Biology subjects

Mitra, K.

Publications and source records attributed to Mitra, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Cryptic Genes as Building Blocks of Antimicrobial Resistance: Evidence from a Novel Marine Imipenem-Hydrolyzing Metallo-β-Lactamase

The emergence of antimicrobial resistant pathogens has led to increase in mortality rate, longer hospital stays, and need for complex and expensive treatment regimens. However, it is not very clear from where does the bacteria get the building blocks to develop resistance to these synthetic antimicrobial agents which have been integral part of modern medicine. One hypothesis is that non-coding cryptic genes without any vital function or positive contribution to fitness act as versatile endogenous genetic repertoires that help in bacterial adaptation and evolution of new phenotypes when conditions change. This study reports the presence of putative cryptic metallo beta lactamase gene in the marine bacterium Cytobacillus oceanisediminis isolated from the Gulf of Mannar, India. Using in silico, in vitro and in vivo assay, we demonstrate that putative cryptic metallo beta lactamase encodes an enzyme that can cleave only imipenem, has two characteristic domains: HXHXDH and TPGH, but does not share homology with the other reported Metallo-beta-Lactamase genes. The findings underscore the importance for comprehensive screening of putative cryptic antimicrobial resistance genes to fully address and mitigate the challenges posed by antimicrobial resistance.

microbiology↗

bioRxiv2026

Interaction of light-dark and dietary cues prime Drosophila ovarian stem cell properties by precisely tuning Drp1 recruitment and organization on mitochondria

The quiescent adult stem cells are activated to maintain tissue integrity. Mitochondria have emerged as critical regulators of stem/progenitor cells. Scattered literature suggests existence of a high-potency stemness state with characteristic mitochondrial structure-function properties that may prime stem cell activation. Precise tuning of mitochondrial structure by the mitochondrial fission protein, Drp1, can prime a stem cell state for neoplasticity in vitro. However, the in vivo significance and regulation of such a proposed primed stemness state remains elusive. We use quantitative genetic and cell biological approaches to demonstrate that light-dark cycling driven precise tuning of Drp1 primes the Drosophila ovarian germline stem cell (GSC) response to protein-rich dietary supplement towards stimulating egg production. This can be reversed by repressing mitochondrial fusion protein, Opa1, and is disrupted in the absence of functional Cry protein that maintains light-dark rhythm. Mechanistically, this is achieved by light or dark allowing protein-rich supplements to stimulate distinct characteristic recruitment and organization of Drp1 on mitochondria in GSC subpopulations. Screening of essential amino acids reveals the importance of interaction between Threonine supplementation, Cry, Drp1 and Opa1 in such priming of the GSCs and egg production. The priming of the ovarian follicle stem cells happens at a different tuned level of Drp1. Such Drp1-tuning driven priming of Drosophila ovarian stem cells reverses the inhibitory impact of diabetes-like condition on egg development in the presence of protein-rich supplement. Thus, our study revealing the paradigm of adult stem cell regulation by integration of light-dark and dietary cues via precise mitochondrial modulation, is significant in regenerative health and degenerative diseases.

developmental biology↗

bioRxiv2026

In search of intralocus sexual conflict in the multivariate and local genetic architecture of metabolic traits in humans

Despite a common genome, males and females are remarkably different across species. Intralocus Sexual Conflict (IASC) arises when selection favours different trait values in the sexes but a shared genetic architecture constrains their evolutionary divergence. IASC is typically studied at the single-trait level, and genome-wide architectures can obscure conflict localized to specific genomic regions. We investigated IASC in the multivariate and local genetic architecture of 17 human metabolic traits and lifetime reproductive success by estimating the sex-stratified additive genetic (co)variance matrix (Gmf) and local cross-sex-cross-trait genetic correlations. Genome-wide, between-sex covariance matrix (B) showed only 1.15% asymmetry, and sexually concordant (SC) genetic variation exceeded sexually antagonistic (SA) variation by 18.5-fold. Indirect evolutionary response to SC selection was 1.6-fold stronger than direct responses to SA selection. In contrast, local analyses revealed a more heterogeneous structure - among the regions with significant local correlations, 12% were consistently SA, showing opposite fitness effects in the sexes, with functional enrichment in WNT signalling, while 20% exhibited both SA and SC effects. Our results demonstrate that in human metabolic traits, SC variation predominates genome-wide, whereas SA variation is concentrated in specific local regions, highlighting the importance of integrating multivariate and local frameworks in understanding conflict.

