Lysophosphatidic acid triggers inflammation in the liver and white adipose tissue in rat models of 1-acyl-sn-glycerol-3-phosphate acyltransferase 2 deficiency and overnutrition.

AGPAT2 (1-acyl-sn-glycerol-3-phosphate-acyltransferase-2) converts lysophosphatidic acid (LPA) into phosphatidic acid (PA), and mutations of the AGPAT2 gene cause the most common form of congenital generalized lipodystrophy which leads to steatohepatitis. The underlying mechanism by which AGPAT2 deficiency leads to lipodystrophy and steatohepatitis has not been elucidated. We addressed this question using an antisense oligonucleotide (ASO) to knockdown expression of Agpat2 in the liver and white adipose tissue (WAT) of adult male Sprague-Dawley rats. Agpat2 ASO treatment induced lipodystrophy and inflammation in WAT and the liver, which was associated with increased LPA content in both tissues, whereas PA content was unchanged. We found that a controlled-release mitochondrial protonophore (CRMP) prevented LPA accumulation and inflammation in WAT whereas an ASO against glycerol-3-phosphate acyltransferase, mitochondrial (Gpam) prevented LPA content and inflammation in the liver in Agpat2 ASO-treated rats. In addition, we show that overnutrition, due to high sucrose feeding, resulted in increased hepatic LPA content and increased activated macrophage content which were both abrogated with Gpam ASO treatment. Taken together, these data identify LPA as a key mediator of liver and WAT inflammation and lipodystrophy due to AGPAT2 deficiency as well as liver inflammation due to overnutrition and identify LPA as a potential therapeutic target to ameliorate these conditions.

Identification of a 1-acyl-glycerol-3-phosphate acyltransferase from Mycobacterium tuberculosis, a key enzyme involved in triacylglycerol biosynthesis.

Latent tuberculosis, caused by dormant Mycobacterium tuberculosis (Mtb), poses a threat to global health through the incubation of undiagnosed infections within the community. Dormant Mtb, which is phenotypically tolerant to antibiotics, accumulates triacylglycerol (TAG) utilizing fatty acids obtained from macrophage lipid droplets. TAG is vital to mycobacteria, serving as a cell envelope component and energy reservoir during latency. TAG synthesis occurs by sequential acylation of glycerol-3-phosphate, wherein the second acylation step is catalyzed by acylglycerol-3-phosphate acyltransferase (AGPAT), resulting in the production of phosphatidic acid (PA), a precursor for the synthesis of TAG and various phospholipids. Here, we have characterized a putative acyltransferase of Mtb encoded by Rv3816c. We found that Rv3816c has all four characteristic motifs of AGPAT, exists as a membrane-bound enzyme, and functions as 1-acylglycerol-3-phosphate acyltransferase. The enzyme could transfer the acyl group to acylglycerol-3-phosphate (LPA) from monounsaturated fatty acyl-coenzyme A of chain length 16 or 18 to produce PA. Complementation of Escherichia coli PlsC mutant in vivo by Rv3816c confirmed that it functions as AGPAT. Its active site mutants, H43A and D48A, were incapable of transferring the acyl group to LPA in vitro and were not able to rescue the growth defect of E. coli PlsC mutant in vivo. Identifying Rv3816c as AGPAT and comparing its properties with other AGPAT homologs is not only a step toward understanding the TAG biosynthesis in mycobacteria but has the potential to explore it as a drug target.

Unmasking crucial residues in adipose triglyceride lipase for coactivation with comparative gene identification-58.

