Biology subjects

Kamat, S. S.

Publications and source records attributed to Kamat, S. S..

At least 19 recordsLinked to original sources

bioRxiv2026

Orthogonal Chemical Proteomic Strategies Reveal the Cholesteryl Ester Interactome in Mammalian Cells

Cholesteryl esters (CEs) are widely regarded as inert storage forms of cholesterol, yet their potential to directly engage cellular proteins remains largely unexplored. Here, we establish an integrated chemical proteomic platform to systematically define CE-protein interactions in mammalian cells. We developed and comparatively deployed three orthogonal photoaffinity labeling strategies: (i) metabolic assembly of a bifunctional CE probe through endogenous acyl-CoA:cholesterol acyltransferase activity, (ii) direct delivery of a structurally defined diazirine-alkyne CE analog, and (iii) fragment-assisted subtraction using sterol and fatty acyl control probes to resolve interactions dependent on the intact esterified scaffold. Each probe system was rigorously validated by lipidomics and UV-dependent crosslinking prior to quantitative proteomic analysis. Integration of these complementary modalities identified 495 CE-associated proteins spanning enzymes, transporters, scaffolding proteins, and canonical sterol-binding families. The limited overlap across strategies reveals that lipid-protein engagement is strongly conditioned by biosynthetic origin, probe topology, and intracellular routing. Functional and database annotations further demonstrate enrichment of druggable and disease-linked proteins, connecting CE interactions to metabolic, neurological, and cardiovascular pathways. Collectively, this work provides the first systems-level map of CE-protein interactions and establishes a generalizable, multimodal framework for chemically resolving lipid-protein interactomes.

biochemistry↗

bioRxiv2026

A lipid acyl code-based Dip2-Pkc1 signalling axis maintains mitochondrial integrity in eukaryotes

Organelle membranes employ diverse lipids to relay key signals for efficient coordination of cellular processes. Diacylglycerol (DAG) is a simple yet critical lipid secondary messenger, but the regulatory mechanisms and functional implications for its distribution remain poorly understood. We have recently shown that Protein Kinase C (Pkc1) activation is driven by selective DAGs (C36:0, C36:1), whose levels are governed by Disco-interacting protein 2 (Dip2) (Shambhavi et al., 2025). Here, through genetic, chemical, and lipidomic screens, we show that the absence of Dip2 leads to specific DAG accumulation on the mitochondrial membrane and impacts its morphology, function, and quality control in yeast. Remarkably, the elevated DAGs in{Delta} dip2 promote translocation of Pkc1 to mitochondria via its DAG-binding C1 domain, but not the HR1 domain, suggesting functional partitioning between the regulatory domains. We also show that only specific DAGs, not the bulk DAGs, are required for Pkc1s targeting and inactivating Phospholipase C (Plc1) restores Pkc1 localisation and the associated mitochondrial defects. In addition, we identify that respiratory growth triggers specific DAG (C36:1) accumulation in the mitochondria, thereby promoting Pkc1 recruitment. Furthermore, we establish that the Psi1-Plc1-Dip2 axis is required for survival under respiratory growth conditions. Taken together, our study uncovers a novel, Dip2-mediated, unconventional Pkc1 signalling axis for maintaining mitochondrial homeostasis under nutrient transition and highlights how distinct lipid fingerprints enable precise control over organellar homeostasis.

cell biology↗

bioRxiv2026

γLinolenic Acid Induces a Vitamin D Receptor-Independent Mineralization Program by Activating CaMKII--SMAD2/3 Pathway in Calvarial Osteoblasts

