Cooperative lipolytic control of neuronal triacylglycerol by spastic paraplegia-associated enzyme DDHD2 and ATGL.

Intracellular lipolysis-the enzymatic breakdown of lipid droplet-associated triacylglycerol (TAG)-depends on the cooperative action of several hydrolytic enzymes and regulatory proteins, together designated as lipolysome. Adipose triglyceride lipase (ATGL) acts as a major cellular TAG hydrolase and core effector of the lipolysome in many peripheral tissues. Neurons initiate lipolysis independently of ATGL via DDHD domain-containing 2 (DDHD2), a multifunctional lipid hydrolase whose dysfunction causes neuronal TAG deposition and hereditary spastic paraplegia. Whether and how DDHD2 cooperates with other lipolytic enzymes is currently unknown. In this study, we further investigated the enzymatic properties and functions of DDHD2 in neuroblastoma cells and primary neurons. We found that DDHD2 hydrolyzes multiple acylglycerols in vitro and substantially contributes to neutral lipid hydrolase activities of neuroblastoma cells and brain tissue. Substrate promiscuity of DDHD2 allowed its engagement at different steps of the lipolytic cascade: In neuroblastoma cells, DDHD2 functioned exclusively downstream of ATGL in the hydrolysis of sn-1,3-diacylglycerol (DAG) isomers but was dispensable for TAG hydrolysis and lipid droplet homeostasis. In primary cortical neurons, DDHD2 exhibited lipolytic control over both, DAG and TAG, and complemented ATGL-dependent TAG hydrolysis. We conclude that neuronal cells use noncanonical configurations of the lipolysome and engage DDHD2 as dual TAG/DAG hydrolase in cooperation with ATGL.

The Patatin-Like Phospholipase Domain Containing Protein 7 Regulates Macrophage Classical Activation through SIRT1/NF-κB and p38 MAPK Pathways.

Lysophosphatidylcholine (LPC) is a bioactive lipid that modulates macrophage polarization during immune responses, inflammation, and tissue remodeling. Patatin-like phospholipase domain containing protein 7 (PNPLA7) is a lysophospholipase with a preference for LPC. However, the role of PNPLA7 in macrophage polarization as an LPC hydrolase has not been explored. In the current study, we found that PNPLA7 is highly expressed in naïve macrophages and downregulated upon lipopolysaccharide (LPS)-induced polarization towards the classically activated (M1) phenotype. Consistently, overexpression of PNPLA7 suppressed the expression of proinflammatory M1 marker genes, including interleukin 1ß (IL-1ß), IL-6, inducible nitric oxide synthase (iNOS), and tumor necrosis factor α (TNF-α), whereas knockdown of PNPLA7 augmented the inflammatory gene expression in LPS-challenged macrophages. PNPLA7 overexpression and knockdown increased and decreased Sirtuin1 (SIRT1) mRNA and protein levels, respectively, and affected the acetylation of the nuclear factor-kappa B (NF-κB) p65 subunit, a key transcription factor in M1 polarization. In addition, the levels of phosphorylated p38 mitogen-activated protein kinase (MAPK) were suppressed and enhanced by PNPLA7 overexpression and knockdown, respectively. Taken together, these findings suggest that PNPLA7 suppresses M1 polarization of LPS-challenged macrophages by modulating SIRT1/NF-κB- and p38 MAPK-dependent pathways.

Drosophila Lipase 3 Mediates the Metabolic Response to Starvation and Aging.

The human LIPA gene encodes for the enzyme lysosomal acid lipase, which hydrolyzes cholesteryl ester and triacylglycerol. Lysosomal acid lipase deficiency results in Wolman disease and cholesteryl ester storage disease. The Drosophila genome encodes for two LIPA orthologs, Magro and Lipase 3. Magro is a gut lipase that hydrolyzes triacylglycerides, while Lipase 3 lacks characterization based on mutant phenotypes. We found previously that Lipase 3 transcription is highly induced in mutants with defects in peroxisome biogenesis, but the conditions that allow a similar induction in wildtypic flies are not known. Here we show that Lipase 3 is drastically upregulated in starved larvae and starved female flies, as well as in aged male flies. We generated a lipase 3 mutant that shows sex-specific starvation resistance and a trend to lifespan extension. Using lipidomics, we demonstrate that Lipase 3 mutants accumulate phosphatidylinositol, but neither triacylglycerol nor diacylglycerol. Our study suggests that, in contrast to its mammalian homolog LIPA, Lipase 3 is a putative phospholipase that is upregulated under extreme conditions like prolonged nutrient deprivation and aging.

