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Sample records for gtpase ns3 acts

  1. A monovalent cation acts as structural and catalytic cofactor in translational GTPases

    PubMed Central

    Kuhle, Bernhard; Ficner, Ralf

    2014-01-01

    Translational GTPases are universally conserved GTP hydrolyzing enzymes, critical for fidelity and speed of ribosomal protein biosynthesis. Despite their central roles, the mechanisms of GTP-dependent conformational switching and GTP hydrolysis that govern the function of trGTPases remain poorly understood. Here, we provide biochemical and high-resolution structural evidence that eIF5B and aEF1A/EF-Tu bound to GTP or GTPγS coordinate a monovalent cation (M+) in their active site. Our data reveal that M+ ions form constitutive components of the catalytic machinery in trGTPases acting as structural cofactor to stabilize the GTP-bound “on” state. Additionally, the M+ ion provides a positive charge into the active site analogous to the arginine-finger in the Ras-RasGAP system indicating a similar role as catalytic element that stabilizes the transition state of the hydrolysis reaction. In sequence and structure, the coordination shell for the M+ ion is, with exception of eIF2γ, highly conserved among trGTPases from bacteria to human. We therefore propose a universal mechanism of M+-dependent conformational switching and GTP hydrolysis among trGTPases with important consequences for the interpretation of available biochemical and structural data. PMID:25225612

  2. Psammaplin A inhibits hepatitis C virus NS3 helicase.

    PubMed

    Salam, Kazi Abdus; Furuta, Atsushi; Noda, Naohiro; Tsuneda, Satoshi; Sekiguchi, Yuji; Yamashita, Atsuya; Moriishi, Kohji; Nakakoshi, Masamichi; Tsubuki, Masayoshi; Tani, Hidenori; Tanaka, Junichi; Akimitsu, Nobuyoshi

    2013-10-01

    Hepatitis C virus (HCV) is the causative agent of hepatitis C, a chronic infectious disease that can lead to development of hepatocellular carcinoma. The NS3 nucleoside triphosphatase (NTPase)/helicase has an essential role in HCV replication, and is therefore an attractive target for direct-acting antiviral strategies. In this study, we employed high-throughput screening using a photo-induced electron transfer (PET) system to identify an inhibitor of NS3 helicase from marine organism extracts. We successfully identified psammaplin A as a novel NS3 inhibitor. The dose-response relationship clearly demonstrates the inhibition of NS3 RNA helicase and ATPase activities by psammaplin A, with IC₅₀ values of 17 and 32 μM, respectively. Psammaplin A has no influence on the apparent Km value (0.4 mM) of NS3 ATPase activity, and acts as a non-competitive inhibitor. Additionally, it inhibits the binding of NS3 to single-stranded RNA in a dose-dependent manner. Furthermore, psammaplin A shows an inhibitory effect on viral replication, with EC₅₀ values of 6.1 and 6.3 μM in subgenomic replicon cells derived from genotypes 1b and 2a, respectively. We postulate that psammaplin A is a potential anti-viral agent through the inhibition of ATPase, RNA binding and helicase activities of NS3. PMID:23359228

  3. Hepatitis C Virus NS3 Inhibitors: Current and Future Perspectives

    PubMed Central

    Akimitsu, Nobuyoshi

    2013-01-01

    Currently, hepatitis C virus (HCV) infection is considered a serious health-care problem all over the world. A good number of direct-acting antivirals (DAAs) against HCV infection are in clinical progress including NS3-4A protease inhibitors, RNA-dependent RNA polymerase inhibitors, and NS5A inhibitors as well as host targeted inhibitors. Two NS3-4A protease inhibitors (telaprevir and boceprevir) have been recently approved for the treatment of hepatitis C in combination with standard of care (pegylated interferon plus ribavirin). The new therapy has significantly improved sustained virologic response (SVR); however, the adverse effects associated with this therapy are still the main concern. In addition to the emergence of viral resistance, other targets must be continually developed. One such underdeveloped target is the helicase portion of the HCV NS3 protein. This review article summarizes our current understanding of HCV treatment, particularly with those of NS3 inhibitors. PMID:24282816

  4. Bluetongue Virus Nonstructural Protein NS3/NS3a Is Not Essential for Virus Replication

    PubMed Central

    van Gennip, René G. P.; van de Water, Sandra G. P.; van Rijn, Piet A.

    2014-01-01

    Orbiviruses form the largest genus of the family Reoviridae consisting of at least 23 different virus species. One of these is the bluetongue virus (BTV) and causes severe hemorrhagic disease in ruminants, and is transmitted by bites of Culicoides midges. BTV is a non-enveloped virus which is released from infected cells by cell lysis and/or a unique budding process induced by nonstructural protein NS3/NS3a encoded by genome segment 10 (Seg-10). Presence of both NS3 and NS3a is highly conserved in Culicoides borne orbiviruses which is suggesting an essential role in virus replication. We used reverse genetics to generate BTV mutants to study the function of NS3/NS3a in virus replication. Initially, BTV with small insertions in Seg-10 showed no CPE but after several passages these BTV mutants reverted to CPE phenotype comparable to wtBTV, and NS3/NS3a expression returned by repair of the ORF. These results show that there is a strong selection for functional NS3/NS3a. To abolish NS3 and/or NS3a expression, Seg-10 with one or two mutated start codons (mutAUG1, mutAUG2 and mutAUG1+2) were used to generate BTV mutants. Surprisingly, all three BTV mutants were generated and the respective AUGMet→GCCAla mutations were maintained. The lack of expression of NS3, NS3a, or both proteins was confirmed by westernblot analysis and immunostaining of infected cells with NS3/NS3a Mabs. Growth of mutAUG1 and mutAUG1+2 virus in BSR cells was retarded in both insect and mammalian cells, and particularly virus release from insect cells was strongly reduced. Our findings now enable research on the role of RNA sequences of Seg-10 independent of known gene products, and on the function of NS3/NS3a proteins in both types of cells as well as in the host and insect vector. PMID:24465709

  5. Potassium Acts as a GTPase-Activating Element on Each Nucleotide-Binding Domain of the Essential Bacillus subtilis EngA

    PubMed Central

    Foucher, Anne-Emmanuelle; Reiser, Jean-Baptiste; Ebel, Christine; Housset, Dominique; Jault, Jean-Michel

    2012-01-01

    EngA proteins form a unique family of bacterial GTPases with two GTP-binding domains in tandem, namely GD1 and GD2, followed by a KH (K-homology) domain. They have been shown to interact with the bacterial ribosome and to be involved in its biogenesis. Most prokaryotic EngA possess a high GTPase activity in contrast to eukaryotic GTPases that act mainly as molecular switches. Here, we have purified and characterized the GTPase activity of the Bacillus subtilis EngA and two shortened EngA variants that only contain GD1 or GD2-KH. Interestingly, the GTPase activity of GD1 alone is similar to that of the whole EngA, whereas GD2-KH has a 150-fold lower GTPase activity. At physiological concentration, potassium strongly stimulates the GTPase activity of each protein construct. Interestingly, it affects neither the affinities for nucleotides nor the monomeric status of EngA or the GD1 domain. Thus, potassium likely acts as a chemical GTPase-activating element as proposed for another bacterial GTPase like MnmE. However, unlike MnmE, potassium does not promote dimerization of EngA. In addition, we solved two crystal structures of full-length EngA. One of them contained for the first time a GTP-like analogue bound to GD2 while GD1 was free. Surprisingly, its overall fold was similar to a previously solved structure with GDP bound to both sites. Our data indicate that a significant structural change must occur upon K+ binding to GD2, and a comparison with T. maritima EngA and MnmE structures allowed us to propose a model explaining the chemical basis for the different GTPase activities of GD1 and GD2. PMID:23056455

  6. ELMOD2 is an Arl2 GTPase-activating protein that also acts on Arfs.

    PubMed

    Bowzard, J Bradford; Cheng, Dongmei; Peng, Junmin; Kahn, Richard A

    2007-06-15

    Regulatory GTPases in the Ras superfamily employ a cycle of alternating GTP binding and hydrolysis, controlled by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs), as essential features of their actions in cells. Studies of these GAPs and guanine nucleotide exchange factors have provided important insights into our understanding of GTPase signaling and biology. Within the Ras superfamily, the Arf family is composed of 30 members in mammals, including 22 Arf-like (Arl) proteins. Much less is known about the mechanisms of cell regulation by Arls than by Arfs. We report the purification from bovine testis of an Arl2 GAP and its identity as ELMOD2, a protein with no previously described function. ELMOD2 is one of six human proteins that contain an ELMO domain, and a second member, ELMOD1, was also found to have Arl2 GAP activity. Surprisingly, ELMOD2 also exhibited GAP activity against Arf proteins even though it does not contain the canonical Arf GAP sequence signature. The broader specificity of ELMOD2, as well as the previously described role for ELMO1 and ELMO2 in linking Arf6 and Rac1 signaling, suggests that ELMO family members may play a more general role in integrating signaling pathways controlled by Arls and other GTPases. PMID:17452337

  7. Pestivirus NS3 (p80) protein possesses RNA helicase activity.

    PubMed Central

    Warrener, P; Collett, M S

    1995-01-01

    The pestivirus bovine viral diarrhea virus (BVDV) p80 protein (referred to here as the NS3 protein) contains amino acid sequence motifs predictive of three enzymatic activities: serine proteinase, nucleoside triphosphatase, and RNA helicase. We have previously demonstrated that the former two enzymatic activities are associated with this protein. Here, we show that a purified recombinant BVDV NS3 protein derived from baculovirus-infected insect cells possesses RNA helicase activity. BVDV NS3 RNA helicase activity was specifically inhibited by monoclonal antibodies to the p80 protein. The activity was dependent on the presence of nucleoside triphosphate and divalent cation, with a preference for ATP and Mn2+. Hydrolysis of the nucleoside triphosphate was necessary for strand displacement. The helicase activity required substrates with an un-base-paired region on the template strand 3' of the duplex region. As few as three un-base-paired nucleotides were sufficient for efficient oligonucleotide displacement. However, the enzyme did not act on substrates having a single-stranded region only to the 5' end of the duplex or on substrates lacking single-stranded regions altogether (blunt-ended duplex substrates), suggesting that the directionality of the BVDV RNA helicase was 3' to 5' with respect to the template strand. The BVDV helicase activity was able to displace both RNA and DNA oligonucleotides from RNA template strands but was unable to release oligonucleotides from DNA templates. The possible role of this activity in pestivirus replication is discussed. PMID:7853509

  8. Non-structural protein NS3/NS3a is required for propagation of bluetongue virus in Culicoides sonorensis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Bluetongue virus (BTV) causes non-contagious haemorrhagic disease in ruminants and is transmitted by Culicoides spp. biting midges. BTV encodes four non-structural proteins of which NS3/NS3a is functional in virus release. NS3/NS3a is not essential for in vitro virus replication. However...

  9. NetSim Project contributions to ns-3

    Energy Science and Technology Software Center (ESTSC)

    2012-05-01

    ns-3 is an external (non-LLNL) open-source framework for modeling computer networks. The LLNL NetSim project uses the ns-3 framework to address specific questions in computer network design, operation, and security. As part of the NetSim work, we develop bug fixes, deature enhancements, and new capabilities for the ns-3 framework. The virtual package referenced here, ns-3-contrib, consists of those developments we have (or will) contribute back to the ns-3 project in source code form, for inclusionmore » in future releases of ns-3.« less

  10. Balance of RNA sequence requirement and NS3/NS3a expression of segment 10 of orbiviruses.

    PubMed

    Feenstra, Femke; van Gennip, René G P; Schreuder, Myrte; van Rijn, Piet A

    2016-02-01

    Orbiviruses are insect-transmitted, non-enveloped viruses with a ten-segmented dsRNA genome of which the bluetongue virus (BTV) is the prototype. Viral non-structural protein NS3/NS3a is encoded by genome segment 10 (Seg-10), and is involved in different virus release mechanisms. This protein induces specific release via membrane disruptions and budding in both insect and mammalian cells, but also the cytopathogenic release that is only seen in mammalian cells. NS3/NS3a is not essential for virus replication in vitro with BTV Seg-10 containing RNA elements essential for virus replication, even if protein is not expressed. Recently, new BTV serotypes with distinct NS3/NS3a sequence and cell tropism have been identified. Multiple studies have hinted at the importance of Seg-10 in orbivirus replication, but the exact prerequisites are still unknown. Here, more insight is obtained with regard to the needs for orbivirus Seg-10 and the balance between protein expression and RNA elements. Multiple silent mutations in the BTV NS3a ORF destabilized Seg-10, resulting in deletions and sequences originating from other viral segments being inserted, indicating strong selection at the level of RNA during replication in mammalian cells in vitro. The NS3a ORFs of other orbiviruses were successfully exchanged in BTV1 Seg-10, resulting in viable chimeric viruses. NS3/NS3a proteins in these chimeric viruses were generally functional in mammalian cells, but not in insect cells. NS3/NS3a of the novel BTV serotypes 25 and 26 affected virus release from Culicoides cells, which might be one of the reasons for their distinct cell tropism. PMID:26644214

  11. Autocatalytic Cleavage within Classical Swine Fever Virus NS3 Leads to a Functional Separation of Protease and Helicase

    PubMed Central

    Lamp, Benjamin; Riedel, Christiane; Wentz, Eveline; Tortorici, Maria-Alejandra

    2013-01-01

    Classical swine fever virus (CSFV) is a positive-stranded RNA virus belonging to the genus Pestivirus within the Flaviviridae family. Pivotal for processing of a large portion of the viral polyprotein is a serine protease activity within nonstructural protein 3 (NS3) that also harbors helicase and NTPase activities essential for RNA replication. In CSFV-infected cells, NS3 appears as two forms, a fully processed NS3 of 80 kDa and the precursor molecule NS2-3 of 120 kDa. Here we report the identification and mapping of additional autocatalytic intramolecular cleavages. One cleavable peptide bond occurs between Leu1781 and Met1782, giving rise to a helicase subunit of 55 kDa and, depending on the substrate, a NS2-3 fragment of 78 kDa (NS2-3p) or a NS3 protease subunit of 26 kDa (NS3p). In trans-cleavage assays using NS4-5 as a substrate, NS3p acts as a fully functional protease that is able to process the polyprotein. NS3p comprises the minimal essential protease, as deletion of Leu1781 results in inactivation. A second intramolecular cleavage was mapped to the Leu1748/Lys1749 peptide bond that yields a proteolytically inactive NS3 fragment. Deletion of either of the cleavage site residues resulted in a loss of RNA infectivity, indicating the functional importance of amino acid identity at the respective positions. Our data suggest that internal cleavage within the NS3 moiety is a common process that further extends the functional repertoires of the multifunctional NS2-3 or NS3 and represents another level of the complex polyprotein processing of Flaviviridae. PMID:23986594

  12. Mechanisms of HCV NS3 Helicase Monitored by Optical Tweezers

    PubMed Central

    Cheng, Wei

    2015-01-01

    As one of the essential enzymes for viral genome replication, the hepatitis C virus NS3 helicase is one of the best characterized RNA helicases to date in understanding the mechanistic cycles in a helicase-catalyzed strand separation reaction. Recently, single-molecule studies on NS3, in particular the use of optical tweezers with sub-base pair spatial resolution, have allowed people to examine the potential elementary steps of NS3 in unwinding the double-stranded RNA fueled by ATP binding and hydrolysis. In this chapter, I detail the essential technical elements involved in conducting a high-resolution optical tweezers study of NS3 helicase, starting from the purification of the recombinant helicase protein from E. coli to setting up a high-resolution single-molecule experiment using optical tweezers. PMID:25579590

  13. Functional differences in hepatitis C virus nonstructural (NS) 3/4A- and 5A-specific T cell responses

    PubMed Central

    Holmström, Fredrik; Chen, Margaret; Balasiddaiah, Anangi; Sällberg, Matti; Ahlén, Gustaf; Frelin, Lars

    2016-01-01

    The hepatitis C virus nonstructural (NS) 3/4A and NS5A proteins are major targets for the new direct-acting antiviral compounds. Both viral proteins have been suggested as modulators of the response to the host cell. We have shown that NS3/4A- and NS5A-specific T cell receptors confer different effector functions, and that killing of NS3/4A-expressing hepatocytes is highly dependent on IFN-γ. We here characterize the functional differences in the T cell responses to NS3/4A and NS5A. NS3/4A- and NS5A-specific T cells could be induced at various frequencies in wild-type-, NS3/4A-, and NS5A-transgenic mice. Priming of NS5A-specific T cells required a high DNA dose, and was unlike NS3/4A dependent on both CD4+ and CD8+ T cells, but less influenced by CD25+/GITR+ regulatory T cells. The presence of IL-12 greatly improved specific CD8+ T cell priming by NS3/4A but not by NS5A, suggesting a less dependence of IFN-γ for NS5A. This notion was supported by the observation that NS5A-specific T cells could eliminate NS5A-expressing hepatocytes also in the absence of IFN-γ-receptor-2. This supports that NS3/4A- and NS5A-specific T cells become activated and eliminate antigen expressing, or infected hepatocytes, by distinct mechanisms, and that NS5A-specific T cells show an overall less dependence of IFN-γ. PMID:27141891

  14. Hepatitis C Virus NS3/4A Protease Inhibitors.

    PubMed

    López-Labrador, Francesc-Xavier

    2008-11-01

    Chronic hepatitis C virus infection is a global problem worldwide due to the lack of an effective therapy (the current standard of care treatment is effective in about 40-50% of the cases), and the difficulties in developing a protective vaccine. Chronic infection progresses to end-stage liver disease and liver failure in a considerable number of infected individuals. Once liver function is compromised, the only reliable therapeutic intervention is liver transplantation. Unfortunately, re-infection of the graft is unavoidable, and a new chronic hepatitis is early established in transplant recipients, that can result in graft loss. Thus, there is an urgent need for new, specifically targeted therapies for the treatment of HCV chronic infection. Among the viral proteins, the NS3/4A protease and the NS5b RNA-dependent RNA-polymerase, essential for the virus life cycle, have concentrated the efforts in the development of new antivirals, and some promising ones have already entered clinical trials. In particular, inhibitors of the HCV NS3/4A protease are the most advanced in clinical development. This review summarizes the available data for the most important HCV NS3/4A protease inhibitors in development, the most recent patents of these type of compounds, the envisioned options for future HCV therapies, and the eventual impact of HCV genetic variability on resistance to new NS3/4A protease inhibitors. PMID:18991798

  15. Dengue NS3, an RNAi suppressor, modulates the human miRNA pathways through its interacting partner.

    PubMed

    Kakumani, Pavan Kumar; Rajgokul, K S; Ponia, Sanket Singh; Kaur, Inderjeet; Mahanty, Srikrishna; Medigeshi, Guruprasad R; Banerjea, Akhil C; Chopra, Arun Prasad; Malhotra, Pawan; Mukherjee, Sunil K; Bhatnagar, Raj K

    2015-10-01

    RNAi acts as a host immune response against non-self molecules, including viruses. Viruses evolved to neutralize this response by expressing suppressor proteins. In the present study, we investigated dengue virus non structural protein 3 (dvNS3), for its RNAi-suppressor activity in human cell lines. Dengue virus (DV) NS3 reverts the GFP expression in GFP-silenced cell lines. Pull-down assays of dvNS3 revealed that it interacts with the host factor human heat shock cognate 70 (hHSC70). Down-regulation of hHSC70 resulted in accumulation of dengue viral genomic RNA. Also, the interaction of dvNS3 with hHSC70 perturbs the formation of RISC (RNA-induced silencing complex)-loading complex (RLC), by displacing TRBP (TAR RNA-binding protein) and possibly impairing the downstream activity of miRNAs. Interestingly, some of these miRNAs have earlier been reported to be down-regulated upon DV infection in Huh7 cells. Further studies on the miRNA-mRNA relationship along with mRNA profiling of samples overexpressing dvNS3 revealed up-regulation of TAZ (tafazzin) and SYNGR1 (synaptogyrin 1), known dengue viral host factors (DVHFs). Importantly, overexpression of dvNS3 in human embryonic kidney (HEK) 293T cells resulted in modulation of both mature and precursor miRNAs in human cell lines. Subsequent analysis suggested that dvNS3 induced stage-specific down-regulation of miRNAs. Taken together, these results suggest that dvNS3 affects biogenesis and function of host miRNAs to regulate DVHFs for favouring DV replication. PMID:26221025

  16. Gem GTPase acts upstream Gmip/RhoA to regulate cortical actin remodeling and spindle positioning during early mitosis.

    PubMed

    Andrieu, Guillaume; Quaranta, Muriel; Leprince, Corinne; Cuvillier, Olivier; Hatzoglou, Anastassia

    2014-11-01

    Gem is a small guanosine triphosphate (GTP)-binding protein within the Ras superfamily, involved in the regulation of voltage-gated calcium channel activity and cytoskeleton reorganization. Gem overexpression leads to stress fiber disruption, actin and cell shape remodeling and neurite elongation in interphase cells. In this study, we show that Gem plays a crucial role in the regulation of cortical actin cytoskeleton that undergoes active remodeling during mitosis. Ectopic expression of Gem leads to cortical actin disruption and spindle mispositioning during metaphase. The regulation of spindle positioning by Gem involves its downstream effector Gmip. Knockdown of Gmip rescued Gem-induced spindle phenotype, although both Gem and Gmip accumulated at the cell cortex. In addition, we implicated RhoA GTPase as an important effector of Gem/Gmip signaling. Inactivation of RhoA by overexpressing dominant-negative mutant prevented normal spindle positioning. Introduction of active RhoA rescued the actin and spindle positioning defects caused by Gem or Gmip overexpression. These findings demonstrate a new role of Gem/Gmip/RhoA signaling in cortical actin regulation during early mitotic stages. PMID:25173885

  17. Dynamin, a membrane remodelling GTPase

    PubMed Central

    Ferguson, Shawn M.; De Camilli, Pietro

    2012-01-01

    Dynamin, the founding member of a family of dynamin-like GTPases (DLPs) implicated in membrane remodelling, has a critical role in endocytic membrane fission events. The use of complementary approaches, including live cell imaging, cell free-studies, X-ray crystallography and genetic studies in mice has greatly advanced our understanding of the mechanisms by which dynamin acts, its essential roles in cell physiology and the specific function of different dynamin isoforms. In addition, several connections between dynamin and human disease have also emerged that highlight specific contributions of this GTPase to the physiology of different tissues. PMID:22233676

  18. Host cell killing by the West Nile Virus NS2B-NS3 proteolytic complex: NS3 alone is sufficient to recruit caspase-8-based apoptotic pathway

    SciTech Connect

    Ramanathan, Mathura P.; Chambers, Jerome A.; Pankhong, Panyupa; Chattergoon, Michael; Attatippaholkun, Watcharee; Dang, Kesen; Shah, Neelima; Weiner, David B. . E-mail: dbweiner@mail.med.upenn.edu

    2006-02-05

    The West Nile Virus (WNV) non-structural proteins 2B and 3 (NS2B-NS3) constitute the proteolytic complex that mediates the cleavage and processing of the viral polyprotein. NS3 recruits NS2B and NS5 proteins to direct protease and replication activities. In an effort to investigate the biology of the viral protease, we cloned cDNA encoding the NS2B-NS3 proteolytic complex from brain tissue of a WNV-infected dead crow, collected from the Lower Merion area (Merion strain). Expression of the NS2B-NS3 gene cassette induced apoptosis within 48 h of transfection. Electron microscopic analysis of NS2B-NS3-transfected cells revealed ultra-structural changes that are typical of apoptotic cells including membrane blebbing, nuclear disintegration and cytoplasmic vacuolations. The role of NS3 or NS2B in contributing to host cell apoptosis was examined. NS3 alone triggers the apoptotic pathways involving caspases-8 and -3. Experimental results from the use of caspase-specific inhibitors and caspase-8 siRNA demonstrated that the activation of caspase-8 was essential to initiate apoptotic signaling in NS3-expressing cells. Downstream of caspase-3 activation, we observed nuclear membrane ruptures and cleavage of the DNA-repair enzyme, PARP in NS3-expressing cells. Nuclear herniations due to NS3 expression were absent in the cells treated with a caspase-3 inhibitor. Expression of protease and helicase domains themselves was sufficient to trigger apoptosis generating insight into the apoptotic pathways triggered by NS3 from WNV.

  19. Analysis of the Enzymatic Activity of an NS3 Helicase Genotype 3a Variant Sequence Obtained from a Relapse Patient

    PubMed Central

    Provazzi, Paola J. S.; Mukherjee, Sourav; Hanson, Alicia M.; Nogueira, Mauricio L.; Carneiro, Bruno M.; Frick, David N.; Rahal, Paula

    2015-01-01

    The hepatitis C virus (HCV) is a species of diverse genotypes that infect over 170 million people worldwide, causing chronic inflammation, cirrhosis and hepatocellular carcinoma. HCV genotype 3a is common in Brazil, and it is associated with a relatively poor response to current direct-acting antiviral therapies. The HCV NS3 protein cleaves part of the HCV polyprotein, and cellular antiviral proteins. It is therefore the target of several HCV drugs. In addition to its protease activity, NS3 is also an RNA helicase. Previously, HCV present in a relapse patient was found to harbor a mutation known to be lethal to HCV genotype 1b. The point mutation encodes the amino acid substitution W501R in the helicase RNA binding site. To examine how the W501R substitution affects NS3 helicase activity in a genotype 3a background, wild type and W501R genotype 3a NS3 alleles were sub-cloned, expressed in E. coli, and the recombinant proteins were purified and characterized. The impact of the W501R allele on genotype 2a and 3a subgenomic replicons was also analyzed. Assays monitoring helicase-catalyzed DNA and RNA unwinding revealed that the catalytic efficiency of wild type genotype 3a NS3 helicase was more than 600 times greater than the W501R protein. Other assays revealed that the W501R protein bound DNA less than 2 times weaker than wild type, and both proteins hydrolyzed ATP at similar rates. In Huh7.5 cells, both genotype 2a and 3a subgenomic HCV replicons harboring the W501R allele showed a severe defect in replication. Since the W501R allele is carried as a minor variant, its replication would therefore need to be attributed to the trans-complementation by other wild type quasispecies. PMID:26658750

  20. VP2 Exchange and NS3/NS3a Deletion in African Horse Sickness Virus (AHSV) in Development of Disabled Infectious Single Animal Vaccine Candidates for AHSV

    PubMed Central

    van de Water, Sandra G. P.; van Gennip, René G. P.; Potgieter, Christiaan A.; Wright, Isabel M.

    2015-01-01

    ABSTRACT African horse sickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae. There are nine serotypes of AHSV showing different levels of cross neutralization. AHSV is transmitted by species of Culicoides biting midges and causes African horse sickness (AHS) in equids, with a mortality rate of up to 95% in naive horses. AHS has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates appear to be competent vectors for the related bluetongue virus (BTV). To control AHS, live-attenuated vaccines (LAVs) are used in Africa. We used reverse genetics to generate “synthetic” reassortants of AHSV for all nine serotypes by exchange of genome segment 2 (Seg-2). This segment encodes VP2, which is the serotype-determining protein and the dominant target for neutralizing antibodies. Single Seg-2 AHSV reassortants showed similar cytopathogenic effects in mammalian cells but displayed different growth kinetics. Reverse genetics for AHSV was also used to study Seg-10 expressing NS3/NS3a proteins. We demonstrated that NS3/NS3a proteins are not essential for AHSV replication in vitro. NS3/NS3a of AHSV is, however, involved in the cytopathogenic effect in mammalian cells and is very important for virus release from cultured insect cells in particular. Similar to the concept of the bluetongue disabled infectious single animal (BT DISA) vaccine platform, an AHS DISA vaccine platform lacking NS3/NS3a expression was developed. Using exchange of genome segment 2 encoding VP2 protein (Seg-2[VP2]), we will be able to develop AHS DISA vaccine candidates for all current AHSV serotypes. IMPORTANCE African horse sickness virus is transmitted by species of Culicoides biting midges and causes African horse sickness in equids, with a mortality rate of up to 95% in naive horses. African horse sickness has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate

  1. The effect of glycosylation on cytotoxicity of Ibaraki virus nonstructural protein NS3

    PubMed Central

    URATA, Maho; WATANABE, Rie; IWATA, Hiroyuki

    2015-01-01

    The cytotoxicity of Ibaraki virus nonstructural protein NS3 was confirmed, and the contribution of glycosylation to this activity was examined by using glycosylation mutants of NS3 generated by site-directed mutagenesis. The expression of NS3 resulted in leakage of lactate dehydrogenase to the culture supernatant, suggesting the cytotoxicity of this protein. The lack of glycosylation impaired the transport of NS3 to the plasma membrane and resulted in reduced cytotoxicity. Combined with the previous observation that NS3 glycosylation was specifically observed in mammalian cells (Urata et al., Virus Research 2014), it was suggested that the alteration of NS3 cytotoxicity through modulating glycosylation is one of the strategies to achieve host specific pathogenisity of Ibaraki virus between mammals and vector arthropods. PMID:26178820

  2. Locking GTPases covalently in their functional states

    NASA Astrophysics Data System (ADS)

    Wiegandt, David; Vieweg, Sophie; Hofmann, Frank; Koch, Daniel; Li, Fu; Wu, Yao-Wen; Itzen, Aymelt; Müller, Matthias P.; Goody, Roger S.

    2015-07-01

    GTPases act as key regulators of many cellular processes by switching between active (GTP-bound) and inactive (GDP-bound) states. In many cases, understanding their mode of action has been aided by artificially stabilizing one of these states either by designing mutant proteins or by complexation with non-hydrolysable GTP analogues. Because of inherent disadvantages in these approaches, we have developed acryl-bearing GTP and GDP derivatives that can be covalently linked with strategically placed cysteines within the GTPase of interest. Binding studies with GTPase-interacting proteins and X-ray crystallography analysis demonstrate that the molecular properties of the covalent GTPase-acryl-nucleotide adducts are a faithful reflection of those of the corresponding native states and are advantageously permanently locked in a defined nucleotide (that is active or inactive) state. In a first application, in vivo experiments using covalently locked Rab5 variants provide new insights into the mechanism of correct intracellular localization of Rab proteins.

  3. Structural and Thermodynamic Effects of Macrocyclization in HCV NS3/4A Inhibitor MK-5172.

    PubMed

    Soumana, Djadé I; Kurt Yilmaz, Nese; Prachanronarong, Kristina L; Aydin, Cihan; Ali, Akbar; Schiffer, Celia A

    2016-04-15

    Recent advances in direct-acting antivirals against Hepatitis C Virus (HCV) have led to the development of potent inhibitors, including MK-5172, that target the viral NS3/4A protease with relatively low susceptibility to resistance. MK-5172 has a P2-P4 macrocycle and a unique binding mode among current protease inhibitors where the P2 quinoxaline packs against the catalytic residues H57 and D81. However, the effect of macrocyclization on this binding mode is not clear, as is the relation between macrocyclization, thermodynamic stabilization, and susceptibility to the resistance mutation A156T. We have determined high-resolution crystal structures of linear and P1-P3 macrocyclic analogs of MK-5172 bound to WT and A156T protease and compared these structures, their molecular dynamics, and experimental binding thermodynamics to the parent compound. We find that the "unique" binding mode of MK-5172 is conserved even when the P2-P4 macrocycle is removed or replaced with a P1-P3 macrocycle. While beneficial to decreasing the entropic penalty associated with binding, the constraint exerted by the P2-P4 macrocycle prevents efficient rearrangement to accommodate the A156T mutation, a deficit alleviated in the linear and P1-P3 analogs. Design of macrocyclic inhibitors against NS3/4A needs to achieve the best balance between exerting optimal conformational constraint for enhancing potency, fitting within the substrate envelope and allowing adaptability to be robust against resistance mutations. PMID:26682473

  4. Review: Translational GTPases.

    PubMed

    Maracci, Cristina; Rodnina, Marina V

    2016-08-01

    Translational GTPases (trGTPases) play key roles in facilitating protein synthesis on the ribosome. Despite the high degree of evolutionary conservation in the sequences of their GTP-binding domains, the rates of GTP hydrolysis and nucleotide exchange vary broadly between different trGTPases. EF-Tu, one of the best-characterized model G proteins, evolved an exceptionally rapid and tightly regulated GTPase activity, which ensures rapid and accurate incorporation of amino acids into the nascent chain. Other trGTPases instead use the energy of GTP hydrolysis to promote movement or to ensure the forward commitment of translation reactions. Recent data suggest the GTPase mechanism of EF-Tu and provide an insight in the catalysis of GTP hydrolysis by its unusual activator, the ribosome. Here we summarize these advances in understanding the functional cycle and the regulation of trGTPases, stimulated by the elucidation of their structures on the ribosome and the progress in dissecting the reaction mechanism of GTPases. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 463-475, 2016. PMID:26971860

  5. Naturally occurring NS3 resistance-associated variants in hepatitis C virus genotype 1: Their relevance for developing countries.

    PubMed

    Echeverría, Natalia; Betancour, Gabriela; Gámbaro, Fabiana; Hernández, Nelia; López, Pablo; Chiodi, Daniela; Sánchez, Adriana; Boschi, Susana; Fajardo, Alvaro; Sóñora, Martín; Moratorio, Gonzalo; Cristina, Juan; Moreno, Pilar

    2016-09-01

    Hepatitis C virus (HCV) is a major cause of global morbidity and mortality, with an estimated 130-150 million infected individuals worldwide. HCV is a leading cause of chronic liver diseases including cirrhosis and hepatocellular carcinoma. Current treatment options in developing countries involve pegylated interferon-α and ribavirin as dual therapy or in combination with one or more direct-acting antiviral agents (DAA). The emergence of resistance-associated variants (RAVs) after treatment reveals the great variability of this virus leading to a great difficulty in developing effective antiviral strategies. Baseline RAVs detected in DAA treatment-naïve HCV-infected patients could be of great importance for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS3 protease inhibitor mutations has been addressed in many countries, there are only a few reports on their prevalence in South America. In this study, we investigated the presence of RAVs in the HCV NS3 serine protease region by analysing a cohort of Uruguayan patients with chronic hepatitis C who had not been treated with any DAAs and compare them with the results found for other South American countries. The results of these studies revealed that naturally occurring mutations conferring resistance to NS3 inhibitors exist in a substantial proportion of Uruguayan treatment-naïve patients infected with HCV genotype 1 enrolled in these studies. The identification of these baseline RAVs could be of great importance for patients' management and outcome prediction in developing countries. PMID:27449600

  6. A tale of two GTPases in cotranslational protein targeting

    PubMed Central

    Saraogi, Ishu; Akopian, David; Shan, Shu-Ou

    2011-01-01

    Guanosine triphosphatases (GTPases) comprise a superfamily of proteins that provide molecular switches to regulate numerous cellular processes. The “GTPase switch” paradigm, in which a GTPase acts as a bimodal switch that is turned “on” and “off” by external regulatory factors, has been used to interpret the regulatory mechanism of many GTPases. Recent work on a pair of GTPases in the signal recognition particle (SRP) pathway has revealed a distinct mode of GTPase regulation. Instead of the classical GTPase switch, the two GTPases in the SRP and SRP receptor undergo a series of conformational changes during their dimerization and reciprocal activation. Each conformational rearrangement provides a point at which these GTPases can communicate with and respond to their upstream and downstream biological cues, thus ensuring the spatial and temporal precision of all the molecular events in the SRP pathway. We suggest that the SRP and SRP receptor represent an emerging class of “multistate” regulatory GTPases uniquely suited to provide exquisite control over complex cellular pathways that require multiple molecular events to occur in a highly coordinated fashion. PMID:21898651

  7. X-Ray Structure of the Pestivirus NS3 Helicase and Its Conformation in Solution

    PubMed Central

    Duquerroy, Stéphane; Kwok, Jane; Vonrhein, Clemens; Perez, Javier; Lamp, Benjamin; Bricogne, Gerard; Rümenapf, Till; Vachette, Patrice

    2015-01-01

    ABSTRACT Pestiviruses form a genus in the Flaviviridae family of small enveloped viruses with a positive-sense single-stranded RNA genome. Viral replication in this family requires the activity of a superfamily 2 RNA helicase contained in the C-terminal domain of nonstructural protein 3 (NS3). NS3 features two conserved RecA-like domains (D1 and D2) with ATPase activity, plus a third domain (D3) that is important for unwinding nucleic acid duplexes. We report here the X-ray structure of the pestivirus NS3 helicase domain (pNS3h) at a 2.5-Å resolution. The structure deviates significantly from that of NS3 of other genera in the Flaviviridae family in D3, as it contains two important insertions that result in a narrower nucleic acid binding groove. We also show that mutations in pNS3h that rescue viruses from which the core protein is deleted map to D3, suggesting that this domain may be involved in interactions that facilitate particle assembly. Finally, structural comparisons of the enzyme in different crystalline environments, together with the findings of small-angle X-ray-scattering studies in solution, show that D2 is mobile with respect to the rest of the enzyme, oscillating between closed and open conformations. Binding of a nonhydrolyzable ATP analog locks pNS3h in a conformation that is more compact than the closest apo-form in our crystals. Together, our results provide new insight and bring up new questions about pNS3h function during pestivirus replication. IMPORTANCE Although pestivirus infections impose an important toll on the livestock industry worldwide, little information is available about the nonstructural proteins essential for viral replication, such as the NS3 helicase. We provide here a comparative structural and functional analysis of pNS3h with respect to its orthologs in other viruses of the same family, the flaviviruses and hepatitis C virus. Our studies reveal differences in the nucleic acid binding groove that could have implications

  8. Natural HCV variants with increased replicative fitness due to NS3 helicase mutations in the C-terminal helix α18

    PubMed Central

    Stross, Claudia; Shimakami, Tetsuro; Haselow, Katrin; Ahmad, Monazza Q.; Zeuzem, Stefan; Lange, Christian M.; Welsch, Christoph

    2016-01-01

    High replicative fitness is a general determinant of a multidrug resistance phenotype and may explain lower sensitivity to direct-acting antiviral agents (DAAs) in some hepatitis C virus genotypes. Genetic diversity in the molecular target site of peptidomimetic NS3 protease inhibitors could impact variant replicative fitness and potentially add to virologic treatment failure. We selected NS3 helicase residues near the protease natural substrate in the NS3 domain interface and identified natural variants from a public database. Sequence diversity among different genotypes was identified and subsequently analyzed for potential effects of helicase variants on protein structure and function, and phenotypic effects on RNA replication and DAA resistance. We found increased replicative fitness in particular for amino acid substitutions at the NS3 helicase C-terminal helix α18. A network of strongly coupled residue pairs is identified. Helix α18 is part of this regulatory network and connects several NS3 functional elements involved in RNA replication. Among all genotypes we found distinct sequence diversity at helix α18 in particular for the most difficult-to-treat genotype 3. Our data suggest sequence diversity with implications for virus replicative fitness due to natural variants in helicase helix α18. PMID:26787124

  9. The NS3 proteinase domain of hepatitis C virus is a zinc-containing enzyme.

    PubMed Central

    Stempniak, M; Hostomska, Z; Nodes, B R; Hostomsky, Z

    1997-01-01

    NS3 proteinase of hepatitis C virus (HCV), contained within the N-terminal domain of the NS3 protein, is a chymotrypsin-like serine proteinase responsible for processing of the nonstructural region of the HCV polyprotein. In this study, we examined the sensitivity of the NS3 proteinase to divalent metal ions, which is unusual behavior for this proteinase class. By using a cell-free coupled transcription-translation system, we found that HCV polyprotein processing can be activated by Zn2+ (and, to a lesser degree, by Cd2+, Pb2+, and Co2+) and inhibited by Cu2+ and Hg2+ ions. Elemental analysis of the purified NS3 proteinase domain revealed the presence of zinc in an equimolar ratio. The zinc content was unchanged in a mutated NS3 proteinase in which active-site residues His-57 and Ser-139 were replaced with Ala, suggesting that the zinc atom is not directly involved in catalysis but rather may have a structural role. Based on data from site-directed mutagenesis combined with zinc content determination, we propose that Cys-97, Cys-99, Cys-145, and His-149 coordinate the structural zinc in the HCV NS3 proteinase. A similar metal binding motif is found in 2A proteinases of enteroviruses and rhinoviruses, suggesting that these 2A proteinases and HCV NS3 proteinase are structurally related. PMID:9060645

  10. Identification of Hydroxyanthraquinones as Novel Inhibitors of Hepatitis C Virus NS3 Helicase

    PubMed Central

    Furuta, Atsushi; Tsubuki, Masayoshi; Endoh, Miduki; Miyamoto, Tatsuki; Tanaka, Junichi; Abdus Salam, Kazi; Akimitsu, Nobuyoshi; Tani, Hidenori; Yamashita, Atsuya; Moriishi, Kohji; Nakakoshi, Masamichi; Sekiguchi, Yuji; Tsuneda, Satoshi; Noda, Naohiro

    2015-01-01

    Hepatitis C virus (HCV) is an important etiological agent of severe liver diseases, including cirrhosis and hepatocellular carcinoma. The HCV genome encodes nonstructural protein 3 (NS3) helicase, which is a potential anti-HCV drug target because its enzymatic activity is essential for viral replication. Some anthracyclines are known to be NS3 helicase inhibitors and have a hydroxyanthraquinone moiety in their structures; mitoxantrone, a hydroxyanthraquinone analogue, is also known to inhibit NS3 helicase. Therefore, we hypothesized that the hydroxyanthraquinone moiety alone could also inhibit NS3 helicase. Here, we performed a structure–activity relationship study on a series of hydroxyanthraquinones by using a fluorescence-based helicase assay. Hydroxyanthraquinones inhibited NS3 helicase with IC50 values in the micromolar range. The inhibitory activity varied depending on the number and position of the phenolic hydroxyl groups, and among different hydroxyanthraquinones examined, 1,4,5,8-tetrahydroxyanthraquinone strongly inhibited NS3 helicase with an IC50 value of 6 µM. Furthermore, hypericin and sennidin A, which both have two hydroxyanthraquinone-like moieties, were found to exert even stronger inhibition with IC50 values of 3 and 0.8 µM, respectively. These results indicate that the hydroxyanthraquinone moiety can inhibit NS3 helicase and suggest that several key chemical structures are important for the inhibition. PMID:26262613

  11. Comparison of structural architecture of HCV NS3 genotype 1 versus Pakistani genotype 3a.

    PubMed

    Fatima, Kaneez; Azhar, Esam; Mathew, Shilu; Damanhouri, Ghazi; Qadri, Ishtiaq

    2014-01-01

    This study described the structural characterization of Pakistani HCV NS3 GT3a in parallel with genotypes 1a and 1b NS3. We investigated the role of amino acids and their interaction patterns in different HCV genotypes by crystallographic modeling. Different softwares were used to study the interaction pattern, for example, CLCBIO sequence viewer, MODELLER, NMRCLUST, ERRAT score, and MODELLER. Sixty models were produced and clustered into groups and the best model of PK-NCVI/Pk3a NS3 was selected and studied further to check the variability with other HCV NS3 genotypes. This study will help in future to understand the structural architecture of HCV genome variability and to further define the conserved targets for antiviral agents. PMID:25401105

  12. Comparison of Structural Architecture of HCV NS3 Genotype 1 versus Pakistani Genotype 3a

    PubMed Central

    Mathew, Shilu; Damanhouri, Ghazi

    2014-01-01

    This study described the structural characterization of Pakistani HCV NS3 GT3a in parallel with genotypes 1a and 1b NS3. We investigated the role of amino acids and their interaction patterns in different HCV genotypes by crystallographic modeling. Different softwares were used to study the interaction pattern, for example, CLCBIO sequence viewer, MODELLER, NMRCLUST, ERRAT score, and MODELLER. Sixty models were produced and clustered into groups and the best model of PK-NCVI/Pk3a NS3 was selected and studied further to check the variability with other HCV NS3 genotypes. This study will help in future to understand the structural architecture of HCV genome variability and to further define the conserved targets for antiviral agents. PMID:25401105

  13. Ebselen Inhibits Hepatitis C Virus NS3 Helicase Binding to Nucleic Acid and Prevents Viral Replication

    PubMed Central

    2015-01-01

    The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previously shown to be a HCV antiviral agent, inhibits the NS3 helicase. Ebselen inhibited the abilities of NS3 to unwind nucleic acids, to bind nucleic acids, and to hydrolyze ATP, and about 1 μM ebselen was sufficient to inhibit each of these activities by 50%. However, ebselen had no effect on the activity of the NS3 protease, even at 100 times higher ebselen concentrations. At concentrations below 10 μM, the ability of ebselen to inhibit HCV helicase was reversible, but prolonged incubation of HCV helicase with higher ebselen concentrations led to irreversible inhibition and the formation of covalent adducts between ebselen and all 14 cysteines present in HCV helicase. Ebselen analogues with sulfur replacing the selenium were just as potent HCV helicase inhibitors as ebselen, but the length of the linker between the phenyl and benzisoselenazol rings was critical. Modifications of the phenyl ring also affected compound potency over 30-fold, and ebselen was a far more potent helicase inhibitor than other, structurally unrelated, thiol-modifying agents. Ebselen analogues were also more effective antiviral agents, and they were less toxic to hepatocytes than ebselen. Although the above structure–activity relationship studies suggest that ebselen targets a specific site on NS3, we were unable to confirm binding to either the NS3 ATP binding site or nucleic acid binding cleft by examining the effects of ebselen on NS3 proteins lacking key cysteines. PMID:25126694

  14. Long non-coding RNA GAS5 inhibited hepatitis C virus replication by binding viral NS3 protein.

    PubMed

    Qian, Xijing; Xu, Chen; Zhao, Ping; Qi, Zhongtian

    2016-05-01

    HCV infection has a complex and dynamic process which involves a large number of viral and host factors. Long non-coding RNA GAS5 inhibits liver fibrosis and liver tumor migration and invasion. However, the contribution of GAS5 on HCV infection remains unknown. In this study, GAS5 was gradually upregulated during HCV infection in Huh7 cells. In addition, GAS5 attenuated virus replication with its 5' end sequences, as confirmed by different GAS5 truncations. Moreover, this 5' end sequences showed RNA-protein interaction with HCV NS3 protein that could act as a decoy to inhibit its functions, which contributed to the suppression of HCV replication. Finally, the innate immune responses remained low in HCV infected Huh7 cells, ruling out the possibility of GAS5 to modulate innate immunity. Thus, HCV stimulated endogenous GAS5 can suppress HCV infection by acting as HCV NS3 protein decoy, providing a potential role of GAS5 as a diagnostic or therapeutic target. PMID:26945984

  15. Human Transbodies to HCV NS3/4A Protease Inhibit Viral Replication and Restore Host Innate Immunity

    PubMed Central

    Jittavisutthikul, Surasak; Seesuay, Watee; Thanongsaksrikul, Jeeraphong; Thueng-in, Kanyarat; Srimanote, Potjanee; Werner, Rolf G.; Chaicumpa, Wanpen

    2016-01-01

    A safe and effective direct acting anti-hepatitis C virus (HCV) agent is still needed. In this study, human single chain variable fragments of antibody (scFvs) that bound to HCV NS3/4A protein were produced by phage display technology. The engineered scFvs were linked to nonaarginines (R9) for making them cell penetrable. HCV-RNA-transfected Huh7 cells treated with the transbodies produced from four different transformed E. coli clones had reduced HCV-RNA inside the cells and in the cell spent media, as well as fewer HCV foci in the cell monolayer compared to the transfected cells in culture medium alone. The transbodies-treated transfected cells also had up-expression of the genes coding for the host innate immune response, including TRIF, TRAF3, IRF3, IL-28B, and IFN-β. Computerized homology modeling and intermolecular docking predicted that the effective transbodies interacted with several critical residues of the NS3/4A protease, including those that form catalytic triads, oxyanion loop, and S1 and S6 pockets, as well as a zinc-binding site. Although insight into molecular mechanisms of the transbodies need further laboratory investigation, it can be deduced from the current data that the transbodies blocked the HCV NS3/4A protease activities, leading to the HCV replication inhibition and restoration of the virally suppressed host innate immunity. The engineered antibodies should be tested further for treatment of HCV infection either alone, in combination with current therapeutics, or in a mixture with their cognates specific to other HCV proteins. PMID:27617013

  16. Human Transbodies to HCV NS3/4A Protease Inhibit Viral Replication and Restore Host Innate Immunity.

    PubMed

    Jittavisutthikul, Surasak; Seesuay, Watee; Thanongsaksrikul, Jeeraphong; Thueng-In, Kanyarat; Srimanote, Potjanee; Werner, Rolf G; Chaicumpa, Wanpen

    2016-01-01

    A safe and effective direct acting anti-hepatitis C virus (HCV) agent is still needed. In this study, human single chain variable fragments of antibody (scFvs) that bound to HCV NS3/4A protein were produced by phage display technology. The engineered scFvs were linked to nonaarginines (R9) for making them cell penetrable. HCV-RNA-transfected Huh7 cells treated with the transbodies produced from four different transformed E. coli clones had reduced HCV-RNA inside the cells and in the cell spent media, as well as fewer HCV foci in the cell monolayer compared to the transfected cells in culture medium alone. The transbodies-treated transfected cells also had up-expression of the genes coding for the host innate immune response, including TRIF, TRAF3, IRF3, IL-28B, and IFN-β. Computerized homology modeling and intermolecular docking predicted that the effective transbodies interacted with several critical residues of the NS3/4A protease, including those that form catalytic triads, oxyanion loop, and S1 and S6 pockets, as well as a zinc-binding site. Although insight into molecular mechanisms of the transbodies need further laboratory investigation, it can be deduced from the current data that the transbodies blocked the HCV NS3/4A protease activities, leading to the HCV replication inhibition and restoration of the virally suppressed host innate immunity. The engineered antibodies should be tested further for treatment of HCV infection either alone, in combination with current therapeutics, or in a mixture with their cognates specific to other HCV proteins. PMID:27617013

  17. Allosteric inhibition of the NS2B-NS3 protease from dengue virus.

    PubMed

    Yildiz, Muslum; Ghosh, Sumana; Bell, Jeffrey A; Sherman, Woody; Hardy, Jeanne A

    2013-12-20

    Dengue virus is the flavivirus that causes dengue fever, dengue hemorrhagic disease, and dengue shock syndrome, which are currently increasing in incidence worldwide. Dengue virus protease (NS2B-NS3pro) is essential for dengue virus infection and is thus a target of therapeutic interest. To date, attention has focused on developing active-site inhibitors of NS2B-NS3pro. The flat and charged nature of the NS2B-NS3pro active site may contribute to difficulties in developing inhibitors and suggests that a strategy of identifying allosteric sites may be useful. We report an approach that allowed us to scan the NS2B-NS3pro surface by cysteine mutagenesis and use cysteine reactive probes to identify regions of the protein that are susceptible to allosteric inhibition. This method identified a new allosteric site utilizing a circumscribed panel of just eight cysteine variants and only five cysteine reactive probes. The allosterically sensitive site is centered at Ala125, between the 120s loop and the 150s loop. The crystal structures of WT and modified NS2B-NS3pro demonstrate that the 120s loop is flexible. Our work suggests that binding at this site prevents a conformational rearrangement of the NS2B region of the protein, which is required for activation. Preventing this movement locks the protein into the open, inactive conformation, suggesting that this site may be useful in the future development of therapeutic allosteric inhibitors. PMID:24164286

  18. RNA-protein interactions: involvement of NS3, NS5, and 3' noncoding regions of Japanese encephalitis virus genomic RNA.

