Biology subjects

Zhu, W.

Publications and source records attributed to Zhu, W..

At least 91 records · Page 5Linked to original sources

bioRxiv2024

Macrophage microRNA-146a is a central regulator of the foreign body response to biomaterial implants

Host recognition and immune-mediated foreign body response (FBR) to biomaterials can adversely affect the functionality of implanted materials. To identify key targets underlying the generation of FBR, here we perform analysis of microRNAs (miR) and mRNAs responses to implanted biomaterials. We found that (a) miR-146a levels inversely affect macrophage accumulation, foreign body giant cell (FBGC) formation, and fibrosis in a murine implant model; (b) macrophage-derived miR-146a is a crucial regulator of the FBR and FBGC formation, as confirmed by global and cell-specific knockout of miR-146a; (c) miR-146a modulates genes related to inflammation, fibrosis, and mechanosensing; (d) miR-146a modulates tissue stiffness near the implant during FBR; and (e) miR-146a is linked to F-actin production and cellular traction force induction, which are vital for FBGC formation. These novel findings suggest that targeting macrophage miR-146a could be a selective strategy to inhibit FBR, potentially improving the biocompatibility of biomaterials.

cell biology↗

bioRxiv2024

TLR2 Supports γδ T cell IL-17A Response to ocular surface commensals by Metabolic Reprogramming

The ocular surface is a mucosal barrier tissue colonized by commensal microbes, which tune local immunity by eliciting IL-17 from conjunctival {gamma}{delta} T cells to prevent pathogenic infection. The commensal Corynebacterium mastitidis (C. mast) elicits protective IL-17 responses from conjunctival V{gamma}4 T cells through a combination of {gamma}{delta} TCR ligation and IL-1 signaling. Here, we identify V{gamma}6 T cells as a major C. mast-responsive subset in the conjunctiva and uncover its unique activation requirements. We demonstrate that V{gamma}6 cells require not only extrinsic (via dendritic cells) but also intrinsic TLR2 stimulation for optimal IL-17A response. Mechanistically, intrinsic TLR2 signaling was associated with epigenetic changes and enhanced expression of genes responsible for metabolic shift to fatty acid oxidation to support Il17a transcription. We identify one key transcription factor, I{kappa}B{zeta}, which is upregulated by TLR2 stimulation and is essential for this program. Our study highlights the importance of intrinsic TLR2 signaling in driving metabolic reprogramming and production of IL-17A in microbiome-specific mucosal {gamma}{delta} T cells. SummaryOcular commensal Corynebacterium mastitidis (C. mast) induces IL-17 responses from {gamma}{delta} T cells by activating TLR2 signaling. {gamma}{delta} T cell-intrinsic TLR2 stimulation can increase Il17a transcription and promote fatty acid oxidation, favoring {gamma}{delta} T cell IL-17A responses. Highlights(1) V{gamma}6 T cells are a major C. mast-responsive subset in the conjunctiva (2) TLR2-deficient mice exhibit reduced {gamma}{delta} T cell responses to ocular commensal bacteria. (3) {gamma}{delta} T cell-intrinsic TLR2 deficiency causes defects of fatty acid oxidation and IL-17A production in a {gamma}{delta} subset-specific manner. (4) The transcription factor, I{kappa}B{zeta} is upregulated by TLR2 stimulation and supports {gamma}{delta} IL-17A production through fatty acid oxidation. O_FIG O_LINKSMALLFIG WIDTH=178 HEIGHT=200 SRC="FIGDIR/small/587519v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1d1ba87org.highwire.dtl.DTLVardef@2d17d9org.highwire.dtl.DTLVardef@78e846org.highwire.dtl.DTLVardef@1d96dc2_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Detecting phenotype-specific tumor microenvironment by merging bulk and single cell expression data to spatial transcriptomics

