOV3 tumor growth in a mouse xenograft model. Our studies also indicated that induction of Rsf-1 expression affected the molecular partnership of hSNF2H and translocated hSNF2H into nuclei where it colocalized with Rsf-1. Furthermore, analysis of Rsf-1 deletion mutants showed that the Rsf-D4 fragment contained the hSNF2H binding site based on coimmunoprecipitation and in vitro competition assays. As compared with other truncated mutants, expression of Rsf-D4 resulted in remarkable growth inhibition in ovarian cancer cells with Rsf-1 gene amplification and overexpression, but not in those without detectable Rsf-1 expression. The above findings suggest that interaction between Rsf-1 and hSNF2H may define a survival signal in those tumors overexpressing Rsf-1.

Cells undergo apoptosis upon exposure to severe DNA damage stress. Under this condition, p73 is phosphorylated and activated by c-Abl. The transcription coactivator Yap1 binds p73 to generate a complex that escapes p73 proteasomal degradation and recruits p300 to support transcription of proapoptotic genes. However, the mechanism of selective activation of proapoptotic genes by Yap1 remained unclear. In this study, we show that c-Abl directly phosphorylates Yap1 at position Y357 in response to DNA damage. Tyrosine-phosphorylated Yap1 is a more stable protein that displays higher affinity to p73 and selectively coactivates p73 proapoptotic target genes. Furthermore, we show that Yap1 switches between p73-mediated proapoptotic and growth arrest target genes based on its phosphorylation state. Thus, our data demonstrate that modification of a transcription coactivator, namely the DNA damage-induced phosphorylation of Yap1 by c-Abl, influences the specificity of target gene activation.

Background: Fibulin-5 is a novel extracellular protein that is thought to act as a bridging peptide between elastin fibers and cell surface integrins in blood vessel wall. Fibulin-5 binding to endothelial cell (EC) surface integrins may effect cell proliferation and cell attachment to extracellular matrix (ECM) or to artificial surfaces. In this paper, we describe the effects of fibulin-5 on attachment, adhesion, and proliferation of primary human EC. After demonstrating that fibulin-5 over-expression inhibited EC proliferation, we tested the hypothesis that co-expression of fibulin-5 and VEGF₁₆₅ will lead to unique EC phenotype that will exhibit increased adherence properties and retain its proliferation capacity. Methods and results: Fibulin-5 and VEGF₁₆₅ gene transfer to primary human saphenous vein endothelial cells was accomplished using retroviral vectors encoding the two genes. Transgene expression was verified using immunohistochemistry, Western blotting, and ELISA. Fibulin 5 over-expression tended to improve immediate EC attachment (30 min after seeding) and improved significantly adhesion (>40%) under shear stress tested 24 h after EC seeding. The effects of fibulin-5 and VEGF₁₆₅ on EC proliferation in the presence or absence of basic FGF were also tested. EC expressing fibulin-5 had reduced proliferation while VEGF₁₆₅ co-expression ameliorated this effect. Conclusion: Fibulin-5 improved EC attachment to artificial surfaces. Dual transfer of fibulin-5 and VEGF₁₆₅ resulted in EC phenotype with increased adhesion and improved proliferation. This unique EC phenotype can be useful for tissue engineering on endovascular prostheses.

The degradation of the majority of cellular proteins is mediated by the proteasomes. Ubiquitin-dependent proteasomal protein degradation is executed by a number of enzymes that interact to modify the substrates prior to their engagement with the 26S proteasomes. Alternatively, certain proteins are inherently unstable and undergo "default" degradation by the 20S proteasomes. Puzzlingly, proteins are by large subjected to both degradation pathways. Proteins with unstructured regions have been found to be substrates of the 20S proteasomes in vitro and, therefore, unstructured regions may serve as signals for protein degradation "by default" in the cell. The literature is loaded with examples where engagement of a protein into larger complexes increases protein stability, possibly by escaping degradation "by default". Our model suggests that formation of protein complexes masks the unstructured regions, making them inaccessible to the 20S proteasomes. This model not only provides molecular explanations for a recent theoretical "cooperative stability" principle, but also provokes new predictions and explanations in the field of protein regulation and functionality.

