bluuebunny/crossref_metadata_2025_split huggingface dataset:
WTAP targets the METTL3 m6A-methyltransferase complex to cytoplasmic hepatitis C virus RNA to regulate infection
Authors: Sacco Matthew T. , Bland Katherine M. , Horner Stacy M. 
Published: June 2022

ABSTRACTModification of the hepatitis C virus (HCV) positive-strand RNA genome by N6-methyladenosine (m6A) regulates the viral lifecycle. This lifecycle takes place solely in the cytoplasm, while m6A addition on cellular mRNA takes place in the nucleus. Thus, the mechanisms by which m6A is deposited on the viral RNA have been unclear. In this work, we find that m6A modification of HCV RNA by the m6A-methyltransferase proteins METTL3 and METTL14 is regulated by WTAP. WTAP, a predominantly nuclear protein, is an essential member of the cellular mRNA m6A-methyltransferase complex and known to target METTL3 to mRNA. We found that HCV infection induces localization of WTAP to the cytoplasm. Importantly, we found that WTAP is required for both METTL3 interaction with HCV RNA and for m6A modification across the viral RNA genome. Further, we found that WTAP, like METTL3 and METTL14, negatively regulates the production of infectious HCV virions, a process that we have previously shown is regulated by m6A. Excitingly, WTAP regulation of both HCV RNA m6A modification and virion production were independent of its ability to localize to the nucleus. Together, these results reveal that WTAP is critical for HCV RNA m6A modification by METTL3 and METTL14 in the cytoplasm.IMPORTANCEPositive-strand RNA viruses such as HCV represent a significant global health burden. Previous work has described how HCV RNA contains the RNA modification m6A and how this modification regulates viral infection. Yet, how this modification is targeted to HCV RNA has remained unclear due to the incompatibility of the nuclear cellular processes that drive m6A modification with the cytoplasmic HCV lifecycle. In this study, we present evidence for how m6A modification is targeted to HCV RNA in the cytoplasm by a mechanism in which WTAP recruits the m6A-methyltransferase METTL3 to HCV RNA. This targeting strategy for m6A modification of cytoplasmic RNA viruses is likely relevant for other m6A-modified positive-strand RNA viruses with cytoplasmic lifecycles such as enterovirus 71 and SARS-CoV-2 and provides an exciting new target for potential antiviral therapies.
Predicting the locations of cryptic pockets from single protein structures using the PocketMiner graph neural network
Authors: Meller Artur , Ward Michael , Borowsky Jonathan , Lotthammer Jeffrey M. , Kshirsagar Meghana , Oviedo Felipe , Ferres Juan Lavista , Bowman Gregory R. 
Published: June 2022

AbstractCryptic pockets expand the scope of drug discovery by enabling targeting of proteins currently considered undruggable because they lack pockets in their ground state structures. However, identifying cryptic pockets is labor-intensive and slow. The ability to accurately and rapidly predict if and where cryptic pockets are likely to form from a protein structure would greatly accelerate the search for druggable pockets. Here, we present PocketMiner, a graph neural network trained to predict where pockets are likely to open in molecular dynamics simulations. Applying PocketMiner to single structures from a newly-curated dataset of 39 experimentally-confirmed cryptic pockets demonstrates that it accurately identifies cryptic pockets (ROC-AUC: 0.87) >1,000-fold faster than existing methods. We apply PocketMiner across the human proteome and show that predicted pockets open in simulations, suggesting that over half of proteins thought to lack pockets based on available structures are likely to contain cryptic pockets, vastly expanding the druggable proteome.
Structural basis for host recognition and superinfection exclusion by bacteriophage T5
Authors: van den Berg Bert , Silale Augustinas , Baslé Arnaud , Mader Sophie L. , Khalid Syma 
Published: June 2022