In addressing the limitations of current multimodal analysis methods that largely ignore phenotypic data, leading to a lack of biological interpretability at the phenotypic level, we developed the Single-Cell and Tissue Phenotype prediction (SCTP), a deep-learning-based multimodal fusion framework. SCTP can simultaneously detect phenotype-specific cells and characterize the tumor microenvironment of pathological tissue by integrating essential information from the bulk sample phenotype, the composition of individual cells, and the spatial distribution of cells. Upon evaluating SCTPs efficiency and robustness against traditional analytical methods, we developed a specialized model, SCTP-CRC, tailored for colorectal cancer (CRC). This model integrates RNA-seq, scRNA-seq, and spatial transcriptomic data to offer a better understanding of CRC. SCTP-CRC has proven effective in accurately identifying tumor-associated cells and clusters and continuously defines boundary regions as well as the spatial organization of the entire tumor microenvironment. This enables a detailed depiction of cellular communication networks, capturing the dynamic shifts that occur during tumor progression. Furthermore, SCTP-CRC extends to the identification of abnormal sub-regions in the early state of CRC and uncovers potential early-warning signature genes such as MMP2, IGKC, and PIGR. These biomarkers are not only important in recognizing the onset of CRC but may also play a crucial role in differentiating between CRC-derived liver metastases and primary liver tumors. SCTP stands as a transformative framework, offering a deeper understanding of the tumor microenvironment through its ability to quantitatively characterize cancers fundamental traits and dissect the intricate molecular and cellular interactions at play. This comprehensive insight supports the early diagnosis and enables personalized treatment strategies, marking a significant stride toward improving patient outcomes and tailoring therapies to individual disease profiles.

bioinformatics↗

bioRxiv2024

Decoding cell identity with multi-scale explainable deep learning

Cells are the fundamental structural and functional units of life. Studying the definition and composition of different cell types can help us understand the complex mechanisms underlying biological diversity and functionality. The increasing volume of extensive single-cell omics data makes it possible to provide detailed characterisations of cell types. Recently, there has been a rise in deep learning-based approaches that generate cell type labels solely through mapping query data to reference data. However, these approaches lack multi-scale descriptions and interpretations of identified cell types. Here, we propose Cell Decoder, a biological prior knowledge informed model to achieve multi-scale representation of cells. We implemented automated machine learning and post-hoc analysis techniques to decode cell identity. We have shown that Cell Decoder compares favourably to existing methods, offering multi-view interpretability for decoding cell identity and data integration. Furthermore, we have showcased its applicability in uncovering novel cell types and states in both human bone and mouse embryonic contexts, thereby revealing the multi-scale heterogeneity inherent in cell identities.

bioinformatics↗

bioRxiv2023

A spectrum of altered non-rapid eye movement sleep in schizophrenia

BackgroundMultiple facets of sleep neurophysiology, including electroencephalography (EEG) metrics such as non-rapid eye movement (NREM) spindles and slow oscillations (SO), are altered in individuals with schizophrenia (SCZ). However, beyond group-level analyses which treat all patients as a unitary set, the extent to which NREM deficits vary among patients is unclear, as are their relationships to other sources of heterogeneity including clinical factors, illness duration and ageing, cognitive profiles and medication regimens. Using newly collected high density sleep EEG data on 103 individuals with SCZ and 68 controls, we first sought to replicate our previously reported (Kozhemiako et. al, 2022) group-level mean differences between patients and controls (original N=130). Then in the combined sample (N=301 including 175 patients), we characterized patient-to-patient variability in NREM neurophysiology. ResultsWe replicated all group-level mean differences and confirmed the high accuracy of our predictive model (Area Under the ROC Curve, AUC = 0.93 for diagnosis). Compared to controls, patients showed significantly increased between-individual variability across many (26%) sleep metrics, with patterns only partially recapitulating those for group-level mean differences. Although multiple clinical and cognitive factors were associated with NREM metrics including spindle density, collectively they did not account for much of the general increase in patient-to-patient variability. Medication regimen was a greater (albeit still partial) contributor to variability, although original group mean differences persisted after controlling for medications. Some sleep metrics including fast spindle density showed exaggerated age-related effects in SCZ, and patients exhibited older predicted biological ages based on an independent model of ageing and the sleep EEG. ConclusionWe demonstrated robust and replicable alterations in sleep neurophysiology in individuals with SCZ and highlighted distinct patterns of effects contrasting between-group means versus within-group variances. We further documented and controlled for a major effect of medication use, and pointed to greater age-related change in NREM sleep in patients. That increased NREM heterogeneity was not explained by standard clinical or cognitive patient assessments suggests the sleep EEG provides novel, nonredundant information to support the goals of personalized medicine. Collectively, our results point to a spectrum of NREM sleep deficits among SCZ patients that can be measured objectively and at scale, and that may offer a unique window on the etiological and genetic diversity that underlies SCZ risk, treatment response and prognosis.