NAD(P)H quinone oxidoreductase 1 (NQO1) is a ubiquitous flavoenzyme that catalyzes two-electron reduction of quinones to hydroquinones utilizing NAD(P)H as an electron donor. NQO1 binds and stabilizes several short-lived proteins including the tumor suppressors p53 and p73 and the enzyme ornithine decarboxylase (ODC). Dicoumarol is a widely used potent competitive inhibitor of NQO1 enzymatic activity, which competes with NAD(P)H for binding to NQO1. Dicoumarol also disrupts the binding of NQO1 to p53, p73, and ODC and induces their ubiquitin-independent proteasomal degradation. We report here the crystal structure of human NQO1 in complex with dicoumarol at 2.75 Å resolution. We have identified the interactions of dicoumarol with the different residues of NQO1 and the conformational changes imposed upon dicoumarol binding. The most prominent conformational changes that occur in the presence of dicoumarol involve Tyr 128 and Phe 232 that are present on the surface of the NQO1 catalytic pocket. On the basis of the comparison of the NQO1 structure in complex with different NQO1 inhibitors and our previous analysis of NQO1 mutants, we propose that the specific conformation of Tyr 128 and Phe 232 is important for NQO1 interaction with p53 and other client proteins.

The hepatitis B virus (HBV) core Ag (HBcAg) serves as the structural subunit of the highly immunogenic capsid shell. HBcAg harbors a unique arginine-rich C terminus that was implicated in immune responses induced by the capsid. In this study, we examined the capacity of the HBV capsid to induce proinflammatory and regulatory cytokines in human THP-1 macrophages and the possible underlying mechanism. Full-length HBc capsids, but not HBc-144 capsids lacking the arginine-rich domain of HBcAg, efficiently bound differentiated THP-1 macrophages and strongly induced TNF-α, IL-6, and IL-12p40. Capsid binding to macrophages and cytokine induction were independent of the RNA associated with the arginine-rich domain. Soluble heparin and heparan sulfate but not chondroitin sulfates greatly diminished cytokine induction through inhibition of capsid binding to THP-1 macrophages. Furthermore, serine phosphorylation in the arginine-rich domain modulates capsid binding to macrophages and the cytokine response. Induction of cytokines by the capsid involved activation of NF-κB, ERK-1/2, and p38 MAPK and did not require endosomal acidification. Finally, NF-κB activation by the capsid in HEK 293 cells specifically required expression of TLR2 and was compromised by soluble heparin. Thus, cytokine induction by the HBV capsid in macrophages is facilitated by interaction of its arginine-rich domain with membrane heparan sulfate and involves signaling through TLR2.

The rate of association of a protein complex is a function of an intrinsic basal rate and of the magnitude of electrostatic steering. In the present study we analyze the contribution of electrostatics towards the association rate of proteins in a database of 68 transient hetero-protein-protein complexes. Our calculations are based on an upgraded version of the computer algorithm PARE, which was shown to successfully predict the impact of mutations on k _on by calculating the difference in Columbic energy of interaction of a pair of proteins. HyPare (http://bip.weizmann.ac.il/HyPare), automatically calculates the impact of mutations on a perresidue basis for all residues of a protein-protein interaction, achieving a precision similar to that of PARE. Our calculations show that electrostatics play a marginal role (

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is a very labile protein. ODC is a homodimeric enzyme that undergoes ubiquitin-independent proteasomal degradation via direct interaction with antizyme, a polyamine-induced protein. Binding of antizyme promotes the dissociation of ODC homodimers and marks ODC for degradation by the 26S proteasomes. We describe here an alternative pathway for ODC degradation that is regulated by NAD(P)H quinone oxidoreductase 1 (NQO1). We show that NQO1 binds and stabilizes ODC. Dicoumarol, an inhibitor of NQO1, dissociates ODC-NQO1 interaction and enhances ubiquitin-independent ODC proteasomal degradation. We further show that dicoumarol sensitizes ODC monomers to proteasomal degradation in an antizyme-independent manner. This process of NQO1-regulated ODC degradation was recapitulated in vitro by using purified 20S proteasomes. Finally, we show that the regulation of ODC stability by NQO1 is especially prominent under oxidative stress. Our findings assign to NQO1 a role in regulating ubiquitin-independent degradation of ODC by the 20S proteasomes.

