Lysophosphatidylserine: A Signaling Lipid with Implications in Human Diseases.

Lysophosphatidylserine (lyso-PS) has emerged as yet another important signaling lysophospholipid in mammals, and deregulation in its metabolism has been directly linked to an array of human autoimmune and neurological disorders. It has an indispensable role in several biological processes in humans, and therefore, cellular concentrations of lyso-PS are tightly regulated to ensure optimal signaling and functioning in physiological settings. Given its biological importance, the past two decades have seen an explosion in the available literature toward our understanding of diverse aspects of lyso-PS metabolism and signaling and its association with human diseases. In this Review, we aim to comprehensively summarize different aspects of lyso-PS, such as its structure, biodistribution, chemical synthesis, and SAR studies with some synthetic analogs. From a biochemical perspective, we provide an exhaustive coverage of the diverse biological activities modulated by lyso-PSs, such as its metabolism and the receptors that respond to them in humans. We also briefly discuss the human diseases associated with aberrant lyso-PS metabolism and signaling and posit some future directions that may advance our understanding of lyso-PS-mediated mammalian physiology.

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 and 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.

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 their protein sequences and available biochemical studies, enzymes with similar catalytic mechanisms have been clustered together into an enzyme superfamily. Typically, enzymes within a superfamily have similar overall three-dimensional structures, conserved 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. Based on known enzymatic activities and protein sequences, they are split almost equally into the serine proteases and metabolic serine hydrolases. Within the metabolic serine hydrolases, there are two outlying members, ABHD14A and ABHD14B, that have high sequence similarity, but their biological 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 often wrongly assign ABHD14A and ABHD14B as the same enzyme, and therefore, annotating functions to them in various organisms has been problematic. In this article, we present a bioinformatics study coupled with biochemical experiments, which identifies key sequence determinants for both ABHD14A and ABHD14B, and enable better classification for them. In addition, we map these enzymes on an evolutionary timescale and provide a much-wanted resource for studying these interesting enzymes in different organisms.

Metabolic regulation of CTCF expression and chromatin association dictates starvation response in mice and flies.

Coordinated temporal control of gene expression is essential for physiological homeostasis, especially during metabolic transitions. However, the interplay between chromatin architectural proteins and metabolism in regulating transcription is less understood. Here, we demonstrate a conserved bidirectional interplay between CTCF (CCCTC-binding factor) expression/function and metabolic inputs during feed-fast cycles. Our results indicate that its loci-specific functional diversity is associated with physiological plasticity in mouse hepatocytes. CTCF differential expression and long non-coding RNA-Jpx mediated changes in chromatin occupancy, unraveled its paradoxical yet tuneable functions, which are governed by metabolic inputs. We illustrate the key role of CTCF in controlling temporal cascade of transcriptional response, with effects on hepatic mitochondrial energetics and lipidome. Underscoring the evolutionary conservation of CTCF-dependent metabolic homeostasis, CTCF knockdown in flies abrogated starvation resistance. In summary, we demonstrate the interplay between CTCF and metabolic inputs that highlights the coupled plasticity of physiological responses and chromatin function.

Mapping lipid pathways during phagocytosis.

Phagocytosis is an evolutionarily conserved important immunological process in higher organisms, and acts as the first line of defense against invading pathogenic microbial infections. Additionally, this dynamic innate immune response is also critical for clearing apoptotic cells and/or tissues, is responsible for maintaining homeostasis and acts as a systemic regulator of critical physiological processes such as wound healing and tissue regeneration. Over the past two decades, numerous studies have shown that phagocytosis occurs in three spatiotemporally distinct steps, namely formation, maturation and resolution of the phagosome, and that, both the protein and lipid composition change as a function of the aforementioned steps during this immunological process. While significant knowledge is now available on the proteomic content of a phagosome during the different stages of phagocytosis, the lipidome however, remained lesser explored, until the past few years. In this review, we summarize recent efforts towards mapping the physiological roles and functions of three lipid classes, the phosphatidylinositols, cholesterol and sphingolipids during the various stages of phagocytosis, and discuss strategies evolved by microbes to hijack and/or disrupt these lipid pathways to evade the immune system. We conclude this review with some potential avenues that may be pursued towards mapping hitherto unknown lipid pathways during phagocytosis, and how this research might be beneficial in our ongoing battle to overcome pathogenic infections.

