Dataset Information

HCV NS5A protein containing potential ligands for both Src homology 2 and 3 domains enhances autophosphorylation of Src family kinase Fyn in B cells.

ABSTRACT: Hepatitis C virus (HCV) infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkin's lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A) protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV) implied that NS5A was tyrosine phosphorylated by pervanadate (PV) treatment of the cells. Therefore we examined pull-down assay by using glutathione S-transferase (GST)-fusion proteins of various Src homology 2 (SH2) domains, which associates with phosphotyrosine within a specific amino acid sequence. The results showed that NS5A specifically bound to SH2 domain of Fyn from PV-treated B cells in addition to Src homology 3 (SH3) domain. Substitution of Arg(176) to Lys in the SH2 domain of Fyn abrogated this interaction. Deletion mutational analysis demonstrated that N-terminal region of NS5A was not required for the interaction with the SH2 domain of Fyn. Tyr(334) was identified as a tyrosine phosphorylation site in NS5A. Far-western analysis revealed that SH2 domain of Fyn directly bound to NS5A. Fyn and NS5A were colocalized in the lipid raft. These results suggest that NS5A directly binds to the SH2 domain of Fyn in a tyrosine phosphorylation-dependent manner. Lastly, we showed that the expression of NS5A in B cells increased phosphorylation of activation loop tyrosine in the kinase domain of Fyn. NS5A containing ligand for both SH2 and SH3 domains enhances an aberrant autophosphorylation and kinase activity of Fyn in B cells.

SUBMITTER: Nakashima K

PROVIDER: S-EPMC3473057 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Publications

HCV NS5A protein containing potential ligands for both Src homology 2 and 3 domains enhances autophosphorylation of Src family kinase Fyn in B cells.

Nakashima Kenji K Takeuchi Kenji K Chihara Kazuyasu K Horiguchi Tomoko T Sun Xuedong X Deng Lin L Shoji Ikuo I Hotta Hak H Sada Kiyonao K

PloS one 20121016 10

Hepatitis C virus (HCV) infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkin's lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A) protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV) implied that NS5A was tyrosine phosphorylated by pervan ...[more]

PMID: 23077515

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Project description:ContextEgypt has a high prevalence of the hepatitis C virus (HCV) genotype 4a (GT-4a). Unfortunately, the high resistance it exhibited still was not given the deserved attention in the scientific community. There is currently no consensus on the NS5A binding site because the crystal structure of HCV NS5A has not been resolved. The prediction of the binding modes of direct-acting antivirals (DAA) with the NS5A is a point of controversy due to the fact that several research groups presented different interaction models to elucidate the NS5A binding site. Consequently, a 3D model of HCV NS5A GT-4a was constructed and evaluated using molecular dynamics (MD) simulations. The generated model implies an intriguing new orientation of the AH relative to domain I. Additionally, the probable binding modes of marketed NS5A inhibitors were explored. MD simulations validated the stability of the predicted protein-ligand complexes. The suggested model predicts that daclatasvir and similar drugs bind symmetrically to HCV NS5A GT-4a. This will allow for the development of new NS5A-directed drugs, which may result in reduced resistance and/or a wider range of effectiveness against HCV.MethodsThe 3D model of HCV NS5A GT-4a was constructed using the comparative modeling approach of the web-based application Robetta. Its stability was tested with 200-ns MD simulations using the Desmond package of Schrodinger. The OPLS2005 force field was assigned for minimization, and the RMSD, RMSF, and rGyr were tracked throughout the MD simulations. Fpocket was used to identify druggable protein pockets (cavities) over the simulation trajectories. The binding modes of marketed NS5A inhibitors were then generated and refined with the aid of docking predictions made by FRED and AutoDock Vina. The stability of these drugs in complex with GT-4a was investigated by using energetic and structural analyses over MD simulations. The Prime MM-GBSA (molecular mechanics/generalized Born surface area) method was used as a validation tool after the docking stage and for the averaged clusters after the MD simulation stage. We utilized PyMOL and VMD to visualize the data.

Dataset Information

HCV NS5A protein containing potential ligands for both Src homology 2 and 3 domains enhances autophosphorylation of Src family kinase Fyn in B cells.

Publications

HCV NS5A protein containing potential ligands for both Src homology 2 and 3 domains enhances autophosphorylation of Src family kinase Fyn in B cells.

Similar Datasets

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