Biology subjects

Kumar, Y.

Publications and source records attributed to Kumar, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Decoding Clade-Specific bNAb-HIV-1 Env Interactions Using Machine Learning

Human immunodeficiency virus type 1 (HIV-1) exhibits extensive genetic diversity, posing a major challenge to AIDS vaccine development. Broadly neutralizing antibodies (bNAbs) isolated from HIV-1-infected individuals offer valuable templates for designing broadly protective immunogens. Here, structural and energetic descriptors extracted from experimentally resolved bNAb/HIV-1 Envelope (Env) protein-protein complexes were used to develop machine-learning (ML) models predicting binding free energies ({triangleup}G) and dissociation constants (KD) across multiple HIV-1 clades. Among the algorithms evaluated, extreme gradient boosting (XGBoost) achieved the best predictive performance, with training and test R2 values of 0.92 and 0.658, respectively. Applying the optimized model to HIV-1 datasets revealed clade-dependent binding patterns, with clade B showing the greatest variability and clade G exhibiting signatures associated with reduced neutralization potency. Predicted {triangleup}G of different bNAb classes followed the trend gp120-gp41 interface {approx} fusion domain > CD4 binding site (CD4bs) > V1V2 loop {approx} V3-glycan, consistent with experimentally reported affinity profiles of benchmark bNAbs. Clade C exhibited intermediate {triangleup}G with PGT121, VRC01, and 10-1074 maintaining high-affinity interactions, whereas PG9 and 2G12 show reduced binding affinity. These findings offer quantitative insight into clade-specific bNAb binding energetics and demonstrate the potential of machine-learning approaches to guide HIV-1 immunogen design and vaccine development.

biochemistry↗

bioRxiv2026

BCOR mutations establish a persistent culture-adaptive state in human induced pluripotent stem cells

Human induced pluripotent stem cells (hiPSCs) are widely used for disease modelling and regenerative medicine, yet their utility depends on maintaining molecular integrity during long-term culture. BCOR (BCL6 Co-Repressor) mutations are among the most recurrent culture-acquired alterations in hiPSCs, but their functional consequences remain poorly understood. Here, we show that hiPSC BCOR mutations are predominantly truncating indels enriched in exon 7, defining a mutational landscape distinct from that observed in cancer. Multi-omics profiling reveals that BCOR loss drives widespread chromatin, transcriptomic and proteomic remodelling, with coordinated activation of developmental, pluripotency-associated and mitochondrial metabolism programmes. To facilitate routine surveillance, we develop a cost-effective TaqMan qPCR assay that accurately identifies BCOR-mutant hiPSCs across independent cell lines. Finally, we demonstrate that correction of BCOR mutation by CRISPR-Cas9 only partially restores the wildtype molecular state, highlighting the importance of early detection and monitoring of adaptive mutations in hiPSC cultures.

cell biology↗

bioRxiv2026

Hepatic Differentiation of Human Pluripotent Stem Cells into Functional In Vitro Models Recapitulating Native Liver Complexity for MASLD Modelling

Human in vitro hepatic models that accurately recapitulate liver function are essential for fundamental and translational research; however, currently utilised models for disease modelling and drug discovery lack physiological fidelity and require prolonged culture time. Here, we present a streamlined 10-day protocol for efficient and reproducible differentiation of human pluripotent stem cells into hepatocyte like cells (HLCs) and hepatic liver organoids (HLOs). Both models exhibited mature hepatocyte differentiation, as evidenced by albumin secretion and CYP3A4 metabolic activity. Interestingly, HLOs display enhanced multicellular complexity incorporating endothelial, stellate, and macrophage populations along with hepatocytes, thereby more closely recapitulating the native liver microenvironment than HLCs. Here, steatosis was induced in both platforms, which resulted in triglyceride accumulation and upregulated lipogenic markers (DGAT1, DGAT2). However, only HLOs recapitulated advanced disease characteristics, including inflammatory (IL-10) and fibrotic (alpha SMA, COL1A1) responses. Resmetirom, a thyroid hormone receptor-{beta} agonist, significantly reduced steatosis and restored molecular signatures in both models. Additionally, transplanted HLOs demonstrated prolonged survival and displayed host-derived vascularization, thereby validating in vivo maturation. Collectively, HLCs provides a rapid and physiologically relevant liver model, with HLOs offering superior utility for disease modelling, therapeutic evaluation, and regenerative applications due to their enhanced functional and physiological relevance.

