Biology subjects

Guy, C. S.

Publications and source records attributed to Guy, C. S..

8 recordsLinked to original sources

bioRxiv2025

Suppression of non-canonical autophagy induces endothelial and cardiac dysfunction

BackgroundWhile roles for canonical autophagy in the pathophysiology of cardiovascular disease have been established, we have limited understanding of the non-canonical functions of autophagy proteins in this context. LC3-asssociated endocytosis (LANDO) is a novel non-canonical function of autophagy proteins, in which LC3 (microtubule-associated protein light chain 3) is conjugated to early endosome membranes using a portion of the canonical autophagy machinery, and functions in the endocytic recycling of several plasma membrane proteins. Here we ask whether perturbation of LANDO can promote cardiovascular pathogenesis. MethodsCardiac and endothelial functions were assessed by echocardiography and flow-mediated dilatation in mice lacking Rubicon (Rubcn-/-) or the WD domain of ATG16L1 (Atg16l1{Delta}WDki), two known effectors of LANDO. Mice with conditional depletion of Rubicon in the endothelial, myeloid and cardiomyocyte compartments were used as well. Three-dimensional murine cardiac vasculature leakiness was investigated by light sheet fluorescence microcopy. Endothelial activation induced by shear stress was characterized in vitro in primary endothelial cells isolated from murine lungs and human aortic endothelial cells. Associations between genetically predicted expression of candidate genes involved in LANDO and human cardiovascular parameters were studied in the Young Finns Study and the UK Biobank. ResultsCompared to littermate controls, young Rubcn-/- and Atg16l1{Delta}WDki mice showed a decrease in cardiac and endothelial functions, as did mice with endothelium-specific deficiency. VEGFR2 recycling to the plasma membrane and nitric oxide pathway during shear stress were disrupted in LANDO-deficient primary murine and human endothelial cells. Proteomic analysis in primary human aortic endothelial cells revealed an upregulation of intracellular hemoglobin subunit alpha (Hb-) upon shear stress, which was blunted when RUBCN was ablated. Genetic expression studies uncovered several candidate genes related to LANDO that correlated with cardiovascular parameters. These included the retromer complex subunit VPS29, disruption of which decreased Hb- expression levels in human endothelial cells. ConclusionsOur data support a pivotal role of non-canonical functions of autophagy proteins in recycling VEGFR2 upon shear stress activation in endothelial cells together with Hb- expression that may contribute to the etiology of cardiovascular diseases.

physiology↗

bioRxiv2025

Transfer learning enables discovery of sub-micromolar antibacterials for ESKAPE pathogens from ultra-large chemical spaces

The rise of antimicrobial resistance, especially among gram-negative ESKAPE pathogens, presents an urgent global health threat. However, the discovery of new antibiotics is hampered by sparse publicly available antibacterial data, complex bacterial defenses, and weak economic incentives. Here, we introduce a transfer learning framework using deep graph neural networks (DGNNs) to identify antibacterials from ultra-large chemical libraries. DGNNs were first pre-trained on large molecular datasets of protein-ligand simulations, binding affinities, and physicochemical properties to learn generalizable chemical features, and then fine-tuned on limited antibacterial screening data. Compared to classical methods, transfer learning significantly improved enrichment factors and predictive performance in cross-dataset benchmarks. Applying this strategy to the ChemDiv and Enamine libraries, we virtually screened over a billion compounds and prioritized 156 candidates. Experimental testing against Escherichia coli revealed that 54% of compounds exhibited antibacterial activity (MIC [≤]64 {micro}g/ml), with several demonstrating sub-micromolar potency and broad-spectrum efficacy against gram-positive and gram-negative pathogens, including three ESKAPE species. Of 18 broad-spectrum candidates, 15 showed minimal cytotoxicity and no hemolytic activity. These results validate our approach for navigating underexplored chemical space and identifying potent, low-toxicity compounds with antibiotic activity. We release open-source models and a scalable workflow to accelerate antibacterial discovery in the face of data scarcity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/669197v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@d1e793org.highwire.dtl.DTLVardef@ba6c85org.highwire.dtl.DTLVardef@12ab303org.highwire.dtl.DTLVardef@4ea019_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