evolutionary biology↗

bioRxiv2026

Host cell plasma membrane-derived vesicles efficiently inhibit in vitro Influenza A virus infection in a size-dependent manner

The influenza virus poses a significant global health threat due to its continuous evolution, immune evasion, and zoonotic spillover. The rise of drug resistance, reduced susceptibility to existing antiviral medications, and the limited effectiveness of annual vaccines underscore the need for new antiviral strategies. To infect, the influenza virus binds to sialic acid (SA)-containing molecules on host cell membranes through hemagglutinin (HA). Blocking this interaction represents a promising antiviral approach. Herein, we report that SA containing plasma membrane-derived vesicles (PMV) efficiently inhibits in vitro Influenza A virus (IAV) infection. Using orthogonal methods, we demonstrate that PMV derived from A549, MDCK, and HEK cells competitively bind to H1N1 (WSN) and H3N2 (X-31) IAV strains, block entry and infection in human respiratory epithelial cells in a dose-dependent manner, without causing significant toxicity. When the size of the vesicles was reduced through extrusion, the antiviral activity was enhanced, and this was found to be correlated with a size-dependent increase in hemagglutination inhibition and reduced IAV internalisation. Plasma membrane-derived vesicles may serve as a novel antiviral strategy against influenza virus infections due to their simple production method and conserved SA binding site on HA.

biochemistry↗

bioRxiv2026

Fall risk-aware adaptation explains suboptimal locomotor performance

AO_SCPLOWBSTRACTC_SCPLOWHuman locomotion requires balancing multiple biological objectives, such as metabolic energy efficiency, stability, and symmetry. While models based on optimization successfully predict how humans walk in familiar settings, they fail to explain why individuals adopt inefficient movement patterns in novel environments, even after extensive practice. Here, we show that such suboptimality in a novel environment arises from a fundamental prioritization of safety. We find that individuals do not simply fail to reach an optimal solution; instead, they navigate an environment-dependent risk landscape by mitigating the statistical probability of falling. We find that this risk-averse strategy is explained by adjusting internal learning parameters: specifically, the learning rate and the tradeoff between metabolic cost and symmetry, in a manner that lowers fall risk. To quantify this process, we developed an inverse adaptation modeling framework; this approach works backwards from locomotor performance data to mathematically infer the underlying internal learning parameters and how they vary with fall risk. Our analysis reveals that the observed motor performance is explained by a global probabilistic fall risk rather than a local step-based measure of instability. Ultimately, these findings reveal that fall risk-aware adaptation explains suboptimal locomotor behavior, providing a new data-driven framework to understand the drivers of motor performance.

neuroscience↗

bioRxiv2025

Organelles harbour pH gradients

Organelle pH is critical to organelle identity and function. Resident proteins that define each organelle modify transiting cargo proteins, with both retention and trafficking between organelles governed by pH-dependent mechanisms. For example, lysosomal enzymes bind mannose-6-phosphate receptors at the higher pH ([~]6.5) of the Golgi and dissociate at the lower pH ([~]5.5) of late endosomes1. Proteins that stray from the endoplasmic reticulum (ER) are captured by KDEL receptors in the acidic Golgi and returned into the neutral ER2,3. This pH-tuned trafficking system compartmentalizes organelle function and prevents mis-localization of critical enzymes4. Dysregulated organelle pH disrupts their function and leads to various diseases. Because protons move rapidly in water, the pH within a single organelle is currently assumed to be spatially uniform5. Here, using a reporter sensitive from pH 5.5 - 10.5 to map a spectrum of organelles at high resolution, we discovered that pH gradients exist within single, large or long organelles such as the ER and mitochondria, and in membrane-less organelles without ion-transporting proteins such as the nucleolus. These new findings upend our understanding of organellar pH, prompting new questions about proton diffusion within the cell, and its potential consequences on organelle function.

cell biology↗

bioRxiv2025

T13F2.2 of C. elegans is Critical for Chromatin Organization, Autophagy, and Longevity

