Biology subjects

Kumar, G.

Publications and source records attributed to Kumar, G..

At least 19 recordsLinked to original sources

bioRxiv2026

Longitudinal single-cell transcriptional profiling identifies dynamic neurodegenerative mechanisms in Christianson Syndrome rat

Endolysosomal dysfunction is a central feature of many neurodegenerative disorders. Loss-of-function mutations in the endosomal Na+/H+ exchanger NHE6 cause lysosomal dysfunction and the early-onset neurodegenerative disorder Christianson syndrome (CS). CS shares pathological features with more common neurodegenerative diseases; however, the extent of mechanistic overlap remains unclear. Using single-nucleus RNA sequencing across the lifespan in a CS rat model, we define the temporal progression of disease at single-cell resolution in the hippocampus. Neurons exhibit early transcriptional signatures of mitochondrial dysfunction. We investigated mitochondrial function in vitro and ex vivo and discover progressive defects in mitochondrial quality control and bioenergetics in NHE6-null tissue. Furthermore, both transcriptomic and proteomic analyses reveal robust activation of innate immunity, including cGAS-STING signaling which localizes to microglia. Therefore, in CS rat brain, early neuronal endosomal and mitochondrial impairments are followed by pronounced microglial and astrocytic activation, as well as age-dependent decline in oligodendrocyte myelinating capacity. Cross species comparison reveals substantial overlap between single-cell analyses in CS rat and in human postmortem Alzheimers disease hippocampus. Together, our findings define the cellular trajectories of neurodegeneration in CS, and identify convergent endolysosomal, mitochondrial, and inflammatory mechanisms that support a broader role for endolysosomal dysfunction in Alzheimers disease pathogenesis.

neuroscience↗

bioRxiv2026

Hepatocyte UBE4A protects against MASLD/MASH by maintaining ER homeostasis via UBXN4 interaction

Molecular understanding of the regulation of endoplasmic reticulum (ER) functions in healthy and MASLD livers is limited. Here, we elucidate that Ube4A, a cytosolic E3 ubiquitin ligase, protects against MASLD development by maintaining ER homeostasis during nutritional stress. Hepatocyte-Ube4A deletion (HUKO) augments Gubra-Amylin-diet (GAN-D)-induced steatosis, inflammation, insulin resistance, ER stress and markers of steatohepatitis of the liver in both sexes of mice. Proteomic studies reveal that in addition to altered metabolic and fibrogenic pathways, levels of many ER proteins are also changed in HUKO livers. Impaired ER functions sensitize HUKO livers to nutrition- or chemical (tunicamycin)-induced ER stress. Moreover, HUKO primary hepatocytes exhibit increased ER stress. Furthermore, Ube4A maintains ER homeostasis by facilitating ER-associated protein degradation (ERAD). The lipogenic protein SCD1 is identified as one of the ERAD targets of Ube4A. SCD1 stability and triglyceride accumulation are amplified in HUKO primary hepatocytes. The liver Ube4A-interactome studies identified the ERAD mediator UBXN4 as a major Ube4A interacting protein. Alphafold3-based molecular modelling and interaction site mapping analyses pinpointed the Ube4A binding residues for UBXN4. ER-localized Ube4A-UBXN4 complex facilitates ERAD (e.g., SCD1) and UBXN4-binding deficient Ube4A mutants fail to enhance this process. Finally, Ube4A and UBXN4 are downregulated in the livers of human MASLD and MASH patients and this complex is diminished in GAN-D-fed mouse livers. In conclusion, Ube4A preserves ER homeostasis by maintaining ERAD via its interaction with UBXN4, which maintains liver health. Enhancing levels and/or preserving interaction of these proteins may offer new therapeutic strategies for MASLD treatment.

physiology↗

bioRxiv2026

Natural Compound-Mediated SUMOylation Rewires Host Immunity Against Klebsiella pneumoniae

