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 TMPRSS2’s 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.

RPS3, a universal core component of the 40S ribosomal subunit, interacts with mRNA at the entry channel. Whether RPS3 mRNA-binding contributes to specific mRNA translation and ribosome specialization in mammalian cells is unknown. Here we mutated RPS3 mRNA-contacting residues R116, R146 and K148 and report their impact on cellular and viral translation. R116D weakened cap-proximal initiation and promoted leaky scanning, while R146D had the opposite effect. Additionally, R146D and K148D displayed contrasting effects on start-codon fidelity. Translatome analysis uncovered common differentially translated genes of which the downregulated set bears long 5'UTR and weak AUG context, suggesting a stabilizing role during scanning and AUG selection. We identified an RPS3-dependent regulatory sequence (RPS3RS) in the sub-genomic 5'UTR of SARS-CoV-2 consisting of a CUG initiation codon and a downstream element that is also the viral transcription regulatory sequence (TRS). Furthermore, RPS3 mRNA-binding residues are essential for SARS-CoV-2 NSP1-mediated inhibition of host translation and for its ribosomal binding. Intriguingly, NSP1-induced mRNA degradation was also reduced in R116D cells, indicating that mRNA decay occurs in the ribosome context. Thus, RPS3 mRNA-binding residues have multiple translation regulatory functions and are exploited by SARS-CoV-2 in various ways to influence host and viral mRNA translation and stability.

The degradation of intrinsically disordered proteins (IDPs) by a non-26S proteasome process does not require proteasomal targeting by polyubiquitin. However, whether and how IDPs are recognized by the non-26S proteasome, including the 20S complex, remains unknown. Analyses of protein interactome datasets revealed that the 20S proteasome subunit, PSMA3, preferentially interacts with many IDPs. In vivo and cell-free experiments revealed that the C-terminus of PSMA3, a 69-amino-acids-long fragment, is an IDP trapper. A recombinant trapper is sufficient to interact with many IDPs, and blocks IDP degradation in vitro by the 20S proteasome, possibly by competing with the native trapper. In addition, over a third of the PSMA3 trapper-binding proteins have previously been identified as 20S proteasome substrates and, based on published datasets, many of the trapper-binding proteins are associated with the intracellular proteasomes. The PSMA3-trapped IDPs that are proteasome substrates have the unique features previously recognized as characteristic 20S proteasome substrates in vitro. We propose a model whereby the PSMA3 C-terminal region traps a subset of IDPs to facilitate their proteasomal degradation.

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.

Hepatitis B virus (HBV) is a small DNA virus responsible for significant morbidity and mortality worldwide. The liver, which is the main target organ for HBV infection, provides the virus with the machinery necessary for persistent infection and propagation, a process that might ultimately lead to severe liver pathologies such as chronic hepatitis, cirrhosis and liver cancer. HBV gene expression is regulated mainly at the transcriptional level by recruitment of a whole set of cellular transcription factors (TFs) and co-activators to support transcription. Over the years, many of these TFs were identified and interestingly enough most are associated with the body's nutritional state. These include the hepatocyte nuclear factors, forkhead Box O1, Farnesoid X receptor, cyclic-AMP response element-binding (CREB), CCAAT/enhancer-binding protein (C/EBP) and glucocorticoid receptor TFs and the transcription coactivator PPARγ coactivator-1α Consequently, HBV gene expression is linked to hepatic metabolic processes such as glucose and fat production and utilization as well as bile acids' production and secretion. Furthermore, recent evidence indicates that HBV actively interferes with some of these hepatic metabolic processes by manipulating key TFs, such as CREB and C/EBP, to meet its requirements. The discovery of the mechanisms by which HBV is controlled by the hepatic metabolic milieu may broaden our understanding of the unique regulation of HBV expression and may also explain the mechanisms by which HBV induces liver pathologies. The emerging principle of the intimate link between HBV and liver metabolism can be further exploited for host-targeted therapeutic strategies.

