Biology subjects

Kamat, S.

Publications and source records attributed to Kamat, S..

16 recordsLinked to original sources

bioRxiv2026

Lysosomal acid lipase regulates cholesterol metabolism during phagosomal maturation

Phagocytosis, a central process in innate immunity, depends on dynamic lipid remodelling, yet how cholesterol accumulates on late phagosomes remains unresolved. Here, we identify lysosomal acid lipase (LIPA) as a key cholesterol ester (CE) hydrolase driving cholesterol mobilization during phagosomal maturation. Using integrated lipidomics, chemoproteomics, and biochemical assays, we show that LIPA exhibits acidic CE hydrolase activity enriched on late phagosomes, generating free cholesterol essential for lipid raft formation. Pharmacologically inhibiting LIPA disrupts cholesterol-rich lipid raft assembly, impairs phagosomal trafficking, and alters pathogen fate - enhancing Staphylococcus aureus persistence, while restricting Mycobacterium tuberculosis survival. These findings reveal that LIPA couples cholesterol metabolism to phagocytosis, defining a mechanistic link between lipid catabolism and antimicrobial defence. By positioning CE hydrolysis as a critical determinant of phagosomal dynamics, our work uncovers a metabolic checkpoint in innate immunity and identifies LIPA as a potential therapeutic node in infection and inflammation.

biochemistry↗

bioRxiv2026

Chaperoning through time - defining DNAJA2 co-chaperone client selectivity via pulse-SILAC and BioID.

Molecular chaperones are a major driving force ensuring efficient protein folding and regulating proteostasis in the cell. However, it remains unclear how their clients are selected in most cases, especially after the release of nascent protein chains from ribosomes. Here, we present a novel approach that combines pulse metabolic labelling with SILAC and BioID mass spectrometry to temporally resolve protein-protein interactions of a co-chaperone. Using the Hsp70 co-chaperone DNAJA2 as a benchmark, we reveal that two distinct pools of proteins are enriched. In particular, DNAJA2 displays a preferential association with highly structured recently synthesized proteins enriched in {beta}-strands. In contrast, pre-existing proteins captured in our assay exhibit higher intrinsic disorder. In both cases, these proteins tend to be longer and contain a lower net charge compared to the proteome. Notably, these preferential associations are retained upon heat-shock, while interactions with the "older" pool of proteins become more prevalent under these conditions. Through this methodology, we gain novel insights on how co-chaperone-client interactions may occur over the lifespan of a protein to preserve proteostasis.

biochemistry↗

bioRxiv2026

The Hunt for Cholesteryl Ester Hydrolases: Identification of Lipoprotein Lipase as a Cholesterylesterase

Cholesteryl esters (CEs) are central intermediates in cholesterol storage and transport, yet the enzymes responsible for their hydrolysis in mammals remain poorly defined. While lysosomal acid lipase is the only well-established acidic CE hydrolase, the molecular identity of physiologically relevant neutral CE hydrolases has remained unresolved. Here, we systematically profiled CE hydrolase activity across mouse tissues and blood using substrate-based LC-MS assays, tissue fractionation, and inhibitor screening. We observed robust CE hydrolase activity in multiple tissues and circulation, with activity predominantly enriched in membrane fractions and strongly sensitive to broad-spectrum metabolic serine hydrolase inhibitors. Pharmacological screening excluded previously proposed neutral CE hydrolases, including NCEH1 and LIPE, and identified tetrahydrolipstatin-sensitive lipoprotein lipase (LPL) as a candidate CE hydrolase. Competitive activity-based protein profiling analyses in RAW264.7 macrophages further supported selective enrichment and inhibition of LPL. Biochemical characterization demonstrated that recombinant wild-type LPL, but not the catalytic S159A variant, efficiently hydrolyzed CEs in vitro. Importantly, this activity required co-expression of the lipase maturation factor 1, indicating that LPL-mediated CE hydrolysis is dependent on proper enzymatic maturation. Together, these findings identify LPL as a previously unrecognized mammalian CE hydrolase and expand its functional role beyond triglyceride metabolism.

