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Kumar, A.

Publications and source records attributed to Kumar, A..

At least 91 records · Page 5Linked to original sources

Consolidation Separates Implicit and Explicit Components of Compound Motor Memories

Motor adaptation involves the parallel operation of implicit recalibration and explicit re-aiming processes. During naturalistic learning, these systems interact, producing compound behavioral outputs that reflect their combined contributions. It remains unclear whether simultaneously engaged implicit and explicit processes form a single unified representation, or generate parallel memory representations that are merely co-expressed, and how consolidation transforms such representations. We addressed these questions across three visuomotor adaptation experiments (n = 120), in which the implicit process was engaged via gradual cursor rotation and the explicit process via target jump, by systematically manipulating the sequence of learning and the timing of expression. Immediately after learning, behavior reflected an inflexible, integrated memory that could not be decomposed by changing task demands. Following 24-hour consolidation, however, expression became component-selective, with implicit or explicit contributions retrieved in response to task demand. This reorganization had direct consequences on relearning, producing facilitation when the expressed and relearned components matched and interference when they mismatched. Moreover, when implicit adaptation was stabilized prior to compound learning, consolidation preserved the updated state rather than the original implicit representation. Together, these findings demonstrate that consolidation does not merely stabilize compound motor memories. Instead, it actively reorganizes them, transforming the initially integrated representations into independent, context-dependent components.

neuroscience↗

Coupling of electron-bifurcation modules powers aromatic ring reduction beyond the biological redox window

Microbial degradation of ubiquitous aromatic compounds is central to the global carbon cycle and bioremediation, yet the intrinsic stability of aromatic rings poses a major barrier to their breakdown. Under strictly anaerobic conditions, class II benzoyl-CoA reductases (BCRII) catalyse the key step of this process, a Birch-like reduction of the aromatic ring to a cyclic diene at a tungsten cofactor. This reaction operates beyond the redox limits of conventional biological electron transfer, yet the mechanism by which BCRII generates such extreme reducing power has remained unclear. Here, high-resolution cryo-electron microscopy, in situ cryo-electron tomography, and enzymatic analyses reveal that the one-MDa BCRII complex from Geobacter metallireducens links two distinct flavin-based electron-bifurcation modules, previously characterised in hydrogenases and heterodisulfide reductases, to drive aromatic ring reduction. Reduced ferredoxin and NADH deliver electrons through sequential confurcation and bifurcation to the catalytic site, while cryo-electron tomography of native cells identifies electron-transferring flavoprotein as the second acceptor directing high-potential electrons into the respiratory chain. These results show that modular FBEB units can be hierarchically assembled to extend metabolic redox capacity, highlighting their versatility as adaptable components for electron transfer pathways.

biochemistry↗

"Editing the conserved IPA1-TB1 regulatory module reshapes plant architecture and enhances tillering in wheat

Plant architecture is a major determinant of yield potential in cereal crops, where tiller number directly influences spike production and grain yield. The transcriptional regulators Ideal Plant Architecture 1 (IPA1) and Teosinte Branched1 (TB1) constitute a conserved genetic module controlling axillary bud activity and branching in grasses; however, their functional contribution to wheat architecture remains largely unexplored. Here, we employed CRISPR/Cas9-mediated genome editing to investigate the roles of TaIPA1 and TaTB1 in regulating tillering in hexaploid wheat (Triticum aestivum L.). Comparative genomic analysis identified conserved IPA1 orthologs across the wheat A, B, and D sub-genomes, with strong conservation of the SQUAMOSA-binding protein domain. Sequencing analysis confirmed targeted mutations, including nucleotide substitutions and insertions that generated frameshift mutations and premature stop codons. Genome-edited lines exhibited enhanced tillering compared with wild-type plants. Several TaIPA1 mutant lines produced up to two-fold higher tiller numbers, while TaTB1 knockout lines showed earlier tiller initiation and [~]50% increased tillering. Notably, enhanced tillering was associated with increased grain weight without affecting spikelet number per spike. Together, these results demonstrate that the conserved TaIPA1-TaTB1 regulatory module plays a pivotal role in shaping wheat plant architecture. Targeted manipulation of this pathway using CRISPR/Cas9 provides a promising strategy for optimizing tillering and developing high-yielding wheat ideotypes.