Klebsiella pneumoniae is a major cause of multidrug-resistant and hypervirulent infections worldwide, highlighting the urgent need for host-directed therapeutic strategies that complement conventional antibiotics. Emerging evidence suggests that K. pneumoniae promotes intracellular persistence by suppressing host SUMOylation, a reversible post-translational modification that regulates protein stability, cellular signaling, and innate immune response. This decrease in SUMOylation perturbs NF-{kappa}B signaling, leading to dysregulation of inflammation that promotes bacterial survival, rather than effective immune clearance. Clove Bud Oil (CBO), a natural product derived from Syzygium aromaticum, has previously been shown to possess anti-virulence as well as immunomodulatory properties. In this regard, we investigated the capability of CBO to restore host SUMOylation and enhance innate immune response during infection with classical as well as hypervirulent K. pneumoniae. In macrophages, classical and hypervirulent K. pneumoniae impaired global host SUMOylation, disrupting IKK{beta}-RelA/NF-{kappa}B signalling and uncoupling inflammatory regulation from effective antimicrobial defense to facilitate intracellular persistence. CBO restored SUMOylation-dependent immune homeostasis by attenuating sustained IKK{beta} phosphorylation and degradation, thereby constraining aberrant RelA nuclear translocation, rebalancing pro-inflammatory cytokine responses, and restricting intracellular bacterial survival. These findings demonstrate that restoration of host SUMOylation acts as a key driver of the immunomodulatory activity of CBO, leading to coordinated innate immune responses and enhanced intracellular bacterial clearance. Collectively, our study identifies host SUMOylation as a critical pathway targeted during K. pneumoniae infection and highlights restoration of SUMOylation-mediated immune homeostasis as a promising host-directed therapeutic strategy against multidrug-resistant and hypervirulent K. pneumoniae.

immunology↗

bioRxiv2026

Microbial diversity and insights into feeding behavior of the two-spot cotton leaf hopper (Amrasca biguttula)

The recent invasion of the two-spot cotton leaf hopper (TSCL) (Amrasca biguttula) into the U.S. now threatens row and vegetable crops as well as ornamentals. TSCL ecology and biology, including microbial diversity, are understudied. Insect-specific microbes could influence host biology and ecology including insect-plant interactions. Here, microbiota (bacteria and viruses) within TSCL along with its feeding behavior were characterized using high throughput sequencing, microscopy, and Electrical penetration graph (EPG). Results revealed low diversity of bacteria dominated by Wolbachia and confirm the absence of obligate (primary) symbionts. Two distinct and differentially abundant Wolbachia strains (B-supergroup) were found co-infecting midgut tissues of 60% of TSCL tested, and the remaining 40% were free of symbionts. Virome analysis uncovered a diverse complex of at least eight dsDNA genomes (12-17 kb) of Adintoviruses (Eupolintoviridae), but no RNA viruses commonly found in other leafhoppers were identified in TSCL. The mesophyll feeding nature of TSCL was identified by observing chlorophyll derived autofluorescence in midguts and higher abundance of chloroplast transcripts (photosystem I, II, Rubisco, cytochrome b6 and ATP synthase complex) in TSCL (whole insects) compared with thrips and aphids. EPG recordings show a cell-rupture style of feeding with differences between adults and nymphs. Lastly, consistent with its cell-rupturing feeding style, no evidence for the presence of plant-pathogenic bacteria or viruses that could induce hopperburn was found. These results overall provide insights into the microbial diversity and feeding pattern of TSCL and lay a foundation for future research. Key messageThis work characterizes the TSCL microbiome and its mesophyll feeding pattern. Symbionts within insects often complement the nutritional deficiencies of its host and can aid insect spread by boosting immunity. The findings of this study lay the foundation for future research on the biology, ecology, and management of this invasive pest.

microbiology↗

bioRxiv2026

Olfactory Search Behavior Across Flow Regimes Supports a Unifying Framework

How animals perform odor-guided search across the broad range of dynamic flow conditions they encounter in nature is not well understood. Prior work treats established strategies such as circling and zigzagging as discrete, context-dependent behaviors. Here, we propose a parsimonious mathematical framework that unites these motifs into a continuum. We show that free flying Drosophila exhibit this continuum by smoothly reshaping search behavior between circling and zigzagging across unsteady flow, still air, and a range of wind speeds. Strikingly, a flow-invariant rhythmic course progression underlies this behavioral spectrum. Available data from moths and sharks across different flow regimes suggest a similarly preserved rhythm, while the rate of this rhythm varies across taxa. Together, these results support a common conceptual basis for olfactory search behavior and explain how search geometry can adapt automatically to local flow.