An immune-sympathetic neuron communication axis guides adipose tissue browning in cancer-associated cachexia.

Cancer-associated cachexia (CAC) is a hypermetabolic syndrome characterized by unintended weight loss due to the atrophy of adipose tissue and skeletal muscle. A phenotypic switch from white to beige adipocytes, a phenomenon called browning, accelerates CAC by increasing the dissipation of energy as heat. Addressing the mechanisms of white adipose tissue (WAT) browning in CAC, we now show that cachexigenic tumors activate type 2 immunity in cachectic WAT, generating a neuroprotective environment that increases peripheral sympathetic activity. Increased sympathetic activation, in turn, results in increased neuronal catecholamine synthesis and secretion, ß-adrenergic activation of adipocytes, and induction of WAT browning. Two genetic mouse models validated this progression of events. 1) Interleukin-4 receptor deficiency impeded the alternative activation of macrophages, reduced sympathetic activity, and restrained WAT browning, and 2) reduced catecholamine synthesis in peripheral dopamine ß-hydroxylase (DBH)-deficient mice prevented cancer-induced WAT browning and adipose atrophy. Targeting the intraadipose macrophage-sympathetic neuron cross-talk represents a promising therapeutic approach to ameliorate cachexia in cancer patients.

Lipid droplet-mitochondria coupling via perilipin 5 augments respiratory capacity but is dispensable for FA oxidation.

Disturbances in lipid homeostasis can cause mitochondrial dysfunction and lipotoxicity. Perilipin 5 (PLIN5) decorates intracellular lipid droplets (LDs) in oxidative tissues and controls triacylglycerol (TG) turnover via its interactions with adipose triglyceride lipase and the adipose triglyceride lipase coactivator, comparative gene identification-58. Furthermore, PLIN5 anchors mitochondria to the LD membrane via the outermost part of the carboxyl terminus. However, the role of this LD-mitochondria coupling (LDMC) in cellular energy catabolism is less established. In this study, we investigated the impact of PLIN5-mediated LDMC in comparison to disrupted LDMC on cellular TG homeostasis, FA oxidation, mitochondrial respiration, and protein interaction. To do so, we established PLIN5 mutants deficient in LDMC whilst maintaining normal interactions with key lipolytic players. Radiotracer studies with cell lines stably overexpressing wild-type or truncated PLIN5 revealed that LDMC has no significant impact on FA esterification upon lipid loading or TG catabolism during stimulated lipolysis. Moreover, we demonstrated that LDMC exerts a minor if any role in mitochondrial FA oxidation. In contrast, LDMC significantly improved the mitochondrial respiratory capacity and metabolic flexibility of lipid-challenged cardiomyocytes, which was corroborated by LDMC-dependent interactions of PLIN5 with mitochondrial proteins involved in mitochondrial respiration, dynamics, and cristae organization. Taken together, this study suggests that PLIN5 preserves mitochondrial function by adjusting FA supply via the regulation of TG hydrolysis and that LDMC is a vital part of mitochondrial integrity.

Optimized expression and purification of adipose triglyceride lipase improved hydrolytic and transacylation activities in vitro.

Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis, the mobilization of stored triacylglycerol. This work provides an important basis for generating reproducible and detailed data on the hydrolytic and transacylation activities of ATGL. We generated full-length and C-terminally truncated ATGL variants fused with various affinity tags and analyzed their expression in different hosts, namely E.coli, the insect cell line Sf9, and the mammalian cell line human embryonic kidney 293T. Based on this screen, we expressed a fusion protein of ATGL covering residues M1-D288 flanked with N-terminal and C-terminal purification tags. Using these fusions, we identified key steps in expression and purification protocols, including production in the E. coli strain ArcticExpress (DE3) and removal of copurified chaperones. The resulting purified ATGL variant demonstrated improved lipolytic activity compared with previously published data, and it could be stimulated by the coactivator protein comparative gene identification-58 and inhibited by the protein G0/G1 switch protein 2. Shock freezing and storage did not affect the basal activity but reduced coactivation of ATGL by comparative gene identification 58. In vitro, the truncated ATGL variant demonstrated acyl-CoA-independent transacylation activity when diacylglycerol was offered as substrate, resulting in the formation of fatty acid as well as triacylglycerol and monoacylglycerol. However, the ATGL variant showed neither hydrolytic activity nor transacylation activity upon offering of monoacylglycerol as substrate. To understand the role of ATGL in different physiological contexts, it is critical for future studies to identify all its different functions and to determine under what conditions these activities occur.