AbstractA key but poorly understood stage of the bacteriophage life cycle is the binding of phage receptor binding proteins (RBPs) to receptors on the host cell surface, leading to injection of the phage genome and, for lytic phages, host cell lysis. To prevent a secondary viral infection by the same or a closely related phage, superinfection exclusion (SE) proteins can prevent the binding of RBPs via modulation of the host receptor structure in ways that are also unclear. Here we present the cryo-EM structure of the phage T5 outer membrane (OM) receptor FhuA in complex with the T5 RBP pb5, and the crystal structure of FhuA complexed to the OM SE lipoprotein Llp. Pb5 inserts four loops deeply into the extracellular lumen of FhuA and contacts the plug, but does not cause any conformational changes in the receptor, supporting the view that DNA translocation does not occur through the lumen of OM channels. The FhuA-Llp structure reveals that Llp is periplasmic and binds to a non-native conformation of the plug of FhuA, causing the inward folding of two extracellular loops via “reverse” allostery. The inward-folded loops of FhuA overlap with the pb5 binding site, explaining how Llp binding to FhuA abolishes further infection of E. coli by phage T5, and suggesting a mechanism for SE via the jamming of TonB-dependent transporters by small phage lipoproteins.
Peptide from NSP7 is able to form amyloid-like fibrils: artifact or challenge to drug design?
Authors: Garmay Yuri , Rubel Aleksandr , Egorov Vladimir 
Published: June 2022

AbstractUsing a set of programs for analyzing the primary structure of amyloidogenic proteins, we analyzed the primary structure of the of the COVID-19 NSP7 protein. Peptides containing potential amyloidogenic determinants in the primary structure have been synthesized and shown to be capable of forming amyloid-like fibrils in vitro. In the future, this peptide can be used as a basis for creating antiviral agents that act directly on the spatial structure of the NSP7 subunit of the COVID-19 polymerase.
Short communication: Antibiotic resistance in Gram-negative bacteria isolated from street-vended foods in Bangladesh
Authors: Meem Fariha Chowdhury , Raihan Topu , Shourove Jahid Hasan , Azad Abul Kalam , Islam GM Rabiul 
Published: June 2022

AbstractAntibiotic-resistant pathogens disseminated through food are a public health concern. Although a significant proportion of the urban population in developing countries consume street-vended foods, their role in spreading antibiotic resistance has been rarely investigated. In this study, we evaluated the antibiotic resistance patterns of bacterial isolates (n = 50) collected from five categories of street-vended foods (phuchka, chatpati, sausage, bun, and salad) in Bangladesh. Antibiotic susceptibility to twelve antibiotics was investigated by the Kirby-Bauer disk diffusion method. We found a high prevalence of E. coli (n = 32) in street-vended foods, with most isolates (65.63%) exhibiting multidrug resistance. The multiple antibiotic resistance (MAR) index showed that 22 isolates had MAR above 0.2, with resistance mostly against oxacillin, ampicillin, and cefuroxime. From the rest, three representative isolates were selected for molecular identification by DNA sequencing of 16S rDNA. Klebsiella oxytoca showed multiple drug resistance (MDR) and was resistant to ampicillin, oxacillin, cefuroxime, and kanamycin. Burkholderia fungorum showed no distinct inhibition zone against ampicillin and chloramphenicol. Additionally, the Serratia nematodiphila isolate showed no distinct inhibition zone against three antibiotics, including ampicillin, oxacillin, and cefuroxime. These findings might contribute to the knowledge of emerging antibiotic-resistant foodborne pathogens and raise concerns about the safety of street-vended foods in Bangladesh.
Mechanical loading is required for initiation of extracellular matrix deposition at the developing murine myotendinous junction
Authors: Lipp Sarah N. , Jacobson Kathryn R. , Colling Haley A. , Tuttle Tyler G. , Miles Dalton T. , McCreery Kaitlin P. , Calve Sarah 
Published: June 2022

AbstractThe myotendinous junction (MTJ) contributes to the generation of motion by connecting muscle to tendon. At the adult MTJ, a specialized extracellular matrix (ECM) is thought to contribute to the mechanical integrity of the muscle-tendon interface, but the factors that influence MTJ formation during mammalian development are unclear. Here, we combined 3D imaging and proteomics with murine models in which muscle contractility and patterning are disrupted to resolve morphological and compositional changes in the ECM during MTJ development. We found that MTJ-specific ECM deposition can be initiated via static loading due to growth; however, it required cyclic loading to develop a mature morphology. Furthermore, the MTJ can mature without the tendon terminating into cartilage. Based on these results, we describe a model wherein MTJ development depends on mechanical loading but not insertion into an enthesis.
Oxidative stress drives mutagenesis through transcription coupled repair in bacteria
Authors: Carvajal-Garcia Juan , Samadpour Ariana N. , Hernandez Viera Angel J. , Merrikh Houra 
Published: June 2022