cell biology↗

bioRxiv2026

Recombinant Photo-tagging Enables Fluorescent Labelling of Biomolecules and Visualization of Liquid-Liquid Phase Separation

Liquid-liquid phase separation (LLPS) of biomolecules has emerged as a fundamental principle governing biological systems. A key aspect to understand LLPS is the ability to visualize these coacervates, which requires labelling strategies. Fluorescent labelling of biomolecules remains one of the widely adopted techniques involving either small-molecule chemical conjugation or recombinant expression alongside a fluorescent protein. These small-molecule fluorophores are typically employed in excess, which further necessitates additional downstream processing steps, including separation of the conjugated molecules from the unreacted fluorophores. On the other hand, recombinantly appended fluorescent protein affects the functioning of the labelled biomolecule owing to its large size. There is still a need to develop a labelling technique with small molecular weight fluorophores that is also genetically encodable. We previously discovered that the C-terminal peptide fragment (CTPF) released during photo-exposure of a green-to-red photoconvertible fluorescent protein (PhoCl1) displayed red fluorescence. Notably, this chromophore displays broad excitation and emission wavelength profiles, rendering it robust under multiple excitation wavelengths. We leverage this phenomenon to achieve post-expression fluorescent labelling by genetically fusing PhoCl1 to the target peptide or protein to be labelled (TPTL). We successfully demonstrated this approach of conferring fluorescence to silica binding peptide, riboflavin kinase enzyme, TEV protease, Lanthanide Binding Peptide and maltose binding protein, all of which exhibited red fluorescence upon photocleavage. Finally, we demonstrate the applicability of this probe to visualize LLPS of CTPF tagged elastin-like polypeptide in the presence of polymeric crowder. We anticipate that this strategy for inducing red fluorescence in non-fluorescent biomolecules via a photocleavable protein will open new avenues for minimally perturbative fluorescent labelling.

bioengineering↗

bioRxiv2025

Polycomb Repressive Complexes 1 and 2 are recruited independently to pericentromeric heterochromatin in response to hypomethylation in mouse embryonic stem cells

Pericentromeric heterochromatin (PCH) is delineated by the enrichment of repressive epigenetic modifications, specifically trimethylated histone H3 at lysine 9 (H3K9me3) and DNA methylation (5-methylcytosine), which establish and maintain a condensed, transcriptionally silenced chromatin state. Depletion of either H3K9me3 or DNA methylation in mouse embryonic stem cells (mESCs) induces a permissive chromatin configuration that permits de novo recruitment and deposition of normally excluded Polycomb Repressive Complexes 1 and 2 (PRC1 and PRC2), characterized by H2AK119ub1 and H3K27me3 modifications, respectively, at PCH. Here, we demonstrate that H2AK119ub1 and H3K27me3 are independently recruited to hypomethylated PCH using a doxycycline-inducible mESC model allowing modulation of Dnmt1 expression levels and catalytic activity. We further investigate the roles of proposed mediators of PRC1/2 targeting, including SCML2, BEND3, KDM2b, and TET enzymes, in this context, our findings indicate that neither PRC1 nor PRC2 recruitment at hypomethylated PCH depends on these factors. Additionally, our data suggest that the permissive chromatin environment resulting from DNA hypomethylation is the principal facilitator of Polycomb complex spreading, offering novel insights into the mechanisms governing epigenetic modifier dynamics and interactions during periods of DNA methylation reprogramming.

molecular biology↗

bioRxiv2025

Engineering Escherichia coli FAD synthetase for the biosynthesis of FAD nucleotide analogues results in aminoglycoside antibiotic tolerance