animal behavior and cognition↗

bioRxiv2026

Rapid coordination of followership and leadership roles in homing pigeons navigating with unfamiliar partners

Collective movement requires coordination between individuals, yet how this emerges during early interactions remains poorly understood. We investigated how partner familiarity influences coordination, leader-follower dynamics, and learning in homing pigeon pairs navigating from novel sites. Birds were released repeatedly with either familiar or unfamiliar partners, followed by solo releases to assess learning. By quantifying bidirectional information flow, we found familiarity influenced information-transfer dynamics during the first release: familiar pairs exhibited more asymmetric information transfer, likely reflecting established leader-follower relationships, whereas unfamiliar pairs showed more symmetric exchange. These differences disappeared after one release. Conversely, familiarity had little effect on cohesion or navigational performance. There was some evidence for an influence on learning: birds from familiar pairings had higher homing efficiency on a subsequent solo release. Finally, across partnerships, followership was more predictable than leadership with respect to individual identity and flight speed, indicating stable variation in individuals tendency to follow rather than lead. This suggests that a shift in emphasis from leadership to followership might enhance our understanding of collective decision-making dynamics. Our results demonstrate how flight partners rapidly coordinate, producing limited downstream effects on navigation and learning, with implications for many animals that travel in fission-fusion transitory collectives.

animal behavior and cognition↗

bioRxiv2026

Genome-wide protein-protein interaction analysis between aquaporins and harpin (HrpZ2) through molecular docking and MD simulations to unravel its role in growth and stress management in tomato.

HrpZ2, a harpin protein produced by Pseudomonas syringae, a gram-negative plant pathogenic bacterium, elicits hypersensitive response and pathogen defense in non-host plants. Harpins from various bacterial sources elicit varying responses in different non-host plants, due to its structural variations, their precise mechanisms of action are not yet completely understood. As per previous reports, harpins from diverse bacterial sources interact with distinctive members of integral membrane proteins, known as aquaporins. For example, harpin (Hpa1Xoo) interacts with OsPIP1;3 in rice, whereas, in Arabidopsis the harpins Hpa1 and HrpZ interacts with AtPIP1;4 and AtPIP1;3 respectively. Here, we conducted the first genome-wide computational screening of protein-protein interactions between HrpZ2 and all 47 members of tomato aquaporins. Molecular docking identified nine interactors across five subfamilies of aquaporins, with HrpZ2 N-terminal residues mediating these interactions. We validated these via molecular dynamics (MD) simulations, principal component analysis, and free energy landscape analysis, assessing the stability (RMSD, RMSF, radius of gyration), dynamics, and affinity (MM-GMSA). PIP complexes, especially PIP2;1 (-460.46 kcal/mol) and PIP1;7 (-303.82 kcal/mol), exhibited superior stability, compactness, and defined energy minima, confirming PIPs as primary sensors of harpins. Non-PIP aquaporins like TIP1;1 and NIP4;1 showed moderate stability, outperforming weaker interactors (SIP2;1, XIP1;5, XIP1;3). These findings provide robust evidence that HrpZ2 preferentially targets PIPs in tomato, while engaging TIPs and NIPs as auxiliary partners. This multifaceted interaction profile of harpins suggests complex plant-pathogen recognition, modulating aquaporin-mediated cellular responses like growth and stress management in plants.

plant biology↗

bioRxiv2026

Endosome motility controls light-responsive reproductive development and secondary metabolite production in Aspergillus