Intricately modulated by a spectrum of proteins, Chromatin structure governs gene expression and cellular homeostasis. In Caenorhabditis elegans, critical components like the TATA-binding protein tbp-1 play pivotal roles in orchestrating chromatin dynamics. While the function of many of the interacting partners of tbp-1 is well-understood, our study brings into focus a lesser-known entity, T13F2.2, an unexplored tbp-1 interacting protein and a putative RNA polymerase II transcriptional coactivator. Employing reverse genetics, we found that RNAi-induced depletion of T13F2.2 resulted in pronounced disruptions to nuclear architecture, evidenced by nuclear staining and transmission electron microscopy. Accompanying these structural anomalies, we observed increased autophagy, pointing to cellular stress and a hyperacetylation of the core histones, suggesting potential chromatin decompaction. Notably, multifaceted functional alterations, upon the partial knockdown of the T13F2.2, culminated in a substantial reduction in the worms lifespan. Intriguingly, interventions such as administering ROS scavengers and autophagy modulators offered a reprieve from this life-shortening effect. Transcriptomic analysis upon T13F2.2 knockdown revealed upregulation of genes related to autophagy and chromatin remodelling, alongside downregulation of genes involved in longevity pathways and oxidative stress response. This study, thus, not only puts forward the functional implication of an uncharacterized gene in C. elegans biology, but also further emphasizes the role of chromatin organization in aging at the organismal level.

genetics↗

bioRxiv2025

Inflammasomes and DNA Damage Orchestrate Divergent Pyroptotic Fates in Neutrophils

The understanding of pyroptotic cell death and its mechanistic insights in neutrophils remains less explored and ambiguous. This study analyzes neutrophil pyroptotic responses under different conditions and dissects distinct pyroptosis-associated events, including IL-1{beta} release and cell death. We decipher the signalling pathways underlying these responses and distinguish them from other cell death mechanisms. Interestingly, we observe IL-1{beta} release and enhanced survival in neutrophils in response to LPS-primed ATP-treatment, in contrast to a combination of IL-1{beta} release with cell death in macrophages. While LPS-primed nigericin-treated neutrophils exhibit IL-1{beta} release and cell death, characterized by nuclear rounding, cell swelling/ ballooning, plasma membrane pore formation, and subsequent cell rupture, confirming the occurrence of pyroptotic cell death. While nigericin alone triggers cytokine uncoupled pyroptosis. Intriguingly, these phenomena do not induce under other death programs, including apoptosis, NETosis. This provides opportunity to unravel the regulation of these pyroptotic responses in neutrophils. Data observed reveal the role of NLRP3 and context-dependent caspase -1 & 11 in IL-1{beta} secretion. While, DNA damage and caspase-7 & 9 regulate pyroptotic death. Intriguingly, forced DNA damage by ATM kinase inhibitor mitigated inflammasome activation and IL-1{beta} release, while spur death. Furthermore, this study identifies perinuclear actomyosin forces driving nuclear rounding and pyroptotic death. Both murine neutrophils exposed to bacteria and human neutrophils treated with nigericin exhibit these pyroptotic processes, highlighting their broad relevance. This study depicts decision whether to secrete cytokines or undergo pyroptotic cell death emphasizing the regulatory role of DNA damage-cytoskeletal axis beyond inflammasome activation. Moreover, LPS and bacteria induced acute lung injury in mice displays nuclear rounding presenting pyroptotic neutrophils and enhanced NLRP3, Caspase-11 activation, and IL-1{beta} release. Together, this study defines intriguing crosstalk of NLRP3, caspases, DNA damage, and actomyosin forces that orchestrate divergent inflammatory fates, potentially leading to opportunities for targeted strategies for combating inflammation. HIGHLIGHTSO_LINeutrophils undergo pyroptotic cell death with nigericin and bacteria, but not with ATP. C_LIO_LILPS priming is essential for IL-1{beta} secretion, while dispensable for cell swelling/ballooning and death. C_LIO_LINLRP3 and Caspase 1/11 regulate IL-1{beta} secretion, while DNA damage and Caspase-7/9 control pyroptotic cell death. C_LIO_LIPerinuclear actomyosin signalling regulates nuclear rounding during pyroptotic cell death. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=174 HEIGHT=200 SRC="FIGDIR/small/673694v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@102da3forg.highwire.dtl.DTLVardef@46838borg.highwire.dtl.DTLVardef@16648faorg.highwire.dtl.DTLVardef@413bc2_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Non-Destructive Larval Genotyping of Danio rerio for Mitochondrial DNA Genetics