Heterocyclic Diaryliodonium-Based Inhibitors of Carbapenem-Resistant Acinetobacter baumannii.

Finding new therapeutic strategies against Gram-negative pathogens such as Acinetobacter baumannii is challenging. Starting from diphenyleneiodonium (dPI) salts, which are moderate Gram-positive antibacterials, we synthesized a focused heterocyclic library and found a potent inhibitor of patient-derived multidrug-resistant Acinetobacter baumannii strains that significantly reduced bacterial burden in an animal model of infection caused by carbapenem-resistant Acinetobacter baumannii (CRAB), listed as a priority 1 critical pathogen by the World Health Organization. Next, using advanced chemoproteomics platforms and activity-based protein profiling (ABPP), we identified and biochemically validated betaine aldehyde dehydrogenase (BetB), an enzyme that is involved in the metabolism and maintenance of osmolarity, as a potential target for this compound. Together, using a new class of heterocyclic iodonium salts, a potent CRAB inhibitor was identified, and our study lays the foundation for the identification of new druggable targets against this critical pathogen. IMPORTANCE Discovery of novel antibiotics targeting multidrug-resistant (MDR) pathogens such as A. baumannii is an urgent, unmet medical need. Our work has highlighted the potential of this unique scaffold to annihilate MDR A. baumannii alone and in combination with amikacin both in vitro and in animals, that too without inducing resistance. Further in depth analysis identified central metabolism to be a putative target. Taken together, these experiments lay down the foundation for effective management of infections caused due to highly MDR pathogens.

Photoreactive bioorthogonal lipid probes and their applications in mammalian biology.

Lipids are an important class of biological molecules that possess many critical physiological functions, which enable the optimal survival of all organisms, including humans. While the role of lipids in the formation of biological cellular membranes and as a source of energy is fairly well understood, the cellular signalling pathways that lipids modulate in mammals are, in comparison, poorly characterized mechanistically and/or largely unknown. In an effort to dissect these mammalian cellular pathways regulated by signalling lipids and map hitherto unknown protein-lipid interactions, the last two decades have seen tremendous progress in the development of multifunctional lipid probes that, in conjunction with well-established bioorthogonal chemistries and chemoproteomics platforms, has almost exponentially expanded our knowledge in this field. In this review, we focus on the various photoreactive bioorthogonal lipid probes described in the literature, and briefly summarize the different photo-crosslinking groups and bioorthogonal chemistries used by them. Furthermore, we report specific case examples of such photoreactive bioorthogonal lipid probes, and discuss the new biological pathways and insights that have emerged from their use through chemoproteomics in mammalian cells. Finally, we highlight the challenges associated with the use of lipid probes in biological systems, and highlight their importance in the discovery and mechanistic understanding of lipid signalling pathways in the years to come.

Respiratory Quinone Switches from Menaquinone to Polyketide Quinone during the Development Cycle in Streptomyces sp. Strain MNU77.