neuroscience↗

bioRxiv2023

The RNA-binding protein RbpB is a central regulator of polysaccharide utilization in gut Bacteroides

Paramount to human health, symbiotic bacteria in the gastrointestinal tract rely on the breakdown of complex polysaccharides to thrive in this sugar-deprived environment. Gut Bacteroides are metabolic generalists and deploy dozens of polysaccharide utilization loci (PULs) to forage diverse dietary and host-derived glycans. The expression of the multi-protein PUL complexes is tightly regulated at the transcriptional level. However, how PULs are orchestrated at translational level in response to the fluctuating levels of their cognate substrates is unknown. Here, we identify the RNA-binding protein RbpB and a family of noncoding RNAs as key players in post-transcriptional PUL regulation. Ablation of RbpB in Bacteroides thetaiotaomicron displays compromised colonization in the mouse gut in a host diet-dependent manner. Current dogma holds that individual PULs are regulated by dedicated transcriptional regulators. We demonstrate that RbpB acts as a global RNA binder that directly interacts with several hundred cellular transcripts. This includes a paralogous noncoding RNA family comprised of 14 members, the FopS (family of paralogous sRNAs) cluster. Through a series of in-vitro and in-vivo assays, we reveal that FopS sRNAs repress the translation of a SusC-like glycan transporter when substrates are limited--an effect antagonized by RbpB. Together, this study implicates RNA-coordinated metabolic control as an important, yet previously overlooked, factor contributing to the in-vivo fitness of predominant microbiota species in dynamic nutrient landscapes.

microbiology↗

bioRxiv2023

Byproducts of inflammatory radical metabolism provide transient nutrient niches for microbes in the inflamed gut

Louis Pasteurs experiments on tartaric acid laid the foundation for our understanding of molecular chirality, but major questions remain. By comparing the optical activity of naturally-occurring tartaric acid with chemically-synthesized paratartaric acid, Pasteur realized that naturally-occurring tartaric acid contained only L-tartaric acid while paratartaric acid consisted of a racemic mixture of D- and L-tartaric acid. Curiously, D-tartaric acid has no known natural source, yet several gut bacteria specifically degrade D-tartaric acid. Here, we investigated the oxidation of monosaccharides by inflammatory reactive oxygen and nitrogen species. We found that this reaction yields an array of alpha hydroxy carboxylic acids, including tartaric acid isomers. Utilization of inflammation- derived D- and L-tartaric acid enhanced colonization by Salmonella Typhimurium and E. coli in murine models of gut inflammation. Our findings suggest that byproducts of inflammatory radical metabolism, such as tartrate and other alpha hydroxy carboxylic acids, create transient nutrient niches for enteric pathogens and other potentially harmful bacteria. Furthermore, this work illustrates that inflammatory radicals generate a zoo of molecules, some of which may erroneously presumed to be xenobiotics.

microbiology↗

bioRxiv2023

AP-2α/AP-2β transcription factors are key regulators of epidermal homeostasis

AP-2 transcription factors regulate ectodermal development but their roles for epidermal homeostasis in the adult skin are unknown. We find that AP-2 is the predominant AP-2 family member in adult epidermis, followed by AP-2{beta}. Through inactivation of AP-2, AP-2{beta}, or both in keratinocytes we assessed the effects of a gradient of epidermal AP-2 activity on skin function. We find that (1) loss of AP-2{beta} in keratinocytes is compensated for by AP-2, (2) loss of AP-2 impairs terminal keratinocyte differentiation and hair morphogenesis, and (3) the combined loss of AP-2/AP-2{beta} results in more severe skin and hair abnormalities. Keratinocyte differentiation defects precede a progressive neutrophilic skin inflammation. Inducible inactivation of AP-2/AP-2{beta} in the adult phenocopies these manifestations. Transcriptomic analyses of epidermis lacking AP-2 or AP-2/AP-2{beta} in keratinocytes demonstrate a terminal keratinocyte differentiation defect with upregulation of alarmin keratins and of several immune pathway regulators. Moreover, our analyses suggest a key role of loss of AP-2-dependent gene expression of CXCL14 and KRT15 as an early pathogenic event towards the manifestation of skin inflammation. Thus, AP-2/AP-2{beta} are critical regulators of epidermal homeostasis in the adult skin.