The rapid advancement of nuclear and mitochondrial genomic editing tools has created an urgent need for efficient, non-lethal larval genotyping methods in zebrafish (Danio rerio) research. This study optimizes and validates a non-destructive proteinase K digestion method for mitochondrial and nuclear DNA genotyping while characterizing its impact on larval survival and gene expression. Using optimized protocol parameters, we demonstrate successful amplification of different mitochondrial and nuclear genetic loci with consistently high sensitivity. Molecular validation through PCR, restriction fragment length polymorphism analysis, and Sanger sequencing confirmed the specificity and reliability of the extracted DNA. The method successfully detected C-to-T base edits in the mt-tl1 gene introduced using the FusX TALE Base editor system, demonstrating its applicability to gene editing studies. Both 48-well and optimized 96-well formats were used, enabling this approach to be deployed at scale. This optimized method enables researchers to correlate genotypes with phenotypes in longitudinal studies while maintaining specimen viability particularly valuable for investigating early-onset mitochondrial diseases and utilizes standard laboratory equipment and reagents, facilitating widespread adoption in zebrafish research while adhering to ethical principles in reducing animal mortality.

genetics↗

bioRxiv2025

M6A reader protein YTHDF3 regulates cardiomyocyte death and atrophy by modulating the alternative splicing program

BackgroundThe functional impact of m6A modifications on RNA is governed by reader proteins that read the m6A marks and process the RNA accordingly. Recent studies have highlighted the importance of m6A reader proteins in the heart. However, the function of a reader protein Ythdf3 in the heart remains completely unknown. ObjectiveWe aim to uncover the function of Ythdf3 in the hearts. Methods& ResultsHere, we show that Ythdf3 is indispensable for the heart, and its knockdown leads to cardiac cachexia evident by cardiomyocyte atrophy, death, and less dense myofibrils visualized by electron microscopy. We also found downregulation of Ythdf3 in response to doxorubicin, and its overexpression rescues doxorubicin-induced cardiomyocyte atrophy and death. Contrary to primarily cytoplasmic localization, we found that Ythdf3 localizes in the cardiomyocyte nucleus. Furthermore, using co-immunoprecipitation coupled with LC-MS/MS, we show that Ythdf3 interacts with splicing proteins like DDX5 and HNRNPU and regulates alternative splicing in the heart. Mechanistically, we found that Ythdf3 regulates the splicing of CaMKII{delta}, and its knockdown leads to an increase in CaMKII{delta}A and CaMKII{delta}C isoforms while a decrease in CaMKII{delta}9 isoform. ConclusionCollectively, Ythdf3 knockdown in the adult heart leads to cardiac cachexia due to the alteration of the splicing program.

cell biology↗

bioRxiv2025

In vitro and in vivo synergy of Vancomycin and β-lactams against drug-resistant Mycobacterium tuberculosis and Non-tuberculous mycobacteria

The unabated increase in antimicrobial resistance has underlined the importance of identifying novel drug combinations which eliminate infections more potently and likely reduce the emergence of resistance. In this context, we have identified Vancomycin and many {beta}-lactams as being potently active against drug-resistant Mycobacterium tuberculosis and non-tuberculous mycobacteria including M. abscessus, emerging as pathogens of concern owing to their inherent drug resistance profile. In this study, we have identified combinations of Vancomycin, a glycopeptide and {beta}-lactams, especially Ceftriaxone, Ceftazidime and Meropenem, in the presence or absence of Sulbactam, a {beta}- lactamase inhibitor, as possessing potent antimicrobial activity against several drug-resistant mycobacterial strains. The combination of Vancomycin and {beta}-lactams exhibited potent bactericidal activity and reduced the bacterial load better than either drug alone. The molecular basis of synergy was mediated by increase in permeability of mycobacterial cell as demonstrated by ethidium bromide assay. In the murine model of mycobacterial infection, synergistic combination of Vancomycin and {beta}-lactams outperformed clinically utilized drugs including Isoniazid, Rifampicin and Ethambutol against M. tuberculosis and Amikacin, Clarithromycin against M. abscessus. The combinations caused a significant reduction in bacterial load in various organs in M. tuberculosis and M. abscessus infected mice. Thus, the synergistic combination of Vancomycin and {beta}-lactams could potentially be utilized for treatment of recalcitrant mycobacterial infection especially those caused due to drug-resistant pathogens.