Vitamin D receptor (VDR) signaling is essential for osteoblast maturation and skeletal mineralization, yet the intracellular mechanisms linking VDR activity to matrix production remain poorly defined. Here, we show that vdr-/- calvarial osteoblasts initiate differentiation but fail to complete the transition to a mature, mineralizing state, exhibiting suppressed late-stage markers such as Dmp1, Phex, and Col1a1 and defective nodule formation both in vivo and in vitro. Transcriptomic profiling revealed a SMAD network imbalance, with elevated inhibitory SMADs and attenuated phosphorylation of both SMAD1/5/9 and SMAD2/3. Whereas SMAD1/5/9 activation was preserved in vivo and restored by exogenous BMP2 in vitro, consistent with in vivo paracrine BMP availability, reduced SMAD2/3 phosphorylation persisted in both contexts, indicating a cell-autonomous defect. Mechanistically, vdr-/- osteoblasts displayed impaired intracellular Ca2+ dynamics and diminished CaMKII activation, with VDR/RXR occupancy detected near the Camk2g locus. Pharmacological CaMKII inhibition (KN-93) recapitulated the phenotype, reducing SMAD2/3 phosphorylation and mineralization and establishing CaMKII as an upstream regulator of SMAD2/3. A milk-based diet rescued skeletal defects in vdr-/- mice in a calcium-independent manner and selectively restored CaMKII-SMAD2/3 signaling. Metabolomic profiling identified the omega-6 fatty acid y-linolenic acid (GLA) as an elevated circulating mediator; exogenous GLA restored Ca2+ flux, CaMKII activation, SMAD2/3 phosphorylation, matrix production, and mineralization, without reactivating Runx2, Sp7, or BMP-SMAD1/5/9 signaling, and dependent on CaMKII activity. These findings reveal a VDR-independent, GLA-inducible CaMKII-SMAD2/3 mineralization program, positioning metabolic modulation of calcium signaling as a strategy to restore osteoblast function under impaired vitamin D signaling.

physiology↗

bioRxiv2026

Wunen(s) help navigate Primordial Germ Cells by attenuating Hedgehog signaling

Directed cell migration is a vital process that depends on the combined activities of attractive and repulsive cues. As it is essential for normal development, the precise identity of guidance signals and the underlying molecular and cellular mechanisms is being rigorously investigated. In a Drosophila embryo, PGC migration is orchestrated by non-cell autonomous repulsive and attractive cues, controlled by Wunen(s) - Wunen and Wunen2 and, HMGCoA-reductase (Hmgcr), respectively. Hedgehog (Hh), a PGC attractant, is potentiated by Hmgcr. We demonstrate that Wunen(s) employ both nonautonomous and autonomous modes to inhibit Hh signaling. Consistently, in embryos maternally compromised for wunen, mesodermal cells and PGCs accumulate excess Hh, leading to precocious clumping of the PGCs. This behaviour is reminiscent of PGC-specific loss of patched (ptc) - the Hh receptor and an antagonist of Smoothened (Smo), a G protein-coupled receptor (GPCR), involved in Hh signal transduction. Consistently, Wunen(s) inhibit membrane localization of Smo. Conversely, simultaneous overexpression of wunen mitigates PGC scattering induced by ectopic hmgcr expression. Finally, unbiased lipidomics of embryonic extracts after maternal knockdown of wunen confirms disruptions in lipid metabolism. We discuss the mechanistic underpinnings of Wunen(s) involvement in repressing Hh signalling to engineer PGC migration.

developmental biology↗

bioRxiv2026

Lysine specific demethylase 1 (LSD1) regulates host alpha-ketoglutarate levels to modulate lipid peroxidation during Mycobacterium tuberculosis infection

Mycobacterium tuberculosis (Mtb) subverts host immune responses via modulation of host epigenome and metabolism. In this study, we underscore a role for the epigenetic modifier, Lysine Specific Demethylase 1 (LSD1), in regulating macrophage metabolism to support mycobacterial pathogenesis. In ex vivo and in vivo infection models, LSD1 inhibition reduced mycobacterial CFU alleviating lung pathology. Metabolomic analysis of Mtb infected, LSD1 deficient macrophages revealed increased levels of alpha-ketoglutarate (AKG), a crucial TCA cycle metabolite via regulating genes implicated in glutamine breakdown. Moreover, exogenous addition of AKG resulted in reduced oxidative stress and attenuated lipid peroxidation (LPO) with a consequent decrease in Mtb survival. Blocking glutamine breakdown in LSD1 deficient macrophages failed to reduce LPO and promoted Mtb intracellular survival, highlighting the role of LSD1-AKG axis in this immunomodulation. Dietary supplementation of AKG to Mtb infected mice improved lung pathology, limited Mtb dissemination and reduced the levels of oxidative Malondialdehyde adducts. Therefore, we highlight a host protective role of AKG during Mtb pathogenesis through suppression of lipid peroxidation and uncover an epigenetic-metabolic axis exploited by Mtb, thereby positing dietary supplementation of AKG as a potential therapeutic strategy against Tuberculosis.