biochemistry↗

bioRxiv2026

Neuro-glial lipid imbalance in a Drosophila model of Amyotrophic Lateral Sclerosis 8

Membrane Contact sites (MCS) have emerged as physiologically relevant zones that coordinate inter-organelle communication and cellular function. VAPB, an ER-resident MCS tethering protein, plays a central role in regulating MCSs through its numerous protein interactors, thereby influencing cellular homeostasis. A pathogenic missense VAPBP56S mutation causes familial Amyotrophic Lateral Sclerosis 8 (ALS8) in humans, with progressive degeneration of motor neurons. The precise mechanisms underlying the motor neurodegeneration remain poorly understood. In this study, we examine lipid imbalance in the brain of a Drosophila model of ALS8 (VAPBP58S). Specifically, we find that lipid homeostasis is disrupted in an age-dependent manner. Strikingly, cholesterol esters and sphingolipids show an age-dependent increase, while cholesterol shows a decrease. Intriguingly, from a cellular perspective, despite the accumulation of triacylglycerols (TAGs) in the brains of VAPBP58S animals, the increased neutral lipid species do not correlate with lipid droplets (LDs), which are fewer in density and smaller in size. Lipid imbalance and progressive motor dysfunction in VAPBP58S animals can be reversed by expressing VAPBWT, suggesting a relationship between VAPB activity and lipid flux. To uncover VAPBs role in lipid homeostasis, we modulate VAPB activity in neurons and glia to dissect out tissue-specific roles. We find that both cell types contribute to lipid homeostasis in differential ways. In glia, LD flux is strongly dependent on VAPB activity, a dependence further recapitulated in cultured human cell lines, suggesting evolutionary conservation of the regulatory mechanism. Thus, we hypothesise that lipid dysregulation constitutes a critical pathogenic feature of ALS8, with the VAPBP56S allele disrupting lipid homeostasis in the neuro-glial axis. Summary StatementThe ER-membrane tethering protein VAPB regulates lipid homeostasis

neuroscience↗

bioRxiv2026

BAG6 and RNF126 promote the degradation of cytosolic misfolded proteins that contain buried degron motifs

Missense mutations account for the majority of catalogued human disease-associated variants, and many are predicted to destabilize proteins and promote their degradation. To characterize the pathways responsible for recognizing and clearing such variants, we employed a two-pronged approach to identify both quality control components mediating turnover of misfolded proteins and the sequence elements within their substrates that drive this process. Using a panel of unstable cytosolic missense variants in proximity-labeling and RNAi-based experiments, we identified the BAG6-RNF126 pathway as contributing to the clearance of a subset of these substrates. Applying a tile-based approach to a model cytosolic protein, we uncovered strong potential degrons, including a C-terminal degron degraded in part in a BAG6- and RNF126-dependent manner. Modeling supports that this degron can be accommodated by BAG6. Together, our findings add to the growing body of evidence implicating the BAG6-RNF126 pathway as a key mediator of cytosolic protein quality control.

molecular biology↗

bioRxiv2025

Biochemical characterization of ABHD14A, an outlying member of the metabolic serine hydrolase family

Certain uncharacterised members of the metabolic serine hydrolase enzyme family remain difficult to annotate due to poor tractability, context-dependent expression, and the absence of defined biochemical activities. Here, we provide the first functional characterization of the human enzyme ABHD14A. By engineering a soluble N-terminally truncated variant, we demonstrate by gel-based activity-based protein profiling and p-nitrophenyl-ester hydrolysis assays that ABHD14A is an active enzyme that preferentially turns over short-chain esters. Notably, ABHD14A exhibits a CoA-dependent enhancement of p-nitrophenyl-acetate hydrolysis, indicative of a ping-pong type acetyltransferase mechanism similar to that previously described for another homologous ABHD14-enzyme, ABHD14B. To investigate endogenous protein levels, we generated a high-affinity polyclonal antibody and found that ABHD14A is undetectable across a panel of immortalized mammalian cell lines and adult mouse tissues, challenging current transcriptomic database predictions. Upon heterologous expression in HEK293T cells, full length ABHD14A is catalytically active and localizes specifically to the Golgi apparatus, suggesting a specialized role in secretory pathway biology. Together, these findings establish the enzymatic activity, mechanistic features, and subcellular localization of ABHD14A, while providing essential biochemical and immunological tools that now enable the systematic discovery of its physiological substrates and regulatory contexts.