    PubMed Central

    Chen, C J; Kuo, M D; Chien, L J; Hsu, S L; Wang, Y M; Lin, J H

    1997-01-01

    The mechanism of replication of the flavivirus Japanese encephalitis virus (JEV) is not well known. The structures at the 3' end of the viral genome are highly conserved among divergent flaviviruses, suggesting that they may function as cis-acting signals for RNA replication and, as such, might specifically bind to cellular or viral proteins. UV cross-linking experiments were performed to identify the proteins that bind with the JEV plus-strand 3' noncoding region (NCR). Two proteins, p71 and p110, from JEV-infected but not from uninfected cell extracts were shown to bind specifically to the plus-strand 3' NCR. The quantities of these binding proteins increased during the course of JEV infection and correlated with the levels of JEV RNA synthesis in cell extracts. UV cross-linking coupled with Western blot and immunoprecipitation analysis showed that the p110 and p71 proteins were JEV NS5 and NS3, respectively, which are proposed as components of the RNA replicase. The putative stem-loop structure present within the plus-strand 3' NCR was required for the binding of these proteins. Furthermore, both proteins could interact with each other and form a protein-protein complex in vivo. These findings suggest that the 3' NCR of JEV genomic RNA may form a replication complex together with NS3 and NS5; this complex may be involved in JEV minus-strand RNA synthesis. PMID:9094618

  19. Hepatitis C virus nonstructural protein NS3 transforms NIH 3T3 cells.

    PubMed Central

    Sakamuro, D; Furukawa, T; Takegami, T

    1995-01-01

    Clinical evidence suggests that hepatitis C virus (HCV) is etiologically involved in hepatic cancer and liver cirrhosis. To investigate whether the HCV nonstructural protein NS3 has oncogenic activity, NIH 3T3 cells were transfected with an expression vector containing cDNA for the 5'- or 3'-half sequence of the HCV genome segment encoding NS3. Only cells transfected with the 5'-half cDNA rapidly proliferated, lost contact inhibition, grew anchorage independently in soft agar, and formed tumors in nude mice. PCR analysis confirmed the presence of the 5'-half DNA in the transfectants. These results suggest that the 5' region of the HCV genome segment encoding NS3 is involved in cell transformation. PMID:7745741

  20. Potent inhibitors of HCV-NS3 protease derived from boronic acids

    SciTech Connect

    Venkatraman, Srikanth; Wu, Wanli; Prongay, Andrew; Girijavallabhan, Viyyoor; Njoroge, F. George

    2009-07-23

    Chronic hepatitis C infection is the leading causes for cirrhosis of the liver and hepatocellular carcinoma, leading to liver failure and liver transplantation. The etiological agent, HCV virus produces a single positive strand of RNA that is processed with the help of serine protease NS3 to produce mature virus. Inhibition of NS3 protease can be potentially used to develop effective drugs for HCV infections. Numerous efforts are now underway to develop potent inhibitors of HCV protease that contain ketoamides as serine traps. Herein we report the synthesis of a series of potent inhibitors that contain a boronic acid as a serine trap. The activity of these compounds were optimized to 200 pM. X-ray structure of compound 17 bound to NS3 protease is also discussed.

  1. Allosteric Inhibitors of the NS3 Protease from the Hepatitis C Virus

    PubMed Central

    Abian, Olga; Vega, Sonia; Sancho, Javier; Velazquez-Campoy, Adrian

    2013-01-01

    The nonstructural protein 3 (NS3) from the hepatitis C virus processes the non-structural region of the viral precursor polyprotein in infected hepatic cells. The NS3 protease activity has been considered a target for drug development since its identification two decades ago. Although specific inhibitors have been approved for clinical therapy very recently, resistance-associated mutations have already been reported for those drugs, compromising their long-term efficacy. Therefore, there is an urgent need for new anti-HCV agents with low susceptibility to resistance-associated mutations. Regarding NS3 protease, two strategies have been followed: competitive inhibitors blocking the active site and allosteric inhibitors blocking the binding of the accessory viral protein NS4A. In this work we exploit the intrinsic Zn+2-regulated plasticity of the protease to identify a new type of allosteric inhibitors. In the absence of Zn+2, the NS3 protease adopts a partially-folded inactive conformation. We found ligands binding to the Zn+2-free NS3 protease, trap the inactive protein, and block the viral life cycle. The efficacy of these compounds has been confirmed in replicon cell assays. Importantly, direct calorimetric assays reveal a low impact of known resistance-associated mutations, and enzymatic assays provide a direct evidence of their inhibitory activity. They constitute new low molecular-weight scaffolds for further optimization and provide several advantages: 1) new inhibition mechanism simultaneously blocking substrate and cofactor interactions in a non-competitive fashion, appropriate for combination therapy; 2) low impact of known resistance-associated mutations; 3) inhibition of NS4A binding, thus blocking its several effects on NS3 protease. PMID:23936097

  2. Three Conformational Snapshots of the Hepatitis Virus NS3 Helicase Reveal a Ratchet Translocation Mechanism

    SciTech Connect

    Gu, M.; Rice, C

    2010-01-01

    A virally encoded superfamily-2 (SF2) helicase (NS3h) is essential for the replication of hepatitis C virus, a leading cause of liver disease worldwide. Efforts to elucidate the function of NS3h and to develop inhibitors against it, however, have been hampered by limited understanding of its molecular mechanism. Here we show x-ray crystal structures for a set of NS3h complexes, including ground-state and transition-state ternary complexes captured with ATP mimics (ADP {center_dot} BeF{sub 3} and ADP {center_dot} AlF{sub 4}{sup -}). These structures provide, for the first time, three conformational snapshots demonstrating the molecular basis of action for a SF2 helicase. Upon nucleotide binding, overall domain rotation along with structural transitions in motif V and the bound DNA leads to the release of one base from the substrate base-stacking row and the loss of several interactions between NS3h and the 3{prime} DNA segment. As nucleotide hydrolysis proceeds into the transition state, stretching of a 'spring' helix and another overall conformational change couples rearrangement of the (d)NTPase active site to additional hydrogen-bonding between NS3h and DNA. Together with biochemistry, these results demonstrate a 'ratchet' mechanism involved in the unidirectional translocation and define the step size of NS3h as one base per nucleotide hydrolysis cycle. These findings suggest feasible strategies for developing specific inhibitors to block the action of this attractive, yet largely unexplored drug target.

  3. Discovery of MK-5172, a Macrocyclic Hepatitis C Virus NS3/4a Protease Inhibitor

    PubMed Central

    2012-01-01

    A new class of HCV NS3/4a protease inhibitors containing a P2 to P4 macrocyclic constraint was designed using a molecular modeling-derived strategy. Building on the profile of previous clinical compounds and exploring the P2 and linker regions of the series allowed for optimization of broad genotype and mutant enzyme potency, cellular activity, and rat liver exposure following oral dosing. These studies led to the identification of clinical candidate 15 (MK-5172), which is active against genotype 1–3 NS3/4a and clinically relevant mutant enzymes and has good plasma exposure and excellent liver exposure in multiple species. PMID:24900473

  4. Mutagenesis of the NS3 Protease of Dengue Virus Type 2

    PubMed Central

    Valle, Rosaura P. C.; Falgout, Barry

    1998-01-01

    The flavivirus protease is composed of two viral proteins, NS2B and NS3. The amino-terminal portion of NS3 contains sequence and structural motifs characteristic of bacterial and cellular trypsin-like proteases. We have undertaken a mutational analysis of the region of NS3 which contains the catalytic serine, five putative substrate binding residues, and several residues that are highly conserved among flavivirus proteases and among all serine proteases. In all, 46 single-amino-acid substitutions were created in a cloned NS2B-NS3 cDNA fragment of dengue virus type 2, and the effect of each mutation on the extent of self-cleavage of the NS2B-NS3 precursor at the NS2B-NS3 junction was assayed in vivo. Twelve mutations almost completely or completely inhibited protease activity, 9 significantly reduced it, 14 decreased cleavage, and 11 yielded wild-type levels of activity. Substitution of alanine at ultraconserved residues abolished NS3 protease activity. Cleavage was also inhibited by substituting some residues that are conserved among flavivirus NS3 proteins. Two (Y150 and G153) of the five putative substrate binding residues could not be replaced by alanine, and only Y150 and N152 could be replaced by a conservative change. The two other putative substrate binding residues, D129 and F130, were more freely substitutable. By analogy with the trypsin model, it was proposed that D129 is located at the bottom of the substrate binding pocket so as to directly interact with the basic amino acid at the substrate cleavage site. Interestingly, we found that significant cleavage activity was displayed by mutants in which D129 was replaced by E, S, or A and that low but detectable protease activity was exhibited by mutants in which D129 was replaced by K, R, or L. Contrary to the proposed model, these results indicate that D129 is not a major determinant of substrate binding and that its interaction with the substrate, if it occurs at all, is not essential. This mutagenesis

  5. RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP

    PubMed Central

    Dumont, Sophie; Cheng, Wei; Serebrov, Victor; Beran, Rudolf K.; Tinoco, Ignacio; Pyle, Anna Marie; Bustamante, Carlos

    2006-01-01

    Helicases are a ubiquitous class of enzymes involved in nearly all aspects of DNA and RNA metabolism. Despite recent progress in understanding their mechanism of action, limited resolution has left inaccessible the detailed mechanisms by which these enzymes couple the rearrangement of nucleic acid structures to the binding and hydrolysis of ATP1,2. Observing individual mechanistic cycles of these motor proteins is central to understanding their cellular functions. Here we follow in real time, at a resolution of two base pairs and 20 ms, the RNA translocation and unwinding cycles of a hepatitis C virus helicase (NS3) monomer. NS3 is a representative superfamily-2 helicase essential for viral replication3, and therefore a potentially important drug target4. We show that the cyclic movement of NS3 is coordinated by ATP in discrete steps of 11 ± 3 base pairs, and that actual unwinding occurs in rapid smaller substeps of 3.6 ± 1.3 base pairs, also triggered by ATP binding, indicating that NS3 might move like an inchworm5,6. This ATP-coupling mechanism is likely to be applicable to other non-hexameric helicases involved in many essential cellular functions. The assay developed here should be useful in investigating a broad range of nucleic acid translocation motors. PMID:16397502

  6. Portulaca oleracea L. as a Prospective Candidate Inhibitor of Hepatitis C Virus NS3 Serine Protease.

    PubMed

    Noreen, Sobia; Hussain, Ishtiaq; Tariq, Muhammad Ilyas; Ijaz, Bushra; Iqbal, Shahid; Qamar-ul-Zaman; Ashfaq, Usman Ali; Husnain, Tayyab

    2015-06-01

    Hepatitis C virus (HCV) infection is a worldwide health problem affecting about 300 million individuals. HCV causes chronic liver disease, liver cirrhosis, hepatocellular carcinoma, and death. Many side effects are associated with the current treatment options. Natural products that can be used as anti-HCV drugs are thus of considerable potential significance. NS3 serine protease (NS3-SP) is a target for the screening of antiviral activity against HCV. The present work explores plants with anti-HCV potential, isolating possible lead compounds. Ten plants, used for medicinal purposes against different infections in rural areas of Pakistan, were collected. The cellular toxicity effects of methanolic extracts of the plants on the viability of Huh-7 cells were studied through the Trypan blue dye exclusion method. Following this, the anti-HCV potential of phytoextracts was assessed by infecting liver cells with HCV-3a-infected serum inoculum. Only the methanolic extract of Portulaca oleracea L. (PO) exhibited more than 70% inhibition. Four fractions were obtained through bioassay-guided extraction of PO. Subsequent inhibition of all organic extract fractions against NS3 serine protease was checked to track the specific target in the virus. The results showed that the PO methanolic crude and ethyl acetate extract specifically abridged the HCV NS3 protease expression in a dose-dependent fashion. Hence, PO extract and its constituents either alone or with interferon could offer a future option to treat chronic HCV. PMID:25871297

  7. Double-stranded DNA-induced localized unfolding of HCV NS3 helicase subdomain 2.

    PubMed

    Liu, Dingjiang; Windsor, William T; Wyss, Daniel F

    2003-12-01

    The NS3 helicase of the hepatitis C virus (HCV) unwinds double-stranded (ds) nucleic acid (NA) in an NTP-dependent fashion. Mechanistic details of this process are, however, largely unknown for the HCV helicase. We have studied the binding of dsDNA to an engineered version of subdomain 2 of the HCV helicase (d(2Delta)NS3h) by NMR and circular dichroism. Binding of dsDNA to d(2Delta)NS3h induces a local unfolding of helix (alpha(3)), which includes residues of conserved helicase motif VI (Q(460)RxxRxxR(467)), and strands (beta(1) and beta(8)) from the central beta-sheet. This also occurs upon lowering the pH (4.4) and introducing an R461A point mutation, which disrupt salt bridges with Asp 412 and Asp 427 in the protein structure. NMR studies on d(2Delta)NS3h in the partially unfolded state at low pH map the dsDNA binding site to residues previously shown to be involved in single-stranded DNA binding. Sequence alignment and structural comparison suggest that these Arg-Asp interactions are highly conserved in SF2 DEx(D/H) proteins. Thus, modulation of these interactions by dsNA may allow SF2 helicases to switch between conformations required for helicase function. PMID:14627736

  8. Small Molecule Pan-dengue and West Nile Virus NS3 Protease Inhibitors

    PubMed Central

    Cregar-Hernandez, Lynne; Jiao, Guan-Sheng; Johnson, Alan T.; Lehrer, Axel T.; Wong, Teri Ann S.; Margosiak, Stephen A.

    2011-01-01

    Background Dengue fever, dengue hemorrhagic fever, and dengue shock syndrome are caused by infections with any of the four serotypes of the dengue virus (DENV) and are an increasing global health risk. The related West Nile Virus (WNV) causes significant morbidity and mortality as well and continues to be a threat in endemic areas. Currently no FDA approved vaccines or therapeutics are available to prevent or treat any of these infections. Like the other members of the Flaviviridae family, DENV and WNV encode a protease (NS3) which is essential for viral replication and therefore is a promising target for developing therapies to treat dengue and West Nile infections. Methods Flaviviral protease inhibitors were identified and biologically characterized for mechanism of inhibition and DENV anti-viral activity. Results A guanidinylated 2, 5-dideoxystreptamine class of compounds was identified that competitively inhibited the NS3 protease from DENV(1-4) and WNV with IC50 values in the 1-70 μM range. Cytotoxicity was low; however, antiviral activity versus DENV-2 on VERO cells was not detectable. Conclusions This class of compounds is the first to demonstrate competitive pan-dengue and WNV NS3 protease inhibition and, given the sequence conservation among flavivirus NS3 proteins, suggests that developing a pan-dengue or possibly pan-flavivirus therapeutic is feasible. PMID:21566267

  9. Recombinant Dengue 2 Virus NS3 Helicase Protein Enhances Antibody and T-Cell Response of Purified Inactivated Vaccine

    PubMed Central

    Simmons, Monika; Sun, Peifang; Putnak, Robert

    2016-01-01

    Dengue virus purified inactivated vaccines (PIV) are highly immunogenic and protective over the short term, but may be poor at inducing cell-mediated immune responses and long-term protection. The dengue nonstructural protein 3 (NS3) is considered the main target for T-cell responses during viral infection. The amino (N)-terminal protease and the carboxy (C)-terminal helicase domains of DENV-2 NS3 were expressed in E. coli and analyzed for their immune-potentiating capacity. Mice were immunized with DENV-2 PIV with and without recombinant NS3 protease or NS3 helicase proteins, and NS3 proteins alone on days 0, 14 and 28. The NS3 helicase but not the NS3 protease was effective in inducing T-cell responses quantified by IFN-γ ELISPOT. In addition, markedly increased total IgG antibody titer against virus antigen was seen in mice immunized with the PIV/NS3 helicase combination in the ELISA, as well as increased neutralizing antibody titer measured by the plaque reduction neutralization test. These results indicate the potential immunogenic properties of the NS3 helicase protein and its use in a dengue vaccine formulation. PMID:27035715

  10. Policresulen, a novel NS2B/NS3 protease inhibitor, effectively inhibits the replication of DENV2 virus in BHK-21 cells

    PubMed Central

    Wu, Deng-wei; Mao, Fei; Ye, Yan; Li, Jian; Xu, Chuan-lian; Luo, Xiao-min; Chen, Jing; Shen, Xu

    2015-01-01

    Aim: Dengue is a severe epidemic disease caused by dengue virus (DENV) infection, for which no effective treatment is available. The protease complex, consisting of nonstructural protein 3 (NS3) and its cofactor NS2B, plays a pivotal role in the replication of DENV, thus may be a potential target for anti-DENV drugs. Here, we report a novel inhibitor of DENV2 NS2B/NS3 protease and its antiviral action. Methods: An enzymatic inhibition assay was used for screening DENV2 NS2B/NS3 inhibitors. Cytotoxicity to BHK-21 cells was assessed with MTT assay. Antiviral activity was evaluated in BHK-21 cells transfected with Rlu-DENV-Rep. The molecular mechanisms of the antiviral action was analyzed using surface plasmon resonance, ultraviolet-visible spectral analysis and differential scanning calorimetry assays, as well as molecular docking analysis combined with site-directed mutagenesis. Results: In our in-house library of old drugs (∼1000 compounds), a topical hemostatic and antiseptic 2-hydroxy-3,5-bis[(4-hydroxy-2-methyl-5-sulfophenyl)methyl]-4-methyl-benzene-sulfonic acid (policresulen) was found to be a potent inhibitor of DENV2 NS2B/NS3 protease with IC50 of 0.48 μg/mL. Furthermore, policresulen inhibited DENV2 replication in BHK-21 cells with IC50 of 4.99 μg/mL, whereas its IC50 for cytotoxicity to BHK-21 cells was 459.45 μg/mL. Policresulen acted as a competitive inhibitor of the protease, and slightly affected the protease stability. Using biophysical technology-based assays and molecular docking analysis combined with site-directed mutagenesis, we demonstrated that the residues Gln106 and Arg133 of DENV2 NS2B/NS3 protease directly interacted with policresulen via hydrogen bonding. Conclusion: Policresulen is a potent inhibitor of DENV2 NS2B/NS3 protease that inhibits DENV2 replication in BHK-21 cells. The binding mode of the protease and policresulen provides useful hints for designing new type of inhibitors against the protease. PMID:26279156

  11. Construction and Immunogenicity Analysis of Hepatitis C Virus (HCV) Truncated Non-Structural Protein 3 (NS3) Plasmid Vaccine

    PubMed Central

    Pouriayevali, Mohammad-Hassan; Bamdad, Taravat; Aghasadeghi, Mohammad-Reza; Sadat, Seyed Mehdi; Sabahi, Farzaneh

    2016-01-01

    Background To develop hepatitis C virus (HCV) vaccine, induction of potent humoral and T cell response against immunogenic targets with conserved region should be achieved. T cell response against NS3 is often associated with complete clearance of the virus. Objectives Herein, we expressed the truncated form of NS3 in a mammalian cell line and evaluated immune responses of NS3 DNA vaccine in BALB/c. Materials and Methods The partial length of NS3 gene, which encodes immunogenic epitopes (1095 - 1379 aa), was amplified by reverse transcription-polymerase chain reaction (RT-PCR) on RNA obtained from a patient with HCV, inserted into pcDNA3.1 plasmid using XhoI/HindIII sites, and finally evaluated by restriction analysis and sequencing. After transfection of the recombinant plasmid into HEK293T cells, the NS3 protein expression was confirmed by western blotting. Mice were immunized intra-dermally close to the base of the mice tail with four doses in two-weeks intervals and the immune responses were assessed using total and subtypes of IgG antibody assay, cell proliferation and cytokine assay. Results The pcDNA3.1 plasmid harboring the coding sequence of NS3 (pc-NS3) was constructed and confirmed with the expected size. Proper expression of the recombinant protein in transfected HEK 293T cells was confirmed using western blotting. The immunization results indicated that pc-NS3 induced significant levels of total antibody, IgG2a subclass antibody, Interferon (IFN)-γ, Interleukin (IL)-4 and proliferation assay compared to the control group (P < 0.05). Conclusions The pc-NS3 possesses the capacity to express NS3 in the mammalian cell line and demonstrated strong immunogenicity in a murine model. Our primary results demonstrated that the immunogenic truncated region of NS3 could be used as a potential vaccine candidate against hepatitis C. PMID:27226878

  12. Evaluating the Role of Cellular Immune Responses in the Emergence of HCV NS3 Resistance Mutations During Protease Inhibitor Therapy.

    PubMed

    Abdel-Hameed, Enass A; Rouster, Susan D; Ji, Hong; Ulm, Ashley; Hetta, Helal F; Anwar, Nadeem; Sherman, Kenneth E; Shata, Mohamed Tarek M

    2016-05-01

    The efficacy of protease inhibitor drugs in hepatitis C virus (HCV) treatment is limited by the selection and expansion of drug-resistant mutations. HCV replication is error-prone and genetic variability within the dominant epitopes ensures its persistence. The aims of this study are to evaluate the role of cellular immune response in the emergence of HCV protease resistance mutations and its effects on treatment outcome. Ten chronically HCV-infected subjects were treated with boceprevir (BOC)-based triple therapy. HCV-RNA was tested for BOC resistance-associated viral variants. HCV protease resistance mutations were investigated pretreatment and 24 weeks post-treatment. Synthetic peptides representing the wild-type and the potential nonstructural (NS)3 variants were used to evaluate T cell responses and human leukocyte antigen binding. Sustained viral response was achieved in 70% of patients, two patients were treatment nonresponders (NRs) and one was classified as a relapse. Pretreatment, the proportion of drug-resistant variants within individuals was higher in sustained viral responders (SVRs) than in NR patients. However, resistance-associated variants increased in NRs after BOC combined triple therapy. In contrast to NR patients, significant stronger cell-mediated immune responses were observed at the baseline among those who achieved sustained viral response for all T cell epitopes tested. Despite the increase in cell-mediated immune responses at week 24 in NRs, they failed to control the virus replication, leading to development of overt drug-resistant variants. Our data suggest that strong NS3-specific T cell immune responses at the baseline may predict a positive outcome of directly acting antiviral-based therapy, and the presence of pre-existent resistance mutations does not play a significant role in the outcome of anti-HCV combined therapy. PMID:26885675

  13. Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays

    PubMed Central

    Mukherjee, Sourav; Hanson, Alicia M.; Shadrick, William R.; Ndjomou, Jean; Sweeney, Noreena L.; Hernandez, John J.; Bartczak, Diana; Li, Kelin; Frankowski, Kevin J.; Heck, Julie A.; Arnold, Leggy A.; Schoenen, Frank J.; Frick, David N.

    2012-01-01

    Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma’s Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC50 = 1.4 μM), suramin sodium salt (IC50 = 3.6 μM), NF 023 hydrate (IC50 = 6.2 μM) and tyrphostin AG 538 (IC50 = 3.6 μM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors. PMID:22740655

  14. Turnover Rate of NS3 Proteins Modulates Bluetongue Virus Replication Kinetics in a Host-Specific Manner

    PubMed Central

    Ftaich, Najate; Ciancia, Claire; Viarouge, Cyril; Barry, Gerald; Ratinier, Maxime; van Rijn, Piet A.; Breard, Emmanuel; Vitour, Damien; Zientara, Stephan; Palmarini, Massimo; Terzian, Christophe

    2015-01-01

    ABSTRACT Bluetongue virus (BTV) is an arbovirus transmitted to livestock by midges of the Culicoides family and is the etiological agent of a hemorrhagic disease in sheep and other ruminants. In mammalian cells, BTV particles are released primarily by virus-induced cell lysis, while in insect cells they bud from the plasma membrane and establish a persistent infection. BTV possesses a ten-segmented double-stranded RNA genome, and NS3 proteins are encoded by segment 10 (Seg-10). The viral nonstructural protein 3 (NS3) plays a key role in mediating BTV egress as well as in impeding the in vitro synthesis of type I interferon in mammalian cells. In this study, we asked whether genetically distant NS3 proteins can alter BTV-host interactions. Using a reverse genetics approach, we showed that, depending on the NS3 considered, BTV replication kinetics varied in mammals but not in insects. In particular, one of the NS3 proteins analyzed harbored a proline at position 24 that leads to its rapid intracellular decay in ovine but not in Culicoides cells and to the attenuation of BTV virulence in a mouse model of disease. Overall, our data reveal that the genetic variability of Seg-10/NS3 differentially modulates BTV replication kinetics in a host-specific manner and highlight the role of the host-specific variation in NS3 protein turnover rate. IMPORTANCE BTV is the causative agent of a severe disease transmitted between ruminants by biting midges of Culicoides species. NS3, encoded by Seg-10 of the BTV genome, fulfills key roles in BTV infection. As Seg-10 sequences from various BTV strains display genetic variability, we assessed the impact of different Seg-10 and NS3 proteins on BTV infection and host interactions. In this study, we revealed that various Seg-10/NS3 proteins alter BTV replication kinetics in mammals but not in insects. Notably, we found that NS3 protein turnover may vary in ovine but not in Culicoides cells due to a single amino acid residue that, most

  15. Locking GTPases covalently in their functional states

    PubMed Central

    Wiegandt, David; Vieweg, Sophie; Hofmann, Frank; Koch, Daniel; Li, Fu; Wu, Yao-Wen; Itzen, Aymelt; Müller, Matthias P.; Goody, Roger S.

    2015-01-01

    GTPases act as key regulators of many cellular processes by switching between active (GTP-bound) and inactive (GDP-bound) states. In many cases, understanding their mode of action has been aided by artificially stabilizing one of these states either by designing mutant proteins or by complexation with non-hydrolysable GTP analogues. Because of inherent disadvantages in these approaches, we have developed acryl-bearing GTP and GDP derivatives that can be covalently linked with strategically placed cysteines within the GTPase of interest. Binding studies with GTPase-interacting proteins and X-ray crystallography analysis demonstrate that the molecular properties of the covalent GTPase–acryl–nucleotide adducts are a faithful reflection of those of the corresponding native states and are advantageously permanently locked in a defined nucleotide (that is active or inactive) state. In a first application, in vivo experiments using covalently locked Rab5 variants provide new insights into the mechanism of correct intracellular localization of Rab proteins. PMID:26178622

  16. Purification and crystallization of dengue and West Nile virus NS2B–NS3 complexes

    SciTech Connect

    D’Arcy, Allan Chaillet, Maxime; Schiering, Nikolaus; Villard, Frederic; Lim, Siew Pheng; Lefeuvre, Peggy; Erbel, Paul

    2006-02-01

    Crystals of dengue serotype 2 and West Nile virus NS2B–NS3 protease complexes have been obtained and the crystals of both diffract to useful resolution. Sample homogeneity was essential for obtaining X-ray-quality crystals of the dengue protease. Controlled proteolysis produced a crystallizable fragment of the apo West Nile virus NS2B–NS3 and crystals were also obtained in the presence of a peptidic inhibitor. Both dengue and West Nile virus infections are an increasing risk to humans, not only in tropical and subtropical areas, but also in North America and parts of Europe. These viral infections are generally transmitted by mosquitoes, but may also be tick-borne. Infection usually results in mild flu-like symptoms, but can also cause encephalitis and fatalities. Approximately 2799 severe West Nile virus cases were reported this year in the United States, resulting in 102 fatalities. With this alarming increase in the number of West Nile virus infections in western countries and the fact that dengue virus already affects millions of people per year in tropical and subtropical climates, there is a real need for effective medicines. A possible therapeutic target to combat these viruses is the protease, which is essential for virus replication. In order to provide structural information to help to guide a lead identification and optimization program, crystallizations of the NS2B–NS3 protease complexes from both dengue and West Nile viruses have been initiated. Crystals that diffract to high resolution, suitable for three-dimensional structure determinations, have been obtained.

  17. Monomeric nature of dengue virus NS3 helicase and thermodynamic analysis of the interaction with single-stranded RNA

    PubMed Central

    Gebhard, Leopoldo G.; Incicco, J. Jeremías; Smal, Clara; Gallo, Mariana; Gamarnik, Andrea V.; Kaufman, Sergio B.

    2014-01-01

    Dengue virus nonstructural protein 3 (NS3) is a multifunctional protein formed by a superfamily-2 RNA helicase linked to a protease domain. In this work, we report results from in vitro experiments designed to determine the oligomeric state of dengue virus NS3 helicase (NS3h) and to characterize fundamental properties of the interaction with single-stranded (ss)RNA. Pulsed field gradient-NMR spectroscopy was used to determine the effective hydrodynamic radius of NS3h, which was constant over a wide range of protein concentrations in the absence and presence of ssRNA. Size exclusion chromatography-static light scattering experiments showed that NS3h eluted as a monomeric molecule even in the presence of ssRNA. Binding of NS3h to ssRNA was studied by quantitative fluorescence titrations using fluorescein-labeled and unlabeled ssRNA oligonucleotides of different lengths, and the effect of the fluorescein label on the interaction parameters was also analyzed. Experimental results were well described by a statistical thermodynamic model based on the theory of non-specific interactions of large ligands to a one-dimensional lattice. We found that binding of NS3h to ssRNA oligonucleotides and to poly(A) is characterized by minimum and occluded binding site sizes both of 10 nucleotides and by a weak positive cooperativity between adjacent proteins. PMID:25223789

  18. Novel Benzoxazole Inhibitor of Dengue Virus Replication That Targets the NS3 Helicase

    PubMed Central

    Grosenbach, Douglas W.; Berhanu, Aklile; Dai, Dongcheng; Jones, Kevin F.; Cardwell, Kara B.; Schneider, Christine; Yang, Guang; Tyavanagimatt, Shanthakumar; Harver, Chris; Wineinger, Kristin A.; Page, Jessica; Stavale, Eric; Stone, Melialani A.; Fuller, Kathleen P.; Lovejoy, Candace; Leeds, Janet M.; Hruby, Dennis E.; Jordan, Robert

    2013-01-01

    Dengue virus (DENV) is the predominant mosquito-borne viral pathogen that infects humans with an estimated 50 to 100 million infections per year worldwide. Over the past 50 years, the incidence of dengue disease has increased dramatically and the virus is now endemic in more than 100 countries. Moreover, multiple serotypes of DENV are now found in the same geographic region, increasing the likelihood of more severe forms of disease. Despite extensive research, there are still no approved vaccines or therapeutics commercially available to treat DENV infection. Here we report the results of a high-throughput screen of a chemical compound library using a whole-virus assay that identified a novel small-molecule inhibitor of DENV, ST-610, that potently and selectively inhibits all four serotypes of DENV replication in vitro. Sequence analysis of drug-resistant virus isolates has identified a single point mutation, A263T, in the NS3 helicase domain that confers resistance to this compound. ST-610 inhibits DENV NS3 helicase RNA unwinding activity in a molecular-beacon-based helicase assay but does not inhibit nucleoside triphosphatase activity based on a malachite green ATPase assay. ST-610 is nonmutagenic, is well tolerated (nontoxic) in mice, and has shown efficacy in a sublethal murine model of DENV infection with the ability to significantly reduce viremia and viral load compared to vehicle controls. PMID:23403421

  19. Long-term functional duration of immune responses to HCV NS3/4A induced by DNA vaccination

    PubMed Central

    Ahlén, G; Holmström, F; Gibbs, A; Alheim, M; Frelin, L

    2014-01-01

    We have investigated the ability of hepatitis C virus non-structural (NS) 3/4A-DNA-based vaccines to activate long-term cell-mediated immune responses in mice. Wild-type and synthetic codon optimized (co) NS3/4A DNA vaccines have previously been shown to be immunogenic in mice, rabbits and humans, although we have very poor knowledge about the longevity of the immune responses primed. We therefore analyzed the functionality of primed NS3/4A-specific immune responses in BALB/c (H-2d) and/or C57BL/6J (H-2b) mice 1, 2, 3, 4, 6, 12 and 16 months after the last immunization. Mice were immunized one, two, three or four times using gene gun delivery to the skin or by intramuscular administration. Immunological responses after immunization were monitored by protection against in vivo challenge of NS3/4A-expressing syngeneic tumor cells. In addition, functionality of the NS3/4A-specific T cells was analyzed by a standard cytotoxicity assay. First, we identified a new unique murine H-2d-restricted NS3/4A cytotoxic T lymphocyte (CTL) epitope, which enabled us to study the epitope-specific immune responses. Our results show that the coNS3/4A vaccine was highly immunogenic by determination of interferon-γ/tumor necrosis factor-α production and lytic cytotoxic T cells, which could efficiently inhibit in vivo tumor growth. Importantly, we showed that one to four monthly immunizations protected mice from tumor development when challenged up to 16 months after the last immunization. When determining the functionality of NS3/4A-specific T cells in vitro, we showed detectable lytic activity up to 12 months after the last immunization. Thus, NS3/4A-based DNA vaccines activate potent cellular immune responses that are present and function in both BALB/c and C57BL/6J mice up to 12–16 months after the last immunization. The induction of long-term immunity after NS3/4A DNA immunization has not been shown previously and supports the use of NS3/4A in hepatitis C virus

  20. Long-term functional duration of immune responses to HCV NS3/4A induced by DNA vaccination.

    PubMed

    Ahlén, G; Holmström, F; Gibbs, A; Alheim, M; Frelin, L

    2014-08-01

    We have investigated the ability of hepatitis C virus non-structural (NS) 3/4A-DNA-based vaccines to activate long-term cell-mediated immune responses in mice. Wild-type and synthetic codon optimized (co) NS3/4A DNA vaccines have previously been shown to be immunogenic in mice, rabbits and humans, although we have very poor knowledge about the longevity of the immune responses primed. We therefore analyzed the functionality of primed NS3/4A-specific immune responses in BALB/c (H-2(d)) and/or C57BL/6J (H-2(b)) mice 1, 2, 3, 4, 6, 12 and 16 months after the last immunization. Mice were immunized one, two, three or four times using gene gun delivery to the skin or by intramuscular administration. Immunological responses after immunization were monitored by protection against in vivo challenge of NS3/4A-expressing syngeneic tumor cells. In addition, functionality of the NS3/4A-specific T cells was analyzed by a standard cytotoxicity assay. First, we identified a new unique murine H-2(d)-restricted NS3/4A cytotoxic T lymphocyte (CTL) epitope, which enabled us to study the epitope-specific immune responses. Our results show that the coNS3/4A vaccine was highly immunogenic by determination of interferon-γ/tumor necrosis factor-α production and lytic cytotoxic T cells, which could efficiently inhibit in vivo tumor growth. Importantly, we showed that one to four monthly immunizations protected mice from tumor development when challenged up to 16 months after the last immunization. When determining the functionality of NS3/4A-specific T cells in vitro, we showed detectable lytic activity up to 12 months after the last immunization. Thus, NS3/4A-based DNA vaccines activate potent cellular immune responses that are present and function in both BALB/c and C57BL/6J mice up to 12-16 months after the last immunization. The induction of long-term immunity after NS3/4A DNA immunization has not been shown previously and supports the use of NS3/4A in hepatitis C virus vaccine

  1. New Details of HCV NS3/4A Proteinase Functionality Revealed by a High-Throughput Cleavage Assay

    PubMed Central

    Cieplak, Piotr; Chudin, Eugene; Cheltsov, Anton V.; Chee, Mark S.; Kozlov, Igor A.; Strongin, Alex Y.

    2012-01-01

    Background The hepatitis C virus (HCV) genome encodes a long polyprotein, which is processed by host cell and viral proteases to the individual structural and non-structural (NS) proteins. HCV NS3/4A serine proteinase (NS3/4A) is a non-covalent heterodimer of the N-terminal, ∼180-residue portion of the 631-residue NS3 protein with the NS4A co-factor. NS3/4A cleaves the polyprotein sequence at four specific regions. NS3/4A is essential for viral replication and has been considered an attractive drug target. Methodology/Principal Findings Using a novel multiplex cleavage assay and over 2,660 peptide sequences derived from the polyprotein and from introducing mutations into the known NS3/4A cleavage sites, we obtained the first detailed fingerprint of NS3/4A cleavage preferences. Our data identified structural requirements illuminating the importance of both the short-range (P1–P1′) and long-range (P6-P5) interactions in defining the NS3/4A substrate cleavage specificity. A newly observed feature of NS3/4A was a high frequency of either Asp or Glu at both P5 and P6 positions in a subset of the most efficient NS3/4A substrates. In turn, aberrations of this negatively charged sequence such as an insertion of a positively charged or hydrophobic residue between the negatively charged residues resulted in inefficient substrates. Because NS5B misincorporates bases at a high rate, HCV constantly mutates as it replicates. Our analysis revealed that mutations do not interfere with polyprotein processing in over 5,000 HCV isolates indicating a pivotal role of NS3/4A proteolysis in the virus life cycle. Conclusions/Significance Our multiplex assay technology in light of the growing appreciation of the role of proteolytic processes in human health and disease will likely have widespread applications in the proteolysis research field and provide new therapeutic opportunities. PMID:22558217

  2. A coiled-coil motif in non-structural protein 3 (NS3) of bluetongue virus forms an oligomer.

    PubMed

    Chacko, Nirmal; Mohanty, Nihar Nalini; Biswas, Sanchay Kumar; Chand, Karam; Yogisharadhya, Revanaiah; Pandey, Awadh Bihari; Mondal, Bimalendu; Shivachandra, Sathish Bhadravati

    2015-10-01

    Bluetongue, an arthropod-borne non-contagious hemorrhagic disease of small ruminants, is caused by bluetongue virus (BTV). Several structural and non-structural proteins encoded by BTV have been associated with virulence mechanisms. In the present study, the NS3 protein sequences of bluetongue viral serotypes were analyzed for the presence of heptad regions and oligomer formation. Bioinformatic analysis of NS3 sequences of all 26 BTV serotypes revealed the presence of at least three coiled-coil motifs (CCMs). A conserved α-helical heptad sequence was identified at 14-26 aa (CCM-I), 185-198aa (CCM-II), and 94-116 aa (CCM-III). Among these, CCM-I occurs close to the N-terminus of NS3 and was presumed to be involved in oligomerization. Furthermore, the N-terminus of NS3 (1M-R117 aa) was over-expressed as a recombinant fusion protein in a prokaryotic expression system. Biochemical characterization of recombinant NS3Nt protein revealed that it forms SDS-resistant dimers and high-order oligomers (hexamer and/or octamer) under reducing or non-reducing conditions. Coiled-coil motifs are believed to be critical for NS protein oligomerization and have potential roles in the formation of viroporin ring/pore either with six/eight subunits and this is the first study toward characterization of CCMs in NS3 of bluetongue virus. PMID:26318174

  3. AmeriFlux CA-NS3 UCI-1964 burn site

    SciTech Connect

    Goulden, Mike

    2016-01-01

    This is the AmeriFlux version of the carbon flux data for the site CA-NS3 UCI-1964 burn site. Site Description - The UCI-1964 site is located in a continental boreal forest, dominated by black spruce trees, within the BOREAS northern study area in central Manitoba, Canada. The site is a member of a chronological series of sites that are representative secondary succession growth stages after large stand replacement fires. Black spruce trees undergo a slow growth process enabling the accurate determination of the chronosequence of stand age disturbance. Additionally, boreal forests make up approximately 25% of forest ecosystems on earth. With both of these in mind, the UCI sites provide an excellent location to study the CO2 exchange between the atmosphere and boreal forest ecosystems as a function of sequential wildfires.