Protein degradation is an essential and highly regulated process. The proteasomal degradation of the tumor suppressors p53 and p73 is regulated by both polyubiquitination and by an ubiquitin-independent process. Here, we show that this ubiquitin-independent process is mediated by the 20S proteasomes and is regulated by NQO1. NQO1 physically interacts with p53 and p73 in an NADH-dependent manner and protects them from 20S proteasomal degradation. Remarkably, the vast majority of NQO1 in cells is found in physical association with the 20S proteasomes, suggesting that NQO1 functions as a gatekeeper of the 20S proteasomes. We further show that this pathway plays a role in p53 accumulation in response to ionizing radiation. Our findings provide the first evidence for in vivo degradation of p53 and p73 by the 20S proteasomes and its regulation by NQO1 and NADH level.

Association of two proteins can be described as a two-step process, with the formation of an encounter complex followed by desolvation and establishment of a tight complex. Here, by using the computer algorithm PARE, we designed a set of mutants of the Ras effector protein Ral guanine nucleotide dissociation stimulator (RalGDS) with optimized electrostatic steering. The fastest binding RalGDS mutant, M26K,D47K,E54K, binds Ras 14-fold faster and 25-fold tighter compared with WT. A linear correlation was found between the calculated and experimental data, with a correlation coefficient of 0.97 and a slope of 0.65 for the 24 mutants produced. The data suggest that increased electrostatic steering specifically stabilizes the encounter complex and transition state. This conclusion is backed up by Φ analysis of the encounter complex and transition state of the RalGDS^{M26K,D47K,E54K}/Ras complex, with both values being close to 1. Upon further formation of the final complex, the increased Coulombic interactions are probably counterbalanced by the cost of desolvation of charges, keeping the dissociation rate constant almost unchanged. This mechanism is also reflected by the mutual compensation of enthalpy and entropy changes quantified by isothermal titration calorimetry. The binding constants of the faster binding RalGDS mutants toward Ras are similar to those of Raf, the most prominent Ras effector, suggesting that the design methodology may be used to switch between signal transduction pathways.

p73 is a structural and functional homologue of the p53 tumor-suppressor protein. Like p53, p73 is activated in response to DNA-damaging insults to induce cell cycle arrest or apoptosis. Under these conditions p73 is tyrosine-phosphorylated by c-Abl, a prerequisite modification for p73 to elicit cell death in fibroblasts. In this study we report that in response to ionizing radiation, p73 undergoes nuclear redistribution and becomes associated with the nuclear matrix. This association is c-Abl-dependent because it was not observed in cells that are defective in c-Abl kinase activation. Moreover, STI-571, a specific c-Abl kinase inhibitor, is sufficient to block significantly p73α nuclear matrix association. The observed c-Abl dependence of nuclear matrix association was recapitulated in the heterologous baculovirus system. Under these conditions p73α but not p53 is specifically tyrosine-phosphorylated by c-Abl. Moreover, the phosphorylated p73α is predominantly found in association with the nuclear matrix. Thus, in response to ionizing radiation p73 is modified in a c-Abl-dependent manner and undergoes nuclear redistribution and translocates to associate with the nuclear matrix. Our data describe a novel mechanism of p73 regulation.

Virus infection is initiated by recognition and attachment of the virus to the cell surface. Despite the fact that this interaction determines the virus-related pathogenesis, its molecular basis remained obscure for HBV. This process is mediated primarily by the viral envelope and the cellular receptors. HBV infection is not exceptional in this regard but its putative receptors have not been identified yet. The recent development of protocols to establish HBV susceptible cell lines and unique tools to measure HBV-cell attachment at a single cell resolution set the stage for the study of HBV-host cell interaction. These studies revealed that the QLDPAF epitope of the HBV surface antigen large protein (LHBsAg) plays a major role in this process. Quantitative measurements suggested the presence of a second player in this process and both act synergistically to improve cell attachment. As the step of virus-cell attachment is potentially susceptible to specific inhibitors, understanding the molecular basis of virus-cell attachment can be expected to have therapeutic impacts.