molecular biology↗

Minoxidil hydrochloride impedes NLRP3 inflammasome activation via upregulation of AMPK-mediated autophagy

NLRP3 inflammasome is a cytosolic multi-protein complex that plays a crucial role in the immune system, responding to various exogenous and endogenous stimuli by triggering protective inflammatory responses. However, aberrant NLRP3 inflammasome activation is implicated in numerous inflammatory diseases. Therefore, the NLRP3 inflammasome is an important pharmacological target for the treatment of multiple diseases. In this context, we screened various US-FDA-approved drugs for NLRP3 inflammasome inhibition. We found that among various drugs, minoxidil hydrochloride (MXL) effectively inhibits NLRP3 inflammasome, evidenced by reduced secretion of IL-1{beta} and IL-18 in J774A.1 cells treated with MXL. The IC50 values of MXL for inhibition of IL-1{beta} and IL-18 were calculated to be 1.2 and 1.06 {micro}M, respectively. MXL was found to prevent ASC oligomerization, thereby inhibiting the NLRP3 inflammasome and leading to CASP1 cleavage. Further investigation revealed that MXL also utilizes AMPK-mediated autophagy to modulate NLRP3 inflammasome activity. Using siAMPK and bafilomycin A1, an end-stage autophagy inhibitor, we elucidated crosstalk between the NLRP3 inflammasome and autophagic pathways, which was modulated by MXL. Furthermore, we demonstrated the efficacy of MXL in two different mouse models of inflammation, involving the NLRP3 inflammasome. MXL at doses of 10 and 20 mg/kg effectively inhibited the activation of NLRP3 inflammasome by monosodium urate in the air pouch model and by ATP in the peritoneal inflammation model, as evidenced by reduced secretion of 1{beta} and IL-18 in the lavage. Our study identifies MXL as a potent NLRP3 inflammasome inhibitor, warranting further investigation as a potential therapeutic agent for inflammatory diseases.

immunology↗

ZDHHC17 Links S-Acylation, Huntington Disease, VCP-associated Multisystem Proteinopathy, and Amyotrophic Lateral Sclerosis.

Protein mislocalization is an important contributor to neurodegeneration. We have identified disrupted S-acylation as a driver of mislocalization. S11acylation, the addition of long-chain fatty acids to cysteine residues, is mediated by the ZDHHC family of S-acyltransferases, and regulates protein localization by modulating protein hydrophobicity. Among these enzymes, we have identified ZDHHC17 as a central node in neurodegenerative disease. Although decreased ZDHHC17 activity is correlated with Huntington disease (HD) pathology, its broader contributions to neurodegeneration remain poorly defined. Here, we present new evidence that ZDHHC17 S-acylates or interacts with multiple proteins implicated in amyotrophic lateral sclerosis (ALS), including Valosin-containing protein (VCP) and TAR DNA Binding Protein (TDP1143). We further confirm that VCP, TDP-43, FUS, C9ORF72, and SQSTM1 are S-acylated across rodent models of multiple neurodegenerative diseases, including HD, VCP-associated multisystem proteinopathy, and ALS. Using biochemical S-acylation assays and confocal microscopy, we show that ZDHHC17 S-acylates VCP, depletes the nuclear localization of both VCP and TDP-43, and modulates VCP-dependent toxicity via ER-stress. We additionally demonstrate that TDP-43 is S-acylated by ZDHHC9. Motivated by these findings, we investigated the functional significance of motor-neuronal (MN) ZDHHC17 by studying dHip14 (the fly analog of ZDHHC17) in Drosophila melanogaster. Here, we found that motor neuron-specific dHip14 knockdown (KD) in flies resulted in impaired motor function, whereas ubiquitous depletion led to pharate-adult lethality. Together, these results highlight a shared mechanism across neurodegenerative diseases. Collectively, our findings position ZDHHC17 as a critical enzyme that links fatty acylation, protein homeostasis, and neurodegeneration. Understanding how ZDHHC17 orchestrates S-acylation across different cellular pathways may reveal new therapeutic strategies to restore proteostasis in neurodegenerative disease.