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.

c-Abl tyrosine kinase is activated by agents that induce double-strand DNA breaks (DSBs) and interacts with key components of the DNA damage response and of the DSB repair machinery. However, the functional significance of c-Abl in these processes, remained unclear. In this study, we demonstrate, using comet assay and pulsed-field gel electrophoresis, that c-Abl inhibited the repair of DSBs induced by ionizing radiation, particularly during the second and slow phase of DSB repair. Pharmacological inhibition of c-Abl and c-Abl depletion by siRNA-mediated knockdown resulted in higher DSB rejoining. c-Abl null MEFs exhibited higher DSB rejoining compared with cells reconstituted for c-Abl expression. Abrogation of c-Abl kinase activation resulted in higher H2AX phosphorylation levels and higher numbers of post-irradiation γH2AX foci, consistent with a role of c-Abl in DSB repair regulation. In conjunction with these findings, transient abrogation of c-Abl activity resulted in increased cellular radioresistance. Our findings suggest a novel function for c-Abl in inhibition of the slow phase of DSB repair.

Intrinsically disordered proteins (IDPs) are subject to ubiquitin-independent degradation, a default and passive process. We describe here a model wherein a group of nanny proteins function to protect newly synthesized IDPs from degradation by default, thereby insuring their maturation into important regulatory molecules.

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.

Protein degradation is a key cellular process involved in almost every aspect of the living cell. The current prevailing concept is that proteins are stable unless marked by poly-ubiquitination for degradation by the proteasomes. Studies on the tumor suppressor p53 have indeed demonstrated that poly-ubiquitination of p53 by different E3 ubiquin ligases targets p53 for degradation by the 26S proteasomes. Recent findings suggest that p53 also undergoes ubiquitin-independent degradation by the 20S proteasomes and that this process is regulated by NAD(P)H quinone oxidoreductase 1 (NQO1) together with NADH. This "degradation by default" mechanism sheds new light on our understanding of p53 degradation and possibly on protein degradation in general and may establish a new principle in protein stability with wide physiological implications.

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.

Neural restrictive silencer factor, NRSF (also known as REST) binds a neuronal cell type selective silencer element to mediate transcriptional repression of neuron-specific genes in non-neuronal cells and neuronal progenitors. Two repression domains (RD-1 and RD-2) occur in its N-terminal and C-terminal regions, respectively. RD-1 recruits mSin3 and HDAC, thereby inhibiting transcription by inducing reorganization of the chromatin structure. However, little is known about how such global repression becomes promoter-specific repression or whether the NRSF-HDAC complex can interact with transcriptional core factors at each specific promoter. Here we show evidence that NRSF interacts with core promoter factors, including TATA-binding protein (TBP). The NRSF-TBP interaction occurred between the linear segments of the N- and C-terminal-most portions of NRSF and the C-terminal half of TBP. A RD-2 mutant of NRSF lost the TBP-binding activity and was unable to repress transcription at an exogenously introduced TGTA promoter. These results indicate that the direct interaction between the NRSF C-terminal domain and TBP is essential for the C-terminal repression mechanism of NRSF. Thus, the RD-1 and RD-2 repression domains of NRSF utilize both chromatin-dependent and chromatin-independent mechanisms, which may be segregated at various stages of neural development and modulation.

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.

Hepatitis B virus (HBV) gene expression is mainly regulated at the transcription initiation level. The viral X protein (pX) is a transcription coactivator/mediator targeting TFIIB for the recruitment of RNA polymerase II. Here we report a novel pX nuclear target designated HBXAP (hepatitis B virus X-associated protein). HBXAP is a novel cellular nuclear protein containing a PHD (plant homology domain) finger, a domain shared by many proteins that play roles in chromatin remodeling, transcription coactivation, and oncogenesis. pX physically interacts with HBXAP in vitro and in vivo via the HBXAP region containing the PHD finger. At the functional level HBXAP increases HBV transcription in a pX-dependent manner suggesting a role for this interaction in the virus life cycle. Interestingly, HBXAP collaborates with pX in coactivating the transcriptional activator NF-κB. Coactivation of NF-κB was also observed in tumor necrosis factor α-treated cells suggesting that pX-HBXAP functional collaboration localized downstream to the NF-κB nuclear import. Collectively our data suggest that pX recruits and potentiates a novel putative transcription coactivator to regulate NF-κB. The implication of pX-HBXAP interaction in the development of hepatocellular carcinoma is discussed.

