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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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ELISA experiment demonstrating that ATGL254 interacts with CGI-58 and G0S2.A. ELISA plates were coated with purified ATGL254-MBP and then incubated with purified His-tagged CGI-58. Purified smt (the fusion tag for CGI-58) was used as a negative control. Detection was performed by anti-his-antibody, HRP-labeled secondary antibody and tetramethyl-benzidine as substrate. B. ELISA plates were coated with purified mG0S2 and then incubated with purified ATGL254-MBP. Purified MBP was used as negative control. Detection was performed using an anti-MBP (HRP conjugated) antibody and otherwise as in A. Data are presented as mean+SD and representative for 2 independent experiments (performed in triplicates). C. SDS-PAGE confirming the expression and purification of respective fusion proteins.
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pone-0026349-g006: ELISA experiment demonstrating that ATGL254 interacts with CGI-58 and G0S2.A. ELISA plates were coated with purified ATGL254-MBP and then incubated with purified His-tagged CGI-58. Purified smt (the fusion tag for CGI-58) was used as a negative control. Detection was performed by anti-his-antibody, HRP-labeled secondary antibody and tetramethyl-benzidine as substrate. B. ELISA plates were coated with purified mG0S2 and then incubated with purified ATGL254-MBP. Purified MBP was used as negative control. Detection was performed using an anti-MBP (HRP conjugated) antibody and otherwise as in A. Data are presented as mean+SD and representative for 2 independent experiments (performed in triplicates). C. SDS-PAGE confirming the expression and purification of respective fusion proteins.

Mentions: To further substantiate our conclusions that ATGL254 is sufficient for regulation by CGI-58 and G0S2, the implied physical interaction between the involved proteins was also determined by ELISA experiments. For this purpose, plates were coated with purified ATGL254 or mG0S2. Then, wells were incubated with purified His6-smt-tagged CGI-58 or maltose binding protein (MBP)-tagged ATGL254. As expected, protein-protein interactions between the ATGL254 and its co-activator CGI-58, as well as its inhibitor G0S2 were detected (Figure 6A and B). These data further support the conclusion that ATGL254 harbors all residues required for catalytic activity as well as interaction with ATGL's regulatory proteins, CGI-58 and G0S2.


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)

ELISA experiment demonstrating that ATGL254 interacts with CGI-58 and G0S2.A. ELISA plates were coated with purified ATGL254-MBP and then incubated with purified His-tagged CGI-58. Purified smt (the fusion tag for CGI-58) was used as a negative control. Detection was performed by anti-his-antibody, HRP-labeled secondary antibody and tetramethyl-benzidine as substrate. B. ELISA plates were coated with purified mG0S2 and then incubated with purified ATGL254-MBP. Purified MBP was used as negative control. Detection was performed using an anti-MBP (HRP conjugated) antibody and otherwise as in A. Data are presented as mean+SD and representative for 2 independent experiments (performed in triplicates). C. SDS-PAGE confirming the expression and purification of respective fusion proteins.
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Related In: Results  -  Collection

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

pone-0026349-g006: ELISA experiment demonstrating that ATGL254 interacts with CGI-58 and G0S2.A. ELISA plates were coated with purified ATGL254-MBP and then incubated with purified His-tagged CGI-58. Purified smt (the fusion tag for CGI-58) was used as a negative control. Detection was performed by anti-his-antibody, HRP-labeled secondary antibody and tetramethyl-benzidine as substrate. B. ELISA plates were coated with purified mG0S2 and then incubated with purified ATGL254-MBP. Purified MBP was used as negative control. Detection was performed using an anti-MBP (HRP conjugated) antibody and otherwise as in A. Data are presented as mean+SD and representative for 2 independent experiments (performed in triplicates). C. SDS-PAGE confirming the expression and purification of respective fusion proteins.
Mentions: To further substantiate our conclusions that ATGL254 is sufficient for regulation by CGI-58 and G0S2, the implied physical interaction between the involved proteins was also determined by ELISA experiments. For this purpose, plates were coated with purified ATGL254 or mG0S2. Then, wells were incubated with purified His6-smt-tagged CGI-58 or maltose binding protein (MBP)-tagged ATGL254. As expected, protein-protein interactions between the ATGL254 and its co-activator CGI-58, as well as its inhibitor G0S2 were detected (Figure 6A and B). These data further support the conclusion that ATGL254 harbors all residues required for catalytic activity as well as interaction with ATGL's regulatory proteins, CGI-58 and G0S2.

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