cell biology↗

bioRxiv2023

Targeted genome editing restores auditory function in adult mice with progressive hearing loss caused by a human microRNA mutation

Mutations in microRNA-96 (MIR96) cause dominant delayed onset hearing loss DFNA50 without treatment. Genome editing has shown efficacy in hearing recovery by intervention in neonatal mice, yet editing in the adult inner ear is necessary for clinical applications. Here, we developed an editing therapy for a C>A point mutation in the seed region of the Mir96 gene, Mir9614C>A associated with hearing loss by screening gRNAs for genome editors and optimizing Cas9 and sgRNA scaffold for efficient and specific mutation editing in vitro. By AAV delivery in pre-symptomatic (3-week-old) and symptomatic (6-week-old) adult Mir9614C>A mutant mice, hair cell on-target editing significantly improved hearing long-term, with an efficacy inversely correlated with injection age. We achieved transient Cas9 expression without the evidence of AAV genomic integration to significantly reduce the safety concerns associated with editing. We developed an AAV-sgmiR96-master system capable of targeting all known human MIR96 mutations. As mouse and human MIR96 sequences share 100% homology, our approach and sgRNA selection for efficient and specific hair cell editing for long-term hearing recovery lays the foundation for future treatment of DFNA50 caused by MIR96 mutations.

bioengineering↗

bioRxiv2023

Adaptive immunity: from CRISPR to CRIHSP?

Clustered regularly interspaced short palindromic repeats (CRISPR) confer adaptive immunity in prokaryotes against viral infection and plasmid transformation. Here, we identified abundant clustered regularly interspaced homologous stem-loop pairs (CRIHSP) within human adaptive immunity-related genes, such as antigen receptor germline genes. By examining genomic stability under activation-induced cytidine deaminase (AID) overexpression, we found that CRIHSP structures are preferred targets for AID. Through collecting and analyzing cancer cell genomic mutation databases, we discovered that these mutation sites tend to cluster toward stem-loop structures. Importantly, the frequency of stem-loop sequences within immunoglobulin heavy chain variable (IGHV) gene segments was over 3-fold higher compared to that within other regions. We concurrently observed stem-loop structures frequently flanking RSS motifs. Conservative estimates indicate a 74.07% probability of immunoglobulin heavy chain diversity (IGHD) gene segment enclosure within stem-loop. These RSS-proximal stem-loops may assemble into homologous stem-loop pair (HSP)-like intermediates and subsequent CRIHSP arrays. Our results implicate CRIHSP architectures as critical enhancer of antigen receptor gene recombination and diversification. Further molecular dissection of these processes could potentially elucidate mechanisms underlying cancer cell genomic instability, chromatin folding and dynamic regulation, gene expression control, etc.

immunology↗

bioRxiv2023

Cardiac disease diagnosis based on GAN in case of missing data

In daily life, two common algorithms are used for collecting medical disease data: data integration of medical institutions and questionnaires. However, these statistical methods require collecting data from the entire research area, which consumes a significant amount of manpower and material resources. Additionally, data integration is difficult and poses privacy protection challenges, resulting in a large number of missing data in the dataset. The presence of incomplete data significantly reduces the quality of the published data, hindering the timely analysis of data and the generation of reliable knowledge by epidemiologists, public health authorities, and researchers. Consequently, this affects the downstream tasks that rely on this data. To address the issue of discrete missing data in cardiac disease, this paper proposes the AGAN (Attribute Generative Adversarial Nets) architecture for missing data filling, based on generative adversarial networks. This algorithm takes advantage of the strong learning ability of generative adversarial networks. Given the ambiguous meaning of filling data in other network structures, the attribute matrix is designed to directly convert it into the corresponding data type, making the actual meaning of the filling data more evident. Furthermore, the distribution deviation between the generated data and the real data is integrated into the loss function of the generative adversarial networks, improving their training stability and ensuring consistency between the generated data and the real data distribution. This approach establishes the missing data filling mechanism based on the generative adversarial networks, which ensures the rationality of the data distribution while filling the missing data samples. The experimental results demonstrate that compared to other filling algorithms, the data matrix filled by the proposed algorithm in this paper has more evident practical significance, fewer errors, and higher accuracy in downstream classification prediction.