Type III polyketide synthases (PKSs) found across Streptomyces species are primarily known for synthesis of a vast repertoire of clinically and industrially relevant secondary metabolites. However, our understanding of the functional relevance of these bioactive metabolites in Streptomyces physiology is still limited. Recently, a role of type III PKS harboring gene cluster in producing alternate electron carrier, polyketide quinone (PkQ) was established in a related member of the Actinobacteria, Mycobacteria, highlighting the critical role these secondary metabolites play in primary cellular metabolism of the producer organism. Here, we report the developmental stage-specific transcriptional regulation of homologous type III PKS containing gene cluster in freshwater Streptomyces sp. strain MNU77. Gene expression analysis revealed the type III PKS gene cluster to be stringently regulated, with significant upregulation observed during the dormant sporulation stage of Streptomyces sp. MNU77. In contrast, the expression levels of only known electron carrier, menaquinone biosynthetic genes were interestingly found to be downregulated. Our liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis of a metabolite extract from the Streptomyces sp. MNU77 spores also showed 10 times more metabolic abundance of PkQs than menaquinones. Furthermore, through heterologous complementation studies, we demonstrate that Streptomyces sp. MNU77 type III PKS rescues a respiratory defect of the Mycobacterium smegmatis type III PKS deletion mutant. Together, our studies reveal that freshwater Streptomyces sp. MNU77 robustly produces novel PkQs during the sporulation stage, suggesting utilization of PkQs as alternate electron carriers across Actinobacteria during dormant hypoxic conditions. IMPORTANCE The complex developmental life cycle of Streptomyces sp. mandates efficient cellular respiratory reconfiguration for a smooth transition from aerated nutrient-rich vegetative hyphal growth to the hypoxic-dormant sporulation stage. Polyketide quinones (PkQs) have recently been identified as a class of alternate electron carriers from a related member of the Actinobacteria, Mycobacteria, that facilitates maintenance of membrane potential in oxygen-deficient niches. Our studies with the newly identified freshwater Streptomyces sp. strain MNU77 show conditional transcriptional upregulation and metabolic abundance of PkQs in the spore state of the Streptomyces life cycle. In parallel, the levels of menaquinones, the only known Streptomyces electron carrier, were downregulated, suggesting deployment of PkQs as universal electron carriers in low-oxygen, unfavorable conditions across the Actinobacteria family.

A multi-omics analysis reveals that the lysine deacetylase ABHD14B influences glucose metabolism in mammals.

The sirtuins and histone deacetylases are the best characterized members of the lysine deacetylase (KDAC) enzyme family. Recently, we annotated the "orphan" enzyme ABHD14B (α/ß-hydrolase domain containing protein # 14B) as a novel KDAC and showed this enzyme's ability to transfer an acetyl-group from protein lysine residue(s) to coenzyme-A to yield acetyl-coenzyme-A, thereby, expanding the repertoire of this enzyme family. However, the role of ABHD14B in metabolic processes is not fully elucidated. Here, we investigated the role of this enzyme using mammalian cell knockdowns in a combined transcriptomics and metabolomics analysis. We found from these complementary experiments in vivo that the loss of ABHD14B results in significantly altered glucose metabolism, specifically the decreased flux of glucose through glycolysis and the citric acid cycle. Further, we show that depleting hepatic ABHD14B in mice also results in defective systemic glucose metabolism, particularly during fasting. Taken together, our findings illuminate the important metabolic functions that the KDAC ABHD14B plays in mammalian physiology and poses new questions regarding the role of this hitherto cryptic metabolism-regulating enzyme.

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 remain 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. DIP2 is associated with vacuoles through mitochondria-vacuole contact sites and 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.

Lipids, such as fats and hormones, constitute one of the main building blocks of cells. There are thousands of different lipids each with distinctive chemical properties that allow them to carry out specific roles. For example, a group of lipids called diacylglycerols help cells perform a myriad of tasks, like sensing external signals, making membranes, and storing energy. The production and breakdown of diacylglycerols is therefore tightly regulated. However, very little is known about the molecules involved in this metabolic process. One possible candidate is the enzyme DIP2 which is comprised of a protein module known as FAAL (short for fatty acyl-AMP ligase). FAAL belongs to a family of enzymes that synthesize lipid-like molecules in bacteria. In 2021, a group of researchers tracked the evolutionary trajectory of these bacterial proteins and found that most of them were lost in eukaryotes, such as animals and fungi. FAAL-like proteins, however, had been retained through evolution and incorporated in to DIP2. Here, Mondal, Kinatukara et al. ­ including some of the researchers involved in the 2021 study ­ have used a combination of genetic and biochemical experiments to investigate whether and how DIP2 contributes to lipid metabolism in eukaryotes. They found that yeast cells without the gene for DIP2 had higher levels of diacylglycerols which hampered the shape and function of certain cellular compartments. The mutant cells were also unable to convert diacylglycerols in to another group of lipids which are involved in energy storage. This effect was observed in fruit flies and mice lacking DIP2, suggesting that this role for DIP2 is conserved across most eukaryotes. Further experiments in yeast cells revealed that unlike other enzymes that metabolize diacylglycerols, DIP2 only acted on a sub-population of diacylglycerols at specific locations and times. Furthermore, yeast cells lacking DIP2 could still grow under ideal conditions, but could not cope with high or low salt concentrations in their surroundings, suggesting that the enzyme helps cells deal with environmental stresses. Since DIP2 is found in most eukaryotes, understanding how it works could be useful for multiple branches of biology. For example, some pathogenic fungi that cause diseases in crop plants and humans also rely on DIP2. Further studies are needed to better understand the role that DIP2 plays in other eukaryotic species which may shed light on other processes the enzyme is involved in.