immunology↗

bioRxiv2025

SLAMF1-peptide mediated epigenetic priming reprograms innate immune responses in sepsis

Sepsis is characterized by profound immune dysregulation, including impaired innate immune responses and epigenetic reprogramming of monocytes. However, strategies to therapeutically restore immune function remain limited. Here, we identify a cell-penetrating peptide, P7-Pen, as a modulator of monocyte epigenetic state and inflammatory responsiveness. Using primary human monocytes and peripheral blood mononuclear cells (PBMCs) from healthy donors and sepsis patients, we demonstrate that P7-Pen enhances cytokine production in response to Toll-like receptor stimulation while having minimal effects under basal conditions. P7-Pen treatment increased global histone acetylation, particularly H3 acetylation, and prevented the development of endotoxin tolerance. Transcriptomic and functional analyses revealed restoration of inflammatory gene expression, including TNF, IL6, and IFNB1, in otherwise hyporesponsive cells. Mechanistically, we identified the lysine deacetylase ABHD14B as a direct binding partner of P7-Pen. Silencing of ABHD14B recapitulated the effects of P7-Pen, leading to enhanced histone acetylation and cytokine production. Importantly, P7-Pen selectively potentiated responses to TLR ligands without inducing basal hyperinflammation. Collectively, our findings identify ABHD14B-dependent epigenetic regulation as a key checkpoint in innate immune tolerance and establish P7-Pen as a novel tool to restore immune responsiveness in sepsis.

immunology↗

bioRxiv2025

A metabolic hierarchy directs cell cycle transition and morphogenesis

To optimize survival, cells must align differentiation and proliferation with metabolic status. Yet, how metabolic cues fine-tune cell cycle programs and morphogenesis remain unclear. Here, we show that cytosolic redox dynamics critically govern cell cycle transitions in Caulobacter crescentus. By integrating evolutionary genetics with fluxomics, we uncover temporally orchestrated shifts in core metabolic pathways that remodel cytosolic redox across the cell cycle. Early stages channel carbon flux toward unsaturated fatty-acid synthesis and a reverse-TCA to drive cytosolic oxidation-coupled G1-S transition. The later stages depend on an enhanced forward-TCA cycle that promote cytosolic reduction-driven proliferation. Strikingly, perturbing fatty-acid synthesis or reverse/forward-TCA uncouples growth from proliferation. Intriguingly, a biphasic glucose uptake program dictates the cell cycle-stage-specific metabolism. These findings reveal an unprecedented metabolic-redox circuitry coordinating morphogenesis with cell cycle progression in a carefully choreographed menage a trois.

microbiology↗

bioRxiv2025

Selective Targeting of Kinesin on Lipid Droplets in the Liver Reduces Serum Lipids