Wild-type p53 is a tumor-suppressor gene that encodes a short-lived protein that, upon accumulation, induces growth arrest or apoptosis. Accumulation of p53 occurs mainly by posttranslational events that inhibit its proteosomal degradation. We have reported previously that inhibition of NAD(P)H: quinone oxidoreductase 1 (NQO1) activity by dicoumarol induces degradation of p53, indicating that NQO1 plays a role in p53 stabilization. We now have found that wild-type NQO1, but not the inactive polymorphic NQO1, can stabilize endogenous as well as transfected wild-type p53. NQO1-mediated p53 stabilization was especially prominent under induction of oxidative stress. NQO1 also partially inhibited p53 degradation mediated by the human papilloma virus E6 protein, but not when mediated by Mdm-2. Inhibitors of heat shock protein 90 (hsp90), radicicol and geldanamycin, induced degradation of p53 and suppressed p53-induced apoptosis in normal thymocytes and myeloid leukemic cells. Differences in the effectiveness of dicoumarol and hsp90 inhibitors to induce p53 degradation and suppress apoptosis in these cell types indicate that NQO1 and hsp90 stabilize p53 through different mechanisms. Our results indicate that NQO1 has a distinct role in the regulation of p53 stability, especially in response to oxidative stress. The present data on the genetic and pharmacologic regulation of the level of p53 have clinical implications for tumor development and therapy.

Lipoprotein(a) [Lp(a)] consists of LDL and apolipoprotein(a), and has been shown to be a major, independent, risk factor for arteriosclerosis and thrombosis in humans. To further elucidate the (patho)physiological function(s) of Lp(a)/apo(a), we searched for new protein ligands, using the yeast two-hybrid system and employing the highly repetitive kringle IV type 2 (KIV-2) sequence from apo(a) as bait. The extracellular matrix protein DANCE [developmental arteries and neural crest epidermal growth factor (EGF)-like] recently implicated in atherogenesis was identified as an interactor. A direct physical interaction between DANCE and apo(a) was confirmed by co-purification of both recombinant proteins derived from culture supernatants of transiently transfected COS-1 cells. Furthermore, binding of human plasma-derived Lp(a) to recombinant DANCE was also observed. Finally, when monoclonal anti-apo(a) and polyclonal anti-DANCE antibodies were applied to tissue slices of atherosclerotic carotid artery, the two proteins were found to be co-localized in endothelial and smooth muscle cells, suggesting that they occur together in the arterial wall. However, as yet, the in vivo relevance and the possible functional role of this newly identified DANCE:Lp(a)/apo(a) interaction remains speculative.

Hepadnaviruses do not infect cultured cells, therefore our knowledge of the mechanism of the early stages of virus-cell interaction is rather poor. In this study, we show that dimethylsulfoxide (DMSO)-treated HepG2 hepatoblastoma cells are infected efficiently by serum-derived hepatitis B virus (HBV) as monitored by viral gene expression and replication markers. To measure virus attachment, a variety of HBV surface proteins (HBsAgs) were conjugated to polystyrene beads and their capacity to attach cells was visualized and quantified by light microscopy at a single-cell resolution. Remarkably, DMSO increases the attachment efficiency by >200-fold. We further identify the QLDPAF sequence within preS1 as the receptor-binding viral domain epitope. Interestingly, a similar sequence is shared by several cellular, bacterial and viral proteins involved in cell adhesion, attachment and fusion. We also found that the small HBsAg contains a secondary attachment site that recognizes a distinct receptor on the cell membrane. Furthermore, we provide evidence in support of multivalent HBV attachment with synergistic interplay. Our data depict a mechanistic view of virus attachment and ingestion.

The HBV X protein (HBx) is implicated in infection and development of hepatocellular carcinoma. HBx has a pleiotropic effect on cells, suggesting multiple targets in the virus-host cell interaction. We employed the cytoplasmic-based two-hybrid screen and identified the HIV Tat-binding protein 1 (Tbp1) as a novel HBx interacting protein. Tbp1 interacts in vivo with HBx both in yeast and in animal cells. This interaction maps to the functionally important ATP-binding motif of Tbp1. Furthermore, HBx and Tbp1 interaction is functionally significant and regulates HBV transcription. Tbp1 homologues, such as Sug1, are known members of the proteasome 19S regulatory cap particle and have also been implicated in transcription coactivation. Remarkably, Tbp1 and Sug1 interact with multiple viral effector proteins including HIV Tat, SV40 large T antigen, and adenovirus E1A, establishing these proteins as important targets of the viral oncogenes.