Lipolysis is an essential metabolic process that releases unesterified fatty acids from neutral lipid stores to maintain energy homeostasis in living organisms. Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis and can be coactivated upon interaction with the protein comparative gene identification-58 (CGI-58). The underlying molecular mechanism of ATGL stimulation by CGI-58 is incompletely understood. Based on analysis of evolutionary conservation, we used site directed mutagenesis to study a C-terminally truncated variant and full-length mouse ATGL providing insights in the protein coactivation on a per-residue level. We identified the region from residues N209-N215 in ATGL as essential for coactivation by CGI-58. ATGL variants with amino acids exchanges in this region were still able to hydrolyze triacylglycerol at the basal level and to interact with CGI-58, yet could not be activated by CGI-58. Our studies also demonstrate that full-length mouse ATGL showed higher tolerance to specific single amino acid exchanges in the N209-N215 region upon CGI-58 coactivation compared to C-terminally truncated ATGL variants. The region is either directly involved in protein-protein interaction or essential for conformational changes required in the coactivation process. Three-dimensional models of the ATGL/CGI-58 complex with the artificial intelligence software AlphaFold demonstrated that a large surface area is involved in the protein-protein interaction. Mapping important amino acids for coactivation of both proteins, ATGL and CGI-58, onto the 3D model of the complex locates these essential amino acids at the predicted ATGL/CGI-58 interface thus strongly corroborating the significance of these residues in CGI-58-mediated coactivation of ATGL.

Saccharomyces cerevisiae Δ9-desaturase Ole1 forms a supercomplex with Slc1 and Dga1.

Biosynthesis of the various lipid species that compose cellular membranes and lipid droplets depends on the activity of multiple enzymes functioning in coordinated pathways. The flux of intermediates through lipid biosynthetic pathways is regulated to respond to nutritional and environmental demands placed on the cell necessitating that there be flexibility in pathway activity and organization. This flexibility can in part be achieved through the organization of enzymes into metabolon supercomplexes. However, the composition and organization of such supercomplexes remain unclear. Here, we identified protein-protein interactions between acyltransferases Sct1, Gpt2, Slc1, Dga1, and the Δ9 acyl-CoA desaturase Ole1 in Saccharomyces cerevisiae. We further determined that a subset of these acyltransferases interact with each other independent of Ole1. We show that truncated versions of Dga1 lacking the carboxyl-terminal 20 amino acid residues are nonfunctional and unable to bind Ole1. Furthermore, charged-to-alanine scanning mutagenesis revealed that a cluster of charged residues near the carboxyl terminus was required for the interaction with Ole1. Mutation of these charged residues disrupted the interaction between Dga1 and Ole1 but allowed Dga1 to retain catalytic activity and to induce lipid droplet formation. These data support the formation of a complex of acyltransferases involved in lipid biosynthesis that interacts with Ole1, the sole acyl-CoA desaturase in S. cerevisiae, that can channel unsaturated acyl chains toward phospholipid or triacylglycerol synthesis. This desaturasome complex may provide the architecture that allows for the necessary flux of de novo-synthesized unsaturated acyl-CoA to phospholipid or triacylglycerol synthesis as demanded by cellular requirements.

AGPAT3 deficiency impairs adipocyte differentiation and leads to a lean phenotype in mice.

Acylglycerophosphate acyltransferases (AGPATs) catalyze the de novo formation of phosphatidic acid to synthesize glycerophospholipids and triglycerides. AGPATs demonstrate unique physiological roles despite a similar biochemical function. AGPAT3 is highly expressed in the testis, kidney, and liver, with intermediate expression in adipose tissue. Loss of AGPAT3 is associated with reproductive abnormalities and visual dysfunction. However, the role of AGPAT3 in adipose tissue and whole body metabolism has not been investigated. We found that male Agpat3 knockout (KO) mice exhibited reduced body weights with decreased white and brown adipose tissue mass. Such changes were less pronounced in the female Agpat3-KO mice. Agpat3-KO mice have reduced plasma insulin growth factor 1 (IGF1) and insulin levels and diminished circulating lipid metabolites. They manifested intact glucose homeostasis and insulin sensitivity despite a lean phenotype. Agpat3-KO mice maintained an energy balance with normal food intake, energy expenditure, and physical activity, except for increased water intake. Their adaptive thermogenesis was also normal despite reduced brown adipose mass and triglyceride content. Mechanistically, Agpat3 was elevated during mouse and human adipogenesis and enriched in adipocytes. Agpat3-knockdown 3T3-L1 cells and Agpat3-deficient mouse embryonic fibroblasts (MEFs) have impaired adipogenesis in vitro. Interestingly, pioglitazone treatment rescued the adipogenic deficiency in Agpat3-deficient cells. We conclude that AGPAT3 regulates adipogenesis and adipose development. It is possible that adipogenic impairment in Agpat3-deficient cells potentially leads to reduced adipose mass. Findings from this work support the unique role of AGPAT3 in adipose tissue.NEW & NOTEWORTHY AGPAT3 deficiency results in male-specific growth retardation. It reduces adipose tissue mass but does not significantly impact glucose homeostasis or energy balance, except for influencing water intake in mice. Like AGPAT2, AGPAT3 is upregulated during adipogenesis, potentially by peroxisome proliferator-activated receptor gamma (PPARγ). Loss of AGPAT3 impairs adipocyte differentiation, which could be rescued by pioglitazone. Overall, AGPAT3 plays a significant role in regulating adipose tissue mass, partially involving its influence on adipocyte differentiation.