The Drosophila model to interrogate triacylglycerol biology.

The deposition of storage fat in the form of triacylglycerol (TAG) is an evolutionarily conserved strategy to cope with fluctuations in energy availability and metabolic stress. Organismal TAG storage in specialized adipose tissues provides animals a metabolic reserve that sustains survival during development and starvation. On the other hand, excessive accumulation of adipose TAG, defined as obesity, is associated with an increasing prevalence of human metabolic diseases. During the past decade, the fruit fly Drosophila melanogaster, traditionally used in genetics and developmental biology, has been established as a versatile model system to study TAG metabolism and the etiology of lipid-associated metabolic diseases. Similar to humans, Drosophila TAG homeostasis relies on the interplay of organ systems specialized in lipid uptake, synthesis, and processing, which are integrated by an endocrine network of hormones and messenger molecules. Enzymatic formation of TAG from sugar or dietary lipid, its storage in lipid droplets, and its mobilization by lipolysis occur via mechanisms largely conserved between Drosophila and humans. Notably, dysfunctional Drosophila TAG homeostasis occurs in the context of aging, overnutrition, or defective gene function, and entails tissue-specific and organismal pathologies that resemble human disease. In this review, we summarize the physiology and biochemistry of TAG in Drosophila and outline the potential of this organism as a model system to understand the genetic and dietary basis of TAG storage and TAG-related metabolic disorders.

Hormone-sensitive lipase couples intergenerational sterol metabolism to reproductive success.

Triacylglycerol (TG) and steryl ester (SE) lipid storage is a universal strategy to maintain organismal energy and membrane homeostasis. Cycles of building and mobilizing storage fat are fundamental in (re)distributing lipid substrates between tissues or to progress ontogenetic transitions. In this study, we show that Hormone-sensitive lipase (Hsl) specifically controls SE mobilization to initiate intergenerational sterol transfer in Drosophila melanogaster. Tissue-autonomous Hsl functions in the maternal fat body and germline coordinately prevent adult SE overstorage and maximize sterol allocation to embryos. While Hsl-deficiency is largely dispensable for normal development on sterol-rich diets, animals depend on adipocyte Hsl for optimal fecundity when dietary sterol becomes limiting. Notably, accumulation of SE but not of TG is a characteristic of Hsl-deficient cells across phyla including murine white adipocytes. In summary, we identified Hsl as an ancestral regulator of SE degradation, which improves intergenerational sterol transfer and reproductive success in flies.

Lipidome remodeling in aging normal and genetically obese Drosophila males.

Lipid homeostasis is essential for insects to maintain phospholipid (PL)-based membrane integrity and to provide on-demand energy supply throughout life. Triacylglycerol (TAG) is the major lipid class used for energy production and is stored in lipid droplets, the universal cellular fat storage organelles. Accumulation and mobilization of TAG are strictly regulated since excessive accumulation of TAG leads to obesity and has been correlated with adverse effects on health- and lifespan across phyla. Little is known, however, about when during adult life and why excessive storage lipid accumulation restricts lifespan. We here used genetically obese Drosophila mutant males, which were all shown to be short-lived compared to control males and applied single fly mass spectrometry-based lipidomics to profile TAG, diacylglycerol and major membrane lipid signatures throughout adult fly life from eclosion to death. Our comparative approach revealed distinct phases of lipidome remodeling throughout aging. Quantitative and qualitative compositional changes of TAG and PL species, which are characterized by the length and saturation of their constituent fatty acids, were pronounced during young adult life. In contrast, lipid signatures of adult and senescent flies were remarkably stable. Genetically obese flies displayed both quantitative and qualitative changes in TAG species composition, while PL signatures were almost unaltered compared to normal flies at all ages. Collectively, this suggests a tight control of membrane composition throughout lifetime largely uncoupled from storage lipid metabolism. Finally, we present first evidence for a characteristic lipid signature of moribund flies, likely generated by a rapid and selective storage lipid depletion close to death. Of note, the analytical power to monitor lipid species profiles combined with high sensitivity of this single fly lipidomics approach is universally applicable to address developmental or behavioral lipid signature modulations of importance for insect life.