Mycobacterium tuberculosis Transcription Factor EmbR Regulates the Expression of Key Virulence Factors That Aid in Ex Vivo and In Vivo Survival.

Mycobacterium tuberculosis encodes ~200 transcription factors that modulate gene expression under different microenvironments in the host. Even though high-throughput chromatin immunoprecipitation sequencing and transcriptome sequencing (RNA-seq) studies have identified the regulatory network for ~80% of transcription factors, many transcription factors remain uncharacterized. EmbR is one such transcription factor whose in vivo regulon and biological function are yet to be elucidated. Previous in vitro studies suggested that phosphorylation of EmbR by PknH upregulates the embCAB operon. Using a gene replacement mutant of embR, we investigated its role in modulating cellular morphology, antibiotic resistance, and survival in the host. Contrary to the prevailing hypothesis, under normal growth conditions, EmbR is neither phosphorylated nor impacted by ethambutol resistance through the regulation of the embCAB operon. The embR deletion mutant displayed attenuated M. tuberculosis survival in vivo. RNA-seq analysis suggested that EmbR regulates operons involved in the secretion pathway, lipid metabolism, virulence, and hypoxia, including well-known hypoxia-inducible genes devS and hspX. Lipidome analysis revealed that EmbR modulates levels of all lysophospholipids, several phospholipids, and M. tuberculosis-specific lipids, which is more pronounced under hypoxic conditions. We found that the EmbR mutant is hypersusceptible to hypoxic stress, and RNA sequencing performed under hypoxic conditions indicated that EmbR majorly regulates genes involved in response to acidic pH, hypoxia, and fatty acid metabolism. We observed condition-specific phosphorylation of EmbR, which contributes to EmbR-mediated transcription of several essential genes, ensuring enhanced survival. Collectively, the study establishes EmbR as a key modulator of hypoxic response that facilitates mycobacterial survival in the host. IMPORTANCE Mycobacterium tuberculosis modulates its transcriptional machinery in response to dynamic microenvironments encountered within the host. In this study, we identified that EmbR, a transcription factor, plays important roles in modulating cellular morphology, antibiotic resistance, and survival in the host. We found that EmbR undergoes condition-specific phosphorylation for its activation. Together, the study establishes a key role of EmbR as a transcriptional activator of genes belonging to multiple pathways, viz., virulence, secretion, or polyketide synthesis, that aid in mycobacterial survival during hypoxia and within the host.

Kupyaphores are zinc homeostatic metallophores required for colonization of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) endures a combination of metal scarcity and toxicity throughout the human infection cycle, contributing to complex clinical manifestations. Pathogens counteract this paradoxical dysmetallostasis by producing specialized metal trafficking systems. Capture of extracellular metal by siderophores is a widely accepted mode of iron acquisition, and Mtb iron-chelating siderophores, mycobactin, have been known since 1965. Currently, it is not known whether Mtb produces zinc scavenging molecules. Here, we characterize low-molecular-weight zinc-binding compounds secreted and imported by Mtb for zinc acquisition. These molecules, termed kupyaphores, are produced by a 10.8 kbp biosynthetic cluster and consists of a dipeptide core of ornithine and phenylalaninol, where amino groups are acylated with isonitrile-containing fatty acyl chains. Kupyaphores are stringently regulated and support Mtb survival under both nutritional deprivation and intoxication conditions. A kupyaphore-deficient Mtb strain is unable to mobilize sufficient zinc and shows reduced fitness upon infection. We observed early induction of kupyaphores in Mtb-infected mice lungs after infection, and these metabolites disappeared after 2 wk. Furthermore, we identify an Mtb-encoded isonitrile hydratase, which can possibly mediate intracellular zinc release through covalent modification of the isonitrile group of kupyaphores. Mtb clinical strains also produce kupyaphores during early passages. Our study thus uncovers a previously unknown zinc acquisition strategy of Mtb that could modulate host-pathogen interactions and disease outcome.