Cofactors such as flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide, coenzyme A, and S-adenosylmethionine cofactors share a common adenosine moiety that is generally not considered to contribute directly to catalysis. In FAD biosynthesis, the adenosine group is installed by the flavin mononucleotide adenylyltransferase (FMNAT) domain of the bifunctional FAD synthetase using FMN and ATP. This study explores the nucleotide selectivity of the Escherichia coli FMNAT domain and physiological effects of FAD nucleotide analogues synthesized with cellular nucleotides other than ATP. First, we engineer the FAD synthetase enzyme at an active site loop to produce FAD nucleotide analogues. Chromosomal substitution of the wild-type E. coli FAD synthetase with the mutated version results in the intracellular synthesis of these noncanonical FAD nucleotide analogues and confers tolerance to aminoglycoside antibiotics. Upon investigating their potential downstream metabolic roles, we find that their biosynthesis perturbs cellular metabolism and they could bind to a range of E. coli enzymes. These findings validate the FMNAT domain loop as a viable site for cofactor engineering, with potential for synthetic biology applications. Finally, the observed correlation between FAD analogue production and increased tolerance to aminoglycosides reveals novel metabolic mechanisms underlying antibiotic sensitivity.

microbiology↗

bioRxiv2025

Clade-specific influences of glycans on the interactions between HIV-1 envelope and broadly neutralizing antibodies

N-linked glycans are important in the elicitation and the activity of many broadly neutralizing antibodies (bNAbs) against HIV-1. The high conformational flexibility of glycans hindered detailed atomistic investigations of glycan-bNAb interactions, including glycan shielding of bN-Abs. Importantly, how these interactions vary across different HIV-1 clades remains unclear. The variability in the number and location of potential N-linked glycosylation sites (PNGS) on the HIV-1 envelope (Env) protein across clades can lead to differences in glycan dynamics and topology, potentially affecting Env-bNAb interactions and the clade-specific efficacy of bNAb-based therapies. Here, we combined comprehensive glycan conformational sampling, using the software glycoSHIELD, and molecular dynamics (MD) simulations to model fully glycosylated trimeric Env for six HIV-1 strains, one from each of the major clades A, B, C, G, CRF01 AE (01 AE) and CRF07 BC (07 BC). We assessed the interactions of 50 different bNAbs, drawn from all the major bNAb classes, with each of these strains, quantifying glycan shielding, glycan-bNAb interactions, and their clade-specific variations for each bNAb in microscopic detail. Our findings reveal that while glycans cover most of the exposed surface area in all clades, the amount of accessible surface varies, with clade B having the minimum and clade 07 BC the maximum antibody accessible surface area. The number of glycan conformers per glycosylation site also varies with clades, even for conserved sites. Overall, we observed that bNAbs interact with more glycans than previously reported in experimental and computational studies. Important variations emerge in Env-bNAb interactions with clade and bNAb-class. These atomic-level insights will be valuable for improving bNAb-based therapies and vaccine design strategies against HIV-1. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/655089v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@eedc04org.highwire.dtl.DTLVardef@1aebf67org.highwire.dtl.DTLVardef@1982a02org.highwire.dtl.DTLVardef@ee08e5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Identifying a novel mechanism of L-leucine uptake in Mycobacterium tuberculosis using a chemical genomic approach

Amino acid biosynthesis is vital for Mycobacterium tuberculosis (Mtb) proliferation and tuberculosis pathogenesis. However, it is not clear how amino acids are transported in Mtb, particularly the branched chain amino acids (BCAAs) that contribute to the production of the cell-wall lipid component precursors such as acetyl-CoA and propionyl-CoA. While performing the screening of an FDA-approved repurposed library of small molecule inhibitors against the auxotrophic strain Mtb mc2 6206, which lacks leuC-leuD and panC-panD genes, we identified a molecule namely semapimod, which exclusively inhibits growth of the auxotrophic strain, whereas no effect is observed against the wild-type Mtb H37Rv. Interestingly, 24 h of exposure of Mtb mc2 6206 to semapimod causes massive transcriptional reprogramming with differential expression of >450 genes associated with a myriad of metabolic activities. By performing a series of experiments, we affirm that semapimod indeed inhibits the L-leucine uptake in Mtb mc2 6206 by targeting a protein involved in the cell-wall lipid biosynthesis pathway. Remarkably, semapimod treatment of mice infected with Mtb H37Rv causes a significant reduction of bacterial load in lungs and spleen, despite showing no efficacy against the pathogenic strain in vitro. Overall findings of our study reveal that together with an endogenous pathway for L-leucine biosynthesis, a well-orchestrated machinery for its uptake is functional in Mtb which is important for intracellular survival of the TB pathogen.

