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Adaptation to the shortage in free amino acids (AA) is mediated by 2 pathways, the integrated stress response (ISR) and the mechanistic target of rapamycin (mTOR). In response to reduced levels, primarily of leucine or arginine, mTOR in its complex 1 configuration (mTORC1) is suppressed leading to a decrease in translation initiation and elongation. The eIF2α kinase general control nonderepressible 2 (GCN2) is activated by uncharged tRNAs, leading to induction of the ISR in response to a broader range of AA shortage. ISR confers a reduced translation initiation, while promoting the selective synthesis of stress proteins, such as ATF4. To efficiently adapt to AA starvation, the 2 pathways are cross-regulated at multiple levels. Here we identified a new mechanism of ISR/mTORC1 crosstalk that optimizes survival under AA starvation, when mTORC1 is forced to remain active. mTORC1 activation during acute AA shortage, augmented ATF4 expression in a GCN2-dependent manner. Under these conditions, enhanced GCN2 activity was not dependent on tRNA sensing, inferring a different activation mechanism. We identified a labile physical interaction between GCN2 and mTOR that results in a phosphorylation of GCN2 on serine 230 by mTOR, which promotes GCN2 activity. When examined under prolonged AA starvation, GCN2 phosphorylation by mTOR promoted survival. Our data unveils an adaptive mechanism to AA starvation, when mTORC1 evades inhibition., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Inositol-requiring enzyme 1 (IRE1) is a transmembrane sensor that is part of a trio of sensors responsible for controlling the unfolded protein response within the endoplasmic reticulum (ER). Upon the accumulation of unfolded or misfolded proteins in the ER, IRE1 becomes activated and initiates the cleavage of a 26-nucleotide intron from human X-box-containing protein 1 (XBP1). The cleavage is mediated by the RtcB ligase enzyme, which splices together two exons, resulting in the formation of the spliced isoform XBP1s. The XBP1s isoform activates the transcription of genes involved in ER-associated degradation to maintain cellular homeostasis. The catalytic activity of RtcB is controlled by the phosphorylation and dephosphorylation of three tyrosine residues (Y306, Y316, and Y475), which are regulated by the ABL1 tyrosine kinase and PTP1B phosphatase, respectively. This study focuses on investigating the mechanism by which the PTP1B phosphatase activates the RtcB ligase using a range of advanced in silico methods. Protein-protein docking identified key interacting residues between RtcB and PTP1B. Notably, the phosphorylated Tyr306 formed hydrogen bonds and salt bridge interactions with the "gatekeeper" residues Arg47 and Lys120 of the inactive PTP1B. Classical molecular dynamics simulation emphasized the crucial role of Asp181 in the activation of PTP1B, driving the conformational change from an open to a closed state of the WPD-loop. Furthermore, QM/MM-MD simulations provided insights into the free energy landscape of the dephosphorylation reaction mechanism of RtcB, which is mediated by the PTP1B phosphatase.

The flavonoid Quercetin (Qe) was identified as an activator of Inositol-requiring enzyme 1 (IRE1) in S. cerevisiae (scIre1p), but its impact on human IRE1 (hIRE1) remains controversial due to the absence of a conserved Qe binding site. We have explored the binding modes and effect of Qe on both scIre1p and hIRE1 dimers using in silico and in vitro approaches. The activation site in scIre1p stably accommodates both Qe and its derivative Quercitrin (Qi), thus enhancing the stability of the RNase pocket. However, the corresponding region in hIRE1 does not bind any of the two molecules. Instead, we show that both Qe and Qi block the RNase activity of hIRE1 in vitro, with sub-micromolar IC 50 values. Our results provide a rationale for why Qe is an activator in scIre1p but a potent inhibitor in hIRE1. The identification of a new allosteric site in hIRE1 opens a promising window for drug development and UPR modulation., (© 2024. The Author(s).)

