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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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Minimal active fragment of ATGL can be inhibited by G0S2.A. ATGL254 was expressed in E. coli, lysates prepared, and TG hydrolase activity assay performed in the presence of purified CGI-58 (*), without and with addition of mG0S2 as described in Figure 2. Data are presented as mean+SD and representative for three independent experiments, each performed in triplicates. *** indicate statistical significant differences as determined by unpaired Student's t-test (two-tailed), p>0.001. B. Western Blotting analysis of ATGL254 expression and SDS-PAGE gel confirming expression of mG0S2 in bacterial lysate.
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pone-0026349-g005: Minimal active fragment of ATGL can be inhibited by G0S2.A. ATGL254 was expressed in E. coli, lysates prepared, and TG hydrolase activity assay performed in the presence of purified CGI-58 (*), without and with addition of mG0S2 as described in Figure 2. Data are presented as mean+SD and representative for three independent experiments, each performed in triplicates. *** indicate statistical significant differences as determined by unpaired Student's t-test (two-tailed), p>0.001. B. Western Blotting analysis of ATGL254 expression and SDS-PAGE gel confirming expression of mG0S2 in bacterial lysate.

Mentions: Next we asked whether ATGL254, the minimal active fragment of ATGL, can also be inhibited by G0S2. As shown in Figure 5, CGI-58 activated ATGL254 can be inhibited by addition of bacterial lysate of G0S2. This also suggests that the protein surfaces for interaction of ATGL with G0S2 reside within the first 254 residues of the protein, similar to the results observed for protein interaction with CGI-58.


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)

Minimal active fragment of ATGL can be inhibited by G0S2.A. ATGL254 was expressed in E. coli, lysates prepared, and TG hydrolase activity assay performed in the presence of purified CGI-58 (*), without and with addition of mG0S2 as described in Figure 2. Data are presented as mean+SD and representative for three independent experiments, each performed in triplicates. *** indicate statistical significant differences as determined by unpaired Student's t-test (two-tailed), p>0.001. B. Western Blotting analysis of ATGL254 expression and SDS-PAGE gel confirming expression of mG0S2 in bacterial lysate.
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Related In: Results  -  Collection

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

pone-0026349-g005: Minimal active fragment of ATGL can be inhibited by G0S2.A. ATGL254 was expressed in E. coli, lysates prepared, and TG hydrolase activity assay performed in the presence of purified CGI-58 (*), without and with addition of mG0S2 as described in Figure 2. Data are presented as mean+SD and representative for three independent experiments, each performed in triplicates. *** indicate statistical significant differences as determined by unpaired Student's t-test (two-tailed), p>0.001. B. Western Blotting analysis of ATGL254 expression and SDS-PAGE gel confirming expression of mG0S2 in bacterial lysate.
Mentions: Next we asked whether ATGL254, the minimal active fragment of ATGL, can also be inhibited by G0S2. As shown in Figure 5, CGI-58 activated ATGL254 can be inhibited by addition of bacterial lysate of G0S2. This also suggests that the protein surfaces for interaction of ATGL with G0S2 reside within the first 254 residues of the protein, similar to the results observed for protein interaction with CGI-58.

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