Mechanical strain stimulates COPII‐dependent secretory trafficking via Rac1

Cells are constantly exposed to various chemical and physical stimuli. While much has been learned about the biochemical factors that regulate secretory trafficking from the endoplasmic reticulum (ER), much less is known about whether and how this trafficking is subject to regulation by mechanical signals. Here, we show that subjecting cells to mechanical strain both induces the formation of ER exit sites (ERES) and accelerates ER‐to‐Golgi trafficking. We found that cells with impaired ERES function were less capable of expanding their surface area when placed under mechanical stress and were more prone to develop plasma membrane defects when subjected to stretching. Thus, coupling of ERES function to mechanotransduction appears to confer resistance of cells to mechanical stress. Furthermore, we show that the coupling of mechanotransduction to ERES formation was mediated via a previously unappreciated ER‐localized pool of the small GTPase Rac1. Mechanistically, we show that Rac1 interacts with the small GTPase Sar1 to drive budding of COPII carriers and stimulates ER‐to‐Golgi transport. This interaction therefore represents an unprecedented link between mechanical strain and export from the ER

QM/MM Well-Tempered Metadynamics Study of the Mechanism of XBP1 mRNA Cleavage by Inositol Requiring Enzyme 1 alpha RNase

International audienceA range of in silico methodologies were herein employed to study the unconventional XBP1 mRNA cleavage mechanism performed by the unfolded protein response (UPR) mediator Inositol Requiring Enzyme 1 alpha (IRE1). Using Protein- RNA molecular docking along with a series of extensive restrained/ unrestrained atomistic molecular dynamics (MD) simulations, the dynamical behavior of the system was evaluated and a reliable model of the IRE1/XBP1 mRNA complex was constructed. From a series of well-converged quantum mechanics molecular mechanics well-tempered metadynamics (QM/MM WT-MetaD) simulations using the Grimme dispersion interaction corrected semiempirical parametrization method 6 level of theory (PM6-D3) and the AMBER14SB-OL3 force field, the free energy profile of the cleavage mechanism was determined, along with intermediates and transition state structures. The results show two distinct reaction paths based on general acid-general base type mechanisms, with different activation energies that perfectly match observations from experimental mutagenesis data. The study brings unique atomistic insights into the cleavage mechanism of XBP1 mRNA by IRE1 and clarifies the roles of the catalytic residues H910 and Y892. Increased understanding of the details in UPR signaling can assist in the development of new therapeutic agents for its modulation

Dataset for Multiscale in silico study of mechanism of activation of RtcB ligase by PTP1B phosphatase.

The pdb structures of the RtcB_PTP1B complex after protein-protein docking and after 1000ns MD simulation, 1000ns MD trajectory, and a video captured from the QM/MM WT-MetaD simulation trajectory showing the reaction mechanis

Sensor dimer disruption as a new mode of action to block the IRE1-mediated unfolded protein response

International audienceThe unfolded protein response (UPR) is activated to cope with an accumulation of improperly folded proteins in the Endoplasmic reticulum (ER). The Inositol requiring enzyme 1α (IRE1α) is the most evolutionary conserved transducer of the UPR. Activated IRE1 forms ’back-to-back’-dimers that enables the unconventional splicing of X-box Binding Protein 1 (XBP1) mRNA. The spliced XBP1 (XBP1s) mRNA is translated into a transcription factor controlling the expression of UPR target genes. Herein, we report a detailed in silico screening specifically targeting for the first time the dimer interface at the IRE1 RNase region. Using the database of FDA approved drugs, we identified four compounds (neomycin, pemetrexed, quercitrin and rutin) that were able to bind to and distort IRE1 RNase cavity. The activity of the compounds on IRE1 phosphorylation was evaluated in HEK293T cells and on IRE1 RNase activity using an in vitro fluorescence assay. These analyzes revealed sub-micromolar IC(50) values. The current study reveals a new and unique mode of action to target and block the IRE1-mediated UPR signaling, whereby we may avoid problems associated with selectivity occurring when targeting the IRE1 kinase pocket as well as the inherent reactivity of covalent inhibitors targeting the RNase pocket

Structural and molecular bases to IRE1 activity modulation

International audienceThe Unfolded Protein response is an adaptive pathway triggered upon alteration of endoplasmic reticulum (ER) homeostasis. It is transduced by three major ER stress sensors, among which the Inositol Requiring Enzyme 1 (IRE1) is the most evolutionarily conserved. IRE1 is an ER-resident type I transmembrane protein exhibiting an ER luminal domain that senses the protein folding status and a catalytic kinase and RNase cytosolic domain. In recent years, IRE1 has emerged as a relevant therapeutic target in various diseases including degenerative, inflammatory and metabolic pathologies and cancer. As such several drugs altering IRE1 activity were developed that target either catalytic activity and showed some efficacy in preclinical pathological mouse models. In this review, we describe the different drugs identified to target IRE1 activity as well as their mode of action from a structural perspective, thereby identifying common and different modes of action. Based on this information we discuss on how new IRE1-targeting drugs could be developed that outperform the currently available molecules

Stress-induced tyrosine phosphorylation of RtcB modulates IRE1 activity and signaling outputs

International audienceER stress is mediated by three sensors and the most evolutionary conserved IRE1α signals through its cytosolic kinase and endoribonuclease (RNase) activities. IRE1α RNase activity can either catalyze the initial step of XBP1 mRNA unconventional splicing or degrade a number of RNAs through regulated IRE1-dependent decay. Until now, the biochemical and biological outputs of IRE1α RNase activity have been well documented; however, the precise mechanisms controlling whether IRE1α signaling is adaptive or pro-death (terminal) remain unclear. We investigated those mechanisms and hypothesized that XBP1 mRNA splicing and regulated IRE1-dependent decay activity could be co-regulated by the IRE1α RNase regulatory network. We identified that RtcB, the tRNA ligase responsible for XBP1 mRNA splicing, is tyrosine-phosphorylated by c-Abl and dephosphorylated by PTP1B. Moreover, we show that the phosphorylation of RtcB at Y306 perturbs RtcB interaction with IRE1α, thereby attenuating XBP1 mRNA splicing. Our results demonstrate that the IRE1α RNase regulatory network is dynamically fine-tuned by tyrosine kinases and phosphatases upon various stresses and that the extent of RtcB tyrosine phosphorylation determines cell adaptive or death outputs