Filamentous fungi, such as Aspergillus species, use microtubule transport to move early endosomes. Other cargos, such as peroxisomes and mRNAs, "hitchhike" on early endosomes to move throughout the long hyphae of these organisms. In Aspergillus nidulans, peroxisomes hitchhike on early endosomes using the endosomal protein PxdA and the peroxisomal protein AcbdA. The HookA adaptor protein links endosomes to microtubule motors. Here, we set out to explore the physiological functions of peroxisome hitchhiking and endosome motility. Aspergillus nidulans has a complex life cycle that includes asexual and sexual reproduction. A. nidulans and other fungi within the Pezizomycotina subphylum are also notable for the vast number of secondary metabolites they produce. Light and other environmental conditions influence developmental decisions and secondary metabolite production. Here, we found that sexual reproduction is favored in the absence of endosome motility, even in the light, which normally promotes asexual reproduction. RNA sequencing of strains lacking PxdA-marked motile early endosomes showed altered expression of genes involved in development. Unexpectedly, we also observed altered expression of genes involved in secondary metabolism in strains lacking endosome motility and peroxisome hitchhiking. Using mass spectrometry, we found that the loss of endosome motility affected the biosynthesis of secondary metabolites, including sterigmatocystin, a carcinogenic mycotoxin that is a food contaminant. Finally, in a pathogenic species, Aspergillus fumigatus, we found that deletion of its pxdA homolog also significantly altered secondary metabolite production. Our work uncovers an unexpected link between organelle motility, developmental decisions in response to light, and secondary metabolite production in filamentous fungi.

cell biology↗

bioRxiv2026

Sticker number modulates pyrenoid condensate assembly to support algal fitness

The valency of intrinsically disordered proteins underpins liquid-liquid phase separation (LLPS), yet how this parameter shapes condensate function and cellular fitness remains poorly understood. Here we exploit the algal pyrenoid-a minimal, two component LLPS system-to directly link condensate properties to physiological performance. Pyrenoid assembly is driven by a disordered, multivalent Linker protein that binds Rubisco at symmetry-related surface sites, with the number of binding motifs ("stickers") varying across species. Using Chlamydomonas reinhardtii, we systematically tuned sticker number from two to nine and examined effects on Rubisco condensation, pyrenoid architecture and CO2 fixation. Three stickers were sufficient for condensation in vitro, but at least four were required for pyrenoid assembly in vivo. Cryo-electron tomography and single-molecule tracking revealed that increasing sticker number enhances Rubisco packing and mobility, while time-resolved imaging and competition assays demonstrated that sticker number governs the kinetics of pyrenoid formation and determines cellular fitness under fluctuating carbon conditions. Our findings establish sticker number as an evolutionary tuning parameter that balances condensate formation, dynamics, and function, providing a quantitative framework for linking the molecular grammar of phase separation to biological fitness.

cell biology↗

bioRxiv2025

Rete Ovarii Epithelial Cells as an Unappreciated Cell of Origin for Pelvic and Ovarian High-Grade Serous Carcinoma

Over the past two decades, converging clinicopathologic, molecular, and evolutionary evidence has established that pelvic and ovarian high-grade serous carcinoma (HGSC) originates predominantly from tubal-type epithelia rather than the ovarian surface epithelium. Consequently, Fallopian tube secretory epithelial cells are widely recognized as the principal cell of origin for HGSC. However, the female reproductive tract contains additional tubular epithelial networks whose potential contributions to HGSC pathogenesis remain unexplored. Here, we identify the rete ovarii (RO), a complex network of intra- and extra-ovarian tubules located within the ovarian hilus and mesovarium, as an alternative candidate tissue of origin for HGSC. We demonstrate that genetic and genomic alterations in rete ovarii epithelial cells (ROECs) can drive their malignant transformation, giving rise to tumors that closely recapitulate the histologic and molecular characteristics of human HGSC. Spatially resolved single-cell transcriptomic analyses of tumors derived from Brca/Trp53/Pten-deficient ROECs (BTP-RO) reveal distinct invasive and immunosuppressive molecular programs. These findings establish ROECs as a previously unrecognized cell of origin for HGSC, expanding the landscape of potential precursor populations beyond the Fallopian tube. The RO-based HGSC model provides a powerful framework for developing origin-informed prevention strategies, early detection approaches, and targeted therapeutics.

cancer biology↗

bioRxiv2025

Engineering NIR probes to enhance affinity and clinical workflow compatibility for prostate cancer imaging

