Biology subjects

Gu, W.

Publications and source records attributed to Gu, W..

At least 91 records · Page 5Linked to original sources

bioRxiv2021

The rs1421085 variant within FTO promotes but not inhibits thermogenesis and is potentially associated with human migration

Disease-associated GWAS loci are predominantly scattered among noncoding regions of the human genome, which impedes causality estimation. One lead risk signal of obesity-rs1421085 T>C within the FTO gene-is reported to functional in vitro but lack of organismal evidence. Here, we established global and the brown-adipocyte specific locus-knock-in mice to recapitulate this homologous variant in humans, and discovered the minor allele (C-allele) as one candidate thermogenic locus. Mice carrying the C-alleles showed increased thermogenic capacity and a resistance to high-fat diet-induced adiposity. In terms of mechanism, the knock-in models showed enhanced FTO expression, while FTO knockdown or inhibition effectively eliminated the increased thermogenic ability of brown adipocytes. In humans, the C-allele was associated with lower birthweight, and its allele frequency increases following the environmental temperature decreases. Cumulatively, these findings demonstrated rs1421085 T>C as a functional variant regulating whole-body thermogenesis, and this variation was possibly related to early human migration from hot to cold environments.

genetics↗

bioRxiv2021

Nicotinic acid mononucleotide is an allosteric SARM1 inhibitor promoting axonal protection

SARM1 is an inducible NAD+ hydrolase that is the central executioner of pathological axon loss. Recently, we elucidated the molecular mechanism of SARM1 activation, demonstrating that SARM1 is a metabolic sensor regulated by the levels of NAD+ and its precursor, nicotinamide mononucleotide (NMN), via their competitive binding to an allosteric site within the SARM1 N-terminal ARM domain. In healthy neurons with abundant NAD+, binding of NAD+ blocks access of NMN to this allosteric site. However, with injury or disease the levels of the NAD+ biosynthetic enzyme NMNAT2 drop, increasing the NMN/ NAD+ ratio and thereby promoting NMN binding to the SARM1 allosteric site, which in turn induces a conformational change activating the SARM1 NAD+ hydrolase. Hence, NAD+ metabolites both regulate the activation of SARM1 and, in turn, are regulated by the SARM1 NAD+ hydrolase. This dual upstream and downstream role for NAD+ metabolites in SARM1 function has hindered mechanistic understanding of axoprotective mechanisms that manipulate the NAD+ metabolome. Here we reevaluate two methods that potently block axon degeneration via modulation of NAD+ related metabolites, 1) the administration of the NMN biosynthesis inhibitor FK866 in conjunction with the NAD+ precursor nicotinic acid riboside (NaR) and 2) the neuronal expression of the bacterial enzyme NMN deamidase. We find that these approaches not only lead to a decrease in the levels of the SARM1 activator NMN, but also an increase in the levels of the NAD+ precursor nicotinic acid mononucleotide (NaMN). We show that NaMN inhibits SARM1 activation, and demonstrate that this NaMN-mediated inhibition is important for the long-term axon protection induced by these treatments. Analysis of the NaMN-ARM domain co-crystal structure shows that NaMN competes with NMN for binding to the SARM1 allosteric site and promotes the open, autoinhibited configuration of SARM1 ARM domain. Together, these results demonstrate that the SARM1 allosteric pocket can bind a diverse set of metabolites including NMN, NAD+, and NaMN to monitor cellular NAD+ homeostasis and regulate SARM1 NAD+ hydrolase activity. The relative promiscuity of the allosteric site may enable the development of potent pharmacological inhibitors of SARM1 activation for the treatment of neurodegenerative disorders. HighlightsO_LINaMN binds to SARM1 N-terminal allosteric site and inhibits SARM1 NAD+ hydrolase C_LIO_LINaMN inhibits SARM1 activation by stabilizing its open, inactive structure C_LIO_LINMN deamidase promotes strong axonal protection by reducing NMN and increasing NaMN C_LI

neuroscience↗

bioRxiv2021

A recombinant protein containing influenza viral conserved epitopes and superantigen induces broad-spectrum protection