The liver controls plasma lipids by secreting lipid-rich very low density lipoproteins (VLDL) into blood. Inside hepatocytes in the liver, Lipid Droplets (LDs) are transported to the smooth Endoplasmic Reticulum (sER) by kinesin-1 motors, and then catabolized in the sER to supply lipids for VLDL assembly. LDs are the only cellular organelle bounded by a phospholipid monolayer, and are thus distinct from all other (bilayer-bounded) organelles. It is therefore plausible that a given protein can bind to the LD membrane using mechanisms that are completely different from all other organelles. Indeed, here we find that kinesin-1 uses its tail domain to bind LDs, but alternative mechanisms to bind other organelles. A peptide corresponding to kinesins tail domain therefore competes with, and removes kinesin-1 selectively from LDs with minimal effect on other organelles. Delivery of lipids for VLDL assembly is consequently reduced, causing a remarkable reduction of [~]50% of secreted lipids (triglycerides and cholesterol) in cell culture. We further develop Orally fed Egg-liposomes as a method to deliver kinesin tail domain peptide to the liver of Zebrafish. The peptide reverses diet-induced hyperlipidaemia in Zebrafish larvae and brings the larvae back to a normolipidaemic state, thus confirming the effectiveness of our method in a physiologically relevant in-vivo situation. Strikingly, the peptide causes no unwanted accumulation of lipids in the liver, no toxicity and no developmental or behavioural defects in Zebrafish. Using a peptide to displace proteins (e.g. kinesin) selectively from LDs provides a conceptually novel and radically different approach against hyperlipidaemia. This monolayer-versus-bilayer strategy can be potentially extended to target other LD-bound proteins that function as key regulators of Lipid metabolism.

cell biology↗

bioRxiv2025

Integrative lipidomics of brain and plasma uncovers sex-specific metabolic signatures in Parkinson's disease

Lipid dysregulation is increasingly recognized as a key feature of Parkinsons disease (PD). A central unresolved question, however, is whether lipidomic signatures identified in accessible peripheral biofluids faithfully recapitulate the pathogenic alterations within the central nervous system (CNS). This gap impedes the development of reliable biomarkers and constrains a comprehensive understanding of PD pathophysiology. To address this challenge, we employed a cross-species lipidomic approach. We modeled PD in both male and female mice by injecting human -synuclein preformed fibrils into the substantia nigra. Three months post-injection, lipidomic profiles of the midbrain and plasma were generated and compared. These findings were further validated in plasma from male and female PD patients and age-matched controls, enabling the identification of conserved alterations. We identified shared dysregulation of sphingolipids, glycerophospholipids, and fatty acids in the brains and plasma of diseased mice as well as in plasma from PD patients. Notably, lipids associated with lipid droplet biogenesis, including triacylglycerols and monoacylglycerols, were elevated in diseased mouse brains and patient plasma. These alterations coincided with a marked accumulation of lipid droplets in the mouse midbrain, and were further corroborated by increased lipid droplet abundance in macrophages derived from PD patients. Interestingly, lipid droplet accumulation exhibited sex-specific patterns: male mice displayed greater microglial accumulation, whereas female mice showed enhanced neuronal deposition. Together, these findings demonstrate that peripheral lipidomic signatures reflect CNS pathology in PD, highlighting new opportunities for biomarker discovery and therapeutic intervention. Furthermore, sex-specific lipid droplet accumulation in innate immune cells and neurons implicates these pathways as mechanistic contributors to PD and underscores the necessity of sex-stratified strategies in biomarker discovery and disease modeling.

neuroscience↗

bioRxiv2025

PE11 promotes intracellular persistence of Mycobacterium tuberculosis by inhibiting autophagy and lysosomal biogenesis by targeting the FLCN-lactate-TFEB signaling axis

Mycobacterium tuberculosis (Mtb) employs multiple virulence factors, including cell wall-associated proteins, to evade host immune responses. PE11, a cell wall-localized esterase, contributes to Mtb persistence by facilitating cell wall remodelling and resistance to acidic and antibiotic stress. Herein we describe a novel role of PE11 in subverting host autophagy through disruption of TFEB-mediated lysosomal function. PE11 promotes FLCN-dependent depletion of intracellular lactate to destabilize TFEB and thereby downregulate genes essential for autophagic flux and lysosomal acidification. Using a PE11-deficient Mtb strain, we demonstrate that PE11 targets the FLCN-lactate axis to regulate TFEB stability. Exogenous lactate supplementation restored TFEB stability, enhanced lysosomal acidification, and significantly reduced intracellular bacterial burden. Lactate also synergized with frontline anti-tubercular drugs to improve Mtb clearance. These findings establish PE11 as a key immune evasion factor and highlight lactate as a promising host-directed therapeutic to enhance bacterial killing and reduce antibiotic-associated toxicity.