  4. Potent ketoamide inhibitors of HCV NS3 protease derived from quaternized P1 groups.

    PubMed

    Venkatraman, Srikanth; Velazquez, Francisco; Wu, Wanli; Blackman, Melissa; Madison, Vincent; Njoroge, F George

    2010-04-01

    Blood borne hepatitis C infections are the primary cause for liver cirrhosis and hepatocellular carcinoma. HCV NS3 protease, a pivotal enzyme in the replication cycle of HCV virus has been the primary target for development of new drug candidates. Boceprevir and telaprevir are two novel ketoamide derived inhibitors that are currently undergoing phase-III clinical trials. These inhibitors include ketoamide functionality as serine trap and have an acidic alpha-ketoamide center that undergoes epimerization under physiological conditions. Our initial attempts to arrest this epimerization by introducing quaternary amino acids at P(1) had resulted in significantly diminished activity. In this manuscript we describe alpha quaternized P(1) group that result in potent inhibitors in the enzyme assay and demonstrate cellular activity comparable to boceprevir. PMID:20226659

  5. HCV-NS3 inhibitors: determination of their kinetic parameters and mechanism.

    PubMed

    Flores, María Victoria; Strawbridge, Joanne; Ciaramella, Giuseppe; Corbau, Romuald

    2009-10-01

    Existing HCV protease inhibitors fall into two categories: reversible and non-covalent, such as BILN-2061, and covalent and reversible, exemplified by SCH-503034 and VX-950. In this work, the characterization of the kinetics of these three inhibitors is presented. SCH-503034 and VX-950 initially bind to the genotype 1b HCV NS3/4A protease to form a low affinity complex, with K(i) values of 5 and 5.8 microM respectively. The ability of those two compounds to form a second covalent complex (EI) results in a potency increase, with overall K(i) values of 20 and 45 nM, respectively. The increase in potency can be explained by their slow dissociation rate, forming complexes with half-lives of 2 h (VX-950) and 5 h (SCH-503034). Although BILN-2061 has been described as a fast reversible, non-covalent inhibitor, our results show a slow binding two-step mechanism. Contrary to SCH-503034 and VX-950, BILN-2061 can form a high affinity first complex with a K(i) value of 3.9 nM, and an overall K(i) of 0.14 nM. The half-life of the BILN-2061 EI complex is shorter (t(1/2) approximately 0.7 h) than that of the other two compounds. The potency of these compounds is genotype dependent, and a kinetic analysis using NS3/4A from genotype 3a indicates that the loss of potency of SCH-503034 and VX-950 relative to genotype 1 is mainly due to the slow on-rate and faster off-rate for the formation of the EI complex. In the case of BILN-2061, a better fit is obtained using a one-step model, indicating that the loss of potency is due to an increase in the off-rate of the EI complex. PMID:19505593

  6. NMR Analysis of a Novel Enzymatically Active Unlinked Dengue NS2B-NS3 Protease Complex*

    PubMed Central

    Kim, Young Mee; Gayen, Shovanlal; Kang, CongBao; Joy, Joma; Huang, Qiwei; Chen, Angela Shuyi; Wee, John Liang Kuan; Ang, Melgious Jin Yan; Lim, Huichang Annie; Hung, Alvin W.; Li, Rong; Noble, Christian G.; Lee, Le Tian; Yip, Andy; Wang, Qing-Yin; Chia, Cheng San Brian; Hill, Jeffrey; Shi, Pei-Yong; Keller, Thomas H.

    2013-01-01

    The dengue virus (DENV) is a mosquito-borne pathogen responsible for an estimated 100 million human infections annually. The viral genome encodes a two-component trypsin-like protease that contains the cofactor region from the nonstructural protein NS2B and the protease domain from NS3 (NS3pro). The NS2B-NS3pro complex plays a crucial role in viral maturation and has been identified as a potential drug target. Using a DENV protease construct containing NS2B covalently linked to NS3pro via a Gly4-Ser-Gly4 linker (“linked protease”), previous x-ray crystal structures show that the C-terminal fragment of NS2B is remote from NS3pro and exists in an open state in the absence of an inhibitor; however, in the presence of an inhibitor, NS2B complexes with NS3pro to form a closed state. This linked enzyme produced NMR spectra with severe signal overlap and line broadening. To obtain a protease construct with a resolved NMR spectrum, we expressed and purified an unlinked protease complex containing a 50-residue segment of the NS2B cofactor region and NS3pro without the glycine linker using a coexpression system. This unlinked protease complex was catalytically active at neutral pH in the absence of glycerol and produced dispersed cross-peaks in a 1H-15N heteronuclear single quantum correlation spectrum that enabled us to conduct backbone assignments using conventional techniques. In addition, titration with an active-site peptide aldehyde inhibitor and paramagnetic relaxation enhancement studies demonstrated that the unlinked DENV protease exists predominantly in a closed conformation in solution. This protease complex can serve as a useful tool for drug discovery against DENV. PMID:23511634

  7. HCV NS3 protease enhances liver fibrosis via binding to and activating TGF-β type I receptor

    NASA Astrophysics Data System (ADS)

    Sakata, Kotaro; Hara, Mitsuko; Terada, Takaho; Watanabe, Noriyuki; Takaya, Daisuke; Yaguchi, So-Ichi; Matsumoto, Takehisa; Matsuura, Tomokazu; Shirouzu, Mikako; Yokoyama, Shigeyuki; Yamaguchi, Tokio; Miyazawa, Keiji; Aizaki, Hideki; Suzuki, Tetsuro; Wakita, Takaji; Imoto, Masaya; Kojima, Soichi

    2013-11-01

    Viruses sometimes mimic host proteins and hijack the host cell machinery. Hepatitis C virus (HCV) causes liver fibrosis, a process largely mediated by the overexpression of transforming growth factor (TGF)-β and collagen, although the precise underlying mechanism is unknown. Here, we report that HCV non-structural protein 3 (NS3) protease affects the antigenicity and bioactivity of TGF-β2 in (CAGA)9-Luc CCL64 cells and in human hepatic cell lines via binding to TGF-β type I receptor (TβRI). Tumor necrosis factor (TNF)-α facilitates this mechanism by increasing the colocalization of TβRI with NS3 protease on the surface of HCV-infected cells. An anti-NS3 antibody against computationally predicted binding sites for TβRI blocked the TGF-β mimetic activities of NS3 in vitro and attenuated liver fibrosis in HCV-infected chimeric mice. These data suggest that HCV NS3 protease mimics TGF-β2 and functions, at least in part, via directly binding to and activating TβRI, thereby enhancing liver fibrosis.

  8. High Throughput Flow Cytometry Bead-based Multiplex Assay for Identification of Rho GTPase Inhibitors

    PubMed Central

    Surviladze, Zurab; Young, Susan M; Sklar, Larry A

    2015-01-01

    Summary Rho family GTPases and their effector proteins regulate a wide range of cell signaling pathways. In normal physiological conditions their activity is tightly controlled and it is not surprising that their aberrant activation contributes to tumorigenesis or other diseases. For this reason, the identification of small, cell permeable molecules capable of inhibition of Rho GTPases can be extraordinarily useful, particularly if they are specific and act reversibly. Herein we describe a flow cytometric assay, which allows us to measure the activity of six small GTPases simultaneously. GST-tagged small GTPases are bound to six glutathione bead sets each set having a different intensity of red fluorescence at a fixed wavelength. The coated bead sets were washed, combined, and dispensed into 384-well plates with test compounds, and fluorescent-GTP binding was used as the read-out. This multiplex bead-based assay was successfully used for to identify both general and selective inhibitors of Rho family GTPases. PMID:22144280

  9. Structure-Based Mutagenesis Study of Hepatitis C Virus NS3 Helicase

    PubMed Central

    Lin, Chao; Kim, Joseph L.

    1999-01-01

    The NS3 protein of hepatitis C virus (HCV) is a bifunctional protein containing a serine protease in the N-terminal one-third, which is stimulated upon binding of the NS4A cofactor, and an RNA helicase in the C-terminal two-thirds. In this study, a C-terminal hexahistidine-tagged helicase domain of the HCV NS3 protein was expressed in Escherichia coli and purified to homogeneity by conventional chromatography. The purified HCV helicase domain has a basal ATPase activity, a polynucleotide-stimulated ATPase activity, and a nucleic acid unwinding activity and binds efficiently to single-stranded polynucleotide. Detailed characterization of the purified HCV helicase domain with regard to all four activities is presented. Recently, we published an X-ray crystallographic structure of a binary complex of the HCV helicase with a (dU)8 oligonucleotide, in which several conserved residues of the HCV helicase were shown to be involved in interactions between the HCV helicase and oligonucleotide. Here, site-directed mutagenesis was used to elucidate the roles of these residues in helicase function. Four individual mutations, Thr to Ala at position 269, Thr to Ala at position 411, Trp to Leu at position 501, and Trp to Ala at position 501, produced a severe reduction of RNA binding and completely abolished unwinding activity and stimulation of ATPase activity by poly(U), although the basal ATPase activity (activity in the absence of polynucleotide) of these mutants remained intact. Alanine substitution at Ser-231 or Ser-370 resulted in enzymes that were indistinguishable from wild-type HCV helicase with regard to all four activities. A mutant bearing Phe at Trp-501 showed wild-type levels of basal ATPase, unwinding activity, and single-stranded RNA binding activity. Interestingly, ATPase activity of this mutant became less responsive to stimulation by poly(U) but not to stimulation by other polynucleotides, such as poly(C). Given the conservation of some of these residues in

  10. Novel Potent Hepatitis C Virus NS3 Serine Protease Inhibitors Derived from Proline-Based Macrocycles

    SciTech Connect

    Chen, Kevin X.; Njoroge, F. George; Arasappan, Ashok; Venkatraman, Srikanth; Vibulbhan, Bancha; Yang, Weiying; Parekh, Tejal N.; Pichardo, John; Prongay, Andrew; Cheng, Kuo-Chi; Butkiewicz, Nancy; Yao, Nanhua; Madison, Vincent; Girijavallabhan, Viyyoor

    2008-06-30

    The hepatitis C virus (HCV) NS3 protease is essential for viral replication. It has been a target of choice for intensive drug discovery research. On the basis of an active pentapeptide inhibitor, 1, we envisioned that macrocyclization from the P2 proline to P3 capping could enhance binding to the backbone Ala156 residue and the S4 pocket. Thus, a number of P2 proline-based macrocyclic {alpha}-ketoamide inhibitors were prepared and investigated in an HCV NS3 serine protease continuous assay (K*{sub i}). The biological activity varied substantially depending on factors such as the ring size, number of amino acid residues, number of methyl substituents, type of heteroatom in the linker, P3 residue, and configuration at the proline C-4 center. The pentapeptide inhibitors were very potent, with the C-terminal acids and amides being the most active ones (24, K*{sub i} = 8 nM). The tetrapeptides and tripeptides were less potent. Sixteen- and seventeen-membered macrocyclic compounds were equally potent, while fifteen-membered analogues were slightly less active. gem-Dimethyl substituents at the linker improved the potency of all inhibitors (the best compound was 45, K*{sub i} = 6 nM). The combination of tert-leucine at P3 and dimethyl substituents at the linker in compound 47 realized a selectivity of 307 against human neutrophil elastase. Compound 45 had an IC{sub 50} of 130 nM in a cellular replicon assay, while IC{sub 50} for 24 was 400 nM. Several compounds had excellent subcutaneous AUC and bioavailability in rats. Although tripeptide compound 40 was 97% orally bioavailable, larger pentapeptides generally had low oral bioavailability. The X-ray crystal structure of compounds 24 and 45 bound to the protease demonstrated the close interaction of the macrocycle with the Ala156 methyl group and S4 pocket. The strategy of macrocyclization has been proved to be successful in improving potency (>20-fold greater than that of 1) and in structural depeptization.

  11. ATP dependent NS3 helicase interaction with RNA: insights from molecular simulations

    PubMed Central

    Pérez-Villa, Andrea; Darvas, Maria; Bussi, Giovanni

    2015-01-01

    Non-structural protein 3 (NS3) helicase from hepatitis C virus is an enzyme that unwinds and translocates along nucleic acids with an ATP-dependent mechanism and has a key role in the replication of the viral RNA. An inchworm-like mechanism for translocation has been proposed based on crystal structures and single molecule experiments. We here perform atomistic molecular dynamics in explicit solvent on the microsecond time scale of the available experimental structures. We also construct and simulate putative intermediates for the translocation process, and we perform non-equilibrium targeted simulations to estimate their relative stability. For each of the simulated structures we carefully characterize the available conformational space, the ligand binding pocket, and the RNA binding cleft. The analysis of the hydrogen bond network and of the non-equilibrium trajectories indicates an ATP-dependent stabilization of one of the protein conformers. Additionally, enthalpy calculations suggest that entropic effects might be crucial for the stabilization of the experimentally observed structures. PMID:26358809

  12. [Detections of hepatitis C virus RNA and NS3 antigen and their relation to liver histopathology].

    PubMed

    Wang, F; Wang, S; Jin, L

    1995-11-01

    To detect the distribution of hepatitis C virus and investigate the pathogenesis mechanisms of the viral infection in the liver tissues of the patients with acute or chronic hepatitis C, we examined HCV antigen expression by using the murine monoclonal antibody against HCV C33c peptide in the paraffin-embedded liver tissues from 28 patients with acute or chronic hepatitis C. The NS3 antigen was detected in 85.7% (24/28) of all the biopsy specimens. The distribution and staining density of the antigen immunoreactive signal varied according to different types of patients and the regions in the liver sections, but they obviously had a topographical relationship with the inflammatory-necrosis areas such as fatty and ballooning degeneration and focal necrosis in the liver tissues of nearly all the patients. In addition, the localization of HCV RNA investigated by in situ hybridization assay in 20 liver tissues the above 28 biopsy HD in the Chinese. They also provide valuable data for HD molecular diagnosis, genetic counselling and genetic health. PMID:8697087

  13. In Silico Identification and Evaluation of Leads for the Simultaneous Inhibition of Protease and Helicase Activities of HCV NS3/4A Protease Using Complex Based Pharmacophore Mapping and Virtual Screening

    PubMed Central

    Wadood, Abdul; Riaz, Muhammad; Uddin, Reaz; ul-Haq, Zaheer

    2014-01-01

    Hepatitis C virus (HCV) infection is an alarming and growing threat to public health. The present treatment gives limited efficacy and is poorly tolerated, recommending the urgent medical demand for novel therapeutics. NS3/4A protease is a significant emerging target for the treatment of HCV infection. This work reports the complex-based pharmacophore modeling to find out the important pharmacophoric features essential for the inhibition of both protease and helicase activity of NS3/4A protein of HCV. A seven featured pharmacophore model of HCV NS3/4A protease was developed from the crystal structure of NS3/4A protease in complex with a macrocyclic inhibitor interacting with both protease and helicase sites residues via MOE pharmacophore constructing tool. It consists of four hydrogen bond acceptors (Acc), one hydrophobic (Hyd), one for lone pair or active hydrogen (Atom L) and a heavy atom feature (Atom Q). The generated pharmacophore model was validated by a test database of seventy known inhibitors containing 55 active and 15 inactive/least active compounds. The validated pharmacophore model was used to virtually screen the ChemBridge database. As a result of screening 1009 hits were retrieved and were subjected to filtering by Lipinski’s rule of five on the basis of which 786 hits were selected for further assessment using molecular docking studies. Finally, 15 hits of different scaffolds having interactions with important active site residues were predicted as lead candidates. These candidates having unique scaffolds have a strong likelihood to act as further starting points in the development of novel and potent NS3/4A protease inhibitors. PMID:24551230

  14. Characterisation of divergent flavivirus NS3 and NS5 protein sequences detected in Rhipicephalus microplus ticks from Brazil

    PubMed Central

    Maruyama, Sandra Regina; Castro-Jorge, Luiza Antunes; Ribeiro, José Marcos Chaves; Gardinassi, Luiz Gustavo; Garcia, Gustavo Rocha; Brandão, Lucinda Giampietro; Rodrigues, Aline Rezende; Okada, Marcos Ituo; Abrão, Emiliana Pereira; Ferreira, Beatriz Rossetti; da Fonseca, Benedito Antonio Lopes; de Miranda-Santos, Isabel Kinney Ferreira

    2013-01-01

    Transcripts similar to those that encode the nonstructural (NS) proteins NS3 and NS5 from flaviviruses were found in a salivary gland (SG) complementary DNA (cDNA) library from the cattle tick Rhipicephalus microplus. Tick extracts were cultured with cells to enable the isolation of viruses capable of replicating in cultured invertebrate and vertebrate cells. Deep sequencing of the viral RNA isolated from culture supernatants provided the complete coding sequences for the NS3 and NS5 proteins and their molecular characterisation confirmed similarity with the NS3 and NS5 sequences from other flaviviruses. Despite this similarity, phylogenetic analyses revealed that this potentially novel virus may be a highly divergent member of the genus Flavivirus. Interestingly, we detected the divergent NS3 and NS5 sequences in ticks collected from several dairy farms widely distributed throughout three regions of Brazil. This is the first report of flavivirus-like transcripts in R. microplus ticks. This novel virus is a potential arbovirus because it replicated in arthropod and mammalian cells; furthermore, it was detected in a cDNA library from tick SGs and therefore may be present in tick saliva. It is important to determine whether and by what means this potential virus is transmissible and to monitor the virus as a potential emerging tick-borne zoonotic pathogen. PMID:24626302

  15. Intradermal delivery of DNA encoding HCV NS3 and perforin elicits robust cell-mediated immunity in mice and pigs.

    PubMed

    Grubor-Bauk, B; Yu, W; Wijesundara, D; Gummow, J; Garrod, T; Brennan, A J; Voskoboinik, I; Gowans, E J

    2016-01-01

    Currently, no vaccine is available against hepatitis C virus (HCV), and although DNA vaccines have considerable potential, this has not been realised. Previously, the efficacy of DNA vaccines for human immunodeficiency virus (HIV) and HCV was shown to be enhanced by including the gene for a cytolytic protein, viz. perforin. In this study, we examined the mechanism of cell death by this bicistronic DNA vaccine, which encoded the HCV non-structural protein 3 (NS3) under the control of the CMV promoter and perforin is controlled by the SV40 promoter. Compared with a canonical DNA vaccine and a bicistronic DNA vaccine encoding NS3 and the proapoptotic gene NSP4, the perforin-containing vaccine elicited enhanced cell-mediated immune responses against the NS3 protein in vaccinated mice and pigs, as determined by ELISpot and intracellular cytokine staining, whereas a mouse challenge model suggested that the immunity was CD8(+) T-cell-dependent. The results of the study showed that the inclusion of perforin in the DNA vaccine altered the fate of NS3-positive cells from apoptosis to necrosis, and this resulted in more robust immune responses in mice and pigs, the latter of which represents an accepted large animal model in which to test vaccine efficacy. PMID:26262584

  16. HCV core and NS3 proteins mediate toll like receptor induced innate immune response in corneal epithelium.

    PubMed

    Rajalakshmy, Ayilam Ramachandran; Malathi, Jambulingam; Madhavan, Hajib Naraharirao

    2014-11-01

    Direct association of dry eye syndrome and hepatitis C virus (HCV) infection is a well established fact. In this context, the current study examines the in vitro corneal inflammatory response with respect to HCV core and NS3 antigens. Toll like receptors (TLRs) are pattern recognition receptors which can mediate innate immune response. In the present study, corneal epithelial cells responded to HCV core and NS3 proteins by secreting pro-inflammatory cytokines IL-8, IL-6 and TNF-α via TLR1, TLR2 and TLR6 mediated innate immune response. MyD88/NF-kB signalling was involved in pro-inflammatory cytokine production. Corneal epithelium synthesised nitric oxide (NO) via iNOS during HCV core and NS3 exposure. On later stages of inflammation, cells underwent apoptosis which lead to cell death. SiRNA mediated silencing of TLR1, TLR2 and TLR6 resulted in a significant down regulation of IL-8 and NO. In conclusion, this study indicates that HCV core and NS3 proteins are capable of inducing immune response in corneal epithelium which can potentiate the pathology of HCV associated dry eye condition. Blocking specific TLR response can have therapeutic application in controlling the inflammatory response associated with this dry eye condition. PMID:25280963

  17. Induction of a Protective Response in Mice by the Dengue Virus NS3 Protein Using DNA Vaccines

    PubMed Central

    Costa, Simone M.; Yorio, Anna Paula; Gonçalves, Antônio J. S.; Vidale, Mariana M.; Costa, Emmerson C. B.; Mohana-Borges, Ronaldo; Motta, Marcia A.; Freire, Marcos S.; Alves, Ada M. B.

    2011-01-01

    The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serino-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work, we investigate the protective efficacy of DNA vaccines based on the NS3 protein from DENV2. Different recombinant plasmids were constructed, encoding either the full-length NS3 protein or only its functional domains (protease and helicase), fused or not to a signal peptide (t-PA). The recombinant proteins were successfully expressed in transfected BHK-21 cells, and only plasmids encoding the t-PA signal sequence mediated protein secretion. Balb/c mice were immunized with the different DNA vaccines and challenged with a lethal dose of DENV2. Most animals immunized with plasmids encoding the full-length NS3 or the helicase domain survived challenge, regardless of the presence of the t-PA. However, some mice presented clinical signs of infection with high morbidity (hind leg paralysis and hunched posture), mainly in animal groups immunized with the DNA vaccines based on the helicase domain. On the other hand, inoculation with plasmids encoding the protease domain did not induce any protection, since mortality and morbidity rates in these mouse groups were similar to those detected in the control animals. The cellular immune response was analyzed by ELISPOT with a specific-CD8+ T cell NS3 peptide. Results revealed that the DNA vaccines based on the full-length protein induced the production of INF-γ, thus suggesting the involvement of this branch of the immune system in the protection. PMID:22031819

  18. Structural Mechanisms and Drug Discovery Prospects of Rho GTPases

    PubMed Central

    Smithers, Cameron C.; Overduin, Michael

    2016-01-01

    Rho GTPases regulate cellular morphology and dynamics, and some are key drivers of cancer progression. This superfamily offers attractive potential targets for therapeutic intervention, with RhoA, Rac1 and Cdc42 being prime examples. The challenges in developing agents that act on these signaling enzymes include the lack of obvious druggable pockets and their membrane-bound activities. However, progress in targeting the similar Ras protein is illuminating new strategies for specifically inhibiting oncogenic GTPases. The structures of multiple signaling and regulatory states of Rho proteins have been determined, and the post-translational modifications including acylation and phosphorylation points have been mapped and their functional effects examined. The development of inhibitors to probe the significance of overexpression and mutational hyperactivation of these GTPases underscores their importance in cancer progression. The ability to integrate in silico, in vitro, and in vivo investigations of drug-like molecules indicates the growing tractability of GTPase systems for lead optimization. Although no Rho-targeted drug molecules have yet been clinically approved, this family is clearly showing increasing promise for the development of precision medicine and combination cancer therapies. PMID:27304967

  19. Structural Mechanisms and Drug Discovery Prospects of Rho GTPases.

    PubMed

    Smithers, Cameron C; Overduin, Michael

    2016-01-01

    Rho GTPases regulate cellular morphology and dynamics, and some are key drivers of cancer progression. This superfamily offers attractive potential targets for therapeutic intervention, with RhoA, Rac1 and Cdc42 being prime examples. The challenges in developing agents that act on these signaling enzymes include the lack of obvious druggable pockets and their membrane-bound activities. However, progress in targeting the similar Ras protein is illuminating new strategies for specifically inhibiting oncogenic GTPases. The structures of multiple signaling and regulatory states of Rho proteins have been determined, and the post-translational modifications including acylation and phosphorylation points have been mapped and their functional effects examined. The development of inhibitors to probe the significance of overexpression and mutational hyperactivation of these GTPases underscores their importance in cancer progression. The ability to integrate in silico, in vitro, and in vivo investigations of drug-like molecules indicates the growing tractability of GTPase systems for lead optimization. Although no Rho-targeted drug molecules have yet been clinically approved, this family is clearly showing increasing promise for the development of precision medicine and combination cancer therapies. PMID:27304967

  20. Rho GTPases and their effector proteins.

    PubMed Central

    Bishop, A L; Hall, A

    2000-01-01

    Rho GTPases are molecular switches that regulate many essential cellular processes, including actin dynamics, gene transcription, cell-cycle progression and cell adhesion. About 30 potential effector proteins have been identified that interact with members of the Rho family, but it is still unclear which of these are responsible for the diverse biological effects of Rho GTPases. This review will discuss how Rho GTPases physically interact with, and regulate the activity of, multiple effector proteins and how specific effector proteins contribute to cellular responses. To date most progress has been made in the cytoskeleton field, and several biochemical links have now been established between GTPases and the assembly of filamentous actin. The main focus of this review will be Rho, Rac and Cdc42, the three best characterized mammalian Rho GTPases, though the genetic analysis of Rho GTPases in lower eukaryotes is making increasingly important contributions to this field. PMID:10816416

  1. Regulating Rho GTPases and their regulators.

    PubMed

    Hodge, Richard G; Ridley, Anne J

    2016-08-01

    Rho GTPases regulate cytoskeletal and cell adhesion dynamics and thereby coordinate a wide range of cellular processes, including cell migration, cell polarity and cell cycle progression. Most Rho GTPases cycle between a GTP-bound active conformation and a GDP-bound inactive conformation to regulate their ability to activate effector proteins and to elicit cellular responses. However, it has become apparent that Rho GTPases are regulated by post-translational modifications and the formation of specific protein complexes, in addition to GTP-GDP cycling. The canonical regulators of Rho GTPases - guanine nucleotide exchange factors, GTPase-activating proteins and guanine nucleotide dissociation inhibitors - are regulated similarly, creating a complex network of interactions to determine the precise spatiotemporal activation of Rho GTPases. PMID:27301673

  2. Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures.

    PubMed Central

    Westaway, E G; Mackenzie, J M; Kenney, M T; Jones, M K; Khromykh, A A

    1997-01-01

    The subcellular location of the nonstructural proteins NS1, NS2B, and NS3 in Vero cells infected with the flavivirus Kunjin was investigated using indirect immunofluorescence and cryoimmunoelectron microscopy with monospecific antibodies. Comparisons were also made by dual immunolabelling using antibodies to double-stranded RNA (dsRNA), the putative template in the flavivirus replication complex. At 8 h postinfection, the immunofluorescent patterns showed NS1, NS2B, NS3, and dsRNA located in a perinuclear rim with extensions into the peripheral cytoplasm. By 16 h, at the end of the latent period, all patterns had changed to some discrete perinuclear foci associated with a thick cytoplasmic reticulum. By 24 h, this localization in perinuclear foci was more apparent and some foci were dual labelled with antibodies to dsRNA. In immuno-gold-labelled cryosections of infected cells at 24 h, all antibodies were associated with clusters of induced membrane structures in the perinuclear region. Two important and novel observations were made. First, one set of induced membranes comprised vesicle packets of smooth membranes dual labelled with anti-dsRNA and anti-NS1 or anti-NS3 antibodies. Second, adjacent masses of paracrystalline arrays or of convoluted smooth membranes, which appeared to be structurally related, were strongly labelled only with anti-NS2B and anti-NS3 antibodies. Paired membranes similar in appearance to the rough endoplasmic reticulum were also labelled, but less strongly, with antibodies to the three nonstructural proteins. Other paired membranes adjacent to the structures discussed above enclosed accumulated virus particles but were not labelled with any of the four antibodies. The collection of induced membranes may represent virus factories in which translation, RNA synthesis, and virus assembly occur. PMID:9261387

  3. Deregulation of Rho GTPases in cancer

    PubMed Central

    Porter, Andrew P.; Papaioannou, Alexandra; Malliri, Angeliki

    2016-01-01

    ABSTRACT In vitro and in vivo studies and evidence from human tumors have long implicated Rho GTPase signaling in the formation and dissemination of a range of cancers. Recently next generation sequencing has identified direct mutations of Rho GTPases in human cancers. Moreover, the effects of ablating genes encoding Rho GTPases and their regulators in mouse models, or through pharmacological inhibition, strongly suggests that targeting Rho GTPase signaling could constitute an effective treatment. In this review we will explore the various ways in which Rho signaling can be deregulated in human cancers. PMID:27104658

  4. Investigation of translocation, DNA unwinding, and protein displacement by NS3h, the helicase domain from the Hepatitis C virus helicase†

    PubMed Central

    Matlock, Dennis L.; Yeruva, Laxmi; Byrd, Alicia K.; Mackintosh, Samuel G.; Langston, Clint; Brown, Carrie; Cameron, Craig E.; Fischer, Christopher J.; Raney, Kevin D.

    2010-01-01

    Helicases are motor proteins that are involved in DNA and RNA metabolism, replication, recombination, transcription and repair. The motors are powered by ATP binding and hydrolysis. Hepatitis C virus encodes a helicase called non-structural protein (NS3). NS3 possesses protease and helicase activities on its N-terminal and C-terminal domains respectively. The helicase domain of NS3 protein is referred as NS3h. In vitro, NS3h catalyzes RNA and DNA unwinding in a 3’ to -5’ direction. The directionality for unwinding is thought to arise in part from the enzyme's ability to translocate along DNA, but translocation has not been shown explicitly. We examined the DNA translocase activity of NS3h by using single-stranded oligonucleotide substrates containing a fluorescent probe on the 5’ end. NS3h can bind to the ssDNA and in the presence of ATP, move towards the 5’-end. When the enzyme encounters the fluorescent probe, a fluorescence change is observed that allows translocation to be characterized. Under conditions that favor binding of one NS3h per DNA substrate (100 nM NS3h, 200 nM oligonucleotide) we find that NS3h translocates on ssDNA at a rate of 46 ± 5 nt s−1 and that it can move for 230 ± 60 nt before dissociating from the DNA. The translocase activity of some helicases is responsible for displacing proteins that are bound to DNA. We studied protein displacement by using a ssDNA oligonucleotide covalently linked to biotin on the 5’-end. Upon addition of streptavidin, a ‘protein-block’ was placed in the pathway of the helicase. Interestingly, NS3h was unable to displace streptavidin from the end of the oligonucleotide, despite its ability to translocate along the DNA. The DNA unwinding activity of NS3h was examined using a 22 bp duplex DNA substrate under conditions that were identical to those used to study translocation. NS3h exhibited little or no DNA unwinding under single cycle conditions, supporting the conclusion that NS3h is a relatively

  5. Flavivirus premembrane protein cleavage and spike heterodimer secretion require the function of the viral proteinase NS3.

    PubMed Central

    Lobigs, M

    1993-01-01

    Flavivirus protein biosynthesis involves the proteolytic processing of a single polyprotein precursor by host- and virus-encoded proteinases. In this study, the requirement for the proteolytic function of the viral proteinase NS3 for correct processing of a polyprotein segment encompassing the Murray Valley encephalitis virus structural proteins is shown. The NS3-mediated cleavage in the structural polyprotein region presumably releases the capsid protein from its membrane anchor and triggers the appearance of the premembrane (prM) protein. This suggests that cleavage of prM by signal peptidase in the lumen of the endoplasmic reticulum is under control of a cytoplasmic cleavage catalyzed by a viral proteinase. The function of the viral proteinase is also essential for secretion of flaviviral spike proteins when expressed from cDNA via vaccinia virus recombinants or in COS cell transfections. This has important implications for the design of flavivirus subunit vaccines. Images Fig. 1 Fig. 2 Fig. 3 PMID:8392191

  6. XAS Characterization of the Zn Site of Non-structural Protein 3 (NS3) from Hepatitis C Virus

    NASA Astrophysics Data System (ADS)

    Ascone, I.; Nobili, G.; Benfatto, M.; Congiu-Castellano, A.

    2007-02-01

    XANES spectra of non structural protein 3 (NS3) have been calculated using 4 Zn coordination models from three crystallographic structures in the Protein Data Base (PDB): 1DY9, subunit B, 1CU1 subunit A and B, and 1JXP subunit B. Results indicate that XANES is an appropriate tool to distinguish among them. Experimental XANES spectra have been simulated refining crystallographic data. The model obtained by XAS is compared with the PDB models.

  7. Inhibitory activity of cyclohexenyl chalcone derivatives and flavonoids of fingerroot, Boesenbergia rotunda (L.), towards dengue-2 virus NS3 protease.

    PubMed

    Kiat, Tan Siew; Pippen, Richard; Yusof, Rohana; Ibrahim, Halijah; Khalid, Norzulaani; Rahman, Noorsaadah Abd

    2006-06-15

    Boesenbergia rotunda (L.) cyclohexenyl chalcone derivatives, 4-hydroxypanduratin A and panduratin A, showed good competitive inhibitory activities towards dengue 2 virus NS3 protease with the Ki values of 21 and 25 microM, respectively, whilst those of pinostrobin and cardamonin were observed to be non-competitive. NMR and GCMS spectroscopic data formed the basis of assignment of structures of the six compounds isolated. PMID:16621533

  8. Sequence analysis and evaluation of the NS3/A gene region of bluetongue virus isolates from South Africa.

    PubMed

    Steyn, Jumari; Venter, Estelle Hildegard

    2016-04-01

    Phylogenetic networks and sequence analysis allow a more accurate understanding of the serotypes, genetic relationships and epidemiology of viruses. Based on gene sequences of the conserved segment 10 (NS3), bluetongue virus (BTV) can be divided into five topotypes. In this molecular epidemiology study, segment 10 sequence data of 11 isolates obtained from the Virology Section of the Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, were analyzed and compared to sequence data of worldwide BTV strains available in the GenBank database. The consensus nucleotide sequences of NS3/A showed intermediate levels of variation, with the nucleotide sequence identity ranging from 79.72 % to 100 %. All 11 strains demonstrated conserved amino acid characteristics. Phylogenetic networks were used to identify BTV topotypes. The phylogeny obtained from the nucleotide sequence data of the NS3/A-encoding gene presented three major and two minor topotypes. The clustering of strains from different geographical areas into the same group indicated spatial spread of the segment 10 genes, either through gene reassortment or through the introduction of new strains from other geographical areas via trade. The effect of reassortment and genetic drift on BTV and the importance of correct serotyping to identify viral strains are highlighted. PMID:26780892

  9. The Spring α-Helix Coordinates Multiple Modes of HCV (Hepatitis C Virus) NS3 Helicase Action.

    PubMed

    Gu, Meigang; Rice, Charles M

    2016-07-01

    Genomic DNA replication requires helicases to processively unwind duplexes. Although helicases encoded by positive-strand RNA viruses are necessary for RNA genome replication, their functions are not well understood. We determined structures of the hepatitis C virus helicase (NS3h) in complex with the transition state ATP mimic ADP·AlF4 (-) and compared them with the previous nucleic acid-associated ternary complexes. The results suggested that nucleic acid binding promotes a structural change of the spring helix at the transition state, optimizing the interaction network centered on the nucleophilic water. Analysis of ATP hydrolysis with and without conformational restraints on the spring helix further supported the importance of its action for both nucleic acid-stimulated and basal catalysis. We further found that an F238P substitution, predicted to destabilize the helix, diminished viral RNA replication without significantly affecting ATP-dependent duplex unwinding. The stability of the secondary structure, thus, seems critical for additional functions of NS3h. Taken together, the results suggest that the spring helix may be central to the coordination of multiple modes of NS3h action. Further characterization centered on this element may help understand the molecular details of how the viral helicase facilitates RNA replication. This new structural information may also aid efforts to develop specific inhibitors targeting this essential viral enzyme. PMID:27226535

  10. Molecular Mechanisms of Viral and Host Cell Substrate Recognition by Hepatitis C Virus NS3/4A Protease

    SciTech Connect

    Romano, Keith P.; Laine, Jennifer M.; Deveau, Laura M.; Cao, Hong; Massi, Francesca; Schiffer, Celia A.

    2011-08-16

    Hepatitis C NS3/4A protease is a prime therapeutic target that is responsible for cleaving the viral polyprotein at junctions 3-4A, 4A4B, 4B5A, and 5A5B and two host cell adaptor proteins of the innate immune response, TRIF and MAVS. In this study, NS3/4A crystal structures of both host cell cleavage sites were determined and compared to the crystal structures of viral substrates. Two distinct protease conformations were observed and correlated with substrate specificity: (i) 3-4A, 4A4B, 5A5B, and MAVS, which are processed more efficiently by the protease, form extensive electrostatic networks when in complex with the protease, and (ii) TRIF and 4B5A, which contain polyproline motifs in their full-length sequences, do not form electrostatic networks in their crystal complexes. These findings provide mechanistic insights into NS3/4A substrate recognition, which may assist in a more rational approach to inhibitor design in the face of the rapid acquisition of resistance.

  11. Comparative analysis of NS3 sequences of temporally separated dengue 3 virus strains isolated from southeast Asia.

    PubMed

    Chow, V T; Seah, C L; Chan, Y C

    1994-01-01

    By a combination of PCR and direct-cycle sequencing using consensus primers, we analyzed approximately 400-bp fragments within the NS3 genes of twenty-one dengue virus type 3 strains isolated from five neighboring Southeast Asian countries at different time intervals from 1956 to 1992. The majority of base disparities were silent mutations, with few predicted amino acid substitutions, thus emphasizing the strict conservation of the NS3 gene. Phylogenetic trees constructed on the basis of these nucleotide differences revealed distinct but related clusters of strains from the Philippines, Indonesia, and strains from Singapore and Malaysia of the 1970s and early 1980s, while the Thai cluster was relatively more distant. This genetic relationship was compatible with that proposed by other workers who have studied other dengue 3 virus genes such as E, M and prM. However, we observed that the more recent, epidemic-associated dengue 3 strains from Singapore and Malaysia of the late 1980s and early 1990s were more closely related to the Thai cluster, implying their evolution from the latter, and emphasizing the importance of viral spread via increasing travel within the Southeast Asian area and elsewhere. Nucleotide sequence analysis of the NS3 genes of dengue viruses can serve to advance the understanding of the epidemiology and evolution of these viruses. PMID:7698880

  12. Discovery of a Non-Peptidic Inhibitor of West Nile Virus NS3 Protease by High-Throughput Docking

    PubMed Central

    Ekonomiuk, Dariusz; Su, Xun-Cheng; Ozawa, Kiyoshi; Bodenreider, Christophe; Lim, Siew Pheng; Yin, Zheng; Keller, Thomas H.; Beer, David; Patel, Viral; Otting, Gottfried; Caflisch, Amedeo; Huang, Danzhi

    2009-01-01

    Background The non-structural 3 protease (NS3pro) is an essential flaviviral enzyme and therefore one of the most promising targets for drug development against West Nile virus (WNV) and dengue infections. Methodology In this work, a small-molecule inhibitor of the WNV NS3pro has been identified by automatic fragment-based docking of about 12000 compounds and testing by nuclear magnetic resonance (NMR) spectroscopy of only 22 molecules. Specific binding of the inhibitor into the active site of NS3pro and its binding mode are confirmed by 15N-HSQC NMR spectra. The inhibitory activity is further validated by an enzymatic assay and a tryptophan fluorescence quenching assay. Conclusion The inhibitor [4-(carbamimidoylsulfanylmethyl)-2,5-dimethylphenyl]-methylsulfanylmethanimidamide has a good ratio of binding affinity versus molecular weight (ligand efficiency of 0.33 kcal/mol per non-hydrogen atom), and thus has good potential as lead compound for further development to combat West Nile virus infections. PMID:19159012

  13. Identification and Biochemical Characterization of Halisulfate 3 and Suvanine as Novel Inhibitors of Hepatitis C Virus NS3 Helicase from a Marine Sponge

    PubMed Central

    Furuta, Atsushi; Abdus Salam, Kazi; Hermawan, Idam; Akimitsu, Nobuyoshi; Tanaka, Junichi; Tani, Hidenori; Yamashita, Atsuya; Moriishi, Kohji; Nakakoshi, Masamichi; Tsubuki, Masayoshi; Peng, Poh Wee; Suzuki, Youichi; Yamamoto, Naoki; Sekiguchi, Yuji; Tsuneda, Satoshi; Noda, Naohiro

    2014-01-01

    Hepatitis C virus (HCV) is an important etiological agent that is responsible for the development of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV nonstructural protein 3 (NS3) helicase is a possible target for novel drug development due to its essential role in viral replication. In this study, we identified halisulfate 3 (hal3) and suvanine as novel NS3 helicase inhibitors, with IC50 values of 4 and 3 µM, respectively, from a marine sponge by screening extracts of marine organisms. Both hal3 and suvanine inhibited the ATPase, RNA binding, and serine protease activities of NS3 helicase with IC50 values of 8, 8, and 14 µM, and 7, 3, and 34 µM, respectively. However, the dengue virus (DENV) NS3 helicase, which shares a catalytic core (consisting mainly of ATPase and RNA binding sites) with HCV NS3 helicase, was not inhibited by hal3 and suvanine, even at concentrations of 100 µM. Therefore, we conclude that hal3 and suvanine specifically inhibit HCV NS3 helicase via an interaction with an allosteric site in NS3 rather than binding to the catalytic core. This led to the inhibition of all NS3 activities, presumably by inducing conformational changes. PMID:24451189

  14. LRRK2 autophosphorylation enhances its GTPase activity.

    PubMed

    Liu, Zhiyong; Mobley, James A; DeLucas, Lawrence J; Kahn, Richard A; West, Andrew B

    2016-01-01

    The leucine-rich repeat kinase (LRRK)-2 protein contains nonoverlapping GTPase and kinase domains, and mutation in either domain can cause Parkinson disease. GTPase proteins are critical upstream modulators of many effector protein kinases. In LRRK2, this paradigm may be reversed, as the kinase domain phosphorylates its own GTPase domain. In this study, we found that the ameba LRRK2 ortholog ROCO4 phosphorylates the GTPase domain [termed Ras-of-complex (ROC) domain in this family] of human LRRK2 on the same residues as the human LRRK2 kinase. Phosphorylation of ROC enhances its rate of GTP hydrolysis [from kcat (catalytic constant) 0.007 to 0.016 min(-1)], without affecting GTP or GDP dissociation kinetics [koff = 0.093 and 0.148 min(-1) for GTP and GDP, respectively). Phosphorylation also promotes the formation of ROC dimers, although GTPase activity appears to be equivalent between purified dimers and monomers. Modeling experiments show that phosphorylation induces conformational changes at the critical p-loop structure. Finally, ROC appears to be one of many GTPases phosphorylated in p-loop residues, as revealed by alignment of LRRK2 autophosphorylation sites with GTPases annotated in the phosphoproteome database. These results provide an example of a novel mechanism for kinase-mediated control of GTPase activity. PMID:26396237

  15. HCV NS3Ag: a reliable and clinically useful predictor of antiviral outcomes in genotype 1b hepatitis C virus-infected patients.

    PubMed

    Ren, S; Jin, Y; Huang, Y; Ma, L; Liu, Y; Meng, C; Guan, S; Xie, L; Chen, X

    2016-07-01

    Since hepatitis C virus (HCV) non-structural 3 (NS3) protease inhibitor (PI) combined with pegylated interferon/ribavirin (PR) has been approved for chronic HCV genotype (GT) 1b infection, a reliable and clinically useful predictor combining with serum HCV RNA to predict early virologic response, breakthrough, and relapse is important during HCV antiviral treatment. We evaluated the role of HCV NS3 antigen (HCV NS3Ag) on the prediction of virologic response in patients with HCV GT1b during PR or PR/simeprevir (triple) therapy. Three hundred patients were recruited, and HCV RNA and HCV NS3Ag were tested at baseline and weeks 2, 4, 12, 24, 48, and 72. NS3Ag and HCV RNA were significantly related (r(2) = 0.67) in the whole patient selection. The kinetic pattern of HCV RNA and HCV NS3Ag during triple treatment was similar. HCV NS3Ag levels in the triple group closely followed those of HCV RNA; the r(2) values were 0.756 (baseline), 0.837 (2 weeks), 0.989 (4 weeks), and 0.993 (12 weeks), respectively. For patients treated with PR, the positive and negative predictive values (PPVs and NPVs) for viral response were 96.31 % and 67.19 %, respectively, at week 4 by using the decrease of NS3Ag (dHCV NS3Ag) combined with HCV RNA. At week 12, the PPV was similar at 94.16 %, while the NPV reached 87.26 %. The PPV and NPV for the prediction of relapse and breakthrough were 90.6 % and 76.7 %, respectively. HCV NS3Ag is a valuable marker and could be a supplementary predictor of HCV RNA for the prediction of antiviral response, breakthrough, or relapse during HCV antiviral treatment. PMID:27173787

  16. Conditional Inducible Triple-Transgenic Mouse Model for Rapid Real-Time Detection of HCV NS3/4A Protease Activity

    PubMed Central

    Yang, Jing; Zhao, Haiwei; Qiao, Qinghua; Han, Peijun; Xu, Zhikai; Yin, Wen

    2016-01-01

    Hepatitis C virus (HCV) frequently establishes persistent infections that can develop into severe liver disease. The HCV NS3/4A serine protease is not only essential for viral replication but also cleaves multiple cellular targets that block downstream interferon activation. Therefore, NS3/4A is an ideal target for the development of anti-HCV drugs and inhibitors. In the current study, we generated a novel NS3/4A/Lap/LC-1 triple-transgenic mouse model that can be used to evaluate and screen NS3/4A protease inhibitors. The NS3/4A protease could be conditionally inducibly expressed in the livers of the triple-transgenic mice using a dual Tet-On and Cre/loxP system. In this system, doxycycline (Dox) induction resulted in the secretion of Gaussia luciferase (Gluc) into the blood, and this secretion was dependent on NS3/4A protease-mediated cleavage at the 4B5A junction. Accordingly, NS3/4A protease activity could be quickly assessed in real time simply by monitoring Gluc activity in plasma. The results from such monitoring showed a 70-fold increase in Gluc activity levels in plasma samples collected from the triple-transgenic mice after Dox induction. Additionally, this enhanced plasma Gluc activity was well correlated with the induction of NS3/4A protease expression in the liver. Following oral administration of the commercial NS3/4A-specific inhibitors telaprevir and boceprevir, plasma Gluc activity was reduced by 50% and 65%, respectively. Overall, our novel transgenic mouse model offers a rapid real-time method to evaluate and screen potential NS3/4A protease inhibitors. PMID:26943641

  17. Conditional Inducible Triple-Transgenic Mouse Model for Rapid Real-Time Detection of HCV NS3/4A Protease Activity.

    PubMed

    Yao, Min; Lu, Xin; Lei, Yingfeng; Yang, Jing; Zhao, Haiwei; Qiao, Qinghua; Han, Peijun; Xu, Zhikai; Yin, Wen

    2016-01-01

    Hepatitis C virus (HCV) frequently establishes persistent infections that can develop into severe liver disease. The HCV NS3/4A serine protease is not only essential for viral replication but also cleaves multiple cellular targets that block downstream interferon activation. Therefore, NS3/4A is an ideal target for the development of anti-HCV drugs and inhibitors. In the current study, we generated a novel NS3/4A/Lap/LC-1 triple-transgenic mouse model that can be used to evaluate and screen NS3/4A protease inhibitors. The NS3/4A protease could be conditionally inducibly expressed in the livers of the triple-transgenic mice using a dual Tet-On and Cre/loxP system. In this system, doxycycline (Dox) induction resulted in the secretion of Gaussia luciferase (Gluc) into the blood, and this secretion was dependent on NS3/4A protease-mediated cleavage at the 4B5A junction. Accordingly, NS3/4A protease activity could be quickly assessed in real time simply by monitoring Gluc activity in plasma. The results from such monitoring showed a 70-fold increase in Gluc activity levels in plasma samples collected from the triple-transgenic mice after Dox induction. Additionally, this enhanced plasma Gluc activity was well correlated with the induction of NS3/4A protease expression in the liver. Following oral administration of the commercial NS3/4A-specific inhibitors telaprevir and boceprevir, plasma Gluc activity was reduced by 50% and 65%, respectively. Overall, our novel transgenic mouse model offers a rapid real-time method to evaluate and screen potential NS3/4A protease inhibitors. PMID:26943641

  18. Priming with two DNA vaccines expressing hepatitis C virus NS3 protein targeting dendritic cells elicits superior heterologous protective potential in mice.

