4j1v | pdb_00004j1v
Functional and structural studies of MOBKL1B, a Salvador/Warts/Hippo tumor suppressor pathway, in HCV replication
Structural highlights
FunctionMOB1A_HUMAN Activator of LATS1/2 in the Hippo signaling pathway which plays a pivotal role in organ size control and tumor suppression by restricting proliferation and promoting apoptosis. The core of this pathway is composed of a kinase cascade wherein STK3/MST2 and STK4/MST1, in complex with its regulatory protein SAV1, phosphorylates and activates LATS1/2 in complex with its regulatory protein MOB1, which in turn phosphorylates and inactivates YAP1 oncoprotein and WWTR1/TAZ. Phosphorylation of YAP1 by LATS1/2 inhibits its translocation into the nucleus to regulate cellular genes important for cell proliferation, cell death, and cell migration. Stimulates the kinase activity of STK38 and STK38L. Acts cooperatively with STK3/MST2 to activate STK38.[1] [2] [3] Publication Abstract from PubMedHepatitis C virus (HCV) is a widespread human pathogen causing liver cirrhosis and cancer. Similar to other viruses, HCV depends on host and viral factors to complete its life cycle. We used proteomic and yeast two-hybrid approaches to elucidate host factors involved in HCV nonstructural protein NS5A function and found that MOBKL1B interacts with NS5A. Initial experiments with siRNA knockdown suggesting a role in HCV replication led us to examine the interaction using biochemical and structural approaches. As revealed by a co-crystal structure of a core MOBKL1B-NS5A peptide complex at 1.95 A, NS5A binds to a hydrophobic patch on the MOBKL1B surface. Biosensor binding assays identified a highly conserved, 18 amino acid binding site in domain II of NS5A, which encompasses residues implicated in cyclophilin A (CypA)-dependent HCV RNA replication. However, a CypA-independent HCV variant had reduced replication in MOBKL1B knockdown cells, even though its NS5A does not interact with MOBKL1B. These discordant results prompted more extensive studies of MOBKL1B gene knockdowns, which included additional siRNAs and specifically matched seed-sequence siRNA controls. We found that reduced virus replication after treating cells with MOBKL1B siRNA was actually due to off-target inhibition, and indicated that the initial finding of virus replication dependence on the MOBKL1B-NS5A interaction was incorrect. Ultimately, using several approaches we found no relationship of the MOBKL1B-NS5A interaction to virus replication. These findings collectively serve as a reminder to investigators and scientific reviewers of the pervasive impact of siRNA off-target effects on interpretation of biological data. IMPORTANCE: Our study illustrates an underappreciated shortcoming of siRNA gene knockdown technology. We initially identified a cellular protein, MOBKL1B, as a binding partner with the NS5A protein of hepatitis C virus (HCV). MOBKL1B siRNA, but not irrelevant RNA treatment was associated with both reduced virus replication and the absence of MOBKL1B. Believing that HCV replication depended on the MOBKL1B-NS5A interaction, we carried out structural and biochemical analyses. Unexpectedly, an HCV variant lacking the MOBKL1B-NS5A interaction could not replicate after cells were treated with MOBKL1B siRNA. By repeating the MOBKL1B siRNA knockdowns, and including seed sequence-matched siRNA instead of irrelevant siRNA as a control, we found that the MOBKL1B siRNAs utilized had off-target inhibitory effects on virus replication. Collectively, our results suggest that stricter controls must be utilized in all RNAi-mediated gene knockdown experiments to ensure sound conclusions and a reliable scientific knowledge database. Seed-sequence matched controls reveal limitations of siRNA knockdown in functional and structural studies of HCV NS5A-MOBKL1B interaction.,Chung HY, Gu M, Buehler E, MacDonald MR, Rice CM J Virol. 2014 Jul 16. pii: JVI.01582-14. PMID:25031347[4] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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