Aging reduces ABHD5 protein content in the adipose tissue of mice: The reversal effect of exercise.

Dysfunction of the adipose tissue metabolism is considered as a significant hallmark of aging. It has been proposed that α-ß hydrolase domain containing 5 (ABHD5) plays a critical role in the control of lipolysis. However, the role of ABHD5 in the control of lipolysis during aging or exercise is unknown. Here we combined the experimental mouse model with transcriptomic analyzes by using murine and human databases to explore the role of ABHD5 in the adipose tissue during aging and in response to exercise. Transcriptomic data revealed a downregulation of Abhd5 messenger RNA levels in the subcutaneous white adipose tissue (scWAT) over time in individuals from 20 to 69 years old. Aged mice displayed dramatic reduction of ABHD5 protein content and lipolytic-related proteins in the scWAT. Interestingly, 4 weeks of high-intensity interval training increased ABHD5 protein level and restored the lipolytic pathway in the scWAT of aged mice. Altogether, our findings demonstrated that aging affects ABHD5 content in the adipose tissue of mice and humans. Conversely, exercise increases ABHD5 activity, recovering the lipolytic activity in aged mice.

ABHD5 suppresses cancer cell anabolism through lipolysis-dependent activation of the AMPK/mTORC1 pathway.

ABHD5 is an essential coactivator of ATGL, the rate-limiting triglyceride (TG) lipase in many cell types. Importantly, ABHD5 also functions as a tumor suppressor, and ABHD5 mRNA expression levels correlate with patient survival for several cancers. Nevertheless, the mechanisms involved in ABHD5-dependent tumor suppression are not known. We found that overexpression of ABHD5 induces cell cycle arrest at the G1 phase and causes growth retardation in a panel of prostate cancer cells. Transcriptomic profiling and biochemical analysis revealed that genetic or pharmacological activation of lipolysis by ABHD5 potently inhibits mTORC1 signaling, leading to a significant downregulation of protein synthesis. Mechanistically, we found that ABHD5 elevates intracellular AMP content, which activates AMPK, leading to inhibition of mTORC1. Interestingly, ABHD5-dependent suppression of mTORC1 was abrogated by pharmacological inhibition of DGAT1 or DGAT2, isoenzymes that re-esterify fatty acids in a process that consumes ATP. Collectively, this study maps out a novel molecular pathway crucial for limiting cancer cell proliferation, in which ABHD5-mediated lipolysis creates an energy-consuming futile cycle between TG hydrolysis and resynthesis, leading to inhibition of mTORC1 and cancer cell growth arrest.

The ATGL lipase cooperates with ABHD5 to mobilize lipids for hepatitis C virus assembly.