Influenza pandemic poses public health threats annually for lacking vaccine which provides cross-protection against novel and emerging influenza viruses. Combining conserved antigens inducing cross-protective antibody response with epitopes activating cross-protective cytotoxic T-cells would offer an attractive strategy for developing universal vaccine. In this study, we constructed a recombinant protein NMHC consisting of influenza viral conserved epitopes and superantigen fragment. NMHC promoted the mature of bone marrow-derived dendritic cells and induced CD4+ T cells to differentiate into Th1, Th2 and Th17 subtypes. Mice vaccinated with NMHC produced high level of immunoglobulins which cross-bound to HA fragments from six influenza virus subtypes with high antibody titers. Anti-NMHC serum showed potent hemagglutinin inhibition effects to highly divergent group 1 (H1 subtypes) and group 2 (H3 subtype) influenza virus strains. And purified anti-NMHC antibodies could bind to multiple HAs with high affinities. NMHC vaccination effectively protected the mice from infection and lung damage challenged by two subtypes of H1N1 influenza virus. Moreover, NMHC vaccination elicited CD4+ and CD8+ T-cell responses to clear the virus from infected tissue and prevent virus spreading. In conclusion, this study provided proof of concept for triggering both B cells and T cells immune responses against multiple influenza virus infection, and NMHC may be a potential candidate of universal broad-spectrum vaccine for various influenza virus prevention and therapy.

epidemiology↗

bioRxiv2021

Sliding of HIV-1 reverse transcriptase over DNA creates a transient P pocket - Targeting P-pocket by fragment screening

HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We report a crystal structure of RT/dsDNA complex in which RT overstepped the primer 3-end of a dsDNA substrate and created a transient P-pocket at the priming site. We performed a high-throughput screening of 300 drug-like fragments by X-ray crystallography and identified binding of two to P-pocket, which is composed of structural elements from polymerase active site, primer grip, and template-primer that are resilient to drugresistance mutations. Analogs of a fragment were synthesized of which two showed noticeable RT inhibition. An engineered RT/DNA aptamer complex trapped the transient P-pocket in solution. Structures of the RT/DNA complex were determined with a fragment and a synthesized analog bound at P-pocket by single-particle cryo-EM. Identification of P-pocket and the devised structurebased platform provide an opportunity for designing new types of polymerase inhibitors.

biophysics↗

bioRxiv2021

COBREXA.jl: constraint-based reconstruction and exascale analysis

COBREXA.jl is a Julia package for scalable, high-performance constraint-based reconstruction and analysis of very large-scale biological models. Its primary purpose is to facilitate the integration of modern high performance computing environments with the processing and analysis of large-scale metabolic models of challenging complexity. We report the architecture of the package, and demonstrate how the design promotes analysis scalability on several use-cases with multi-organism community models. Availability and implementationhttps://doi.org/10.17881/ZKCR-BT30. Contactchristophe.trefois@uni.lu, wei.gu@uni.lu

systems biology↗

bioRxiv2021

The middle lipin (M-Lip) domain is a new dimeric protein fold that binds membranes

Phospholipid synthesis and fat storage as triglycerides is regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized and novel protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in full-length lipin 1 influences PAP activity, membrane binding, subcellular localization, oligomerization, and adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a new dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.

biochemistry↗

bioRxiv2021

PredictProtein - Predicting Protein Structure and Function for 29 Years

Since 1992 PredictProtein (https://predictprotein.org) is a one-stop online resource for protein sequence analysis with its main site hosted at the Luxembourg Centre for Systems Biomedicine (LCSB) and queried monthly by over 3,000 users in 2020. PredictProtein was the first Internet server for protein predictions. It pioneered combining evolutionary information and machine learning. Given a protein sequence as input, the server outputs multiple sequence alignments, predictions of protein structure in 1D and 2D (secondary structure, solvent accessibility, transmembrane segments, disordered regions, protein flexibility, and disulfide bridges) and predictions of protein function (functional effects of sequence variation or point mutations, Gene Ontology (GO) terms, subcellular localization, and protein-, RNA-, and DNA binding). PredictProteins infrastructure has moved to the LCSB increasing throughput; the use of MMseqs2 sequence search reduced runtime five-fold; user interface elements improved usability, and new prediction methods were added. PredictProtein recently included predictions from deep learning embeddings (GO and secondary structure) and a method for the prediction of proteins and residues binding DNA, RNA, or other proteins. PredictProtein.org aspires to provide reliable predictions to computational and experimental biologists alike. All scripts and methods are freely available for offline execution in high-throughput settings. AvailabilityFreely accessible webserver PredictProtein.org; Source and docker images: github.com/rostlab