biochemistry↗

bioRxiv2025

Understudied and underprotected: biodiversity and conservation challenges in the Konkan region of Maharashtra

In biodiversity hotspots, such as the Western Ghats, periodic synthesis of existing ecological research can help identify knowledge gaps and address critical threats to biodiversity. The Maharashtra part of the Konkan region, situated between the Sahyadri foothills and the west coast, with diverse tropical vegetation, harbours unique open ecosystems, such as lateritic plateaus, and supports a multitude of threatened species, including hornbills and tigers. However, the region remains relatively understudied and has not received adequate protection through state or national conservation policies and laws. The area is undergoing rapid human-driven land-use change. These activities can impact the regions biodiversity, necessitating an effort to identify knowledge gaps and critical threats. Through a combination of a literature review and focus group discussions with 44 participants from various institutions and non-governmental organisations, we synthesised existing published information on the Konkan region of Maharashtra and identified key research gaps and conservation challenges. Our review of 138 studies found that while agroforestry and human-wildlife interactions have received some research attention, the effects of climate change on the regions biodiversity remain poorly understood. Focus group discussions highlighted major threats, including land-use changes due to expanding monoculture plantations, clear-felling of forests, forest fires, environmental pollution, and rapid infrastructure development, which are leading to habitat loss and fragmentation. Some of these concerns were validated by publicly available regional data, which revealed a 30% increase in roads, a 14% expansion of cashew plantations, and associated forest loss. This synthesis offers valuable insights for government and non-governmental organisations to inform future research and conservation efforts in the region.

ecology↗

bioRxiv2025

Bioinformatics analysis identifies sequence determinants of enzymatic activity for the PHARC associated lipase ABHD12

In humans, PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract) is an early onset autosomal recessive neurological disorder caused by deleterious mutations to ABHD12 (/{beta}-hydrolase domain protein # 12). Biochemically, ABHD12 functions as a lipase, and catalyzes the hydrolysis of lysophosphatidylserine (lyso-PS) (lyso-PS lipase). By doing so, it controls the concentrations and signaling pathways regulated by this potent signaling lysophospholipid in the mammalian brain. While genetic mapping efforts have identified over 30 mutations in ABHD12 from human PHARC subjects, the biochemical activity of these pathogenic mutants remains unknown. To understand this, here, we performed an exhaustive bioinformatics survey, and collated ABHD12 protein sequences from various organisms across evolution. Next, based on sequence alignments and structural modeling, we identified functionally relevant conserved residues in the ABHD12 protein sequence that are potentially important for its enzymatic activity. To validate these in silico findings, we generated numerous mutants of murine ABHD12, including those associated with human PHARC subjects, and assayed them for their enzymatic activity. Taken together, these complementary in silico and biochemical studies provide the first thorough sequence-function relationship for mammalian ABHD12, especially relevant in the context of PHARC. Finally, our evolutionary analysis identified CG15111 as an ABHD12 ortholog in the fruit fly (Drosophila melanogaster), and enzymatic assays indeed confirmed that recombinant CG15111 has robust lyso-PS lipase activity. Flies serve as an excellent animal system to model various human neurological diseases, and the identification of CG15111 as a Drosophila melanogaster ABHD12 ortholog opens new avenues to study PHARC in fly models.