molecular biology↗

Myeloid Gi signaling acts as a weight-independent immunometabolic switch controlling systemic insulin sensitivity

Metabolic dysfunction does not necessarily correlate with adiposity. Metabolically healthy obese individuals and insulin-resistant lean individuals represent a fundamental paradox that implicates immune cell intrinsic mechanisms in the pathogenesis of type 2 diabetes. Here, we identify myeloid Gi signaling as a previously unrecognized determinant of whole-body glucose homeostasis. Single-cell transcriptomic analysis of adipose tissue macrophages from obese mice and humans reveals marked alteration in Gnai isoform, suggesting that myeloid Gi signaling is functionally engaged during metabolic disease. Using complementary myeloid-specific rodent models of Gi inhibition (pertussis toxin) and chemogenetic Gi activation (DREADD), we demonstrate that inhibition of Gi signaling improves glucose tolerance and enhances insulin sensitivity under both regular chow and high-fat diet conditions, independent of body weight and energy expenditure. Whereas acute Gi activation in lean mice modestly enhances glucose disposal, the same intervention during diet-induced obesity markedly impairs systemic glucose homeostasis, revealing context-dependent pathway function. Mechanistically, Gi inhibition amplifies macrophage cAMP-CREB signaling to drive IL-6 production, engaging STAT3- and AMPK-dependent pathways in adipose tissue and skeletal muscle to support insulin action. Conversely, Gi activation engages a previously uncharacterized G{beta}{gamma}-mTOR/AKT-JNK cascade, driving IL-1{beta} secretion that directly impairs insulin signaling in adipocytes and myotubes. Pharmacological IL-6 receptor blockade abolishes the metabolic benefits of Gi inhibition, whereas IL-1 receptor antagonism fully rescues Gi activation-induced metabolic dysfunction, establishing these cytokines as obligate downstream effectors. This signaling architecture is conserved in human macrophages, and ATAC-seq profiling reveals chromatin remodeling at cAMP-CREB and IL-6 regulatory pathway loci, consistent with the observed transcriptional reprogramming. Together, these findings establish myeloid Gi signaling as a weight-independent immunometabolic switch that couples opposing cytokine programs to systemic insulin sensitivity and identify this pathway as a therapeutic target in obesity-associated metabolic disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/713834v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@15e4576org.highwire.dtl.DTLVardef@224cd5org.highwire.dtl.DTLVardef@1e24888org.highwire.dtl.DTLVardef@1080bf3_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

physiology↗

Folate Receptor α Contributes to Radiation Resistance in Neuroendocrine Prostate Cancer by Regulating Redox Homeostasis

Ionizing radiation can be an effective therapy for prostate cancer. Unfortunately, however, more aggressive prostate cancers such as neuroendocrine prostate cancer (NEPC) are often radiation resistant, which contributes to their high degree of morbidity and mortality. In this study, we used an unbiased approach to identify novel mechanisms that contribute to resistance to radiation and that are associated with neuroendocrine differentiation. Specifically, we compared the expression of cell surface proteins by mass spectrometry in prostate cancer cell lines that had been either untreated or treated with radiation to induce resistance, a process that also promotes neuroendocrine differentiation. Among the proteins identified by this screen, we focused on folate receptor (FR) because of its known biological functions and the fact that it is a validated therapeutic target. Our data reveal that FR has a causal role in enabling prostate cancer cells to resist radiation. Importantly, we also demonstrate that the expression of FR is regulated by HIF-1, which also has a causal role in radiation resistance and neuroendocrine differentiation. Given that the ability of cells to resist damage and death in response to ionizing radiation depends largely on their ability to buffer the substantial increase in reactive oxygen species (ROS) that is generated by radiation, we also demonstrate that the folate-FR axis promotes radiation resistance by sustaining intracellular glutathione levels that buffer this increase in ROS. In summary, the data reported here highlight a novel role for FR in resistance to ionizing radiation that is intimately associated with the hypoxic microenvironment of NEPC and the ability of the folate-FRa axis to maintain redox homeostasis.

