Leger, P. | bioRxiv Science

bioRxiv2018

High-quality SNPs from genic regions highlight introgression patterns among European white oaks (Quercus petraea and Q. robur).

The Src homology-2 domain containing phosphatase SHP2 is a critical regulator of signal transduction, being implicated in cell growth and differentiation. Activating mutations cause developmental disorders and act as oncogenic drivers in hematologic cancers. SHP2 is activated by phosphopeptide binding to the N-SH2 domain, triggering the release of N-SH2 from the catalytic PTP domain. Based on early crystallographic data, it has been widely accepted that opening of the binding cleft of N-SH2 serves as the key "allosteric switch" driving SHP2 activation. To test the putative coupling between binding cleft opening and SHP2 activation as assumed by the "allosteric switch" model, we critically reviewed structural data of SHP2 and we used extensive molecular dynamics (MD) simulation and free energy calculations of isolated N-SH2 in solution, SHP2 in solution, and SHP2 in a crystal environment. Our results demonstrate that the binding cleft in N-SH2 is constitutively flexible and open in solution, and that a closed cleft found in certain structures is a consequence of crystal contacts. The degree of opening of the binding cleft has only a negligible effect on the free energy of SHP2 activation. Instead, SHP2 activation is greatly favored by the opening of the central {beta}- sheet of N-SH2. We conclude that opening of the N-SH2 binding cleft is not the key allosteric switch triggering SHP2 activation. Significance StatementSHP2 is a multi-domain protein, playing an important role in up-regulating cellular processes such as cell survival, proliferation, and programmed cell death. SHP2 mutations cause developmental disorders and were found in many cancer types, including neuroblastoma, breast cancer, and leukemia. In healthy cells, SHP2 mainly takes an autoinhibited, inactive form, and SHP2 is activated upon binding of phosphopeptides to the N-SH2 domain. For the past two decades, the widening of the binding cleft upon peptide binding has been considered as the key event driving SHP2 activation. Here, by analyzing crystallographic data and molecular simulations, we demonstrate that the binding cleft in N-SH2 is, instead, already open and accessible in solution, and its degree of opening does not influence SHP2 activation.

evolutionary biology↗

bioRxiv2017

DDX3 is exploited by Arenaviruses to suppress type I interferons and favor their replication

Several arenaviruses cause hemorrhagic fever (HF) diseases that are associated with high morbidity and mortality in humans. Accordingly, HF arenaviruses have been listed as top-priority emerging diseases for which countermeasures are urgently needed. Because arenavirus nucleoprotein (NP) plays critical roles in both virus multiplication and immune-evasion, we used an unbiased proteomic approach to identify NP-interacting proteins in human cells. DDX3, a DEAD-box ATP-dependent-RNA-helicase, interacted with NP in both NP-transfected and virus-infected cells. Importantly, DDX3 deficiency compromised the propagation of both Old and New World arenaviruses, including the HF arenaviruses Lassa and Junin viruses. The DDX3 role in promoting arenavirus multiplication correlated with both a previously un-recognized DDX3 contribution to type I interferon suppression in arenavirus infected cells and a positive effect of DDX3 on viral RNA synthesis. Our results uncover novel mechanisms used by arenavirus to exploit the host machinery and subvert immunity, singling out DDX3 as a potential host target for developing new therapies against highly pathogenic arenaviruses.\n\nAUTHOR SUMMARYArenaviruses include severe clinical pathogens causing hemorrhagic fevers and have been recently incorporated by the World Health Organization in a list of critical emerging diseases for which additional research and identification of clinical targets is urgently required. A better understanding of how viral proteins interact with host cellular factors to favor arenavirus multiplication can illuminate novel pipelines on therapeutic strategies. Here we demonstrated that the ATP-dependent RNA helicase DDX3 interacted with the arenavirus nucleoprotein, which displays fundamental functions in different steps of the viral-cycle. Our work also revealed an unexpected new biology on the role that DDX3 might play during viral infections. In sharp contrast to previous studies showing DDX3 enhancement of IFN-I induction, we demonstrated that DDX3 suppressed IFN-I production at late time points after arenavirus infection, contributing to a DDX3 pro-viral effect. We also showed that early after infection, DDX3 pro-viral role was IFN-I independent and was mediated by DDX3 facilitation of viral RNA synthesis without affecting RNA translation. Altogether, our study established DDX3 as a critical host interacting partner of the arenavirus nucleoprotein and demonstrated two previously unrecognized DDX3-dependent strategies by which these deadly viruses exploit the host cellular machinery and suppress immunity.

microbiology↗