Lipid droplets are essential cellular organelles for storage of fatty acids and triglycerides. The hepatitis C virus (HCV) translocates several of its proteins onto their surface and uses them for production of infectious progeny. We recently reported that the lipid droplet-associated α/ß hydrolase domain-containing protein 5 (ABHD5/CGI-58) participates in HCV assembly by mobilizing lipid droplet-associated lipids. However, ABHD5 itself has no lipase activity and it remained unclear how ABHD5 mediates lipolysis critical for HCV assembly. Here, we identify adipose triglyceride lipase (ATGL) as ABHD5 effector and new host factor involved in the hepatic lipid droplet degradation as well as in HCV and lipoprotein morphogenesis. Modulation of ATGL protein expression and lipase activity controlled lipid droplet lipolysis and virus production. ABHD4 is a paralog of ABHD5 unable to activate ATGL or support HCV assembly and lipid droplet lipolysis. Grafting ABHD5 residues critical for activation of ATGL onto ABHD4 restored the interaction between lipase and co-lipase and bestowed the pro-viral and lipolytic functions onto the engineered protein. Congruently, mutation of the predicted ABHD5 protein interface to ATGL ablated ABHD5 functions in lipid droplet lipolysis and HCV assembly. Interestingly, minor alleles of ABHD5 and ATGL associated with neutral lipid storage diseases in human, are also impaired in lipid droplet lipolysis and their pro-viral functions. Collectively, these results show that ABHD5 cooperates with ATGL to mobilize triglycerides for HCV infectious virus production. Moreover, viral manipulation of lipid droplet homeostasis via the ABHD5-ATGL axis, akin to natural genetic variation in these proteins, emerges as a possible mechanism by which chronic HCV infection causes liver steatosis.

Lysocardiolipin acyltransferase regulates NSCLC cell proliferation and migration by modulating mitochondrial dynamics.

Lysocardiolipin acyltransferase (LYCAT), a cardiolipin (CL)-remodeling enzyme, is crucial for maintaining normal mitochondrial function and vascular development. Despite the well-characterized role for LYCAT in the regulation of mitochondrial dynamics, its involvement in lung cancer, if any, remains incompletely understood. In this study, in silico analysis of TCGA lung cancer data sets revealed a significant increase in LYCAT expression, which was later corroborated in human lung cancer tissues and immortalized lung cancer cell lines via indirect immunofluorescence and immunoblotting, respectively. Stable knockdown of LYCAT in NSCLC cell lines not only reduced CL and increased monolyso-CL levels but also reduced in vivo tumor growth, as determined by xenograft studies in athymic nude mice. Furthermore, blocking LYCAT activity using a LYCAT mimetic peptide attenuated cell migration, suggesting a novel role for LYCAT activity in promoting NSCLC. Mechanistically, the pro-proliferative effects of LYCAT were mediated by an increase in mitochondrial fusion and a G1/S cell cycle transition, both of which are linked to increased cell proliferation. Taken together, these results demonstrate a novel role for LYCAT in promoting NSCLC and suggest that targeting LYCAT expression or activity in NSCLC may provide new avenues for the therapeutic treatment of lung cancer.

Dengue virus reduces AGPAT1 expression to alter phospholipids and enhance infection in Aedes aegypti.

More than half of the world population is at risk of dengue virus (DENV) infection because of the global distribution of its mosquito vectors. DENV is an envelope virus that relies on host lipid membranes for its life-cycle. Here, we characterized how DENV hijacks the mosquito lipidome to identify targets for novel transmission-blocking interventions. To describe metabolic changes throughout the mosquito DENV cycle, we deployed a Liquid chromatography-high resolution mass spectrometry (LC-HRMS) workflow including spectral similarity annotation in cells, midguts and whole mosquitoes at different times post infection. We revealed a major aminophospholipid reconfiguration with an overall early increase, followed by a reduction later in the cycle. We phylogenetically characterized acylglycerolphosphate acyltransferase (AGPAT) enzyme isoforms to identify those that catalyze a rate-limiting step in phospholipid biogenesis, the acylation of lysophosphatidate to phosphatidate. We showed that DENV infection decreased AGPAT1, but did not alter AGPAT2 expression in cells, midguts and mosquitoes. Depletion of either AGPAT1 or AGPAT2 increased aminophospholipids and partially recapitulated DENV-induced reconfiguration before infection in vitro. However, only AGPAT1 depletion promoted infection by maintaining high aminophospholipid concentrations. In mosquitoes, AGPAT1 depletion also partially recapitulated DENV-induced aminophospholipid increase before infection and enhanced infection by maintaining high aminophospholipid concentrations. These results indicate that DENV inhibition of AGPAT1 expression promotes infection by increasing aminophospholipids, as observed in the mosquito's early DENV cycle. Furthermore, in AGPAT1-depleted mosquitoes, we showed that enhanced infection was associated with increased consumption/redirection of aminophospholipids. Our study suggests that DENV regulates aminophospholipids, especially phosphatidylcholine and phosphatidylethanolamine, by inhibiting AGPAT1 expression to increase aminophospholipid availability for virus multiplication.