Adipose triglyceride lipase activity regulates cancer cell proliferation via AMP-kinase and mTOR signaling.

Aberrant fatty acid (FA) metabolism is a hallmark of proliferating cells, including untransformed fibroblasts or cancer cells. Lipolysis of intracellular triglyceride (TG) stores by adipose triglyceride lipase (ATGL) provides an important source of FAs serving as energy substrates, signaling molecules, and precursors for membrane lipids. To investigate if ATGL-mediated lipolysis impacts cell proliferation, we modified ATGL activity in murine embryonic fibroblasts (MEFs) and in five different cancer cell lines to determine the consequences on cell growth and metabolism. Genetic or pharmacological inhibition of ATGL in MEFs causes impaired FA oxidation, decreased ROS production, and a substrate switch from FA to glucose leading to decreased AMPK-mTOR signaling and higher cell proliferation rates. ATGL expression in these cancer cells is low when compared to MEFs. Additional ATGL knockdown in cancer cells did not significantly affect cellular lipid metabolism or cell proliferation whereas the ectopic overexpression of ATGL increased lipolysis and reduced proliferation. In contrast to ATGL silencing, pharmacological inhibition of ATGL by Atglistatin© impeded the proliferation of diverse cancer cell lines, which points at an ATGL-independent effect. Our data indicate a crucial role of ATGL-mediated lipolysis in the regulation of cell proliferation. The observed low ATGL activity in cancer cells may represent an evolutionary selection process and mechanism to sustain high cell proliferation rates. As the increasing ATGL activity decelerates proliferation of five different cancer cell lines this may represent a novel therapeutic strategy to counteract uncontrolled cell growth.

Enhanced monoacylglycerol lipolysis by ABHD6 promotes NSCLC pathogenesis.

BACKGROUND: Tumor cells display metabolic changes that correlate with malignancy, including an elevated hydrolysis of monoacylglycerol (MAG) in various cancer types. However, evidence is absent for the relationship between MAG lipolysis and NSCLC. METHODS: MAG hydrolase activity assay, migration, invasion, proliferation, lipids quantification, and transactivation assays were performed in vitro. Tumor xenograft studies and lung metastasis assays were examined in vivo. The correlations of MAGL/ABHD6 expression in cancerous tissues with the clinicopathological characteristics and survival of NSCLC patients were validated. FINDINGS: ABHD6 functions as the primary MAG lipase and an oncogene in NSCLC. MAG hydrolase activities were more than 11-fold higher in cancerous lung tissues than in paired non-cancerous tissues derived from NSCLC patients. ABHD6, instead of MAGL, was significantly associated with advanced tumor node metastasis (TNM) stage (HR, 1.382; P = 0.004) and had a negative impact on the overall survival of NSCLC patients (P = 0.001). ABHD6 silencing reduced migration and invasion of NSCLC cells in vitro as well as metastatic seeding and tumor growth in vivo. Conversely, ectopic overexpression of ABHD6 provoked the pathogenic potential. ABHD6 blockade significantly induced intracellular MAG accumulation which activated PPARα/γ signaling and inhibited cancer pathophysiology. INTERPRETATION: The present study provide evidence for a previously uncovered pro-oncogenic function of ABHD6 in NSCLC, with the outlined metabolic mechanisms shedding light on new potential strategies for anticancer therapy. FUND: This work was supported by the Project for Major New Drug Innovation and Development (2015ZX09501010 and 2018ZX09711001-002-003).

Interaction of the Lysophospholipase PNPLA7 with Lipid Droplets through the Catalytic Region.