bioinformatics↗

bioRxiv2021

SearcHPV: a novel approach to identify and assemble human papillomavirus-host genomic integration events in cancer

BackgroundHuman papillomavirus (HPV) is a well-established driver of malignant transformation in a number of sites including head and neck, cervical, vulvar, anorectal and penile squamous cell carcinomas; however, the impact of HPV integration into the host human genome on this process remains largely unresolved. This is due to the technical challenge of identifying HPV integration sites, which includes limitations of existing informatics approaches to discover viral-host breakpoints from low read coverage sequencing data. MethodsTo overcome this limitation, we developed a new HPV detection pipeline called SearcHPV based on targeted capture technology and applied the algorithm to targeted capture data. We performed an integrated analysis of SearcHPV-defined breakpoints with genome-wide linked read sequencing to identify potential HPV-related structural variations. ResultsThrough analysis of HPV+ models, we show that SearcHPV detects HPV-host integration sites with a higher sensitivity and specificity than two other commonly used HPV detection callers. SearcHPV uncovered HPV integration sites adjacent to known cancer-related genes including TP63 and MYC, as well as near regions of large structural variation. We further validated the junction contig assembly feature of SearcHPV, which helped to accurately identify viral-host junction breakpoint sequences. We found that viral integration occurred through a variety of DNA repair mechanisms including non-homologous end joining, alternative end joining and microhomology mediated repair. ConclusionsIn summary, we show that SearcHPV is a new optimized tool for the accurate detection of HPV-human integration sites from targeted capture DNA sequencing data.

bioinformatics↗

bioRxiv2021

Crystal structure determination of the armadillo repeat domain of Drosophila SARM1 using MIRAS phasing

We describe the crystal structure determination of the ARM domain of Drosophila SARM1 (dSARM1ARM), which required combination of a number of sources of phase information in order to obtain interpretable electron density maps. SARM1 is a central executioner of the process of axon degeneration, a common feature of the early phase of a range of neurodegenerative diseases. SARM1 is held in the inactive state in healthy axons by its N-terminal auto-inhibitory ARM domain, and is activated to cleave NAD+ upon injury, triggering the subsequent axon degeneration. To characterize the molecular mechanism of SARM1 activation, we sought to determine the crystal structure of the SARM1 ARM domain. Here we describe the recombinant production and crystallization of dSARM1ARM, as well as unconventional process used for structure determination. Crystals were obtained in the presence of NMN, a precursor of NAD+ and a potential activator of SARM1, only after in situ proteolysis of the N-terminal 63 residues. After molecular replacement attempts failed, we determined the crystal structure of dSARM1ARM at 1.65 [A] resolution using the MIRAS phasing technique with the program autoSHARP, combining data from the native, SeMet-labelled, and Br-soaked crystals. The structure will further our understanding of the regulation of SARM1.

biophysics↗

bioRxiv2021

Evolutionary selection on synonymous codons in RNA G-quadruplex structural region

In addition to the amino acid sequence information, synonymous codons can encode multiple regulatory and structural signals in protein coding region. In this study, we investigated how synonymous codons have been adapted to the formation of RNA G-quadruplex (rG4) structure. We found a universal selective pressure acting on synonymous codons to facilitate rG4 formation in five eukaryotic organisms. While G-rich codons are preferred in rG4 structural region, C-rich codons are selectively unpreferred for rG4 structures. Genes codon usage bias, nucleotide composition and evolutionary rate can account for the selective variations on synonymous codons among rG4 structures within a species. Moreover, rG4 structures in translational initiation region showed significantly higher selective pressures than those in translational elongation region. These results bring us another dimension of evolutionary selection on synonymous codons for proper RNA structure and function.