AbstractIn bacteria, mutations lead to the evolution of antibiotic resistance, which is one the main public health problems of the 21stcentury. Therefore, determining which cellular processes most frequently contribute to mutagenesis, especially in cells that have not been exposed to exogenous DNA damage, is critical. Here, we show that endogenous oxidative stress is a key driver of mutagenesis and the subsequent development of antibiotic resistance. This is the case for all classes of antibiotics tested and across highly divergent species, including patient-derived strains. We show that the transcription-coupled repair pathway, which uses the nucleotide excision repair proteins (TC-NER), is responsible for endogenous oxidative stress-dependent mutagenesis and subsequent evolution. This strongly suggests that a majority of mutations arise through transcription-associated processes rather than the replication fork. In addition to determining that the NER proteins play a critical role in mutagenesis and evolution, we also identify the DNA polymerases responsible for this process. Our data strongly suggest that cooperation between three different mutagenic DNA polymerases, likely at the last step of TC-NER, is responsible for mutagenesis and evolution. Overall, our work identifies that a highly conserved pathway drives mutagenesis due to endogenous oxidative stress, which has broad implications for all diseases of evolution, including antibiotic resistance development.
Optimal reliability, robustness and control of nucleus centering in fission yeast is contingent on nonequilibrium force patterning
Authors: Jain Ishutesh , Rao Madan , Tran Phong T. 
Published: June 2022

AbstractCells, such as fission yeast, center their division apparatus to ensure symmetric cell division, a challenging task when the governing dynamics is stochastic. Here we show that the spindle pole body (SPB) positioning, which defines the division septum, is controlled by the patterning of nonequilibrium polymerization forces of microtubule (MT) bundles. We define two cellular objectives, reliability, the mean SPB position relative to the geometric center, and robustness, the variance of the SPB position, which are sensitive to genetic perturbations that change cell length, MT-bundle number/orientation and MT dynamics. We show that the optimal control of reliability and robustness required to minimize septum positioning error is achieved by the wild-type (WT). A stochastic model for the MT-based nucleus centering, with parameters measured directly or estimated using Bayesian inference, recapitulates WT optimality. We use this to perform a sensitivity analysis of the parameters that control nuclear centering.
Pou3f1 orchestrates a gene regulatory network controlling contralateral retinogeniculate projections
Authors: Fries Michel , Brown Thomas W. , Jolicoeur Christine , Boudreau-Pinsonneault Camille , Javed Awais , Abram Pénélope , Cayouette Michel 
Published: June 2022

AbstractThe balance of contralateral and ipsilateral retinogeniculate projections is critical for the establishment of binocular vision, but the transcriptional programs regulating this process remain ill-defined. In this study, we show that the Pou class homeobox protein POU3F1 is selectively expressed in developing mouse contralateral retinal ganglion cells (cRGCs). Inactivation of Pou3f1 increases the number of ipsilateral RGCs produced, leading to abnormal ipsilateral to contralateral projection ratio at the optic chiasm, whereas expression of Pou3f1 in retinal progenitors increases the production of cRGCs. Using Cut&Run and RNA sequencing in wildtype and Pou3f1 mouse knockout retinas, we demonstrate that Pou3f1 is necessary for the establishment of a cRGC gene regulatory network through activation and repression of several known contralateral and ipsilateral determinants, respectively. Finally, we report that POU3F1 is sufficient to induce production of RGC-like cells sending projections to the optic nerve, even in late-stage retinal progenitors not normally competent to generate RGCs. This work uncovers POU3F1 as a master regulator of the cRGC transcriptional program and opens new possibilities for optic nerve regenerative therapies.
Neutralizing monoclonal antibodies elicited by mosaic RBD nanoparticles bind conserved sarbecovirus epitopes
Authors: Fan Chengcheng , Cohen Alexander A. , Park Miso , Hung Alfur Fu-Hsin , Keeffe Jennifer R. , Gnanapragasam Priyanthi N.P. , Lee Yu E. , Kakutani Leesa M. , Wu Ziyan , Malecek Kathryn E. , Williams John C. , Bjorkman Pamela J. 
Published: June 2022