biochemistry↗

bioRxiv2024

Wag31, a membrane tether, is crucial for lipid homeostasis in mycobacteria

The mycobacterial cytoskeletal protein Wag31 is necessary for maintaining cell shape and directing cellular growth and elongation. Wag31 has a characteristic N-terminal DivIVA-domain and a C-terminal coiled-coil domain. While the role of Wag31 in polar elongation is known, there is limited mechanistic insight on how it orchestrates growth and elongation. In this report, we delineate roles of the N- and C-terminal domains of Wag31 using genetics, state-of-the-art multi-omics, biochemical, and imaging approaches. We show that Wag31 predominantly interacts with several membrane-associated proteins involved in lipid metabolism, cell wall synthesis and division. Native levels of Wag31 are critical for the maintenance and distribution of membrane lipids. Both depletion and overexpression of Wag31 perturbs lipid homeostasis, leading to the formation of intracellular lipid inclusions (ILIs). Protein-lipid crosslinking and imaging studies reveal that purified Wag31 can bind and effectively tether Cardiolipin (CL)-containing liposomes. We further show that the tethering activity lies in the DivIVA-domain containing N-terminal of Wag31 while the C-terminal mediates protein-protein interactions of Wag31. Despite retaining its ability to interact with partner proteins, the DivIVA domain-deleted Wag31 mutant shows defects in liposome tethering in vitro and non-polar localization of CL in vivo, which eventually causes lethality. Our study suggests that membrane tethering licenses Wag31 to form scaffolds that help orchestrate protein-lipid and protein-protein interactions necessary for mycobacterial growth and survival.

microbiology↗

bioRxiv2024

Statins attenuate Wnt/β-catenin signaling by targeting SATB family proteins in colorectal cancer

Colorectal cancer is the second leading cause of cancer-related deaths worldwide, highlighting the need for improved treatments and advanced molecular research. A recent therapeutic approach focuses on repurposing drugs to target dysregulated pathways involved in tumorigenesis. Among these, statins, commonly known for lowering cholesterol, have attracted attention for their potential anti-cancer properties. Here, we provide direct evidence for the same by assessing the impact of statin treatment on lipid, transcript, and protein levels. Our findings reveal that statins specifically target key components of the Wnt/{beta}-catenin pathway, a major factor in adenoma formation, including the SATB (Special AT-rich Binding protein) family proteins. While SATB1 is recognized as a regulator of tumorigenesis, particularly under Wnt signaling, SATB2 appears to exert an opposing role. We demonstrate that statin treatment reciprocally alters the expression pattern of these proteins. Furthermore, a human clinical trial evaluating statins as an anti-cancer therapy supports the hypothesis that differential expression of SATB proteins is crucial in tumorigenic outcomes. In conclusion, this modulation by statin treatment suggests promising new therapeutic avenues through drug repurposing.

cancer biology↗

bioRxiv2024

Identification of ABHD6 as a regulator of lysophosphatidylserines in the mammalian liver and kidneys

Lysophosphatidylserine (lyso-PS) is a potent hormone-like signaling lysophospholipid, which regulates many facets of mammalian biology and dysregulation in its metabolism is associated with several human neurological and autoimmune diseases. Despite the physiological importance and causal relation with human pathophysiology, little is known about the metabolism of lyso-PS in tissues other than the nervous and immune systems. To address this problem, here, we attempted to identify one (or more) lipase(s) capable of degrading lyso-PS in different mammalian tissues. We found that the membrane proteomic fraction of most mammalian tissues possess lyso-PS lipase activity, yet interestingly, the only bona fide lyso-PS lipase ABHD12 displays this enzymatic activity and has control over lyso-PS metabolism only in the mammalian brain. Using an in vitro inhibitor screen against membrane proteomic fractions of different tissues, we find that another lipase from the metabolic serine hydrolase family, ABHD6, is a putative lyso-PS lipase in the mouse liver and kidney. Finally, using pharmacological tools, we validate the lyso-PS lipase activity of ABHD6 in vivo, and functionally designate this enzyme as a major lyso-PS lipase in primary hepatocytes, and the mammalian liver and kidneys. HIGHLIGHTSO_LILyso-PS lipase enzymatic activity is present in the membrane proteomic fraction of most mammalian tissues C_LIO_LIAn in vitro inhibitor screen identifies ABHD6 as a putative lyso-PS lipase in the liver and kidney C_LIO_LIABHD6 functions as a lyso-PS lipase in primary hepatocytes C_LIO_LIABHD6 also performs lyso-PS lipase activity in vivo and regulates lyso-PS levels in the liver and kidney C_LI

