Biology subjects

Garg, A.

Publications and source records attributed to Garg, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Dynamic Disorder in Chlorophyll Aggregation and Light-Harvesting Complex II in the Plant Thylakoid Membrane using Coarse-Grained Simulations

The dynamics of the aggregated light-harvesting complex (LHCII) associated with its antennae pigments can be crucial for a transition between light harvesting and dissipative states pivotal for non-photochemical quenching (NPQ). To this end, aggregation of chlorophyll-a (CLA) without the LHCII and pigment binding LHCII monomers in the plant thylakoid membranes have been investigated using coarse-grained molecular dynamics simulations at 293 K. Both CLA without the LHCII and pigment-binding LHCII monomers dynamically form and break dimers and higher-order aggregates in thylakoids within the simulation time. The contact lifetime and waiting time distributions of CLA dimers exhibit multiple time scales including most populated fast time scales and less populated slow time scales. The survival probability of CLA dimer in the absence of the LHCII follows a non-exponential decay with multiple residence time scales, leading to a time-dependent rate, unlike conventional rate theory. Such non-exponential decay of survival manifests the emergence of dynamic disorder in CLA without the LHCII resulting from the coupling between time scales of dimer formation and higher-order aggregates. The conformational fluctuations of the LHCII known for inter-CLA coupling variation occur on multiple time scales comparable to the LHCII dimer residence time scales leading to less probable but comparable and more probable slower inter-CLA fluctuations. This indicates the dynamic coupling in the LHCII conformations and their aggregates with the antennae pigments can result in dynamic disorder which will be highly relevant for the light-harvesting efficiency and regulation of NPQ. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/638782v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@5fd895org.highwire.dtl.DTLVardef@8456cforg.highwire.dtl.DTLVardef@5f5c69org.highwire.dtl.DTLVardef@abf6d0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

The atypical filament assembly underpins the inflammasome-independent functions of IFI16.

Inflammasomes trigger cell death upon sensing various intracellular maladies. These supra-structures relay upstream signals by sequentially assembling architecturally congruent filaments via their pyrin domains (PYDs). Interferon Inducible Protein 16 (IFI16) is an innate immune sensor that detects dysregulated nucleic acids. Once presumed as an inflammasome receptor due to its PYD, the role of IFI16 has been much more appreciated in other innate immune pathways such as regulating interferon production and viral replication restriction. Here, a cryo-EM structure of the filament assembled by the PYD of IFI16(PYD) shows a helical architecture distinct from inflammasome PYD filaments. In silico Rosetta interaction energy calculations suggest that the helical architecture of the IFI16PYD filament is incompatible with those assembled by the central inflammasome adaptor ASC and its interacting partners. Cellular experiments further support that IFI16 fails to interact with ASC. Together, we provide the structural basis for the inflammasome-independent functions of IFI16.

biophysics↗

bioRxiv2024

Light Harvesting Complex II Resists Non-Bilayer Lipid-Induced Polymorphism in Plant Thylakoid Membranes via Lipid Redistribution

The plant thylakoid membrane hosting the light-harvesting complex (LHCII) is the site of oxygenic photosynthesis. Contrary to the earlier consensus of a protein-driven single lamellar phase of the thylakoid despite containing 40% non-bilayer-forming lipids, recent experiments confirm the polymorphic state of the functional thylakoid. What, then, is the origin of this polymorphism, and what factors control it? The current Letter addresses the question using a total of 617.8 s long coarse-grained simulations of thylakoids with and without LHCII and varying concentrations of non-bilayer lipids using Martini-2.2 and 3.0 at 323 K. The LHCII redistributes the non-bilayer lipids into its annular region, increases the bending modulus and the stalk formation free energy, reduces the non-zero mean curvature propensity, and resists the polymorphism these lipids promote. The thermodynamic trade-off between non-bilayer lipids and LHCII dictates the degree of nanoscopic curvature leading to the polymorphism crucial for non-photochemical quenching under excess heat conditions. Graphical TOC Entry O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/623718v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@7eab4forg.highwire.dtl.DTLVardef@20dd1forg.highwire.dtl.DTLVardef@19558c2org.highwire.dtl.DTLVardef@1f64f52_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Shc1 cooperates with Frs2 and Shp2 to recruit Grb2 in FGF-induced lens development