Resting skeletal muscle PNPLA2 (ATGL) and CPT1B are associated with peak fat oxidation rates in men and women but do not explain observed sex differences.

NEW FINDINGS: What is the central question of this study? What is the relationship between proteins in skeletal muscle and adipose tissue determined at rest and at peak rates of fat oxidation in men and women? What is the main finding and its importance? The resting contents of proteins in skeletal muscle involved in triglyceride hydrolysis and mitochondrial lipid transport were more strongly associated with peak fat oxidation rates than proteins related to lipid transport or hydrolysis in adipose tissue. Although females displayed higher relative rates of fat oxidation than males, this was not explained by the proteins measured in this study, suggesting that other factors determine sex differences in fat metabolism. ABSTRACT: We explored key proteins involved in fat metabolism that might be associated with peak fat oxidation (PFO) and account for sexual dimorphism in fuel metabolism during exercise. Thirty-six healthy adults [15 women; 40 ± 11 years of age; peak oxygen consumption 42.5 ± 9.5 ml (kg body mass)-1  min-1 ; mean ± SD] completed two exercise tests to determine PFO via indirect calorimetry. Resting adipose tissue and/or skeletal muscle biopsies were obtained to determine the adipose tissue protein content of PLIN1, ABHD5 (CGI-58), LIPE (HSL), PNPLA2 (ATGL), ACSL1, CPT1B and oestrogen receptor α (ERα) and the skeletal muscle protein content of FABP 3 (FABPpm), PNPLA2 (ATGL), ACSL1, CTP1B and ESR1 (ERα). Moderate strength correlations were found between PFO [in milligrams per kilogram of fat-free mass (FFM) per minute] and the protein content of PNPLA2 (ATGL) [rs  = 0.41 (0.03-0.68), P < 0.05] and CPT1B [rs  = 0.45 (0.09-0.71), P < 0.05] in skeletal muscle. No other statistically significant bivariate correlations were found consistently. Females had a greater relative PFO than males [7.1 ± 1.9 vs. 4.5 ± 1.3 and 7.3 ± 1.7 vs. 4.8 ± 1.2 mg (kg FFM)-1  min-1 in the adipose tissue (n = 14) and skeletal muscle (n = 12) subgroups, respectively (P < 0.05)]. No statistically significant sex differences were found in the content of these proteins. The regulation of PFO might involve processes relating to intramyocellular triglyceride hydrolysis and mitochondrial fatty acid transport, and adipose tissue is likely to play a more minor role than muscle. Sex differences in fat metabolism are likely to be attributable to factors other than the resting content of proteins in skeletal muscle and adipose tissue relating to triglyceride hydrolysis and fatty acid transport.

Cardiac endurance training alters plasma profiles of circular RNA MBOAT2.

Marathon running is an extreme physical activity, which determines cardiopulmonary adaption of athletes. Circular RNAs (circRNAs) as potential biomarkers in the blood stream have so far not been tested after such strenuous activities. In silico approaches were performed to identify the potential candidate circRNA MBOAT2. Next, we demonstrated high stability and conservation of circRNA MBOAT2 as well as its abundancy in human plasma. In addition to Sanger sequencing of the circRNA specific head-to-tail junction, or back-splice site, we established a synthetic plasmid standard which allowed exact copy number calculations of circRNA MBOAT2. We then analyzed plasmatic circRNA MBOAT2 and observed a significantly lower level 24 h after the marathon. Such alterations were correlated to physical exercise parameters confirming the role of circRNA MBOAT2 as a promising noncoding RNA biomarker detecting cardiopulmonary adaption.NEW & NOTEWORTHY In brief, we herein report a timeline of circulating circular RNA (circRNA) MBOAT2 in a cohort of marathon runners. Time-course analysis of plasmatic circRNA MBOAT2 demonstrated a significantly lowered level 24 h after the marathon. Abundancy of circRNA was correlated to physical exercise parameters highlighting the role of circRNA MBOAT2 as a valuable noncoding RNA biomarker detecting and following up cardiopulmonary adaption.