Positive surgical margins following radical prostatectomy increase the risk of biochemical recurrence and subsequent disease progression. Fluorescence guided surgery (FGS) using targeted contrast agents has shown clinical benefits for several cancer types. However, current prostate cancer targeted imaging probes exhibit long pharmacokinetic (PK) profiles, necessitating extended waiting periods or repeated hospital visits, limiting their integration into standard clinical workflow. To overcome this critical clinical compatibility challenge, we developed an innovative tri-compartment, chemistry-driven probe design strategy. Specifically, we developed a congeneric library of near infrared (NIR) water soluble fluorescent probes incorporating: (1) a glutamic acid-urea-lysine (EuK) ligand targeting prostate specific membrane antigen (PSMA); (2) a NIR heptamethine cyanine fluorophore optimized for enhanced PSMA binding via secondary binding sites interactions; and (3) distinct PK modulators residing outside the PSMA binding pocket to promote rapid off-target tissue clearance. While molecular docking scores, photophysical properties and live-cell staining results showed similar overall performance, probes bearing PK modulators produced stronger tumor-specific fluorescence in vivo than the control lacking a PK modulator. This effort enabled identification of a lead probe with robust tumor targeting and accelerated off-target clearance, providing optimal tumor-specific signal and contrast in a timeframe, fully compatible with robotic-assisted radical prostatectomy (RARP) timelines.

pharmacology and toxicology↗

bioRxiv2025

Targeting the amino acid metabolism in Lung Sarcopenia: A Systems Engineering Approach

Non-small cell lung cancer (NSCLC) is the most prevalent subtype of lung cancer and a leading cause of cancer-related mortality worldwide. Literature evidences indicates a strong association between systemic inflammation, driven by cytokines such as Interleukin-6 (IL-6), and the development of NSCLC-associated sarcopenia. However, the immuno-metabolic underpinnings that link tumor-derived IL-6 signaling to skeletal muscle degradation remain incompletely understood. We developed a comprehensive immuno-metabolic mathematical model to investigate how IL-6 signaling influences branched-chain amino acids (BCAA) metabolism and redox homeostasis in the context of NSCLC-induced sarcopenia by understanding two key causes of sarcopenia which are malnutrition and redox homeostasis. Our model integrates IL-6-mediated activation of the Janus Kinase (JAK)/Signal transducer and activator of transcription 3 (STAT3) pathway, highlighting the differential roles of STAT3 phosphorylation at S727 (mitochondrial electron transport chain (ETC) activation) and Y705 (nuclear translocation and transcriptional regulation of oncogenes Hypoxia-Inducible Factor 1, alpha (HIF-1) and cellular Myc (c-Myc). These transcription factors reprogram tumor metabolism by upregulating glucose and amino acid transporters, including those specific to BCAAs. In skeletal muscle, elevated IL-6 disrupts insulin signaling via Suppressor of cytokine signaling 3 (SOCS3), suppresses Mechanistic Target of Rapamycin Complex 1 (mTORC1) activation, and enhances oxidative stress, collectively contributing to impaired protein synthesis, increased proteolysis, and muscle wasting. BCAA dysregulation further impairs metabolic balance and exacerbates sarcopenia through defective Tricarboxylic Acid Cycle (TCA) input and epigenetic modulation via acetyl coenzyme A (acetyl-CoA) and S-Adenosylmethionine (SAM). Our model reveals a central role for IL-6-driven metabolic rewiring, particularly BCAA utilization and redox imbalance, in promoting NSCLC-induced sarcopenia. These findings underscore the dual impact of IL-6 on tumor progression and systemic muscle degradation, and provide a framework for evaluating therapeutic strategies that target IL-6/STAT3 signaling and amino acid metabolism via STAT3 and acetyl-CoA cross talk to mitigate NSCLC-induced sarcopenia. Authors SummaryLung cancer is the leading cause of cancer-related deaths worldwide, and NSCLC accounts for approximately 85% of all cases. Beyond its direct effects, NSCLC can induce systemic conditions such as sarcopenia which is a condition leading to progressive loss of skeletal muscle mass and function through chronic inflammation. A key player in this process is IL-6, a cytokine overexpressed and secreted by tumor cells and cells in the tumor microenvironment. In this study, we developed a systems-level mathematical model to explore how IL-6 contributes to NSCLC-induced sarcopenia by disrupting the metabolism of BCAAs, which are essential for muscle protein synthesis and energy homeostasis. Our model shows that IL-6 activates the JAK/STAT3 signaling pathway in cancer cells, promoting tumor survival and metabolic reprogramming which may simultaneously impair nutritional uptake in muscle cells. IL-6 expressed by Tumor micro environment (TME) may disrupt BCAA uptake and utilization, inhibit anabolic signaling, and increase oxidative stress in muscle tissue, thereby accelerating muscle atrophy. By highlighting the interlinked roles of IL-6 signaling and BCAA metabolism in tumor progression and metabolic reprogramming by cancer cells, our findings offer new insights into the metabolic basis of NSCLC-induced sarcopenia and potential intervention points for therapy.