microbiology↗

bioRxiv2024

Quantitative analyses of structure-function heterogeneity of individual mitochondrial components reveal an early role of small-mitochondrial-networks in priming neoplastic transformation

Lack of quantitative understanding of the marked heterogeneity of multifaceted mitochondria poses challenges in unraveling their translatable role. Here, we hierarchically untangled multilevel heterogeneity of mitochondrial networks and non-networks by simultaneously analysing their structure-function within single components and their units, in cells and tissues. Such mito-SinComp quantitative analyses revealed that redox levels of mitochondrial networks and their dramatic intra-network heterogeneity can be predicted by structural features through a translatable machine-learning approach. Mito-SinComp identified and quantitatively characterized a redox-tuned subpopulation of Small-Mitochondrial-Networks(SMNs) that specifies stemness. These SMNs are generated by severing of nodes of oxidized Hyperfused-Mitochondrial-Networks(HMNs), are ten-folds smaller with specific network complexity and elevated mt-DNA nucleoid abundance. Thus, HMN to SMN conversion supports elevated expression of mtDNA genes in establishing MT-ND1(redox)-KRT15(stemness) transcriptomic interaction, as revealed by coupled scRNA-seq. This stemness priming of non-transformed cells is sustained after neoplastic transformation and is detected in patient transcriptome, demonstrating translational relevance of SMNs.

cell biology↗

bioRxiv2024

Sca-1 expression depicts pro-inflammatory murine neutrophils under steady state and pathological conditions

Neutrophils play a crucial role in various pathophysiological conditions, yet targeting them for therapeutic intervention has been discouraged due to the associated risk of infections. Thus, identification of neutrophil subsets and their involvement in inflammatory conditions is warranted for targeted therapeutic strategies. This study, through screening of surface proteins on neutrophils isolated from different tissue microenvironments, identified a distinct neutrophil subset, CD11b+Ly6G+Sca1+ neutrophils, expressing Stem cell antigen-1 (Sca-1). Interestingly, these Sca1pos neutrophils were more abundant in the liver than BM, blood, and lungs. Further analysis revealed that Sca1pos neutrophils are mature and activated with enhanced effector functions, including superoxide generation, phagocytosis, degranulation, and NETosis. Tracing studies demonstrated ageing-independent characteristics of Sca1pos neutrophils. Remarkably, Sca1pos pro-inflammatory neutrophils promote T cell proliferation through ROS, while inhibition of Sca-1 restores T cell proliferation and ROS generation. Intriguingly, inflammatory as well as metabolic cues induce the transition of conventional neutrophils (Sca1neg) to Sca1pos neutrophils and differentiation of progenitors (granulocyte monocyte progenitors, GMPs) into Sca1pos neutrophils. Furthermore, in vivo models of acute inflammation, peritonitis, and chronic inflammatory condition, non-alcoholic steatohepatitis (NASH), exhibit an increase of Sca1pos neutrophils at inflammatory sites, while the pharmacological approach using NAC specifically mitigates the expansion of these pro-inflammatory neutrophils. Collectively, our findings unveil a novel subset of Sca1pos neutrophils with implications for inflammation. Significance StatementNeutrophilic inflammation remains the leading driver in infectious and inflammatory diseases. Targeting neutrophil populations remained un-recommended due to hampering the immunological functions of neutrophils. The heterogeneity of neutrophils provides the perspective to target altered neutrophil subsets, but subtle changes defining neutrophil subsets make it complex and ambiguous. Our study identified abundant expression of Sca1on distinct neutrophils under steady state and inflammation. Thus, we reported previously undefined Sca1pos pro-inflammatory neutrophil subsets and elucidated their regulation. This study further established their involvement in acute and chronic inflammatory conditions. This understanding may further pave the way toward targeting specific neutrophil subsets in pathologies characterized by neutrophilic inflammation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/613221v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@62f3eaorg.highwire.dtl.DTLVardef@7baed3org.highwire.dtl.DTLVardef@12da63org.highwire.dtl.DTLVardef@1bfcce9_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LICD11b+Ly6G+Sca1+ neutrophil subset identified with <1% presence in BM and >40% frequency in the liver. C_LIO_LIThese neutrophils are mature and activated, demonstrating enhanced effector functions. C_LIO_LISca1pos neutrophils promote T cell proliferation and display pro-inflammatory characteristics. C_LIO_LIConventional Sca1neg neutrophils transition into Sca1pos neutrophils in response to inflammatory signals, while progenitors undergo differentiation. C_LIO_LIBoth acute and chronic inflammatory models show the expansion of CD11b+Ly6G+Sca1+ neutrophils. C_LI