pX, the hepatitis B virus-encoded transcription coactivator, is involved in viral infection in vivo. pX stimulates the activity of several transcription factors including nuclear factor-kappaB (NF-kappaB), but the mechanism of activation is poorly understood. The I kappaB kinase complex (IKK) mediates activation of NF-kappaB in response to various extracellular stimuli, including inflammatory cytokines like tumor necrosis factor and interleukin 1, human T cell lymphoma virus 1 Tax protein, and tumor promoters like phorbol esters. It is not known whether IKK also mediates activation of NF-kappaB by pX. Here we report that IKK was not essential for activation of NF-kappaB by pX. Expression of pX resulted in the degradation of I kappaB alpha in the absence of its phosphorylation at Ser(32) and Ser(36) residues. Although pX stimulated the activity of cotransfected IKK-beta when it was overexpressed, it failed to activate endogenous IKK. Furthermore, expression of pX stimulated NF-kappaB nuclear translocation and transcriptional activity in IKK-gamma -null fibroblast 5R cells. Our data indicate that pX stimulates NF-kappaB activity through a mechanism that is dependent on I kappaB alpha degradation but not on IKK activation.

Hepatitis B, one of the most common infectious diseases in the world, is closely associated with acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Many clinical investigations have revealed that hepatic fibrosis is an important component of these liver diseases caused by chronic hepatitis B. TGF-β signaling plays an important role in the pathogenesis of fibrosis in chronic hepatitis and cirrhosis. As these diseases are associated with hepatitis B virus (HBV) infection, we examined the possibility that the HBV-encoded pX oncoprotein regulates TGF-β signaling. We show that pX enhances transcriptional activity in response to TGF-β, BMP-2, and activin by stabilizing the complex of Smad4 with components of the basic transcriptional machinery. Additionally, confocal microscopic studies suggest that pX facilitates and potentiates the nuclear translocation of Smads, further enhancing TGF-β signaling. Our studies suggest a new paradigm for amplification of Smad-mediated signaling by an oncoprotein and suggest that enhanced Smad-mediated signaling may contribute to HBV-associated liver fibrosis.

The tumor suppressor gene wild-type p53 encodes a labile protein that accumulates in cells after different stress signals and can cause either growth arrest or apoptosis. One of the p53 target genes, p53-inducible gene 3 (PIG3), encodes a protein with significant homology to oxidoreductases, enzymes involved in cellular responses to oxidative stress and irradiation. This fact raised the possibility that cellular oxidation-reduction events controlled by such enzymes also may regulate the level of p53. Here we show that NADH quinone oxidoreductase 1 (NQO1) regulates p53 stability. The NQO1 inhibitor dicoumarol caused a reduction in the level of both endogenous and γ-irradiation-induced p53 in HCT116 human colon carcinoma cells. This reduction was prevented by the proteasome inhibitors MG132 and lactacystin, suggesting enhanced p53 degradation in the presence of dicoumarol. Dicoumarol-induced degradation of p53 also was prevented in the presence of simian virus 40 large T antigen, which is known to bind and to stabilize p53. Cells overexpressing NQO1 were resistant to dicoumarol, and this finding indicates the direct involvement of NQO1 in p53 stabilization. NQO1 inhibition induced p53 degradation and blocked wild-type p53-mediated apoptosis in γ-irradiated normal thymocytes and in M1 myeloid leukemic cells that overexpress wild-type p53. Dicoumarol also reduced the level of p53 in its mutant form in M1 cells. The results indicate that NQO1 plays an important role in regulating p53 functions by inhibiting its degradation.