SummaryProtection from SARS-related coronaviruses with spillover potential and SARS-CoV-2 variants could prevent and/or end pandemics. We show that mice immunized with nanoparticles co-displaying spike receptor-binding domains (RBDs) from eight sarbecoviruses (mosaic-8 RBD-nanoparticles) efficiently elicit cross-reactive anti-sarbecovirus antibodies against conserved class 1/4 and class 3 RBD epitopes. Monoclonal antibodies (mAbs) identified from initial screening of <10,000 single B-cells secreting IgGs binding two or more sarbecovirus RBDs showed cross-reactive binding and neutralization of SARS-CoV-2 variants and animal sarbecoviruses. Single-particle cryo-EM structures of antibody–spike complexes, including a Fab-Omicron complex, mapped neutralizing mAbs to conserved class 1/4 RBD epitopes and revealed neutralization mechanisms, potentials for intra-spike trimer crosslinking by single IgGs, and induced changes in trimer upon Fab binding. In addition, we identified a mAb resembling Bebtelovimab, an EUA-approved human class 3 anti-RBD mAb. These results support using mosaic RBD-nanoparticles to identify therapeutic pan-sarbecovirus and pan-variant mAbs and to elicit them by vaccination.
Quantitative prediction and measurement of Piezo’s membrane footprint
Authors: Haselwandter Christoph A. , Guo Yusong R. , Fu Ziao , MacKinnon Roderick 
Published: June 2022

AbstractPiezo proteins are mechanosensitive ion channels that can locally curve the membrane into a dome shape (Y. R. Guo, R. MacKinnon, 2017). The curved shape of the Piezo dome is expected to deform the surrounding lipid bilayer membrane into a membrane footprint, which may serve to amplify Piezo’s sensitivity to applied forces (C. A. Haselwandter, R. MacKinnon, 2018). If Piezo proteins are embedded in lipid bilayer vesicles, the membrane shape deformations induced by the Piezo dome depend on the vesicle size. We employ here membrane elasticity theory to predict, with no free parameters, the shape of such Piezo vesicles outside the Piezo dome, and show that the predicted vesicle shapes agree quantitatively with the corresponding measured vesicle shapes obtained through cryo-electron tomography, for a range of vesicle sizes (Helfrich W. 1973). On this basis, we explore the coupling between Piezo and membrane shape, and demonstrate that the features of the Piezo dome affecting Piezo’s membrane footprint follow approximately a spherical cap geometry. Our work puts into place the foundation for deducing key elastic properties of the Piezo dome from membrane shape measurements and provides a general framework for quantifying how proteins deform bilayer membranes.ClassificationBiophysicss
Structural basis for FLCN RagC GAP activation in TFEB substrate-selective mTORC1 regulation
Authors: Jansen Rachel M. , Peruzzo Roberta , Fromm Simon A. , Yokom Adam L. , Zoncu Roberto , Hurley James H. 
Published: June 2022

AbstractmTORC1 regulates cell growth and catabolism in response to fluctuations in nutrients through phosphorylation of key substrates. The tumor suppressor FLCN is a RagC GTPase activating protein (GAP) that regulates mTORC1 phosphorylation of TFEB, controlling lysosome biogenesis and autophagy. Here, we determined the cryo-EM structure of the active FLCN complex (AFC) containing FLCN, FNIP2, the N-terminal tail of SLC38A9, the RagAGDP:RagCGDP.BeFx- GTPase dimer, and the Ragulator scaffold. Relative to the inactive lysosomal FLCN complex (LFC) structure, FLCN reorients by 90°, breaks its contacts with RagA, and makes new contacts with RagC that position its Arg164 finger for catalysis. Disruption of the AFC-specific interfaces of FLCN and FNIP2 with RagC eliminated GAP activity in vitro and led to nuclear retention of TFE3, with no effect on mTORC1 phosphorylation of S6K or 4E-BP1. The structure thus provides a roadmap to discover TFEB-selective mTORC1 antagonists.One-Sentence SummaryThe cryo-EM structure of the active FLCN RagC GAP complex provides a structural basis for TFEB/TFE3 substrate-selective targeting of mTORC1.
Connecting sequence features within the disordered C-terminal linker ofB. subtilisFtsZ to functions and bacterial cell division
Authors: Shinn Min Kyung , Cohan Megan C. , Bullock Jessie L. , Ruff Kiersten M. , Levin Petra A. , Pappu Rohit V. 
Published: June 2022