microbiology↗

bioRxiv2025

Developing epidermis acquires nutrients from the external milieu by mTOR-dependent macropinocytosis

The vertebrate epidermis transitions from a simple bilayer to a complex, multilayered epithelium during development. Periderm, the outermost layer of the bilayered epidermis, is in direct contact with the external aquatic surrounding. While the barrier function of the epidermis is well established, the functional repertoire of the developing epidermis remains unclear. Using zebrafish, we uncover a function of the periderm in nutrient acquisition. Our findings reveal a developmentally regulated wave of macropinocytosis, which is further amplified by extracellular nutrients, leading to the formation of large endo-lysosomal compartments termed Stimulated Nutrient Activated Compartments (SNACs). We identify mTOR as a key integrator of developmental and nutrient-derived signals that modulate SNAC biogenesis. A global lipidomics analysis demonstrates that macropinocytosis-mediated uptake drives the production of metabolically relevant lipids and enhances animal survival when gut function is compromised. Our study identifies a hitherto unappreciated route of nutrient acquisition by the metabolically responsive developing epidermis. We propose that this mechanism fulfils a vital nutritional role, particularly during the transitional phase when maternally provided resources are depleting and the gut is not yet fully operational.

developmental biology↗

bioRxiv2024

Chemical Proteomics Identifies Protein Ligands for Monoacylglycerol Lipids

Signaling lipids are hormone-like small biomolecules that regulate many critical facets of physiology in mammals, including humans. Given their biomedical importance, the past few decades have seen a tremendous increase in our mechanistic understanding of the physiological processes regulated by a handful of such signaling lipids (e.g.: endocannabinoids, lysophospholipids, prostaglandins). However, a significant number of signaling lipid classes still remain poorly characterized, despite their direct associations to human pathophysiology and disease. Over the past decade, the advent of chemical proteomics technologies coupled with the development of multifunctional lipid probes has rapidly expanded our knowledge in terms of the protein ligands and biological pathways that the different signaling lipids interact with and modulate respectively. While the signaling pathways regulated by the endocannabinoid 2-arachidonoyl-glycerol in mammals are extensively characterized, the same cannot be said for the other members of the monoacylglycerol (MAG) family of signaling lipids. To understand this, here, we report the synthesis of a bifunctional MAG probe, containing a photoreactive group and a biorthogonal handle. Using established chemical proteomics approaches, we profile this bifunctional MAG probe in mouse brain and mammalian cell lysates, and leveraging probe competition experiments identify hitherto unknown protein ligands for MAG lipids. Finally, we biochemically validate the neuronal calcium sensor Hippocalcin as a putative MAG protein ligand, and show for the first time, that MAG may have a role to play in calcium sensing and downstream signaling in the mammalian brain.

biochemistry↗

bioRxiv2024

Emergence of Dip2-mediated Specific DAG-based PKC Signalling Axis in Eukaryotes

Diacylglycerols (DAGs) are used for metabolic purposes and are tightly regulated secondary lipid messengers in eukaryotes. DAG subspecies with different fatty-acyl chains are proposed to be involved in the activation of distinct PKC isoforms, resulting in diverse physiological outcomes. However, the molecular players and the regulatory origin for fine-tuning the PKC pathway are unknown. Here, we show that Dip2, a conserved DAG regulator across Fungi and Animalia, has emerged as a modulator of PKC signalling in yeast. Dip2 maintains the level of a specific DAG subpopulation, required for the activation of PKC-mediated cell wall integrity pathway. Interestingly, the canonical DAG-metabolism pathways, being promiscuous, are decoupled from PKC signalling. We demonstrate that these DAG subspecies are sourced from a phosphatidylinositol pool generated by the acyl-chain remodelling pathway. Furthermore, we provide insights into the intimate coevolutionary relationship between the regulator (Dip2) and the effector (PKC) of DAG-based signalling. Hence, our study underscores the establishment of Dip2-PKC axis about 1.2 billion years ago in Opisthokonta, which marks the rooting of the first specific DAG-based signalling module of eukaryotes.