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.

Humanized BALB/c mice (termed trimera mice) conditioned by lethal total body irradiation and bone marrow transplantation from SCID mice have been described to support rapid engraftment of human peripheral blood mononuclear cells (PBMC) and the induction of strong B and T cell responses after immunization in vivo. Moreover, these mice can be infected with the hepatitis B and C viruses (HBV, HCV). The current study employed this model to study therapeutic vaccination approaches against the HBV. Thus, strong primary Th cell responses against the HBV core (HBc) and the Borrelia burgdorferi control antigen were induced by transfer of antigen-loaded dendritic cells together with autologous PBMC from HBV-naive donors as well as by vaccination with high doses of antigen or a DNA plasmid encoding for HBcAg. Moreover, primary peptide-specific CTL responses against the immunodominant epitope HBc_18-27 were induced by HBc particle or DNA vaccination of chimera engrafted with HBV-naive PBMC. Finally, strong HBc-specific Th cell and antibody responses were induced by HBc or DNA vaccination of mice reconstituted with PBMC from a chronic HBV patient. Thus, since HBc represents the immunodominant antigen in self-limited HBV infection, HBc particles or DNA vectors are good candidates for therapeutic vaccination, that will be further studied in our model and clinical studies.

Microemulsions containing octanol, decanol, or dodecanol as the oil phase and oligomeric, grafted nonionic amphiphiles based on ethoxylated polymethylsiloxanes (Silwets) have been studied. It was demonstrated that significant amounts of water can be solubilized only when the hydrophobic siliconic backbone is very short (trimers). The water solubilization was evaluated using SAXS, DSC, and conductivity measurements. It was found that up to 40 wt% of water can be solubilized in dodecanol and Silwet L-7607 (MW 1000 and 75 wt% ethylene oxide (EO)). Surprisingly, no free water was detected in the aggregate core. All the solubilized water was confined in the vicinity of the interphasal region and froze at -10°C and below. Up to three molecules of water can be associated with each EO headgroup. Based on SAXS measurements, the structural units of the microemulsions were interpreted to be lamellar-like, a form previously found for the related monomeric microemulsions.

A variant polyadenylation signal, which is conserved and employed by mammalian hepadnaviruses, has a sequence resembling that of the TATA box. We report here that this composite box manifests all the promoter characteristics. It binds effectively TATA-binding protein with TFIIB and TFIIA in a synergistic manner. This capacity, however, is lost when the box is converted to a canonical and simple poly(A) signal. Furthermore, we show that it has promoter activity and supports transcription of reporter genes preferentially in liver-derived cells, a characteristic behavior of the hepatitis B virus (HBV) promoters. In addition, we show that the HBV noncanonical poly(A) signal supports transcription initiation from the viral genome, suggesting that it is a genuine promoter, possibly of the polymerase/reverse transcriptase gene. Finally, we found that this deviant poly(A) signal is crucial for HBV replication since a viral mutant with a canonical poly(A) box is impaired in replication. Our data, therefore, raise the interesting and novel possibility that a composite poly(A) box might have a dual function. At the level of DNA it functions as a promoter to initiate transcription, whereas at the level of RNA it serves as a poly(A) signal to process RNA. An interesting outcome of this strategy of gene expression is that it provides a novel mechanism for the synthesis of an approximately genome length transcript.