AbstractIntrinsically disordered regions (IDRs) can function as autoregulators of folded enzymes to which they are tethered. One example is the bacterial cell division protein, FtsZ. This includes a folded core and a C-terminal tail (CTT) that encompasses a poorly conserved, disordered C-terminal linker (CTL) and a well-conserved 17-residue C-terminal peptide (CT17). Sites for GTPase activity of FtsZs are formed at the interface between GTP binding sites and T7 loops on cores of adjacent subunits within dimers. Here, we explore the basis of autoregulatory functions of the CTT inBacillus subtilisFtsZ (Bs-FtsZ). Molecular simulations show that the CT17 ofBs-FtsZ makes statistically significant CTL-mediated contacts with the T7 loop. Statistical Coupling Analysis of more than 103sequences from FtsZ orthologs reveals clear covariation of the T7 loop and the CT17 with most of the core domain whereas the CTL is under independent selection. Despite this, we discover the conservation of non-random sequence patterns within CTLs across orthologs. To test how the non-random patterns of CTLs mediate CTT-core interactions and modulate FtsZ functionalities, we designedBs-FtsZ variants by altering the patterning of oppositely charged residues within the CTL. Such alterations disrupt the core-CTT interactions, lead to anomalous assembly and inefficient GTP hydrolysisin vitroand protein degradation, aberrant assembly, and disruption of cell divisionin vivo. Our findings suggest that viable CTLs in FtsZs are likely to be IDRs that encompass non-random, functionally relevant sequence patterns that also preserve three-way covariation of the CT17, the T7 loop, and core domain.Significance StatementZ-ring formation by the protein FtsZ controls cell division in rod-shaped bacteria. The C-terminus of FtsZ encompasses a disordered C-terminal linker (CTL) and a conserved CT17 motif. Both modules are essential for Z-ring formation and proper localization of FtsZ in cells. Previous studies suggested that generic intrinsically disordered regions (IDRs) might be suitable functional replacements for naturally occurring CTLs. Contrary to this suggestion, we find that the sequence-encoded conformational properties of CTLs help mediate autoregulatory interactions between covarying regions within FtsZ. Functional properties of the CTL are encoded via evolutionarily conserved, non-random sequence patterns. Disruption of these patterns impair molecular functions and cellular phenotypes. Our findings have broad implications for discovering functionally consequential sequence features within IDRs of other proteins.
Mitochondria directly sense osmotic stress to trigger rapid metabolic remodeling via PDH regulation
Authors: Ikizawa Takeshi , Ikeda Kazutaka , Arita Makoto , Kitajima Shojiro , Soga Tomoyoshi , Ichijo Hidenori , Naguro Isao 
Published: June 2022

AbstractA high-salt diet significantly impacts various diseases, including cancer and immune diseases. Recent studies suggest that the high-salt/hyperosmotic environment in the body may alter the chronic properties of cancer and immune cells in the disease context. However, little is known about the acute metabolic changes in hyperosmotic stress. Here, we found that hyperosmotic stress for a few minutes induces Warburg-like metabolic remodeling in HeLa and Raw264.7 cells and suppresses fatty acid oxidation. Regarding Warburg-like remodeling, the PDH phosphorylation status was altered bidirectionally (high in hyperosmolarity and low in hypoosmolarity) to osmotic stress in isolated mitochondria, suggesting that mitochondria themselves have an acute osmo-sensing mechanism. Warburg-like remodeling is required for HeLa cells to maintain ATP levels and survive under hyperosmotic conditions. Our findings suggest that cells exhibit acute metabolic remodeling under osmotic stress via the regulation of PDH phosphorylation by direct osmosensing within mitochondria.
Dynamic market complexity and service value advantage: an empirical investigation of business process management
Authors: Hong Paul C. , Hejazi Mohammed Taj , Deng Xiyue , Jagani Sandeep 
Published: June 2022

PurposeHaving complexity theory as the overarching conceptual rationale, this paper presents a research model that defines external drivers, strategic and internal business practices and performance outcomes in service firm contexts.Design/methodology/approachUsing an original survey instrument, the authors empirically examine business process management (1) adopting the mediating roles of shared goal practices and technology imperative practices (2) involving moderating roles of competitive market environments and joint implementation practices.FindingsIn response to dynamic market complexity, firms implement business process management through shared goal practices and technology imperative practices for achieving customer service outcomes. The findings also suggest the moderating roles of competitive pressure and joint implementation practices.Research limitations/implicationsGeneralizations here are limited to service firms. Increasing customer service expectations (e.g. simplicity, convenience, visual images and rapid responses) require firms to involve cross-functional work throughout their organizational processes.Practical implicationsIn digital environments, business process management requires socio-technological synergy through shared goal practices and technology imperative practices.Originality/valueBuilding on a theory-driven research model, a survey instrument provides tools to examine business process management of service firms that sense dynamic market complexity challenges and translate them to achieve desirable customer service outcomes.