physiology↗

bioRxiv2023

Metabolomics-assisted mechanism analysis of gamma-PGA synthesis regulated by PhoP in B. licheniformis

Poly {gamma}-glutamic acid ({gamma}-PGA) is a widely used biopolymer whose synthesis relies on external nitrogen sources. PhoP is a global transcription factor that has been reported to be involved in regulation of phosphorus and nitrogen metabolisms, whether PhoP regulates {gamma}-PGA synthesis is worthy of further study. In this study, {gamma}-PGA yield was decreased by 19.4% in phoP deletion strain, while PhoP overexpression benefited {gamma}-PGA synthesis in Bacillus licheniformis, and the results of transcriptional level, electrophoretic mobility shift (EMSA) and GFP expression assays confirmed the direct positive regulation on {gamma}-PGA synthetase gene pgsB by PhoP. Furthermore, based on metabolomic and physiological analysis, we dissected three aspects that {gamma}-PGA synthesis indirectly regulated by PhoP. (i) PhoP influences glutamate transport through positively regulating glutamate transporter GltT. (ii) PhoP influences nitrogen source utilization through negatively regulating nitrogen metabolic repressor TnrA and positively regulating GlnR. (iii) PhoP influences ammonia assimilation through GlnR and TnrA. Together, our study improved metabolic regulatory network of {gamma}-PGA synthesis, and laid a foundation for PhoP regulation nitrogen metabolic network in Bacillus.

microbiology↗

bioRxiv2023

Single cell RNA analysis uncovers the cell differentiation trajectories and functionalization for air breathing of frog lung

The evolution and development of vertebrate lungs have received extensive concerns for the significance in terrestrial adaptation. Amphibians possess the most primitive lungs among tetrapods, underscoring the evolutionary importance of lungs in bridging the transition from aquatic to terrestrial life. However, the intricate process of cell differentiation during amphibian lung development is still lacking. Using single cell RNA-seq, we identified 21 cell types in the developing lung of a land-dwelling frog (Microhyla fissipes). We elucidated that single type of alveolar epithelial cells (AECs) existed in amphibian and the diversity of AECs may correspond to the capacity for terrestrial adaptation in tetrapods. Based on pseudotime trajectories analysis, we revealed previously unrecognized developmental-specific transition cell states of epithelial and endothelial cells supporting the rapid morphogenesis of lung during metamorphic climax. We illustrated the cellular and molecular processes during lung functionalization. These findings uncover the cell differentiation trajectories and functionalization for air breathing of frog lungs and highlight the evolutionary peculiarity of the primitive lungs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/549571v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@d3188aorg.highwire.dtl.DTLVardef@f049feorg.highwire.dtl.DTLVardef@189f98borg.highwire.dtl.DTLVardef@15e3857_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Iron acquisition by a commensal bacterium modifies host nutritional immunity during Salmonella infection

During intestinal inflammation, host nutritional immunity starves microbes of essential micronutrients such as iron. Pathogens scavenge iron using siderophores, which is counteracted by the host using lipocalin-2, a protein that sequesters iron-laden siderophores, including enterobactin. Although the host and pathogens compete for iron in the presence of gut commensal bacteria, the roles of commensals in nutritional immunity involving iron remain unexplored. Here, we report that the gut commensal Bacteroides thetaiotaomicron acquires iron in the inflamed gut by utilizing siderophores produced by other bacteria including Salmonella, via a secreted siderophore-binding lipoprotein termed XusB. Notably, XusB-bound siderophores are less accessible to host sequestration by lipocalin-2 but can be "re-acquired" by Salmonella, allowing the pathogen to evade nutritional immunity. As the host and pathogen have been the focus of studies of nutritional immunity, this work adds commensal iron metabolism as a previously unrecognized mechanism modulating the interactions between pathogen and host nutritional immunity.