Fibroblast growth factor (FGF) signaling elicits multiple downstream pathways, most notably the Ras/MAPK cascade facilitated by the adaptor protein Grb2. However, the mechanism by which Grb2 is recruited to the FGF signaling complex remains unresolved. Here we showed that genetic ablation of FGF signaling prevented lens induction by disrupting transcriptional regulation and actin cytoskeletal arrangements, which could be reproduced by deleting the juxtamembrane region of the FGF receptor and rescued by Kras activation. Conversely, mutations affecting the Frs2-binding site on the FGF receptor or the deletion of Frs2 and Shp2 primarily impact later stages of lens vesicle development involving lens fiber cell differentiation. Our study further revealed that the loss of Grb2 abolished MAPK signaling, resulting in a profound arrest of lens development. However, removing Grb2s putative Shp2 dephosphorylation site (Y209) neither produced a detectable phenotype nor impaired MAPK signaling during lens development. Furthermore, the catalytically inactive Shp2 mutation (C459S) only modestly impaired FGF signaling, whereas replacing Shp2s C-terminal phosphorylation sites (Y542/Y580) previously implicated in Grb2 binding only caused placental defects, perinatal lethality, and reduced lacrimal gland branching without impacting lens development, suggesting that Shp2 only partially mediates Grb2 recruitment. In contrast, we observed that FGF signaling is required for the phosphorylation of the Grb2-binding sites on Shc1 and the deletion of Shc1 exacerbates the lens vesicle defect caused by Frs2 and Shp2 deletion. These findings establish Shc1 as a critical collaborator with Frs2 and Shp2 in targeting Grb2 during FGF signaling.

developmental biology↗

bioRxiv2024

The molecular basis of Human FN3K mediated phosphorylation of glycated substrate

Glycation, a non-enzymatic post-translational modification occurring on proteins, can be actively reversed via site-specific phosphorylation of the fructose-lysine moiety by FN3K kinase, to impact the cellular function of target protein. A regulatory axis between FN3K and glycated protein targets has been associated with conditions like diabetes and cancer. However the molecular basis of this relationship has not been explored so far. Here, we determined a series of crystal structures of HsFN3K in apo-state, and in complex with different nucleotide analogs together with a sugar substrate mimic to reveal the features important for its kinase activity and substrate recognition. Additionally, the dynamics in sugar substrate binding during the kinase catalytic cycle provide important mechanistic insights into HsFN3K function. Our structural work provides the molecular basis for rationale small molecule design targeting FN3K.

biochemistry↗

bioRxiv2024

Selective promotion of oligonucleotides in the course of evolution

Evolution shapes the structure and content of genomes, yet the contribution of local sequence composition to variant selection remains poorly understood. While traditional models emphasize protein function or cross-species conservation, we propose that intra-genomic patterns of oligonucleotide (k-mer) frequencies also reflect selective forces. To explore this, we developed kGain score, a metric that quantifies the frequency shift of a k-mer upon single-nucleotide substitution, using the surrounding genomic context as a baseline. We hypothesize that variants arising in high-kGain contexts are more likely to persist due to evolutionary favorability. We validated this hypothesis across multiple systems. In E. coli and S. cerevisiae long-term evolution experiments, we found that fixed, essential, and parallel mutations consistently show elevated kGain scores. This trend held in SARS-CoV-2 variants of concern and in an in-house antibiotic adaptation experiment, where a high-kGain fusA Y515N mutation conferred resistance and maintained fitness when overexpressed, demonstrating a causal link between kGain and adaptive potential. To enable cross-species generalization, we trained a transformer-based neural network regressor on LTEE-derived mutations to predict kGain from sequence alone. The model achieved high correlation in held-out in-domain data (Pearson r = 0.81) and accurately predicted kGain trends in out-of-domain data (Pearson r = 0.82), demonstrating that k-mer-based sequence constraints learned from one genome can be effectively transferred to others. Together, our results establish kGain as a biologically meaningful, scalable metric for probing within-genome sequence selection, offering a complementary lens to existing conservation-based frameworks for understanding evolutionary fitness and variant persistence.