The loss of enzymatic activity of the PHARC-associated lipase ABHD12 results in increased phagocytosis that causes neuroinflammation.

Phagocytosis is an important evolutionary conserved process, essential for clearing pathogens and cellular debris in higher organisms, including humans. This well-orchestrated innate immunological response is intricately regulated by numerous cellular factors, important amongst which are the immunomodulatory lysophosphatidylserines (lyso-PSs) and the pro-apoptotic oxidized phosphatidylserines (PSs) signalling lipids. Interestingly, in mammals, both these signalling lipids are physiologically regulated by the lipase ABHD12, mutations of which cause the human neurological disorder PHARC. Despite the biomedical significance of this lipase, detailed mechanistic studies and the specific contribution of ABHD12 to innate processes like phagocytosis remain poorly understood. Here, by immunohistochemical and immunofluorescence approaches, using the murine model of PHARC, we show, that upon an inflammatory stimulus, activated microglial cells in the cerebellum of mice deficient in ABHD12 have an amoeboid morphology, increased soma size and display heightened phagocytosis activity. We also report that upon an inflammatory stimulus, cerebellar levels of ABHD12 increase to possibly metabolize the heightened oxidized PS levels, temper phagocytosis and, in turn, control neuroinflammation during oxidative stress. Next, to complement these findings, with the use of biochemical approaches in cultured microglial cells, we show that the pharmacological inhibition and/or genetic deletion of ABHD12 results in increased phagocytic uptake in a fluorescent bead uptake assay. Together, our studies provide compelling evidence that ABHD12 plays an important role in regulating phagocytosis in cerebellar microglial cells and provides a possible explanation, as to why human PHARC subjects display neuroinflammation and atrophy in the cerebellum.

Leveraging an enzyme/artificial substrate system to enhance cellular persulfides and mitigate neuroinflammation.

Persulfides and polysulfides, collectively known as the sulfane sulfur pool along with hydrogen sulfide (H2S), play a central role in cellular physiology and disease. Exogenously enhancing these species in cells is an emerging therapeutic paradigm for mitigating oxidative stress and inflammation that are associated with several diseases. In this study, we present a unique approach of using the cell's own enzyme machinery coupled with an array of artificial substrates to enhance the cellular sulfane sulfur pool. We report the synthesis and validation of artificial/unnatural substrates specific for 3-mercaptopyruvate sulfurtransferase (3-MST), an important enzyme that contributes to sulfur trafficking in cells. We demonstrate that these artificial substrates generate persulfides in vitro as well as mediate sulfur transfer to low molecular weight thiols and to cysteine-containing proteins. A nearly 100-fold difference in the rates of H2S production for the various substrates is observed supporting the tunability of persulfide generation by the 3-MST enzyme/artificial substrate system. Next, we show that the substrate 1a permeates cells and is selectively turned over by 3-MST to generate 3-MST-persulfide, which protects against reactive oxygen species-induced lethality. Lastly, in a mouse model, 1a is found to significantly mitigate neuroinflammation in the brain tissue. Together, the approach that we have developed allows for the on-demand generation of persulfides in vitro and in vivo using a range of shelf-stable, artificial substrates of 3-MST, while opening up possibilities of harnessing these molecules for therapeutic applications.

Mapping Sphingolipid Metabolism Pathways during Phagosomal Maturation.