cancer biology↗

Diet-conditioned microbiota enhances fecal microbiota transplantation efficacy in alcoholic liver disease through caproic acid-PPARα signaling

Background and AimFecal microbiota transplantation (FMT) in Alcohol-related liver disease (ALD) has shown therapeutic potential, with variable efficacy and unclear mechanism. Because dietary protein influences gut microbiota composition, we hypothesized that donor dietary preconditioning could enhance FMT efficacy. We therefore examined in a murine ALD model if high-protein donor diet improves FMT outcome. MethodsALD was induced in C57BL/6N mice using a Lieber-DeCarli ethanol diet combined with thioacetamide administration for 12 weeks. FMT was performed using stool from diet-modulated donors, and recovery was assessed on day7 post-FMT. Multi-omics analysis using 16s rRNA and mass spectroscopy was performed for Gut microbiota composition, plasma- and stool-metabolome, and hepatic proteomes. Multi-omics outcomes were validated in ALD animal and Huh7 hepatocytes. ResultsBoth protein-based FMTs improved ALD recovery; Veg-FMT demonstrated superior efficacy, significantly reducing hepatic injury (AST 1.2-fold, p=0.002; bilirubin 1.2-fold, p=0.03; steatosis 1.7-fold,p=0.01) and restoring gut barrier integrity (occludin 1.5-fold,p=0.04; mucin 2 2.2-fold, p=002; and plasma endotoxin 1.7-fold, p=0.02). A significant 2-fold increase was observed in Lachnospiraceae NK4A136, Coriobacteriaceae UCG-002, and short-chain fatty acids, particularly caproic acid. Functional validation confirmed that caproic acid promoted hepatic fatty acid {beta}-oxidation through PPAR-dependent mechanisms, reducing triglyceride accumulation and lipogenesis in both cellular and animal models. ConclusionDonor preconditioning with a plant-protein enriched diet enhances FMT efficacy in ALD by gut microbiota modulation with increased metabolites like caproic acid. These findings highlight a microbiota-metabolite-host axis through which diet-modulated FMT improves hepatic lipid metabolism and injury, and identifies a pathway via which FMT imparts its effect. SignificanceThis study identifies a mechanistic basis for improving fecal microbiota transplantation (FMT) efficacy in alcohol-related liver disease (ALD) by demonstrating that dietary preconditioning of donor microbiota improves therapeutic outcomes. We show that plant protein-modulated donor microbiota supplements abstinence-associated recovery through increased production of the microbial metabolite caproic acid, which promotes hepatic fatty acid {beta}-oxidation via PPAR signaling. These findings highlight donor dietary conditioning and microbiota-derived metabolites, rather than microbial composition alone, as important determinants of FMT efficacy. The results suggest that microbial metabolites such as caproic acid may represent potential therapeutic targets or biomarkers to enhance and standardize microbiota-based interventions in ALD. Although the current work is based on a murine model, the identified microbiota-metabolite-host metabolic axis provides a framework for future translational studies aimed at optimizing FMT strategies in liver disease.

microbiology↗

SUMO modulates meiotic crossover rates between and within vertebrate species

Crossing over during meiosis drives genetic diversity and ensures the accurate segregation of homologous chromosomes. Variation in the rate of crossing over has been linked to evolutionary divergence and environmental adaptability, shaping fitness and responses to selective pressures. Despite its significance, the molecular mechanisms underlying this variation remain poorly understood. Crossover sites are selected from a large pool of potential sites initiated by programmed DNA double-strand breaks. Post-translational modification by SUMO (Small Ubiquitin-like Modifier) has been implicated in this process. Here, we show that crossover rate, chromosome length, and abundance of chromosome-associated SUMO are positively correlated across a range of vertebrate species, including mouse, chicken, pig, cattle, sheep, and goat. Crossover variation between goat breeds across the Indian subcontinent was also positively correlated with chromosomal SUMO level. Furthermore, modulating SUMO levels in cultured goat spermatocytes altered crossover frequency. Cumulatively, these observations point to a central role for SUMO in mediating crossover variation both between and within vertebrate species.