biochemistry↗

bioRxiv2024

The co-chaperone DNAJA2 buffers proteasomal degradation of cytosolic proteins with missense mutations

Mutations can result in the loss of a proteins native function due to protein misfolding, which is generally handled by an intricate protein quality control network. To better understand the triaging mechanisms of misfolded cytosolic proteins, we screened a human mutation library to identify a panel of unstable mutations. The degradation of these mutated cytosolic proteins is largely dependent on the ubiquitin proteasome system. Using BioID proximity labelling, we found that the co-chaperones DNAJA1 and DNAJA2 are key interactors of one of the mutated proteins. Notably, the absence of DNAJA2 increases the turnover of the mutant protein but not of the wild-type protein. Our work indicates that missense mutations in cytosolic proteins can promote interactions with molecular chaperones that normally do not occur. Assessment of the broader panel of cytosolic mutant proteins shows that the co-chaperone DNAJA2 exhibits three distinct behaviours: acting to stabilize solely the mutant, both the wild-type and mutant proteins, or being dispensable. Our work illustrates how distinct elements of the protein homeostasis network are utilized in the presence of a cytosolic misfolded protein. Summary StatementWe identified a panel of cytosolic mutant proteins degraded by the proteasome. DNAJA2 is often required to prevent mutant protein turnover, even if it is sometimes dispensable for the wild-type protein.

cell biology↗

bioRxiv2023

MORPHOGENIC VERSUS MITOGENIC ROLES OF SHH ARE SEGREGATED ON DISTINCT EXOSOMES REGULATED BY CELLULAR RAB7 LEVELS

The secreted signaling molecule, Sonic hedgehog (Shh) is involved in patterning and growth of various embryonic tissues across species. This lipid-anchored protein is secreted on extracellular vesicles to activate signaling. Shh has two functions during spinal cord development, acting as a morphogen to pattern the ventral neural tube and a mitogen to maintain neural progenitors. Here, we find that these activities are segregated on to two distinct pools of exosomes. Shh secreted on a classical exosomal pool (Shh-P150) is able to activate ventral threshold targets in the neural tube. In contrast, the mitogenic activity, is elicited by a lighter exosomal pool (Shh-P450). We further show that cellular environment plays a major role in regulating the biogenesis of P150 versus P450 by modulating Rab7 levels. We find that active Rab7 is necessary for ventral neural tube patterning through the regulation of Shh-P150 secretion. The cellular mechanisms involved in packaging and secretion of Shh with different partners on P150 or P450 pools may be more general, enabling the separation of functional activities of signaling molecules during development and disease.

developmental biology↗

bioRxiv2023

Metabolic transitions regulate global protein fatty acylation

Intermediary metabolites and flux through various pathways have emerged as key determinants of post-translational modifications. Independently, dynamic fluctuations in their concentrations are known to drive cellular energetics in a bi-directional manner. Notably, intracellular fatty acid pools that drastically change during fed and fasted states act as precursors for both ATP production and fatty acylation of proteins. Protein fatty acylation is well regarded for its role in regulating structure and functions of diverse proteins, however the effect of intracellular concentrations of fatty acids on protein modification is less understood. In this regard, we unequivocally demonstrate that metabolic contexts, viz. fed and fasted states, dictate the extent of global fatty acylation. Moreover, we show that presence or absence of glucose, that influences cellular and mitochondrial uptake/utilization of fatty acids, affects palmitoylation and oleoylation, which is consistent with their intracellular abundance in fed and fasted states. Employing complementary approaches including click-chemistry, lipidomics and imaging, we show the top-down control of cellular metabolic state. Importantly, our results establish the crucial role of mitochondria and retrograde signaling components like SIRT4, AMPK and mTOR in orchestrating protein fatty acylation at a whole cell level. Specifically, pharmacogenetic perturbations that alter either mitochondrial functions and/or retrograde signaling affect protein fatty acylation. Besides illustrating the cross-talk between carbohydrate and lipid metabolism in mediating bulk post-translational modification, our findings also highlight the involvement of mitochondrial energetics.