    PubMed

    Guan, Jie; Deng, Yao; Chen, Hong; Yin, Xiao; Yang, Yang; Tan, Wenjie

    2015-10-01

    Development an effective vaccine may offer an alternative preventive and therapeutic strategy against HCV infection. DNA vaccination has been shown to induce robust humoral and cellular immunity and overcome many problems associated with conventional vaccines. In this study, mice were primed with either conventional pVRC-based or suicidal pSC-based DNA vaccines carrying DEC-205-targeted NS3 antigen (DEC-NS3) and boosted with type 5 adenoviral vectors encoding the partial NS3 and core antigens (C44P). The prime boost regimen induced a marked increase in antigen-specific humoral and T-cell responses in comparison with either rAd5-based vaccines or DEC-205-targeted DNA immunization in isolation. The protective effect against heterogeneous challenge was correlated with high levels of anti-NS3 IgG and T-cell-mediated immunity against NS3 peptides. Moreover, priming with a suicidal DNA vaccine (pSC-DEC-NS3), which elicited increased TNF-α-producing CD4+ and CD8+ T-cells against NS3-2 peptides (aa 1245-1461), after boosting, showed increased heterogeneous protective potential compared with priming with a conventional DNA vaccine (pVRC-DEC-NS3). In conclusion, a suicidal DNA vector (pSC-DEC-NS3) expressing DEC-205-targeted NS3 combined with boosting using an rAd5-based HCV vaccine (rAd5-C44P) is a good candidate for a safe and effective vaccine against HCV infection. PMID:26215441

  19. The Macroscopic Rate of Nucleic Acid Translocation by Hepatitis C virus Helicase NS3h is Dependent on Both the Sugar and Base Moieties

    PubMed Central

    Khaki, Ali R.; Field, Cassandra; Malik, Shuja; Niedziela-Majka, Anita; Leavitt, Stephanie A.; Wang, Ruth; Hung, Magdeleine; Sakowicz, Roman; Brendza, Katherine M.; Fischer, Christopher J.

    2010-01-01

    The NS3 helicase (NS3h) of hepatitis C virus (HCV) is a 3′ to 5′ SF2 RNA and DNA helicase that is essential for the replication of HCV. We have examined the kinetic mechanism of translocation of NS3h along single-stranded nucleic acid with bases rU, dU and dT and have found that the macroscopic rate of translocation is dependent upon both the base and sugar moieties of the nucleic acid, with approximate macroscopic translocation rates of 3 nt/s (oligo-dT), 35 nt/s (oligo-dU), and 42 nt/s (oligo-rU), respectively. We found a strong correlation between the macroscopic translocation rates and the binding affinity of the translocating NS3h protein to the respective substrates such that weaker affinity corresponded to faster translocation. The values of K0.5 for NS3h translocation at a saturating ATP concentration are: (3.3 ± 0.4) μM nucleotide (poly-dT), (27 ± 2) μM nucleotide (poly-dU), and (36 ± 2) μM nucleotide (poly-rU). Furthermore, the results of isothermal titration of NS3h with these oligonucleotides suggest that differences in TΔS° are the principal source of the differences in the affinity of NS3h binding to these substrates. Interestingly, despite the differences in macroscopic translocation rates and binding affinities, the ATP coupling stoichiometry for NS3h translocation was identical for all three substrates, ~0.5 ATP molecules consumed per nucleotide translocated. This similar periodicity of ATP consumption implies a similar mechanism for NS3h translocation along RNA and DNA substrates. PMID:20451531

  20. Molecular mechanisms of Sar/Arf GTPases in vesicular trafficking in yeast and plants

    PubMed Central

    Yorimitsu, Tomohiro; Sato, Ken; Takeuchi, Masaki

    2014-01-01

    Small GTPase proteins play essential roles in the regulation of vesicular trafficking systems in eukaryotic cells. Two types of small GTPases, secretion-associated Ras-related protein (Sar) and ADP-ribosylation factor (Arf), act in the biogenesis of transport vesicles. Sar/Arf GTPases function as molecular switches by cycling between active, GTP-bound and inactive, GDP-bound forms, catalyzed by guanine nucleotide exchange factors and GTPase-activating proteins, respectively. Activated Sar/Arf GTPases undergo a conformational change, exposing the N-terminal amphipathic α-helix for insertion into membranes. The process triggers the recruitment and assembly of coat proteins to the membranes, followed by coated vesicle formation and scission. In higher plants, Sar/Arf GTPases also play pivotal roles in maintaining the dynamic identity of organelles in the secretory pathway. Sar1 protein strictly controls anterograde transport from the endoplasmic reticulum (ER) through the recruitment of plant COPII coat components onto membranes. COPII vesicle transport is responsible for the organization of highly conserved polygonal ER networks. In contrast, Arf proteins contribute to the regulation of multiple trafficking routes, including transport through the Golgi complex and endocytic transport. These transport systems have diversified in the plant kingdom independently and exhibit several plant-specific features with respect to Golgi organization, endocytic cycling, cell polarity and cytokinesis. The functional diversification of vesicular trafficking systems ensures the multicellular development of higher plants. This review focuses on the current knowledge of Sar/Arf GTPases, highlighting the molecular details of GTPase regulation in vesicle formation in yeast and advances in knowledge of the characteristics of vesicle trafficking in plants. PMID:25191334

  1. Formins as effector proteins of Rho GTPases

    PubMed Central

    Kühn, Sonja; Geyer, Matthias

    2014-01-01

    Formin proteins were recognized as effectors of Rho GTPases some 15 years ago. They contribute to different cellular actin cytoskeleton structures by their ability to polymerize straight actin filaments at the barbed end. While not all formins necessarily interact with Rho GTPases, a subgroup of mammalian formins, termed Diaphanous-related formins or DRFs, were shown to be activated by small GTPases of the Rho superfamily. DRFs are autoinhibited in the resting state by an N- to C-terminal interaction that renders the central actin polymerization domain inactive. Upon the interaction with a GTP-bound Rho, Rac, or Cdc42 GTPase, the C-terminal autoregulation domain is displaced from its N-terminal recognition site and the formin becomes active to polymerize actin filaments. In this review we discuss the current knowledge on the structure, activation, and function of formin-GTPase interactions for the mammalian formin families Dia, Daam, FMNL, and FHOD. We describe both direct and indirect interactions of formins with GTPases, which lead to formin activation and cytoskeletal rearrangements. The multifaceted function of formins as effector proteins of Rho GTPases thus reflects the diversity of the actin cytoskeleton in cells. PMID:24914801

  2. Interaction of LRRK2 with kinase and GTPase signaling cascades

    PubMed Central

    Boon, Joon Y.; Dusonchet, Julien; Trengrove, Chelsea; Wolozin, Benjamin

    2014-01-01

    LRRK2 is a protein that interacts with a plethora of signaling molecules, but the complexity of LRRK2 function presents a challenge for understanding the role of LRRK2 in the pathophysiology of Parkinson’s disease (PD). Studies of LRRK2 using over-expression in transgenic mice have been disappointing, however, studies using invertebrate systems have yielded a much clearer picture, with clear effects of LRRK2 expression, knockdown or deletion in Caenorhabditis elegans and Drosophila on modulation of survival of dopaminergic neurons. Recent studies have begun to focus attention on particular signaling cascades that are a target of LRRK2 function. LRRK2 interacts with members of the mitogen activated protein kinase (MAPK) pathway and might regulate the pathway action by acting as a scaffold that directs the location of MAPK pathway activity, without strongly affecting the amount of MAPK pathway activity. Binding to GTPases, GTPase-activating proteins and GTPase exchange factors are another strong theme in LRRK2 biology, with LRRK2 binding to rac1, cdc42, rab5, rab7L1, endoA, RGS2, ArfGAP1, and ArhGEF7. All of these molecules appear to feed into a function output for LRRK2 that modulates cytoskeletal outgrowth and vesicular dynamics, including autophagy. These functions likely impact modulation of α-synuclein aggregation and associated toxicity eliciting the disease processes that we term PD. PMID:25071441

  3. Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein.

    PubMed

    Cui, T; Sugrue, R J; Xu, Q; Lee, A K; Chan, Y C; Fu, J

    1998-07-01

    The full-length dengue virus NS3 protein has been successfully expressed as a 94-kDa GST fusion protein in Escherichia coli. Treatment of the purified fusion protein with thrombin released a 68-kDa protein which is the expected molecular mass for the DEN1 NS3 protein. The identity of this protein was confirmed by Western blotting using dengue virus antisera. Two related activities of the recombinant NS3 protein were characterized, which were the binding of the protein to the 3'-noncoding region of the dengue virus RNA genome and NTPase activity. We demonstrated using a band shift assay that the DEN1 NS3 protein could form a complex with the stem-loop structure in the 3'-noncoding region (3'-NCR), although sites outside the stem-loop may also participate in binding. Using various unlabeled homopolymeric and heteropolymeric RNAs as competitors for binding, it was further shown that the DEN1 NS3 protein exhibits preferential binding to a 94-nt RNA transcript from the 3'-NCR of the dengue virus. The NTPase activity of the recombinant DEN1 NS3 protein was characterized using a thin-layer chromatography assay. We found that the DEN1 NS3 protein possesses some aspects of NTPase activity, which are distinct from those found in other flaviviruses. Although the NS3 protein was able to utilize all four ribonucleoside triphosphates as its substrates, the NS3 protein showed a distinct preference for purine triphosphates (i.e., ATP and GTP). The addition of poly(U) did not stimulate NTPase activity in DEN1 NS3 protein, which contrasts with the reports for other flaviviral NS3 proteins. However, NTPase activity was specifically stimulated by the viral NS5 protein, which was manifested by a more than twofold increase in the rate of ATP hydrolysis and a 25% increase in the yield of ADP at the end of a 120-min reaction. These data suggest that the NTPase activity of the NS3 protein may be regulated by the viral NS5 protein during virus replication. PMID:9657959

  4. Naturally occurring NS3-protease-inhibitor resistant mutant A156T in the liver of an untreated chronic hepatitis C patient.

    PubMed

    Cubero, Maria; Esteban, Juan Ignacio; Otero, Teresa; Sauleda, Silvia; Bes, Marta; Esteban, Rafael; Guardia, Jaume; Quer, Josep

    2008-01-20

    An increasing number of new hepatitis C virus NS3-protease inhibitors are being evaluated for the treatment of chronic hepatitis C. Treatment-induced selection of mutants conferring resistance to protease inhibitors has been shown both in vivo and in vitro. A specific mutation, A156T has been shown to confer high-level resistance to several such agents (BILN2061, VX-950, SCH446211 (SCH6) and SCH503034). Here we report the presence of the A156T mutation in close to 1% of NS3 sequences within the liver quasispecies of a chronic hepatitis C patient never treated with anti-NS3-protease inhibitors. PMID:18006035

  5. A comparison of 454 sequencing and clonal sequencing for the characterization of hepatitis C virus NS3 variants.

    PubMed

    Ho, Cynthia K Y; Welkers, Matthijs R A; Thomas, Xiomara V; Sullivan, James C; Kieffer, Tara L; Reesink, Henk W; Rebers, Sjoerd P H; de Jong, Menno D; Schinkel, Janke; Molenkamp, Richard

    2015-07-01

    We compared 454 amplicon sequencing with clonal sequencing for the characterization of intra-host hepatitis C virus (HCV) NS3 variants. Clonal and 454 sequences were obtained from 12 patients enrolled in a clinical phase I study for telaprevir, an NS3-4a protease inhibitor. Thirty-nine datasets were used to compare the consensus sequence, average pairwise distance, normalized Shannon entropy, phylogenetic tree topology and the number and frequency of variants derived from both sequencing techniques. In general, a good concordance was observed between both techniques for the majority of datasets. Discordant results were observed for 5 out of 39 clonal and 454 datasets, which could be attributed to primer-related selective amplification used for clonal sequencing. Both 454 and clonal datasets consisted of a few major variants and a large number of low-frequency variants. Telaprevir resistance-associated variants were observed in low frequencies and were detected more often by 454. We conclude that performance of 454 and clonal sequencing is comparable for the characterization of intra-host virus populations. Not surprisingly, 454 is superior for the detection of low frequency resistance-associated variants. However, despite the greater coverage, 454 failed to detect some low frequency variants detected by clonal sequencing. PMID:25818622

  6. Resistance Analyses of HCV NS3/4A Protease and NS5B Polymerase from Clinical Studies of Deleobuvir and Faldaprevir

    PubMed Central

    Berger, Kristi L.; Sarrazin, Christoph; Nelson, David R.; Scherer, Joseph; Sha, Nanshi; Marquis, Martin; Côté-Martin, Alexandra; Vinisko, Richard; Stern, Jerry O.; Mensa, Federico J.; Kukolj, George

    2016-01-01

    Background & Aim The resistance profile of anti-hepatitis C virus (HCV) agents used in combination is important to guide optimal treatment regimens. We evaluated baseline and treatment-emergent NS3/4A and NS5B amino-acid variants among HCV genotype (GT)-1a and -1b-infected patients treated with faldaprevir (HCV protease inhibitor), deleobuvir (HCV polymerase non-nucleoside inhibitor), and ribavirin in multiple clinical studies. Methods HCV NS3/4A and NS5B population sequencing (Sanger method) was performed on all baseline plasma samples (n = 1425 NS3; n = 1556 NS5B) and on post-baseline plasma samples from patients with virologic failure (n = 113 GT-1a; n = 221 GT-1b). Persistence and time to loss of resistance-associated variants (RAVs) was estimated using Kaplan–Meier analysis. Results Faldaprevir RAVs (NS3 R155 and D168) and deleobuvir RAVs (NS5B 495 and 496) were rare (<1%) at baseline. Virologic response to faldaprevir/deleobuvir/ribavirin was not compromised by common baseline NS3 polymorphisms (e.g. Q80K in 17.5% of GT-1a) or by NS5B A421V, present in 20% of GT-1a. In GT-1b, alanine at NS5B codon 499 (present in 15% of baseline sequences) was associated with reduced response. Treatment-emergent RAVs consolidated previous findings: NS3 R155 and D168 were key faldaprevir RAVs; NS5B A421 and P495 were key deleobuvir RAVs. Among on-treatment virologic breakthroughs, RAVs emerged in both NS3 and NS5B (>90%). Virologic relapse was associated with RAVs in both NS3 and NS5B (53% GT-1b; 52% GT-1b); some virologic relapses had NS3 RAVs only (47% GT-1a; 17% GT-1b). Median time to loss of GT-1b NS5B P495 RAVs post-treatment (5 months) was less than that of GT-1b NS3 D168 (8.5 months) and GT-1a R155 RAVs (11.5 months). Conclusion Faldaprevir and deleobuvir RAVs are more prevalent among virologic failures than at baseline. Treatment response was not compromised by common NS3 polymorphisms; however, alanine at NS5B amino acid 499 at baseline (wild-type in GT-1a

  7. The macroscopic rate of nucleic acid translocation by hepatitis C virus helicase NS3h is dependent on both sugar and base moieties.

    PubMed

    Khaki, Ali R; Field, Cassandra; Malik, Shuja; Niedziela-Majka, Anita; Leavitt, Stephanie A; Wang, Ruth; Hung, Magdeleine; Sakowicz, Roman; Brendza, Katherine M; Fischer, Christopher J

    2010-07-16

    The nonstructural protein 3 helicase (NS3h) of hepatitis C virus is a 3'-to-5' superfamily 2 RNA and DNA helicase that is essential for the replication of hepatitis C virus. We have examined the kinetic mechanism of the translocation of NS3h along single-stranded nucleic acid with bases uridylate (rU), deoxyuridylate (dU), and deoxythymidylate (dT), and have found that the macroscopic rate of translocation is dependent on both the base moiety and the sugar moiety of the nucleic acid, with approximate macroscopic translocation rates of 3 nt s(-1) (oligo(dT)), 35 nt s(-1) (oligo(dU)), and 42 nt s(-1) (oligo(rU)), respectively. We found a strong correlation between the macroscopic translocation rates and the binding affinity of the translocating NS3h protein for the respective substrates such that weaker affinity corresponded to faster translocation. The values of K(0.5) for NS3h translocation at a saturating ATP concentration are as follows: 3.3+/-0.4 microM nucleotide (poly(dT)), 27+/-2 microM nucleotide (poly(dU)), and 36+/-2 microM nucleotide (poly(rU)). Furthermore, results of the isothermal titration of NS3h with these oligonucleotides suggest that differences in TDeltaS(0) are the principal source of differences in the affinity of NS3h binding to these substrates. Interestingly, despite the differences in macroscopic translocation rates and binding affinities, the ATP coupling stoichiometries for NS3h translocation were identical for all three substrates (approximately 0.5 ATP molecule consumed per nucleotide translocated). This similar periodicity of ATP consumption implies a similar mechanism for NS3h translocation along RNA and DNA substrates. PMID:20451531

  8. Novel fullerene derivatives as dual inhibitors of Hepatitis C virus NS5B polymerase and NS3/4A protease.

    PubMed

    Kataoka, Hiroki; Ohe, Tomoyuki; Takahashi, Kyoko; Nakamura, Shigeo; Mashino, Tadahiko

    2016-10-01

    We evaluated the Hepatitis C virus (HCV) NS5B polymerase and HCV NS3/4A protease inhibition activities of a new set of proline-type fullerene derivatives. All of the compounds had the potential to inhibit both the enzymes, indicating that the fullerene derivatives may be dual inhibitors against NS5B and NS3/4A and could be novel lead compounds for the treatment of HCV infections. PMID:27597249

  9. Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication.

    PubMed Central

    Xu, J; Mendez, E; Caron, P R; Lin, C; Murcko, M A; Collett, M S; Rice, C M

    1997-01-01

    Members of the Flaviviridae encode a serine proteinase termed NS3 that is responsible for processing at several sites in the viral polyproteins. In this report, we show that the NS3 proteinase of the pestivirus bovine viral diarrhea virus (BVDV) (NADL strain) is required for processing at nonstructural (NS) protein sites 3/4A, 4A/4B, 4B/5A, and 5A/5B but not for cleavage at the junction between NS2 and NS3. Cleavage sites of the proteinase were determined by amino-terminal sequence analysis of the NS4A, NS4B, NS5A, and NS5B proteins. A conserved leucine residue is found at the P1 position of all four cleavage sites, followed by either serine (3/4A, 4B/5A, and 5A/5B sites) or alanine (4A/4B site) at the P1' position. Consistent with this cleavage site preference, a structural model of the pestivirus NS3 proteinase predicts a highly hydrophobic P1 specificity pocket. trans-Processing experiments implicate the 64-residue NS4A protein as an NS3 proteinase cofactor required for cleavage at the 4B/5A and 5A/5B sites. Finally, using a full-length functional BVDV cDNA clone, we demonstrate that a catalytically active NS3 serine proteinase is essential for pestivirus replication. PMID:9188600

  10. Hepatitis C virus NS3/4A protein interacts with ATM, impairs DNA repair and enhances sensitivity to ionizing radiation

    SciTech Connect

    Lai, Chao-Kuen; Jeng, King-Song; Machida, Keigo; Cheng, Yi-Sheng; Lai, Michael M.C.

    2008-01-20

    Hepatitis C virus (HCV) infection is frequently associated with the development of hepatocellular carcinomas and non-Hodgkin's B-cell lymphomas. Nonstructural protein 3 (NS3) of HCV possesses serine protease, nucleoside triphosphatase, and helicase activities, while NS4A functions as a cofactor for the NS3 serine protease. Here, we show that HCV NS3/4A interacts with the ATM (ataxia-telangiectasia mutated), a cellular protein essential for cellular response to irradiation. The expression of NS3/4A caused cytoplasmic translocation of either endogenous or exogenous ATM and delayed dephosphorylation of the phosphorylated ATM and {gamma}-H2AX following ionizing irradiation. As a result, the irradiation-induced {gamma}-H2AX foci persisted longer in the NS3/4A-expressing cells. Furthermore, these cells showed increased comet tail moment in single-cell electrophoresis assay, indicating increased double-strand DNA breaks. The cells harboring an HCV replicon also exhibited cytoplasmic localization of ATM and increased sensitivity to irradiation. These results demonstrate that NS3/4A impairs the efficiency of DNA repair by interacting with ATM and renders the cells more sensitive to DNA damage. This effect may contribute to HCV oncogenesis.

  11. Detection of dengue NS1 and NS3 proteins in placenta and umbilical cord in fetal and maternal death.

    PubMed

    Nunes, Priscila Conrado Guerra; Paes, Marciano Viana; de Oliveira, Carlos Alberto Basilio; Soares, Ana Carla Gomes; de Filippis, Ana Maria Bispo; Lima, Monique da Rocha Queiroz; de Barcelos Alves, Ada Maria; da Silva, Juliana Fernandes Amorim; de Oliveira Coelho, Janice Mery Chicarino; de Carvalho Rodrigues, Francisco das Chagas; Nogueira, Rita Maria Ribeiro; Dos Santos, Flávia Barreto

    2016-08-01

    In Brazil, dengue is a public health problem with the occurrence of explosive epidemics. This study reports maternal and fetal deaths due to dengue and which tissues of placenta and umbilical cord were analyzed by molecular methods and immunohistochemistry. The dengue NS3 and NS1 detection revealed the viral presence in different cells from placenta and umbilical cord. In the latter, DENV-2 was detected at a viral titer of 1,02 × 10(4) amounts of viral RNA. It was shown that the DENV markers analyzed here may be an alternative approach for dengue fatal cases investigation, especially involving maternal and fetal death. J. Med. Virol. 88:1448-1452, 2016. © 2016 Wiley Periodicals, Inc. PMID:26792253

  12. Crystal structure of Zika virus NS2B-NS3 protease in complex with a boronate inhibitor.

    PubMed

    Lei, Jian; Hansen, Guido; Nitsche, Christoph; Klein, Christian D; Zhang, Linlin; Hilgenfeld, Rolf

    2016-07-29

    The ongoing Zika virus (ZIKV) outbreak is linked to severe neurological disorders. ZIKV relies on its NS2B/NS3 protease for polyprotein processing; hence, this enzyme is an attractive drug target. The 2.7 angstrom; crystal structure of ZIKV protease in complex with a peptidomimetic boronic acid inhibitor reveals a cyclic diester between the boronic acid and glycerol. The P2 4-aminomethylphenylalanine moiety of the inhibitor forms a salt-bridge with the nonconserved Asp(83) of NS2B; ion-pairing between Asp(83) and the P2 residue of the substrate likely accounts for the enzyme's high catalytic efficiency. The unusual dimer of the ZIKV protease:inhibitor complex seen in the crystal may provide a model for assemblies formed at high local concentrations of protease at the endoplasmatic reticulum membrane, the site of polyprotein processing. PMID:27386922

  13. Further theoretical insight into the reaction mechanism of the hepatitis C NS3/NS4A serine protease

    NASA Astrophysics Data System (ADS)

    Martínez-González, José Ángel; Rodríguez, Alex; Puyuelo, María Pilar; González, Miguel; Martínez, Rodrigo

    2015-01-01

    The main reactions of the hepatitis C virus NS3/NS4A serine protease are studied using the second-order Møller-Plesset ab initio method and rather large basis sets to correct the previously reported AM1/CHARMM22 potential energy surfaces. The reaction efficiencies measured for the different substrates are explained in terms of the tetrahedral intermediate formation step (the rate-limiting process). The energies of the barrier and the corresponding intermediate are so close that the possibility of a concerted mechanism is open (especially for the NS5A/5B substrate). This is in contrast to the suggested general reaction mechanism of serine proteases, where a two-step mechanism is postulated.

  14. Molecular principles behind Boceprevir resistance due to mutations in hepatitis C NS3/4A protease.

    PubMed

    Nagpal, Neha; Goyal, Sukriti; Wahi, Divya; Jain, Ritu; Jamal, Salma; Singh, Aditi; Rana, Preeti; Grover, Abhinav

    2015-10-01

    The hepatitis C virus (HCV) infection is a primary cause of chronic hepatitis which eventually progresses to cirrhosis and in some instances might advance to hepatocellular carcinoma. According to the WHO report, HCV infects 130-150 million people globally and every year 350,000 to 500,000 people die from hepatitis C virus infection. Great achievement has been made in viral treatment evolution, after the development of HCV NS3/4A protease inhibitor (Boceprevir). However, efficacy of Boceprevir is compromised by the emergence of drug resistant variants. The molecular principle behind drug resistance of the protease mutants such as (V36M, T54S and R155K) is still poorly understood. Therefore in this study, we employed a series of computational strategies to analyze the binding of antiviral drug, Boceprevir to HCV NS3/4A protease mutants. Our results clearly demonstrate that the point mutations (V36M, T54S and R155K) in protease are associated with lowering of its binding affinity with Boceprevir. Exhaustive analysis of the simulated Boceprevir-bound wild and mutant complexes revealed variations in hydrophobic interactions, hydrogen bond occupancy and salt bridge interactions. Also, substrate envelope analysis scrutinized that the studied mutations reside outside the substrate envelope which may affect the Boceprevir affinity towards HCV protease but not the protease enzymatic activity. Furthermore, structural analyses of the binding site volume and flexibility show impairment in flexibility and stability of the binding site residues in mutant structures. In order to combat Boceprevir resistance, renovation of binding interactions between the drug and protease may be valuable. The structural insight from this study reveals the mechanism of the Boceprevir resistance and the results can be valuable for the design of new PIs with improved efficiency. PMID:26055089

  15. Isoform-specific roles of the GTPase activating protein Nadrin in cytoskeletal reorganization of platelets.

    PubMed

    Beck, S; Fotinos, A; Lang, F; Gawaz, M; Elvers, M

    2013-01-01

    Cytoskeletal reorganization of activated platelets plays a crucial role in hemostasis and thrombosis and implies activation of Rho GTPases. Rho GTPases are important regulators of cytoskeletal dynamics and function as molecular switches that cycle between an inactive and an active state. They are regulated by GTPase activating proteins (GAPs) that stimulate GTP hydrolysis to terminate Rho signaling. The regulation of Rho GTPases in platelets is not explored. A detailed characterization of Rho regulation is necessary to understand activation and inactivation of Rho GTPases critical for platelet activation and aggregation. Nadrin is a RhoGAP regulating cytoplasmic protein explored in the central nervous system. Five Nadrin isoforms are known that share a unique GAP domain, a serine/threonine/proline-rich domain, a SH3-binding motif and an N-terminal BAR domain but differ in their C-terminus. Here we identified Nadrin in platelets where it co-localizes to actin-rich regions and Rho GTPases. Different Nadrin isoforms selectively regulate Rho GTPases (RhoA, Cdc42 and Rac1) and cytoskeletal reorganization suggesting that - beside the GAP domain - the C-terminus of Nadrin determines Rho specificity and influences cell physiology. Furthermore, Nadrin controls RhoA-mediated stress fibre and focal adhesion formation. Spreading experiments on fibrinogen revealed strongly reduced cell adhesion upon Nadrin overexpression. Unexpectedly, the Nadrin BAR domain controls Nadrin-GAP activity and acts as a guidance domain to direct this GAP to its substrate at the plasma membrane. Our results suggest a critical role for Nadrin in the regulation of RhoA, Cdc42 and Rac1 in platelets and thus for platelet adhesion and aggregation. PMID:22975681

  16. The Combination of Grazoprevir, a Hepatitis C Virus (HCV) NS3/4A Protease Inhibitor, and Elbasvir, an HCV NS5A Inhibitor, Demonstrates a High Genetic Barrier to Resistance in HCV Genotype 1a Replicons.

    PubMed

    Lahser, Frederick C; Bystol, Karin; Curry, Stephanie; McMonagle, Patricia; Xia, Ellen; Ingravallo, Paul; Chase, Robert; Liu, Rong; Black, Todd; Hazuda, Daria; Howe, Anita Y M; Asante-Appiah, Ernest

    2016-05-01

    The selection of resistance-associated variants (RAVs) against single agents administered to patients chronically infected with hepatitis C virus (HCV) necessitates that direct-acting antiviral agents (DAAs) targeting multiple viral proteins be developed to overcome failure resulting from emergence of resistance. The combination of grazoprevir (formerly MK-5172), an NS3/4A protease inhibitor, and elbasvir (formerly MK-8742), an NS5A inhibitor, was therefore studied in genotype 1a (GT1a) replicon cells. Both compounds were independently highly potent in GT1a wild-type replicon cells, with 90% effective concentration (EC90) values of 0.9 nM and 0.006 nM for grazoprevir and elbasvir, respectively. No cross-resistance was observed when clinically relevant NS5A and NS3 RAVs were profiled against grazoprevir and elbasvir, respectively. Kinetic analyses of HCV RNA reduction over 14 days showed that grazoprevir and elbasvir inhibited prototypic NS5A Y93H and NS3 R155K RAVs, respectively, with kinetics comparable to those for the wild-type GT1a replicon. In combination, grazoprevir and elbasvir interacted additively in GT1a replicon cells. Colony formation assays with a 10-fold multiple of the EC90 values of the grazoprevir-elbasvir inhibitor combination suppressed emergence of resistant colonies, compared to a 100-fold multiple for the independent agents. The selected resistant colonies with the combination harbored RAVs that required two or more nucleotide changes in the codons. Mutations in the cognate gene caused greater potency losses for elbasvir than for grazoprevir. Replicons bearing RAVs identified from resistant colonies showed reduced fitness for several cell lines and may contribute to the activity of the combination. These studies demonstrate that the combination of grazoprevir and elbasvir exerts a potent effect on HCV RNA replication and presents a high genetic barrier to resistance. The combination of grazoprevir and elbasvir is currently approved for

  17. Dengue protease activity: the structural integrity and interaction of NS2B with NS3 protease and its potential as a drug target.

    PubMed

    Phong, Wai Y; Moreland, Nicole J; Lim, Siew P; Wen, Daying; Paradkar, Prasad N; Vasudevan, Subhash G

    2011-10-01

    Flaviviral NS3 serine proteases require the NS2B cofactor region (cNS2B) to be active. Recent crystal structures of WNV (West Nile virus) protease in complex with inhibitors revealed that cNS2B participates in the formation of the protease active site. No crystal structures of ternary complexes are currently available for DENV (dengue virus) to validate the role of cNS2B in active site formation. In the present study, a GST (glutathione transferase) fusion protein of DENV-2 cNS2B49-95 was used as a bait to pull down DENV-2 protease domain (NS3pro). The affinity of NS3pro for cNS2B was strong (equilibrium-binding constant <200 nM) and the heterodimeric complex displayed a catalytic efficiency similar to that of single-chain DENV-2 cNS2B/NS3pro. Various truncations and mutations in the cNS2B sequence showed that conformational integrity of the entire 47 amino acids is critical for protease activity. Furthermore, DENV-2 NS3 protease can be pulled down and transactivated by cNS2B cofactors from DENV-1, -3, -4 and WNV, suggesting that mechanisms for activation are conserved across the flavivirus genus. To validate NS2B as a potential target in allosteric inhibitor development, a cNS2B-specific human monoclonal antibody (3F10) was utilized. 3F10 disrupted the interaction between cNS2B and NS3 in vitro and reduced DENV viral replication in HEK (human embryonic kidney)-293 cells. This provides proof-of-concept for developing assays to find inhibitors that block the interaction between NS2B and NS3 during viral translation. PMID:21329491

  18. Binding specificity of polypeptide substrates in NS2B/NS3pro serine protease of dengue virus type 2: A molecular dynamics Study.

    PubMed

    Yotmanee, Pathumwadee; Rungrotmongkol, Thanyada; Wichapong, Kanin; Choi, Sy Bing; Wahab, Habibah A; Kungwan, Nawee; Hannongbua, Supot

    2015-07-01

    The pathogenic dengue virus (DV) is a growing global threat, particularly in South East Asia, for which there is no specific treatment available. The virus possesses a two-component (NS2B/NS3) serine protease that cleaves the viral precursor proteins. Here, we performed molecular dynamics simulations of the NS2B/NS3 protease complexes with six peptide substrates (capsid, intNS3, 2A/2B, 4B/5, 3/4A and 2B/3 containing the proteolytic site between P(1) and P(1)' subsites) of DV type 2 to compare the specificity of the protein-substrate binding recognition. Although all substrates were in the active conformation for cleavage reaction by NS2B/NS3 protease, their binding strength was somewhat different. The simulated results of intermolecular hydrogen bonds and decomposition energies suggested that among the ten substrate residues (P(5)-P(5)') the P(1) and P(2) subsites play a major role in the binding with the focused protease. The arginine residue at these two subsites was found to be specific preferential binding at the active site with a stabilization energy of <-10 kcal mol(-1). Besides, the P(3), P(1)', P(2)' and P(4)' subsites showed a less contribution in binding interaction (<-2 kcal mol(-1)). The catalytic water was detected nearby the carbonyl oxygen of the P(1) reacting center of the capsid, intNS3, 2A/2B and 4B/5 peptides. These results led to the order of absolute binding free energy (ΔGbind) between these substrates and the NS2B/NS3 protease ranked as capsid>intNS3>2A/2B>4B/5>3/4A>2B/3 in a relative correspondence with previous experimentally derived values. PMID:26086900

  19. Rho GTPase Transcriptome Analysis Reveals Oncogenic Roles for Rho GTPase-Activating Proteins in Basal-like Breast Cancers.

    PubMed

    Lawson, Campbell D; Fan, Cheng; Mitin, Natalia; Baker, Nicole M; George, Samuel D; Graham, David M; Perou, Charles M; Burridge, Keith; Der, Channing J; Rossman, Kent L

    2016-07-01

    The basal-like breast cancer (BLBC) subtype accounts for a disproportionately high percentage of overall breast cancer mortality. The current therapeutic options for BLBC need improvement; hence, elucidating signaling pathways that drive BLBC growth may identify novel targets for the development of effective therapies. Rho GTPases have previously been implicated in promoting tumor cell proliferation and metastasis. These proteins are inactivated by Rho-selective GTPase-activating proteins (RhoGAP), which have generally been presumed to act as tumor suppressors. Surprisingly, RNA-Seq analysis of the Rho GTPase signaling transcriptome revealed high expression of several RhoGAP genes in BLBC tumors, raising the possibility that these genes may be oncogenic. To evaluate this, we examined the roles of two of these RhoGAPs, ArhGAP11A (also known as MP-GAP) and RacGAP1 (also known as MgcRacGAP), in promoting BLBC. Both proteins were highly expressed in human BLBC cell lines, and knockdown of either gene resulted in significant defects in the proliferation of these cells. Knockdown of ArhGAP11A caused CDKN1B/p27-mediated arrest in the G1 phase of the cell cycle, whereas depletion of RacGAP1 inhibited growth through the combined effects of cytokinesis failure, CDKN1A/p21-mediated RB1 inhibition, and the onset of senescence. Random migration was suppressed or enhanced by the knockdown of ArhGAP11A or RacGAP1, respectively. Cell spreading and levels of GTP-bound RhoA were increased upon depletion of either RhoGAP. We have established that, via the suppression of RhoA, ArhGAP11A and RacGAP1 are both critical drivers of BLBC growth, and propose that RhoGAPs can act as oncogenes in cancer. Cancer Res; 76(13); 3826-37. ©2016 AACR. PMID:27216196

  20. Therapeutic effects of the Rho GTPase modulator CNF1 in a model of Parkinson's disease.

    PubMed

    Musilli, Marco; Ciotti, Maria Teresa; Pieri, Massimo; Martino, Assunta; Borrelli, Sonia; Dinallo, Vincenzo; Diana, Giovanni

    2016-10-01

    Recent evidence suggests an early involvement of dopaminergic (DA) processes and terminals in Parkinson's disease (PD). The arborization of neurons depends on the actin cytoskeleton, which in turn is regulated by small GTPases of the Rho family, encompassing Rho, Rac and Cdc42 subfamilies. Indeed, some reports point to a role for Rac and Cdc42 signaling in the pathophysiology of inherited parkinsonisms. We thus investigated the potential therapeutic effect of the modulation of cerebral Rho GTPases in PD. Cytotoxic necrotizing factor 1 (CNF1), a 114 kDa protein toxin produced by Escherichia coli, permanently activates RhoA, Rac1 and Cdc42 in intact cells. We report that the modulation of Rho GTPases by CNF1 results in hypertrophy of DA cell processes of cultured substantia nigra neurons, including increase in length, branching and varicosity. In vivo, the treatment corrects long-standing motor and biochemical asymmetries and restores degenerated nigrostriatal DA tissue after 6-hydroxydopamine lesion. We conclude that the pharmacological modulation of Rho GTPases shows neurorestorative potential and represents a promising avenue in the treatment PD. The study also suggests that naturally occurring molecules acting on Rho GTPase signaling, such as some bacterial protein toxins, might play a role in the development of PD. PMID:27350290

  1. Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on Its GTPase Activity

    PubMed Central

    Li, Lian-Feng; Yu, Jiahui; Li, Yongfeng; Wang, Jinghan; Li, Su; Zhang, Lingkai; Xia, Shui-Li; Yang, Qian; Wang, Xiao; Yu, Shaoxiong; Luo, Yuzi; Sun, Yuan; Zhu, Yan; Munir, Muhammad

    2016-01-01

    ABSTRACT Many viruses trigger the type I interferon (IFN) pathway upon infection, resulting in the transcription of hundreds of interferon-stimulated genes (ISGs), which define the antiviral state of the host. Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious viral disease endangering the pig industry in many countries. However, anti-CSFV ISGs are poorly documented. Here we screened 20 ISGs that are commonly induced by type I IFNs against CSFV in lentivirus-delivered cell lines, resulting in the identification of guanylate-binding protein 1 (GBP1) as a potent anti-CSFV ISG. We observed that overexpression of GBP1, an IFN-induced GTPase, remarkably suppressed CSFV replication, whereas knockdown of endogenous GBP1 expression by small interfering RNAs significantly promoted CSFV growth. Furthermore, we demonstrated that GBP1 acted mainly on the early phase of CSFV replication and inhibited the translation efficiency of the internal ribosome entry site of CSFV. In addition, we found that GBP1 was upregulated at the transcriptional level in CSFV-infected PK-15 cells and in various organs of CSFV-infected pigs. Coimmunoprecipitation and glutathione S-transferase (GST) pulldown assays revealed that GBP1 interacted with the NS5A protein of CSFV, and this interaction was mapped in the N-terminal globular GTPase domain of GBP1. Interestingly, the K51 of GBP1, which is crucial for its GTPase activity, was essential for the inhibition of CSFV replication. We showed further that the NS5A-GBP1 interaction inhibited GTPase activity, which was critical for its antiviral effect. Taking our findings together, GBP1 is an anti-CSFV ISG whose action depends on its GTPase activity. IMPORTANCE Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, only a few host restriction factors against CSFV

  2. Japanese encephalitis virus NS2B-NS3 protease induces caspase 3 activation and mitochondria-mediated apoptosis in human medulloblastoma cells.

    PubMed

    Yang, Tsuey-Ching; Shiu, Su-Lian; Chuang, Pei-Hsin; Lin, Ying-Ju; Wan, Lei; Lan, Yu-Ching; Lin, Cheng-Wen

    2009-07-01

    Japanese encephalitis virus (JEV) causes severe neurological diseases with a high fatality rate. Clinical, neurophysiological and radiological features of Japanese encephalitis JE patients showed that JEV infection resulted in widespread involvement of the nervous system, including thalamus, basal ganglia, brainstem, cerebellum, cerebral cortex and spinal cord. In this study, we characterized the apoptotic effect of JEV infection and its viral proteins on the TE671 human medulloblastoma cells. JEV replicated in TE671 cells, inducing caspase 3-mediated apoptosis in MOI- and time-dependent manners. Of viral proteins, co-expression of JEV NS3 protease with NS2B cofactor significantly induced higher degrees of apoptosis and triggered higher caspase 3 activities than single expression of E, NS1, NS2B or NS3 protease in human medulloblastoma cells. Moreover, JEV NS2B-NS3 protease induced reduction of mitochondrial membrane potential and release of mitochondrial cytochrome C, which were responsible for the mitochondria-mediated apoptosis. In addition, the production of reactive oxygen species production and activation of ASK1-p38 MAPK signaling pathway might be associated with JEV NS2B-NS3 protease-induced mitochondria-mediated apoptosis. The results demonstrated that the JEV infection and the co-expression of JEV NS3 protease with NS2B cofactor induced caspase 3 activation and mitochondria-mediated apoptosis in human medulloblastoma cells, being valuable insight for cellular and molecular levels of JEV pathogenesis. PMID:19463724

  3. In Situ Hepatitis C NS3 Protein Detection Is Associated with High Grade Features in Hepatitis C-Associated B-Cell Non-Hodgkin Lymphomas

    PubMed Central

    Rabiega, Pascaline; Molina, Thierry J.; Charlotte, Frédéric; Lazure, Thierry; Davi, Frédéric; Settegrana, Catherine; Berger, Françoise; Alric, Laurent; Cacoub, Patrice; Terrier, Benjamin; Suarez, Felipe; Sibon, David; Dupuis, Jehan; Feray, Cyrille; Tilly, Hervé; Pol, Stanislas; Deau Fischer, Bénédicte; Roulland, Sandrine; Thieblemont, Catherine; Leblond, Véronique; Carrat, Fabrice; Hermine, Olivier; Besson, Caroline

    2016-01-01

    Hepatitis C Virus (HCV) infection is associated with the B-cell non-Hodgkin lymphomas (NHL), preferentially marginal zone lymphomas (MZL) and diffuse large B-cell lymphomas (DLBCL). While chronic antigenic stimulation is a main determinant of lymphomagenesis in marginal zone lymphomas (MZL), a putative role of HCV infection of B-cells is supported by in vitro studies. We performed a pathological study within the "ANRS HC-13 LymphoC" observational study focusing on in situ expression of the oncogenic HCV non structural 3 (NS3) protein. Lympho-C study enrolled 116 HCV-positive patients with B-NHL of which 86 histological samples were collected for centralized review. Main histological subtypes were DLBCL (36%) and MZL (34%). Almost half of DLBCL (12/26) were transformed from underlying small B-cell lymphomas. NS3 immunostaining was found positive in 17 of 37 tested samples (46%). There was a striking association between NS3 detection and presence of high grade lymphoma features: 12 out of 14 DLBCL were NS3+ compared to only 4 out of 14 MZL (p = 0.006). Moreover, 2 among the 4 NS3+ MZL were enriched in large cells. Remarkably, this study supports a new mechanism of transformation with a direct oncogenic role of HCV proteins in the occurrence of high-grade B lymphomas. PMID:27257992

  4. Infectious Bovine Viral Diarrhea Virus (Strain NADL) RNA from Stable cDNA Clones: a Cellular Insert Determines NS3 Production and Viral Cytopathogenicity

    PubMed Central

    Mendez, Ernesto; Ruggli, Nicolas; Collett, Marc S.; Rice, Charles M.

    1998-01-01

    Bovine viral diarrhea virus (BVDV), strain NADL, was originally isolated from an animal with fatal mucosal disease. This isolate is cytopathic in cell culture and produces two forms of NS3-containing proteins: uncleaved NS2-3 and mature NS3. For BVDV NADL, the production of NS3, a characteristic of cytopathic BVDV strains, is believed to be a consequence of an in-frame insertion of a 270-nucleotide cellular mRNA sequence (called cIns) in the NS2 coding region. In this study, we constructed a stable full-length cDNA copy of BVDV NADL in a low-copy-number plasmid vector. As assayed by transfection of MDBK cells, uncapped RNAs transcribed from this template were highly infectious (>105 PFU/μg). The recovered virus was similar in plaque morphology, growth properties, polyprotein processing, and cytopathogenicity to the BVDV NADL parent. Deletion of cIns abolished processing at the NS2/NS3 site and produced a virus that was no longer cytopathic for MDBK cells. This deletion did not affect the efficiency of infectious virus production or viral protein production, but it reduced the level of virus-specific RNA synthesis and accumulation. Thus, cIns not only modulates NS3 production but also upregulates RNA replication relative to an isogenic noncytopathic derivative lacking the insert. These results raise the possibility of a linkage between enhanced BVDV NADL RNA replication and virus-induced cytopathogenicity. PMID:9573238

  5. The NS3 protein of Rice hoja blanca tenuivirus suppresses RNA silencing in plant and insect hosts by efficiently binding both siRNAs and miRNAs

    PubMed Central

    Hemmes, Hans; Lakatos, Lóránt; Goldbach, Rob; Burgyán, József; Prins, Marcel

    2007-01-01

    RNA silencing plays a key role in antiviral defense as well as in developmental processes in plants and insects. Negative strand RNA viruses such as the plant virus Rice hoja blanca tenuivirus (RHBV) replicate in plants and in their insect transmission vector. Like most plant-infecting viruses, RHBV encodes an RNA silencing suppressor, the NS3 protein, and here it is demonstrated that this protein is capable of suppressing RNA silencing in both plants and insect cells. Biochemical analyses showed that NS3 efficiently binds siRNA as well as miRNA molecules. Binding of NS3 is greatly influenced by the size of small RNA molecules, as 21 nucleotide (nt) siRNA molecules are bound > 100 times more efficiently than 26 nt species. Competition assays suggest that the activity of NS3 is based on binding to siRNAs prior to strand separation during the assembly of the RNA-induced silencing complex. In addition, NS3 has a high affinity for miRNA/miRNA* duplexes, indicating that its activity might also interfere with miRNA-regulated gene expression in both insects and plants. PMID:17513697

  6. Rho GTPase signalling in cell migration

    PubMed Central

    Ridley, Anne J

    2015-01-01

    Cells migrate in multiple different ways depending on their environment, which includes the extracellular matrix composition, interactions with other cells, and chemical stimuli. For all types of cell migration, Rho GTPases play a central role, although the relative contribution of each Rho GTPase depends on the environment and cell type. Here, I review recent advances in our understanding of how Rho GTPases contribute to different types of migration, comparing lamellipodium-driven versus bleb-driven migration modes. I also describe how cells migrate across the endothelium. In addition to Rho, Rac and Cdc42, which are well known to regulate migration, I discuss the roles of other less-well characterized members of the Rho family. PMID:26363959

  7. Cleavage of the dengue virus polyprotein at the NS3/NS4A and NS4B/NS5 junctions is mediated by viral protease NS2B-NS3, whereas NS4A/NS4B may be processed by a cellular protease.

