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Hepatic expression of proteasome subunit alpha type-6 is upregulated during viral hepatitis and putatively regulates the expression of ISG15 ubiquitin-like modifier, a proviral host gene in hepatitis C virus infection

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Abstract

The interferon-stimulated gene 15 (ISG15) plays an important role in the pathogenesis of hepatitis C virus (HCV) infection. ISG15-regulated proteins have previously been identified that putatively affect this proviral interaction. The present observational study aimed to elucidate the relation between ISG15 and these host factors during HCV infection. Transcriptomic and proteomic analyses were performed using liver samples of HCV-infected (n = 54) and uninfected (n = 10) or HBV-infected controls (n = 23). Primary human hepatocytes (PHH) were treated with Toll-like receptor ligands, interferons and kinase inhibitors. Expression of ISG15 and proteasome subunit alpha type-6 (PSMA6) was suppressed in subgenomic HCV replicon cell lines using specific siRNAs. Comparison of hepatic expression patterns revealed significantly increased signals for ISG15, IFIT1, HNRNPK and PSMA6 on the protein level as well as ISG15, IFIT1 and PSMA6 on the mRNA level in HCV-infected patients. In contrast to interferon-stimulated genes, PSMA6 expression occurred independent of HCV load and genotype. In PHH, the expression of ISG15 and PSMA6 was distinctly induced by poly(I:C), depending on IRF3 activation or PI3K/AKT signalling, respectively. Suppression of PSMA6 in HCV replicon cells led to significant induction of ISG15 expression, thus combined knock-down of both genes abrogated the antiviral effect induced by the separate suppression of ISG15. These data indicate that hepatic expression of PSMA6, which is upregulated during viral hepatitis, likely depends on TLR3 activation. PSMA6 affects the expression of immunoregulatory ISG15, a proviral factor in the pathogenesis of HCV infection. Therefore, the proteasome might be involved in the enigmatic interaction between ISG15 and HCV.

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... 12 Furthermore, upregulated expression of ISG15 was observed at both the mRNA and protein levels in liver tissue samples from patients with HCV infection compared to the levels detected in uninfected controls. 13 Similarly, the abundance of ISG15 transcripts was found to be increased in human PBMCs and liver cells in patients with HCV infection who were unresponsive to IFN treatment compared to the levels in corresponding IFN-responsive patients. 14 In addition, high levels of ISG15 in the liver of patients with HCV infection were associated with an unfavourable HCV genotype 1, a high hepatic HCV load and a low antiviral response to IFN compared to patients who did not present such characteristics. ...
... 14 In addition, high levels of ISG15 in the liver of patients with HCV infection were associated with an unfavourable HCV genotype 1, a high hepatic HCV load and a low antiviral response to IFN compared to patients who did not present such characteristics. 13 These findings indicate the potential of ISG15 as a biomarker of IFN treatment response in patients with HCV infection. ...
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... In contrast, our study showed that ISG15 levels were positively correlated with viral loads, implying a contradictory effect of ISG15 on antiviral activities. This is consistent with studies showing that ISG15 can promote HCV replication [19,35,36]. ...
... In contrast, our study showed that ISG15 levels were positively correlated with viral loads, implying a contradictory effect of ISG15 on antiviral activities. This is consistent with studies showing that ISG15 can promote HCV replication [19,35,36]. Host immune factors are essential in the immunepathogenesis of HBV infection through genetic and epigenetic modifications [37,38] and via the effects of cytokines [39]. ...
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The lack of an efficient system to produce hepatitis C virus (HCV) particles has impeded the analysis of the HCV life cycle. Recently, we along with others demonstrated that transfection of Huh7 hepatoma cells with a novel HCV isolate (JFH1) yields infectious viruses. To facilitate studies of HCV replication, we generated JFH1-based bicistronic luciferase reporter virus genomes. We found that RNA replication of the reporter construct was only slightly attenuated and that virus titers produced were only three- to fivefold lower compared to the parental virus, making these reporter viruses an ideal tool for quantitative analyses of HCV infections. To expand the scope of the system, we created two chimeric JFH1 luciferase reporter viruses with structural proteins from the Con1 (genotype 1b) and J6CF (genotype 2a) strains. Using these and the authentic JFH1 reporter viruses, we analyzed the early steps of the HCV life cycle. Our data show that the mode of virus entry is conserved between these isolates and involves CD81 as a key receptor for pH-dependent virus entry. Competition studies and time course experiments suggest that interactions of HCV with cell surface-resident glycosaminoglycans aid in efficient infection of Huh7 cells and that CD81 acts during a postattachment step. The reporter viruses described here should be instrumental for investigating the viral life cycle and for the development of HCV inhibitors.