immunology↗

bioRxiv2024

Mitochondrial calcium uniporter regulates human fibroblast-like synoviocytes invasion via altering mitochondrial dynamics and dictates rheumatoid arthritis pathogenesis

Rheumatoid arthritis (RA) is characterized by the aggressive migration and invasion of fibroblast-like synoviocytes (FLS) into cartilage and bone, a process that is significantly influenced by mitochondrial calcium uptake. This study highlights the critical role of mitochondrial calcium uniporter (MCU) in regulating FLS migration and mitochondrial dynamics, and its potential as a therapeutic target in RA. Notably, RA-FLS exhibited increased MCU expression and mitochondrial dysfunction compared to controls. Treatment with Ru360, a potent MCU inhibitor significantly reduced RA-FLS migration, calcium influx, and mitochondrial reactive oxygen species (ROS) levels, while restoring mitochondrial morphology and enhancing ATP production. Further analysis of MCU complex expression revealed elevated levels of MCU and other regulatory subunits (EMRE, MICU1, MICU2) in RA-FLS compared to controls, indicating mitochondrial dysfunction in RA. Mechanistically, MCU inhibition altered gene expression related to cytoskeletal dynamics, focal adhesion, and metabolic pathways. In human RA-FLS, MCU inhibition suppressed migration and invasion both in vitro and in vivo. The interaction between MCU and Miro1, essential for mitochondrial transport, was validated, and its disruption impaired FLS migration. Our findings highlight MCU as a promising therapeutic target to inhibit FLS migration and ameliorate RA progression.

immunology↗

bioRxiv2024

Small Extracellular Vesicle Signaling and Mitochondrial Transfer Reprograms T Helper Cell Function in Human Asthma

Small extracellular vesicles (sEVs) are known to orchestrate cell-cell communication, but the role of sEV signaling via mitochondria in perpetuating asthmatic airway inflammation is unknown. Myeloid-derived regulatory cells (MDRCs) are known to control CD4+ T cell responses in asthma. We demonstrate that airway MDRC-derived sEVs from asthmatics mediate T cell receptor engagement and transfer of mitochondria that induce antigen-specific activation and polarization of Th17 and Th2 cells; these cells are drivers of chronic airway inflammation in asthma. CD4+ T cells internalize sEVs containing mitochondria predominantly by membrane fusion, and blocking mitochondrial oxidant signaling in MDRC-derived sEVs mitigates T cell activation. Reactive oxygen species-mediated signaling that elicits T cell activation in asthmatics is sEV-dependent. Additionally, a Drp1-dependent mechanism in pro-inflammatory MDRCs promotes mitochondrial packaging within sEVs, which then co-localize with the polarized cytoskeleton and mitochondrial networks in recipient T cells. Importantly, intranasal transfer of mitochondria packaged sEVs enhances airway inflammation and Th polarization in vivo in a murine model of asthma. Thus, our studies indicate a previously unrecognized role for mitochondrial fission and sEV-mediated mitochondrial transfer-mediated signaling in dysregulated T cell activation and Th cell polarization in asthma which could constitute a novel therapeutic target.

immunology↗

bioRxiv2023

Protein-Ligand Binding Site Prediction and de Novo Ligand Generation from Cryo-EM Maps