    PubMed Central

    Cahour, A; Falgout, B; Lai, C J

    1992-01-01

    The cleavage mechanism utilized for processing of the NS3-NS4A-NS4B-NS5 domain of the dengue virus polyprotein was studied by using the vaccinia virus expression system. Recombinant vaccinia viruses vNS2B-NS3-NS4A-NS4B-NS5, vNS3-NS4A-NS4B-NS5, vNS4A-NS4B-NS5, and vNS4B-NS5 were constructed. These recombinants were used to infect cells, and the labeled lysates were analyzed by immunoprecipitation. Recombinant vNS2B-NS3-NS4A-NS4B-NS5 expressed the authentic NS3 and NS5 proteins, but the other recombinants produced uncleaved polyproteins. These findings indicate that NS2B is required for processing of the downstream nonstructural proteins, including the NS3/NS4A and NS4B/NS5 junctions, both of which contain a dibasic amino acid sequence preceding the cleavage site. The flavivirus NS4A/NS4B cleavage site follows a long hydrophobic sequence. The polyprotein NS4A-NS4B-NS5 was cleaved at the NS4A/NS4B junction in the absence of other dengue virus functions. One interpretation for this finding is that NS4A/NS4B cleavage is mediated by a host protease, presumably a signal peptidase. Although vNS3-NS4A-NS4B-NS5 expressed only the polyprotein, earlier results demonstrated that cleavage at the NS4A/NS4B junction occurred when an analogous recombinant, vNS3-NS4A-84%NS4B, was expressed. Thus, it appears that uncleaved NS3 plus NS5 inhibit NS4A/NS4B cleavage presumably because the putative signal sequence is not accessible for recognition by the responsible protease. Finally, recombinants that expressed an uncleaved NS4B-NS5 polyprotein, such as vNS4A-NS4B-NS5 or vNS4B-NS5, produced NS5 when complemented with vNS2B-30%NS3 or with vNS2B plus v30%NS3. These results indicate that cleavage at the NS4B/NS5 junction can be mediated by NS2B and NS3 in trans. Images PMID:1531368

  8. The metabolism and disposition of a potent inhibitor of hepatitis C virus NS3/4A protease.

    PubMed

    Monteagudo, E; Fonsi, M; Chu, X; Bleasby, K; Evers, R; Pucci, V; Orsale, M V; Cianetti, S; Ferrara, M; Harper, S; Laufer, R; Rowley, M; Summa, V

    2010-12-01

    Compound A ((1aR,5S,8S,10R,22aR)-5-tert-butyl-N-{(1R,2S)-1-[(cyclopropylsulfonyl)carbamoyl]-2-ethenylcyclopropyl}-14-methoxy-3,6-dioxo-1,1a,3,4,5,6,9,10,18,19,20,21,22,22a-tetradecahydro-8H-7,10-methanocyclopropa[18,19][1,10,3,6]dioxadiazacyclononadecino[12,11-b]quinoline-8-carboxamide) is a prototype of a series of subnanomolar inhibitors of genotypes 1, 2, and 3 hepatitis C virus (HCV) NS3/4A proteases. HCV NS3/4A protease inhibitors have demonstrated high antiviral effects in patients with chronic HCV infection and are likely to form a key component of future HCV therapy. Compound A showed excellent liver exposure in rats, which is essential for compounds intended to treat HCV. The compound was mainly eliminated intact in bile and showed greater than dose proportional systemic exposure in rats. Compound A demonstrated time- and temperature-dependent uptake into rat and human hepatocytes and proved to be a substrate for rat hepatic uptake transporter Oatp1b2 and for human hepatic uptake transporters OATP1B1 and OATP1B3. The liver selectivity observed for this compound is likely to be due to transporter-mediated hepatic uptake together with moderate passive permeability. Metabolism was mainly CYP3A-mediated and generated a reactive epoxide on the vinylcyclopropyl sulfonamide moiety that could be quenched by glutathione. Similar metabolic profiles of Compound A were obtained in liver microsomes of rats and humans. The oral bioavailability at 5 mg/kg was low due to extensive hepatic first-pass effect but clearly the intestinal absorption was enough to deliver a high amount of the compound to the liver. The metabolism and disposition properties of Compound A are particularly attractive to support its evaluation as a drug candidate for the treatment of hepatitis C. PMID:20925584

  9. Are There Rab GTPases in Archaea?

    PubMed Central

    Surkont, Jaroslaw; Pereira-Leal, Jose B.

    2016-01-01

    A complex endomembrane system is one of the hallmarks of Eukaryotes. Vesicle trafficking between compartments is controlled by a diverse protein repertoire, including Rab GTPases. These small GTP-binding proteins contribute identity and specificity to the system, and by working as molecular switches, trigger multiple events in vesicle budding, transport, and fusion. A diverse collection of Rab GTPases already existed in the ancestral Eukaryote, yet, it is unclear how such elaborate repertoire emerged. A novel archaeal phylum, the Lokiarchaeota, revealed that several eukaryotic-like protein systems, including small GTPases, are present in Archaea. Here, we test the hypothesis that the Rab family of small GTPases predates the origin of Eukaryotes. Our bioinformatic pipeline detected multiple putative Rab-like proteins in several archaeal species. Our analyses revealed the presence and strict conservation of sequence features that distinguish eukaryotic Rabs from other small GTPases (Rab family motifs), mapping to the same regions in the structure as in eukaryotic Rabs. These mediate Rab-specific interactions with regulators of the REP/GDI (Rab Escort Protein/GDP dissociation Inhibitor) family. Sensitive structure-based methods further revealed the existence of REP/GDI-like genes in Archaea, involved in isoprenyl metabolism. Our analysis supports a scenario where Rabs differentiated into an independent family in Archaea, interacting with proteins involved in membrane biogenesis. These results further support the archaeal nature of the eukaryotic ancestor and provide a new insight into the intermediate stages and the evolutionary path toward the complex membrane-associated signaling circuits that characterize the Ras superfamily of small GTPases, and specifically Rab proteins. PMID:27034425

  10. Are There Rab GTPases in Archaea?

    PubMed

    Surkont, Jaroslaw; Pereira-Leal, Jose B

    2016-07-01

    A complex endomembrane system is one of the hallmarks of Eukaryotes. Vesicle trafficking between compartments is controlled by a diverse protein repertoire, including Rab GTPases. These small GTP-binding proteins contribute identity and specificity to the system, and by working as molecular switches, trigger multiple events in vesicle budding, transport, and fusion. A diverse collection of Rab GTPases already existed in the ancestral Eukaryote, yet, it is unclear how such elaborate repertoire emerged. A novel archaeal phylum, the Lokiarchaeota, revealed that several eukaryotic-like protein systems, including small GTPases, are present in Archaea. Here, we test the hypothesis that the Rab family of small GTPases predates the origin of Eukaryotes. Our bioinformatic pipeline detected multiple putative Rab-like proteins in several archaeal species. Our analyses revealed the presence and strict conservation of sequence features that distinguish eukaryotic Rabs from other small GTPases (Rab family motifs), mapping to the same regions in the structure as in eukaryotic Rabs. These mediate Rab-specific interactions with regulators of the REP/GDI (Rab Escort Protein/GDP dissociation Inhibitor) family. Sensitive structure-based methods further revealed the existence of REP/GDI-like genes in Archaea, involved in isoprenyl metabolism. Our analysis supports a scenario where Rabs differentiated into an independent family in Archaea, interacting with proteins involved in membrane biogenesis. These results further support the archaeal nature of the eukaryotic ancestor and provide a new insight into the intermediate stages and the evolutionary path toward the complex membrane-associated signaling circuits that characterize the Ras superfamily of small GTPases, and specifically Rab proteins. PMID:27034425

  11. Phylogenetic distribution of translational GTPases in bacteria

    PubMed Central

    Margus, Tõnu; Remm, Maido; Tenson, Tanel

    2007-01-01

    Background Translational GTPases are a family of proteins in which GTPase activity is stimulated by the large ribosomal subunit. Conserved sequence features allow members of this family to be identified. Results To achieve accurate protein identification and grouping we have developed a method combining searches with Hidden Markov Model profiles and tree based grouping. We found all the genes for translational GTPases in 191 fully sequenced bacterial genomes. The protein sequences were grouped into nine subfamilies. Analysis of the results shows that three translational GTPases, the translation factors EF-Tu, EF-G and IF2, are present in all organisms examined. In addition, several copies of the genes encoding EF-Tu and EF-G are present in some genomes. In the case of multiple genes for EF-Tu, the gene copies are nearly identical; in the case of multiple EF-G genes, the gene copies have been considerably diverged. The fourth translational GTPase, LepA, the function of which is currently unknown, is also nearly universally conserved in bacteria, being absent from only one organism out of the 191 analyzed. The translation regulator, TypA, is also present in most of the organisms examined, being absent only from bacteria with small genomes. Surprisingly, some of the well studied translational GTPases are present only in a very small number of bacteria. The translation termination factor RF3 is absent from many groups of bacteria with both small and large genomes. The specialized translation factor for selenocysteine incorporation – SelB – was found in only 39 organisms. Similarly, the tetracycline resistance proteins (Tet) are present only in a small number of species. Proteins of the CysN/NodQ subfamily have acquired functions in sulfur metabolism and production of signaling molecules. The genes coding for CysN/NodQ proteins were found in 74 genomes. This protein subfamily is not confined to Proteobacteria, as suggested previously but present also in many other

  12. Discovery of pyrazinone based compounds that potently inhibit the drug-resistant enzyme variant R155K of the hepatitis C virus NS3 protease.

    PubMed

    Belfrage, Anna Karin; Abdurakhmanov, Eldar; Kerblom, Eva; Brandt, Peter; Oshalim, Anna; Gising, Johan; Skogh, Anna; Neyts, Johan; Danielson, U Helena; Sandström, Anja

    2016-06-15

    Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors with variations in the C-terminus. Biochemical evaluation was performed using genotype 1a, both the wild-type and the drug resistant enzyme variant, R155K. Surprisingly, compounds without an acidic sulfonamide retained good inhibition, challenging our previous molecular docking model. Moreover, selected compounds in this series showed nanomolar potency against R155K NS3 protease; which generally confer resistance to all HCV NS3 protease inhibitors approved or in clinical trials. These results further strengthen the potential of this novel substance class, being very different to the approved drugs and clinical candidates, in the development of inhibitors less sensitive to drug resistance. PMID:27160057

  13. [Immunogenicity and heterologous protection in mice with a recombinant adenoviral-based vaccine carrying a hepatitis C virus truncated NS3 and core fusion protein].

    PubMed

    Guan, Jie; Deng, Yao; Chen, Hong; Yang, Yang; Wen, Bo; Tan, Wenjie

    2015-01-01

    To develop a safe and broad-spectrum effective hepatitis C virus (HCV) T cell vaccine,we constructed the recombinant adenovirus-based vaccine that carried the hepatitis C virus truncated NS3 and core fusion proteins. The expression of the fusion antigen was confirmed by in vitro immunofluorescence and western blotting assays. Our results indicated that this vaccine not only stimulated antigen-specific antibody responses,but also activated strong NS3-specific T cell immune responses. NS3-specific IFN-γ+ and TNF-α+ CD4+ T cell subsets were also detected by a intracellular cytokine secretion assay. In a surrogate challenge assay based on a recombinant heterologous HCV (JFH1,2a) vaccinia virus,the recombinant adenovirus-based vaccine was capable of eliciting effective levels of cross-protection. These findings have im- portant implications for the study of HCV immune protection and the future development of a novel vaccine. PMID:25997323

  14. Small RAB GTPases Regulate Multiple Steps of Mitosis.

    PubMed

    Miserey-Lenkei, Stéphanie; Colombo, María I

    2016-01-01

    GTPases of the RAB family are key regulators of multiple steps of membrane trafficking. Several members of the RAB GTPase family have been implicated in mitotic progression. In this review, we will first focus on the function of endosome-associated RAB GTPases reported in early steps of mitosis, spindle pole maturation, and during cytokinesis. Second, we will discuss the role of Golgi-associated RAB GTPases at the metaphase/anaphase transition and during cytokinesis. PMID:26925400

  15. Small RAB GTPases Regulate Multiple Steps of Mitosis

    PubMed Central

    Miserey-Lenkei, Stéphanie; Colombo, María I.

    2016-01-01

    GTPases of the RAB family are key regulators of multiple steps of membrane trafficking. Several members of the RAB GTPase family have been implicated in mitotic progression. In this review, we will first focus on the function of endosome-associated RAB GTPases reported in early steps of mitosis, spindle pole maturation, and during cytokinesis. Second, we will discuss the role of Golgi-associated RAB GTPases at the metaphase/anaphase transition and during cytokinesis. PMID:26925400

  16. Clinical pharmacology profile of boceprevir, a hepatitis C virus NS3 protease inhibitor: focus on drug-drug interactions.

    PubMed

    Khalilieh, Sauzanne; Feng, Hwa-Ping; Hulskotte, Ellen G J; Wenning, Larissa A; Butterton, Joan R

    2015-06-01

    Boceprevir is a potent, orally administered ketoamide inhibitor that targets the active site of the hepatitis C virus (HCV) non-structural (NS) 3 protease. The addition of boceprevir to peginterferon plus ribavirin resulted in higher rates of sustained virologic response (SVR) than for peginterferon plus ribavirin alone in phase III studies in both previously treated and untreated patients with HCV infection. Because boceprevir is metabolized by metabolic routes common to many other drugs, and is an inhibitor of cytochrome P450 (CYP) 3A4/5, there is a high potential for drug-drug interactions when boceprevir is administered with other therapies, particularly when treating patients with chronic HCV infection who are often receiving other medications concomitantly. Boceprevir is no longer widely used in the US or EU due to the introduction of second-generation treatments for HCV infection. However, in many other geographic regions, first-generation protease inhibitors such as boceprevir continue to form an important treatment option for patients with HCV infection. This review summarizes the interactions between boceprevir and other therapeutic agents commonly used in this patient population, indicating dose adjustment requirements where needed. Most drug interactions do not affect boceprevir plasma concentrations to a clinically meaningful extent, and thus efficacy is likely to be maintained when boceprevir is coadministered with the majority of other therapeutics. Overall, the drug-drug interaction profile of boceprevir suggests that this agent is suitable for use in a wide range of HCV-infected patients receiving concomitant therapies. PMID:25787025

  17. In Silico Screening, Alanine Mutation, and DFT Approaches for Identification of NS2B/NS3 Protease Inhibitors

    PubMed Central

    Balajee, R.; Srinivasadesikan, V.; Sakthivadivel, M.; Gunasekaran, P.

    2016-01-01

    To identify the ligand that binds to a target protein with high affinity is a nontrivial task in computer-assisted approaches. Antiviral drugs have been identified for NS2B/NS3 protease enzyme on the mechanism to cleave the viral protein using the computational tools. The consequence of the molecular docking, free energy calculations, and simulation protocols explores the better ligand. It provides in-depth structural insights with the catalytic triad of His51, Asp75, Ser135, and Gly133. The MD simulation was employed here to predict the stability of the complex. The alanine mutation has been performed and its stability was monitored by using the molecular dynamics simulation. The minimal RMSD value suggests that the derived complexes are close to equilibrium. The DFT outcome reveals that the HOMO-LUMO gap of Ligand19 is 2.86 kcal/mol. Among the considered ligands, Ligand19 shows the lowest gap and it is suggested that the HOMO of Ligand19 may transfer the electrons to the LUMO in the active regions. The calculated binding energy of Ligand19 using the DFT method is in good agreement with the docking studies. The pharmacological activity of ligand was performed and satisfies Lipinski rule of 5. Moreover, the computational results are compared with the available IC50 values of experimental results. PMID:27057355

  18. Boceprevir, an NS3 serine protease inhibitor of hepatitis C virus, for the treatment of HCV infection.

    PubMed

    Mederacke, Ingmar; Wedemeyer, Heiner; Manns, Michael P

    2009-02-01

    Boceprevir is an HCV NS3 (non-structural protein 3) serine protease inhibitor being developed by Schering-Plough Corp as a capsule formulation. In pharmacokinetic studies, boceprevir was adequately absorbed, with the most effective mode of administration appearing to be a three-times-daily regimen. In phase I clinical trials, monotherapy with boceprevir led to a distinct viral load reduction. In phase Ib combination trials of boceprevir with PEGylated IFNalpha2b and ribavirin, the reduction in viral replication was further increased. Early data reported from phase II clinical trials have been promising, suggesting a rapid early HCV-RNA reduction. Phase III trials for the drug began in 2008. Results available to date have demonstrated the compound to be well tolerated, with adverse events that were within the range of current standard-of-care therapy. Thus, boceprevir may have the potential to increase sustained virological response rates and possibly also to shorten duration of therapy; data from ongoing clinical trials are awaited. PMID:19197796

  19. Approaches of targeting Rho GTPases in cancer drug discovery

    PubMed Central

    Lin, Yuan; Zheng, Yi

    2016-01-01

    Introduction Rho GTPases are master regulators of actomyosin structure and dynamics and play pivotal roles in a variety of cellular processes including cell morphology, gene transcription, cell cycle progression and cell adhesion. Because aberrant Rho GTPase signaling activities are widely associated with human cancer, key components of Rho GTPase signaling pathways have attracted increasing interest as potential therapeutic targets. Similar to Ras, Rho GTPases themselves were, until recently, deemed “undruggable” because of structure-function considerations. Several approaches to interfere with Rho GTPase signaling have been explored and show promise as new ways for tackling cancer cells. Areas covered This review focuses on the recent progress in targeting the signaling activities of three prototypical Rho GTPases, i.e. RhoA, Rac1, and Cdc42. The authors describe the involvement of these Rho GTPases, their key regulators and effectors in cancer. Furthermore, the authors discuss the current approaches for rationally targeting aberrant Rho GTPases along their signaling cascades, upstream and downstream of Rho GTPases and posttranslational modifications at a molecular level. Expert opinion To date, while no clinically effective drugs targeting Rho GTPase signaling for cancer treatment are available, tool compounds and lead drugs that pharmacologically inhibit Rho GTPase pathways have shown promise. Small molecule inhibitors targeting Rho GTPase signaling may add new treatment options for future precision cancer therapy, particularly in combination with other anti-cancer agents. PMID:26087073

  20. Identification of amino acids involved in recognition by dengue virus NS3-specific, HLA-DR15-restricted cytotoxic CD4+ T-cell clones.

    PubMed Central

    Zeng, L; Kurane, I; Okamoto, Y; Ennis, F A; Brinton, M A

    1996-01-01

    The majority of T-cell clones derived from a donor who experienced dengue illness following receipt of a live experimental dengue virus type 3 (DEN3) vaccine cross-reacted with all four serotypes of dengue virus, but some were serotype specific or only partially cross-reactive. The nonstructural protein, NS3, was immuno-dominant in the CD4+ T-cell response of this donor. The epitopes of four NS3-specific T-cell clones were analyzed. JK15 and JK13 recognized only DEN3 NS3, while JK44 recognized DEN1, DEN2, and DEN3 NS3 and JK5 recognized DEN1, DEN3, and West Nile virus NS3. The epitopes recognized by these clones on the DEN3 NS3 protein were localized with recombinant vaccinia viruses expressing truncated regions of the NS3 gene, and then the minimal recognition sequence was mapped with synthetic peptides. Amino acids critical for T-cell recognition were assessed by using peptides with amino acid substitutions. One of the serotype-specific clones (JK13) and the subcomplex- and flavivirus-cross-reactive clone (JK5) recognized the same core epitope, WITDFVGKTVW. The amino acid at the sixth position of this epitope is critical for recognition by both clones. Sequence analysis of the T-cell receptors of these two clones showed that they utilize different VP chains. The core epitopes for the four HLA-DR15-restricted CD4+ CTL clones studied do not contain motifs similar to those proposed by previous studies on endogenous peptides eluted from HLA-DR15 molecules. However, the majority of these dengue virus NS3 core epitopes have a positive amino acid (K or R) at position 8 or 9. Our results indicate that a single epitope can induce T cells with different virus specificities despite the restriction of these T cells by the same HLA-DR15 allele. This finding suggests a previously unappreciated level of complexity for interactions between human T-cell receptors and viral epitopes with very similar sequences on infected cells. PMID:8627790

  1. Potent Inhibitors of the Hepatitis C Virus NS3 Protease: Design and Synthesis of Macrocyclic Substrate-Based [beta]-Strand Mimics

    SciTech Connect

    Goudreau, Nathalie; Brochu, Christian; Cameron, Dale R.; Duceppe, Jean-Simon; Faucher, Anne-Marie; Ferland, Jean-Marie; Grand-Maître, Chantal; Poirier, Martin; Simoneau, Bruno; Tsantrizos, Youla S.

    2008-06-30

    The virally encoded NS3 protease is essential to the life cycle of the hepatitis C virus (HCV), an important human pathogen causing chronic hepatitis, cirrhosis of the liver, and hepatocellular carcinoma. The design and synthesis of 15-membered ring {beta}-strand mimics which are capable of inhibiting the interactions between the HCV NS3 protease enzyme and its polyprotein substrate will be described. The binding interactions between a macrocyclic ligand and the enzyme were explored by NMR and molecular dynamics, and a model of the ligand/enzyme complex was developed.

  2. Invited review: Small GTPases and their GAPs.

    PubMed

    Mishra, Ashwini K; Lambright, David G

    2016-08-01

    Widespread utilization of small GTPases as major regulatory hubs in many different biological systems derives from a conserved conformational switch mechanism that facilitates cycling between GTP-bound active and GDP-bound inactive states under control of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which accelerate slow intrinsic rates of activation by nucleotide exchange and deactivation by GTP hydrolysis, respectively. Here we review developments leading to current understanding of intrinsic and GAP catalyzed GTP hydrolytic reactions in small GTPases from structural, molecular and chemical mechanistic perspectives. Despite the apparent simplicity of the GTPase cycle, the structural bases underlying the hallmark hydrolytic reaction and catalytic acceleration by GAPs are considerably more diverse than originally anticipated. Even the most fundamental aspects of the reaction mechanism have been challenging to decipher. Through a combination of experimental and in silico approaches, the outlines of a consensus view have begun to emerge for the best studied paradigms. Nevertheless, recent observations indicate that there is still much to be learned. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 431-448, 2016. PMID:26972107

  3. In-depth phylogenetic analysis of hepatitis C virus subtype 1a and occurrence of 80K and associated polymorphisms in the NS3 protease.

    PubMed

    Santos, André F; Bello, Gonzalo; Vidal, Luãnna L; Souza, Suiane L; Mir, Daiana; Soares, Marcelo A

    2016-01-01

    HCV genetic diversity is high and impacts disease progression, treatment and drug resistance. HCV subtype 1a is divided in two clades (I and II), and the 80 K natural polymorphism in the viral NS3 protease is prevalent in clade I. Paradoxically, countries dominated by this clade have contrasting frequencies of 80 K. Over 2,000 HCV 1a NS3 sequences were retrieved from public databases representing Europe, Oceania and the Americas. Sequences were aligned with HCV reference sequences and subjected to phylogenetic analysis to investigate the relative presence of different subtype 1a clades and NS3 protease mutations. HCV-1a sequences split into clades I and II. Clade I was further structured into three subclades, IA to C. Sub-clade IA prevailed in the U.S., while subclade IC was major in Brazil. The NS3 80 K polymorphism was associated with subclade IA, but nearly absent in subclades IB and IC, a pattern similarly seen for the 91S/T compensatory mutation. Three HCV-1a-I sub-clades have been identified, with different frequencies in distinct regions. The 80 K and 91A/S mutations were associated with subclade IA, which provide an explanation for the disparities seen in simeprevir resistance profiles of countries dominated by HCV 1a-I, like the U.S. and Brazil. PMID:27531254

  4. In-depth phylogenetic analysis of hepatitis C virus subtype 1a and occurrence of 80K and associated polymorphisms in the NS3 protease

    PubMed Central

    Santos, André F.; Bello, Gonzalo; Vidal, Luãnna L.; Souza, Suiane L.; Mir, Daiana; Soares, Marcelo A.

    2016-01-01

    HCV genetic diversity is high and impacts disease progression, treatment and drug resistance. HCV subtype 1a is divided in two clades (I and II), and the 80 K natural polymorphism in the viral NS3 protease is prevalent in clade I. Paradoxically, countries dominated by this clade have contrasting frequencies of 80 K. Over 2,000 HCV 1a NS3 sequences were retrieved from public databases representing Europe, Oceania and the Americas. Sequences were aligned with HCV reference sequences and subjected to phylogenetic analysis to investigate the relative presence of different subtype 1a clades and NS3 protease mutations. HCV-1a sequences split into clades I and II. Clade I was further structured into three subclades, IA to C. Sub-clade IA prevailed in the U.S., while subclade IC was major in Brazil. The NS3 80 K polymorphism was associated with subclade IA, but nearly absent in subclades IB and IC, a pattern similarly seen for the 91S/T compensatory mutation. Three HCV-1a-I sub-clades have been identified, with different frequencies in distinct regions. The 80 K and 91A/S mutations were associated with subclade IA, which provide an explanation for the disparities seen in simeprevir resistance profiles of countries dominated by HCV 1a-I, like the U.S. and Brazil. PMID:27531254

  5. A Pan-GTPase Inhibitor as a Molecular Probe

    PubMed Central

    Hong, Lin; Guo, Yuna; BasuRay, Soumik; Agola, Jacob O.; Romero, Elsa; Simpson, Denise S.; Schroeder, Chad E.; Simons, Peter; Waller, Anna; Garcia, Matthew; Carter, Mark; Ursu, Oleg; Gouveia, Kristine; Golden, Jennifer E.; Aubé, Jeffrey; Wandinger-Ness, Angela; Sklar, Larry A.

    2015-01-01

    Overactive GTPases have often been linked to human diseases. The available inhibitors are limited and have not progressed far in clinical trials. We report here a first-in-class small molecule pan-GTPase inhibitor discovered from a high throughput screening campaign. The compound CID1067700 inhibits multiple GTPases in biochemical, cellular protein and protein interaction, as well as cellular functional assays. In the biochemical and protein interaction assays, representative GTPases from Rho, Ras, and Rab, the three most generic subfamilies of the GTPases, were probed, while in the functional assays, physiological processes regulated by each of the three subfamilies of the GTPases were examined. The chemical functionalities essential for the activity of the compound were identified through structural derivatization. The compound is validated as a useful molecular probe upon which GTPase-targeting inhibitors with drug potentials might be developed. PMID:26247207

  6. Processing of the yellow fever virus nonstructural polyprotein: a catalytically active NS3 proteinase domain and NS2B are required for cleavages at dibasic sites.

    PubMed Central

    Chambers, T J; Grakoui, A; Rice, C M

    1991-01-01

    The vaccinia virus-T7 transient expression system was used to further examine the role of the NS3 proteinase in processing of the yellow fever (YF) virus nonstructural polyprotein in BHK cells. YF virus-specific polyproteins and cleavage products were identified by immunoprecipitation with region-specific antisera, by size, and by comparison with authentic YF virus polypeptides. A YF virus polyprotein initiating with a signal sequence derived from the E protein fused to the N terminus of NS2A and extending through the N-terminal 356 amino acids of NS5 exhibited processing at the 2A-2B, 2B-3, 3-4A, 4A-4B, and 4B-5 cleavage sites. Similar results were obtained with polyproteins whose N termini began within NS2A (position 110) or with NS2B. When the NS3 proteinase domain was inactivated by replacing the proposed catalytic Ser-138 with Ala, processing at all sites was abolished. The results suggest that an active NS3 proteinase domain is necessary for cleavage at the diabasic nonstructural cleavage sites and that cleavage at the proposed 4A-4B signalase site requires prior cleavage at the 4B-5 site. Cleavages were not observed with a polyprotein whose N terminus began with NS3, but cleavage at the 4B-5 site could be restored by supplying the the NS2B protein in trans. Several experimental results suggested that trans cleavage at the 4B-5 site requires association of NS2B and the NS3 proteinase domain. Coexpression of different proteinases and catalytically inactive polyprotein substrates revealed that trans cleavage at the 2B-3 and 4B-5 sites was relatively efficient when compared with trans cleavage at the 2A-2B and 3-4A sites. Images PMID:1833562

  7. Design and docking studies of peptide inhibitors as potential antiviral drugs for dengue virus ns2b/ns3 protease.

    PubMed

    Velmurugan, Devadasan; Mythily, Udhayakumar; Rao, Kutumba

    2014-01-01

    Dengue virus (DENV), one of the members of genus Flavivirus is emerging as a global threat to human health. It had led to the emergence of dengue fever (flu-like illness), dengue shock syndrome, and the most severe dengue hemorrhagic fever (severe dengue with bleeding abnormalities). As Dengue hemorrhage diseases are the life-threatening ones, attempts are being made worldwide to design inhibitors for DENV-2 NS2B-NS3 protease. NS2B/NS3 protease plays a vital role in the replication of dengue virus. The trypsin-like serine protease domain of NS3 contains the functional catalytic triad His-51, Asp-75, and Ser-135 in the N-terminal region. Inhibition of the NS3 protease activity is expected to prevent the propagation of dengue virus. Current drug discovery methods are largely inefficient and thus relatively ineffective in tackling the growing threat to public health presented by emerging and remerging viral pathogens. Recently, there has been a need of interest in peptides and their mimetics as potential antagonists for dengue protease because these small peptides are unlikely to invoke an immune response since they fall below the immunogenic threshold. They are often potent and display fewer toxicity issues than small-molecule compounds as a result of high specificity. This study was conducted to design peptides as enzyme inhibitors of dengue virus NS3 protease through computational approach. Crystallographic structure of dengue protease was retrieved from Protein Data Bank (PDBID: 2FOM) and docked with the peptides and the results are analyzed. From the docking studies reported in this paper, tetrapeptide (Lys-Gly-Pro-Glu), pentapeptide (Ser-Ile-Lys-Phe-Ala) and hexapeptide (Ala-Ile-Lys-Lys-Phe-Ser) with glide energy -70.0 kcal/mol, -72.2 kcal/mol and - 80.4 kcal/mol respectively show promising results which can be considered for further optimization and in vitro studies. PMID:23855663

  8. Preclinical Characterization of BI 201335, a C-Terminal Carboxylic Acid Inhibitor of the Hepatitis C Virus NS3-NS4A Protease ▿ †

    PubMed Central

    White, Peter W.; Llinàs-Brunet, Montse; Amad, Ma'an; Bethell, Richard C.; Bolger, Gordon; Cordingley, Michael G.; Duan, Jianmin; Garneau, Michel; Lagacé, Lisette; Thibeault, Diane; Kukolj, George

    2010-01-01

    BI 201335 is a hepatitis C virus (HCV) NS3-NS4A (NS3 coexpressed with NS4A) protease inhibitor that has been shown to have potent clinical antiviral activity. It is a highly optimized noncovalent competitive inhibitor of full-length NS3-NS4A proteases of HCV genotypes 1a and 1b with Ki values of 2.6 and 2.0 nM, respectively. Ki values of 2 to 230 nM were measured against the NS3-NS4A proteases of HCV genotypes 2 to 6, whereas it was a very weak inhibitor of cathepsin B and showed no measurable inhibition of human leukocyte elastase. BI 201335 was also shown to be a potent inhibitor of HCV RNA replication in vitro with 50% effective concentrations (EC50s) of 6.5 and 3.1 nM obtained in genotype 1a and 1b replicon assays. Combinations of BI 201335 with either interferon or ribavirin had additive effects in replicon assays. BI 201335 had good permeability in Caco-2 cell assays and high metabolic stability after incubation with human, rat, monkey, and dog liver microsomes. Its good absorption, distribution, metabolism, and excretion (ADME) profile in vitro, as well as in rat, monkey, and dog, predicted good pharmacokinetics (PK) in humans. Furthermore, drug levels were significantly higher in rat liver than in plasma, suggesting that distribution to the target organ may be especially favorable. BI 201335 is a highly potent and selective NS3-NS4A protease inhibitor with good in vitro and animal ADME properties, consistent with its good human PK profile, and shows great promise as a treatment for HCV infection. PMID:20823284

  9. Preclinical characterization of BI 201335, a C-terminal carboxylic acid inhibitor of the hepatitis C virus NS3-NS4A protease.

    PubMed

    White, Peter W; Llinàs-Brunet, Montse; Amad, Ma'an; Bethell, Richard C; Bolger, Gordon; Cordingley, Michael G; Duan, Jianmin; Garneau, Michel; Lagacé, Lisette; Thibeault, Diane; Kukolj, George

    2010-11-01

    BI 201335 is a hepatitis C virus (HCV) NS3-NS4A (NS3 coexpressed with NS4A) protease inhibitor that has been shown to have potent clinical antiviral activity. It is a highly optimized noncovalent competitive inhibitor of full-length NS3-NS4A proteases of HCV genotypes 1a and 1b with K(i) values of 2.6 and 2.0 nM, respectively. K(i) values of 2 to 230 nM were measured against the NS3-NS4A proteases of HCV genotypes 2 to 6, whereas it was a very weak inhibitor of cathepsin B and showed no measurable inhibition of human leukocyte elastase. BI 201335 was also shown to be a potent inhibitor of HCV RNA replication in vitro with 50% effective concentrations (EC(50)s) of 6.5 and 3.1 nM obtained in genotype 1a and 1b replicon assays. Combinations of BI 201335 with either interferon or ribavirin had additive effects in replicon assays. BI 201335 had good permeability in Caco-2 cell assays and high metabolic stability after incubation with human, rat, monkey, and dog liver microsomes. Its good absorption, distribution, metabolism, and excretion (ADME) profile in vitro, as well as in rat, monkey, and dog, predicted good pharmacokinetics (PK) in humans. Furthermore, drug levels were significantly higher in rat liver than in plasma, suggesting that distribution to the target organ may be especially favorable. BI 201335 is a highly potent and selective NS3-NS4A protease inhibitor with good in vitro and animal ADME properties, consistent with its good human PK profile, and shows great promise as a treatment for HCV infection. PMID:20823284

  10. Coevolution of RAC Small GTPases and their Regulators GEF Proteins

    PubMed Central

    Jiménez-Sánchez, Alejandro

    2016-01-01

    RAC proteins are small GTPases involved in important cellular processes in eukaryotes, and their deregulation may contribute to cancer. Activation of RAC proteins is regulated by DOCK and DBL protein families of guanine nucleotide exchange factors (GEFs). Although DOCK and DBL proteins act as GEFs on RAC proteins, DOCK and DBL family members are evolutionarily unrelated. To understand how DBL and DOCK families perform the same function on RAC proteins despite their unrelated primary structure, phylogenetic analyses of the RAC, DBL, and DOCK families were implemented, and interaction patterns that may suggest a coevolutionary process were searched. Interestingly, while RAC and DOCK proteins are very well conserved in humans and among eukaryotes, DBL proteins are highly divergent. Moreover, correlation analyses of the phylogenetic distances of RAC and GEF proteins and covariation analyses between residues in the interacting domains showed significant coevolution rates for both RAC–DOCK and RAC–DBL interactions. PMID:27226705

  11. Mitochondrial Fusion in Yeast Requires the Transmembrane GTPase Fzo1p

    PubMed Central

    Hermann, Greg J.; Thatcher, John W.; Mills, John P.; Hales, Karen G.; Fuller, Margaret T.; Nunnari, Jodi; Shaw, Janet M.

    1998-01-01

    Membrane fusion is required to establish the morphology and cellular distribution of the mitochondrial compartment. In Drosophila, mutations in the fuzzy onions (fzo) GTPase block a developmentally regulated mitochondrial fusion event during spermatogenesis. Here we report that the yeast orthologue of fuzzy onions, Fzo1p, plays a direct and conserved role in mitochondrial fusion. A conditional fzo1 mutation causes the mitochondrial reticulum to fragment and blocks mitochondrial fusion during yeast mating. Fzo1p is a mitochondrial integral membrane protein with its GTPase domain exposed to the cytoplasm. Point mutations that alter conserved residues in the GTPase domain do not affect Fzo1p localization but disrupt mitochondrial fusion. Suborganellar fractionation suggests that Fzo1p spans the outer and is tightly associated with the inner mitochondrial membrane. This topology may be required to coordinate the behavior of the two mitochondrial membranes during the fusion reaction. We propose that the fuzzy onions family of transmembrane GTPases act as molecular switches to regulate a key step in mitochondrial membrane docking and/or fusion. PMID:9786948

  12. Rag GTPase in amino acid signaling.

    PubMed

    Kim, Joungmok; Kim, Eunjung

    2016-04-01

    Rag small GTPases were identified as the sixth subfamily of Ras-related GTPases. Compelling evidence suggests that Rag heterodimer (RagA/B and RagC/D) plays an important role in amino acid signaling toward mechanistic target of rapamycin complex 1 (mTORC1), which is a central player in the control of cell growth in response to a variety of environmental cues, including growth factors, cellular energy/oxygen status, and amino acids. Upon amino acid stimulation, active Rag heterodimer (RagA/B(GTP)-RagC/D(GDP)) recruits mTORC1 to the lysosomal membrane where Rheb resides. In this review, we provide a current understanding on the amino acid-regulated cell growth control via Rag-mTORC1 with recently identified key players, including Ragulator, v-ATPase, and GATOR complexes. Moreover, the functions of Rag in physiological systems and in autophagy are discussed. PMID:26781224

  13. Small GTPases as regulators of cell division

    PubMed Central

    Militello, Rodrigo; Colombo, María I.

    2013-01-01

    The superfamily of small GTPases serves as a signal transducer to regulate a diverse array of cellular functions. The members of this superfamily are structurally and functionally classified into at least 5 groups (Ras, Rho/Rac, Rab, Arf, and Ran) and they are involved in the control of cell proliferation and differentiation, regulation of the actin cytoskeleton, membrane trafficking, and nuclear transport. It is widely reported that members of the Rab family participate in the control of intracellular membrane trafficking through the interaction with specific effector molecules. However, many Rabs and other small GTPases have also been shown to function in cell division. In this review, we discuss current knowledge about Rab proteins regulating different stages of the cell cycle, such as the congregation and segregation of chromosomes (during metaphase) and the final stage of cell division known as cytokinesis, in which a cell is cleaved originating 2 daughter cells. PMID:24265858

  14. Key steps in the structure-based optimization of the hepatitis C virus NS3/4A protease inhibitor SCH503034.

    PubMed

    Madison, Vincent; Prongay, Andrew J; Guo, Zhuyan; Yao, Nanhua; Pichardo, John; Fischmann, Thierry; Strickland, Corey; Myers, Joseph; Weber, Patricia C; Beyer, Brian M; Ingram, Richard; Hong, Zhi; Prosise, Winifred W; Ramanathan, Lata; Taremi, S Shane; Yarosh-Tomaine, Taisa; Zhang, Rumin; Senior, Mary; Yang, Rong Sheng; Malcolm, Bruce; Arasappan, Ashok; Bennett, Frank; Bogen, Stephane L; Chen, Kevin; Jao, Edwin; Liu, Yi Tsung; Lovey, Raymond G; Saksena, Anil K; Venkatraman, Srikanth; Girijavallabhan, Viyyoor; Njoroge, F George

    2008-05-01

    The structures of both native and S139A holo-HCV NS3/4A protease domain were solved to high resolution. Subsequently, structures were determined for a series of ketoamide inhibitors in complex with the protease. The changes in the inhibitor potency were correlated with changes in the buried surface area upon binding the inhibitor to the active site. The largest contributions to the binding energy arise from the hydrophobic interactions of the P1 and P2 groups as they bind to the S1 and S2 pockets. This correlation of the changes in potency with increased buried surface area contributed directly to the design of a potent tripeptide inhibitor of the HCV NS3/4A protease, which is currently in clinical trials. PMID:18421139

  15. Key steps in the structure-based optimization of the hepatitis C virus NS3/4A protease inhibitor SCH503034

    PubMed Central

    Madison, Vincent; Prongay, Andrew J.; Guo, Zhuyan; Yao, Nanhua; Pichardo, John; Fischmann, Thierry; Strickland, Corey; Myers Jr, Joseph; Weber, Patricia C.; Beyer, Brian M.; Ingram, Richard; Hong, Zhi; Prosise, Winifred W.; Ramanathan, Lata; Taremi, S. Shane; Yarosh-Tomaine, Taisa; Zhang, Rumin; Senior, Mary; Yang, Rong-Sheng; Malcolm, Bruce; Arasappan, Ashok; Bennett, Frank; Bogen, Stephane L.; Chen, Kevin; Jao, Edwin; Liu, Yi-Tsung; Lovey, Raymond G.; Saksena, Anil K.; Venkatraman, Srikanth; Girijavallabhan, Viyyoor; Njoroge, F. George

    2008-01-01

    The structures of both native and S139A holo-HCV NS3/4A protease domain were solved to high resolution. Subsequently, structures were determined for a series of ketoamide inhibitors in complex with the protease. The changes in the inhibitor potency were correlated with changes in the buried surface area upon binding the inhibitor to the active site. The largest contributions to the binding energy arise from the hydrophobic interactions of the P1 and P2 groups as they bind to the S1 and S2 pockets. This correlation of the changes in potency with increased buried surface area contributed directly to the design of a potent tripeptide inhibitor of the HCV NS3/4A protease, which is currently in clinical trials. PMID:18421139

  16. Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

    PubMed Central

    Romano, Keith P.; Ali, Akbar; Royer, William E.; Schiffer, Celia A.

    2010-01-01

    Hepatitis C virus infects an estimated 180 million people worldwide, prompting enormous efforts to develop inhibitors targeting the essential NS3/4A protease. Resistance against the most promising protease inhibitors, telaprevir, boceprevir, and ITMN-191, has emerged in clinical trials. In this study, crystal structures of the NS3/4A protease domain reveal that viral substrates bind to the protease active site in a conserved manner defining a consensus volume, or substrate envelope. Mutations that confer the most severe resistance in the clinic occur where the inhibitors protrude from the substrate envelope, as these changes selectively weaken inhibitor binding without compromising the binding of substrates. These findings suggest a general model for predicting the susceptibility of protease inhibitors to resistance: drugs designed to fit within the substrate envelope will be less susceptible to resistance, as mutations affecting inhibitor binding would simultaneously interfere with the recognition of viral substrates. PMID:21084633

  17. Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions.