microbiology↗

bioRxiv2025

Intermittent Fasting Reprograms Chromatin Accessibility to Modulate Gene Expression in Brain and Muscle

Intermittent fasting (IF), a dietary regimen that mimics the natural feeding patterns observed across diverse organisms--from single-celled life to mammals--is thought to activate systemic survival responses and confer health benefits. However, the molecular mechanisms underlying these effects remain poorly understood. In this study, we utilized ATAC-Seq and RNA-Seq to investigate how IF influences chromatin accessibility and gene expression in brain and muscle tissues of mice, compared to ad libitum feeding. Our results reveal that IF induces significant changes in chromatin accessibility, modulating pathways related to metabolism, ribosome function, HIF-1 signaling, and glycolysis. Motif analysis identified tissue-specific transcription factors enriched in IF-regulated regions, including Sp1, Mef2a, NeuroD2, Banp, and NFIA in the brain, and SMAD4, TCF4, STAT5B, NKX3-1, and ZEB2 in muscle. Integrative analysis of ATAC-Seq and RNA-Seq data demonstrated that IF upregulates 50 genes and downregulates 15 in the cortex, while upregulating 31 genes and downregulating 10 in muscle. These gene expression changes are linked to pathways associated with neuroprotection and enhanced muscle function, offering mechanistic insights into the health benefits of IF. Our findings underscore the role of IF-induced chromatin remodeling in driving adaptive gene regulation.

bioinformatics↗

bioRxiv2025

siRNA complexation with Galactose-Functionalized Dendrimer: PAMAM vs PETIM

siRNA-based therapies hold promise for treating various diseases, but efficient and targeted delivery remains challenging. This study explored the potential of galactose-functionalized poly(amidoamine) (Gal-PAMAM) and poly(propyl ether amine) (Gal-PETIM) dendrimers as siRNA carriers using all-atom molecular dynamics simulations. This study revealed distinct complexation behavior for both the system in physiological (pH [~] 7) and high (pH [~] 10) environments. Electrostatic interactions dominated complexation, with PMF profiles revealing stronger binding affinity for Gal-PAMAM/siRNA at pH 7. siRNA displayed lower flexibility at pH 7 for both complexes, indicating enhanced stability. These findings suggest that Gal-PAMAM dendrimers at physiological pH hold promise for efficient siRNA delivery due to their superior complex stability and interaction strength. However, in polar solvents, Gal-PETIM exhibits hydrophobic behavior, enabling rapid delivery.

biophysics↗

bioRxiv2024

Arv1 interacts with and regulates the first step of GPI biosynthesis in Candida albicans

The ubiquitous ARV1 gene shows significant functional conservation across eukaryotes. In humans, it is implicated in early onset epileptic encephalopathy. Evidence suggests that the phenotypes manifested in affected patients are probably due to the deficiency in expression of cell surface GPI anchored proteins. S. cerevisiae Arv1 is proposed to be the elusive GPI flippase that delivers the GPI intermediate from the cytoplasmic face to the luminal side of the ER for further elaboration by the first mannosyltransferase of the pathway. Human and fungal ARV1 complement S. cerevisiae ARV1. Overexpressing some of the GPI-N-acetylglucosamine transferase (GPI-GnT) subunits rescues the null strain of S. cerevisiae ARV1. In mammals and in T. brucei Arv1 co-immunoprecipitates with one or more subunits of the GPI-GnT. Based on these reports we hypothesized a cross-talk for ARV1 with the GPI biosynthetic pathway in the human pathogenic fungus, C. albicans. Using super resolution radial fluctuation (SRRF) analysis for co-localization, co-immunoprecipitation assays, and acceptor-photobleaching Forster resonance energy transfer (FRET) studies, we show that C. albicans Arv1 physically interacts with the GPI-GnT. It also transcriptionally regulates the expression of the GPI-GnT genes to control the GPI biosynthetic pathway via its very first step. Overexpression of one of the GPI-GnT subunits, CaGpi19, in C. albicans ARV1 null strain rescues its cold-sensitive growth, azole sensitivity, cell wall phenotype and GPI-GnT activity. Thus, our results suggest extensive interactions between Arv1 and GPI biosynthesis in C. albicans.