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.

c-Abl, the non-receptor tyrosine kinase is associated with EP, a DNA element found in promoters/enhancers of different viruses and cell-cycle regulated genes. EP-DNA binds RFXI, a member of a novel family of DNA-binding proteins that is conserved through evolution and in yeast, it controls differentiation and exit from the mitotic cycle to G₀. EP-associated proteins are preferentially tyrosine phosphorylated and the associated c-Abl has strong tyrosine kinase activity. Here we investigated the molecular mechanism underlying this c-Abl kinase activity. We show that RFXI and c-Abl are in direct interaction, in vitro and in cell extracts, through the RFXI proline rich (PxxP) motif and the c-Abl SH3 domain. Remarkably, this interaction significantly potentiates c-Abl but not v-Abl auto-kinase activity. Collectively, we describe a novel mechanism of c-Abl recruitment to a defined DNA-cis element with its concomitant kinase activation. We propose that this mechanism may act to regulate cell-cycle control genes.

The enhancer of hepatitis B (HBV) virus displays a liver-specific activity that determines the postreceptor virus-host tropism. Despite the detailed study of this enhancer our knowledge of the mechanisms underlying this behavior is very limited. Here we report that the hepatocyte nuclear factor 3 (HNF3) is at least in part responsible for the liver-specific activity of the enhancer. We demonstrate that recombinant HNF3α binds the enhancer at two sites with different affinity. Transfection studies have demonstrated that the enhancer is active only in liver cells and that integrity of the HNF3 binding sites is important for its full activity. In vitro transcription assays revealed that the enhancer is active only in liver extracts but not in extracts prepared from HeLa cells. Furthermore, the latter extract cannot be activated by addition of recombinant HNF3α. A similar behavior is manifested in transfected cells and, here again, the inactive enhancer is not activated by cotransfected HNF3β and α. Collectively, our study shows that HNF3 activators are required but not sufficient for full activation of the HBV enhancer and there is a need for additional liver-specific activators or coactivators.

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 DNA‐binding domain of the C/EBP. This gene efficiently represses the enhancer activity of the XRE by competitive displacement of the XRE‐binding factors. Under these conditions, X was found to have only a partially stimulatory effect on transcription, suggesting that the XRE‐binding proteins are required for the activity of X. In contrast, an X‐DB hybrid protein that binds to the XRE is a strong transcription factor and acts without additional XRE‐binding proteins. Furthermore, studies of X mutants revealed that the carboxy‐terminus of the protein is required for this activation. These data show that X directly stimulates the cellular transcription machinery, possibly by protein‐protein interaction with the XRE‐binding factors.

The hepatitis B virus enhancer can be dissected into multiple functional elements, one of which is the E element. We show here that the E element binds multiple nuclear proteins that are essential for its enhancer activity. These findings, together with the ability of this element to respond to at least two different viral transactivators, suggest that the E element is an enhancer modulator capable of binding different factors and responding to multiple stimuli.

The S promoter, one of the major hepatitis B virus (HBV) promoters, directs the synthesis of mRNA for surface antigen. Transient expression studies revealed that this promoter is highly active in the Alexander hepatoma cell line but not in SK-Hep1 and HeLa cells. We found that a distal element of the promoter (-103 to -48) confers this cell-type-specific behavior through a mechanism in which the promoter activity is repressed in HeLa and SK-Hep1 cells but increased in Alexander cells. By using an inhibitor of protein synthesis, we obtained evidence that a labile repressor(s) confers the negative effect in SK-Hep1 cells. We also found an enhancerlike activity associated with a small DNA segment of the S promoter (-27 to + 30). This proximal element was active in HeLa and SK-Hep1 cells only in the absence of the distal negative element. Finally, analysis of S promoter deletion mutants demonstrated that the -27 to -17 region of the S promoter is crucial for its activity.

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 12‐O‐tetradecanoyl‐phorbol 13‐acetate. 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 Me₂SO‐treated cells. On the other hand, when differentiation was followed by determination of globin mRNA accumulation, it had a pleiotropic effect on Me₂SO‐treated cells. At the early phase of differentiation (2–3 days) the rate of globin mRNA accumulation was higher in the promoter‐treated 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 4–5). 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 half‐life 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 cell‐free protein synthesis were not affected by treatment with the promoter throughout the differentiation process.