    PubMed Central

    Bartenschlager, R; Ahlborn-Laake, L; Mous, J; Jacobsen, H

    1993-01-01

    We have studied processing of the nonstructural (NS) polyprotein of the hepatitis C virus. A series of cDNAs corresponding to predicted NS2/3/4 or NS3/4 regions were constructed, and processing of the polyproteins was studied in an in vitro transcription-translation system. We report that a catalytically active serine-type proteinase is encoded by the NS3 region. Substitution of the serine residue of the putative catalytic triad (H, D, and S) by alanine blocked cleavage at the NS3/4 junction, while processing between NS2 and NS3 was not affected. Thus, cleavage at the NS2/3 junction is mediated either by cellular enzymes or by an NS-2 inherent proteinase activity. Deletion analysis of an NS3/4 cDNA construct mapped the amino terminus of the enzymatically active proteinase between amino acids 1049 and 1065 of the polyprotein. As internal deletions of variable segments of the presumed helicase domain prevented processing at the NS314 junction, a continuous NS3 region appears to be required for processing at this site. To analyze hepatitis C virus polyprotein cleavage in vivo, recombinant vaccinia viruses expressing NS2/3/4 or NS3/4/5 proteins were generated. In agreement with the in vitro data, cleavage between NS2 and NS3 was independent of a catalytically active NS3 proteinase, whereas substitution of the active-site serine residue by the amino acid alanine completely blocked processing at the NS3/4 and NS4/5 junctions. These results demonstrate that NS3 encodes the viral proteinase essential for generating the amino termini of NS4 and NS5. Images PMID:8389908

  18. Sustained Virologic Response at 24 Weeks after the End of Treatment Is a Better Predictor for Treatment Outcome in Real-World HCV-Infected Patients Treated by HCV NS3/4A Protease Inhibitors with Peginterferon plus Ribavirin

    PubMed Central

    Kanda, Tatsuo; Nakamoto, Shingo; Sasaki, Reina; Nakamura, Masato; Yasui, Shin; Haga, Yuki; Ogasawara, Sadahisa; Tawada, Akinobu; Arai, Makoto; Mikami, Shigeru; Imazeki, Fumio; Yokosuka, Osamu

    2016-01-01

    Background. Direct-acting antiviral agents against HCV with or without peginterferon plus ribavirin result in higher eradication rates of HCV and shorter treatment duration. We examined which is better for predicting persistent virologic response, the assessment of serum HCV RNA at 12 or 24 weeks after the end of treatment for predicting sustained virologic response (SVR12 or SVR24, respectively) in patients treated by HCV NS3/4A protease inhibitors with peginterferon plus ribavirin. Methods. In all, 149 Japanese patients infected with HCV genotype 1b treated by peginterferon plus ribavirin with telaprevir or simeprevir were retrospectively analyzed: 59 and 90 patients were treated with telaprevir- and simeprevir-including regimens, respectively. HCV RNA was measured by TaqMan HCV Test, version 2.0, real-time PCR assay. SVR12 or SVR24, respectively, was defined as HCV RNA negativity at 12 or 24 weeks after ending treatment. Results. Total SVR rates were 78.0% and 66.7% in the telaprevir and simeprevir groups, respectively. In the telaprevir group, all 46 patients with SVR12 finally achieved SVR24. In the simeprevir group, 60 (93.8%) of the total 64 patients with SVR12 achieved SVR24, with the other 4 patients all being previous-treatment relapsers. Conclusions. SVR12 was suitable for predicting persistent virologic response in almost all cases. In simeprevir-including regimens, SVR12 could not always predict persistent virologic response. Clinicians should use SVR24 for predicting treatment outcome in the use of HCV NS3/4A protease inhibitors with peginterferon plus ribavirin for any group of real-world patients chronically infected with HCV. PMID:27076789

  19. Crystallization and preliminary X-ray analysis of RabX3, a tandem GTPase from Entamoeba histolytica.

    PubMed

    Kumar Srivastava, Vijay; Chandra, Mintu; Datta, Sunando

    2014-07-01

    Ras superfamily GTPases regulate signalling pathways that control multiple biological processes by modulating the GTP/GDP cycle. Various Rab GTPases, which are the key regulators of vesicular trafficking pathways, play a vital role in the survival and virulence of the enteric parasite Entamoeba histolytica. The Rab GTPases act as binary molecular switches that utilize the conformational changes associated with the GTP/GDP cycle to elicit responses from target proteins and thereby regulate a broad spectrum of cellular processes including cell proliferation, cytoskeletal assembly, nuclear transport and intracellular membrane trafficking in eukaryotes. Entamoeba histolytica RabX3 (EhRabX3) is a unique GTPase in the amoebic genome, the only member in the eukaryotic Ras superfamily that harbours tandem G-domains and shares only 8-16% sequence identity with other GTPases. Recent studies suggested that EhRabX3 binds to a single guanine nucleotide through its N-terminal G-domain (NTD), while the C-terminal G-domain (CTD) plays a potential role in binding of the nucleotide to the NTD. Thus, understanding the intermolecular regulation between the two GTPase domains is expected to reveal valuable information on the overall action of EhRabX3. To provide structural insights into the inclusive action of this unique GTPase, EhRabX3 was crystallized by successive micro-seeding using the vapour-diffusion method. A complete data set was collected to 3.3 Å resolution using a single native EhRabX3 crystal at 100 K on BM14 at the ESRF, Grenoble, France. The crystal belonged to monoclinic space group C2, with unit-cell parameters a=198.6, b=119.3, c=89.2 Å, β=103.1°. Preliminary analysis of the data using the Matthews Probability Calculator suggested the presence of four to six molecules in the asymmetric unit. PMID:25005092

  20. Simple Indirect Enzyme-Linked Immunosorbent Assay to Detect Antibodies Against Bovine Viral Diarrhea Virus, Based on Prokaryotically Expressed Recombinant MBP-NS3 Protein

    PubMed Central

    Mahmoodi, Pezhman; Seyfi Abad Shapouri, Masoud Reza; Ghorbanpour, Masoud; Haji Hajikolaei, Mohammad Rahim; Lotfi, Mohsen; Pourmahdi Boroujeni, Mahdi; Daghari, Maryam

    2015-01-01

    Background: Bovine viral diarrhea (BVD) is an economically important disease of cattle distributed worldwide. Diagnosis of BVD relies on laboratory-based detection of its viral causing agent or virus specific antibodies and the most common laboratory method for this purpose is Enzyme-Linked Immunosorbent Assay (ELISA). Objectives: The current study was aimed to develop a simple indirect ELISA to detect antibodies against Bovine Viral Diarrhea Virus (BVDV) in the sera of infected cattle. Materials and Methods: A new simple indirect ELISA method was developed to detect BVDV infection by prokaryotically (Escherichia coli, BL21 strain) expressed recombinant whole nonstructural protein 3 (NS3) of BVDV (NADL strain). Four hundred bovine serum samples were evaluated by the newly developed NS3-ELISA and virus neutralization test (VNT) as the gold standard method to diagnose BVD. Among these samples, 289 sera had been previously tested by a commercial ELISA kit. Results: Statistical analyses showed a very high correlation between the results of the developed NS3-ELISA and VNT (kappa coefficient = 0.935, P < 0.001), with the relative sensitivity and specificity of 94% and 98.8%, respectively. There was also a high correlation between the results of NS3-ELISA and the commercial ELISA kit (kappa coefficient = 0.802, P < 0.001) with the relative sensitivity and specificity of 90.72% and 91.15%, respectively. Conclusions: The newly developed simple indirect ELISA showed high sensitivity and specificity with respect to VNT. Developing such a simple, sensitive, and specific ELISA which is much less expensive than the available commercial ELISA kits can improve the detection of BVDV infections, help to eliminate the disease from herds, and decrease economic losses caused by this disease. PMID:25964844

  1. Expression, Purification, and Evaluation of Diagnostic Potential and Immunogenicity of a Recombinant NS3 Protein from All Serotypes of Dengue Virus

    PubMed Central

    Álvarez-Rodríguez, Laura Mónica; Ramos-Ligonio, Angel; Rosales-Encina, José Luis; Martínez-Cázares, María Teresa; Parissi-Crivelli, Aurora; López-Monteon, Aracely

    2012-01-01

    Dengue is one of the major public health concerns in the world. Since all the four serotypes are actively circulating in Mexico, there is a need to develop an efficient diagnosis system to improve case management of the patients. There exist few studies evaluating the use of the NS3 protein as a protective antigen against dengue virus (DENV). In this paper we show the expression of a recombinant NS3 protein from all serotypes of dengue virus (GST-DVNS3-1-4) and report a reliable “in-house detection system” for the diagnosis of dengue infection which was field-tested in a small village (Tezonapa) in the state of Veracruz, Mexico. The fusion proteins were immunogenic, inducing antibodies to be able to recognize to antigens up to a 1 : 3200 dilution. The purified proteins were used to develop an in-house detection system (ELISA) and were further tested with a panel of 239 serum samples. The in-house results were in excellent agreement with the commercial kits with κ = 0.934 ± 0.064 (95%  CI = 0.808–1.061), and κ = 0.872 ± 0.048 (95%  CI = 0.779–0.965) for IgM and IgG, respectively. The agreement between the NS1 antigen detection versus the rNS3 ELISA, κ = 0.837 ± 0.066 (95%  CI = 0.708–0.966), was very good. Thus, these results demonstrate that recombinant NS3 proteins have potential in early diagnosis of dengue infections. PMID:23258983

  2. Expression of NS3/NS4A Proteins of Hepatitis C Virus in Huh7 Cells Following Engineering Its Eukaryotic Expression Vector

    PubMed Central

    Behzadi, Mohammad Amin; Alborzi, Abdolvahab; Pouladfar, Gholamreza; Dianatpour, Mehdi; Ziyaeyan, Mazyar

    2015-01-01

    Background: Although the development of novel therapeutic regimens to combat hepatitis C virus (HCV) infection have been speeded up with successful results, no efficient vaccines exist yet. Objectives: This study aimed to construct a eukaryotic expression vector encoding nonstructural proteins, NS3/NS4A, of HCV genotype 3a, and evaluate its expression on Huh7 cell surface. Materials and Methods: The NS3/NS4A sequence was isolated from a patient with HCV-3a chronic infection, cloned into intermediate vector pTZ57R/T, and then used for engineering a mammalian expression vector, pDisplay, to direct the respective protein to the secretory pathway and anchor it to the plasma membrane. The expression of the protein in Huh7 cell, which was transiently transfected with the vector using Lipofectamine, was determined by immunocytochemical staining assay with fluorescein isothiocyanate (FITC)-conjugated antibodies to the HA/myc tags located besides the fusion fragment. Results: The results showed that the fragment was successfully amplified and cloned into a eukaryotic expression vector. Sequencing and enzyme digestion analysis confirmed the cloned gene completion and its correct position in the pDisply-NS3/NS4A plasmid. Immunocytochemical staining revealed that the target protein was expressed as a membrane-anchored protein in the Huh7 cells. Conclusions: This study can serve as a fundamental experiment for the construction of a NS3/NS4A eukaryotic expression vector and its expression in mammalian cells. Further research is underway to evaluate the fragment immunogenicity in lab animal models. PMID:26862385

  3. Preclinical Profile of VX-950, a Potent, Selective, and Orally Bioavailable Inhibitor of Hepatitis C Virus NS3-4A Serine Protease

    PubMed Central

    Perni, Robert B.; Almquist, Susan J.; Byrn, Randal A.; Chandorkar, Gurudatt; Chaturvedi, Pravin R.; Courtney, Lawrence F.; Decker, Caroline J.; Dinehart, Kirk; Gates, Cynthia A.; Harbeson, Scott L.; Heiser, Angela; Kalkeri, Gururaj; Kolaczkowski, Elaine; Lin, Kai; Luong, Yu-Ping; Rao, B. Govinda; Taylor, William P.; Thomson, John A.; Tung, Roger D.; Wei, Yunyi; Kwong, Ann D.; Lin, Chao

    2006-01-01

    VX-950 is a potent, selective, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3-4A serine protease, and it demonstrated excellent antiviral activity both in genotype 1b HCV replicon cells (50% inhibitory concentration [IC50] = 354 nM) and in human fetal hepatocytes infected with genotype 1a HCV-positive patient sera (IC50 = 280 nM). VX-950 forms a covalent but reversible complex with the genotype 1a HCV NS3-4A protease in a slow-on, slow-off process with a steady-state inhibition constant (Ki*) of 7 nM. Dissociation of the covalent enzyme-inhibitor complex of VX-950 and genotype 1a HCV protease has a half-life of almost an hour. A >4-log10 reduction in the HCV RNA levels was observed after a 2-week incubation of replicon cells with VX-950, with no rebound of viral RNA observed after withdrawal of the inhibitor. In several animal species, VX-950 exhibits a favorable pharmacokinetic profile with high exposure in the liver. In a recently developed HCV protease mouse model, VX-950 showed excellent inhibition of HCV NS3-4A protease activity in the liver. Therefore, the overall preclinical profile of VX-950 supports its candidacy as a novel oral therapy against hepatitis C. PMID:16495249

  4. Two Distinct Hepatitis C Virus Genotype 1a Clades Have Different Geographical Distribution and Association With Natural Resistance to NS3 Protease Inhibitors.

    PubMed

    De Luca, Andrea; Di Giambenedetto, Simona; Lo Presti, Alessandra; Sierra, Saleta; Prosperi, Mattia; Cella, Eleonora; Giovanetti, Marta; Torti, Carlo; Caudai, Cinzia; Vicenti, Ilaria; Saladini, Francesco; Almi, Paolo; Grima, Pierfrancesco; Blanc, Pierluigi; Fabbiani, Massimiliano; Rossetti, Barbara; Gagliardini, Roberta; Kaiser, Rolf; Ciccozzi, Massimo; Zazzi, Maurizio

    2015-04-01

    Background.  Hepatitis C virus (HCV) genotype 1 is the most prevalent worldwide. Subtype 1a, compared with 1b, shows lower response rates and higher propensity to select for drug resistance to NS3 and selected NS5A and nonnucleoside NS5B inhibitors. Two distinct clades of subtype 1a have been described. Methods.  Using Bayesian methodology, we performed a time-scaled phylogeny reconstruction of clade separation and characterized the geographic distribution, phylodynamics, and association with natural resistance variants of NS3 sequences from 362 patients carrying subtype 1a HCV. Results.  All sequences segregated in 2 clearly distinct clades. Clade I showed an earlier origin from the common ancestor compared with clade II. Clade I virus was more prevalent in non-European countries, represented mostly by United States, compared with European (75.7% vs 49.3%; P < .001). The prevalence of the natural NS3 variant Q80K, associated with resistance to the macrocyclic protease inhibitor simeprevir, was detected in 51.6% of clade I and 0% of clade II (P < .001); clade I showed a lower genetic barrier for Q80K, whereas no sign of selective pressure at any protease inhibitor resistance-associated codon was detected. Conclusions.  Hepatitis C virus subtype 1a clades have a clearly different distribution in Europe and the United States, and the natural resistance mutation Q80K is exclusively associated with clade I. PMID:26213689

  5. Two Distinct Hepatitis C Virus Genotype 1a Clades Have Different Geographical Distribution and Association With Natural Resistance to NS3 Protease Inhibitors

    PubMed Central

    De Luca, Andrea; Di Giambenedetto, Simona; Lo Presti, Alessandra; Sierra, Saleta; Prosperi, Mattia; Cella, Eleonora; Giovanetti, Marta; Torti, Carlo; Caudai, Cinzia; Vicenti, Ilaria; Saladini, Francesco; Almi, Paolo; Grima, Pierfrancesco; Blanc, Pierluigi; Fabbiani, Massimiliano; Rossetti, Barbara; Gagliardini, Roberta; Kaiser, Rolf; Ciccozzi, Massimo; Zazzi, Maurizio

    2015-01-01

    Background. Hepatitis C virus (HCV) genotype 1 is the most prevalent worldwide. Subtype 1a, compared with 1b, shows lower response rates and higher propensity to select for drug resistance to NS3 and selected NS5A and nonnucleoside NS5B inhibitors. Two distinct clades of subtype 1a have been described. Methods. Using Bayesian methodology, we performed a time-scaled phylogeny reconstruction of clade separation and characterized the geographic distribution, phylodynamics, and association with natural resistance variants of NS3 sequences from 362 patients carrying subtype 1a HCV. Results. All sequences segregated in 2 clearly distinct clades. Clade I showed an earlier origin from the common ancestor compared with clade II. Clade I virus was more prevalent in non-European countries, represented mostly by United States, compared with European (75.7% vs 49.3%; P < .001). The prevalence of the natural NS3 variant Q80K, associated with resistance to the macrocyclic protease inhibitor simeprevir, was detected in 51.6% of clade I and 0% of clade II (P < .001); clade I showed a lower genetic barrier for Q80K, whereas no sign of selective pressure at any protease inhibitor resistance-associated codon was detected. Conclusions. Hepatitis C virus subtype 1a clades have a clearly different distribution in Europe and the United States, and the natural resistance mutation Q80K is exclusively associated with clade I. PMID:26213689

  6. Exploring resistance mechanisms of HCV NS3/4A protease mutations to MK5172: insight from molecular dynamics simulations and free energy calculations.

    PubMed

    Guan, Yan; Sun, Huiyong; Pan, Peichen; Li, Youyong; Li, Dan; Hou, Tingjun

    2015-09-01

    Mutations at a number of key positions (Ala156, Asp168 and Arg155) of the HCV NS3/4A protease can induce medium to high resistance to MK5172. The emergence of the resistant mutations seriously compromises the antiviral therapy efficacy to hepatitis C. In this study, molecular dynamics (MD) simulations, free energy calculations and free energy decomposition were used to explore the interaction profiles of MK5172 with the wild-type (WT) and four mutated (R155K, D168A, D168V and A156T) HCV NS3/4A proteases. The binding free energies predicted by Molecular Mechanics/Generalized Born Solvent Area (MM/GBSA) are consistent with the trend of the experimental drug resistance data. The free energy decomposition analysis shows that the resistant mutants may change the protein-MK5172 interaction profiles, resulting in the unbalanced energetic distribution amongst the catalytic triad, the strand 135-139 and the strand 154-160. Moreover, umbrella sampling (US) simulations were employed to elucidate the unbinding processes of MK5172 from the active pockets of the WT HCV NS3/4A protease and the four mutants. The US simulations demonstrate that the dissociation pathways of MK5172 escaping from the binding pockets of the WT and mutants are quite different, and it is quite possible that MK5172 will be harder to get access to the correct binding sites of the mutated proteases, thereafter leading to drug resistance. PMID:26219385

  7. Association of hepatitis C virus replication complexes with microtubules and actin filaments is dependent on the interaction of NS3 and NS5A.

    PubMed

    Lai, Chao-Kuen; Jeng, King-Song; Machida, Keigo; Lai, Michael M C

    2008-09-01

    The hepatitis C virus (HCV) RNA replication complex (RC), which is composed of viral nonstructural (NS) proteins and host cellular proteins, replicates the viral RNA genome in association with intracellular membranes. Two viral NS proteins, NS3 and NS5A, are essential elements of the RC. Here, by using immunoprecipitation and fluorescence resonance energy transfer assays, we demonstrated that NS3 and NS5A interact with tubulin and actin. Furthermore, immunofluorescence microscopy and electron microscopy revealed that HCV RCs were aligned along microtubules and actin filaments in both HCV replicon cells and HCV-infected cells. In addition, the movement of RCs was inhibited when microtubules or actin filaments were depolymerized by colchicine and cytochalasin B, respectively. Based on our observations, we propose that microtubules and actin filaments provide the tracks for the movement of HCV RCs to other regions in the cell, and the molecular interactions between RCs and microtubules, or RCs and actin filaments, are mediated by NS3 and NS5A. PMID:18562541

  8. Rho GTPases at the crossroad of signaling networks in mammals

    PubMed Central

    Wojnacki, José; Quassollo, Gonzalo; Marzolo, María-Paz; Cáceres, Alfredo

    2014-01-01

    Microtubule (MT) organization and dynamics downstream of external cues is crucial for maintaining cellular architecture and the generation of cell asymmetries. In interphase cells RhoA, Rac, and Cdc42, conspicuous members of the family of small Rho GTPases, have major roles in modulating MT stability, and hence polarized cell behaviors. However, MTs are not mere targets of Rho GTPases, but also serve as signaling platforms coupling MT dynamics to Rho GTPase activation in a variety of cellular conditions. In this article, we review some of the key studies describing the reciprocal relationship between small Rho-GTPases and MTs during migration and polarization. PMID:24691223

  9. Structure-guided mutagenesis of active site residues in the dengue virus two-component protease NS2B-NS3

    PubMed Central

    2010-01-01

    Background The dengue virus two-component protease NS2B/NS3 mediates processing of the viral polyprotein precursor and is therefore an important determinant of virus replication. The enzyme is now intensively studied with a view to the structure-based development of antiviral inhibitors. Although 3-dimensional structures have now been elucidated for a number of flaviviral proteases, enzyme-substrate interactions are characterized only to a limited extend. The high selectivity of the dengue virus protease for the polyprotein precursor offers the distinct advantage of designing inhibitors with exquisite specificity for the viral enzyme. To identify important determinants of substrate binding and catalysis in the active site of the dengue virus NS3 protease, nine residues, L115, D129, G133, T134, Y150, G151, N152, S163 and I165, located within the S1 and S2 pockets of the enzyme were targeted by alanine substitution mutagenesis and effects on enzyme activity were fluorometrically assayed. Methods Alanine substitutions were introduced by site-directed mutagenesis at residues L115, D129, G133, T134, Y150, G151, N152, S163 and I165 and recombinant proteins were purified from overexpressing E. coli. Effects of these substitutions on enzymatic activity of the NS3 protease were assayed by fluorescence release from the synthetic model substrate GRR-amc and kinetic parameters Km, kcat and kcat/Km were determined. Results Kinetic data for mutant derivatives in the active site of the dengue virus NS3 protease were essentially in agreement with a functional role of the selected residues for substrate binding and/or catalysis. Only the L115A mutant displayed activity comparable to the wild-type enzyme, whereas mutation of residues Y150 and G151 to alanine completely abrogated enzyme activity. A G133A mutant had an approximately 10-fold reduced catalytic efficiency thus suggesting a critical role for this residue seemingly as part of the oxyanion binding hole. Conclusions Kinetic

  10. Genetic interactions in yeast between Ypt GTPases and Arf guanine nucleotide exchangers.

    PubMed Central

    Jones, S; Jedd, G; Kahn, R A; Franzusoff, A; Bartolini, F; Segev, N

    1999-01-01

    Two families of GTPases, Arfs and Ypt/rabs, are key regulators of vesicular transport. While Arf proteins are implicated in vesicle budding from the donor compartment, Ypt/rab proteins are involved in the targeting of vesicles to the acceptor compartment. Recently, we have shown a role for Ypt31/32p in exit from the yeast trans-Golgi, suggesting a possible function for Ypt/rab proteins in vesicle budding as well. Here we report the identification of a new member of the Sec7-domain family, SYT1, as a high-copy suppressor of a ypt31/32 mutation. Several proteins that belong to the Sec7-domain family, including the yeast Gea1p, have recently been shown to stimulate nucleotide exchange by Arf GTPases. Nucleotide exchange by Arf GTPases, the switch from the GDP- to the GTP-bound form, is thought to be crucial for their function. Sec7p itself has an important role in the yeast secretory pathway. However, its mechanism of action is not yet understood. We show that all members of the Sec7-domain family exhibit distinct genetic interactions with the YPT genes. Biochemical assays demonstrate that, although the homology between the members of the Sec7-domain family is relatively low (20-35%) and limited to a small domain, they all can act as guanine nucleotide exchange factors (GEFs) for Arf proteins, but not for Ypt GTPases. The Sec7-domain of Sec7p is sufficient for this activity. Interestingly, the Sec7 domain activity is inhibited by brefeldin A (BFA), a fungal metabolite that inhibits some of the Arf-GEFs, indicating that this domain is a target for BFA. These results demonstrate that the ability to act as Arf-GEFs is a general property of all Sec7-domain proteins in yeast. The genetic interactions observed between Arf GEFs and Ypt GTPases suggest the existence of a Ypt-Arf GTPase cascade in the secretory pathway. PMID:10430582

  11. Regulation of phagocytosis by Rho GTPases

    PubMed Central

    Mao, Yingyu; Finnemann, Silvia C

    2015-01-01

    Phagocytosis is defined as a cellular uptake pathway for particles of greater than 0.5 μm in diameter. Particle clearance by phagocytosis is of critical importance for tissue health and homeostasis. The ultimate goal of anti-pathogen phagocytosis is to destroy engulfed bacteria or fungi and to stimulate cell-cell signaling that mount an efficient immune defense. In contrast, clearance phagocytosis of apoptotic cells and cell debris is anti-inflammatory. High capacity clearance phagocytosis pathways are available to professional phagocytes of the immune system and the retina. Additionally, a low capacity, so-called bystander phagocytic pathway is available to most other cell types. Different phagocytic pathways are stimulated by particle ligation of distinct surface receptors but all forms of phagocytosis require F-actin recruitment beneath tethered particles and F-actin re-arrangement promoting engulfment, which are controlled by Rho family GTPases. The specificity of Rho GTPase activity during the different forms of phagocytosis by mammalian cells is the subject of this review. PMID:25941749

  12. A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication

    PubMed Central

    Chatel-Chaix, Laurent; Fischl, Wolfgang; Scaturro, Pietro; Cortese, Mirko; Kallis, Stephanie; Bartenschlager, Marie; Fischer, Bernd

    2015-01-01

    ABSTRACT Dengue virus (DENV) infection causes the most prevalent arthropod-borne viral disease worldwide. Approved vaccines are not available, and targets suitable for the development of antiviral drugs are lacking. One possible drug target is nonstructural protein 4B (NS4B), because it is absolutely required for virus replication; however, its exact role in the DENV replication cycle is largely unknown. With the aim of mapping NS4B determinants critical for DENV replication, we performed a reverse genetic screening of 33 NS4B mutants in the context of an infectious DENV genome. While the majority of these mutations were lethal, for several of them, we were able to select for second-site pseudoreversions, most often residing in NS4B and restoring replication competence. To identify all viral NS4B interaction partners, we engineered a fully viable DENV genome encoding an affinity-tagged NS4B. Mass spectrometry-based analysis of the NS4B complex isolated from infected cells identified the NS3 protease/helicase as a major interaction partner of NS4B. By combining the genetic complementation map of NS4B with a replication-independent expression system, we identified the NS4B cytosolic loop—more precisely, amino acid residue Q134—as a critical determinant for NS4B-NS3 interaction. An alanine substitution at this site completely abrogated the interaction and DENV RNA replication, and both were restored by pseudoreversions A69S and A137V. This strict correlation between the degree of NS4B-NS3 interaction and DENV replication provides strong evidence that this viral protein complex plays a pivotal role during the DENV replication cycle, hence representing a promising target for novel antiviral strategies. IMPORTANCE With no approved therapy or vaccine against dengue virus infection, the viral nonstructural protein 4B (NS4B) represents a possible drug target, because it is indispensable for virus replication. However, little is known about its precise structure and

  13. Investigation of NS3 Protease Resistance-Associated Variants and Phenotypes for the Prediction of Treatment Response to HCV Triple Therapy

    PubMed Central

    Susser, Simone; Vermehren, Johannes; Peiffer, Kai-Henrik; Filmann, Natalie; Bon, Dimitra; Kuntzen, Thomas; Mauss, Stefan; Grammatikos, Georgios; Perner, Dany; Berkowski, Caterina; Herrmann, Eva; Zeuzem, Stefan; Bartenschlager, Ralf; Sarrazin, Christoph

    2016-01-01

    Triple therapy of chronic hepatitis C virus (HCV) infection with boceprevir (BOC) or telaprevir (TVR) leads to virologic failure in many patients which is often associated with the selection of resistance-associated variants (RAVs). These resistance profiles are of importance for the selection of potential rescue treatment options. In this study, we sequenced baseline NS3 RAVs population-based and investigated the sensitivity of NS3 phenotypes in an HCV replicon assay together with clinical factors for a prediction of treatment response in a cohort of 165 German and Swiss patients treated with a BOC or TVR-based triple therapy. Overall, the prevalence of baseline RAVs was low, although the frequency of RAVs was higher in patients with virologic failure compared to those who achieved a sustained virologic response (SVR) (7% versus 1%, P = 0.06). The occurrence of RAVs was associated with a resistant NS3 quasispecies phenotype (P<0.001), but the sensitivity of phenotypes was not associated with treatment outcome (P = 0.2). The majority of single viral and host predictors of SVR was only weakly associated with treatment response. In multivariate analyses, low AST levels, female sex and an IFNL4 CC genotype were independently associated with SVR. However, a combined analysis of negative predictors revealed a significantly lower overall number of negative predictors in patients with SVR in comparison to individuals with virologic failure (P<0.0001) and the presence of 2 or less negative predictors was indicative for SVR. These results demonstrate that most single baseline viral and host parameters have a weak influence on the response to triple therapy, whereas the overall number of negative predictors has a high predictive value for SVR. PMID:27281344

  14. Molecular docking investigation of the binding interactions of macrocyclic inhibitors with HCV NS3 protease and its mutants (R155K, D168A and A156V).

    PubMed

    Ezat, Ahmed A; El-Bialy, Nihal S; Mostafa, Hamdy I A; Ibrahim, Medhat A

    2014-02-01

    Hepatitis C Virus (HCV) non-structural protein 3 (NS3) protease drug resistance poses serious challenges on the design of an effective treatment. Substrate Envelope Hypothesis, "the substrates of HCV NS3/4A protease have a consensus volume inside the active site called substrate envelope" is used to design potent and specific drugs to overcome this problem. Using molecular docking, we studied the binding interaction of the different inhibitors and protein and evaluated the effect of three different mutations (R155K, D168A and A156V) on the binding of inhibitors. P2-P4 macrocycles of 5A/5B and modified 5A/5B hexapeptide sequences have the best scores against the wild-type protein -204.506 and -206.823 kcal/mole, respectively. Also, charged P2-P4 macrocycles of 3/4A and 4A/4B hexapeptide sequences have low scores with the wild-type protein -200.467 and -203.186 kcal/mole, respectively. R155K mutation greatly affects the conformation of the compounds inside the active site. It inverts its orientations, and this is because the large and free side chain of K155 which restricts the conformation of the large P2-P4 macrocycle. The conformation of charged P2-P4 macrocycle of 3/4A hexapeptide sequence in wild-type, A156V and D168A proteins is nearly equal; while that of charged P2-P4 macrocycle of 4A/4B hexapeptide sequence is different. Nevertheless, these compounds have a slight increase of Van der Waals volume compared to that of substrates, they are potent against mutations and have good scores. Therefore, the suggested drugs can be used as an effective treatment solving HCV NS3/4A protease drug resistance problem. PMID:24374429

  15. Discovery of small-molecule inhibitors of HCV NS3-4A protease as potential therapeutic agents against HCV infection.

    PubMed

    Chen, Shu-Hui; Tan, Seng-Lai

    2005-01-01

    Chronic infection with hepatitis C virus (HCV) is associated with liver cirrhosis that often leads to hepatic failure and hepatocellular carcinoma (HCC). HCV infection has become a global health threat and the main cause of adult liver transplants in developed nations. Current approved anti-HCV therapies (interferon and pegylated interferon alone or in combination with ribavirin) are not effective in eliminating the viral infection in a significant population of patients (e.g., those infected with HCV genotype 1). Furthermore, these therapies are plagued with many undesirable side effects. Therefore, the HCV epidemic represents a huge unmet medical need that has triggered intensive research efforts towards the development of more effective drugs. Given its essential role in the process of HCV replication, the viral NS3/4A serine protease is arguably the most thoroughly characterized HCV enzyme and the most intensively pursued anti-HCV target for drug development. This is further fueled by the successful use of small-molecule inhibitors of the human immunodeficiency virus (HIV) viral protease, which have had an impressive effect on HIV-related morbidity and mortality, offering hope that analogous drugs might also have a similar impact against HCV. Here, we review the recent progress and development of small-molecule inhibitors of the HCV NS3/4A protease. In particular, we focus on the discovery of VX-950, the latest HCV NS3-4A protease inhibitor to be advanced to clinical studies. While the challenges of designing potent inhibitors of the viral protease have been solved, as highlighted by BILN 2061 and VX-950, it is still too early to determine whether these efforts will eventually yield promising drug candidates. For the emerging small-molecule HCV inhibitors, viral resistance will likely be a big problem. Thus, combination therapy of different drugs with different targets/mechanisms will be necessary to effectively inhibit HCV replication. It is also hoped that a

  16. Upregulation of signalase processing and induction of prM-E secretion by the flavivirus NS2B-NS3 protease: roles of protease components.

    PubMed Central

    Yamshchikov, V F; Trent, D W; Compans, R W

    1997-01-01

    Recently, we have shown that the ability of the flavivirus NS2B-NS3 protease complex to promote efficient signalase processing of the C-prM precursor, as well as secretion of prM and E, does not appear to depend strictly on cleavage of the precursor at its Lys-Arg-Gly dibasic site by the protease. We suggested that the association of the protease with the precursor via NS2B may be sufficient by itself for the above effects. To study the proposed association in more detail, we have developed an assay in which processing at the C-prM dibasic cleavage site is abolished by Lys-->Gly conversion. We constructed deletion mutants and chimeras of the West Nile (WN) flavivirus NS2B protein and expressed them in the context of [5'-C-->NS3(243)] containing either wild-type C-prM or its cleavage site mutant. All NS2B variants were able to form active protease complexes. Deletion of the carboxy-terminal cluster of hydrophobic amino acids in NS2B had no apparent effect on the formation of prM and prM-E secretion for the cassettes containing either wild-type or mutated C-prM precursor. Deletion of the amino-terminal hydrophobic cluster in NS2B did not affect prM-E secretion for the cassettes with wild-type C-prM but abrogated prM-E secretion for the cassettes with the mutated dibasic cleavage site in C-prM. Similarly, the NS2B-NS3(178) protease of Japanese encephalitis (JE) virus, when substituted for the WN virus NS2B-NS3(243) protease, was able to promote prM-E secretion for the cassette with the wild-type C-prM precursor but not with the mutated one. Replacement of the deleted amino-terminal hydrophobic cluster in the WN virus NS2B protein with an analogous JE virus sequence restored the ability of the protease to promote prM-E secretion. On the basis of these observations, roles of individual protease components in upregulation of C-prM signalase processing are discussed. PMID:9151825

  17. The introduction of P4 substituted 1-methylcyclohexyl groups into Boceprevir: a change in direction in the search for a second generation HCV NS3 protease inhibitor.

    PubMed

    Bennett, Frank; Huang, Yuhua; Hendrata, Siska; Lovey, Raymond; Bogen, Stephane L; Pan, Weidong; Guo, Zhuyan; Prongay, Andrew; Chen, Kevin X; Arasappan, Ashok; Venkatraman, Srikanth; Velazquez, Francisco; Nair, Latha; Sannigrahi, Mousumi; Tong, Xiao; Pichardo, John; Cheng, Kuo-Chi; Girijavallabhan, Viyyoor M; Saksena, Anil K; Njoroge, F George

    2010-04-15

    In the search for a second generation HCV protease inhibitor, molecular modeling studies of the X-ray crystal structure of Boceprevir1 bound to the NS3 protein suggest that expansion into the S4 pocket could provide additional hydrophobic Van der Waals interactions. Effective replacement of the P4 tert-butyl with a cyclohexylmethyl ligand led to inhibitor 2 with improved enzyme and replicon activities. Subsequent modeling and SAR studies led to the pyridine 38 and sulfone analogues 52 and 53 with vastly improved PK parameters in monkeys, forming a new foundation for further exploration. PMID:20303756

  18. Novel Activities of Select NSAID R-Enantiomers against Rac1 and Cdc42 GTPases.

    PubMed

    Oprea, Tudor I; Sklar, Larry A; Agola, Jacob O; Guo, Yuna; Silberberg, Melina; Roxby, Joshua; Vestling, Anna; Romero, Elsa; Surviladze, Zurab; Murray-Krezan, Cristina; Waller, Anna; Ursu, Oleg; Hudson, Laurie G; Wandinger-Ness, Angela

    2015-01-01

    Rho family GTPases (including Rac, Rho and Cdc42) collectively control cell proliferation, adhesion and migration and are of interest as functional therapeutic targets in numerous epithelial cancers. Based on high throughput screening of the Prestwick Chemical Library® and cheminformatics we identified the R-enantiomers of two approved drugs (naproxen and ketorolac) as inhibitors of Rac1 and Cdc42. The corresponding S-enantiomers are considered the active component in racemic drug formulations, acting as non-steroidal anti-inflammatory drugs (NSAIDs) with selective activity against cyclooxygenases. Here, we show that the S-enantiomers of naproxen and ketorolac are inactive against the GTPases. Additionally, more than twenty other NSAIDs lacked inhibitory action against the GTPases, establishing the selectivity of the two identified NSAIDs. R-naproxen was first identified as a lead compound and tested in parallel with its S-enantiomer and the non-chiral 6-methoxy-naphthalene acetic acid (active metabolite of nabumetone, another NSAID) as a structural series. Cheminformatics-based substructure analyses-using the rotationally constrained carboxylate in R-naproxen-led to identification of racemic [R/S] ketorolac as a suitable FDA-approved candidate. Cell based measurement of GTPase activity (in animal and human cell lines) demonstrated that the R-enantiomers specifically inhibit epidermal growth factor stimulated Rac1 and Cdc42 activation. The GTPase inhibitory effects of the R-enantiomers in cells largely mimic those of established Rac1 (NSC23766) and Cdc42 (CID2950007/ML141) specific inhibitors. Docking predicts that rotational constraints position the carboxylate moieties of the R-enantiomers to preferentially coordinate the magnesium ion, thereby destabilizing nucleotide binding to Rac1 and Cdc42. The S-enantiomers can be docked but are less favorably positioned in proximity to the magnesium. R-naproxen and R-ketorolac have potential for rapid translation and

  19. Novel Activities of Select NSAID R-Enantiomers against Rac1 and Cdc42 GTPases

    PubMed Central

    Oprea, Tudor I.; Sklar, Larry A.; Agola, Jacob O.; Guo, Yuna; Silberberg, Melina; Roxby, Joshua; Vestling, Anna; Romero, Elsa; Surviladze, Zurab; Murray-Krezan, Cristina; Waller, Anna; Ursu, Oleg; Hudson, Laurie G.; Wandinger-Ness, Angela

    2015-01-01

    Rho family GTPases (including Rac, Rho and Cdc42) collectively control cell proliferation, adhesion and migration and are of interest as functional therapeutic targets in numerous epithelial cancers. Based on high throughput screening of the Prestwick Chemical Library® and cheminformatics we identified the R-enantiomers of two approved drugs (naproxen and ketorolac) as inhibitors of Rac1 and Cdc42. The corresponding S-enantiomers are considered the active component in racemic drug formulations, acting as non-steroidal anti-inflammatory drugs (NSAIDs) with selective activity against cyclooxygenases. Here, we show that the S-enantiomers of naproxen and ketorolac are inactive against the GTPases. Additionally, more than twenty other NSAIDs lacked inhibitory action against the GTPases, establishing the selectivity of the two identified NSAIDs. R-naproxen was first identified as a lead compound and tested in parallel with its S-enantiomer and the non-chiral 6-methoxy-naphthalene acetic acid (active metabolite of nabumetone, another NSAID) as a structural series. Cheminformatics-based substructure analyses—using the rotationally constrained carboxylate in R-naproxen—led to identification of racemic [R/S] ketorolac as a suitable FDA-approved candidate. Cell based measurement of GTPase activity (in animal and human cell lines) demonstrated that the R-enantiomers specifically inhibit epidermal growth factor stimulated Rac1 and Cdc42 activation. The GTPase inhibitory effects of the R-enantiomers in cells largely mimic those of established Rac1 (NSC23766) and Cdc42 (CID2950007/ML141) specific inhibitors. Docking predicts that rotational constraints position the carboxylate moieties of the R-enantiomers to preferentially coordinate the magnesium ion, thereby destabilizing nucleotide binding to Rac1 and Cdc42. The S-enantiomers can be docked but are less favorably positioned in proximity to the magnesium. R-naproxen and R-ketorolac have potential for rapid translation and

  20. Small GTPase regulation of GPCR anterograde trafficking

    PubMed Central

    Wang, Guansong; Wu, Guangyu

    2011-01-01

    The physiological functions of heterotrimeric G protein-coupled receptors (GPCRs) are dictated by their intracellular trafficking and precise targeting to the functional destinations. Over the past decades, most studies on the trafficking of GPCRs have focused on the events involved in endocytosis and recycling. In contrast, the molecular mechanisms underlying anterograde transport of newly synthesized GPCRs from the endoplasmic reticulum (ER) to the cell surface have just begun to be revealed. In this review, we will discuss current advances in understanding the role of Ras-like GTPases, specifically the Rab and Sar1/ARF subfamilies, in regulating cell-surface transport of GPCRs en route from the ER and the Golgi. PMID:22015208

  1. Solubilization and partial characterization of a microsomal high affinity GTPase

    SciTech Connect

    Nicchitta, C.; Williamson, J.R.

    1987-05-01

    Isolated rat liver microsomes release sequestered Ca/sup 2 +/ following addition of GTP. In contrast to permeabilized cells, GTP dependent microsomal Ca/sup 2 +/ release requires low concentrations of polyethylene glycol (PEG). They have identified a microsomal, PEG-sensitive high affinity GTPase which shares a number of characteristics with the GTP-dependent Ca/sup 2 +/ release system. To aid in further characterization of this activity they have initiated studies on the solubilization and purification of the microsomal GTPases. When microsomes are solubilized under the following conditions (150 mM NaCl, 5 mg protein/ml, 1% Triton X-114) PEG sensitive GTPase activity selectively partitions into the detergent rich phase of the Triton X-114 extract. As observed in intact microsomal membranes the Triton X-114 soluble GTPase is maximally stimulated by 3% PEG. Half maximal stimulation is observed at 1% PEG. PEG increases the Vmax of this activity; no effects on Km were observed. The Km for GTP of the detergent soluble GTPase is 5 ..mu..M. This GTPase is sensitive to inhibition by sulfhydryl reagents. PEG-sensitive GTPase activity was completely inhibited in the presence of 25 ..mu..M p-hydroxymercuribenzoate (PHMB); half maximal inhibition was observed at 5 ..mu..M. Labeling of the Triton X-114 extract with the photosensitive compound (/sup 32/P) 8-azido GTP indicated the presence of two prominent GTP binding proteins of approximate molecular weights 17 and 54 kD.

  2. Interferon-Inducible GTPases in Host Resistance, Inflammation and Disease.

    PubMed

    Pilla-Moffett, Danielle; Barber, Matthew F; Taylor, Gregory A; Coers, Jörn

    2016-08-28

    Cell-autonomous immunity is essential for host organisms to defend themselves against invasive microbes. In vertebrates, both the adaptive and the innate branches of the immune system operate cell-autonomous defenses as key effector mechanisms that are induced by pro-inflammatory interferons (IFNs). IFNs can activate cell-intrinsic host defenses in virtually any cell type ranging from professional phagocytes to mucosal epithelial cells. Much of this IFN-induced host resistance program is dependent on four families of IFN-inducible GTPases: the myxovirus resistance proteins, the immunity-related GTPases, the guanylate-binding proteins (GBPs), and the very large IFN-inducible GTPases. These GTPase families provide host resistance to a variety of viral, bacterial, and protozoan pathogens through the sequestration of microbial proteins, manipulation of vesicle trafficking, regulation of antimicrobial autophagy (xenophagy), execution of intracellular membranolytic pathways, and the activation of inflammasomes. This review discusses our current knowledge of the molecular function of IFN-inducible GTPases in providing host resistance, as well as their role in the pathogenesis of autoinflammatory Crohn's disease. While substantial advances were made in the recent past, few of the known functions of IFN-inducible GTPases have been explored in any depth, and new functions await discovery. This review will therefore highlight key areas of future exploration that promise to advance our understanding of the role of IFN-inducible GTPases in human diseases. PMID:27181197

  3. Timing Is Everything: GTPase Regulation in Phototransduction

    PubMed Central

    Arshavsky, Vadim Y.; Wensel, Theodore G.

    2013-01-01

    As the molecular mechanisms of vertebrate phototransduction became increasingly clear in the 1980s, a persistent problem was the discrepancy between the slow GTP hydrolysis catalyzed by the phototransduction G protein, transducin, and the much more rapid physiological recovery of photoreceptor cells from light stimuli. Beginning with a report published in 1989, a series of studies revealed that transducin GTPase activity could approach the rate needed to explain physiological recovery kinetics in the presence of one or more factors present in rod outer segment membranes. One by one, these factors were identified, beginning with PDEγ, the inhibitory subunit of the cGMP phosphodiesterase activated by transducin. There followed the discovery of the crucial role played by the regulator of G protein signaling, RGS9, a member of a ubiquitous family of GTPase-accelerating proteins, or GAPs, for heterotrimeric G proteins. Soon after, the G protein β isoform Gβ5 was identified as an obligate partner subunit, followed by the discovery or R9AP, a transmembrane protein that anchors the RGS9 GAP complex to the disk membrane, and is essential for the localization, stability, and activity of this complex in vivo. The physiological importance of all of the members of this complex was made clear first by knockout mouse models, and then by the discovery of a human visual defect, bradyopsia, caused by an inherited deficiency in one of the GAP components. Further insights have been gained by high-resolution crystal structures of subcomplexes, and by extensive mechanistic studies both in vitro and in animal models. PMID:24265205

  4. Influence of bacterial toxins on the GTPase activity of transducin from bovine retinal rod outer segments

    SciTech Connect

    Rybin, V.O.; Gureeva, A.A.

    1986-05-10

    The action of cholera toxin, capable of ADP-ribosylation of the activator N/sub s/ protein, and pertussis toxin, capable of ADP-ribosylation of the inhibitor N/sub i/ protein of the adenylate cyclase complex, on transducin, the GTP-binding protein of the rod outer segments of the retina, was investigated. It was shown that under the action of pertussis and cholera toxins, the GTPase activity of transducin is inhibited. Pertussin toxin inhibits the GTPase of native retinal rod outer segments by 30-40%, while GTPase of homogeneous transducin produces a 70-80% inhibition. The action of toxins on transducin depends on the presence and nature of the guanylic nucleotide with which incubation is performed. On the basis of the data obtained it is suggested that pertussis toxin interacts with pretransducin and with the transducin-GDP complex, while cholera toxin ADP-ribosylates the transducin-GTP complex and does not act on transducin lacking GTP.

  5. Roles of Aspergillus nidulans Cdc42/Rho GTPase regulators in hyphal morphogenesis and development.

    PubMed

    Si, Haoyu; Rittenour, William R; Harris, Steven D

    2016-01-01

    The Rho-related family of GTPases are pivotal regulators of morphogenetic processes in diverse eukaryotic organisms. In the filamentous fungi two related members of this family, Cdc42 and Rac1, perform particularly important roles in the establishment and maintenance of hyphal polarity. The activity of these GTPases is tightly controlled by two sets of regulators: guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Despite the importance of Cdc42 and Rac1 in polarized hyphal growth, the morphogenetic functions of their cognate GEFs and GAPs have not been widely characterized in filamentous fungi outside the Saccharomycotina. Here we present a functional analysis of the Aspergillus nidulans homologs of the yeast GEF Cdc24 and the yeast GAP Rga1. We show that Cdc24 is required for the establishment of hyphal polarity and localizes to hyphal tips. We also show that Rga1 is necessary for the suppression of branching in developing conidiophores. During asexual development Rga1 appears to act primarily via Cdc42 and in doing so serves as a critical determinant of conidiophore architecture. Our results provide new insight into the roles of Cdc42 during development in A nidulans. PMID:26932184

  6. MK-5172, a Selective Inhibitor of Hepatitis C Virus NS3/4a Protease with Broad Activity across Genotypes and Resistant Variants

    PubMed Central

    Summa, Vincenzo; McCauley, John A.; Fandozzi, Christine; Burlein, Christine; Claudio, Giuliano; Coleman, Paul J.; DiMuzio, Jillian M.; Ferrara, Marco; Di Filippo, Marcello; Gates, Adam T.; Graham, Donald J.; Harper, Steven; Hazuda, Daria J.; McHale, Carolyn; Monteagudo, Edith; Pucci, Vincenzo; Rowley, Michael; Rudd, Michael T.; Soriano, Aileen; Stahlhut, Mark W.; Vacca, Joseph P.; Olsen, David B.; Liverton, Nigel J.; Carroll, Steven S.