systems biology↗

bioRxiv2025

PatternExtract: A facile, scalable pipeline for point pattern generation from spatial imaging data

We present PatternExtract, an open-source pipeline that generates accurate spatial point patterns from RGB pathology images and cell coordinate data without relying on composite channels or proprietary software. Using a novel two-kernel tissue segmentation method combined with automated pixel classification in QuPath, PatternExtract precisely excludes tissue artifacts such as necrosis and blood vessels to define spatial windows. Optimized on 568 diffuse large B-cell lymphoma images and validated on an independent cohort, the pipeline enables robust spatial analyses.

bioinformatics↗

bioRxiv2025

Concurrent maternal stress and THC exposure during pregnancy alters adolescent behavioral outcomes and corticolimbic molecular programs

Cannabis use during pregnancy is increasing, often to alleviate stress and anxiety, yet the long-term effect of prenatal cannabis exposure alone or in combination with psychosocial stress on offspring neurodevelopment or maternal behaviors remains unclear. Here, we developed a translational rodent model combining prenatal {Delta}-tetrahydrocannabinol (THC) exposure with chronic psychosocial stress using the maternal witness defeat stress (MWDS) paradigm. Pregnant C57BL/6 mice were exposed to MWDS from gestational day (GD) 3-12 and received daily subcutaneous THC (2 mg/kg) or vehicle until birth. All exposure groups showed impaired maternal behavior, with negative postnatal outcomes and caregiving, with additive effects observed in the combined exposure group. In adolescence, male and female offspring exhibited exposure-specific behavioral alterations. Prenatal stress and combined exposures led to increased anxiety-like behavior and reduced motivated behavior in both sexes, while THC alone primarily impacted female self-care and social behavior. Transcriptomic profiling of the prefrontal cortex (PFC) and nucleus accumbens (NAc) of adolescent offspring revealed sex- and region-specific gene expression changes across all exposure groups. Prenatal THC-, stress-, and combined exposures each altered distinct molecular pathways related to mitochondrial function, synaptic organization, and glial signaling. Comparative analysis with a perinatal fentanyl model revealed shared transcriptional substrates involved in synaptic signaling and circadian regulation. These findings indicate that THC and stress independently and additively impair maternal behaviors with lasting neurodevelopment signatures in offspring.

neuroscience↗

bioRxiv2025

Nucleus accumbens neuron subtype translatome signatures in socially stress females