physiology↗

bioRxiv2018

SOD1 activity thresholds and TOR signalling modulate VAP(P58S) aggregation via ROS-induced proteasomal degradation in a Drosophila model of Amyotrophic Lateral Sclerosis

Familial Amyotrophic Lateral Sclerosis (F-ALS) is an incurable, late onset motor neuron disease, linked strongly to various causative genetic loci. ALS8 codes for a missense mutation, P56S, in VAMP-associated Protein B (VAPB) that causes the protein to misfold and form cellular aggregates. Uncovering genes and mechanisms that affect aggregation dynamics would greatly help increase our understanding of the disease and lead to potential therapeutics.\n\nHere, we develop a quantitative high-throughput, Drosophila S2R+ cell-based kinetic assay coupled with fluorescent microscopy to score for genes involved in the modulation of aggregates of fly ortholog, VAP(P58S), tagged with GFP. As proof of principle, we conducted a targeted RNAi screen against 900 genes, consisting of VAP genetic interactors, other ALS loci, as also genes involved in proteostasis. The screen identified 150 hits that modify aggregation, including the ALS loci SOD1, TDP43 and also genes belonging to the TOR pathway.\n\nTo validate these modifiers, we developed a system to measure the extent of VAP(P58S) aggregation in the Drosophila third instar larval brain using the UAS-GAL4 system, followed by quantitative imaging of cellular inclusions. We find that reduction of SOD1 activity or decreased TOR signalling reduces aggregation. Interestingly, we find that increase in cellular reactive oxygen species (ROS) levels, assessed by measuring oxidation of cellular lipids and proteins, in response to SOD1 knockdown or by inhibition of TOR signalling appears to be the trigger for clearing of aggregates. The mechanism of aggregate clearance is, primarily, the proteasomal machinery, and not autophagy. Increase in VAP, but not VAP(P58S) levels, appears to elevate ROS, which may in turn regulate VAP transcription in a feedback loop.\n\nWe have thus uncovered an interesting interplay between SOD1, ROS and TOR signalling that regulates the dynamics of VAP aggregation. Mechanistic processes underlying such cellular regulatory networks will lead us to a better understanding of initiation and progression of ALS.

neuroscience↗

bioRxiv2018

Drosophila DNA/RNA methyltransferase contributes to robust host defense in ageing animals by regulating sphingolipid metabolism

Drosophila methyltransferase (Mt2) has been implicated in methylation of both DNA and tRNA. In this study, we demonstrate that loss of Mt2 activity leads to an age dependent decline of immune function in the adult fly. A newly eclosed adult has mild immune defects that exacerbate in a fifteen-day old Mt2-/- fly. The age dependent effects appear to be systemic, including disturbances in lipid metabolism, changes in cell shape of hemocytes and significant fold changes in levels of transcripts related to host defense. Lipid imbalance, as measured by quantitative lipidomics, correlates with immune dysfunction with high levels of immunomodulatory lipids, sphingosine-1phosphate (S1P) and ceramides, along with low levels of storage lipids. Activity assays on fly lysates confirm the age dependent increase in S1P and concomitant reduction of S1P lyase activity. We hypothesize that Mt2 functions to regulate genetic loci such as S1P lyase and this regulation is essential for robust host defense as the animal ages. Our study uncovers novel links between age dependent Mt2 function, innate immune response and lipid homeostasis.

immunology↗