microbiology↗

bioRxiv2024

Self-assembly of mycolic acid in water: monolayer or bilayer

The enduring pathogenicity of Mycobacterium tuberculosis can be attributed to its lipid-rich cell wall, with mycolic acids (MAs) being a significant constituent. Different MAs fluidity and structural adaptability within the bacterial cell envelope significantly influence their physicochemical properties, operational capabilities, and pathogenic potential. Therefore, an accurate conformational representation of various MAs in aqueous media can provide insights into their potential role within the intricate structure of the bacterial cell wall. We have carried out the MD simulations of MAs in an aqueous solution and shed light on the various structural properties such as thickness, order parameters, area-per-MAs, conformational changes, and principle component (PC) in the single component and mixed MAs monolayer. The different conformational populations in the monolayer have been estimated using the distance-based analysis between the function groups represented as W, U, and Z-conformations that lead to the fold of the MAs chain in the monolayer. Additionally, we have also simulated the mixture of alpha-MA (-MA or AMA), methoxy-MA (MMA), and keto-MA (KMA) with 56% AMA, 40 % MMA, and 14 % KMA composition. The thickness of the MAs monolayer was observed to range from 5 nm to 7 nm with an average 820 kg/m3 density for -MA, MMA, and KMA quantitative agreement with experimental results. The mero-chain (long chain), consisting of a functional group at the proximal and distal positions, tends to fold and exhibit a more disordered phase than the short chain. The keto-MA showed the greatest WUZ total conformations (35.32 %) with decreasing trend of eZ > eU > aU > aZ folds in both single component and mixture. Our results are in quantitative agreement with the experimental observations. The most minor population of sZ folds (0.75 % in a single component and 1.1 % in a mixture). However, eU and aU folds are most probable for the AMA and MMA. One striking observation is the abundance of MA conformers beyond the known WUZ convention because of the wide range distribution of intramolecular distances and change in dihedral angles. From the thermodynamic viewpoint, all monolayers were found to be stable in nature. The results from enhanced simulation revealed that changes in the magnitude of minimum free energy of the MAs are in order of KMA > AMA > MMA, and keto-MA exhibits more resistance (based on free energy values) for the drug molecules to permeate through the monolayer.

biophysics↗

bioRxiv2024

pH dependent direct sulfhydrylation pathway is required for pathogenesis of Mycobacterium tuberculosis

Methionine is essential for the survival of Mycobacterium tuberculosis (M. tuberculosis) inside the host. However, the transsulfuration pathway, a major contributor of methionine, is dispensable for the growth of M. tuberculosis suggesting redundancy in the methionine biosynthesis pathway. Orthologues of MetZTB in other bacterial species are known to operate a redundant single-step methionine biosynthesis pathway called direct sulfhydrylation. In this study, we demonstrate that genetic disruption of the metZ-mediated direct sulfhydrylation pathway in M. tuberculosis hinders growth at low pH, an effect mitigated by methionine supplementation. Computational analyses, including in-silico molecular docking and molecular dynamics (MD) simulations, reveal enhanced binding of the MetZ substrate, O-succinyl homoserine (OSH), to the active site of MetZ at acidic pH. Intriguingly, despite increased intracellular ATP levels, a relative decrease in the frequency of Bedaquiline (BDQ)-induced persisters is observed in metZ-deficient strain, suggesting a role of direct sulfhydrylation pathway in modulating BDQ sensitivity. Finally, we demonstrated that the absence of metZ impedes the ability of M. tuberculosis to grow inside the host.

microbiology↗

bioRxiv2024

MAGMa: Your Comprehensive Tool for Differential Expression Analysis in Mass-Spectrometry Proteomic Data.