Major depressive disorder (MDD) disproportionately affects women, yet the molecular mechanisms underlying female vulnerability remain poorly understood. The nucleus accumbens (NAc), a reward circuitry hub, is central to stress- and depression-related pathology. While NAc medium spiny neuron (MSN) transcriptional adaptations have been characterized in stressed male rodents, much less is known about female adaptations. To address this, we exposed female D1-Cre-RiboTag and A2A-Cre-RiboTag mice to chronic witness defeat stress (CWDS) and classified them as high- or low-social interactors. Ribosome-associated mRNA was isolated from MSN subtypes and analyzed by RNA sequencing, differential gene expression analysis (DEG), and weighted gene co-expression network analysis (WGCNA). Cross-species consensus WGCNA with male mouse and human transcriptomic datasets was performed to identify conserved, sex-specific signatures. We detected 9 differentially expressed genes (DEGs) in D1-MSNs and 630 in A2A-MSNs (FDR < 0.05). In D1-MSNs, DEGs were primarily upregulated in low interactors and enriched for energy homeostasis and cell adhesion. A2A-MSN DEGs included upregulated structural genes and downregulated neurotransmission-related genes. WGCNA identified 9 significant D1- and 5 significant A2A-MSN modules, with the most impacted enriched for PI3K-Akt-mTOR signaling and regulated by Nf1. Consensus analysis revealed a stress-associated, sex- and subtype-specific module enriched for PI3K-Akt-mTOR signaling conserved across mice and humans. These findings reveal female-specific MSN transcriptional adaptations to chronic social stress and implicate PI3K-Akt-mTOR signaling as a convergent molecular pathway. By highlighting MSN subtype-specific vulnerabilities, this work suggests potential therapeutic targets for alleviating stress-induced pathology in women with MDD.

neuroscience↗

bioRxiv2025

Division of labor in cargo and membrane recognition by SNX1-SNX5: Insights from multiscale modelling

Sorting Nexins (SNXs) are a large group of diverse cellular trafficking proteins that play essential roles in membrane remodeling and cargo sorting between organelles. SNX proteins comprise a banana-shaped BAR domain that acts as a curvature-inducing scaffold and a phosphoinositide lipids sensitive Phox-Homology Domain (PXD) that interacts with the membrane to ensure specific and efficient organelle binding. In concert with the larger retromer machinery, these proteins traffic and recycle cargo between the endosomal membrane, trans-Golgi network, and plasma membrane. Interestingly, the SNX1-SNX5 heterodimeric construct forms a part of the newly discovered pathway where cargo sorting and membrane remodeling can take place in a retromer-independent fashion. In this work, we use molecular dynamics and continuum mechanics simulations to understand the features of SNX1-SNX5 heterodimer, especially the molecular determinants at PXDs, which impart organelle membrane specificity and retromer-independent cargo recognition ability to these proteins. Our all-atom molecular dynamics simulations with isolated PXDs and full-length SNX1-SNX5 on bilayers show that SNX1-PXD has robust membrane-binding features that are largely insensitive to single or double mutation of the basic residues on its surface. Comparing the simulation-based binding poses against the recently solved cryo-EM structures of tubular membrane-bound SNX1 homodimer and SNX1-SNX5 heterodimer also provided interesting insights into the association profile of isolated PXDs when they have the freedom to explore different membrane-binding poses. Our protein-protein simulations of SNX5-PXD with the tail region of the CI-MPR transmembrane cargo protein using metadynamics simulations reveal aromatic residue-rich{pi} - {pi} interactions between the two proteins, and a favorable and kinetically accessible binding free energy profile for SNX5. To model the emergent behavior of cargo sequestration and endosomal tube formation by SNX1-SNX5 heterodimer, we also performed Dynamically Triangulated Surface (DTS) based mesoscopic simulations by developing an augmented Helfrich-like continuum-mechanics Hamiltonian to incorporate transmembrane proteins in DTS models. SIGNIFICANCESorting Nexins (SNXs) are a conserved family of endosomal coat proteins that drive cargo-rich tubule formation for receptor transport and recycling within the cell. The cargo-recognition and sorting in these pathways are mediated by the associated retromer complex. SNX1-SNX5 heterodimer is unique to the SNXs family as it has been shown to sequester cargo for trafficking in a retromer-independent manner. The canonical membrane adaptor Phox-homology domain of SNX5 has evolved to carry out this special function. Using molecular dynamics and advanced sampling metadynamics calculations as well as Helfrich-like Hamiltonian-based mesoscopic modelling, we explore the molecular and thermodynamic driving forces that makes the SNX1-SNX5 heterodimeric constructs multifunctional in character.

biophysics↗

bioRxiv2025

TGF-β inhibitor SB431542 suppresses coronavirus replication through multistep inhibition