Mammalian patatin-like phospholipase domain containing proteins (PNPLAs) play critical roles in triglyceride hydrolysis, phospholipids metabolism, and lipid droplet (LD) homeostasis. PNPLA7 is a lysophosphatidylcholine hydrolase anchored on the endoplasmic reticulum which associates with LDs through its catalytic region (PNPLA7-C) in response to increased cyclic nucleotide levels. However, the interaction of PNPLA7 with LDs through its catalytic region is unknown. Herein, we demonstrate that PNPLA7-C localizes to the mature LDs ex vivo and also colocalizes with pre-existing LDs. Localization of PNPLA7-C with LDs induces LDs clustering via non-enzymatic intermolecular associations, while PNPLA7 alone does not induce LD clustering. Residues 742-1016 contains four putative transmembrane domains which act as a LD targeting motif and are required for the localization of PNPLA7-C to LDs. Furthermore, the N-terminal flanking region of the LD targeting motif, residues 681-741, contributes to the LD targeting, whereas the C-terminal flanking region (1169-1326) has an anti-LD targeting effect. Interestingly, the LD targeting motif does not exhibit lysophosphatidylcholine hydrolase activity even though it associates with LDs phospholipid membranes. These findings characterize the specific functional domains of PNPLA7 mediating subcellular positioning and interactions with LDs, as wells as providing critical insights into the structure of this evolutionarily conserved phospholipid-metabolizing enzyme family.

Low cardiac lipolysis reduces mitochondrial fission and prevents lipotoxic heart dysfunction in Perilipin 5 mutant mice.

AIMS: Lipotoxic cardiomyopathy in diabetic and obese patients typically encompasses increased cardiac fatty acid (FA) uptake eventually surpassing the mitochondrial oxidative capacity. Lowering FA utilization via inhibition of lipolysis represents a strategy to counteract the development of lipotoxic heart dysfunction. However, defective cardiac triacylglycerol (TAG) catabolism and FA oxidation in humans (and mice) carrying mutated ATGL alleles provokes lipotoxic heart dysfunction questioning a therapeutic approach to decrease cardiac lipolysis. Interestingly, decreased lipolysis via cardiac overexpression of Perilipin 5 (Plin5), a binding partner of ATGL, is compatible with normal heart function and lifespan despite massive cardiac lipid accumulation. Herein, we decipher mechanisms that protect Plin5 transgenic mice from the development of heart dysfunction. METHODS AND RESULTS: We generated mice with cardiac-specific overexpression of Plin5 encoding a serine-155 to alanine exchange (Plin5-S155A) of the protein kinase A phosphorylation site, which has been suggested as a prerequisite to stimulate lipolysis and may play a crucial role in the preservation of heart function. Plin5-S155A mice showed a substantial increase in cardiac TAG and ceramide levels, which was comparable to mice overexpressing non-mutated Plin5. Lipid accumulation was compatible with normal heart function even under mild stress. Plin5-S155A mice showed reduced cardiac FA oxidation but normal ATP production and changes in the Plin5-S155A phosphoproteome compared to Plin5 transgenic mice. Interestingly, mitochondrial recruitment of dynamin-related protein 1 (Drp1) was markedly reduced in cardiac muscle of Plin5-S155A and Plin5 transgenic mice accompanied by decreased phosphorylation of mitochondrial fission factor, a mitochondrial receptor of Drp1. CONCLUSIONS: This study suggests that low cardiac lipolysis is associated with reduced mitochondrial fission and may represent a strategy to combat the development of lipotoxic heart dysfunction.

Characterization of the Interaction of Neuropathy Target Esterase with the Endoplasmic Reticulum and Lipid Droplets.

: Neuropathy target esterase (NTE) is an endoplasmic reticulum (ER)-localized phospholipase that deacylates phosphatidylcholine (PC) and lysophosphatidylcholine (LPC). Loss-of-function mutations in the human NTE gene have been associated with a spectrum of neurodegenerative disorders such as hereditary spastic paraplegia, ataxia and chorioretinal dystrophy. Despite this, little is known about structure-function relationships between NTE protein domains, enzymatic activity and the interaction with cellular organelles. In the current study we show that the C-terminal region of NTE forms a catalytically active domain that exhibits high affinity for lipid droplets (LDs), cellular storage organelles for triacylglycerol (TAG), which have been recently implicated in the progression of neurodegenerative diseases. Ectopic expression of the C domain in cultured cells decreases cellular PC, elevates TAG and induces LD clustering. LD interactions of NTE are inhibited by default by a non-enzymatic regulatory (R) region with three putative nucleotide monophosphate binding sites. Together with a N-terminal TMD the R region promotes proper distribution of the catalytic C-terminal region to the ER network. Taken together, our data indicate that NTE may exhibit dynamic interactions with the ER and LDs depending on the interplay of its functional regions. Mutations that disrupt this interplay may contribute to NTE-associated disorders by affecting NTE positioning.