Fluorinated trehalose analogues for cell surface engineering and imaging of Mycobacterium tuberculosis

The sensitive, rapid and accurate diagnosis of Mycobacterium tuberculosis (Mtb) infection is a central challenge in controlling the global tuberculosis (TB) pandemic. Yet the detection of mycobacteria is often made difficult by the low sensitivity of current diagnostic tools, with over 3.6 million TB cases missed each year. To overcome these limitations there is an urgent need for next-generation TB diagnostic technologies. Here we report the use of a discrete panel of native 19F-trehalose (F-Tre) analogues to label and directly visualise Mtb by exploiting the uptake of fluorine-modified trehalose analogues via the mycobacterial trehalose LpqY-SugABC ATP-binding cassette (ABC) importer. We discovered the extent of modified F-Tre uptake correlates with LpqY substrate recognition and characterisation of the interacting sites by saturation transfer difference NMR coupled with molecular dynamics provides a unique glimpse into the molecular basis of fluorine-modified trehalose import in Mtb. Lipid profiling demonstrated that F-Tre analogues modified at positions 2, 3 and 6 are incorporated into mycobacterial cell-surface trehalose-containing glycolipids. This rapid one-step labelling approach facilitates the direct visualisation of F-Tre-labelled Mtb by focused ion beam (FIB) secondary ion mass spectrometry (SIMS), enabling pathogen specific detection. Collectively, our findings highlight that F-Tre analogues have potential as tools to probe and unravel Mtb biology and can be exploited to detect and image TB.

microbiology↗

bioRxiv2023

Phosphatidylserine clustering by membrane receptors triggers LC3-associated phagocytosis.

LC3-associated phagocytosis (LAP) represents a non-canonical function of autophagy proteins in which ATG8 family proteins (LC3 and GABARAP proteins) are lipidated onto single-membrane phagosomes as particles are engulfed by phagocytic cells1-4. LAP plays roles in innate immunity5, inflammation and anti-cancer6 responses and is initiated upon phagocytosis of particles that stimulate Toll-like receptors (TLR), Fc-receptors, and upon engulfment of dying cells6. However, how this molecular route is initiated remains elusive. Here we report that receptors that engage LAP enrich phosphatidylserine (PS) in the phagosome membrane via membrane-proximal domains that are necessary and sufficient for LAP to proceed. Subsequently, PS recruits the Rubicon-containing PI3-kinase complex to initiate the enzymatic cascade leading to LAP. Manipulation of plasma membrane PS content, PS-binding by Rubicon, or the PS-clustering domains of receptors prevents LAP and phagosome maturation. We found that pharmacologic inhibition of PS clustering promotes the ability of dendritic cells to induce anti-cancer responses to engulfed tumor cells. Therefore, the initiation of LAP represents a novel mechanism of PS-mediated signal transduction upon ligation of surface receptors.

cell biology↗

bioRxiv2022

Anti-apoptotic BH3-only proteins inhibit Bak

Bcl-2 family proteins regulate induction of intrinsic apoptosis through initiating mitochondrial outer membrane permeabilization (MOMP). Activation of the MOMP effectors Bax and Bak is controlled by interplay levels of anti-apoptotic Bcl-2 proteins (e.g. Mcl-1) and pro-apoptotic BH3-only proteins (e.g. BIM). Using a genome-wide CRISPR-dCas9 transactivation screen we identified two Bcl-2 family proteins, BNIP5 and Bcl-G, as inhibitors of Bak, but not Bax induced apoptosis. BNIP5 was able to block Bak activation in different cell types and in response to various cytotoxic therapies. The BH3 domain of BNIP5 was both necessary and sufficient to block Bak activation. Mechanistically, the BH3 domains of BNIP5 and Bcl-G act as a selective Bak activators, while not inhibiting anti-apoptotic proteins. This led to increased binding of activated Bak to Mcl-1, which prevented apoptosis engagement, identifying BNIP5 and Bcl-G as anti-apoptotic BH3-only proteins.