evolutionary biology↗

bioRxiv2024

The structural landscape of Microprocessor Mediated pri-let-7 miRNAs processing

miRNA biogenesis is initiated upon cleavage of a primary miRNA (pri-miRNA) hairpin by the Microprocessor (MP), composed of the Drosha RNase III enzyme and its partner DGCR8. Multiple pri-miRNA sequence motifs affect MP recognition, fidelity, and efficiency. Here, we performed cryo-EM and biochemical studies of several let-7 family pri-miRNAs in complex with human MP. We show that MP has the structural plasticity to accommodate a range of pri-miRNAs. These structures revealed key features of the 5 UG sequence motif, more comprehensively represented as the "fUN" motif. Our analysis explains how cleavage of class-II pri-let-7 members harboring a bulged nucleotide generates a noncanonical precursor with a 1-nt 3 overhang. Finally, the MP-SRSF3-pri-let-7f1 structure reveals how SRSF3 contributes to MP fidelity by interacting with the CNNC-motif and Droshas PAZ-like domain. Overall, this study sheds light on the mechanisms for flexible recognition, accurate cleavage, and regulated processing of different pri-miRNAs by MP.

biochemistry↗

bioRxiv2024

Identification and Characterization of Novel Metagenome Assembled Uncultivated Virus Genomes from Human Gut

Metagenomics has revealed an unprecedented viral diversity in human gut although, most of the sequence data remains to be characterized. In this study, we mined a collection of 1090 metagenome assembled "high quality" genomes of human gut viruses. Sequence analysis has revealed eight new species from seven genera of the class, Caudoviricetes and nineteen new species from fourteen genera of the ssDNA virus family, Microviridae. In addition, four "high quality" genomes were identified, which do not show similarity to sequences present in any of the four major viral databases, NCBI viral RefSeq, IMG-VR, Gut Phage Database (GPD) and Gut Virome Database (GVD). Further, annotation of the "high-quality" genomes and KEGG pathway analysis has identified antB, dnaB, DNMT1, DUT, xlyAB, xtmB and xtmA as the most widespread viral and Auxiliary Metabolic Genes (AMGs). Genes for virulence, host-takeover, drug resistance, tRNA, tmRNA and CRISPR elements were also found. Bacterial hosts are predicted for around 40% of the analyzed genomes. Overall, we report identification of new viral genomes and genome analyses of human gut viruses, which will be useful for biological characterization to establish their significance in physiology. IMPORTANCEMultiple studies have found that dysbiosis of gut virome is associated with conditions such as metabolic syndromes, autoimmune disorders and infectious diseases. In the interest of its therapeutic and diagnostic potential, intestinal virome warrants detailed investigation. However, limited ability to culture gut viruses becomes one of the challenges for their biological characterization and fully understanding their role in physiology. Sequence analysis and host prediction methods provide opportunities to understand gut viruses, their functional potential and devise ways for further characterization.

genomics↗

bioRxiv2024

Oxygen partitioning into biomolecular condensates is governed by excluded volume

Biomolecular condensates form through the self-assembly of proteins and nucleic acids to create dynamic compartments in cells. By concentrating specific molecules, condensates establish distinct microenvironments that regulate biochemical reactions in time and space. Macromolecules and metabolites partition into condensates depending on their interactions with the macromolecular constituents, however, the partitioning of gases has not been explored. We investigated oxygen partitioning into condensates formed by intrinsically disordered repeat proteins with systematic sequence variations using microelectrodes and phosphorescence lifetime imaging microscopy (PLIM). Unlike other hydrophobic metabolites, oxygen is partially excluded from the condensate with partitioning constants more strongly modulated by changes in protein length than hydrophobicity. For repeat proteins, the dense phase protein concentration drops with chain length resulting in a looser condensate. We found that oxygen partitioning is anti-correlated with dense phase protein concentration. Several mechanisms could explain such an anti-correlation including excluded volume or salting out effects. Molecular dynamics simulations suggest that oxygen does not form strong and specific interactions with the scaffold and is dynamic on the nanosecond timescale. Biomolecular condensates thus result in variation of oxygen concentrations on nanometer length-scales, which may tune the oxygen concentration available for biochemical reactions within the cell.