Identification of protein-ligand binding sites is one of the most challenging tasks in drug discovery and design. Recent advances in machine learning community, particularly deep learning, inspired considerable research into deep learning-based methods for protein ligand binding site prediction (PLBP) and have achieved promising results. However one limitation of these methods and models is that they are developed and evaluated using conventional databases consisting of relatively small protein structures determined from X-ray crystallography and NMR with high resolution. When current PLBP methods are directly applied to large protein complexes, they either fail or produce very poor results. Given the increasing popularity of protein structures determined from Cryo-EM maps, which usually capture large protein complexes at relatively low resolution, there is a strong need to apply PLBP methods to Cryo-EM maps. For this purpose, we created a novel database (EMD-Ligand Dataset) consisting of Cryo-EM maps and information about their ligand-binding partners. Our study of several state-of-the-art PLBP methods show that even though they perform reasonably well on conventional databases, they do poorly on the novel EMD-Ligand Dataset, which calls for more research and work to be done by the community. As current methods for determining protein structures experimentally from Cryo-EM maps are time-consuming and expensive, we integrated PLBP methods with DeepTracer, an automatic and fast, de novo Cryo-EM protein structure modeling method, and established an end-to-end system capable of predicting ligand binding sites directly from Cryo-EM maps. Additionally, we leveraged an existing ligand generator to generate drug-like ligands from our predicted ligand-binding pockets and demonstrated its effectiveness.

bioinformatics↗

bioRxiv2023

SLC12A9 is a lysosome-detoxifying ammonium - chloride co-transporter

Ammonia is a ubiquitous, toxic by-product of cell metabolism. Its high membrane permeability and proton affinity causes ammonia to accumulate inside acidic lysosomes in its poorly membrane-permeant form: ammonium (NH4+). Ammonium buildup compromises lysosomal function, suggesting the existence of mechanisms that protect cells from ammonium toxicity. Here, we identified SLC12A9 as a lysosomal ammonium exporter that preserves lysosomal homeostasis. SLC12A9 knockout cells showed grossly enlarged lysosomes and elevated ammonium content. These phenotypes were reversed upon removal of the metabolic source of ammonium or dissipation of the lysosomal pH gradient. Lysosomal chloride increased in SLC12A9 knockout cells and chloride binding by SLC12A9 was required for ammonium transport. Our data indicate that SLC12A9 is a chloride-driven ammonium co-transporter that is central in an unappreciated, fundamental mechanism of lysosomal physiology that may have special relevance in tissues with elevated ammonia, such as tumors.

cell biology↗

bioRxiv2023

Musashi-2 causes cardiac hypertrophy and heart failure by inducing mitochondrial dysfunction through destabilizing Cluh and Smyd1 mRNA

Regulation of RNA stability and translation by RNA-binding proteins (RBPs) is a crucial process altering gene expression. Musashi family of RBPs comprising Msi1 and Msi2 are known to control RNA stability and translation. However, despite the presence of MSI2 in the heart, its function remains entirely unknown. Here, we aim to explore the cardiac functions of MSI2. We confirmed the presence of MSI2 in the adult mouse, rat heart, and neonatal rat cardiomyocytes. Furthermore, Msi2 was significantly enriched in the hearts cardiomyocyte fraction. Next, using RNA-seq data and isoform-specific PCR primers, we identified, Msi2 isoforms 1, 4, and 5 and two novel putative isoforms labeled as Msi2 isoforms 6 and 7 to be expressed in the heart. Overexpression of Msi2 isoforms led to cardiac hypertrophy in cultured cardiomyocytes. Additionally, Msi2 was also found to be significantly increased in a pressure-overload model of cardiac hypertrophy. To validate the hypertrophic effects, we selected isoforms 4 and 7 due to their unique alternative splicing patterns. AAV9-mediated overexpression of Msi2 isoforms 4 and 7 in murine hearts led to cardiac hypertrophy, dilation, heart failure, and eventually early death, confirming a pathological function for Msi2. Using global proteomics, gene ontology, transmission electron microscopy, and transmembrane potential measurement assays increased MSI2 was found to cause mitochondrial dysfunction in the heart. Mechanistically, we identified Cluh and Smyd1 as direct downstream targets of Msi2. Overexpression of Cluh or Smyd1 inhibited Msi2-induced hypertrophy and mitochondrial dysfunction in cardiomyocytes. Collectively, we show that Msi2 induces hypertrophy, mitochondrial dysfunction, and heart failure.

cell biology↗