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The minimal domain of adipose triglyceride lipase (ATGL) ranges until leucine 254 and can be activated and inhibited by CGI-58 and G0S2, respectively.

Cornaciu I, Boeszoermenyi A, Lindermuth H, Nagy HM, Cerk IK, Ebner C, Salzburger B, Gruber A, Schweiger M, Zechner R, Lass A, Zimmermann R, Oberer M - PLoS ONE (2011)

Bottom Line: 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.Based on these data, we generated a 3D homology model for the minimal domain.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biosciences, University of Graz, Graz, Austria.

ABSTRACT
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.

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The N-terminal region of ATGL up to residue 255 is necessary for enzymatic activity.A. C-terminally truncated ATGL variants were expressed in E.coli, lysates prepared, and TG hydrolase activity determined in vitro using radiolabeled triolein as substrate. The determined activity was normalized to bacterial lysate protein contents. The experiment was performed in triplicates. A representative result of three independent experiments is shown. B. Expression was assessed by Western blotting and ATGL in the soluble fraction of the bacterial lysates was quantified by densitometric analysis. C. TG hydrolase activities obtained in A were normalized to expression levels of ATGL variants as assessed in B. Data are presented as mean+SD.
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pone-0026349-g003: The N-terminal region of ATGL up to residue 255 is necessary for enzymatic activity.A. C-terminally truncated ATGL variants were expressed in E.coli, lysates prepared, and TG hydrolase activity determined in vitro using radiolabeled triolein as substrate. The determined activity was normalized to bacterial lysate protein contents. The experiment was performed in triplicates. A representative result of three independent experiments is shown. B. Expression was assessed by Western blotting and ATGL in the soluble fraction of the bacterial lysates was quantified by densitometric analysis. C. TG hydrolase activities obtained in A were normalized to expression levels of ATGL variants as assessed in B. Data are presented as mean+SD.

Mentions: Results of TG hydrolase assays clearly demonstrated that truncated proteins of mATGL up to Leu254 (termed ATGL254 throughout this paper) retained the ability to hydrolyze TG (Figure 3A). In contrast, mATGL variants with truncations closer to the N-terminus, at residues 253, 252, 245 and 235, lost their TG hydrolyzing activity. Thus, we can conclude that ATGL254 represents the shortest fragment of ATGL which retains TG hydrolase activity (Figure 3A). Next, we correlated specific activities of truncated variants to full-length ATGL. The rational for this approach is that enzymatic activities measured in protein lysates obviously depend on the amount of the protein in the soluble fraction. In addition, this procedure allowed us to place these data in context with previous reports, which showed higher activities of C-terminally truncated ATGL variants [20]. Western Blot analysis confirmed the presence of soluble ATGL variants in the lysates. Differences in the expression and solubility levels were evident (Figure 3B). To account for these differences in expression/solubility levels, results of the Western Blot was analyzed densitometrically and used for calculating relative activity rates. These relative activities, as depicted in Figure 3C, clearly demonstrate that shorter fragments of ATGL have higher intrinsic in vitro TG hydrolyzing activity when compared to full-length ATGL. The activity of C-terminal ATGL truncations was ablated when shorter fragments than ATGL254 were tested (Figure 3C), in line with ATGL254 as the minimal fragment required for ATGL activity.


The minimal domain of adipose triglyceride lipase (ATGL) ranges until leucine 254 and can be activated and inhibited by CGI-58 and G0S2, respectively.

Cornaciu I, Boeszoermenyi A, Lindermuth H, Nagy HM, Cerk IK, Ebner C, Salzburger B, Gruber A, Schweiger M, Zechner R, Lass A, Zimmermann R, Oberer M - PLoS ONE (2011)

The N-terminal region of ATGL up to residue 255 is necessary for enzymatic activity.A. C-terminally truncated ATGL variants were expressed in E.coli, lysates prepared, and TG hydrolase activity determined in vitro using radiolabeled triolein as substrate. The determined activity was normalized to bacterial lysate protein contents. The experiment was performed in triplicates. A representative result of three independent experiments is shown. B. Expression was assessed by Western blotting and ATGL in the soluble fraction of the bacterial lysates was quantified by densitometric analysis. C. TG hydrolase activities obtained in A were normalized to expression levels of ATGL variants as assessed in B. Data are presented as mean+SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198459&req=5

pone-0026349-g003: The N-terminal region of ATGL up to residue 255 is necessary for enzymatic activity.A. C-terminally truncated ATGL variants were expressed in E.coli, lysates prepared, and TG hydrolase activity determined in vitro using radiolabeled triolein as substrate. The determined activity was normalized to bacterial lysate protein contents. The experiment was performed in triplicates. A representative result of three independent experiments is shown. B. Expression was assessed by Western blotting and ATGL in the soluble fraction of the bacterial lysates was quantified by densitometric analysis. C. TG hydrolase activities obtained in A were normalized to expression levels of ATGL variants as assessed in B. Data are presented as mean+SD.
Mentions: Results of TG hydrolase assays clearly demonstrated that truncated proteins of mATGL up to Leu254 (termed ATGL254 throughout this paper) retained the ability to hydrolyze TG (Figure 3A). In contrast, mATGL variants with truncations closer to the N-terminus, at residues 253, 252, 245 and 235, lost their TG hydrolyzing activity. Thus, we can conclude that ATGL254 represents the shortest fragment of ATGL which retains TG hydrolase activity (Figure 3A). Next, we correlated specific activities of truncated variants to full-length ATGL. The rational for this approach is that enzymatic activities measured in protein lysates obviously depend on the amount of the protein in the soluble fraction. In addition, this procedure allowed us to place these data in context with previous reports, which showed higher activities of C-terminally truncated ATGL variants [20]. Western Blot analysis confirmed the presence of soluble ATGL variants in the lysates. Differences in the expression and solubility levels were evident (Figure 3B). To account for these differences in expression/solubility levels, results of the Western Blot was analyzed densitometrically and used for calculating relative activity rates. These relative activities, as depicted in Figure 3C, clearly demonstrate that shorter fragments of ATGL have higher intrinsic in vitro TG hydrolyzing activity when compared to full-length ATGL. The activity of C-terminal ATGL truncations was ablated when shorter fragments than ATGL254 were tested (Figure 3C), in line with ATGL254 as the minimal fragment required for ATGL activity.

Bottom Line: 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.Based on these data, we generated a 3D homology model for the minimal domain.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biosciences, University of Graz, Graz, Austria.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus