SUMMARY Numerous studies in humans link a nonsynonymous genetic polymorphism (I148M) in adiponutrin (ADPN) to various forms of fatty liver disease and liver cirrhosis. Despite its high clinical relevance, the molecular function of ADPN and the mechanism by which I148M variant affects hepatic metabolism are unclear. Here we show that ADPN promotes cellular lipid synthesis by converting lysophosphatidic acid (LPA) into phosphatidic acid. The ADPN-catalyzed LPA acyltransferase (LPAAT) reaction is specific for LPA and long-chain acyl-CoAs. Wild-type mice receiving a high-sucrose diet exhibit substantial upregulation of Adpn in the liver and a concomitant increase in LPAAT activity. In Adpn-deficient mice, this diet-induced increase in hepatic LPAAT activity is reduced. Notably, the I148M variant of human ADPN exhibits increased LPAAT activity leading to increased cellular lipid accumulation. This gain of function provides a plausible biochemical mechanism for the development of liver steatosis in subjects carrying the I148M variant.
Adipose triglyceride lipase (ATGL) catalyzes the first step in the hydrolysis of triacylglycerol (TG) generating diacylglycerol and free fatty acids. The enzyme requires the activator protein CGI-58 (or ABHD5) for full enzymatic activity. Defective ATGL function causes a recessively inherited disorder named neutral lipid storage disease that is characterized by systemic TG accumulation and myopathy. In this study, we investigated the functional defects associated with mutations in the ATGL gene that cause neutral lipid storage disease. We show that these mutations lead to the expression of either inactive enzymes localizing to lipid droplets (LDs) or enzymatically active lipases with defective LD binding. Additionally, our studies assign important regulatory functions to the C-terminal part of ATGL. Truncated mutant ATGL variants lacking ϳ220 amino acids of the C-terminal protein region do not localize to LDs. Interestingly, however, these mutants exhibit substantially increased TG hydrolase activity in vitro (up to 20-fold) compared with the wild-type enzyme, indicating that the C-terminal region suppresses enzyme activity. Protein-protein interaction studies revealed an increased binding of truncated ATGL to CGI-58, suggesting that the C-terminal part interferes with CGI-58 interaction and enzyme activation. Compared with the human enzyme, the C-terminal region of mouse ATGL is much less effective in suppressing enzyme activity, implicating species-dependent differences in enzyme regulation. Together, our results demonstrate that the C-terminal region of ATGL is essential for proper localization of the enzyme and suppresses enzyme activity.
In mammals, excess energy is stored in the form of triacylglycerol primarily in lipid droplets of white adipose tissue. The first step of lipolysis (i.e. the mobilization of fat stores) is catalyzed by adipose triglyceride lipase (ATGL). The enzymatic activity of ATGL is strongly enhanced by CGI-58 (comparative gene identification-58), and the loss of either ATGL or CGI-58 function causes systemic triglyceride accumulation in humans and mice. However, the mechanism by which CGI-58 stimulates ATGL activity is unknown. To gain insight into CGI-58 function using structural features of the protein, we generated a threedimensional homology model based on sequence similarity with other proteins. Interestingly, the model of CGI-58 revealed that the N terminus forms an extension of the otherwise compact structure of the protein. This N-terminal region (amino acids 1-30) harbors a lipophilic tryptophan-rich stretch, which affects the localization of the protein.1 H NMR experiments revealed strong interaction between the N-terminal peptide and dodecylphosphocholine micelles as a lipid droplet-mimicking system. A role for this N-terminal region of CGI-58 in lipid droplet binding was further strengthened by localization studies in cultured cells. Although wild-type CGI-58 localizes to the lipid droplet, the N-terminally truncated fragments of CGI-58 are dispersed in the cytoplasm. Moreover, CGI-58 lacking the N-terminal extension loses the ability to stimulate ATGL, implying that the ability of CGI-58 to activate ATGL is linked to correct localization. In summary, our study shows that the N-terminal, Trp-rich region of CGI-58 is essential for correct localization and ATGL-activating function of CGI-58.The protein CGI-58 (comparative gene identification-58; identical to ABHD5 (abhydrolase domain-containing protein 5)) plays an important role in mammalian fatty acid metabolism (1, 2). Interaction of CGI-58 with adipose triglyceride lipase (ATGL), 2 the enzyme catalyzing the first step of lipolysis, enhances the hydrolytic activity of ATGL up to 20-fold (1, 3). CGI-58 is predicted to harbor an ␣/-hydrolase domain. Typically, ␣/-hydrolases exert their catalytic activity via a catalytic triad. In the case of CGI-58, however, the putative active site serine is replaced by an asparagine residue. Consequently, no triglyceride (TG)-hydrolyzing activity could be detected for the protein.Very recently, lysophosphatidic acid acyltransferase (LPAAT) activity of CGI-58 was discovered and kinetically characterized (4, 5). In humans, mutations in CGI-58 are associated with the development of neutral lipid storage disease with ichthyosis, which is also known as Chanarin Dorfman syndrome. These patients accumulate neutral lipids in various tissues and cell types, including granulocytes, and suffer from non-bullous congenital ichthyosiform erythroderma (2, 6, 7). Mutant forms of CGI-58 detected in patients with neutral lipid storage disease with ichthyosis harboring nonsense or missense mutations (recently reviewed in Ref. 8) are not compet...
Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme of lipolysis. ATGL specifically hydrolyzes triacylglycerols (TGs), thereby generating diacylglycerols and free fatty acids. ATGL's enzymatic activity is co-activated by the protein comparative gene identification-58 (CGI-58) and inhibited by the protein G0/G1 switch gene 2 (G0S2). The enzyme is predicted to act through a catalytic dyad (Ser47, Asp166) located within the conserved patatin domain (Ile10-Leu178). Yet, neither an experimentally determined 3D structure nor a model of ATGL is currently available, which would help to understand how CGI-58 and G0S2 modulate ATGL's activity. In this study we determined the minimal active domain of ATGL. This minimal fragment of ATGL could still be activated and inhibited by CGI-58 and G0S2, respectively. Furthermore, we show that this minimal domain is sufficient for protein-protein interaction of ATGL with its regulatory proteins. Based on these data, we generated a 3D homology model for the minimal domain. It strengthens our experimental finding that amino acids between Leu178 and Leu254 are essential for the formation of a stable protein domain related to the patatin fold. Our data provide insights into the structure-function relationship of ATGL and indicate higher structural similarities in the N-terminal halves of mammalian patatin-like phospholipase domain containing proteins, (PNPLA1, -2,- 3 and -5) than originally anticipated.
Supplementary key words comparative gene identifi cation-58 • human lipolysis • regulation • insulin resistanceIn periods of nutrient scarcity or in response to increased energy demand, triglyceride (TG) stores are mobilized to provide free fatty acids (FFAs) as energy fuel. The mobilization of TGs is performed in three consecutive reactions, catalyzed by three lipases: adipose triglyceride lipase (ATGL) ( 1-3 ), hormone-sensitive lipase (HSL) ( 4 ), and monoglyceride lipase (MGL) ( 5 ). The crucial physiological role of ATGL (also annotated as patatin-like phospholipase domain containing 2, desnutrin, phospholipase A2 , and transport secretion protein 2.2) in lipolysis became evident by the phenotype of ATGL-defi cient (ATGL-ko) mice. ATGL-ko mice display increased whole-body fat mass, enlarged adipose fat depots, and TG accumulation in many tissues. Massive TG deposition in cardiomyocytes leads to cardiac insuffi ciency and premature death ( 6 ). In humans, the lack of ATGL activity, caused by mutations in the ATGL gene, is associated with a rare inherited disorder, annotated as neutral lipid storage disease with myopathy (NLSDM) ( 7 ). This disease is characterized by TG deposition in multiple tissues and cardiac myopathy.ATGL activity is strongly infl uenced by regulatory proteins. In 2006, Lass et al. identifi ed comparative gene identifi cation-58 (CGI-58, also known as ABHD5) as coactivator of Abstract The hydrolysis of triglycerides in adipocytes, termed lipolysis, provides free fatty acids as energy fuel. Murine lipolysis largely depends on the activity of adipose triglyceride lipase (ATGL), which is regulated by two proteins annotated as comparative gene identifi cation-58 (CGI-58) and G0/G1 switch gene-2 (G0S2). CGI-58 activates and G0S2 inhibits ATGL activity. In contrast to mice, the functional role of G0S2 in human adipocyte lipolysis is poorly characterized. Here we show that overexpression or silencing of G0S2 in human SGBS adipocytes decreases and increases lipolysis, respectively. Human G0S2 is upregulated during adipocyte differentiation and inhibits ATGL activity in a dose-dependent manner. Interestingly, C-terminally truncated ATGL mutants, which fail to localize to lipid droplets, translocate to the lipid droplet upon coexpression with G0S2, suggesting that G0S2 anchors ATGL to lipid droplets independent of ATGL's C-terminal lipid binding domain. Taken together, our results indicate that G0S2 also regulates human lipolysis by affecting enzyme activity and intracellular localization of ATGL. Increased lipolysis is known to contribute to the pathogenesis of insulin resistance, and G0S2 expression has been shown to be reduced in poorly controlled type 2 diabetic patients. Our data indicate that downregulation of G0S2 in adipose tissue could represent one of the underlying causes leading to increased lipolysis in the insulin-resistant state. -Schweiger, M., M. Paar, C. Eder, J. Brandis, E. Moser, G. Gorkiewicz, S. Grond, F. P. W. Radner, I. Cerk, I. Cornaciu, M. Oberer, S. Kersten, R. Zechner, ...
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