bioinformatics↗

bioRxiv2021

Genome recombination-mediated tRNA up-regulation conducts general antibiotic resistance of bacteria at early stage

Bacterial antibiotic resistance sets a great challenge to human health. It seems that the bacteria can spontaneously evolve resistance against any antibiotic within short time without the horizontal transfer of heterologous genes and before accumulating drug-resistant mutations. We have shown that the tRNA-mediated translational regulation counteracts the reactive oxygen species in bacteria. In this study, we demonstrated that isolated and subcultured Escherichia coli elevated its tRNAs under antibiotic stress to rapidly provide antibiotic resistance, especially at the early stage, before upregulating the efflux pump and evolving resistance mutations. The DNA recombination system repaired the antibiotic-induced DNA breakage in the genome, causing numerous structural variations. These structural variations are overrepresented near the tRNA genes, which indicated the cause of tRNA up-regulation. The strains knocking out the recombination system could not up-regulate tRNAs, and coincidently, they could hardly evolve antibiotic resistance in multiple antibiotics, respectively. With these results, we proposed a multi-stage model of bacterial antibiotic resistance in an isolated scenario: the early stage (recombination - tRNA up-regulation - translational regulation); the medium stage (up-regulation of efflux pump); the late stage (resistance mutations). These results also indicated that the bacterial DNA recombination system and tRNA could be targeted to retard the bacterial spontaneous drug resistance.

microbiology↗

bioRxiv2020

Effect of the BMPR-II-SMAD3/MRTF complex on proliferation and migration of ASMCs and the mechanism in asthma.

Phenotype transformation of airway smooth muscle (ASM) is the key feature of airway remodelling in asthma. A variety of smooth muscle-specific genes and proteins, including SMAD3, BMPR-II, and MRTF, are involved in airway remodelling in asthma. Bone morphogenetic protein (BMP) signalling plays an important role in the physiological and pathological processes of asthma. As a receptor of BMP signalling, BMPR-II has important roles in variety of cellular processes. However, the understanding of the roles and underlying mechanism of BMPR-II in airway smooth muscle cells (ASMCs) in asthma remains incomplete. First, we observed significant increases in BMPR-II, SMAD3 and MRTF in asthmatic ASMCs at both the mRNA and protein levels. Second, we observed that silencing of siBMPR-II and siMRTF inhibits proliferation, migratory capacity and intracellular Ca2+ concentration in ASMCs. Furthermore, our results revealed these three factors, BMPR-II, SMAD3 and MRTF, form a complex that affects the bioactivities of ASMCs. Taken together, this study indicates that the BMPR-II-SMAD3/MRTF signalling pathway is involved in the process of ASM remodelling, providing novel avenues for the identification of new therapeutic modalities.

cell biology↗

bioRxiv2020

Enhancement of nitrous oxide emissions in soil microbial consortia via copper competition between proteobacterial methanotrophs and denitrifiers