cell biology↗

bioRxiv2024

Breast cancer spheroids prefer activated macrophages as an accomplice: An in vitro study

Cancer, a heterogeneous disease in nature, often requires help from diverse pro-tumor or tumor-associated- cells, which are recruited and persevered within the stroma. Pro-tumor stromal cells provide the essential support needed for tumor growth, metastasis, and development of drug resistance in due time. Tumor-associated macrophages, one of such cells, are essential to tumor microenvironment and tumor survival. In recent years, TAMs have been identified as potential drug targets and therapeutic agents, which encourages the in-depth characterization of their crosstalk with the tumors. The current study has successfully developed a cost-effective in vitro platform for Chemokine Assisted Recruitment of Macrophages to spheroids mimicking the physiology of TAM recruitment. Firstly, monocytic cell line (U937) were converted into activated naive macrophages (M0) and pro-and anti-inflammatory (M1 and M2) subtypes. Monocytes, M0, M1, and M2 macrophages are characterized extensively. Secondly, the naive and polarized macrophages were subjected to chemokine-dependent recruitment into monotypic and heterotypic breast cancer spheroids. The nature of the recruitment is further investigated by assessing the profile of chemokines and chemokine receptors. Recruited macrophages are also observed to manipulate spheroid behavior in many ways. The recruited macrophages also exhibit an increased level of Siglec-1 (CD169), one of the potential TAM markers. The current platforms potential for application can be extended to understand the recruitment process of other immune/stromal cells to solid tumors. It could be a potential addition to the arrays of in vitro platforms developed to screen the efficiency of cell-based immunotherapeutics in the future.

cancer biology↗

bioRxiv2024

A Facile LC-MS Method for Profiling Cholesterol and Cholesteryl Esters in Mammalian Cells and Tissues

Cholesterol is central to mammalian lipid metabolism and serves many critical functions in the regulation of diverse physiological processes. Dysregulation in cholesterol metabolism is causally linked to numerous human diseases, and therefore, in vivo, the concentrations and flux of cholesterol and cholesteryl esters (fatty acid esters of cholesterol) are tightly regulated. While mass spectrometry has been an analytical method of choice for detecting cholesterol and cholesteryl esters in biological samples, the hydrophobicity, chemically inert nature and poor ionization of these neutral lipids has often proved a challenge in developing lipidomics compatible liquid chromatography-mass spectrometry (LC-MS) methods to study them. To overcome this problem, here, we report a reverse-phase LC-MS method that is compatible with existing high-throughput lipidomics strategies, and capable of identifying and quantifying cholesterol and cholesteryl esters from mammalian cells and tissues. Using this sensitive yet robust LC-MS method, we profiled different mammalian cell lines and tissues, and provide a comprehensive picture of cholesterol and cholesteryl esters content in them. Specifically, amongst cholesteryl esters, we find that mammalian cells and tissues largely possess monounsaturated and polyunsaturated variants. Taken together, our lipidomics compatible LC-MS method to study this lipid class opens new avenues in understanding systemic and tissue-level cholesterol metabolism under various physiological conditions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/587668v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@badb0dorg.highwire.dtl.DTLVardef@1833f2aorg.highwire.dtl.DTLVardef@1a7e868org.highwire.dtl.DTLVardef@8b0ad9_HPS_FORMAT_FIGEXP M_FIG TOC Graphic C_FIG

biochemistry↗

bioRxiv2024

PPARγ mediated enhanced lipid biogenesis fuels Mycobacterium tuberculosis growth in hepatocytes.