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Infection with hepatitis C virus (HCV), which is distributed worldwide, often becomes persistent, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma. For many years, the characterization of the HCV genome and its products has been done by heterologous expression systems because of the lack of a productive cell culture system. The development of the HCV replicon system is a highlight of HCV research and has allowed examination of the viral RNA replication in cell culture. Recently, a robust system for production of recombinant infectious HCV has been established, and classical virological techniques are now able to be applied to HCV. This development of reverse genetics-based experimental tools in HCV research can bring a greater understanding of the viral life cycle and pathogenesis of HCV-induced diseases. This review summarizes the current knowledge of cell culture systems for HCV research and recent advances in the investigation of the molecular virology of HCV.
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Chronic hepatitis C infection is associated with increased expression of interferon-sensitive genes (ISGs) in the liver, which is, paradoxically, correlated with the nonresponse to interferon (IFN)-based therapies. In the present study PHHs were isolated from HCV-infected or uninfected patients and stimulated with the TLR1-9 ligands for 6-24 h. Expression of cytokines and ISGs was determined by ELISA and qRT-PCR. A comparative analysis was performed for TLR3 signalling, which was also correlated with single nucleotide polymorphisms (SNPs) related to HCV pathogenesis. TLR-activated PHHs produced pro-inflammatory and anti-inflammatory cytokines, whereas IFNs were exclusively induced by TLR3 stimulation. Here, IL-29 and IL-28A were significantly highly expressed than IFN-α and IFN-β. TLR3-induced IFN response was enhanced in hepatocytes isolated from patients with HCV infection. This hyper-responsiveness could be mimicked in naïve PHHs consistently stimulated with low dose of poly I:C, but not Guardiquimod. The higher responsiveness in PHH isolated from HCV-infected patients could be partially explained by higher frequencies of unfavourable SNP alleles of different SNPs associated with HCV progression and treatment outcome. These data suggest that durable activation of TLR3 but not TLR7, by low doses of viral replicative intermediates, increases the sensitivity to viral invasion. These findings shed new light on the relevance of TLR3 in the pathogenesis of HCV and may provide a possible explanation for the increased ISG expression during chronic HCV infection, the so-called IFN paradox.
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Abstract In previous studies we identified the interferon stimulated gene 15 (ISG15) as a pro-viral host factor in the pathogenesis of hepatitis C virus (HCV) infection. However, the functional link between ISG15 and the HCV replication cycle is not well understood. Aim of the present study was to functionally analyze the role of ISG15 and to identify possible HCV promoting effector molecules. Isg15 suppression was investigated in the murine subgenomic HCV replicon (MH1) transfected with Isg15-specific siRNA and in C57BL/6 mice intravenously injected with lipid nanoparticles (LNP)-formulated siRNA. Interestingly, the LNP-formulated siRNA led to hepatocyte-specific knockdown of Isg15 in vivo, which mediated a hypo-responsiveness to endogenous and exogenous interferon. A label free proteome analysis accompanied by western blot and quantitative RT-PCR techniques led to identification of five candidate proteins (Heterogeneous nuclear ribonucleoprotein A3 (HnrnpA3), Heterogeneous nuclear ribonucleoprotein K (HnrnpK), Hydroxymethylglutaryl-CoA synthase (Hmgcs1), Isocitrate dehydrogenase cytoplasmic (Idh1) and Thioredoxin domain-containing protein 5 (Txndc5)) that are either involved in lipid metabolism or belong to the family of Heterogeneous nuclear ribonucleoprotein (Hnrnp). All candidate proteins are likely to be associated with the HCV replication complex. Furthermore treatment with HnrnpK-specific siRNA directly suppressed HCV replication in vitro. Taken together these data suggest that targeting Isg15 may represent an attractive novel therapeutic option for the treatment of chronic HCV infection.