Post-translational modifications (PTMs) add a major degree of complexity to the proteome and are essential controllers of protein homeostasis. Amongst the hundreds of PTMs identified, ubiquitin and ubiquitin-like (UBL) modifications are recognized as key regulators of cellular processes through their ability to affect protein-protein interactions, protein stability, and thus the functions of their protein targets. Here, we focus on the most recently identified UBL, ubiquitin-fold modifier 1 (UFM1), and the machinery responsible for its transfer to substrates (UFMylation) or its removal (deUFMylation). We first highlight the biochemical peculiarities of these processes, then we develop on how UFMylation and its machinery control various intertwined cellular processes and we highlight some of the outstanding research questions in this emerging field., Competing Interests: Declaration of interests E.C. is a founder of Thabor therapeutics (www.thabor-tx.com). L.A.E. and S.J.M. are co-founders of ANYO Labs (www.anyolabs.com). L.A.E. is co-founder of Cell Stress Discoveries Ltd. (www.cellstressdiscoveries.com). No conflicts of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Exotoxin A (ETA) is an extracellular secreted toxin and a single-chain polypeptide with A and B fragments that is produced by Pseudomonas aeruginosa . It catalyzes the ADP-ribosylation of a post-translationally modified histidine (diphthamide) on eukaryotic elongation factor 2 (eEF2), which results in the inactivation of the latter and the inhibition of protein biosynthesis. Studies show that the imidazole ring of diphthamide plays an important role in the ADP-ribosylation catalyzed by the toxin. In this work, we employ different in silico molecular dynamics (MD) simulation approaches to understand the role of diphthamide versus unmodified histidine in eEF2 on the interaction with ETA. Crystal structures of the eEF2-ETA complexes with three different ligands NAD + , ADP-ribose, and βTAD were selected and compared in the diphthamide and histidine containing systems. The study shows that NAD + bound to ETA remains very stable in comparison with other ligands, enabling the transfer of ADP-ribose to the N3 atom of the diphthamide imidazole ring in eEF2 during ribosylation. We also show that unmodified histidine in eEF2 has a negative impact on ETA binding and is not a suitable target for the attachment of ADP-ribose. Analyzing of radius of gyration and COM distances for NAD + , βTAD, and ADP-ribose complexes revealed that unmodified His affects the structure and destabilizes the complex with all different ligands throughout the MD simulations.

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., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

A 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 1α (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.

ER 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., (© 2022 Papaioannou et al.)

The 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., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The death-inducing signaling complex (DISC) is a fundamental multiprotein complex, which triggers the extrinsic apoptosis pathway through stimulation by death ligands. DISC consists of different death domain (DD) and death effector domain (DED) containing proteins such as the death receptor Fas (CD95) in complex with FADD, procaspase-8, and cFLIP. Despite many experimental and theoretical studies in this area, there is no global agreement neither on the DISC architecture nor on the mechanism of action of the involved species. In the current work, we have tried to reconstruct the DISC structure by identifying key protein interactions using a new protein-protein docking meta-approach. We combined the benefits of five of the most employed protein-protein docking engines, HADDOCK, ClusPro, HDOCK, GRAMM-X, and ZDOCK, in order to improve the accuracy of the predicted docking complexes. Free energy of binding and hot spot interacting residues were calculated and determined for each protein-protein interaction using molecular mechanics generalized Born surface area and alanine scanning techniques, respectively. In addition, a series of in-cellulo protein-fragment complementation assays were conducted to validate the protein-protein docking procedure. The results show that the DISC formation initiates by dimerization of adjacent Fas DD trimers followed by recruitment of FADD through homotypic DD interactions with the oligomerized death receptor. Furthermore, the in-silico outcomes indicate that cFLIP cannot bind directly to FADD; instead, cFLIP recruitment to the DISC is a hierarchical and cooperative process where FADD initially recruits procaspase-8, which in turn recruits and heterodimerizes with cFLIP. Finally, a possible structure of the entire DISC is proposed based on the docking results.