biophysics↗

bioRxiv2024

Dilated cardiomyopathy-associated skeletal muscle actin (ACTA1) mutation R256H disrupts actin structure and function and causes cardiomyocyte hypocontractility

Skeletal muscle actin (ACTA1) mutations are a prevalent cause of skeletal myopathies consistent with ACTA1s high expression in skeletal muscle. Rare de novo mutations in ACTA1 associated with combined cardiac and skeletal myopathies have been reported, but ACTA1 represents only [~]20% of the total actin pool in cardiomyocytes, making its role in cardiomyopathy controversial. Here we demonstrate how a mutation in an actin isoform expressed at low levels in cardiomyocytes can cause cardiomyopathy by focusing on a unique ACTA1 mutation, R256H. We previously identified this mutation in multiple family members with dilated cardiomyopathy (DCM), who had reduced systolic function without clinical skeletal myopathy. Using a battery of multiscale biophysical tools, we show that R256H has potent functional effects on ACTA1 function at the molecular scale and in human cardiomyocytes. Importantly, we demonstrate that R256H acts in a dominant manner, where the incorporation of small amounts of mutant protein into thin filaments is sufficient to disrupt molecular contractility, and that this effect is dependent on the presence of troponin and tropomyosin. To understand the structural basis of this change in regulation, we resolved a structure of R256H filaments using Cryo-EM, and we see alterations in actins structure that have the potential to disrupt interactions with tropomyosin. Finally, we show that ACTA1R256H/+ human induced pluripotent stem cell cardiomyocytes demonstrate reduced contractility and sarcomeric disorganization. Taken together, we demonstrate that R256H has multiple effects on ACTA1 function that are sufficient to cause reduced contractility and establish a likely causative relationship between ACTA1 R256H and clinical cardiomyopathy. Significance StatementSkeletal muscle actin mutations are well-known to cause skeletal myopathies, but their role in cardiomyopathies have been controversial as skeletal muscle actin is only expressed at modest levels in the heart. Here, we demonstrate that a skeletal muscle actin mutation potently causes multiple defects in actin function at the atomic and molecular scales, and it functions in a dominant fashion, leading to cardiomyocyte contractile defects. Our results establish how skeletal muscle actin mutations may cause cardiomyocyte dysfunction and lay the foundation for future studies of the role of skeletal muscle actin in cardiomyopathy.

biophysics↗

bioRxiv2024

Systemic Dosing of Virus-derived Serpin Improves Survival and Immunothrombotic Damage in Murine Colitis

Inflammatory bowel disease (IBD) is potentially life-threatening, with risk of bleeding, clotting, infection, sepsis, cancer and toxic megacolon. Systemic and local immune and coagulation dysfunction increase IBD severity. Current treatments are partially effective, but there is no definitive cure. Serine protease cascades activate thrombotic, thrombolytic and complement pathways and are regulated by inhibitors, serpins. Viruses encode proteins evolved from endogenous central regulatory pathways. A purified secreted Myxomavirus-derived serpin, Serp-1, dosed as a systemic anti-inflammatory drug, has proven efficacy in vascular and inflammatory disorders. PEGylated Serp-1 protein (PEGSerp-1) has improved efficacy in lupus and SARS-CoV-2 models. We examined PEGSerp-1 treatment in a mouse Dextran Sodium Sulfate (DSS) colitis model. Prophylactic PEGSerp-1 significantly improved survival in acute severe 4-5% DSS colitis, reducing inflammation and crypt damage in acute 4-5% DSS induced colitis and when dosed as a chronic delayed treatment for recurrent 2% DSS colitis. PEGSerp-1 reduced iNOS+ M1 macrophage invasion, damage to crypt architecture and vascular inflammation with decreased uPAR, fXa, fibrinogen and complement activation. This work supports PEGSerp-1 as a tissue targeting serpin therapeutic.

molecular biology↗

bioRxiv2023

Anti-cancer drug Tamoxifen interferes with Mycobacterium tuberculosis PhoPR mediated signaling and inhibits mycobacterial growth