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TLR3 and TLR4 utilize adaptor TRIF to activate interferon regulatory factor 3 (IRF3), resulting in interferon β (IFN-β) production to mediate anti-viral infection. In this report, we analyzed the effect of two known phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 and wortmannin on LPS- and poly(I:C)-induced IFN-β production in peritoneal macrophages. LY294002 inhibited LPS- and poly(I:C)-induced IFN-β transcription and secretion. In contrast, wortmannin could not inhibit IFN-β production. Furthermore, IRF3 transcriptional activation and binding to IFN-β promoter were found to be inhibited by LY294002. Therefore, our findings demonstrate LY294002 negatively regulates LPS- and poly(I:C)-induced IFN-β production through inhibition of IRF3 activation in a PI3K-independent manner.
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S6K1, a critical downstream substrate of mTORC1, has been implicated in regulating protein synthesis and a variety of processes that impinge upon cell growth and proliferation. While the role of the cytoplasmic p70(S6K1) isoform in the regulation of translation has been intensively studied, the targets and function of the nuclear p85(S6K1) isoform remain unclear. Therefore, we carried out a phospho-proteomic screen to identify novel p85(S6K1) substrates. Four novel putative p85(S6K1) substrates, GRP75, CCTβ, PGK1 and RACK1, and two mTORC1 substrates, ANXA4 and PSMA6 were identified, with diverse roles in chaperone function, ribosome maturation, metabolism, vesicle trafficking and the proteasome, respectively. The chaperonin subunit CCTβ was further investigated and the site of phosphorylation mapped to serine 260, a site located in the chaperonin apical domain. Consistent with this domain being involved in folding substrate interactions, we found that phosphorylation of serine 260 modulates chaperonin folding activity.
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The hnRNPs (heterogeneous nuclear ribonucleoproteins) are RNA-binding proteins with important roles in multiple aspects of nucleic acid metabolism, including the packaging of nascent transcripts, alternative splicing and translational regulation. Although they share some general characteristics, they vary greatly in terms of their domain composition and functional properties. Although the traditional grouping of the hnRNPs as a collection of proteins provided a practical framework, which has guided much of the research on them, this approach is becoming increasingly incompatible with current knowledge about their structural and functional divergence. Hence, we review the current literature to examine hnRNP diversity, and discuss how this impacts upon approaches to the classification of RNA-binding proteins in general.
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Unlabelled: We have reported previously that the proteasome activator PA28gamma participates not only in degradation of hepatitis C virus (HCV) core protein in the nucleus but also in the pathogenesis in transgenic mice expressing HCV core protein. However, the biological significance of PA28gamma in the propagation of HCV has not been clarified. PA28gamma is an activator of proteasome responsible for ubiquitin-independent degradation of substrates in the nucleus. In the present study, knockdown of PA28gamma in cells preinfection or postinfection with the JFH-1 strain of HCV impaired viral particle production but exhibited no effect on viral RNA replication. The particle production of HCV in PA28gamma knockdown cells was restored by the expression of an small interfering RNA (siRNA)-resistant PA28gamma. Although viral proteins were detected in the cytoplasm of cells infected with HCV, suppression of PA28gamma expression induced accumulation of HCV core protein in the nucleus. HCV core protein was also degraded in the cytoplasm after ubiquitination by an E3 ubiquitin ligase, E6AP. Knockdown of PA28gamma enhanced ubiquitination of core protein and impaired virus production, whereas that of E6AP reduced ubiquitination of core protein and enhanced virus production. Furthermore, virus production in the PA28gamma knockdown cells was restored through knockdown of E6AP or expression of the siRNA-resistant wild-type but not mutant PA28gamma incapable of activating proteasome activity. Conclusion: Our results suggest that PA28gamma participates not only in the pathogenesis but also in the propagation of HCV by regulating the degradation of the core protein in both a ubiquitin-dependent and ubiquitin-independent manner.