Multicomponent gas separation and purification is an important pre- or post-processing step in industry. Herein, we employed a multiscale computational approach to investigate the possibility of multicomponent low-weight gas (H 2 , O 2 , N 2 , CO 2 , CH 4 ) separation and purification using novel porous 2D carbonaceous nanomaterials, namely Graphdiyne (GD), Graphenylene (GN), and Rhombic-Graphyne (RG). The dispersion-corrected plane-wave density functional theory (DFT) calculation combined with the Climbing Image Nudged Elastic Band (CI-NEB) method was employed to study the gas/membrane interaction energy and diffusion barrier of different gases passing through the geometrically optimized membranes. The results from CI-NEB calculations were then fitted to the Morse potential function to construct a bridge between quantum mechanics calculations and non-equilibrium molecular dynamics (NEMD) simulation. The selectivity of each membrane for all binary mixtures was calculated using the estimated diffusion energy barriers based on the Arrhenius equation. Finally, a series of extensive NEMD simulations were carried out to evaluate the real word and time dependent separation process. According to the results, CH 4 molecules can be completely separated from the other gases using a GD membrane, O 2 molecules from CH 4 , N 2 , and CO 2 by a GN membrane, and H 2 molecules from all other gases using a RG membrane., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The selectivity of the ligand MKC9989, as inhibitor of the Inositol-Requiring Enzyme 1α (IRE1α) transmembrane kinase/ribonuclease protein, towards the residue K907 in the context of Schiff base formation, has been investigated by employing an array of in silico techniques including Multi-Conformation Continuum Electrostatics (MCCE) simulations, Quantum Mechanics/Molecular Mechanics (QM/MM) calculations, covalent docking, and Molecular Dynamics (MD) simulations. According to the MCCE results, K907 displays the lowest p K a value among all 23 lysine residues in IRE1α. The MMCE simulations also indicate a critical interaction between K907 and D885 within the hydrophobic pocket which increases significantly at low protein dielectric constants. The QM/MM calculations reveal a spontaneous proton transfer from K907 to D885, consistent with the low p K a value of K907. A Potential Energy Surface (PES) scan confirms the lack of energy barrier and transition state associated with this proton transfer reaction. Covalent docking and MD simulations verify that the protein pocket containing K907 can effectively stabilize the inhibitor by strong π-π and hydrogen bonding interactions. In addition, Radial Distribution Function (RDF) analysis shows that the imine group formed in the chemical reaction between MKC9989 and K907 is inaccessible to water molecules and thus the probability of imine hydrolysis is almost zero. The results of the current study explain the high selectivity of the MKC9989 inhibitor towards the K907 residue of IRE1α., Competing Interests: The authors declare no conflicting interests., (This journal is © The Royal Society of Chemistry.)

Herein, the applicability and efficiency of two types of pillared graphene nanostructures, namely, (6,6)@G and (7,7)@G, were investigated as membranes in reverse osmosis seawater desalination using extensive nonequilibrium molecular dynamics simulations. The water permeability for (6,6)@G and (7,7)@G membranes was estimated at 4.2 and 6.6 L h-1 cm-2 MPa-1, respectively. According to the results, a complete (100%) and pressure-independent salt rejection was estimated for both membranes. In addition, the mechanism of seawater desalination through the pillared graphene membranes was investigated via the density distribution profile of water molecules inside the pillar channels. Furthermore, a series of steered MD simulations were performed to construct the potential of mean force (PMF) profile of water molecules and salt ions passing through the membranes channels. The passing free energy barriers of Na+ and Cl- ions and water molecules are 0.86, 0.62, and 0.22 eV, respectively, for the (6,6)@G membrane. The corresponding quantities for the (7,7)@G membrane are 0.71, 0.44, and 0.11 eV, respectively.

In the current work, the issue of re-parameterization of Tersoff empirical potential model was addressed for 2D nanomaterial 'germanene' to be applied in molecular dynamics simulation based studies. The well-known chi-square minimization procedure was used to optimize the original Tersoff potential parameters. Many properties of germanene were extracted using both original and optimized Tersoff potentials and they compared with the corresponding density functional theory data. According to the results, the optimized Tersoff potential provides a significant improvement in many structural, thermodynamic, mechanical, and thermal properties of geramanene., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Thermo-mechanical properties of boron nitride nanoribbons (BNNRs) were computed using molecular dynamics simulation with optimized Tersoff empirical potential. Thermal conductivity (TC) and heat transport properties of BNNRs were calculated as functions of both temperature and nanoribbon's length. The results show that TC of BNNRs decreases with raising temperature by T(-1.5) up to 1000K. The phonon-phonon scattering relaxation time, mean free path of phonons, and contribution of high frequency optical phonons in TC of BNNRs were calculated at various temperatures. TC decreases as nanoribbon size increases and it converges to ∼500Wm(-1)K(-1) for nanoribbons with length longer than 30nm. The mechanical properties, including Gruneisen parameter, stress-strain response curves, Young's modulus, intrinsic strength, critical strain, and poisson's ratio were calculated in the temperature range of 137-1000K. The simulation results show that Gruneisen parameter and poisson's ratio of BNNRs are -0.092 and 0.245, respectively. The Young's modulus of BNNRs decreases with raising temperature and its value is 630GPa at 300K. According to the results, BNNRs duo to their extraordinary thermo-mechanical properties, are the promising candidate for the future nano-device manufacturing., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In the present study, the thermal conductivity (TC) and heat transport properties of nitrogen doped graphene (N-graphene) were investigated as a function of temperature (107-400K) and N-doped concentration (0.0-7.0%) using equilibrium molecular dynamics simulation based on Green-Kubo method. According to the results, a drastic decline in TC of graphene observed at very low N-doped concentration (0.5 and 1.0%). Substitution of just 1.0% of carbon atoms with nitrogens causes a 77.2, 65.4, 59.2, and 53.7% reduction in TC at 107, 200, 300, and 400K, respectively. The values of TC of N-graphene at different temperatures approach to each other as N-doped concentration increases. The results also indicate that TC of N-graphene is much less sensitive to temperature compared with pristine graphene and the sensitivity decreases as N-doped concentration increases. The phonon-phonon scattering relaxation times and the phonon mean free path of phonons were also calculated. The contribution of high frequency optical phonons for pristine graphene and N-graphene with 7.0% N-doped concentration is 0-2% and 4-8%, respectively. These findings imply that it is potentially feasible to control heat transfer on the nanoscale when designing N-graphene based thermal devices., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The physisorption of methane in homogeneous armchair open-ended SWBNNT triangular arrays was evaluated using grand canonical ensemble Monte Carlo simulation for tubes 11.08, 13.85, 16.62, and 19.41 Å [(8,8), (10,10), (12,12), and (14,14), respectively] in diameter, at temperatures of 273, 298, 323, and 373 K, and at fugacities of 0.5-9.0 Mpa. The intermolecular forces were modeled using the Lennard-Jones potential model. The absolute, excess, and delivery adsorption isotherms of methane were calculated for the various boron nitride nanotube arrays. The specific surface areas and the isosteric heats of adsorption, Q(st), were also studied, different isotherm models were fitted to the simulated adsorption data, and the model parameters were correlated. According to the results, it is possible to reach 108% and 140% of the US Department of Energy's target for CH(4) storage (180 v/v at 298 K and 35 bar) using the SWBNNT array with nanotubes 16.62 and 19.41 Å in diameter, respectively, as adsorbent. The results show that for a van der Waals gap of 3.4 Å, there is no interstitial adsorption except for arrays containing nanotubes with diameters of >15.8 Å. Multilayer adsorption starts to occur in arrays containing nanotubes with diameters of >16.62 Å, and the minimum pressure required for multilayer adsorption is 1.0 MPa. A brief comparison of the methane adsorption capacities of single-walled carbon and boron nitride nanotube arrays was also performed.