Phagocytosis is an important physiological process, which, in higher organisms, is a means of fighting infections and clearing cellular debris. During phagocytosis, detrimental foreign particles (e.g. pathogens and apoptotic cells) are engulfed by phagocytes (e.g. macrophages), enclosed in membrane-bound vesicles called phagosomes, and transported to the lysosome for eventual detoxification. During this well-choreographed process, the nascent phagosome (also called early phagosome, EP) undergoes a series of spatiotemporally regulated changes in its protein and lipid composition and matures into a late phagosome (LP), which subsequently fuses with the lysosomal membrane to form the phagolysosome. While several elegant proteomic studies have identified the role of unique proteins during phagosomal maturation, the corresponding lipidomic studies are sparse. Recently, we reported a comparative lipidomic analysis between EPs and LPs and showed that ceramides are enriched on the LPs. Further, we found that this ceramide accumulation on LPs was orchestrated by ceramide synthase 2, inhibition of which hampers phagosomal maturation. Following up on this study, here, using biochemical assays, we first show that the increased ceramidase activity on EPs also significantly contributes to the accumulation of ceramides on LPs. Next, leveraging lipidomics, we show that de novo ceramide synthesis does not significantly contribute to the ceramide accumulation on LPs, while concomitant to increased ceramides, glucosylceramides are substantially elevated on LPs. We validate this interesting finding using biochemical assays and show that LPs indeed have heightened glucosylceramide synthase activity. Taken together, our studies provide interesting insights and possible new roles of sphingolipid metabolism during phagosomal maturation.

A Superfamily-wide Activity Atlas of Serine Hydrolases in Drosophila melanogaster.

The serine hydrolase (SH) superfamily is, perhaps, one of the largest functional enzyme classes in all forms of life and consists of proteases, peptidases, lipases, and carboxylesterases as representative members. Consistent with the name of this superfamily, all members, without any exception to date, use a nucleophilic serine residue in the enzyme active site to perform hydrolytic-type reactions via a two-step ping-pong mechanism involving a covalent enzyme intermediate. Given the highly conserved catalytic mechanism, this superfamily has served as a classical prototype in the development of several platforms of chemical proteomics techniques, activity-based protein profiling (ABPP), to globally interrogate the functions of its different members in various native, yet complex, biological settings. While ABPP-based proteome-wide activity atlases for SH activities are available in numerous organisms, including humans, to the best of our knowledge, such an analysis for this superfamily is lacking in any insect model. To address this, we initially report a bioinformatics analysis toward the identification and categorization of nonredundant SHs in Drosophila melanogaster. Following up on this in silico analysis, leveraging discovery chemoproteomics, we identify and globally map the full complement of SH activities during various developmental stages and in different adult tissues of Drosophila. Finally, as a proof of concept of the utility of this activity atlas, we highlight sexual dimorphism in SH activities across different tissues in adult D. melanogaster, and we propose new research directions, resources, and tools that this study can provide to the fly community.

Fatty acid chain length drives lysophosphatidylserine-dependent immunological outputs.

In humans, lysophosphatidylserines (lyso-PSs) are potent lipid regulators of important immunological processes. Given their structural diversity and commercial paucity, here we report the synthesis of methyl esters of lyso-PS (Me-lyso-PSs) containing medium- to very-long-chain (VLC) lipid tails. We show that Me-lyso-PSs are excellent substrates for the lyso-PS lipase ABHD12, and that these synthetic lipids are acted upon by cellular carboxylesterases to produce lyso-PSs. Next, in macrophages we demonstrate that VLC lyso-PSs orchestrate pro-inflammatory responses and in turn neuroinflammation via a Toll-like receptor 2 (TLR2)-dependent pathway. We also show that long-chain (LC) lyso-PSs robustly induce intracellular cyclic AMP production, cytosolic calcium influx, and phosphorylation of the nodal extracellular signal-regulated kinase to regulate macrophage activation via a TLR2-independent pathway. Finally, we report that LC lyso-PSs potently elicit histamine release during the mast cell degranulation process, and that ABHD12 is the major lyso-PS lipase in these immune cells.