Proteomics, the study of proteins and their functions, plays a vital role in understanding biological processes. In this study, we sought to address the challenges in analyzing complex proteomic datasets, where subtle changes in protein abundance are difficult to detect. Utilizing a newly developed tool, Maximal Aggregation of Good protein signal from Mass spectrometric data (MAGMa), we demonstrated its superior performance in accurately identifying true signals while effectively filtering out noise. Here we show that MAGMa strikes a balance between sensitivity and specificity on benchmarking datasets, offering a robust solution for analyzing various quantitative proteomic datasets. These findings advance the field by providing researchers with a powerful tool to uncover subtle changes in protein abundance, contributing to our understanding of complex biological systems and potentially facilitating the discovery of new therapeutic targets.

bioinformatics↗

bioRxiv2024

Phosphoglucomutase A mediated regulation of carbon flux is essential for antibiotic and disease persistence in Mycobacterium tuberculosis

The long-term survival of Mtb mandates judicious utilization of the available resources inside the host. Uninterrupted access to host-derived nutrients holds the key to the success of Mtb. Phosphoglucomutase enzyme besides synthesizing glycogen, which serves as a nutrient reservoir, also helps modulate the carbon flux in different pathogens. Studies on the role of glycogen metabolism in disease progression, reactivation, and drug susceptibility in tuberculosis are severely lacking. To investigate this, we generated an Mtb strain ({Delta}pgmA) devoid of the gene that encodes for the enzyme phosphoglucomutase A (pgmA). The absence of pgmA impedes the ability of the pathogen to survive under nutrient-limiting and reactivation conditions. In the current study, we demonstrate that the absence of cell membrane-associated glycolipids in {Delta}pgmA compromised the cell wall integrity and increased the susceptibility of {Delta}pgmA to various stresses. Interestingly, in comparison to the wild type, low cAMP levels in {Delta}pgmA imparted an enhanced growth phenotype on cholesterol. Differential gene expression and carbon flux analysis suggest that stored carbon in the form of glycogen is essential for the survival of Mtb under nutrient-limiting conditions. Finally, we demonstrate that the pgmA gene of Mtb is essential for the growth of Mtb inside the host. Overall, this study unveils the significance of pgmA-mediated regulation of membrane glycolipids and its implication on antibiotic and disease persistence in tuberculosis. Additionally, information derived from this study will help design anti-TB strategies that are novel, short, and more efficient.

microbiology↗

bioRxiv2024

(p)ppGpp and DksA play crucial role in reducing the efficacy of b-lactam antibiotics by modulating bacterial membrane permeability

The key signaling molecules in the bacterial stress sensing pathway, the alarmone (p)ppGpp and transcription factor DksA, help in survival during nutritional deprivation and exposure to xenobiotics by modulating cellular metabolic pathways. In Vibrio cholerae, (p)ppGpp metabolism is solely linked with the functions of three proteins: RelA, SpoT, and RelV. At threshold or elevated concentrations of (p)ppGpp, the level of cellular metabolites and proteins in the presence and absence of DksA in V. cholerae and other bacteria has not yet been comprehensively studied. We engineered the genome of V. cholerae to develop DksA null mutants in the presence and absence of (p)ppGpp biosynthetic enzymes. We observed a higher sensitivity of the (p)ppGpp0{Delta}dksA V. cholerae mutant to different -lactam antibiotics compared to the wild-type (WT) strain. Our whole-cell metabolomic and proteome analysis revealed that the cell membrane and peptidoglycan biosynthesis pathways are significantly altered in the (p)ppGpp0, {Delta}dksA, and (p)ppGpp0{Delta}dksA V. cholerae strains. Further, the mutant strains displayed enhanced inner and outer membrane permeability in comparison to the WT strains. These results directly correlate with the tolerance and survival of V. cholerae to -lactam antibiotics. These findings may help in the development of adjuvants for -lactam antibiotics by inhibiting the functions of stringent response modulators. ImportanceThe (p)ppGpp biosynthetic pathway is widely conserved in bacteria. Intracellular levels of (p)ppGpp and the transcription factor DksA play crucial roles in bacterial multiplication and viability in the presence of antibiotics and/or other xenobiotics. The present findings have shown that (p)ppGpp and DksA significantly reduces the efficacy of -lactam and other antibiotics by modulating the availability of peptidoglycan and cell membrane-associated metabolites by reducing membrane permeability. Nevertheless, the whole-cell proteome analysis of (p)ppGpp0, {Delta}dksA, and (p)ppGpp0{Delta}dksA strains identified the biosynthetic pathways and associated enzymes that are directly modulated by the stringent response effector molecules. Thus, the (p)ppGpp metabolic pathways and DksA could be a potential target for increasing the efficacy of antibiotics and developing antibiotic adjuvants.