cell biology↗

Subunit selective modulation of GABAA receptors using pharmacogenetically tethered neurosteroids

Neurosteroids are powerful endogenous modulators of inhibition and emerging therapeutics for anxiety, epilepsy, and mood disorders, yet their actions at defined receptor subtypes and within specific neuronal populations remain poorly resolved. Here, we engineer a neurosteroid DART (Drug Acutely Restricted by Tethering) platform to deliver neuroactive steroid (NAS) activity with cellular precision and receptor-subunit selectivity. From a screen of seventeen NAS analogs, we identified seven scaffolds suitable for further engineering, and we discovered that linker attachment at the steroid C11 position uniquely preserves NAS positive allosteric modulation of GABAA receptors, whereas C2 and C17 attachment abolished activity. C11-linked NAS-DARTs slowed IPSC decay kinetics and showed variable off-target modulation of NMDA and AMPA EPSCs. The lead DART compound, YX85.1DART.2, enhanced GABA-evoked currents in neurons expressing engineered 4/{delta}-containing GABAA receptors but spared {gamma}2-containing receptors. A complementary benzodiazepine DART BZP.1DART.2 showed the opposite selectivity. Together, these tools enable cell-restricted, subunit-resolved interrogation of neurosteroid action on inhibitory microcircuits and provide a strategy to dissect how distinct GABAA receptor subclasses contribute to circuit function and therapeutic outcomes.

neuroscience↗

Integrated Artificial Intelligence and Quantum Chemistry Approach for the Rational Design of Novel Antibacterial Agents against Ralstonia solanacearum.

Antimicrobial resistance (AMR) in plant pathogenic bacteria poses a serious threat to global agriculture, necessitating the development of novel antibacterial agents targeting virulence mechanisms. This study presents an integrated bioinformatics-driven framework for the rational design and computational validation of Solres, a newly designed small molecule targeting key virulence proteins in phytopathogenic bacteria. Approximately 10,000 active compounds from PubChem BioAssay (AID: 588726) were analyzed using structural clustering and scaffold mining to identify conserved molecular motifs associated with antibacterial activity. Guided by high-frequency substructures, Solres was designed de novo and screened for structural novelty against PubChem, ChEMBL, and WIPO databases. Drug-likeness evaluation using Lipinskis Rule of Five confirmed favorable physicochemical properties. Molecular docking was performed against essential virulence regulators, including PhcA, PhcR, HrpB, PehA, and Egl from Ralstonia solanacearum and Xanthomonas spp., with active sites predicted using CaspFold. Docking analyses revealed strong binding affinities and stable interactions with key catalytic and regulatory residues. Complex stability and conformational integrity were further validated through molecular dynamics simulations. Quantum chemical descriptors, including HOMO-LUMO energy gap and dipole moment, supported the electronic suitability and reactivity profile of Solres. Collectively, this study demonstrates the effective integration of cheminformatics, structural bioinformatics, molecular simulations, and quantum chemical analyses for plant-focused antibacterial discovery. The compound Solres represents a promising lead candidate for mitigating bacterial wilt disease and provides a computational framework for future experimental validation and sustainable crop protection strategies against AMR-driven phytopathogens.

bioinformatics↗

Knockdown of the long isoform of the prolactin receptor selectively targets pathogenic immune cells and averts lupus nephritis