Glycerol-3-phosphate acyltransferases 3 and 4 direct glycerolipid synthesis and affect functionality in activated macrophages.

Macrophage classical M1 activation via TLR4 triggers a variety of responses to achieve the elimination of foreign pathogens. During this process, there is also an increase in lipid droplets which contain large quantities of triacylglycerol (TAG) and phospholipid (PL). The functional consequences of this increment in lipid mass are poorly understood. Here, we studied the contribution of glycerolipid synthesis to lipid accumulation, focusing specifically on the first and rate-limiting enzyme of the pathway: glycerol-3-phosphate acyltransferase (GPAT). Using bone marrow-derived macrophages (BMDMs) treated with Kdo2-lipid A, we showed that glycerolipid synthesis is induced during macrophage activation. GPAT4 protein level and GPAT3/GPAT4 enzymatic activity increase during this process, and these two isoforms were required for the accumulation of cell TAG and PL. The phagocytic capacity of Gpat3-/- and Gpat4-/- BMDM was impaired. Additionally, inhibiting fatty acid ß-oxidation reduced phagocytosis only partially, suggesting that lipid accumulation is not necessary for the energy requirements for phagocytosis. Finally, Gpat4-/- BMDM expressed and released more pro-inflammatory cytokines and chemokines after macrophage activation, suggesting a role for GPAT4 in suppressing inflammatory responses. Together, these results provide evidence that glycerolipid synthesis directed by GPAT4 is important for the attenuation of the inflammatory response in activated macrophages.

CGI-58: Versatile Regulator of Intracellular Lipid Droplet Homeostasis.

Comparative gene identification-58 (CGI-58), also known as α/ß-hydrolase domain-containing 5 (ABHD5), is a member of a large family of proteins containing an α/ß-hydrolase-fold. CGI-58 is well-known as the co-activator of adipose triglyceride lipase (ATGL), which is a key enzyme initiating cytosolic lipid droplet lipolysis. Mutations in either the human CGI-58 or ATGL gene cause an autosomal recessive neutral lipid storage disease, characterized by the excessive accumulation of triglyceride (TAG)-rich lipid droplets in the cytoplasm of almost all cell types. CGI-58, however, has ATGL-independent functions. Distinct phenotypes associated with CGI-58 deficiency commonly include ichthyosis (scaly dry skin), nonalcoholic steatohepatitis, and hepatic fibrosis. Through regulated interactions with multiple protein families, CGI-58 controls many metabolic and signaling pathways, such as lipid and glucose metabolism, energy balance, insulin signaling, inflammatory responses, and thermogenesis. Recent studies have shown that CGI-58 regulates the pathogenesis of common metabolic diseases in a tissue-specific manner. Future studies are needed to molecularly define ATGL-independent functions of CGI-58, including the newly identified serine protease activity of CGI-58. Elucidation of these versatile functions of CGI-58 may uncover fundamental cellular processes governing lipid and energy homeostasis, which may help develop novel approaches that counter against obesity and its associated metabolic sequelae.

LYSOPHOSPHATIDIC ACID ACYLTRANSFERASES 4 and 5 are involved in glycerolipid metabolism and nitrogen starvation response in Arabidopsis.

Nitrogen (N) deficiency triggers an accumulation of a storage lipid triacylglycerol (TAG) in seed plants and algae. Whereas the metabolic pathway and regulatory mechanism to synthesize TAG from diacylglycerol are well known, enzymes involved in the supply of diacylglycerol remain elusive under N starvation. Lysophosphatidic acid acyltransferase (LPAT) catalyzes an important step of the de novo phospholipid biosynthesis pathway and thus has a strong flux control in the biosynthesis of phospholipids and TAG. Five LPAT isoforms are known in Arabidopsis; however, the functions of LPAT4 and LPAT5 remain elusive. Here, we show that LPAT4 and LPAT5 are functional endoplasmic-reticulum-localized LPATs. Seedlings of the double knockout mutant lpat4-1 lpat5-1 showed reduced content of phospholipids and TAG under normal growth condition. Under N starvation, lpat4-1 lpat5-1 seedlings showed severer growth defect than the wild-type in shoot. The phenotype was similar to dgat1-4, which affects a major TAG biosynthesis pathway and showed similarly reduced TAG content as the lpat4-1 lpat5-1. We suggest that LPAT4 and LPAT5 may redundantly function in endoplasmic-reticulum-localized de novo glycerolipid biosynthesis for phospholipids and TAG, which is important for the N starvation response in Arabidopsis.