Peri-natal growth retardation rate and fat mass accumulation in mice lacking Dip2A is dependent on the dietary composition.

Disco-interacting protein 2 is a highly conserved three-domain protein with two tandem Adenylate-forming domains. It is proposed to influence the processes involved in neuronal development by influencing lipid metabolism and remains to be characterized. In this study, we show that Disco-interacting protein 2a null mice do not exhibit overt phenotype defects. However, the body composition differences were observed in these mice under different dietary regimens. The neutral lipid composition of two different diets was characterized, and it was observed that the new-born mice grow relatively slower than the wild-type mice with delayed appearance of features such as dentition when fed with high-triacylglycerol NIN-formulation diet. The high-diacylglycerol Safe-formulation diet was found to accumulate more fat mass in mice than those fed with high-triacylglycerol NIN-formulation diet beyond 10 months. These findings point to a proposed relationship between dietary components (particularly the lipid composition) and body composition along with the growth of neonates in mice lacking the gene Disco-interacting protein 2a.

The Lysophosphatidylserines-An Emerging Class of Signalling Lysophospholipids.

Lysophospholipids are potent hormone-like signalling biological lipids that regulate many important biological processes in mammals (including humans). Lysophosphatidic acid and sphingosine-1-phosphate represent the best studied examples for this lipid class, and their metabolic enzymes and/or cognate receptors are currently under clinical investigation for treatment of various neurological and autoimmune diseases in humans. Over the past two decades, the lysophsophatidylserines (lyso-PSs) have emerged as yet another biologically important lysophospholipid, and deregulation in its metabolism has been linked to various human pathophysiological conditions. Despite its recent emergence, an exhaustive review summarizing recent advances on lyso-PSs and the biological pathways that this bioactive lysophospholipid regulates has been lacking. To address this, here, we summarize studies that led to the discovery of lyso-PS as a potent signalling biomolecule, and discuss the structure, its detection in biological systems, and the biodistribution of this lysophospholipid in various mammalian systems. Further, we describe in detail the enzymatic pathways that are involved in the biosynthesis and degradation of this lipid and the putative lyso-PS receptors reported in the literature. Finally, we discuss the various biological pathways directly regulated by lyso-PSs in mammals and prospect new questions for this still emerging biomedically important signalling lysophospholipid.

Mapping the Neuroanatomy of ABHD16A, ABHD12, and Lysophosphatidylserines Provides New Insights into the Pathophysiology of the Human Neurological Disorder PHARC.

Lysophosphatidylserine (lyso-PS), a lysophospholipid derived from phosphatidylserine (PS), has emerged as a potent signaling lipid in mammalian physiology. In vivo, the metabolic serine hydrolases ABHD16A and ABHD12 are major lipases that biosynthesize and degrade lyso-PS, respectively. Of biomedical relevance, deleterious mutations to ABHD12 cause accumulation of lyso-PS in the brain, and this deregulated lyso-PS metabolism leads to the human genetic neurological disorder PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract). While the roles of ABHD16A and ABHD12 in lyso-PS metabolism in the mammalian brain are well established, the anatomical and (sub)cellular localizations of both lipases and the functional cross-talk between them with respect to regulating lyso-PS lipids remain under investigated. Here, using subcellular organelle fractionation, biochemical assays, and immunofluorescence-based high-resolution microscopy, we show that the PS lipase ABHD16A is an endoplasmic reticulum-localized enzyme, an organelle intricately regulating cellular PS levels. In addition, leveraging immunohistochemical analysis using genetic ABHD16A and ABHD12 knockout mice as important controls, we map the anatomical distribution of both of these lipases in tandem in the murine brain and show for the first time the distinct localization of these lipases to different regions and cells of the cerebellum. We complement the aforementioned immunohistochemical studies by quantitatively measuring lyso-PS concentrations in various brain regions using mass spectrometry and find that the cerebellar lyso-PS levels are most affected by deletion of ABHD16A (decreased) or ABHD12 (increased). Taken together, our studies provide new insights into lyso-PS signaling in the cerebellum, the most atrophic brain region in human PHARC subjects.