Unique means of copper scavenging have been identified in proteobacterial methanotrophs, particularly the use of methanobactin, a novel ribosomally synthesized post-translationally modified polypeptide that binds copper with very high affinity. The possibility that copper sequestration strategies of methanotrophs may interfere with copper uptake of denitrifiers in situ and thereby enhance N2O emissions was examined using a suite of laboratory experiments performed with rice paddy microbial consortia. Addition of purified methanobactin from Methylosinus trichosporium OB3b to denitrifying rice paddy soil microbial consortia resulted in substantially increased N2O production, with more pronounced responses observed for soils with lower copper content. The N2O emission-enhancing effect of the soils native mbnA-expressing Methylocystaceae methanotrophs on the native denitrifiers was then experimentally verified with a Methylocystaceae-dominant chemostat culture prepared from a rice paddy microbial consortium as the inoculum. Lastly, with microcosms amended with varying cell numbers of methanobactin-producing Methylosinus trichosporium OB3b before CH4 enrichment, microbiomes with different ratios of methanobactin-producing Methylocystaceae to gammaproteobacterial methanotrophs incapable of methanobactin production were simulated. Significant enhancement of N2O production from denitrification was evident in both Methylocystaceae-dominant and Methylococcaceae-dominant enrichments, albeit to a greater extent in the former, signifying the comparative potency of methanobactin-mediated copper sequestration while implying the presence of alternative copper abstraction mechanisms for Methylococcaceae. These observations support that copper-mediated methanotrophic enhancement of N2O production from denitrification is plausible where methanotrophs and denitrifiers cohabit. ImportanceProteobacterial methanotrophs, groups of microorganisms that utilize methane as source of energy and carbon, have been known to utilize unique mechanisms to scavenge copper, namely utilization of methanobactin, a polypeptide that binds copper with high affinity and specificity. Previously the possibility that copper sequestration by methanotrophs may lead to alteration of cuproenzyme-mediated reactions in denitrifiers and consequently increase emission of potent greenhouse gas N2O has been suggested in axenic and co-culture experiments. Here, a suite of experiments with rice paddy soil slurry cultures with complex microbial compositions were performed to corroborate that such copper-mediated interplay may actually take place in environments co-habited by diverse methanotrophs and denitrifiers. As spatial and temporal heterogeneity allow for spatial coexistence of methanotrophy (aerobic) and denitrification (anaerobic) in soils, the results from this study suggest that this previously unidentified mechanism of N2O production may account for significant proportion of N2O efflux from agricultural soils.

microbiology↗

bioRxiv2020

The RNA phosphatase PIR-1 regulates endogenous small RNA pathways in C. elegans

Eukaryotic cells regulate 5 triphosphorylated (ppp-) RNAs to promote cellular functions and prevent recognition by antiviral RNA sensors. For example, RNA capping enzymes possess triphosphatase domains that remove the {gamma} phosphates of ppp-RNAs during RNA capping. Members of the closely related PIR1 family of RNA polyphosphatases remove both the {beta} and {gamma} phosphates from ppp-RNAs. Here we show that C. elegans PIR-1 dephosphorylates ppp-RNAs made by cellular RdRPs and is required for the maturation of 26G-RNAs, Dicer-dependent small RNAs that regulate thousands of genes during spermatogenesis and embryogenesis. PIR-1 also regulates the CSR-1 22G-RNA pathway and has critical functions in both somatic and germline development. Our findings suggest that PIR-1 modulates both Dicer-dependent and - independent Argonaute pathways, and provide insight into how cells and viruses use a conserved RNA phosphatase to regulate and respond to ppp-RNA species.

molecular biology↗

bioRxiv2020

Conservative route to extreme genome compaction in a miniature annelid

The causes and consequences of genome reduction in animals are unclear, because our understanding of this process mostly relies on lineages with often exceptionally high rates of evolution. Here, we decode the compact 73.8 Mb genome of Dimorphilus gyrociliatus, a meiobenthic segmented worm. The D. gyrociliatus genome retains traits classically associated with larger and slower-evolving genomes, such as an ordered, intact Hox cluster, a generally conserved developmental toolkit, and traces of ancestral bilaterian linkage. Unlike some other animals with small genomes, the analysis of the D. gyrociliatus epigenome revealed canonical features of genome regulation, excluding the presence of operons and trans-splicing. Instead, the gene dense D. gyrociliatus genome presents a divergent Myc pathway, a key physiological regulator of growth, proliferation, and genome stability in animals. Altogether, our results uncover a conservative route to genome compaction in annelids, reminiscent of that observed in the vertebrate Takifugu rubripes.

genomics↗

bioRxiv2020

Predicting the Viability of Beta-Lactamase: How Folding and Binding Free Energies Correlate with Beta-Lactamase Fitness