The α/ß-hydrolase domain-containing 4- and 5-related phospholipase Pummelig controls energy storage in Drosophila.

Triglycerides (TGs) are the main energy storage form that accommodates changing organismal energy demands. In Drosophila melanogaster, the TG lipase Brummer is centrally important for body fat mobilization. Its gene brummer (bmm) encodes the ortholog of mammalian adipose TG lipase, which becomes activated by α/ß-hydrolase domain-containing 5 (ABHD5/CGI-58), one member of the paralogous gene pair, α/ß-hydrolase domain-containing 4 (ABHD4) and ABHD5 In Drosophila, the pummelig (puml) gene encodes the single sequence-related protein to mammalian ABHD4/ABHD5 with unknown function. We generated puml deletion mutant flies, that were short-lived as a result of lipid metabolism changes, stored excess body fat at the expense of glycogen, and exhibited ectopic fat storage with altered TG FA profile in the fly kidneys, called Malpighian tubules. TG accumulation in puml mutants was not associated with increased food intake but with elevated lipogenesis; starvation-induced lipid mobilization remained functional. Despite its structural similarity to mammalian ABHD5, Puml did not stimulate TG lipase activity of Bmm in vitro. Rather, Puml acted as a phospholipase that localized on lipid droplets, mitochondria, and peroxisomes. Together, these results show that the ABHD4/5 family member Puml is a versatile phospholipase that regulates Drosophila body fat storage and energy metabolism.

Chronic dysfunction of Stromal interaction molecule by pulsed RNAi induction in fat tissue impairs organismal energy homeostasis in Drosophila.

Obesity is a progressive, chronic disease, which can be caused by long-term miscommunication between organs. It remains challenging to understand how chronic dysfunction in a particular tissue remotely impairs other organs to eventually imbalance organismal energy homeostasis. Here we introduce RNAi Pulse Induction (RiPI) mediated by short hairpin RNA (shRiPI) or double-stranded RNA (dsRiPI) to generate chronic, organ-specific gene knockdown in the adult Drosophila fat tissue. We show that organ-restricted RiPI targeting Stromal interaction molecule (Stim), an essential factor of store-operated calcium entry (SOCE), results in progressive fat accumulation in fly adipose tissue. Chronic SOCE-dependent adipose tissue dysfunction manifests in considerable changes of the fat cell transcriptome profile, and in resistance to the glucagon-like Adipokinetic hormone (Akh) signaling. Remotely, the adipose tissue dysfunction promotes hyperphagia likely via increased secretion of Akh from the neuroendocrine system. Collectively, our study presents a novel in vivo paradigm in the fly, which is widely applicable to model and functionally analyze inter-organ communication processes in chronic diseases.

Triacylglycerol Metabolism in Drosophila melanogaster.

Triacylglycerol (TAG) is the most important caloric source with respect to energy homeostasis in animals. In addition to its evolutionarily conserved importance as an energy source, TAG turnover is crucial to the metabolism of structural and signaling lipids. These neutral lipids are also key players in development and disease. Here, we review the metabolism of TAG in the Drosophila model system. Recently, the fruit fly has attracted renewed attention in research due to the unique experimental approaches it affords in studying the tissue-autonomous and interorgan regulation of lipid metabolism in vivo Following an overview of the systemic control of fly body fat stores, we will cover lipid anabolic, enzymatic, and regulatory processes, which begin with the dietary lipid breakdown and de novo lipogenesis that results in lipid droplet storage. Next, we focus on lipolytic processes, which mobilize storage TAG to make it metabolically accessible as either an energy source or as a building block for biosynthesis of other lipid classes. Since the buildup and breakdown of fat involves various organs, we highlight avenues of lipid transport, which are at the heart of functional integration of organismic lipid metabolism. Finally, we draw attention to some "missing links" in basic neutral lipid metabolism and conclude with a perspective on how fly research can be exploited to study functional metabolic roles of diverse lipids.