The RFX protein family includes members from yeast to humans, which function in various biological systems, and share a DNA-binding domain and a conserved C-terminal region. In the human transcription regulator RFX1, the conserved C terminus is an independent functional domain, which mediates dimerization and transcriptional repression. This dimerization domain has a unique ability to mediate the formation of two alternative homodimeric DNA-protein complexes, the upper of which has been linked to repression. Here, we localize the complex formation capacity to several different RFX1 C-terminal subregions, each of which can function independently to generate the upper complex and repress transcription, thus correlating complex formation with repression. To gain an evolutionary perspective, we have examined whether the different properties of the RFX1 C terminus exist in the two yeast RFX proteins, which are involved in signaling pathways. Replacement of the RFX1 C terminus with those of Saki and Crt1, its orthologues from Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively, and analysis of fusions with the Gal4 DNA-binding domain, revealed that the ability to generate the two alternative complexes is conserved in the RFX family, from S. cerevisiae to man. While sharing this unique biochemical property, the three C termini differed from each other in their ability to mediate dimerization and transcriptional repression. In both functions, RFX1, Saki, and Crt1 showed high capacity, moderate capacity, and no capacity, respectively. This comparative analysis of the RFX proteins, representing different evolutionary stages, suggests a gradual development of the conserved C terminus, from the appearance of the ancestral motif (Crt1), to the later acquisition of the dimerization/repression functions (Sak1), and finally to the enhancement of these functions to generate a domain mediating highly stable protein-protein interactions and potent transcriptional r

In contrast to c-jun and junB, the junD gene is constitutively expressed in quiescent cells. The junD promoter, therefore, may provide a paradigm for promoters mostly active in growth arrested cells. We report here that the human junD promoter is repressed by serum and TPA. Also, the ability of JunD to positively autoregulate its promoter is abolished under these conditions. The obtained promoter repression depends on the junD promoter TRE, suggesting the involvement of bZip proteins in this process. We found that c-Fos, a bZip protein known to be induced by serum and TPA, is sufficient to antagonize the JunD function. Furthermore, selective activation of the junD promoter by JunD is abolished by c-Fos with concomitant activation of the collagenase promoter. The latter contains a TRE that is transcriptionally activated in proliferating cells. We propose that c-Fos plays a novel role in intergenic promoter switching, downregulating quiescent-state related genes while simultaneously upregulating proliferation-state specific genes.

Hepatitis B virus (HBV) infects humans and causes a wide range of clinical manifestations, from acute hepatitis to hepatocellular carcinoma (HCC). The HBV genome contains multiple promoters with gene expression regulated predominantly by the cellular transcription initiation machinery. Accordingly, the HBV-encoded pX, the only known vital regulator, is a potent transcription coactivator. We investigated the relationship between pX and cellular coactivators. We show that pX restores wild-type activity to inactive TBP(AS) mutants with poor TAF(II)250 and activator-binding activity. This pX-mediated recovery, however, is not obtained with inactive TBP(AS) mutants in binding of other general transcription factors. Remarkably, ts13, a cell line temperature sensitive for TAF(II)250 function, exhibiting growth arrest and apoptosis at the restrictive temperature, is rescued partially by pX expression, thus generating a pX-dependent cell growth. Collectively, our results suggest that pX suppresses some of the phenotypes of TBP and TAF(II)250 mutations, implying that pX circumvents the need for a holo-TFIID complex for transcription activation to proceed.

We have previously shown that monomeric p53 can transactivate target genes in vivo and that C-terminal fragments of p53 are oncogenic. To further elaborate these findings a series of C-terminal truncations of p53 was generated. The transactivation capacity and the ability of the truncated p53 to suppress oncogene-mediated transformation were studied. We found that p53 truncated at amino acid 303 (p53wtdl303) can still function in both assays, though less efficiently than full length wild type (wt) p53. Transforming C-terminal fragments inhibited transactivation induced by full length wt p53. Surprisingly, they also inhibited transactivation by wtdl303, with which they do not share e any overlapping sequences. Furthermore, the C-terminal fragments repressed the transactivation domains of several viral and cellular transcriptional activators. These data raise the possibility that the C-terminal domain of p53 may compete with the p53 transactivation domain for a common basal transcription factor.

One of the four genes encoded by hepatitis B virus (HBV) is the regulatory 17 kDa protein called HBx (or pX). HBx is a transcription transactivator of many cellular and viral regulatory elements. We report here that recombinant HBx supports transcription in vitro and has phosphotransfer enzymatic activity. In the presence of EDTA, a phosphoryl-HBx is formed that releases the phosphate residue upon the addition of Mg²⁺. This two-step NTP hydrolysis reaction is characteristic of a group of enzymes termed nucleoside diphosphate kinases (NDPKs). Remarkably, structural similarity between HBx and NDPKs is also evident. Our findings suggest that HBx has evolved from this group of enzymes but acquired additional activities that satisfy the viral needs.