cell biology↗

bioRxiv2021

Neuroblastoma formation requires unconventional CD4 T cells and myeloid amino acid metabolism

By mirroring their function as tissue repair organizers in normal tissues, immune cells regulate tumor growth. To understand the different facets of immune-tumor collaboration through genetics, spatial transcriptomics, and immunological manipulation with non-invasive, longitudinal imaging, we generated a penetrant double oncogene-driven autochthonous model of neuroblastoma. Using spatial transcriptomic analysis, we co-localized CD4+ and myeloid populations within the tumor parenchyma, while CD8+ T cells and B cells were peripherally dispersed. Depletion of CD4+ T cells or CCR2+ macrophages, but not B cells, CD8+, or NK cells, prevented tumor formation. Tumor CD4+ T cells displayed unconventional phenotypes, were clonotypically diverse, and antigen-independent. Within the myeloid fraction, tumor growth required myeloid cells expressing Arginase-1. Overall, our results suggest that arginine-metabolizing myeloid cells conspire with pathogenic CD4+ T cells to create permissive conditions for tumor formation, and therefore suggest that these pro-tumorigenic pathways can be disabled by targeting myeloid amino acid metabolism.

cancer biology↗

bioRxiv2019

Structural basis of glycerophosphodiester recognition by the Mycobacterium tuberculosis substrate-binding protein UgpB

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB) and has evolved an incredible ability to survive latently within the human host for decades. The Mtb pathogen encodes for a low number of ATP-binding cassette (ABC) importers for the acquisition of carbohydrates that may reflect the nutrient poor environment within the host macrophages. Mtb UgpB (Rv2833) is the substrate binding domain of the UgpABCE transporter that recognises glycerophosphocholine (GPC) indicating a potential role in glycerophospholipid recycling. By using a combination of saturation transfer difference (STD) NMR and X-ray crystallography we report the structural analysis of Mtb UgpB complexed with GPC and have identified that Mtb UgpB is promiscuous for other glycerophosphodiesters. Complementary biochemical analyses and site-directed mutagenesis define the molecular basis and specificity of glycerophosphodiester recognition. Our results provide critical insights into the structural and functional role of the Mtb UgpB transporter and reveal that the specificity of this ABC-transporter is not limited to GPC therefore optimising the ability of Mtb to scavenge scarce nutrients and essential glycerophospholipid metabolites during intracellular infection.

biochemistry↗

bioRxiv2019

Targeting extracellular glycans: Tuning multimeric boronic acids for pathogen-selective killing of Mycobacterium tuberculosis

Innovative chemotherapeutic agents that are active against Mycobacterium tuberculosis (Mtb) are urgently required to control the tuberculosis (TB) epidemic. The Mtb cell envelope has distinct (lipo)polysaccharides and glycolipids that play a critical role in Mtb survival and pathogenesis and disruption of pathways involved in the assembly of the Mtb cell envelope are the primary target of anti-tubercular agents. Here we introduce a previously unexplored approach whereby chemical agents directly target the extracellular glycans within the unique Mtb cell envelope, rather than the intracellular biosynthetic machinery. We designed and synthesised multimeric boronic acids that are selectivity lethal to Mtb and function by targeting these structurally unique and essential Mtb cell envelope glycans. By tuning the number of, and distance between, boronic acid units high selectivity to Mtb, low cytotoxicity against mammalian cells and no observable resistance was achieved. This non-conventional approach may prevent the development of drug-resistance and will act as a platform for the design of improved, pathogen-specific, next generation antibiotics.

biochemistry↗