microbiology↗

bioRxiv2024

TPR is required for cytoplasmic chromatin fragment formation during senescence

During oncogene-induced senescence there are striking changes in the structure of the nucleus and the organisation of heterochromatin. This is accompanied by activation of a pro-inflammatory gene expression programme - the senescence associated secretory phenotype (SASP) - driven by transcription factors such as NF-{kappa}B. Here we show that TPR, a protein of the nuclear pore complex basket, is required for the very early activation of NF-{kappa}B signalling during the stress-response phase of oncogene-induced senescence. This is prior to activation of the SASP and occurs without affecting NF-{kappa}B nuclear import. We show that TPR is required for the activation of TBK1 signalling at these early stages of senescence and we link this to the formation of heterochromatin-enriched cytoplasmic chromatin fragments thought to bleb off from the nuclear periphery. These cytoplasmic chromatin fragments appear to lack nuclear pore components. Our data suggest that TPR at the nuclear pore is involved in the loss of structural integrity of the nuclear periphery during senescence. We propose that this acts as a trigger for activation of cytoplasmic nucleic acid sensing, NF-{kappa}B signalling, and activation of the SASP, during senescence.

cell biology↗

bioRxiv2024

The ER-resident Ras Inhibitor 1 (Eri1) of Candida albicans inhibits hyphal morphogenesis via the Ras-independent cAMP-PKA pathway

Ras signaling and glycosylphosphatidylinositol (GPI) biosynthesis are mutually inhibitory in S. cerevisiae. The inhibition is mediated via an interaction of yeast Ras2 with the Eri1 subunit of its GPI-N-acetylglucosaminyl transferase (GPI-GnT), the enzyme catalyzing the very first GPI biosynthetic step. In contrast, Ras signaling and GPI biosynthesis in C. albicans are mutually activated and together control the virulence traits of the human fungal pathogen. What might be the role of Eri1 in this pathogen? The present manuscript addresses this question while simultaneously characterizing the cellular role of CaEri1. It is either non-essential or required at very low levels for cell viability in C. albicans. Severe depletion of CaEri1 results in reduced GPI biosynthesis and cell wall defects. It also produces hyperfilamentation phenotypes in Spider medium as well as in bicarbonate medium containing 5% CO2, suggesting that both the Ras-dependent and Ras-independent cAMP-PKA pathways for hyphal morphogenesis are activated in these cells. Pull-down and acceptor-photobleaching FRET experiments suggest that CaEri1 does not directly interact with CaRas1, but does so through CaGpi2, another GPI-GnT subunit. CaGpi2 is also downstream of CaEri1 in cross-talk with CaRas1 and control of hyphal growth in Spider medium. However, CaEri1 is downstream of all GPI-GnT subunits in inhibiting Ras-independent filamentation. CaERI1 also participates in the inter-subunit transcriptional cross-talk within the GPI-GnT, a feature unique to C. albicans. Virulence studies using G. mellonella larvae show that a heterozygous strain of CaERI1 is better cleared by the host and is attenuated in virulence.

microbiology↗