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Non-response to combination therapy by patients with hepatitis C virus (HCV) has previously been associated with a strong hepatic upregulation of interferon stimulated genes (ISGs) including ISG15. Therefore, the aim of this study was to further elucidate the functional role of this molecule. ISG15 expression was suppressed by siRNAs or enhanced by over-expression in genomic and subgenomic human or murine HCV replicon systems. In addition, ISG15 expression was analysed in liver samples of patients with HCV prior to antiviral therapy and correlated with clinical and virological parameters. Short- or long-term knockdown of ISG15 expression suppressed HCV replication comparable to IFNs without evidence for the induction of resistant mutations. Triple therapy consisting of ISG15 knockdown, interferon alpha (IFNalpha) and ribavirin led to complete suppression of the HCV NS5A protein, corresponding to 99% suppression of HCV-RNA compared to 75% suppression by IFNalpha and ribavirin only. Combination treatment of ISG15 knockdown and IFN was associated with enhanced and prolonged expression of selected ISGs. Consistent with these in vitro data, high hepatic ISG15 levels correlated with the unfavourable HCV genotype 1, a high hepatic HCV load and a low antiviral response to IFN during the initial phase of treatment. ISG15 plays an important role in the HCV replication cycle. Therefore, therapies based on the suppression of ISG15 may provide a promising strategy to overcome non-response to standard combination treatment in the future. Furthermore, analysis of ISG15 prior to therapy may be useful to predict short-term and long-term outcome and thus tailor antiviral therapy with pegIFN and ribavirin.
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ISG15 has recently been reported to possess antiviral properties against viruses, both in vivo and in vitro. Knock-down of ISG15 gene expression by small interfering RNA followed by alpha interferon (IFN-alpha) treatment in Huh-7 cells resulted in an increased phenotypic sensitivity to IFN-alpha, as determined by measuring hepatitis C virus (HCV) RNA replication inhibition in stably transfected HCV replicon cells and in cells infected with genotype 1a HCVcc (infectious HCV). This IFN-alpha-specific effect, which was not observed with IFN-gamma, correlated with an increase in expression of the IFN-alpha-inducible genes IFI6, IFITM3, OAS1 and MX1, whereas the expression of the non-IFN-alpha-inducible genes PTBP-1 and JAK1 remained unchanged. It has previously been reported that, unlike ISG15 knock-down, increased sensitivity to IFN-alpha after knock-down of USP18 occurs through the prolonged phosphorylation of STAT-1. Combination knock-down of ISG15 and USP18 resulted in a moderate increase in IFN-alpha-inducible gene expression compared with single ISG15 or USP18 knock-down. Furthermore, the phenotype of increased gene expression after ISG15 knock-down and IFN-alpha treatment was also observed in non-hepatic cell lines A549 and HeLa. Taken together, these results reveal a novel function for ISG15 in the regulation of the IFN-alpha pathway and its antiviral effect.
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Viral life cycle as that of the hepatitis C virus (HCV) completely relies on host cell infrastructure, presupposing that the virus has evolved mechanisms to utilize and control all cellular molecules or pathways required for viral life cycle. Hence, HCV must have acquired the ability to gain access to key pathways controlling processes, such as cell growth, apoptosis and protein synthesis, which are all considered to also be crucial for liver regeneration. This occurs in a balanced way permitting persistent replication of viral genomes and production of infectious particles without endangering host cell viability and survival. In particular during the last decade, accumulating evidence indicates that HCV utilizes signaling pathways of the host with major impact on cellular growth, viability, cell cycle or cellular metabolism, such as epidermal growth factor-receptor mediated signals, the PI3K/Akt cascade or the family of Src kinases. Furthermore, HCV specifically interacts with parts of the cellular machinery involved in protein translation, processing, maturation and transport, such as components of the translation complex, the heat shock protein family, the immunophilins or the vesicle-associated membrane protein-associated proteins A and B. The present review focuses on the interplay between viral proteins and these factors of the host cell enabling the virus to utilize host cell infrastructure.
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The versatility of ubiquitin in regulating protein function and cell behaviour through post-translational protein modification makes it a particularly attractive target for viruses. Here we review how viruses manipulate the ubiquitin system to favour their propagation by redirecting cellular ubiquitin enzymes or encoding their own ubiquitin components to enable replication, egress and immune evasion. These studies not only reveal the many cellular processes requiring ubiquitin but also illustrate how viruses usurp their host cells.
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The hnRNP A/B proteins are among the most abundant RNA-binding proteins, forming the core of the ribonucleoprotein complex that associates with nascent transcripts in eukaryotic cells. There are several paralogs in this subfamily, each of which is subject to alternative transcript splicing and post-translational modifications. The structural diversity of these proteins generates a multitude of functions that involve interactions with DNA or, more commonly, RNA. They also recruit regulatory proteins associated with pathways related to DNA and RNA metabolism, and appear to accompany transcripts throughout the life of the mRNA. We have highlighted here recent progress in elucidation of molecular mechanisms underlying the roles of these hnRNPs in a wide range of nuclear processes, including DNA replication and repair, telomere maintenance, transcription, pre-mRNA splicing, and mRNA nucleo-cytoplasmic export.