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by chronic inflammation in multiple organ systems. While a clinical association between elevated levels of the hormone/cytokine, prolactin (PRL), and exacerbation of SLE has been recognized for some time, little is known about the mechanisms through which PRL affects the course of this disease. Here, we show that immune cells in SLE have aberrant splicing of the prolactin receptor (PRLR) such that the ratio of the long to short splice variants is increased. To determine whether the change in PRLR isoform expression was causal in this disease, we used a splice-modulating oligomer (SMO) that knocked down expression of the long splice variant (LFPRLR). Using patient samples ex vivo, SLE-prone mice in vivo, high-dimensional flow cytometry and single-cell RNA-sequencing, we demonstrate that the aberrant PRLR isoform expression in SLE both directly and indirectly drives production of autoreactive immune cell phenotypes. Thus, LFPRLR knockdown decreased the expression of type I interferon signaling/response genes known to be biomarkers and hub genes for SLE, reduced immunoglobulins with signatures considered autoreactive, and averted glomerular kidney damage in SLE-prone mice. Importantly, LFPRLR knockdown reduced pathogenic B cells and other immune subsets that drive B-cell activation, without negative impact on healthy donor counterparts. Current treatments for SLE adversely affect healthy cells and do not concurrently eradicate multiple pathogenic immune subsets. Since LFPRLR SMO does not share these disadvantages, knockdown of the LFPRLR represents a potential treatment strategy for SLE that merits further investigation. Graphical AbstractThe LFPRLR represents an attractive therapeutic target in SLE. (Left) In addition to increased pituitary/circulating PRL, individuals with SLE exhibit aberrant increases in the production of autocrine/paracrine PRL by immune cells, and in their expression of specifically the long isoform (LF) of the PRLR. (Middle) Expression of the LFPRLR specifically enhances autoreactive immunophenotypes and promotes lupus nephritis. (Right) A splice modulating oligomer (SMO), that prevents synthesis of only the LFPRLR but not the short PRLR isoforms, reduces pathogenic immunophenotypes without affecting the normal counterparts of immune cells.

immunology↗

DisGeneFormer: Precise Disease Gene Prioritization by Integrating Local and Global Graph Attention

Identifying genes associated with human diseases is essential for effective diagnosis and treatment. Experimentally identifying disease-causing genes is time-consuming and expensive. Computational prioritization methods aim to streamline this process by ranking genes based on their likelihood of association with a given disease. However, existing methods often report long ranked lists consisting of thousands of potential disease genes, often containing a high number of false positives. This fails to meet the practical needs of clinicians who require shorter, more precise candidate lists. To address this problem, we introduce DisGeneFormer (DGF), an end-to-end disease-gene prioritization pipeline. Our approach is based on two distinct graph representations, modeling gene and disease relationships, respectively. Each graph is first processed separately by graph attention and then jointly by a transformer module to combine within-graph and cross-graph knowledge through local and global attention. We propose an evaluation pipeline based on the precision of a top K ranked gene list, with K set to clinically feasible values between 5 and 50, relying solely on experimentally verified associations as ground truth. Our evaluation demonstrates that DGF substantially outperforms existing methods. We additionally assessed the influence of the negative data sampling strategy as well as analyses of the effect of graph topology and features on the performance of our model.

bioinformatics↗

ACTIVATED CASO₄-INDUCED VACUOLATION AS A QUANTITATIVE PLATFORM FOR PHAGOCYTOSIS-DRIVEN DRUG SCREENING

Cytoplasmic vacuolization is a fundamental process associated with phagocytosis, lysosomal acidification, and autophagy, yet robust in-vitro models for its quantification and pharmacological screening remainlimitedor insufficiently established. In this study, we demonstrate that thermally activated calcium sulfate (ACS) induces extensive vacuolation across mammalian cell lines including HeLa, RAW 264.7, 3T3-L1, and SH-SY5Y, thereby establishing a versatile platform to study vacuole biogenesis. To ensure reproducibility, particle heterogeneity was addressed using sedimentation-based fractionation, with homogeneous suspensions obtained at the 5th minute producing stable and consistent vacuole formation. Vacuolation was subsequently quantified by Neutral Red (NR) uptake, dose and time dependent response analyses confirmed direct correlation between ACS concentration and vacuole induction. The assay was validated with bafilomycin A1 (BFA1), a selective V-ATPase inhibitor, which served as a positive control and demonstrated concentration and time dependent inhibition of vacuole formation and acidification. Building on this framework, ten commercially available drugs were screened, revealing distinct profiles ranging from early cytotoxicity, strong vacuole inhibition to partial suppression or negligible effects. This dual capacity to discriminate between vacuole inhibition and cytotoxic responses highlights the utility of ACS-induced vacuolization as a sensitive and scalable in vitro platform. Collectively, our findings position this system as a tractable assay for mechanistic studies of vacuole biology and a functional screening tool for identifying modulators of lysosomal and phagocytic pathways relevant to infection, Lysosomal Disorders, and Phagocytotic dysfunction disorders. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/711143v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@e5b920org.highwire.dtl.DTLVardef@1dd1072org.highwire.dtl.DTLVardef@62ae59org.highwire.dtl.DTLVardef@a468bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