Hypoxia-inducible lipid droplet-associated protein inhibits adipose triglyceride lipase.

Elaborate control mechanisms of intracellular triacylglycerol (TAG) breakdown are critically involved in the maintenance of energy homeostasis. Hypoxia-inducible lipid droplet-associated protein (HILPDA)/hypoxia-inducible gene-2 (Hig-2) has been shown to affect intracellular TAG levels, yet, the underlying molecular mechanisms are unclear. Here, we show that HILPDA inhibits adipose triglyceride lipase (ATGL), the enzyme catalyzing the first step of intracellular TAG hydrolysis. HILPDA shares structural similarity with G0/G1 switch gene 2 (G0S2), an established inhibitor of ATGL. HILPDA inhibits ATGL activity in a dose-dependent manner with an IC50 value of ∼2 µM. ATGL inhibition depends on the direct physical interaction of both proteins and involves the N-terminal hydrophobic region of HILPDA and the N-terminal patatin domain-containing segment of ATGL. Finally, confocal microscopy combined with Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicated that HILPDA and ATGL colocalize and physically interact intracellularly. These findings provide a rational biochemical explanation for the tissue-specific increased TAG accumulation in HILPDA-overexpressing transgenic mouse models.

The phospholipase PNPLA7 functions as a lysophosphatidylcholine hydrolase and interacts with lipid droplets through its catalytic domain.

Mammalian patatin-like phospholipase domain-containing proteins (PNPLAs) are lipid-metabolizing enzymes with essential roles in energy metabolism, skin barrier development, and brain function. A detailed annotation of enzymatic activities and structure-function relationships remains an important prerequisite to understand PNPLA functions in (patho-)physiology, for example, in disorders such as neutral lipid storage disease, non-alcoholic fatty liver disease, and neurodegenerative syndromes. In this study, we characterized the structural features controlling the subcellular localization and enzymatic activity of PNPLA7, a poorly annotated phospholipase linked to insulin signaling and energy metabolism. We show that PNPLA7 is an endoplasmic reticulum (ER) transmembrane protein that specifically promotes hydrolysis of lysophosphatidylcholine in mammalian cells. We found that transmembrane and regulatory domains in the PNPLA7 N-terminal region cooperate to regulate ER targeting but are dispensable for substrate hydrolysis. Enzymatic activity is instead mediated by the C-terminal domain, which maintains full catalytic competence even in the absence of N-terminal regions. Upon elevated fatty acid flux, the catalytic domain targets cellular lipid droplets and promotes interactions of PNPLA7 with these organelles in response to increased cAMP levels. We conclude that PNPLA7 acts as an ER-anchored lysophosphatidylcholine hydrolase that is composed of specific functional domains mediating catalytic activity, subcellular positioning, and interactions with cellular organelles. Our study provides critical structural insights into an evolutionarily conserved class of phospholipid-metabolizing enzymes.

Molecular identification of transmembrane protein 68 as an endoplasmic reticulum-anchored and brain-specific protein.

Acyltransferases catalyze essential reactions in the buildup and remodeling of glycerophospholipids and contribute to the maintenance and diversity of cellular membranes. Transmembrane protein 68 (TMEM68) is an evolutionarily conserved protein of unknown function, that forms a distinct subgroup within the glycerophospholipid acyltransferase family. In the current study we expressed murine TMEM68 for the first time in mammalian cells to characterize its subcellular localization, topology, and possible biological function(s). We show that TMEM68 is an integral membrane protein and orients both, the N- and C-terminus towards the cytosol. Live cell imaging demonstrated that TMEM68 is localized mainly at the endoplasmic reticulum (ER), but not at cellular lipid droplets (LDs). The positioning of TMEM68 at the ER was dependent on its first transmembrane domain (TMD), which by itself was sufficient to target cytosolic green fluorescence protein (GFP) to the ER. In contrast, a second TMD was dispensable for ER localization of TMEM68. Finally, we found that among multiple murine tissues the expression level of TMEM68 transcripts was highest in brain. We conclude that TMEM68 is an integral ER membrane protein and a putative acyltransferase involved in brain glycerolipid metabolism.