A three-dimensional ParF meshwork assembles through the nucleoid to mediate plasmid segregation.

Genome segregation is a fundamental step in the life cycle of every cell. Most bacteria rely on dedicated DNA partition proteins to actively segregate chromosomes and low copy-number plasmids. Here, by employing super resolution microscopy, we establish that the ParF DNA partition protein of the ParA family assembles into a three-dimensional meshwork that uses the nucleoid as a scaffold and periodically shuttles between its poles. Whereas ParF specifies the territory for plasmid trafficking, the ParG partner protein dictates the tempo of ParF assembly cycles and plasmid segregation events by stimulating ParF adenosine triphosphate hydrolysis. Mutants in which this ParG temporal regulation is ablated show partition deficient phenotypes as a result of either altered ParF structure or dynamics and indicate that ParF nucleoid localization and dynamic relocation, although necessary, are not sufficient per se to ensure plasmid segregation. We propose a Venus flytrap model that merges the concepts of ParA polymerization and gradient formation and speculate that a transient, dynamic network of intersecting polymers that branches into the nucleoid interior is a widespread mechanism to distribute sizeable cargos within prokaryotic cells.

ABHD5 stimulates PNPLA1-mediated ω-O-acylceramide biosynthesis essential for a functional skin permeability barrier.

Mutations in the genes coding for patatin-like phospholipase domain-containing 1 (PNPLA1) and α/ß-hydrolase domain-containing 5 (ABHD5), also known as comparative gene identification 58, are causative for ichthyosis, a severe skin barrier disorder. Individuals with mutations in either of these genes show a defect in epidermal ω-O-acylceramide (AcylCer) biosynthesis, suggesting that PNPLA1 and ABHD5 act in the same metabolic pathway. In this report, we identified ABHD5 as a coactivator of PNPLA1 that stimulates the esterification of ω-hydroxy ceramides with linoleic acid for AcylCer biosynthesis. ABHD5 interacts with PNPLA1 and recruits the enzyme to its putative triacylglycerol substrate onto cytosolic lipid droplets. Conversely, alleles of ABHD5 carrying point mutations associated with ichthyosis in humans failed to accelerate PNPLA1-mediated AcylCer biosynthesis. Our findings establish an important biochemical function of ABHD5 in interacting with PNPLA1 to synthesize crucial epidermal lipids, emphasizing the significance of these proteins in the formation of a functional skin permeability barrier.

Metabolic Circuit Involving Free Fatty Acids, microRNA 122, and Triglyceride Synthesis in Liver and Muscle Tissues.

BACKGROUND & AIMS: Effective treatments are needed for hepatic steatosis characterized by accumulation of triglycerides in hepatocytes, which leads to hepatocellular carcinoma. MicroRNA 122 (MIR122) is expressed only in the liver, where it regulates lipid metabolism. We investigated the mechanism by which free fatty acids (FFAs) regulate MIR122 expression and the effect of MIR122 on triglyceride synthesis. METHODS: We analyzed MIR122 promoter activity and validated its target mRNAs by transfection of Luciferase reporter plasmids into Huh7, BNL-1ME, and HEK293 cultured cell lines. We measured levels of microRNAs and mRNAs by quantitative real-time PCR analysis of RNA extracted from plasma, liver, muscle, and adipose tissues of C57BL/6 mice given the FFA-inducer CL316243. MIR122 was inhibited using an inhibitor of MIR122. Metabolic profiles of mice were determined using metabolic chambers and by histologic analyses of liver tissues. We performed RNA sequence analyses to identify metabolic pathways involving MIR122. RESULTS: We validated human Agpat1 and Dgat1 mRNAs, involved in triglyceride synthesis, as targets of MIR122. FFAs increased MIR122 expression in livers of mice by activating the retinoic acid-related orphan receptor alpha, and induced secretion of MIR122 from liver to blood. Circulating MIR122 entered muscle and adipose tissues of mice, reducing mRNA levels of genes involved in triglyceride synthesis. Mice injected with an inhibitor of MIR122 and then given CL316243, accumulated triglycerides in liver and muscle tissues, and had reduced rates of ß-oxidation. There was a positive correlation between level of FFAs and level of MIR122 in plasma samples from 6 healthy individuals, collected before and during fasting. CONCLUSIONS: In biochemical and histologic studies of plasma, liver, muscle, and adipose tissues from mice, we found that FFAs increase hepatic expression and secretion of MIR122, which regulates energy storage vs expenditure in liver and peripheral tissues. Strategies to reduce triglyceride levels, by increasing MIR122, might be developed for treatment of metabolic syndrome.