TSC2 acting as a transcription factor for miR-514b-3p and regulating PI3K-AKT-MTOR pathway via nucleus

The PI3K-AKT-MTOR signalling pathway is pivotal in regulating cell survival, proliferation, and growth. TSC2 or tuberin acts as a key negative regulator of this pathway by forming a cytoplasmic complex with TSC1 and TBC1D7. While the cytoplasmic role of TSC2 is well established, emerging evidence suggests its nuclear functions. Previously, we have identified TSC2 as a transcriptional repressor of the EREG gene. Building on this foundation, the present study has investigated the transcriptional role of TSC2 in miRNA gene regulation. A genome-wide miRNA microarray profiling of TSC2-depleted SCC131 cells identified 19 upregulated and 24 downregulated miRNAs. Of these, miR-514b-3p emerged as one of the most upregulated miRNAs. The RT-qPCR analysis showed that TSC2 knockdown results in a robust miR-514b-3p upregulation, whereas TSC2 overexpression suppresses its expression in SCC131 cells. Moreover, TSC2 negatively regulates the MIR514B promoter activity in an NLS-dependent manner. Our ChIP analysis confirmed the direct binding of TSC2 to the MIR514B promoter, establishing miR-514b-3p as a transcriptional target of TSC2. We further identified TSPAN9 as a direct downstream target of miR-514b-3p. TSC2 positively regulates TSPAN9 levels by repressing miR-514b-3p, thereby establishing a novel TSC2/miR-514b-3p/TSPAN9 regulatory axis. We further uncovered a crosstalk between TSC2/miR-514b-3p/TSPAN9 axis and the canonical PI3K-AKT-MTOR signalling pathway, where miR-514b-3p positively and TSPAN9 negatively regulates this pathway. Interestingly, AKT functions as an upstream regulator of this axis by modulating the nuclear localization of TSC2. Collectively, this study provides new insights into the non-canonical, nucleus-dependent transcriptional functions of TSC2, thus expanding its role beyond cytoplasmic signalling regulation and underscoring its significance in the cellular signalling networks. Graphical abstractThe diagrammatic representation of the TSC2/miR-514b-3p/TSPAN9 axis and its interaction with the canonical PI3K-AKT-MTOR pathway. Abbreviations: PI3K, Phosphatidylinositol-3 kinase; TSC1, Tuberous sclerosis complex subunit 1; TSC2, Tuberous sclerosis complex subunit 2; TBC1D7, Tre2-Bub2-CDC16 domain family member 7; RHEB, Ras homolog enriched in brain; GTP, Guanosine tri phosphate; GDP, Guanosine di phosphate; MTORC1, Mechanistic target of rapamycin kinase complex 1; S6K1, p70 ribosomal protein S6 kinase; 4E-BP-1, eIF4E-binding protein 1; and TSPAN9, Tetraspanin 9. This figure was created in BioRender (https://app.biorender.com/).

cancer biology↗

A novel lipid-triggered allosteric site modulates LC3-LIR receptor binding activity

Membrane recruitment is a fundamental regulator of protein function. However, the allosteric mechanisms by which lipid binding controls protein activity remain poorly understood. In autophagy, the ubiquitin-like protein LC3 is lipid-anchored to autophagosomes, where it is essential for receptor recruitment and vesicle formation. While LC3-receptor interactions are structurally well defined, how membrane engagement governs LC3 functional dynamics has remained enigmatic. Here, we uncover that membrane binding triggers a major conformational transition in LC3, exposing functional pockets that are occluded in its cytosolic form. We demonstrate that this shift is mediated by dynamic coupling between the allosteric site (3- loop5-{beta}3-loop6) and the functional binding pockets. To conclusively test this mechanism, we utilised an ensemble-based protein design strategy guided by molecular dynamics to engineer the allosteric site. From a series of mutants, two variants emerged that stabilized LC3 conformation in either active or inactive state on the membrane. X-ray crystal structures of mutant LC3, biophysical assays, super-resolution microscopy, and TEM confirmed that the activated allosteric site mutant facilitates receptor binding and cargo capture. In contrast, the inactive variant is functionally inert on the membrane. Our work identifies a fundamental lipid-triggered allosteric site in LC3 that is critical for autophagy regulation and broader implication of membrane-dependent reprogrammable protein activities.