Mycobacterium tuberculosis (Mtb) infection of the lungs, besides producing prolonged cough with mucus, also causes progressive fatigue and cachexia with debilitating loss of muscle mass. While anti-tuberculosis (TB) drug therapy is directed toward eliminating bacilli, the treatment regimen ignores the systemic pathogenic derailments that probably dictate TB-associated mortality and morbidity. Presently, it is not understood whether Mtb spreads to metabolic organs and brings about these impairments. Here we show that Mtb creates a replication-conducive milieu of lipid droplets in hepatocytes by upregulating transcription factor PPAR{gamma} and scavenging lipids from the host cells. In hepatocytes, Mtb shields itself against the common anti-TB drugs by inducing drug-metabolizing enzymes. Infection of the hepatocytes in the in vivo aerosol mice model can be consistently observed post-week 4 along with enhanced expression of PPAR{gamma} and drug-metabolizing enzymes. Moreover, histopathological analysis indeed shows the presence of Mtb in hepatocytes along with granuloma-like structures in human biopsied liver sections. Hepatotropism of Mtb during the chronic infectious cycle results in immuno-metabolic dysregulation that could magnify local and systemic pathogenicity, altering clinical presentations.

microbiology↗

bioRxiv2023

Identification of Sequence Determinants for the ABHD14 Enzymes

Over the course of evolution, enzymes have developed remarkable functional diversity in catalyzing important chemical reactions across various organisms, and understanding how new enzyme functions might have evolved remains an important question in modern enzymology. To systematically annotate functions, based on protein sequences and available biochemical studies, enzymes with similar catalytic mechanisms and/or aspects of catalysis have been clustered together into an enzyme superfamily. Typically, enzymes within a superfamily have similar overall three-dimensional structures, conserved key catalytic residues, but large variations in substrate recognition sites and residues to accommodate the diverse biochemical reactions that are catalyzed within the superfamily. The serine hydrolases are an excellent example of such an enzyme superfamily, that based on known enzymatic activities and protein sequences, is split almost equally into the serine proteases and metabolic serine hydrolases. Within the metabolic serine hydrolases, are two outlying members, ABHD14A and ABHD14B, that have high sequence similarity, but their functions remained cryptic till recently. While ABHD14A still lacks any functional annotation to date, we recently showed that ABHD14B functions as a lysine deacetylase in mammals. Given their high sequence similarity, automated databases wrongly assign ABHD14A and ABHD14B as the same enzyme, and therefore, annotating functions to them in various organisms maybe problematic. In this paper, we present a bioinformatics study coupled to biochemical experiments, that identifies key sequence determinants for both ABHD14A and ABHD14B, and enables better classification for them. Additionally, we map these enzymes on an evolutionary timescale, and provide a resource in studying these interesting enzymes in different organisms.

biochemistry↗

bioRxiv2022

DIP2 is a unique regulator of diacylglycerol lipid homeostasis in eukaryotes

Chain-length specific subsets of diacylglycerol (DAG) lipids are proposed to regulate differential physiological responses ranging from signal transduction to modulation of the membrane properties. However, the mechanism or molecular players regulating the subsets of DAG species remains unknown. Here, we uncover the role of a conserved eukaryotic protein family, DISCO-interacting protein 2 (DIP2) as a homeostatic regulator of a chemically distinct subset of DAGs using yeast, fly and mouse models. Genetic and chemical screens along with lipidomics analysis in yeast reveal that DIP2 prevents the toxic accumulation of specific DAGs in the logarithmic growth phase, which otherwise leads to endoplasmic reticulum stress. We also show that the fatty acyl-AMP ligase-like domains of DIP2 are essential for the redirection of the flux of DAG subspecies to storage lipid, triacylglycerols. Such modulation of selective DAG abundance by DIP2 is found to be crucial for optimal vacuole-membrane fusion and consequently osmoadaptation in yeast. Thus, the study illuminates an unprecedented DAG metabolism route and provides new insights on how cell fine-tunes DAG subspecies for cellular homeostasis and environmental adaptation.

biochemistry↗