The X protein of hepatitis B virus (HBV) stimulates transcription of a large number of viral enhancers. This protein augments the activity of the HBV enhancer through a specific cis element, termed X responsive element (XRE). Multimers of XRE exhibit enhancer activity which is further stimulated by X. XRE binds multiple cellular transcription factors one of which is the C/EBP. We have constructed the DB gene containing the DNAbinding domain of the C/EBP. This gene efficiently represses the enhancer activity of the XRE by competitive displacement of the XREbinding factors. Under these conditions, X was found to have only a partially stimulatory effect on transcription, suggesting that the XREbinding proteins are required for the activity of X. In contrast, an XDB hybrid protein that binds to the XRE is a strong transcription factor and acts without additional XREbinding proteins. Furthermore, studies of X mutants revealed that the carboxyterminus of the protein is required for this activation. These data show that X directly stimulates the cellular transcription machinery, possibly by proteinprotein interaction with the XREbinding factors.

We used the enhancer-binding protein C/EBP as a model to study the nature and the complexity of interaction of an enhancer-binding protein with its target DNA. We found that bacterially expressed C/EBP binds the hepatitis B virus enhancer at multiple sites in a hierarchic and cooperative manner. At low concentrations, only the E element is occupied, but at higher concentrations, additional sites are filled including a site that binds EP, a crucial enhancer-activating protein. This pattern of C/EBP binding may explain the concentration-dependent effect of C/EBP on enhancer activity.

The hepatitis B virus (HBV) genome contains a specific DNA binding site for the glucocorticoid receptor. Using DNase I footprinting, this binding site was localized at HBV map positions 341370 clockwise from theEcoRI site. The DNA sequence protected in the footprint contains two tandem copies of the GRE core hexanucleotide 5-TGT_c^TCT-3. Deletion analysis and reconstruction experiments in plasmid expression vectors demonstrated that this glucocorticoid receptor binding sequence serves as a signal for augmenting glucocorticoid-dependent activity of the HBV enhancer, which is located ∼ 730 nucleotides downstream in the HBV genome. Even though it does not serve as an independent enhancer element, the HBV glucocorticoid receptor domain can therefore be categorized as a functional GRE.

We previously reported the cloning and detailed analysis of the integrated hepatitis B virus sequences in a human hepatoma cell line. We report here the integration of at least one of hepatitis B virus at human satellite DNA sequences. The majority of the cellular sequences identified by this satellite DNA were organized as a multimeric composition of a 0.6-kilobase EcoRI fragment. This clone hybridized in situ almost exclusively to the centromeric heterochromatin of chromosomes 1 and 16 and to a lower extent to chromosome 2 and to the heterochromatic region of the Y chromosome. The immediate flanking host sequence appeared as a hierarchy of repeating units which were almost identical to a previously reported human satellite III DNA sequence.

Friend erythroleukemic cells were induced to differentiate by dimethylsulfoxide (Me₂SO) in the absence or presence of the tumor promoter 12Otetradecanoylphorbol 13acetate. The effects of the latter on the molecular parameters related to globin mRNA metabolism were examined. When differentiation was scored by benzidine staining, it had an inhibitory effect on Me₂SOtreated cells. On the other hand, when differentiation was followed by determination of globin mRNA accumulation, it had a pleiotropic effect on Me₂SOtreated cells. At the early phase of differentiation (23 days) the rate of globin mRNA accumulation was higher in the promotertreated cells than in the control. This unexpectedly high level of accumulation was followed by a sharp reduction and most of the globin RNA sequences disappeared at later stages of differentiation (days 45). The reduction can be related to the effect of the promoter on the stability of globin RNA in the cytoplasm which was reduced from a halflife of 16 h to that of 8 h only. Other parameters, such as the rate of globin mRNA synthesis and its capability to serve as a template for cellfree protein synthesis were not affected by treatment with the promoter throughout the differentiation process.