biophysics↗

From Stress to Survival: Trophoblast-Derived Extracellular Vesicle Proteome Captures Aspirin-Driven Cellular Reprogramming in a Preeclampsia Model.

BackgroundLow-dose aspirin (LDA) reduces preeclampsia (PE) risk by up to 40%, yet its molecular effects on chorion trophoblast cells (CTCs) a fetal membrane lineage at the feto-maternal interface remain obscure. CTCs form a structural and immunoregulatory barrier whose dysfunction drives inflammation-associated membrane pathology in PE. Extracellular vesicles (EVs) released by CTCs may encode cellular stress and adaptation states, offering a molecular window into aspirins timing-dependent effects on PE risk modification. MethodsHuman CTCs were challenged with cigarette smoke extract (CSE) to model oxidative stress-driven PE pathology. Two paradigms were tested: (1) prophylactic aspirin (4 and 40 {micro}g/ml) before and/or flanking CSE, and (2) therapeutic aspirin after CSE challenge. EVs were isolated via ultracentrifugation and size exclusion chromatography, characterized by nanoparticle tracking and immunoblotting, and profiled by quantitative mass spectrometry. Network pathway analysis and machine-learning biomarker selection defined EV-encoded molecular states. ResultsCTC-derived EVs from CSE-exposed cells carried a PE-like proteomic signature marked by suppressed VEGF/ECM remodeling, activated TNF-p53 apoptotic signaling, and heightened inflammation. Prophylactic low-dose aspirin shifted EV cargo toward preserved angiogenic capacity (VEGFA, COL1A1, MMP14) with attenuated apoptotic and NF-{kappa}B signatures. High-dose aspirin produced broad transcriptional suppression without vascular benefit in EVs. Therapeutic aspirin partially rescued injury-associated EV cargo but failed to restore angiogenic signatures. Machine-learning analysis of EV proteomes identified a prophylactic biomarker panel anchored by HSPA8, SERPINF2, COL4A1, and PLOD1, linked to angiogenic recovery and redox balance. ConclusionsCTC-derived EV proteomic signatures capture dose-and timing-dependent aspirin effects, positioning the chorion as a pharmacological "secondary responder" favoring cellular resilience over classical anti-inflammatory suppression. EV-based molecular profiling might offer a framework for stratifying aspirin responders from non-responders toward personalized PE prevention.

cell biology↗

Chromatin tethering to the nuclear envelope enhances its accessibility to RNAPII and promotes chromatin asymmetric organization

The intrinsic tendency of chromatin to self-attract competes with its association with RNA Polymerase II (RNAPII), a prerequisite for efficient transcription. Using high-resolution live imaging of chromatin and RNAPII organization in Drosophila larval muscle nuclei, we demonstrate that chromatin tethering to the nuclear envelope via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex is essential for maintaining chromatin three-dimensional organization. Disruption of chromatin-lamina interactions either in LINC mutants or following knockdown of Barrier-to-Autointegration Factor (BAF) results in enhanced chromatin clustering in the nucleoplasm and reduced RNAPII-chromatin interaction. Consistent with these observations, computer simulations revealed an inverse relationship between the chromatin cluster size and the degree of chromatin tethering to the nuclear lamina. We also measured chromatin distribution with respect to the nuclear lamina and found asymmetry of RNAPII distributions within chromatin clusters, which correlated with their proximity to the nuclear envelope, a relationship that is lost in nuclei lacking a functional LINC complex. Our findings demonstrate that chromatin association with the nuclear envelope counteracts chromatin self-attraction and facilitates RNAPII binding to DNA.

molecular biology↗