The COVID-19 pandemic highlighted the critical need for broad-spectrum antivirals with high resistance barriers. Here, we demonstrate that SB431542, a selective TGF-{beta} receptor I (ALK5) inhibitor, exhibits potent antiviral activity against SARS-CoV-2 through unprecedented multitargeted mechanisms. Through comprehensive in vitro, and in silico analyses, we identified that SB431542 directly binds to SARS-CoV-2 ORF3a and disrupt its canonical function in inhibiting autophagosome-lysosome fusion. This interaction restored lysosomal acidification and normalized perinuclear LAMP-1 localization, significantly impairing virion assembly as evidenced by disrupted nucleocapsid-RNA association and reduced intracellular viral titers. Additionally, SB431542 downregulated the CLEAR network genes responsible for lysosomal biogenesis, further restricting viral egress pathways. Our temporal analyses revealed that at later infection stages (36-48 hpi), SARS-CoV-2 exploits TGF-{beta}-induced lysosomal membrane permeabilization (LMP) and apoptosis for viral release--processes effectively inhibited by SB431542 through suppression of GADD45b and BAX expression. These multiple mechanisms resulted in an exceptional EC50 of 515 nM against SARS-CoV-2. In vivo efficacy was demonstrated in embryonated chicken eggs, where SB431542 conferred dose-dependent protection against lethal infectious bronchitis virus (IBV) challenge, with a favourable therapeutic index of 34.54. Remarkably, sequential passaging of SARS-CoV-2 for 50 generations under SB431542 selection pressure failed to generate resistant variants, contrasting sharply with the rapid resistance emergence typical of direct-acting antivirals. These findings establish SB431542 as a promising broad-spectrum coronavirus inhibitor with a unique triple-mechanism approach that simultaneously targets viral entry via TGF-{beta}/Smad modulation, disrupts ORF3a-mediated lysosomal dysfunction affecting assembly, and attenuates TGF-{beta}-induced apoptosis during late-stage infection-- collectively imposing multiple selective constraints that impede escape mutation development. ImportanceThe COVID-19 pandemic highlighted the urgent need for antiviral drugs with high barriers to resistance. This study reveals that SB431542, a drug previously developed to inhibit TGF-{beta} signaling, exhibits remarkable effectiveness against SARS-CoV-2 through an unprecedented triple-mechanism approach. Unlike conventional antivirals that target a single viral component, SB431542 simultaneously disrupts viral entry, assembly, and release by binding to the viral ORF3a protein and modulating host cellular processes. Most importantly, SARS-CoV-2 failed to develop resistance against SB431542 even after 50 generations of exposure--a significant advantage over current therapeutics that quickly lose effectiveness due to viral mutations. Our findings also uncover that coronaviruses exploit both lysosomal dysfunction and programmed cell death to spread efficiently, providing new targets for therapeutic intervention. This research establishes SB431542 as a promising broad-spectrum coronavirus inhibitor and demonstrates the value of targeting host-virus interactions to overcome antiviral resistance.

microbiology↗

bioRxiv2025

DZNep-induced single point mutation (M236I) in poxviral 2'-O-methyltransferase enhances mRNA stability and translation efficiency

Poxviruses encode two highly conserved S-adenosylmethionine (SAM)-dependent methyltransferases for mRNA capping. While D1L facilitates translation via cap-0 formation, the role of VP39-mediated cap-1 modification has been canonically restricted to shield viral mRNA from cytoplasmic pattern-recognition receptors (PRRs). Here, we uncover a novel function of VP39 by demonstrating that prolonged exposure to the SAM cycle inhibitor DZNep selected a virus escape mutant harbouring a point mutation (M236I) in VP39. VP39_mut bypasses S-adenosylhomocysteine (SAH)-mediated feedback inhibition, enhancing 2-O-methylation of viral transcripts and promoting their association with active translation machinery. Consequently, VP39_mut significantly improves viral protein translation, replicative fitness, and yields across species. Notably, VP39_mut-capped in vitro transcribed (IVT) mRNA exhibited [~]2.7-fold higher protein expression compared to VP39_wt, the standard tool for IVT applications. These findings reveal a unique resistance mechanism and provide critical insights into poxvirus mRNA biology while underscoring the potential of optimized VP39 for advancing mRNA-based therapeutics and vaccine production.

microbiology↗