Two-component signaling (TCS) systems empower all bacteria, including intracellular pathogens like Mycobacterium tuberculosis (M. tb) to regulate key pathways governing growth, physiology and virulence. Amongst all M. tb TCS systems, PhoPR and DevRS have been studied extensively for their roles in regulating persistence and virulence. Here, we report that besides its cognate response regulator PhoP, the PhoR sensor kinase displays several non-cognate interactions that augment its role in pathogenesis. We demonstrate that PhoR phosphorylates the DevR response regulator and furthermore, is itself subjected to O-phosphorylation by PknK, a Ser/Thr protein kinase (STPK), connecting TCS pathways with "eukaryotic-like" STPK driven phosphosignaling. This intersection of non-canonical regulatory pathways and the coregulation of PhoP and DevR regulons make M. tb PhoR a potentially attractive drug target. We rationalized that disruption of PhoPR signaling cascade and the resulting dysregulation may result in decreased virulence of M. tb. We tested this hypothesis by performing a high-throughput screen for compounds that inhibit autophosphorylation of PhoR sensor kinase. Screening of pharmacologically active, small molecule libraries yielded 11 potential inhibitors, of which one compound, Tamoxifen was able to attenuate PhoR autophosphorylation at micromolar concentrations in vitro and in vivo. Tamoxifen not only inhibited growth of Mycobacterium bovis BCG in culture but also interrupted PhoPR-mediated downstream signaling. Quantitative expression analysis revealed suppression of target gene, aprA under acidic conditions. Our findings highlight TCS sensor kinases as promising drug targets and underscore the applicability of clinically relevant anti-cancer drug tamoxifen as a repurposed anti-TB drug.

biochemistry↗

bioRxiv2023

Ser/Thr Phosphorylation of Mycobacterium tuberculosis Type II Rel TA module by Protein Kinase K interferes with toxin neutralization: A novel mode of TA regulation.

Toxin-Antitoxin (TA) modules represent genetic elements implicated in bacterial persistence and antibiotic tolerance. Remarkably, Mycobacterium tuberculosis encodes 90+ TA modules, the majority of which are Type II TA comprising of a toxin component and an antitoxin counterpart that neutralizes the toxin. Upon exposure to stress, the antitoxin is degraded, releasing the toxin which then acts to halt cellular growth. Given that TA modules dictate social behavior of a population, we hypothesize that their regulation must be exquisitely controlled to avoid superfluous growth inhibition and initiation of persistence. However, the regulation and coordination of TA modules is poorly understood. Herein, we describe for the first time, a novel regulatory mechanism for Type II TA modules involving post-translational modification (PTM). Using computational tools, we observed that over 85 % of the M. tuberculosis TA proteins possess potential Ser/Thr phosphosites highlighting them as putative substrates for M. tuberculosis Ser/Thr protein kinases (STPK). We demonstrate that members of the RelBE family are subjected to O-phosphorylation by PknK, a stress-responsive growth regulatory STPK. Mass spectrometry confirmed multiple sites of PknK-mediated phosphorylation in the RelJK TA module. To gain insights into the functional impact of this PTM, we conducted in vitro binding and phenotypic growth studies with the wild type and mutant RelJK proteins. Our findings indicate that phosphorylation of Thr77 residue in RelK toxin compromises its binding to the RelJ antitoxin. These results suggest a potential role for O-phosphorylation in influencing the interaction dynamics of the TA module components. ImportanceBacterial pathogens rely on the phenomenon of persistence as a survival strategy to combat the adverse environmental conditions encountered during infection. As a stochastic process, the driving force(s) that potentiate the formation of persisters in a bacterial population are largely unclear. This study is a step towards the discovery of intricate regulatory mechanisms that coordinate a synchronized TA cellular program. We propose a model wherein the TA module is regulated post translationally, specifically via Ser/Thr phosphorylation disrupting the interaction between the toxin and antitoxin proteins as a mechanism to regulate TA function.

biochemistry↗

bioRxiv2022

Inorganic polyphosphate abets silencing of a sub-telomeric gene cluster in fission yeast