Article
The 20S proteasome (prosome) is a highly organized multiprotein complex with approximate molecular weight of about 700 kDa. Whilst the role of the proteasome in the processing and turnover of cellular proteins is becoming clearer, its relationship with RNA remains still obscure. Here we focus on the nature and function of proteasome associated endonuclease activity. Thus the involvement of a proteasome alpha-type subunit in RNA-degradation, the catalytic requirements, the interaction of proteasomes with their RNA-substrate and the identification of a well defined cleavage site in the 3'UTR of short-lived cellular mRNAs will be described in detail. All data indicate that proteasomes associated endonuclease activity could be involved in post-transcriptional gene control at the level of translation.
Article
Ribozymes are small catalytic RNA molecules that can be engineered to enzymatically cleave RNA transcripts in a sequence-specific fashion and thereby inhibit expression and function of the corresponding gene product. With their simple structures and site-specific cleavage activity, they have been exploited as potential therapeutic agents in a variety of human disorders, including hepatitis C virus (HCV) infection. We have designed a hairpin ribozyme (Rz3'X) targeting the HCV minus-strand replication intermediate at position 40 within the 3'X tail. Surprisingly, Rz3'X was found to induce ganciclovir (GCV)-resistant colonies in a bicistronic cellular reporter system with HCV internal ribosome entry site (IRES)-dependent translation of herpes simplex virus thymidine kinase (TK). Rz3'X-transduced GCV-resistant HeLa reporter cells showed substantially reduced IRES-mediated HCV core protein translation compared with control vector-transduced cells. Since these reporter systems do not contain the HCV 3'X tail sequences, the results indicate that Rz3'X probably exerted an inhibitory effect on HCV IRES activity fortuitously through another gene target. A novel technique of ribozyme cleavage-based target gene identification (cleavage-specific amplification of cDNA ends) (M. Krüger, C. Beger, P. J. Welch, J. R. Barber, and F. Wong-Staal, Nucleic Acids Res. 29:e94, 2001) revealed that human 20S proteasome alpha-subunit PSMA7 mRNA was a target RNA recognized and cleaved by Rz3'X. We then showed that additional ribozymes directed against PSMA7 RNA inhibited HCV IRES activity in two assay systems: GCV resistance in the HeLa IRES TK reporter cell system and a transient transfection assay performed with a bicistronic Renilla-HCV IRES-firefly luciferase reporter in Huh7 cells. In contrast, ribozymes were inactive against IRES of encephalomyocarditis virus and human rhinovirus. Additionally, proteasome inhibitor MG132 exerted a dose-dependent inhibitory effect on HCV IRES-mediated translation but not on cap-dependent translation. These data suggest a principal role for PSMA7 in regulating HCV IRES activity, a function essential for HCV replication.
Article
Hepatitis C virus (HCV), the global leading cause of chronic liver disease, has a positive-sense, ssRNA genome that encodes a large polyprotein. HCV polyprotein translation is initiated by an internal ribosome-entry site (IRES) located at the 5' end of the viral genome, in a cap-independent manner, but the regulatory mechanism of this process remains poorly understood. In this study, we characterized the effect of HCV nonstructural proteins on HCV IRES-directed translation in both HCV replicon cells and transiently transfected human liver cells expressing HCV nonstructural proteins. Using bicistronic reporter gene constructs carrying either HCV or other viral IRES sequences, we found that the HCV IRES-mediated translation was specifically upregulated in HCV replicon cells. This enhancement of HCV IRES-mediated translation by the replicon cells was inhibited by treatment with either type I interferon or ribavirin, drugs that perturb HCV genome replication, suggesting that the enhancement is probably due to HCV-encoded protein function(s). Reduced phosphorylation levels of both eIF2alpha and eIF4E were observed in the replicon cells, which is consistent with our previous findings and indicates that the NS5A nonstructural protein may be involved in the regulatory mechanism(s). Indeed, transient expression of NS5A or NS4B in human liver cells stimulated HCV IRES activity. Interestingly, mutation in the ISDR of NS5A perturbed this stimulation of HCV IRES activity. All these results suggest, for the first time, that HCV nonstructural proteins preferentially stimulate the viral cap-independent, IRES-mediated translation.