One of the long-standing holy grails of molecular evolution has been the ability to predict an organisms fitness directly from its genotype. With such predictive abilities in hand, researchers would be able to more accurately forecast how organisms will evolve and how proteins with novel functions could be engineered, leading to revolutionary advances in medicine and biotechnology. In this work, we assemble the largest reported set of experimental TEM-1 {beta}-lactamase folding free energies and use this data in conjunction with previously acquired fitness data and computational free energy predictions to determine how much of the fitness of {beta}-lactamase can be directly predicted by thermodynamic folding and binding free energies. We focus upon {beta}-lactamase because of its long history as a model enzyme and its central role in antibiotic resistance. Based upon a set of 21 {beta}-lactamase single and double mutants expressly designed to influence protein folding, we first demonstrate that modeling software such as FoldX and PyRosetta designed to compute folding free energies can meaningfully, although not perfectly, predict the experimental folding free energies of single mutants. Interestingly, while these techniques also yield sensible double mutant free energies, we show that they do so for the wrong physical reasons. We then go on to assess how well both experimental and computational folding free energies explain single mutant fitness. We find that folding free energies account for, at most, 24% of the variance in {beta}-lactamase fitness values according to linear models and, somewhat surprisingly, complementing folding free energies with computationally-predicted binding free energies of residues near the active site only increases the folding-only figure by a few percent. This strongly suggests that the majority of {beta}-lactamases fitness is controlled by factors other than free energies. Overall, our results shed a bright light on to what extent the community is justified in using thermodynamic measures to infer protein fitness as well as how applicable modern computational techniques for predicting free energies will be to the large data sets of multiply-mutated proteins forthcoming.

biophysics↗

bioRxiv2020

The hexosamine biosynthesis pathway is a targetable liability in lung cancers with concurrent KRAS and LKB1 mutations.

In non-small cell lung cancer (NSCLC), concurrent mutations in the oncogene KRAS and the tumor suppressor STK11 encoding the kinase LKB1 result in aggressive tumors prone to metastasis but with liabilities arising from reprogrammed metabolism. We previously demonstrated perturbed nitrogen metabolism and addiction to an unconventional pathway of pyrimidine synthesis in KRAS/LKB1 co-mutant (KL) cancer cells. To gain broader insight into metabolic reprogramming in NSCLC, we analyzed tumor metabolomes in a series of genetically engineered mouse models with oncogenic KRAS combined with mutations in LKB1 or p53. Metabolomics and gene expression profiling pointed towards an activation of the hexosamine biosynthesis pathway (HBP), another nitrogen-related metabolic pathway, in both mouse and human KL mutant tumors. KL cells contain high levels of HBP metabolites, higher flux through the HBP pathway and elevated dependence on the HBP enzyme Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2). GFPT2 inhibition selectively reduced KL cell growth in culture and xenografts. Our results define a new metabolic vulnerability in KL tumors and provide a rationale for targeting GFPT2 in this aggressive NSCLC subtype.

cancer biology↗

bioRxiv2020

Expanding the chemical space of synthetic cyclic peptides using a promiscuous macrocyclase from prenylagaramide biosynthesis

Cyclic peptides are excellent drug candidates, placing macrocyclization reactions at the apex of drug development. PatG and related dual-action proteases from cyanobactin biosynthesis are responsible for cleaving off the C-terminal recognition sequence and macrocyclizing the substrate to provide cyclic peptides. This reaction has found use in the enzymatic synthesis of diverse macrocycles. However, these enzymes function best on substrates that terminate with the non-proteinogenic thiazole/thiazoline residue, complicating synthetic strategies. Here, we biochemically characterize a new class of PatG-like macrocyclases that natively use proline, obviating the necessity of additional chemical or biochemical steps. We experimentally define the biochemical steps involved in synthesizing the widespread prenylagaramide-like natural products, including macrocyclization and prenylation. Using saturation mutagenesis, we show that macrocyclase PagG and prenyltransferase PagF are highly promiscuous, producing a library of more than 100 cyclic peptides and their prenylated derivatives in vitro. By comparing our results to known cyanobactin macrocyclase enzymes, we catalog a series of enzymes that collectively should synthesize most small macrocycles. Collectively, these data reveal that, by selecting the right cyanobactin macrocyclase, a large array of enzymatically synthesized macrocycles are accessible.

biochemistry↗