microbiology↗

bioRxiv2023

GDF15 is a Critical Renostat in the Defense Against Hypoglycemia

Episodic hypoglycemia is one of the best honed, evolutionary conserved phenomena in biology, because of the constant feast-fast cycles that have characterized most of history. The counterregulatory response to hypoglycemia, mobilizing substrate stores to produce glucose, is the primary adaptive mechanism to enable survival. Catecholamines and glucagon have long been considered the key hypoglycemia counterregulatory hormones, but here we identify a new hypoglycemia counterregulatory factor. We employed the insulin tolerance test (ITT) and hyperinsulinemic-hypoglycemic clamp to mimic the two common settings in which hypoglycemia can occur in patients: postprandial insulin overdose and elevated basal insulin administration, respectively. We found that Growth Differentiation Factor 15 (GDF15) production is induced in the S3 segment of the renal proximal tubules and its release increases hepatic gluconeogenesis by increasing intrahepatic lipolysis in a beta-adrenergic receptor-2 (Adrb2)-dependent manner. In addition, mice exposed to recurrent hypoglycemia and patients with T1D exhibit impaired GDF15 production in the setting of hypoglycemia. These data demonstrate that GDF15 acts acutely as a gluco-counterregulatory hormone and identify a critical role for kidney-derived GDF15 in glucose homeostasis under physiological and pathophysiological conditions.

physiology↗

bioRxiv2023

Soil intake modifies the gut microbiota and alleviates ovalbumin-induced mice asthma inflammation

BackgroundLow cleanliness living environment (LCLE) can increase gut microbial diversity and prevent allergic diseases, whereas gut microbial dysbiosis is closely related to the pathogenesis of asthma. Our previous studies suggested that soil in the LCLE is a key factor in shaping intestinal microbiota. ObjectiveWe aimed to explore if sterilized soil intake as prebiotics while being incubated with microbes in the air can attenuate mice asthma symptoms by modifying gut microbiota. Methods16S rRNA gene sequencing was used to analyze the gut microbial composition, in combination with immune parameters measured in the lung and serum samples. Results16S rRNA gene sequencing results showed significant differences in the fecal microbiota composition between the test and control mice, with a higher abundance of Allobaculum, Alistipes, and Lachnospiraceae_UCG-001, which produce short-chain fatty acids and are beneficial for health in the test mice. Soil intake downregulated the concentrations of IL-6, IL-4, IL-17F, TNF-, and IL-22 in serum and increased the expression of IFN-{gamma}, which regulated the Th1/Th2 balance in lung by polarizing the immune system toward Th1, strongly alleviating ovalbumin-induced asthma inflammation. The effect of sensitization on gut microbiota was greater than that of air microbes and age together, but weaker than that of soil. ConclusionSoil intake had a significant therapeutic effect on mouse asthma, possibly by promoting the growth of multiple beneficial bacteria. The results indicated that the development of soil-based prebiotic products might be used for allergic asthma management and our study provides further evidence for the hygiene hypothesis. ImportanceExposure to a low cleanliness living environment (LCLE), of which soil is an important component, can shape the gut microbiota and support immune tolerance, preventing allergic diseases such as eczema and asthma. However, with the rapid progress of urbanization, it is impossible to return to farm-like living and we are becoming disconnected from the soil. Here, our study found that ingesting sterilized soil and living in an LCLE have the same protective effects on asthma inflammation. Ingestion of sterilized soil significantly altered the gut microbial composition and exerted significant therapeutic effects on asthmatic mice. However, edible sterilized soil possesses more advantages than LCLE exposure, such as the absence of pathogenic bacteria, safer, and convenience. The results indicate that the development of soil-based prebiotic products might be used for allergic asthma management and our study further supports the hygiene hypothesis. NotificationThe article is currently undergoing peer review in the World Allergy Organization Journal.