Expression of AGPAT2, an enzyme involved in the glycerophospholipid/triacylglycerol biosynthesis pathway, is directly regulated by HIF-1 and promotes survival and etoposide resistance of cancer cells under hypoxia.

Hypoxia inducible factor-1 (HIF-1) supports survival of normal cells under low oxygen concentration and cancer cells in the hypoxic tumor microenvironment. This involves metabolic reprogramming via upregulation of glycolysis, downregulation of oxidative phosphorylation and, less well documented, effects on lipid metabolism. To investigate the latter, we examined expression of relevant enzymes in cancer cells grown under hypoxia. We show that expression of acylglycerol-3-phosphate acyltransferase 2 (AGPAT2), also known as lysophosphatidic acid acyltransferase ß (LPAATß), was upregulated under hypoxia and this was impaired by siRNA-mediated knockdown of HIF-1α. Moreover, a sequence of the AGPAT2 gene promoter region, containing 6 putative Hypoxia Response Elements (HREs), activated transcription of a reporter gene under hypoxic conditions or in normoxic cells over-expressing HIF-1α. Chromatin immunoprecipitation experiments confirmed binding of HIF-1α to one of these HREs, mutation of which abolished hypoxic activation of the AGPAT2 promoter. Knockdown of AGPAT2 by siRNA reduced lipid droplet accumulation and cell viability under hypoxia and increased cancer cell sensitivity to the chemotherapeutic etoposide. In conclusion, our findings demonstrate that AGPAT2, which is mutated in patients with congenital generalized lipodystrophy and over-expressed in different types of cancer, is a direct transcriptional target of HIF-1, suggesting that upregulation of lipid storage by HIF-1 plays an important role in adaptation and survival of cancer cells under low oxygen conditions.

ABHD5/CGI-58, the Chanarin-Dorfman Syndrome Protein, Mobilises Lipid Stores for Hepatitis C Virus Production.

Hepatitis C virus (HCV) particles closely mimic human very-low-density lipoproteins (VLDL) to evade humoral immunity and to facilitate cell entry. However, the principles that govern HCV association with VLDL components are poorly defined. Using an siRNA screen, we identified ABHD5 (α/ß hydrolase domain containing protein 5, also known as CGI-58) as a new host factor promoting both virus assembly and release. ABHD5 associated with lipid droplets and triggered their hydrolysis. Importantly, ABHD5 Chanarin-Dorfman syndrome mutants responsible for a rare lipid storage disorder in humans were mislocalised, and unable to consume lipid droplets or support HCV production. Additional ABHD5 mutagenesis revealed a novel tribasic motif that does not influence subcellular localization but determines both ABHD5 lipolytic and proviral properties. These results indicate that HCV taps into the lipid droplet triglyceride reservoir usurping ABHD5 lipase cofactor function. They also suggest that the resulting lipid flux, normally devoted to VLDL synthesis, also participates in the assembly and release of the HCV lipo-viro-particle. Altogether, our study provides the first association between the Chanarin-Dorfman syndrome protein and an infectious disease and sheds light on the hepatic manifestations of this rare genetic disorder as well as on HCV morphogenesis.