Inorganic polyphosphate (PolyP) is a ubiquitous polymer that plays sundry roles in cell and organismal physiology. Whereas there is evidence for polyphosphate in the cell nucleus, it is unclear whether and how physiological levels of PolyP impact transcriptional regulation in eukarya. To address this issue, we performed transcriptional profiling of fission yeast vtc4{Delta} cells, which lack the catalytic subunit of the Vtc4/Vtc2 polyphosphate polymerase complex and thus have no detectable intracellular PolyP. Deleting Vtc4 elicited the de-repression of four protein-coding genes - SPAC186.04c, gdt1/SPAC186.05, SPAC186.06, and SPAC750.01 - located within a sub-telomeric region of the right arm of chromosome I that is known to be transcriptionally silenced by the TORC2 complex. These sub-telomeric genes were equally de-repressed in vtc2{Delta} cells and in cells expressing polymerase-dead Vtc4, signifying that PolyP synthesis is necessary to abet TORC2-dependent locus-specific gene silencing.

genetics↗

bioRxiv2022

Treatment with anti-inflammatory viral serpin modulates immuno-thrombotic responses and improves outcomes in SARS-CoV-2 infected mice

1.Severe acute respiratory distress syndrome (ARDS) during SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) infection, manifests as uncontrolled lung inflammation and systemic thrombosis with high mortality. Anti-viral drugs and monoclonal antibodies can reduce COVID-19 severity if administered in the early viremic phase, but treatments for later stage immuno-thrombotic syndrome and long COVID are limited. Serine protease inhibitors (SERPINS) regulate activated proteases during thrombotic, thrombolytic and immune responses. The myxoma poxvirus-derived Serp-1 protein is a secreted immunomodulatory serpin that targets activated coagulation and complement protease pathways as part of a self-defense strategy to combat viral clearance by the innate immune system. When purified and utilized as an anti-immune therapeutic, Serp-1 is effective as an anti-inflammatory drug in multiple animal models of inflammatory lung disease and vasculitis. Here, we describe systemic treatment with purified PEGylated Serp-1 (PEGSerp-1) as a therapy for immuno-thrombotic complications during ARDS. Treatment with PEGSerp-1 in two distinct mouse-adapted SARS-CoV-2 models in C57Bl/6 and BALB/c mice reduced lung and heart inflammation, with improved clinical outcomes. PEGSerp-1 significantly reduced M1 macrophage invasion in the lung and heart by modifying urokinase-type plasminogen activator receptor (uPAR) and complement membrane attack complex (MAC). Sequential changes in urokinase-type plasminogen activator receptor (uPAR) and serpin gene expression were observed in lung and heart with PEGSerp-1 treatment. PEGSerp-1 is a highly effective immune-modulator with therapeutic potential for treatment of severe viral ARDS with additional potential to reduce late SARS-CoV-2 complications related to immune-thrombotic events that persist during long COVID. SignificanceSevere acute respiratory distress syndrome (ARDS) in SARS-CoV-2 infection manifests as uncontrolled tissue inflammation and systemic thrombosis with high mortality. Anti-viral drugs and monoclonal antibodies reduce COVID-19 severity if administered early, but treatments for later stage immuno-thrombosis are limited. Serine protease inhibitors (SERPINS) regulate thrombotic, thrombolytic and complement pathways. We investigate here systemic treatment with purified poxvirus-derived PEGSerp-1 as a therapeutic for immuno-thrombotic complications in viral ARDS. PEGSerp-1 treatment in two mouse-adapted SARS-CoV-2 models (C57Bl/6 and BALB/c) significantly reduced lung and heart inflammation and improved clinical outcomes, with sequential changes in thrombolytic (uPAR) and complement expression. PEGSerp-1 is a highly effective immune-modulator with therapeutic potential for immune-thrombotic complications in severe viral ARDS and has potential benefit for long COVID.