Article
Small interfering RNAs (siRNAs) are an efficient tool to specifically inhibit gene expression by RNA interference. Since hepatitis C virus (HCV) replicates in the cytoplasm of liver cells without integration into the host genome, RNA-directed antiviral strategies are likely to successfully block the HCV replication cycle. Additional benefit might arise from inhibition of cellular cofactors of HCV replication, such as proteasome alpha-subunit 7 (PSMA7) or Hu antigen R (HuR). In this study, we investigated direct and cofactor-mediated inhibition of HCV by a panel of DNA-based retroviral vectors expressing siRNAs against highly conserved HCV sequences or the putative HCV cofactors PSMA7 and HuR. Effects were determined in HCV IRES-mediated translation assays and subgenomic HCV replicon cells. PSMA7- and HuR-directed siRNAs successfully inhibited expression of the endogenous genes, and PSMA7 and HuR silencing significantly diminished HCV replicon RNA and NS5B protein levels. HCV-directed siRNAs substantially inhibited HCV IRES-mediated translation and subgenomic HCV replication. Combinations of PSMA7- and HuR-directed siRNAs with HCV-directed siRNAs revealed additive HCV RNA inhibitory effects in monocistronic replicon cells. A dual approach of direct- and cofactor-mediated inhibition of HCV replication might avoid selection of mutants and thereby become a powerful strategy against HCV.
Article
The mRNA level for cytosolic NADP-dependent isocitrate dehydrogenase (IDH1) increases 2.3-fold, and enzyme activity of NADP-isocitrate dehydrogenase (IDH) 63%, in sterol-deprived HepG2 cells. The mRNA levels of the NADP- and NAD-dependent mitochondrial enzymes show limited or lack of regulation under the same conditions. Nucleotide sequences that are required, and sufficient, for the sterol regulation of transcription are located within a 67 bp region of an IDH1-secreted alkaline phosphatase promoter-reporter gene. The IDH1 promoter is fully activated by the expression of SREBP-1a in the cells and, to a lesser degree, by that of SREBP-2. A 5′-end truncation of 23 bp containing a CAAT and a GC-Box results in 6.5% residual activity. The promoter region involved in the activation by the sterol regulatory element binding proteins (SREBPs) is located at nucleotides −44 to −25. Mutagenesis analysis identified within this region the IDH1-SRE sequence element GTGGGCTGAG, which binds the SREBPs. Similar to the promoter activation, electrophoretic mobility shifts of probes containing the IDH1-SRE element exhibit preferential binding to SREBP-1a, as compared with SREBP-2. These results indicate that IDH1 activity is coordinately regulated with the cholesterol and fatty acid biosynthetic pathways and suggest that it is the source for the cytosolic NADPH required by these pathways.
Article
Current understanding of the mechanisms by which cell growth is regulated lags significantly behind our knowledge of the complex processes controlling cell cycle progression. Recent studies suggest that the mammalian target of rapamycin (mTOR) pathway is a key regulator of cell growth via the regulation of protein synthesis. The key mTOR effectors of cell growth are eukaryotic initiation factor 4E-binding protein 1 (4EBP-1) and the ribosomal protein S6 kinase (S6K). Here we will review the current models for mTOR dependent regulation of ribosome function and biogenesis as well as its role in coordinating growth factor and nutrient signaling to facilitate homeostasis of cell growth and proliferation. We will place particular emphasis on the role of S6K1 signaling and will highlight the points of cross talk with other key growth control pathways. Finally, we will discuss the impact of S6K signaling and the consequent feedback regulation of the PI3K/Akt pathway on disease processes including cancer.
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RNAs in cells are associated with RNA-binding proteins (RBPs) to form ribonucleoprotein (RNP) complexes. The RBPs influence the structure and interactions of the RNAs and play critical roles in their biogenesis, stability, function, transport and cellular localization. Eukaryotic cells encode a large number of RBPs (thousands in vertebrates), each of which has unique RNA-binding activity and protein-protein interaction characteristics. The remarkable diversity of RBPs, which appears to have increased during evolution in parallel to the increase in the number of introns, allows eukaryotic cells to utilize them in an enormous array of combinations giving rise to a unique RNP for each RNA. In this short review, we focus on the RBPs that interact with pre-mRNAs and mRNAs and discuss their roles in the regulation of post-transcriptional gene expression.