    2012-01-01

    HCV NS3/4a protease inhibitors are proven therapeutic agents against chronic hepatitis C virus infection, with boceprevir and telaprevir having recently received regulatory approval as add-on therapy to pegylated interferon/ribavirin for patients harboring genotype 1 infections. Overcoming antiviral resistance, broad genotype coverage, and a convenient dosing regimen are important attributes for future agents to be used in combinations without interferon. In this communication, we report the preclinical profile of MK-5172, a novel P2-P4 quinoxaline macrocyclic NS3/4a protease inhibitor currently in clinical development. The compound demonstrates subnanomolar activity against a broad enzyme panel encompassing major hepatitis C virus (HCV) genotypes as well as variants resistant to earlier protease inhibitors. In replicon selections, MK-5172 exerted high selective pressure, which yielded few resistant colonies. In both rat and dog, MK-5172 demonstrates good plasma and liver exposures, with 24-h liver levels suggestive of once-daily dosing. When administered to HCV-infected chimpanzees harboring chronic gt1a or gt1b infections, MK-5172 suppressed viral load between 4 to 5 logs at a dose of 1 mg/kg of body weight twice daily (b.i.d.) for 7 days. Based on its preclinical profile, MK-5172 is anticipated to be broadly active against multiple HCV genotypes and clinically important resistance variants and highly suited for incorporation into newer all-oral regimens. PMID:22615282

  7. An updated evolutionary study of Flaviviridae NS3 helicase and NS5 RNA-dependent RNA polymerase reveals novel invariable motifs as potential pharmacological targets.

    PubMed

    Papageorgiou, Louis; Loukatou, Styliani; Sofia, Kossida; Maroulis, Dimitrios; Vlachakis, Dimitrios

    2016-06-21

    The rate of Flaviviridae family virus infections worldwide has increased dramatically in the last few years. In addition, infections caused by arthropod vector viruses including Hepatitis C, West Nile, Dengue fever, Yellow fever and Japanese encephalitis are emerging throughout the world. Based on a recent taxon update, the Flaviviridae family comprises four main genera; Flavivirus, Hepacivirus, Pestivirus and a recent genus Pegivirus. Although the new scientific classification plays a key role in providing useful information about the relationships between viruses, many new documented viruses remain unclassified. Furthermore, based on the different results of several studies the classification is unclear. In an effort to provide more insights into the classification of viruses, a holistic evolutionary study of the two viral enzymes NS3 helicase and NS5 RNA-dependent RNA polymerase (RdRp) has been conducted in this study. These two viral enzymes are very crucial for the inhibition of viruses due to the fact that they are involved in the survival, proliferation and transmission of viruses. The main goal of this study is the presentation of two novel updated phylogenetic trees of the enzymes NS3 helicase and NS5 RdRp as a reliable phylogeny "map" to correlate the information of the closely related viruses and identify new possible targets for the Flaviviridae family virus inhibition. Despite the earliest trials for drugs against Flaviviridae related viruses, no antiviral drug vaccine has been available to date. Therefore there is an urgent need for research towards the development of efficient antiviral agents. PMID:26864387

  8. Evaluating Andrographolide as a Potent Inhibitor of NS3-4A Protease and Its Drug-Resistant Mutants Using In Silico Approaches.

    PubMed

    Chandramohan, Vivek; Kaphle, Anubhav; Chekuri, Mamatha; Gangarudraiah, Sindhu; Bychapur Siddaiah, Gowrishankar

    2015-01-01

    Current combination therapy of PEG-INF and ribavirin against the Hepatitis C Virus (HCV) genotype-1 infections is ineffective in maintaining sustained viral response in 50% of the infection cases. New compounds in the form of protease inhibitors can complement the combination therapy. Asunaprevir is new to the drug regiment as the NS3-4A protease inhibitor, but it is susceptible to two mutations, namely, R155K and D168A in the protein. Thus, in our study, we sought to evaluate Andrographolide, a labdane-diterpenoid from the Andrographis paniculata plant as an effective compound for inhibiting the NS3-4A protease as well as its concomitant drug-resistant mutants by using molecular docking and dynamic simulations. Our study shows that Andrographolide has best docking scores of -15.0862, -15.2322, and -13.9072 compared to those of Asunaprevir -3.7159, -2.6431, and -5.4149 with wild-type R155K and D168A mutants, respectively. Also, as shown in the MD simulations, the compound was good in binding the target proteins and maintains strong bonds causing very less to negligible perturbation in the protein backbone structures. Our results validate the susceptibility of Asunaprevir to protein variants as seen from our docking studies and trajectory period analysis. Therefore, from our study, we hope to add one more option in the drug regiment to tackle drug resistance in HCV infections. PMID:26587022

  9. Evaluating Andrographolide as a Potent Inhibitor of NS3-4A Protease and Its Drug-Resistant Mutants Using In Silico Approaches

    PubMed Central

    Chandramohan, Vivek; Kaphle, Anubhav; Chekuri, Mamatha; Gangarudraiah, Sindhu; Bychapur Siddaiah, Gowrishankar

    2015-01-01

    Current combination therapy of PEG-INF and ribavirin against the Hepatitis C Virus (HCV) genotype-1 infections is ineffective in maintaining sustained viral response in 50% of the infection cases. New compounds in the form of protease inhibitors can complement the combination therapy. Asunaprevir is new to the drug regiment as the NS3-4A protease inhibitor, but it is susceptible to two mutations, namely, R155K and D168A in the protein. Thus, in our study, we sought to evaluate Andrographolide, a labdane-diterpenoid from the Andrographis paniculata plant as an effective compound for inhibiting the NS3-4A protease as well as its concomitant drug-resistant mutants by using molecular docking and dynamic simulations. Our study shows that Andrographolide has best docking scores of −15.0862, −15.2322, and −13.9072 compared to those of Asunaprevir −3.7159, −2.6431, and −5.4149 with wild-type R155K and D168A mutants, respectively. Also, as shown in the MD simulations, the compound was good in binding the target proteins and maintains strong bonds causing very less to negligible perturbation in the protein backbone structures. Our results validate the susceptibility of Asunaprevir to protein variants as seen from our docking studies and trajectory period analysis. Therefore, from our study, we hope to add one more option in the drug regiment to tackle drug resistance in HCV infections. PMID:26587022

  10. Discovery of MK-8831, A Novel Spiro-Proline Macrocycle as a Pan-Genotypic HCV-NS3/4a Protease Inhibitor.

    PubMed

    Neelamkavil, Santhosh F; Agrawal, Sony; Bara, Thomas; Bennett, Chad; Bhat, Sathesh; Biswas, Dipshikha; Brockunier, Linda; Buist, Nicole; Burnette, Duane; Cartwright, Mark; Chackalamannil, Samuel; Chase, Robert; Chelliah, Mariappan; Chen, Austin; Clasby, Martin; Colandrea, Vincent J; Davies, Ian W; Eagen, Keith; Guo, Zhuyan; Han, Yongxin; Howe, John; Jayne, Charles; Josien, Hubert; Kargman, Stacia; Marcantonio, Karen; Miao, Shouwu; Miller, Randy; Nolting, Andrew; Pinto, Patrick; Rajagopalan, Murali; Ruck, Rebecca T; Shah, Unmesh; Soriano, Aileen; Sperbeck, Donald; Velazquez, Francisco; Wu, Jin; Xia, Yan; Venkatraman, Srikanth

    2016-01-14

    We have been focused on identifying a structurally different next generation inhibitor to MK-5172 (our Ns3/4a protease inhibitor currently under regulatory review), which would achieve superior pangenotypic activity with acceptable safety and pharmacokinetic profile. These efforts have led to the discovery of a novel class of HCV NS3/4a protease inhibitors containing a unique spirocyclic-proline structural motif. The design strategy involved a molecular-modeling based approach, and the optimization efforts on the series to obtain pan-genotypic coverage with good exposures on oral dosing. One of the key elements in this effort was the spirocyclization of the P2 quinoline group, which rigidified and constrained the binding conformation to provide a novel core. A second focus of the team was also to improve the activity against genotype 3a and the key mutant variants of genotype 1b. The rational application of structural chemistry with molecular modeling guided the design and optimization of the structure-activity relationships have resulted in the identification of the clinical candidate MK-8831 with excellent pan-genotypic activity and safety profile. PMID:26819676

  11. Analysis of the Small GTPase Gene Superfamily of Arabidopsis1

    PubMed Central

    Vernoud, Vanessa; Horton, Amy C.; Yang, Zhenbiao; Nielsen, Erik

    2003-01-01

    Small GTP-binding proteins regulate diverse processes in eukaryotic cells such as signal transduction, cell proliferation, cytoskeletal organization, and intracellular membrane trafficking. These proteins function as molecular switches that cycle between “active” and “inactive” states, and this cycle is linked to the binding and hydrolysis of GTP. The Arabidopsis genome contains 93 genes that encode small GTP-binding protein homologs. Phylogenetic analysis of these genes shows that plants contain Rab, Rho, Arf, and Ran GTPases, but no Ras GTPases. We have assembled complete lists of these small GTPases families, as well as accessory proteins that control their activity, and review what is known of the functions of individual members of these families in Arabidopsis. We also discuss the possible roles of these GTPases in relation to their similarity to orthologs with known functions and localizations in yeast and/or animal systems. PMID:12644670

  12. Rag GTPases are cardioprotective by regulating lysosomal function

    PubMed Central

    Kim, Young Chul; Mo, Jung-Soon; Jewell, Jenna L.; Russell, Ryan C.; Wu, Xiaohui; Sadoshima, Junichi; Guan, Kun-Liang

    2014-01-01

    The Rag family proteins are Ras-like small GTPases that play a critical role in amino acid-stimulated mTORC1 activation by recruiting mTORC1 to lysosome. Despite progress in the mechanistic understanding of Rag GTPases in mTORC1 activation, little is known about the physiological function of Rag GTPases in vivo. Here, we show that loss of RagA and RagB (RagA/B) in cardiomyocytes results in hypertrophic cardiomyopathy and phenocopies lysosomal storage diseases although mTORC1 activity is not substantially impaired in vivo. We demonstrate that despite upregulation of lysosomal protein expression by constitutive activation of the transcription factor EB (TFEB) in RagA/B knockout mouse embryonic fibroblasts, lysosomal acidification is compromised due to decreased v-ATPase level in the lysosome fraction. Our study uncovers RagA/B GTPases as key regulators of lysosomal function and cardiac protection. PMID:24980141

  13. Exploring potassium-dependent GTP hydrolysis in TEES family GTPases.

    PubMed

    Rafay, Abu; Majumdar, Soneya; Prakash, Balaji

    2012-01-01

    GTPases are important regulatory proteins that hydrolyze GTP to GDP. A novel GTP-hydrolysis mechanism is employed by MnmE, YqeH and FeoB, where a potassium ion plays a role analogous to the Arginine finger of the Ras-RasGAP system, to accelerate otherwise slow GTP hydrolysis rates. In these proteins, two conserved asparagines and a 'K-loop' present in switch-I, were suggested as attributes of GTPases employing a K(+)-mediated mechanism. Based on their conservation, a similar mechanism was suggested for TEES family GTPases. Recently, in Dynamin, Fzo1 and RbgA, which also conserve these attributes, a similar mechanism was shown to be operative. Here, we probe K(+)-activated GTP hydrolysis in TEES (TrmE-Era-EngA-YihA-Septin) GTPases - Era, EngB and the two contiguous G-domains, GD1 and GD2 of YphC (EngA homologue) - and also in HflX, another GTPase that also conserves the same attributes. While GD1-YphC and Era exhibit a K(+)-mediated activation of GTP hydrolysis, surprisingly GD2-YphC, EngB and HflX do not. Therefore, the attributes identified thus far, do not necessarily predict a K(+)-mechanism in GTPases and hence warrant extensive structural investigations. PMID:23650596

  14. Reverse engineering GTPase programming languages with reconstituted signaling networks.

    PubMed

    Coyle, Scott M

    2016-07-01

    The Ras superfamily GTPases represent one of the most prolific signaling currencies used in Eukaryotes. With these remarkable molecules, evolution has built GTPase networks that control diverse cellular processes such as growth, morphology, motility and trafficking. (1-4) Our knowledge of the individual players that underlie the function of these networks is deep; decades of biochemical and structural data has provided a mechanistic understanding of the molecules that turn GTPases ON and OFF, as well as how those GTPase states signal by controlling the assembly of downstream effectors. However, we know less about how these different activities work together as a system to specify complex dynamic signaling outcomes. Decoding this molecular "programming language" would help us understand how different species and cell types have used the same GTPase machinery in different ways to accomplish different tasks, and would also provide new insights as to how mutations to these networks can cause disease. We recently developed a bead-based microscopy assay to watch reconstituted H-Ras signaling systems at work under arbitrary configurations of regulators and effectors. (5) Here we highlight key observations and insights from this study and propose extensions to our method to further study this and other GTPase signaling systems. PMID:27128855

  15. Discovery of SCH446211 (SCH6): A New Ketoamide Inhibitor of the HCV NS3 Serine Protease and HCV Subgenomic RNA Replication

    SciTech Connect

    Bogen, Stephane L.; Arasappan, Ashok; Bennett, Frank; Chen, Kevin; Jao, Edwin; Liu, Yi-Tsung; Lovey, Raymond G.; Venkatraman, Srikanth; Pan, Weidong; Parekh, Tajel; Pike, Russel E.; Ruan, Sumei; Liu, Rong; Baroudy, Bahige; Agrawal, Sony; Chase, Robert; Ingravallo, Paul; Pichardo, John; Prongay, Andrew; Brisson, Jean-Marc; Hsieh, Tony Y.; Cheng, Kuo-Chi; Kemp, Scott J.; Levy, Odile E.; Lim-Wilby, Marguerita; Tamura, Susan Y.; Saksena, Anil K.; Girijavallabhan, Viyyoor; Njoroge, F. George

    2008-06-30

    Introduction of various modified prolines at P{sub 2} and optimization of the P{sub 1} side chain led to the discovery of SCH6 (24, Table 2), a potent ketoamide inhibitor of the HCV NS3 serine protease. In addition to excellent enzyme potency (K*{sub i} = 3.8 nM), 24 was also found to be a potent inhibitor of HCV subgenomic RNA replication with IC{sub 50} and IC{sub 90} of 40 and 100 nM, respectively. Recently, antiviral activity of 24 was demonstrated with inhibition of the full-length genotype 2a HCV genome. In addition, 24 was found to restore the responsiveness of the interferon regulatory factor 3 (IRF-3) in cells containing HCV RNA replicons.

  16. The discovery of asunaprevir (BMS-650032), an orally efficacious NS3 protease inhibitor for the treatment of hepatitis C virus infection.

    PubMed

    Scola, Paul M; Sun, Li-Qiang; Wang, Alan Xiangdong; Chen, Jie; Sin, Ny; Venables, Brian L; Sit, Sing-Yuen; Chen, Yan; Cocuzza, Anthony; Bilder, Donna M; D'Andrea, Stanley V; Zheng, Barbara; Hewawasam, Piyasena; Tu, Yong; Friborg, Jacques; Falk, Paul; Hernandez, Dennis; Levine, Steven; Chen, Chaoqun; Yu, Fei; Sheaffer, Amy K; Zhai, Guangzhi; Barry, Diana; Knipe, Jay O; Han, Yong-Hae; Schartman, Richard; Donoso, Maria; Mosure, Kathy; Sinz, Michael W; Zvyaga, Tatyana; Good, Andrew C; Rajamani, Ramkumar; Kish, Kevin; Tredup, Jeffrey; Klei, Herbert E; Gao, Qi; Mueller, Luciano; Colonno, Richard J; Grasela, Dennis M; Adams, Stephen P; Loy, James; Levesque, Paul C; Sun, Huabin; Shi, Hong; Sun, Lucy; Warner, William; Li, Danshi; Zhu, Jialong; Meanwell, Nicholas A; McPhee, Fiona

    2014-03-13

    The discovery of asunaprevir (BMS-650032, 24) is described. This tripeptidic acylsulfonamide inhibitor of the NS3/4A enzyme is currently in phase III clinical trials for the treatment of hepatitis C virus infection. The discovery of 24 was enabled by employing an isolated rabbit heart model to screen for the cardiovascular (CV) liabilities (changes to HR and SNRT) that were responsible for the discontinuation of an earlier lead from this chemical series, BMS-605339 (1), from clinical trials. The structure-activity relationships (SARs) developed with respect to CV effects established that small structural changes to the P2* subsite of the molecule had a significant impact on the CV profile of a given compound. The antiviral activity, preclincial PK profile, and toxicology studies in rat and dog supported clinical development of BMS-650032 (24). PMID:24564672

  17. Discovery and early clinical evaluation of BMS-605339, a potent and orally efficacious tripeptidic acylsulfonamide NS3 protease inhibitor for the treatment of hepatitis C virus infection.

    PubMed

    Scola, Paul M; Wang, Alan Xiangdong; Good, Andrew C; Sun, Li-Qiang; Combrink, Keith D; Campbell, Jeffrey A; Chen, Jie; Tu, Yong; Sin, Ny; Venables, Brian L; Sit, Sing-Yuen; Chen, Yan; Cocuzza, Anthony; Bilder, Donna M; D'Andrea, Stanley; Zheng, Barbara; Hewawasam, Piyasena; Ding, Min; Thuring, Jan; Li, Jianqing; Hernandez, Dennis; Yu, Fei; Falk, Paul; Zhai, Guangzhi; Sheaffer, Amy K; Chen, Chaoqun; Lee, Min S; Barry, Diana; Knipe, Jay O; Li, Wenying; Han, Yong-Hae; Jenkins, Susan; Gesenberg, Christoph; Gao, Qi; Sinz, Michael W; Santone, Kenneth S; Zvyaga, Tatyana; Rajamani, Ramkumar; Klei, Herbert E; Colonno, Richard J; Grasela, Dennis M; Hughes, Eric; Chien, Caly; Adams, Stephen; Levesque, Paul C; Li, Danshi; Zhu, Jialong; Meanwell, Nicholas A; McPhee, Fiona

    2014-03-13

    The discovery of BMS-605339 (35), a tripeptidic inhibitor of the NS3/4A enzyme, is described. This compound incorporates a cyclopropylacylsulfonamide moiety that was designed to improve the potency of carboxylic acid prototypes through the introduction of favorable nonbonding interactions within the S1' site of the protease. The identification of 35 was enabled through the optimization and balance of critical properties including potency and pharmacokinetics (PK). This was achieved through modulation of the P2* subsite of the inhibitor which identified the isoquinoline ring system as a key template for improving PK properties with further optimization achieved through functionalization. A methoxy moiety at the C6 position of this isoquinoline ring system proved to be optimal with respect to potency and PK, thus providing the clinical compound 35 which demonstrated antiviral activity in HCV-infected patients. PMID:24555570

  18. Image Annotation and Database Mining to Create a Novel Screen for the Chemotype-Dependent Crystallization of HCV NS3 Protease

    SciTech Connect

    H Klei; K Kish; M Russo; S Michalczyk; M Cahn; J Tredup; C Chang; J Khan; E Baldwin

    2011-12-31

    An effective process for screening, imaging, and optimizing crystallization trials using a combination of external and internal hardware and software has been deployed. The combination of this infrastructure with a vast annotated crystallization database enables the creation of custom crystallization screening strategies. Because of the strong chemotype-dependent crystallization observed with HCV NS3 protease (HCVPr), this strategy was applied to a chemotype resistant to all prior crystallization efforts. The crystallization database was mined for ingredients used to generate earlier HCVPr/inhibitor co-crystals. A random screen was created from the most prolific ingredients. A previously untested combination of proven ingredients was identified that led to a successful crystallization condition for the resistant chemotype.

  19. Towards the second generation of Boceprevir: Dithianes as an alternative P2 substituent for 2,2-dimethyl cycloproyl proline in HCV NS3 protease inhibitors.

    PubMed

    Nair, Latha G; Bogen, Stephane; Ruan, Sumei; Pan, Weidong; Pike, Russel; Tong, Xiao; Cheng, Kuo-Chi; Guo, Zhuyan; Doll, Ronald J; Njoroge, F George

    2010-03-01

    Hepatitis C (HCV) infection is a global health crisis leading to chronic liver disease. In our efforts towards a second generation HCV NS3 serine protease inhibitor with improved profile, we have undertaken SAR studies in various regions of Boceprevir including P2. Herein, we report the synthesis and structure-activity relationship studies of inhibitors with (S)-1,4-dithia-7-azaspiro[4.4]nonane-8-carboxylic acid 2 as P2 substituent replacing the (1R,2S,5S)-6,6-dimethyl 3-azabicyclo[3.1.0]hexane-2-carboxylic acid. The systematic investigation led to the discovery of highly potent inhibitor 25 (K(i)( *)=7nM, EC(90)=30nM) with improved rat exposure of 2.56microM h. PMID:20149655

  20. RAC/ROP GTPases and auxin signaling.

    PubMed

    Wu, Hen-ming; Hazak, Ora; Cheung, Alice Y; Yalovsky, Shaul

    2011-04-01

    Auxin functions as a key morphogen in regulating plant growth and development. Studies on auxin-regulated gene expression and on the mechanism of polar auxin transport and its asymmetric distribution within tissues have provided the basis for realizing the molecular mechanisms underlying auxin function. In eukaryotes, members of the Ras and Rho subfamilies of the Ras superfamily of small GTPases function as molecular switches in many signaling cascades that regulate growth and development. Plants do not have Ras proteins, but they contain Rho-like small G proteins called RACs or ROPs that, like fungal and metazoan Rhos, are regulators of cell polarity and may also undertake some Ras functions. Here, we discuss the advances made over the last decade that implicate RAC/ROPs as mediators for auxin-regulated gene expression, rapid cell surface-located auxin signaling, and directional auxin transport. We also describe experimental data indicating that auxin-RAC/ROP crosstalk may form regulatory feedback loops and theoretical modeling that attempts to connect local auxin gradients with RAC/ROP regulation of cell polarity. We hope that by discussing these experimental and modeling studies, this perspective will stimulate efforts to further refine our understanding of auxin signaling via the RAC/ROP molecular switch. PMID:21478442

  1. NMR and MD Studies Reveal That the Isolated Dengue NS3 Protease Is an Intrinsically Disordered Chymotrypsin Fold Which Absolutely Requests NS2B for Correct Folding and Functional Dynamics

    PubMed Central

    Gupta, Garvita; Lim, Liangzhong; Song, Jianxing

    2015-01-01

    Dengue genome encodes a two component protease complex (NS2B-NS3pro) essential for the viral maturation/infectivity, thus representing a key drug target. Previously, due to its “complete insolubility”, the isolated NS3pro could not be experimentally studied and it remains elusive what structure it adopts without NS2B and why NS2B is indispensable. Here as facilitated by our previous discovery, the isolated NS3pro has been surprisingly deciphered by NMR to be the first intrinsically-disordered chymotrypsin-like fold, which exists in a loosely-packed state with non-native long-range interactions as revealed by paramagnetic relaxation enhancement (PRE). The disordered NS3pro appears to be needed for binding a human host factor to trigger the membrane remodeling. Moreover, we have in vitro refolded the NS3pro in complex with either NS2B (48–100) or the full-length NS2B (1–130) anchored into the LMPC micelle, and the two complexes have similar activities but different dynamics. We also performed molecular dynamics (MD) simulations and the results revealed that NS2B shows the highest structural fluctuations in the complex, thus providing the dynamic basis for the observation on its conformational exchange between open and closed states. Remarkably, the NS2B cofactor plays a central role in maintaining the correlated motion network required for the catalysis as we previously decoded for the SARS 3CL protease. Indeed, a truncated NS2B (48–100;Δ77–84) with the flexible loop deleted is able to trap the NS2B-NS3pro complex in a highly dynamic and catalytically-impotent state. Taken together, our study implies potential strategies to perturb the NS2B-NS3pro interface for design of inhibitors for treating dengue infection. PMID:26258523

  2. The GTPase-Activating Protein Rga1 Interacts with Rho3 GTPase and May Regulate Its Function in Polarized Growth in Budding Yeast

    PubMed Central

    He, Fei; Nie, Wen-Chao; Tong, Zongtian; Yuan, Si-Min; Gong, Ting; Liao, Yuan; Bi, Erfei; Gao, Xiang-Dong

    2015-01-01

    In budding yeast, Rga1 negatively regulates the Rho GTPase Cdc42 by acting as a GTPase-activating protein (GAP) for Cdc42. To gain insight into the function and regulation of Rga1, we overexpressed Rga1 and an N-terminally truncated Rga1-C538 (a.a. 538-1007) segment. Overexpression of Rga1-C538 but not full-length Rga1 severely impaired growth and cell morphology in wild-type cells. We show that Rga1 is phosphorylated during the cell cycle. The lack of phenotype for full-length Rga1 upon overexpression may result from a negative regulation by G1-specific Pho85, a cyclin-dependent kinase (CDK). From a high-copy suppressor screen, we isolated RHO3, SEC9, SEC1, SSO1, SSO2, and SRO7, genes involved in exocytosis, as suppressors of the growth defect caused by Rga1-C538 overexpression. Moreover, we detected that Rga1 interacts with Rho3 in two-hybrid and bimolecular fluorescence complementation (BiFC) assays. Rga1 preferentially interacts with the GTP-bound form of Rho3 and the interaction requires the GAP domain and additional sequence upstream of the GAP domain. Our data suggest that the interaction of Rga1 with Rho3 may regulate Rho3’s function in polarized bud growth. PMID:25860339

  3. Thousands of Rab GTPases for the Cell Biologist

    PubMed Central

    Diekmann, Yoan; Seixas, Elsa; Gouw, Marc; Tavares-Cadete, Filipe; Seabra, Miguel C.; Pereira-Leal, José B.

    2011-01-01

    Rab proteins are small GTPases that act as essential regulators of vesicular trafficking. 44 subfamilies are known in humans, performing specific sets of functions at distinct subcellular localisations and tissues. Rab function is conserved even amongst distant orthologs. Hence, the annotation of Rabs yields functional predictions about the cell biology of trafficking. So far, annotating Rabs has been a laborious manual task not feasible for current and future genomic output of deep sequencing technologies. We developed, validated and benchmarked the Rabifier, an automated bioinformatic pipeline for the identification and classification of Rabs, which achieves up to 90% classification accuracy. We cataloged roughly 8.000 Rabs from 247 genomes covering the entire eukaryotic tree. The full Rab database and a web tool implementing the pipeline are publicly available at www.RabDB.org. For the first time, we describe and analyse the evolution of Rabs in a dataset covering the whole eukaryotic phylogeny. We found a highly dynamic family undergoing frequent taxon-specific expansions and losses. We dated the origin of human subfamilies using phylogenetic profiling, which enlarged the Rab repertoire of the Last Eukaryotic Common Ancestor with Rab14, 32 and RabL4. Furthermore, a detailed analysis of the Choanoflagellate Monosiga brevicollis Rab family pinpointed the changes that accompanied the emergence of Metazoan multicellularity, mainly an important expansion and specialisation of the secretory pathway. Lastly, we experimentally establish tissue specificity in expression of mouse Rabs and show that neo-functionalisation best explains the emergence of new human Rab subfamilies. With the Rabifier and RabDB, we provide tools that easily allows non-bioinformaticians to integrate thousands of Rabs in their analyses. RabDB is designed to enable the cell biology community to keep pace with the increasing number of fully-sequenced genomes and change the scale at which we perform

  4. Thousands of rab GTPases for the cell biologist.

    PubMed

    Diekmann, Yoan; Seixas, Elsa; Gouw, Marc; Tavares-Cadete, Filipe; Seabra, Miguel C; Pereira-Leal, José B

    2011-10-01

    Rab proteins are small GTPases that act as essential regulators of vesicular trafficking. 44 subfamilies are known in humans, performing specific sets of functions at distinct subcellular localisations and tissues. Rab function is conserved even amongst distant orthologs. Hence, the annotation of Rabs yields functional predictions about the cell biology of trafficking. So far, annotating Rabs has been a laborious manual task not feasible for current and future genomic output of deep sequencing technologies. We developed, validated and benchmarked the Rabifier, an automated bioinformatic pipeline for the identification and classification of Rabs, which achieves up to 90% classification accuracy. We cataloged roughly 8.000 Rabs from 247 genomes covering the entire eukaryotic tree. The full Rab database and a web tool implementing the pipeline are publicly available at www.RabDB.org. For the first time, we describe and analyse the evolution of Rabs in a dataset covering the whole eukaryotic phylogeny. We found a highly dynamic family undergoing frequent taxon-specific expansions and losses. We dated the origin of human subfamilies using phylogenetic profiling, which enlarged the Rab repertoire of the Last Eukaryotic Common Ancestor with Rab14, 32 and RabL4. Furthermore, a detailed analysis of the Choanoflagellate Monosiga brevicollis Rab family pinpointed the changes that accompanied the emergence of Metazoan multicellularity, mainly an important expansion and specialisation of the secretory pathway. Lastly, we experimentally establish tissue specificity in expression of mouse Rabs and show that neo-functionalisation best explains the emergence of new human Rab subfamilies. With the Rabifier and RabDB, we provide tools that easily allows non-bioinformaticians to integrate thousands of Rabs in their analyses. RabDB is designed to enable the cell biology community to keep pace with the increasing number of fully-sequenced genomes and change the scale at which we perform

  5. Brassica napus Rop GTPases and their expression in microspore cultures.

    PubMed

    Chan, John; Peter Pauls, K

    2007-01-01

    Androgenesis in plants involves a shift in development that causes cultured microspore cells to form embryos rather than continue to develop pollen. In Brassica napus microspore culture a mild heat stress is used to switch on embryo development. An early hallmark of embryogenesis in this system is a symmetrical division of the nucleus instead of the asymmetric division that occurs during pollen formation. ROP GTPases act as molecular switches in a variety of developmental processes; therefore, the current study was initiated to examine whether they might be involved in androgenesis. Five distinct Rop genes with nucleic acid similarities ranging from 82 to 93% to Arabidopsis Rop1 were isolated from B. napus cv Topas. A Southern blot hybridization with a BnRop sequence probe suggested that there are 11-15 ROP gene family members in B. napus. RT-PCR reactions with PCR primers specific to BnRop5, BnRop6, BnRop9 and BnRop10 showed that expression of the BnRop5 was restricted to pollen but the others were detected in leaf, root, stem and pollen tissue. Pollen-like cells obtained from 3-day-old cultures by flow cytometric sorting had BnRop5 transcript levels that were 2.8 times higher than in flow sorted embryogenic microspores. Conversely, the BnRop9 transcript levels were 2.5-fold higher in the embryogenic cells than in the pollen-like cells. The potential involvement of specific ROPs in early stage microspore culture responses is discussed. PMID:16896789

  6. GTPases mechanisms and functions of translation factors on the ribosome.

    PubMed

    Rodnina, M V; Stark, H; Savelsbergh, A; Wieden, H J; Mohr, D; Matassova, N B; Peske, F; Daviter, T; Gualerzi, C O; Wintermeyer, W

    2000-01-01

    The elongation factors (EF) Tu and G and initiation factor 2 (IF2) from bacteria are multidomain GTPases with essential functions in the elongation and initiation phases of translation. They bind to the same site on the ribosome where their low intrinsic GTPase activities are strongly stimulated. The factors differ fundamentally from each other, and from the majority of GTPases, in the mechanisms of GTPase control, the timing of Pi release, and the functional role of GTP hydrolysis. EF-Tu x GTP forms a ternary complex with aminoacyl-tRNA, which binds to the ribosome. Only when a matching codon is recognized, the GTPase of EF-Tu is stimulated, rapid GTP hydrolysis and Pi release take place, EF-Tu rearranges to the GDP form, and aminoacyl-tRNA is released into the peptidyltransferase center. In contrast, EF-G hydrolyzes GTP immediately upon binding to the ribosome, stimulated by ribosomal protein L7/12. Subsequent translocation is driven by the slow dissociation of Pi, suggesting a mechano-chemical function of EF-G. Accordingly, different conformations of EF-G on the ribosome are revealed by cryo-electron microscopy. GTP hydrolysis by IF2 is triggered upon formation of the 70S initiation complex, and the dissociation of Pi and/or IF2 follows a rearrangement of the ribosome into the elongation-competent state. PMID:10937868

  7. Ral-GTPases: approaching their 15 minutes of fame.

    PubMed

    Feig, Larry A

    2003-08-01

    Andy Warhol, the famous pop artist, once claimed that "in the future everyone will be famous for 15 minutes". The same, it seems, can be said of proteins, because at any given time some proteins become more "fashionable" to study than others. But most proteins have been highly conserved throughout millions of years of evolution, which implies that they all have essential roles in cell biology. Thus, each one will no doubt enter the limelight if the right experiment in the right cell type is done. A good example of this is the Ras-like GTPases (Ral-GTPases), which until recently existed in the shadow of their close cousins--the Ras proto-oncogenes. Recent studies have yielded insights into previously unappreciated roles for Ral-GTPases in intensively investigated disciplines such as vesicle trafficking, cell morphology, transcription and possibly even human oncogenesis. PMID:12888294

  8. Production of recombinant non-structural protein-3 hydrophobic domain deletion (NS3ΔHD) protein of bluetongue virus from prokaryotic expression system as an efficient diagnostic reagent.

    PubMed

    Mohanty, Nihar Nalini; Chacko, Nirmal; Biswas, Sanchay Kumar; Chand, Karam; Pandey, Awadh Bihari; Mondal, Bimalendu; Hemadri, Divakar; Shivachandra, Sathish Bhadravati

    2016-09-01

    Serological diagnostics for bluetongue (BT), which is an infectious, non-contagious and arthropod-borne virus disease of ruminants, are primarily dependent on availability of high quality native or recombinant antigen(s) based on either structural/non-structural proteins in sufficient quantity. Non-structural proteins (NS1-NS4) of BT virus are presumed candidate antigens in development of DIVA diagnostics. In the present study, NS3 fusion gene encoding for NS3 protein containing the N- and C-termini with a deletion of two hydrophobic domains (118A to S141 aa and 162S to A182 aa) and intervening variable central domain (142D to K161 aa) of bluetongue virus 23 was constructed, cloned and over-expressed using prokaryotic expression system. The recombinant NS3ΔHD fusion protein (∼38 kDa) including hexa-histidine tag on its both termini was found to be non-cytotoxic to recombinant Escherichia coli cells and purified by affinity chromatography. The purified rNS3ΔHD fusion protein was found to efficiently detect BTV-NS3 specific antibodies in indirect-ELISA format with diagnostic sensitivity (DSn = 94.4%) and specificity (DSp = 93.9%). The study indicated the potential utility of rNS3ΔHD fusion protein as candidate diagnostic reagent in developing an indirect-ELISA for sero-surveillance of animals for BTV antibodies under DIVA strategy, wherever monovalent/polyvalent killed BT vaccine formulations devoid of NS proteins are being practiced for immunization. PMID:27448505

  9. Screening of antiviral activities in medicinal plants extracts against dengue virus using dengue NS2B-NS3 protease assay.

    PubMed

    Rothan, H A; Zulqarnain, M; Ammar, Y A; Tan, E C; Rahman, N A; Yusof, R

    2014-06-01

    Dengue virus infects millions of people worldwide and there is no vaccine or anti-dengue therapeutic available. Screening large numbers of medicinal plants for anti-dengue activities is an alternative strategy in order to find the potent therapeutic compounds. Therefore, this study was designed to identify anti-dengue activities in nineteen medicinal plant extracts that are used in traditional medicine. Local medicinal plants Vernonia cinerea, Hemigraphis reptans, Hedyotis auricularia, Laurentia longiflora, Tridax procumbers and Senna angustifolia were used in this study. The highest inhibitory activates against dengue NS2B-NS3pro was observed in ethanolic extract of S. angustifolia leaves, methanolic extract of V. cinerea leaves and ethanol extract of T. procumbens stems. These findings were further verified by in vitro viral inhibition assay. Methanolic extract of V. cinerea leaves, ethanol extract of T. procumbens stems and at less extent ethanolic extract of S. angustifolia leaves were able to maintain the normal morphology of DENV2-infected Vero cells without causing much cytopathic effects (CPE). The percentage of viral inhibition of V. cinerea and T. procumbens extracts were significantly higher than S. angustifolia extract as measured by plaque formation assay and RT-qPCR. In conclusion, The outcome of this study showed that the methanolic extract of V. cinerea leaves and ethanol extract of T. procumbens stems possessed high inhibitory activates against dengue virus that worth more investigation. PMID:25134897

  10. Monitoring antibody titers to recombinant Core-NS3 fusion polypeptide is useful for evaluating hepatitis C virus infection and responses to interferon-alpha therapy.

    PubMed

    Park, Y M; Byun, B H; Choi, J Y; Bae, S H; Kim, B S; So, H S; Ryu, W S

    1999-04-01

    To evaluate the clinical feasibility of the antibody titer against a chimeric polypeptide (named Core 518), in which a domain of Core and NS3 of hepatitis C virus (HCV) was fused, ELISA was performed in a total of 76 serum samples. Each serum was serially diluted using two-fold dilution method with distilled water into 10 concentrations. They were all positive for second generation anti-HCV assay (HCV EIA II; Abbott Laboratories). Genotyping RT-PCR, quantitative competitive RT-PCR, and RIBA (Lucky Confirm; LG Biotech) were also assayed. Anti-Core 518 antibody was detected in x 12800 or higher dilutions of sera from 35 of 43 chronic hepatitis C (81.4%) and nine of 16 hepatocellular carcinoma sera (56.3%), one of four cirrhosis (25%), 0 of four acute hepatitis C, and one of nine healthy isolated anti-HCV-positive subjects (p=0.0000). The anti-Core 518 antibody titers were well correlated with the presence of HCV RNA in serum (p=0.002). The anti-Core 518 antibody titers decreased significantly in nine of ten responders to IFN-alpha treatment. Monitoring anti-Core 518 titers may be helpful not only for differentiating the status of HCV infection among patients with various type C viral liver diseases, but also for predicting responses to IFN-alpha treatment. PMID:10331562

  11. Dual inhibitors of the dengue and West Nile virus NS2B-NS3 proteases: Synthesis, biological evaluation and docking studies of novel peptide-hybrids.

    PubMed

    Bastos Lima, Allan; Behnam, Mira A M; El Sherif, Yasmin; Nitsche, Christoph; Vechi, Sergio M; Klein, Christian D

    2015-09-01

    Dengue virus (DENV) and West Nile virus (WNV) are mosquito-borne arboviruses responsible for causing acute systemic diseases and severe health conditions in humans. The discovery of therapies capable to prevent infections or treat infected individuals remains an important challenge, since no vaccine or specific efficient treatment could be developed so far. In this context, we present herein the synthesis, characterization, biological evaluation and docking studies of novel peptide-hybrids based on 2,4-thiazolidinedione scaffolds containing non-polar groups. The most promising compound has an IC50 of 0.75 μM against WNV protease, which represents a seventyfold improvement in activity compared to our previously reported compounds. Experimental results and docking studies are in agreement with the hypothesis that a non-polar group in the scaffold is important to obtain interactions between the inhibitors and a hydrophobic pocket in the substrate recognition region of the DENV and WNV NS2B-NS3 serine proteases. PMID:26233795

  12. A novel HCV NS3 protease mutation selected by combination treatment of the protease inhibitor boceprevir and NS5B polymerase inhibitors.

    PubMed

    Chase, Robert; Skelton, Angela; Xia, Ellen; Curry, Stephanie; Liu, Shaotang; McMonagle, Patricia; Huang, H-C; Tong, Xiao

    2009-11-01

    Boceprevir (SCH 503034) is an orally active novel inhibitor of the hepatitis C virus (HCV) NS3 protease currently in clinical development for the treatment of hepatitis C. In this in vitro study, we demonstrate that combination of boceprevir with a nucleoside analog or a non-nucleoside HCV NS5B polymerase inhibitor was superior to treatment by single agents in inhibiting viral RNA replication in replicon cells. In the presence of boceprevir (at 5xEC(90)), the addition of 2'-C-methyl-adenosine or an indole-N-acetamide targeting the polymerase finger-loop site (at 1xEC(90)) significantly reduced the emergence of resistant replicon colonies. A higher dose (5xEC(90)) of either of the polymerase inhibitors in combination with boceprevir suppressed replicon resistance further to below detectable levels. Sequencing analysis of replicon cells selected by the combination treatment revealed known resistance mutations to the two polymerase inhibitors but no previously reported resistance mutations to boceprevir. Interestingly, a novel mutation (M175L) in the protease domain was identified. The dually resistant replicon cells were monitored for over 30 passages and sensitivity to polymerase inhibitors was found to decrease over time in a manner that correlated with the increasing prevalence of specific resistance mutations. Importantly, these cells remained sensitive to interferon-alpha and different classes of polymerase inhibitors. These findings support the rationale for clinical evaluation of combination treatment of HCV protease and polymerase inhibitors. PMID:19747948

  13. Influence of lipidation of GBV-C/HGV NS3 (513-522) and (505-514) peptide sequences on its interaction with mono and bilayers.

    PubMed

    Weroński, Konrad; Busquets, M Antónia; Girona, Victória; Prat, Josefina

    2007-05-15

    Two decapeptide fragments of the non-structural hepatitis G NS3 protein (GBV-C/HGV), 513-522 (RGRTGRGRSG) and 505-514 (SAELSMQRRG), as well as their palmitoylated derivatives were synthesized. The physico-chemical properties of the peptides were analyzed in both the absence and presence of the zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), the negative 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) and the positive 1,2-dioeloyl-3-trimethylammonium-propane (DOTAP) lipid monolayers. Based on their high hydrophilic properties, neither parent peptide presented surface activity and their incorporation into lipid monolayers was low. In contrast, their palmitoylated derivatives showed concentration-dependent surface activity and could be inserted into lipid monolayers to varying degrees depending on their sequence. Compression isotherms showed that the presence of palmitoylated peptides in the subphase resulted in a molecular arrangement less condensed than that corresponding to the pure phospholipid. In concordance with the monolayer results, differential scanning calorimetry (DSC) demonstrated that the parent peptides did not have any effect on the thermograms, while the palmitoylated derivatives affected the thermotropic properties of DPPC bilayers. PMID:17292591

  14. Sar1 GTPase Activity Is Regulated by Membrane Curvature*♦

    PubMed Central

    Hanna, Michael G.; Mela, Ioanna; Wang, Lei; Henderson, Robert M.; Chapman, Edwin R.; Edwardson, J. Michael; Audhya, Anjon

    2016-01-01

    The majority of biosynthetic secretory proteins initiate their journey through the endomembrane system from specific subdomains of the endoplasmic reticulum. At these locations, coated transport carriers are generated, with the Sar1 GTPase playing a critical role in membrane bending, recruitment of coat components, and nascent vesicle formation. How these events are appropriately coordinated remains poorly understood. Here, we demonstrate that Sar1 acts as the curvature-sensing component of the COPII coat complex and highlight the ability of Sar1 to bind more avidly to membranes of high curvature. Additionally, using an atomic force microscopy-based approach, we further show that the intrinsic GTPase activity of Sar1 is necessary for remodeling lipid bilayers. Consistent with this idea, Sar1-mediated membrane remodeling is dramatically accelerated in the presence of its guanine nucleotide-activating protein (GAP), Sec23-Sec24, and blocked upon addition of guanosine-5′-[(β,γ)-imido]triphosphate, a poorly hydrolysable analog of GTP. Our results also indicate that Sar1 GTPase activity is stimulated by membranes that exhibit elevated curvature, potentially enabling Sar1 membrane scission activity to be spatially restricted to highly bent membranes that are characteristic of a bud neck. Taken together, our data support a stepwise model in which the amino-terminal amphipathic helix of GTP-bound Sar1 stably penetrates the endoplasmic reticulum membrane, promoting local membrane deformation. As membrane bending increases, Sar1 membrane binding is elevated, ultimately culminating in GTP hydrolysis, which may destabilize the bilayer sufficiently to facilitate membrane fission. PMID:26546679

  15. Invited review: Microtubule severing enzymes couple atpase activity with tubulin GTPase spring loading.

    PubMed

    Bailey, Megan E; Jiang, Nan; Dima, Ruxandra I; Ross, Jennifer L

    2016-08-01

    Microtubules are amazing filaments made of GTPase enzymes that store energy used for their own self-destruction to cause a stochastically driven dynamics called dynamic instability. Dynamic instability can be reproduced in vitro with purified tubulin, but the dynamics do not mimic that observed in cells. This is because stabilizers and destabilizers act to alter microtubule dynamics. One interesting and understudied class of destabilizers consists of the microtubule-severing enzymes from the ATPases Associated with various cellular Activities (AAA+) family of ATP-enzymes. Here we review current knowledge about GTP-driven microtubule dynamics and how that couples to ATP-driven destabilization by severing enzymes. We present a list of challenges regarding the mechanism of severing, which require development of experimental and modeling approaches to shed light as to how severing enzymes can act to regulate microtubule dynamics in cells. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 547-556, 2016. PMID:27037673

  16. Crystal structure of the Ego1-Ego2-Ego3 complex and its role in promoting Rag GTPase-dependent TORC1 signaling.

    PubMed

    Powis, Katie; Zhang, Tianlong; Panchaud, Nicolas; Wang, Rong; De Virgilio, Claudio; Ding, Jianping

    2015-09-01

    The target of rapamycin complex 1 (TORC1) integrates various hormonal and nutrient signals to regulate cell growth, proliferation, and differentiation. Amino acid-dependent activation of TORC1 is mediated via the yeast EGO complex (EGOC) consisting of Gtr1, Gtr2, Ego1, and Ego3. Here, we identify the previously uncharacterized Ycr075w-a/Ego2 protein as an additional EGOC component that is required for the integrity and localization of the heterodimeric Gtr1-Gtr2 GTPases, equivalent to mammalian Rag GTPases. We also report the crystal structure of the Ego1-Ego2-Ego3 ternary complex (EGO-TC) at 2.4 Å resolution, in which Ego2 and Ego3 form a heterodimer flanked along one side by Ego1. Structural data also reveal the structural conservation of protein components between the yeast EGO-TC and the human Ragulator, which acts as a GEF for Rag GTPases. Interestingly, however, artificial tethering of Gtr1-Gtr2 to the vacuolar membrane is sufficient to activate TORC1 in response to amino acids even in the absence of the EGO-TC. Our structural and functional data therefore support a model in which the EGO-TC acts as a scaffold for Rag GTPases in TORC1 signaling. PMID:26206314

  17. Mass Balance and Metabolite Profiling of Steady-State Faldaprevir, a Hepatitis C Virus NS3/4 Protease Inhibitor, in Healthy Male Subjects

    PubMed Central

    Rose, Peter; Mao, Yanping; Yong, Chan-Loi; St. George, Roger; Huang, Fenglei; Latli, Bachir; Mandarino, Debra; Li, Yongmei

    2014-01-01

    The pharmacokinetics, mass balance, and metabolite profiles of faldaprevir, a selective peptide-mimetic hepatitis C virus NS3/NS4 protease inhibitor, were assessed at steady state in 7 healthy male subjects. Subjects received oral doses of 480 mg faldaprevir on day 1, followed by 240 mg faldaprevir on days 2 to 8 and 10 to 15. [14C]faldaprevir (240 mg containing 100 μCi) was administered on day 9. Blood, urine, feces, and saliva samples were collected at intervals throughout the study. Metabolite profiling was performed using radiochromatography, and metabolite identification was conducted using liquid chromatography-tandem mass spectrometry. The overall recovery of radioactivity was high (98.8%), with the majority recovered from feces (98.7%). There was minimal radioactivity in urine (0.113%) and saliva. Circulating radioactivity was predominantly confined to plasma with minimal partitioning into red blood cells. The terminal half-life of radioactivity in plasma was approximately 23 h with no evidence of any long-lasting metabolites. Faldaprevir was the predominant circulating form, accounting for 98 to 100% of plasma radioactivity from each subject. Faldaprevir was the only drug-related component detected in urine. Faldaprevir was also the major drug-related component in feces, representing 49.8% of the radioactive dose. The majority of the remainder of radioactivity in feces (41% of the dose) was accounted for in almost equal quantities by 2 hydroxylated metabolites. The most common adverse events were nausea, diarrhea, and constipation, all of which were related to study drug. In conclusion, faldaprevir is predominantly excreted in feces with negligible urinary excretion. PMID:24514093

  18. Preclinical Characterization of the Antiviral Activity of SCH 900518 (Narlaprevir), a Novel Mechanism-Based Inhibitor of Hepatitis C Virus NS3 Protease▿

    PubMed Central

    Tong, X.; Arasappan, A.; Bennett, F.; Chase, R.; Feld, B.; Guo, Z.; Hart, A.; Madison, V.; Malcolm, B.; Pichardo, J.; Prongay, A.; Ralston, R.; Skelton, A.; Xia, E.; Zhang, R.; Njoroge, F. G.

    2010-01-01

    Small-molecule hepatitis C virus (HCV) NS3 protease inhibitors such as boceprevir (SCH 503034) have been shown to have antiviral activity when they are used as monotherapy and in combination with pegylated alpha interferon and ribavirin in clinical trials. Improvements in inhibitor potency and pharmacokinetic properties offer opportunities to increase drug exposure and to further increase the sustained virological response. Exploration of the structure-activity relationships of ketoamide inhibitors related to boceprevir has led to the discovery of SCH 900518, a novel ketoamide protease inhibitor which forms a reversible covalent bond with the active-site serine. It has an overall inhibition constant (K*i) of 7 nM and a dissociation half-life of 1 to 2 h. SCH 900518 inhibited replicon RNA at a 90% effective concentration (EC90) of 40 nM. In biochemical assays, SCH 900518 was active against proteases of genotypes 1 to 3. A 2-week treatment with 5× EC90 of the inhibitor reduced the replicon RNA level by 3 log units. Selection of replicon cells with SCH 900518 resulted in the outgrowth of several resistant mutants (with the T54A/S and A156S/T/V mutations). Cross-resistance studies demonstrated that the majority of mutations for resistance to boceprevir and telaprevir caused similar fold losses of activity against all three inhibitors; however, SCH 900518 retained more activity against these mutants due to its higher intrinsic potency. Combination treatment with alpha interferon enhanced the inhibition of replicon RNA and suppressed the emergence of resistant replicon colonies, supporting the use of SCH 900518-pegylated alpha interferon combination therapy in the clinic. In summary, the results of the preclinical characterization of the antiviral activity of SCH 900518 support its evaluation in clinical studies. PMID:20308381

  19. Preclinical characterization of the antiviral activity of SCH 900518 (narlaprevir), a novel mechanism-based inhibitor of hepatitis C virus NS3 protease.

    PubMed

    Tong, X; Arasappan, A; Bennett, F; Chase, R; Feld, B; Guo, Z; Hart, A; Madison, V; Malcolm, B; Pichardo, J; Prongay, A; Ralston, R; Skelton, A; Xia, E; Zhang, R; Njoroge, F G

    2010-06-01

    Small-molecule hepatitis C virus (HCV) NS3 protease inhibitors such as boceprevir (SCH 503034) have been shown to have antiviral activity when they are used as monotherapy and in combination with pegylated alpha interferon and ribavirin in clinical trials. Improvements in inhibitor potency and pharmacokinetic properties offer opportunities to increase drug exposure and to further increase the sustained virological response. Exploration of the structure-activity relationships of ketoamide inhibitors related to boceprevir has led to the discovery of SCH 900518, a novel ketoamide protease inhibitor which forms a reversible covalent bond with the active-site serine. It has an overall inhibition constant (K*(i)) of 7 nM and a dissociation half-life of 1 to 2 h. SCH 900518 inhibited replicon RNA at a 90% effective concentration (EC(90)) of 40 nM. In biochemical assays, SCH 900518 was active against proteases of genotypes 1 to 3. A 2-week treatment with 5x EC(90) of the inhibitor reduced the replicon RNA level by 3 log units. Selection of replicon cells with SCH 900518 resulted in the outgrowth of several resistant mutants (with the T54A/S and A156S/T/V mutations). Cross-resistance studies demonstrated that the majority of mutations for resistance to boceprevir and telaprevir caused similar fold losses of activity against all three inhibitors; however, SCH 900518 retained more activity against these mutants due to its higher intrinsic potency. Combination treatment with alpha interferon enhanced the inhibition of replicon RNA and suppressed the emergence of resistant replicon colonies, supporting the use of SCH 900518-pegylated alpha interferon combination therapy in the clinic. In summary, the results of the preclinical characterization of the antiviral activity of SCH 900518 support its evaluation in clinical studies. PMID:20308381

  20. Transglutaminase 2 Regulates the GTPase-activating Activity of Bcr*

    PubMed Central

    Yi, Sun-Ju; Groffen, John; Heisterkamp, Nora

    2009-01-01

    Transglutaminase 2 (TG2) is a multifunctional protein that has been implicated in numerous pathologies including that of neurodegeneration and celiac disease, but the molecular interactions that mediate its diverse activities are largely unknown. Bcr and the closely related Abr negatively regulate the small G-protein Rac: loss of their combined function in vivo results in increased reactivity of innate immune cells. Bcr and Abr are GTPase-activating proteins that catalyze the hydrolysis of the GTP bound to Rac. However, how the Bcr and Abr GTPase-activating activity is regulated is not precisely understood. We here report a novel mechanism of regulation through direct protein-protein interaction with TG2. TG2 bound to the Rac-binding pocket in the GTPase-activating domains of Bcr and Abr, blocked Bcr activity and, through this mechanism, increased levels of active GTP-bound Rac and EGF-stimulated membrane ruffling. TG2 exists in at least two different conformations. Interestingly, experiments using TG2 mutants showed that Bcr exhibits preferential binding to the non-compacted conformation of TG2, in which its catalytic domain is exposed, but transamidation is not needed for the interaction. Thus, TG2 regulates levels of cellular GTP-bound Rac and actin cytoskeletal reorganization through a new mechanism involving direct inhibition of Bcr GTPase-activating activity. PMID:19840940

  1. The interdependence of the Rho GTPases and apicobasal cell polarity

    PubMed Central

    Mack, Natalie Ann; Georgiou, Marios

    2014-01-01

    Signaling via the Rho GTPases provides crucial regulation of numerous cell polarization events, including apicobasal (AB) polarity, polarized cell migration, polarized cell division and neuronal polarity. Here we review the relationships between the Rho family GTPases and epithelial AB polarization events, focusing on the 3 best-characterized members: Rho, Rac and Cdc42. We discuss a multitude of processes that are important for AB polarization, including lumen formation, apical membrane specification, cell-cell junction assembly and maintenance, as well as tissue polarity. Our discussions aim to highlight the immensely complex regulatory mechanisms that encompass Rho GTPase signaling during AB polarization. More specifically, in this review we discuss several emerging common themes, that include: 1) the need for Rho GTPase activities to be carefully balanced in both a spatial and temporal manner through a multitude of mechanisms; 2) the existence of signaling feedback loops and crosstalk to create robust cellular responses; and 3) the frequent multifunctionality that exists among AB polarity regulators. Regarding this latter theme, we provide further discussion of the potential plasticity of the cell polarity machinery and as a result the possible implications for human disease. PMID:25469537

  2. G domain dimerization controls dynamin's assembly-stimulated GTPase activity

    SciTech Connect

    Chappie, Joshua S.; Acharya, Sharmistha; Leonard, Marilyn; Schmid, Sandra L.; Dyda, Fred

    2010-06-14

    Dynamin is an atypical GTPase that catalyses membrane fission during clathrin-mediated endocytosis. The mechanisms of dynamin's basal and assembly-stimulated GTP hydrolysis are unknown, though both are indirectly influenced by the GTPase effector domain (GED). Here we present the 2.0 {angstrom} resolution crystal structure of a human dynamin 1-derived minimal GTPase-GED fusion protein, which was dimeric in the presence of the transition state mimic GDP.AlF{sub 4}{sup -}. The structure reveals dynamin's catalytic machinery and explains how assembly-stimulated GTP hydrolysis is achieved through G domain dimerization. A sodium ion present in the active site suggests that dynamin uses a cation to compensate for the developing negative charge in the transition state in the absence of an arginine finger. Structural comparison to the rat dynamin G domain reveals key conformational changes that promote G domain dimerization and stimulated hydrolysis. The structure of the GTPase-GED fusion protein dimer provides insight into the mechanisms underlying dynamin-catalysed membrane fission.

  3. RhoGAPs and Rho GTPases in platelets.

    PubMed

    Elvers, Margitta

    2016-08-01

    Platelet cytoskeletal reorganization is essential for platelet adhesion and thrombus formation in hemostasis and thrombosis. The Rho GTPases RhoA, Rac1 and Cdc42 are the main players in cytoskeletal dynamics of platelets responsible for the formation of filopodia and lamellipodia to strongly increase the platelet surface upon activation. They are involved in platelet activation and aggregate formation including platelet secretion, integrin activation and arterial thrombus formation. The activity of Rho GTPases is tightly controlled by different proteins such as GTPase-activating proteins (GAPs). GAPs stimulate GTP hydrolysis to terminate Rho signaling. The role and impact of GAPs in platelets is not well-defined and many of the RhoGAPs identified are not known to be present in platelets or to have any function in platelets. The recently identified RhoGAPs Oligophrenin1 (OPHN1) and Nadrin regulate the activity of RhoA, Rac1 and Cdc42 and subsequent platelet cytoskeletal reorganization, platelet activation and thrombus formation. In the last years, the analysis of genetically modified mice helped to gain the understanding of Rho GTPases and their regulators in cytoskeletal rearrangements and other Rho mediated cellular processes in platelets. PMID:25639730

  4. Control of T lymphocyte morphology by the GTPase Rho

    NASA Technical Reports Server (NTRS)

    Woodside, Darren G.; Wooten, David K.; Teague, T. Kent; Miyamoto, Yuko J.; Caudell, Eva G.; Udagawa, Taturo; Andruss, Bernard F.; McIntyre, Bradley W.

    2003-01-01

    BACKGROUND: Rho family GTPase regulation of the actin cytoskeleton governs a variety of cell responses. In this report, we have analyzed the role of the GTPase Rho in maintenance of the T lymphocyte actin cytoskeleton. RESULTS: Inactivation of the GTPase Rho in the human T lymphocytic cell line HPB-ALL does not inhibit constitutively high adhesion to the integrin beta1 substrate fibronectin. It did however result in the aberrant extension of finger-like dendritic processes on the substrates VCAM-1, Fn, and mAb specific to beta1 integrins. Time-lapse video microscopy demonstrated that C3 induced extensions were primarily the result of an altered pseudopod elongation rather than retraction. Once the stellate pseudopodia extended, none retracted, and cells became completely immobile. Filipodial structures were absent and the dendritic-like processes in C3 treated cells were rich in filamentous actin. Immunolocalization of RhoA in untreated HPB-ALL cells spreading on fibronectin demonstrated a diffuse staining pattern within the pseudopodia. In C3 treated cells, clusters of RhoA were pronounced and localized within the altered extensions. CONCLUSIONS: GTPase Rho is actively involved in the regulation of T lymphocyte morphology and motility.

  5. AMPylation of Rho GTPases Subverts Multiple Host Signaling Processes*

    PubMed Central

    Woolery, Andrew R.; Yu, Xiaobo; LaBaer, Joshua; Orth, Kim

    2014-01-01

    Rho GTPases are frequent targets of virulence factors as they are keystone signaling molecules. Herein, we demonstrate that AMPylation of Rho GTPases by VopS is a multifaceted virulence mechanism that counters several host immunity strategies. Activation of NFκB, Erk, and JNK kinase signaling pathways were inhibited in a VopS-dependent manner during infection with Vibrio parahaemolyticus. Phosphorylation and degradation of IKBα were inhibited in the presence of VopS as was nuclear translocation of the NFκB subunit p65. AMPylation also prevented the generation of superoxide by the phagocytic NADPH oxidase complex, potentially by inhibiting the interaction of Rac and p67. Furthermore, the interaction of GTPases with the E3 ubiquitin ligases cIAP1 and XIAP was hindered, leading to decreased degradation of Rac and RhoA during infection. Finally, we screened for novel Rac1 interactions using a nucleic acid programmable protein array and discovered that Rac1 binds to the protein C1QA, a protein known to promote immune signaling in the cytosol. Interestingly, this interaction was disrupted by AMPylation. We conclude that AMPylation of Rho Family GTPases by VopS results in diverse inhibitory consequences during infection beyond the most obvious phenotype, the collapse of the actin cytoskeleton. PMID:25301945

  6. The interdependence of the Rho GTPases and apicobasal cell polarity.

    PubMed

    Mack, Natalie Ann; Georgiou, Marios

    2014-01-01

    Signaling via the Rho GTPases provides crucial regulation of numerous cell polarization events, including apicobasal (AB) polarity, polarized cell migration, polarized cell division and neuronal polarity. Here we review the relationships between the Rho family GTPases and epithelial AB polarization events, focusing on the 3 best-characterized members: Rho, Rac and Cdc42. We discuss a multitude of processes that are important for AB polarization, including lumen formation, apical membrane specification, cell-cell junction assembly and maintenance, as well as tissue polarity. Our discussions aim to highlight the immensely complex regulatory mechanisms that encompass Rho GTPase signaling during AB polarization. More specifically, in this review we discuss several emerging common themes, that include: 1) the need for Rho GTPase activities to be carefully balanced in both a spatial and temporal manner through a multitude of mechanisms; 2) the existence of signaling feedback loops and crosstalk to create robust cellular responses; and 3) the frequent multifunctionality that exists among AB polarity regulators. Regarding this latter theme, we provide further discussion of the potential plasticity of the cell polarity machinery and as a result the possible implications for human disease. PMID:25469537

  7. Epithelial junctions and Rho family GTPases: the zonular signalosome

    PubMed Central

    Citi, Sandra; Guerrera, Diego; Spadaro, Domenica; Shah, Jimit

    2014-01-01

    The establishment and maintenance of epithelial cell-cell junctions is crucially important to regulate adhesion, apico-basal polarity and motility of epithelial cells, and ultimately controls the architecture and physiology of epithelial organs. Junctions are supported, shaped and regulated by cytoskeletal filaments, whose dynamic organization and contractility are finely tuned by GTPases of the Rho family, primarily RhoA, Rac1 and Cdc42. Recent research has identified new molecular mechanisms underlying the cross-talk between these GTPases and epithelial junctions. Here we briefly summarize the current knowledge about the organization, molecular evolution and cytoskeletal anchoring of cell-cell junctions, and we comment on the most recent advances in the characterization of the interactions between Rho GTPases and junctional proteins, and their consequences with regards to junction assembly and regulation of cell behavior in vertebrate model systems. The concept of “zonular signalosome” is proposed, which highlights the close functional relationship between proteins of zonular junctions (zonulae occludentes and adhaerentes) and the control of cytoskeletal organization and signaling through Rho GTPases, transcription factors, and their effectors. PMID:25483301

  8. Pattern formation of Rho GTPases in single cell wound healing

    PubMed Central

    Simon, Cory M.; Vaughan, Emily M.; Bement, William M.; Edelstein-Keshet, Leah

    2013-01-01

    The Rho GTPases—Rho, Rac, and Cdc42—control an enormous variety of processes, many of which reflect activation of these GTPases in spatially confined and mutually exclusive zones. By using mathematical models and experimental results to establish model parameters, we analyze the formation and segregation of Rho and Cdc42 zones during Xenopus oocyte wound repair and the role played by Abr, a dual guanine nucleotide exchange factor–GTPase-activating protein, in this process. The Rho and Cdc42 zones are found to be best represented as manifestations of spatially modulated bistability, and local positive feedback between Abr and Rho can account for the maintenance and dynamic properties of the Rho zone. In contrast, the invocation of an Abr-independent positive feedback loop is required to account for Cdc42 spatial bistability. In addition, the model replicates the results of previous in vivo experiments in which Abr activity is manipulated. Further, simulating the model with two closely spaced wounds made nonintuitive predictions about the Rho and Cdc42 patterns; these predictions were confirmed by experiment. We conclude that the model is a useful tool for analysis of Rho GTPase signaling and that the Rho GTPases can be fruitfully considered as components of intracellular pattern formation systems. PMID:23264464

  9. MIRO GTPases in Mitochondrial Transport, Homeostasis and Pathology

    PubMed Central

    Tang, Bor Luen

    2015-01-01

    The evolutionarily-conserved mitochondrial Rho (MIRO) small GTPase is a Ras superfamily member with three unique features. It has two GTPase domains instead of the one found in other small GTPases, and it also has two EF hand calcium binding domains, which allow Ca2+-dependent modulation of its activity and functions. Importantly, it is specifically associated with the mitochondria and via a hydrophobic transmembrane domain, rather than a lipid-based anchor more commonly found in other small GTPases. At the mitochondria, MIRO regulates mitochondrial homeostasis and turnover. In metazoans, MIRO regulates mitochondrial transport and organization at cellular extensions, such as axons, and, in some cases, intercellular transport of the organelle through tunneling nanotubes. Recent findings have revealed a myriad of molecules that are associated with MIRO, particularly the kinesin adaptor Milton/TRAK, mitofusin, PINK1 and Parkin, as well as the endoplasmic reticulum-mitochondria encounter structure (ERMES) complex. The mechanistic aspects of the roles of MIRO and its interactors in mitochondrial homeostasis and transport are gradually being revealed. On the other hand, MIRO is also increasingly associated with neurodegenerative diseases that have roots in mitochondrial dysfunction. In this review, I discuss what is currently known about the cellular physiology and pathophysiology of MIRO functions. PMID:26729171

  10. MicroRNAs as key regulators of GTPase-mediated apical actin reorganization in multiciliated epithelia

    PubMed Central

    Mercey, Olivier; Kodjabachian, Laurent; Barbry, Pascal; Marcet, Brice

    2016-01-01

    ABSTRACT Multiciliated cells (MCCs), which are present in specialized vertebrate tissues such as mucociliary epithelia, project hundreds of motile cilia from their apical membrane. Coordinated ciliary beating in MCCs contributes to fluid propulsion in several biological processes. In a previous work, we demonstrated that microRNAs of the miR-34/449 family act as new conserved regulators of MCC differentiation by specifically repressing cell cycle genes and the Notch pathway. Recently, we have shown that miR-34/449 also modulate small GTPase pathways to promote, in a later stage of differentiation, the assembly of the apical actin network, a prerequisite for proper anchoring of centrioles-derived neo-synthesized basal bodies. We characterized several miR-34/449 targets related to small GTPase pathways including R-Ras, which represents a key and conserved regulator during MCC differentiation. Direct RRAS repression by miR-34/449 is necessary for apical actin meshwork assembly, notably by allowing the apical relocalization of the actin binding protein Filamin-A near basal bodies. Our studies establish miR-34/449 as central players that orchestrate several steps of MCC differentiation program by regulating distinct signaling pathways. PMID:27144998

  11. Novel 2-oxoimidazolidine-4-carboxylic acid derivatives as Hepatitis C virus NS3-4A serine protease inhibitors: synthesis, activity, and X-ray crystal structure of an enzyme inhibitor complex

    SciTech Connect

    Arasappan, Ashok; Njoroge, F. George; Parekh, Tejal N.; Yang, Xiaozheng; Pichardo, John; Butkiewicz, Nancy; Prongay, Andrew; Yao, Nanhua; Girijavallabhan, Viyyoor

    2008-06-30

    Synthesis and HCV NS3 serine protease inhibitory activity of some novel 2-oxoimidazolidine-4-carboxylic acid derivatives are reported. Inhibitors derived from this new P2 core exhibited activity in the low {micro}M range. X-ray structure of an inhibitor, 15c bound to the protease is presented.

  12. Osteoblast differentiation and migration are regulated by dynamin GTPase activity.

    PubMed

    Eleniste, Pierre P; Huang, Su; Wayakanon, Kornchanok; Largura, Heather W; Bruzzaniti, Angela

    2014-01-01

    Bone formation is controlled by osteoblasts, but the signaling proteins that control osteoblast differentiation and function are still unclear. We examined if the dynamin GTPase, which is associated with actin remodeling and migration in other cells, plays a role in osteoblast differentiation and migration. Dynamin mRNA was expressed in primary osteoblasts throughout differentiation (0-21 days). However, alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was decreased in osteoblasts over-expressing dynamin. Conversely, ALP activity was increased following shRNA-mediated knockdown of dynamin and in osteoblasts treated with the dynamin inhibitor, dynasore. Dynasore also reduced c-fos and osterix expression, markers of early osteoblasts, suggesting a role for dynamin in pre-osteoblast to osteoblast differentiation. Since dynamin GTPase activity is regulated by tyrosine phosphorylation, we examined the mechanism of dynamin dephosphorylation in osteoblasts. Dynamin formed a protein complex with the tyrosine phosphatase PTP-PEST and inhibition of phosphatase activity increased the level of phosphorylated dynamin. Further, PTP-PEST blocked the Src-mediated increase in the phosphorylation and GTPase activity of wild-type dynamin but not the phosphorylation mutant dynY231F/Y597F. Although ALP activity was increased in osteoblasts expressing GTPase-defective dynK44A, and to a lesser extent dynY231F/Y597F, osteoblast migration was significantly inhibited by dynK44A and dynY231F/Y597F. These studies demonstrate a novel role for dynamin GTPase activity and phosphorylation in osteoblast differentiation and migration, which may be important for bone formation. PMID:24387844

  13. Osteoblast differentiation and migration are regulated by Dynamin GTPase activity

    PubMed Central

    Eleniste, Pierre P.; Huang, Su; Wayakanon, Kornchanok; Largura, Heather W.; Bruzzaniti, Angela

    2013-01-01

    Bone formation is controlled by osteoblasts but the signaling proteins that control osteoblast differentiation and function are still unclear. We examined if the dynamin GTPase, which is associated with actin remodeling and migration in other cells, plays a role in osteoblast differentiation and migration. Dynamin mRNA was expressed in primary osteoblasts throughout differentiation (0–21 days). However, alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was decreased in osteoblasts over-expressing dynamin. Conversely, ALP activity was increased following shRNA-mediated knockdown of dynamin and in osteoblasts treated with the dynamin inhibitor, dynasore. Dynasore also reduced c-fos and osterix expression, markers of early osteoblasts, suggesting a role for dynamin in pre-osteoblast to osteoblast differentiation. Since dynamin GTPase activity is regulated by tyrosine phosphorylation, we examined the mechanism of dynamin dephosphorylation in osteoblasts. Dynamin formed a protein complex with the tyrosine phosphatase PTP-PEST and inhibition of phosphatase activity increased the level of phosphorylated dynamin. Further, PTP-PEST blocked the Src-mediated increase in the phosphorylation and GTPase activity of wild-type dynamin but not the phosphorylation mutant dynY231F/Y597F. Although ALP activity was increased in osteoblasts expressing GTPase-defective dynK44A, and to a lesser extent dynY231F/Y597F, osteoblast migration was significantly inhibited by dynK44A and dynY231F/Y597F. These studies demonstrate a novel role for dynamin GTPase activity and phosphorylation in osteoblast differentiation and migration, which may be important for bone formation. PMID:24387844

  14. G-receptor antagonists increased the activating effect of mastoparan on low Km GTPase of mouse PAG.

    PubMed

    Martínez-Peña, Y; Sánchez-Blázquez, P; Garzón, J

    1995-02-01

    Mastoparan activated in a concentration-dependent manner the low Km GTPase activity in P2 fractions from mouse periaquedultal grey matter (PAG). This peptide at 1-10 mM produced increases of 30-70% over the basal value of 90-120 pmol Pi/mg/min. A series of substances displaying antagonist activity at cellular receptors and not modifying the GTPase function, when used at nanomolar and micromolar concentrations enhanced the effect of mastoparan upon this enzyme. These included antagonists of receptors coupling G proteins: naloxone (non selective opioid antagonist), CTOP (m opioid receptors), ICI 174,864 (d opioid receptors), nor-BNI (k opioid receptors), sulpiride (D2 dopaminergic antagonist), idazoxan (a2 adrenergic antagonist). Bicuculline, antagonist of a receptor not linked to G proteins, GABAA, did not alter the effect of mastoparan on the GTPase. The m opioid agonist, DAMGO, prevented naloxone from increasing the function of the mastoparan-activated enzyme. Thus, mastoparan appears to act on Gi/Go proteins at a site not directly related to the receptor binding domain. PMID:7794687

  15. The dynamics of spatio-temporal Rho GTPase signaling: formation of signaling patterns

    PubMed Central

    Fritz, Rafael Dominik; Pertz, Olivier

    2016-01-01

    Rho GTPases are crucial signaling molecules that regulate a plethora of biological functions. Traditional biochemical, cell biological, and genetic approaches have founded the basis of Rho GTPase biology. The development of biosensors then allowed measuring Rho GTPase activity with unprecedented spatio-temporal resolution. This revealed that Rho GTPase activity fluctuates on time and length scales of tens of seconds and micrometers, respectively. In this review, we describe Rho GTPase activity patterns observed in different cell systems. We then discuss the growing body of evidence that upstream regulators such as guanine nucleotide exchange factors and GTPase-activating proteins shape these patterns by precisely controlling the spatio-temporal flux of Rho GTPase activity. Finally, we comment on additional mechanisms that might feed into the regulation of these signaling patterns and on novel technologies required to dissect this spatio-temporal complexity. PMID:27158467

  16. The function of RhoGTPases in axon ensheathment and myelination

    PubMed Central

    Feltri, M. Laura; Suter, Ueli; Relvas, João B.

    2008-01-01

    RhoGTPases are molecular switches that integrate extracellular signals to perform diverse cellular responses. This ability relies on the network of proteins regulating RhoGTPases activity and localization, and on the interaction of RhoGTPases with many different cellular effectors. Myelination is an ideal place for RhoGTPases regulation, as it is the result of fine orchestration of many stimuli from at least two cell types. Recent work has revealed that RhoGTPases are required for Schwann cells to sort, ensheath and myelinate axons. Here we will review recent advances showing the critical roles for RhoGTPases in various aspects of Schwann development and myelination, including the recent discovery of their involvement in Charcot-Marie-Tooth disease. Comparison with potential roles of RhoGTPases in central nervous system myelination will be drawn. PMID:18803320

  17. Immune control of phagosomal bacteria by p47 GTPases.

    PubMed

    MacMicking, John D

    2005-02-01

    Sequestered from the action of complement, antibody and lytic peptides, phagosomal pathogens pose a unique problem for the innate immune system both in terms of detection and disposal. An immunologically induced 47-kDa (p47) GTPase family recruited to nascent phagosomes (PGs) has provided new insights into how vertebrates deal with facultative bacteria occupying a vacuolar niche. Research over the past 2 years in particular has identified several molecular determinants that underlie the membrane trafficking functions of LRG-47 and other p47 GTPases as part of a PG remodeling program. When coupled to signals issuing from pathogen-specific Toll-like receptors, the p47 proteins may constitute a novel sensory system enlisted by mammals, birds and fish to decode the language of immune recognition against this particular class of infectious agents. PMID:15694860

  18. Regulation of autophagy by the Rab GTPase network

    PubMed Central

    Ao, X; Zou, L; Wu, Y

    2014-01-01

    Autophagy (macroautophagy) is a highly conserved intracellular and lysosome-dependent degradation process in which autophagic substrates are enclosed and degraded by a double-membrane vesicular structure in a continuous and dynamic vesicle transport process. The Rab protein is a small GTPase that belongs to the Ras-like GTPase superfamily and regulates the vesicle traffic process. Numerous Rab proteins have been shown to be involved in various stages of autophagy. Rab1, Rab5, Rab7, Rab9A, Rab11, Rab23, Rab32, and Rab33B participate in autophagosome formation, whereas Rab9 is required in non-canonical autophagy. Rab7, Rab8B, and Rab24 have a key role in autophagosome maturation. Rab8A and Rab25 are also involved in autophagy, but their role is unknown. Here, we summarize new findings regarding the involvement of Rabs in autophagy and provide insights regarding future research on the mechanisms of autophagy regulation. PMID:24440914

  19. Modelling Rho GTPase biochemistry to predict collective cell migration

    NASA Astrophysics Data System (ADS)

    Merchant, Brian; Feng, James

    The collective migration of cells, due to individual cell polarization and intercellular contact inhibition of locomotion, features prominently in embryogenesis and metastatic cancers. Existing methods for modelling collectively migrating cells tend to rely either on highly abstracted agent-based models, or on continuum approximations of the group. Both of these frameworks represent intercellular interactions such as contact inhibition of locomotion as hard-coded rules defining model cells. In contrast, we present a vertex-dynamics framework which predicts polarization and contact inhibition of locomotion naturally from an underlying model of Rho GTPase biochemistry and cortical mechanics. We simulate the interaction between many such model cells, and study how modulating Rho GTPases affects migratory characteristics of the group, in the context of long-distance collective migration of neural crest cells during embryogenesis.

  20. Emerging nexus between RAB GTPases, autophagy and neurodegeneration.

    PubMed

    Jain, Navodita; Ganesh, Subramaniam

    2016-05-01

    The RAB class of small GTPases includes the major regulators of intracellular communication, which are involved in vesicle generation through fusion and fission, and vesicular trafficking. RAB proteins also play an imperative role in neuronal maintenance and survival. Recent studies in the field of neurodegeneration have also highlighted the process of autophagy as being essential for neuronal maintenance. Here we review the emerging roles of RAB proteins in regulating macroautophagy and its impact in the context of neurodegenerative diseases. PMID:26985808

  1. Ral GTPases regulate exocyst assembly through dual subunit interactions.

    PubMed

    Moskalenko, Serge; Tong, Chao; Rosse, Carine; Mirey, Gladys; Formstecher, Etienne; Daviet, Laurent; Camonis, Jacques; White, Michael A

    2003-12-19

    Ral GTPases have been implicated in the regulation of a variety of dynamic cellular processes including proliferation, oncogenic transformation, actin-cytoskeletal dynamics, endocytosis, and exocytosis. Recently the Sec6/8 complex, or exocyst, a multisubunit complex facilitating post-Golgi targeting of distinct subclasses of secretory vesicles, has been identified as a bona fide Ral effector complex. Ral GTPases regulate exocyst-dependent vesicle trafficking and are required for exocyst complex assembly. Sec5, a membrane-associated exocyst subunit, has been identified as a direct target of activated Ral; however, the mechanism by which Ral can modulate exocyst assembly is unknown. Here we report that an additional component of the exocyst, Exo84, is a direct target of activated Ral. We provide evidence that mammalian exocyst components are present as distinct subcomplexes on vesicles and the plasma membrane and that Ral GTPases regulate the assembly interface of a full octameric exocyst complex through interaction with Sec5 and Exo84. PMID:14525976

  2. Targeting Rho-GTPases in immune cell migration and inflammation

    PubMed Central

    Biro, Maté; Munoz, Marcia A; Weninger, Wolfgang

    2014-01-01

    Leukocytes are unmatched migrators capable of traversing barriers and tissues of remarkably varied structural composition. An effective immune response relies on the ability of its constituent cells to infiltrate target sites. Yet, unwarranted mobilization of immune cells can lead to inflammatory diseases and tissue damage ranging in severity from mild to life-threatening. The efficacy and plasticity of leukocyte migration is driven by the precise spatiotemporal regulation of the actin cytoskeleton. The small GTPases of the Rho family (Rho-GTPases), and their immediate downstream effector kinases, are key regulators of cellular actomyosin dynamics and are therefore considered prime pharmacological targets for stemming leukocyte motility in inflammatory disorders. This review describes advances in the development of small-molecule inhibitors aimed at modulating the Rho-GTPase-centric regulatory pathways governing motility, many of which stem from studies of cancer invasiveness. These inhibitors promise the advent of novel treatment options with high selectivity and potency against immune-mediated pathologies. Linked Articles This article is part of a themed section on Cytoskeleton, Extracellular Matrix, Cell Migration, Wound Healing and Related Topics. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-24 PMID:24571448

  3. Modulation of Rab GTPase function by a protein phosphocholine transferase.

    PubMed

    Mukherjee, Shaeri; Liu, Xiaoyun; Arasaki, Kohei; McDonough, Justin; Galán, Jorge E; Roy, Craig R

    2011-09-01

    The intracellular pathogen Legionella pneumophila modulates the activity of host GTPases to direct the transport and assembly of the membrane-bound compartment in which it resides. In vitro studies have indicated that the Legionella protein DrrA post-translationally modifies the GTPase Rab1 by a process called AMPylation. Here we used mass spectrometry to investigate post-translational modifications to Rab1 that occur during infection of host cells by Legionella. Consistent with in vitro studies, DrrA-mediated AMPylation of a conserved tyrosine residue in the switch II region of Rab1 was detected during infection. In addition, a modification to an adjacent serine residue in Rab1 was discovered, which was independent of DrrA. The Legionella effector protein AnkX was required for this modification. Biochemical studies determined that AnkX directly mediates the covalent attachment of a phosphocholine moiety to Rab1. This phosphocholine transferase activity used CDP-choline as a substrate and required a conserved histidine residue located in the FIC domain of the AnkX protein. During infection, AnkX modified both Rab1 and Rab35, which explains how this protein modulates membrane transport through both the endocytic and exocytic pathways of the host cell. Thus, phosphocholination of Rab GTPases represents a mechanism by which bacterial FIC-domain-containing proteins can alter host-cell functions. PMID:21822290

  4. P2-quinazolinones and bis-macrocycles as new templates for next-generation hepatitis C virus NS3/4a protease inhibitors: discovery of MK-2748 and MK-6325.

    PubMed

    Rudd, Michael T; Butcher, John W; Nguyen, Kevin T; McIntyre, Charles J; Romano, Joseph J; Gilbert, Kevin F; Bush, Kimberly J; Liverton, Nigel J; Holloway, M Katharine; Harper, Steven; Ferrara, Marco; DiFilippo, Marcello; Summa, Vincenzo; Swestock, John; Fritzen, Jeff; Carroll, Steven S; Burlein, Christine; DiMuzio, Jillian M; Gates, Adam; Graham, Donald J; Huang, Qian; McClain, Stephanie; McHale, Carolyn; Stahlhut, Mark W; Black, Stuart; Chase, Robert; Soriano, Aileen; Fandozzi, Christine M; Taylor, Anne; Trainor, Nicole; Olsen, David B; Coleman, Paul J; Ludmerer, Steven W; McCauley, John A

    2015-04-01

    With the goal of identifying inhibitors of hepatitis C virus (HCV) NS3/4a protease that are potent against a wide range of genotypes and clinically relevant mutant viruses, several subseries of macrocycles were investigated based on observations made during the discovery of MK-5172. Quinazolinone-containing macrocycles were identified as promising leads, and optimization for superior cross-genotype and mutant enzyme potency as well as rat liver and plasma concentrations following oral dosing, led to the development of MK-2748. Additional investigation of a series of bis-macrocycles containing a fused 18- and 15-membered ring system were also optimized for the same properties, leading to the discovery of MK-6325. Both compounds display the broad genotype and mutant potency necessary for clinical development as next-generation HCV NS3/4a protease inhibitors. PMID:25759009

  5. Mutations Conferring Resistance to SCH6, a Novel Hepatitis C Virus NS3/4A Protease Inhibitor: Reduced DNA Replication Fitness and Partial Rescue by Second-Site Mutations

    SciTech Connect

    Yi, MinKyung; Tong, Xiao; Skelton, Angela; Chase, Robert; Chen, Tong; Prongay, Andrew; Bogen, Stephane L.; Saksena, Anil K.; Njoroge, F. George; Veselenak, Ronald L.; Pyles, Richard B.; Bourne, Nigel; Malcolm, Bruce A.; Lemon, Stanley M.

    2008-06-30

    Drug resistance is a major issue in the development and use of specific antiviral therapies. Here we report the isolation and characterization of hepatitis C virus RNA replicons resistant to a novel ketoamide inhibitor of the NS3/4A protease, SCH6 (originally SCH446211). Resistant replicon RNAs were generated by G418 selection in the presence of SCH6 in a dose-dependent fashion, with the emergence of resistance reduced at higher SCH6 concentrations. Sequencing demonstrated remarkable consistency in the mutations conferring SCH6 resistance in genotype 1b replicons derived from two different strains of hepatitis C virus, A156T/A156V and R109K. R109K, a novel mutation not reported previously to cause resistance to NS3/4A inhibitors, conferred moderate resistance only to SCH6. Structural analysis indicated that this reflects unique interactions of SCH6 with P{prime}-side residues in the protease active site. In contrast, A156T conferred high level resistance to SCH6 and a related ketoamide, SCH503034, as well as BILN 2061 and VX-950. Unlike R109K, which had minimal impact on NS3/4A enzymatic function, A156T significantly reduced NS3/4A catalytic efficiency, polyprotein processing, and replicon fitness. However, three separate second-site mutations, P89L, Q86R, and G162R, were capable of partially reversing A156T-associated defects in polyprotein processing and/or replicon fitness, without significantly reducing resistance to the protease inhibitor.

  6. Depeptidization efforts on P[subscript 3]-P[prime subscript 2] [alpha]-ketoamide inhibitors of HCV NS3-4A serine protease: Effect on HCV replicon activity

    SciTech Connect

    Bogen, Stephane L.; Ruan, Sumei; Liu, Rong; Agrawal, Sony; Pichardo, John; Prongay, Andrew; Baroudy, Bahige; Saksena, Anil K.; Girijavallabhan, Viyyoor; Njoroge, F. George

    2008-06-30

    Depeptidization efforts of the P{sub 3}-P{sub 2} region of P{sub 3} capped {alpha}-ketoamide inhibitor of HCV NS3 serine protease 1 are reported. We clearly established that N-methylation of the P{sub 2} nitrogen and modification of the P{prime}{sub 2} carboxylic acid terminus were essential for activity in the replicon assay.

  7. Single point mutations in the helicase domain of the NS3 protein enhance dengue virus replicative capacity in human monocyte-derived dendritic cells and circumvent the type I interferon response.

    PubMed

    Silveira, G F; Strottmann, D M; de Borba, L; Mansur, D S; Zanchin, N I T; Bordignon, J; dos Santos, C N Duarte

    2016-01-01

    Dengue is the most prevalent arboviral disease worldwide. The outcome of the infection is determined by the interplay of viral and host factors. In the present study, we evaluated the cellular response of human monocyte-derived DCs (mdDCs) infected with recombinant dengue virus type 1 (DV1) strains carrying a single point mutation in the NS3hel protein (L435S or L480S). Both mutated viruses infect and replicate more efficiently and produce more viral progeny in infected mdDCs compared with the parental, non-mutated virus (vBACDV1). Additionally, global gene expression analysis using cDNA microarrays revealed that the mutated DVs induce the up-regulation of the interferon (IFN) signalling and pattern recognition receptor (PRR) canonical pathways in mdDCs. Pronounced production of type I IFN were detected specifically in mdDCs infected with DV1-NS3hel-mutated virus compared with mdDCs infected with the parental virus. In addition, we showed that the type I IFN produced by mdDCs is able to reduce DV1 infection rates, suggesting that cytokine function is effective but not sufficient to mediate viral clearance of DV1-NS3hel-mutated strains. Our results demonstrate that single point mutations in subdomain 2 have important implications for adenosine triphosphatase (ATPase) activity of DV1-NS3hel. Although a direct functional connection between the increased ATPase activity and viral replication still requires further studies, these mutations speed up viral RNA replication and are sufficient to enhance viral replicative capacity in human primary cell infection and circumvent type I IFN activity. This information may have particular relevance for attenuated vaccine protocols designed for DV. PMID:26340409

  8. Regulation of vesicular trafficking and leukocyte function by Rab27 GTPases and their effectors

    PubMed Central

    Catz, Sergio Daniel

    2013-01-01

    The Rab27 family of GTPases regulates the efficiency and specificity of exocytosis in hematopoietic cells, including neutrophils, CTLs, NK cells, and mast cells. However, the mechanisms regulated by Rab27 GTPases are cell-specific, as they depend on the differential expression and function of particular effector molecules that are recruited by the GTPases. In addition, Rab27 GTPases participate in multiple steps of the regulation of the secretory process, including priming, tethering, docking, and fusion through sequential interaction with multiple effector molecules. Finally, recent reports suggest that Rab27 GTPases and their effectors regulate vesicular trafficking mechanisms other than exocytosis, including endocytosis and phagocytosis. This review focuses on the latest discoveries on the function of Rab27 GTPases and their effectors Munc13-4 and Slp1 in neutrophil function comparatively to their functions in other leukocytes. PMID:23378593

  9. Beyond Rab GTPases Legionella activates the small GTPase Ran to promote microtubule polymerization, pathogen vacuole motility, and infection

    PubMed Central

    Hilbi, Hubert; Rothmeier, Eva; Hoffmann, Christine; Harrison, Christopher F

    2014-01-01

    Legionella spp. are amoebae-resistant environmental bacteria that replicate in free-living protozoa in a distinct compartment, the Legionella-containing vacuole (LCV). Upon transmission of Legionella pneumophila to the lung, the pathogens employ an evolutionarily conserved mechanism to grow in LCVs within alveolar macrophages, thus triggering a severe pneumonia termed Legionnaires’ disease. LCV formation is a complex and robust process, which requires the bacterial Icm/Dot type IV secretion system and involves the amazing number of 300 different translocated effector proteins. LCVs interact with the host cell's endosomal and secretory vesicle trafficking pathway. Accordingly, in a proteomics approach as many as 12 small Rab GTPases implicated in endosomal and secretory vesicle trafficking were identified and validated as LCV components. Moreover, the small GTPase Ran and its effector protein RanBP1 have been found to decorate the pathogen vacuole. Ran regulates nucleo-cytoplasmic transport, spindle assembly, and cytokinesis, as well as the organization of non-centrosomal microtubules. In L. pneumophila-infected amoebae or macrophages, Ran and RanBP1 localize to LCVs, and the small GTPase is activated by the Icm/Dot substrate LegG1. Ran activation by LegG1 leads to microtubule stabilization and promotes intracellular pathogen vacuole motility and bacterial growth, as well as chemotaxis and migration of Legionella-infected cells. PMID:25496424

  10. Rho family GTPases: key players in neuronal development, neuronal survival, and neurodegeneration

    PubMed Central

    Stankiewicz, Trisha R.; Linseman, Daniel A.

    2014-01-01

    The Rho family of GTPases belongs to the Ras superfamily of low molecular weight (∼21 kDa) guanine nucleotide binding proteins. The most extensively studied members are RhoA, Rac1, and Cdc42. In the last few decades, studies have demonstrated that Rho family GTPases are important regulatory molecules that link surface receptors to the organization of the actin and microtubule cytoskeletons. Indeed, Rho GTPases mediate many diverse critical cellular processes, such as gene transcription, cell–cell adhesion, and cell cycle progression. However, Rho GTPases also play an essential role in regulating neuronal morphology. In particular, Rho GTPases regulate dendritic arborization, spine morphogenesis, growth cone development, and axon guidance. In addition, more recent efforts have underscored an important function for Rho GTPases in regulating neuronal survival and death. Interestingly, Rho GTPases can exert either a pro-survival or pro-death signal in neurons depending upon both the cell type and neurotoxic insult involved. This review summarizes key findings delineating the involvement of Rho GTPases and their effectors in the regulation of neuronal survival and death. Collectively, these results suggest that dysregulation of Rho family GTPases may potentially underscore the etiology of some forms of neurodegenerative disease such as amyotrophic lateral sclerosis. PMID:25339865