microbiology↗

bioRxiv2022

Design Principles for Inflammasome Inhibition by Pyrin-Only-Proteins

Inflammasomes are filamentous signaling platforms essential for host defense against various intracellular calamities such as pathogen invasion and genotoxic stresses. However, dysregulated inflammasomes cause an array of human diseases including autoinflammatory disorders and cancer. It was recently identified that endogenous pyrin-only-proteins (POPs) regulate inflammasomes by directly inhibiting their filament assembly. Here, by combining Rosetta in silico, in vitro, and in cellulo methods, we investigate the target specificity and inhibition mechanisms of POPs. In contrast to a previous report, we find that POP1 is a poor inhibitor of the central inflammasome adaptor ASC. Instead, POP1 inhibits the assembly of upstream receptor PYD filaments such as those of AIM2, IFI16, NLRP3, and NLRP6. Moreover, not only does POP2 directly suppress the nucleation of ASC, but it can also inhibit the elongation of receptor filaments. In addition to inhibiting the elongation of AIM2 and NLRP6 filaments, POP3 potently suppresses the nucleation of ASC. Our Rosetta analyses and biochemical experiments consistently suggest that a combination of favorable and unfavorable interactions between POPs and PYDs is necessary for effective recognition and inhibition. Together, we reveal the intrinsic target redundancy of POPs and their inhibitory mechanisms.

biochemistry↗

bioRxiv2022

Investigating the eukaryotic host-like SLiMs in microbial mimitopes and their potential as novel drug targets for treating autoimmune diseases

Several pathogens sustain themselves inside the host by mimicking short linear motifs (SLiMs) of the host proteins. SLiMs are short stretches of 3-10 amino acids which are functionally diverse and mediate various signaling and protein-protein interactions (PPIs). Hence, mimicry of the host- SLiMs helps the microbes in modulating/disrupting the host defense mechanisms. This is the first report investigating the evolutionary characteristics and presence of eukaryotic host-like SLiMs in microbial peptides (mimitopes). Evaluation of the selection pressure revealed that 60% of the bacterial and 25% of the viral mimitopes which overlapped with the host-like SLiMs were evolutionarily conserved ({omega} < 1). Interestingly, host-like SLiMs were abundant in mimicry proteins but were less frequent in microbial mimitopes. This reflects that the majority of the pathogens cannot potentially rewire the host PPI networks for their advantage, but some can. Of the 152 bacterial and 43 viral mimitopes investigated only 10 bacterial and 4 viral mimitopes showed SLiMs. This indicates that mimitopes of some pathogens can be explored as novel drug targets for eliminating the etiopathological agent and treating the autoimmune disease, thereof. The repertoire of mimitopes identified here might provide important clues for the discovery of new drugs/protein-based immune-modulatory molecules against the pathogens. Key pointsO_LIMimicry of the host- SLiMs helps the microbes in modulating/disrupting the host defense mechanisms. C_LIO_LIHost-like SLiMs were abundant in mimicry proteins but were less frequent in microbial mimitopes. C_LIO_LIEvaluation of the selection pressure revealed that 60% of the bacterial and 25% of the viral mimitopes which overlapped with the host-like SLiMs were evolutionarily conserved. C_LI

bioinformatics↗

bioRxiv2022

Insights into the Human Gut Virome by Sampling a Population from the Indian Subcontinent

Gut virome plays an important role in human physiology but remains poorly understood. This study reports an investigation of the human gut DNA-virome of a previously unexplored ethnic population through metagenomics of faecal samples collected from individuals residing in Northern India. Analysis shows that, similar to the populations investigated earlier, majority of the identified virome belongs to bacteriophages and a smaller fraction (< 20%) consists of viruses that infect animals, archaea, protists, multiple domains or plants. However, crAss-like phages, in this population, are dominated by the genera VII, VIII and VI. Interestingly, it also reveals the presence of a virus family, Sphaerolipoviridae, which has not been detected in the human gut earlier. Viral families, Siphoviridae, Myoviridae, Podoviridae, Microviridae, Herelleviridae and Phycodnaviridae are detected in all of the analyzed individuals, which supports the existence of a core virome. Lysogeny-associated genes were found in less than 10% of the assembled genomes and a negative correlation was observed in the richness of bacterial and free-viral species, suggesting that the dominant lifestyle of gut phage is not lysogenic. This is in contrast to some of the earlier studies. Further, several hundred high-quality viral genomes were recovered. Detailed characterization of these genomes would be useful for understanding the biology of these viruses and their significance in human physiology. ImportanceViruses are important constituents of the human gut microbiome but it remains poorly understood. The Indian subcontinent is a unique biogeographic region and the Indian population is known to harbour a distinct bacterial microbiome. However, the gut virome in this population has not been investigated earlier. Therefore, in this study, we investigated fecal samples of 12 healthy individuals to analyze their gut virome, through metagenomics.

microbiology↗