microbiology↗

bioRxiv2023

Cryo-EM and Molecular Dynamics Simulations Reveal Hidden Conformational Dynamics Controlling Ammonia Transport in Human Asparagine Synthetase

How motions in enzymes might be linked to catalytic function is of considerable general interest. Advances in X-ray crystallography and cryogenic electron microscopy (cryo-EM) offer the promise of elucidating functionally relevant conformational changes that are not easily studied by other biophysical methods. Here we use 3D variability analysis (3DVA) of the cryo-EM map for wild-type (WT) human asparagine synthetase (ASNS) to identify a functional role for the Arg-142 side chain as a gate that mediates ammonia access to a catalytically relevant intramolecular tunnel. Our 3DVA-derived hypothesis is assessed experimentally, using the R142I variant in which Arg-142 is replaced by isoleucine, and by molecular dynamics (MD) simulations on independent, computational models of the WT human ASNS monomer and its catalytically relevant, ternary complex with {beta}-aspartyl-AMP and MgPPi. Residue fluctuations in the MD trajectories for the human ASNS monomer are consistent with those determined for 3DVA-derived structures. These MD simulations also indicate that the gating function of Arg-142 is separate from the molecular events that form a continuous tunnel linking the two active sites. Experimental support for Arg-142 playing a role in intramolecular ammonia translocation is provided by the glutamine-dependent synthetase activity of the R142 variant relative to WT ASNS. MD simulations of computational models for the R142I variant and the R142I/{beta}-aspartyl-AMP/MgPPi ternary complex provide a possible molecular basis for this observation. Overall, the combination of 3DVA with MD simulations is a generally applicable approach to generate testable hypotheses of how conformational changes in buried side chains might regulate function in enzymes.

molecular biology↗

bioRxiv2023

Endothelial-specific reduction in Arf6 impairs insulin-stimulated vasodilation and skeletal muscle blood flow resulting in systemic insulin resistance

BackgroundMuch of what we know about insulin resistance is based on studies from metabolically active tissues such as liver, adipose tissue, and skeletal muscle. Emerging evidence suggests that the vascular endothelium plays a crucial role in systemic insulin resistance, however, the underlying mechanisms remain incompletely understood. ADP ribosylation factor 6 (Arf6) is a small GTPase that plays a critical role in endothelial cell (EC) function. Here, we tested the hypothesis that the deletion of endothelial Arf6 will result in systemic insulin resistance. MethodsWe used mouse models of constitutive EC-specific Arf6 deletion (Arf6f/- Tie2Cre) and tamoxifen inducible Arf6 knockout (Arf6f/f Cdh5Cre). Endothelium-dependent vasodilation was assessed using pressure myography. Metabolic function was assessed using a battery of metabolic assessments including glucose- and insulin-tolerance tests and hyperinsulinemic-euglycemic clamps. A fluorescence microsphere-based technique was used to measure tissue blood flow. Intravital microscopy was used to assess skeletal muscle capillary density. ResultsEndothelial Arf6 deletion impaired insulin-stimulated vasodilation in white adipose tissue (WAT) and skeletal muscle feed arteries. The impairment in vasodilation was primarily due to attenuated insulin-stimulated nitric oxide (NO) bioavailability but independent of altered acetylcholine- or sodium nitroprusside-mediated vasodilation. In vitro Arf6 inhibition resulted in suppressed insulin stimulated phosphorylation of Akt and endothelial NO synthase. Endothelial cell-specific deletion of Arf6 also resulted in systematic insulin resistance in normal chow fed mice and glucose intolerance in high fat diet fed obese mice. The underlying mechanisms of glucose intolerance were reductions in insulin-stimulated blood flow and glucose uptake in the skeletal muscle and were independent of changes in capillary density or vascular permeability. ConclusionResults from this study support the conclusion that endothelial Arf6 signaling is essential for maintaining insulin sensitivity. Reduced expression of endothelial Arf6 impairs insulin-mediated vasodilation and results in systemic insulin resistance. These results have therapeutic implications for diseases that are associated with endothelial cell dysfunction and insulin resistance such as diabetes.

physiology↗