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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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TG hydrolase activity of full length ATGL is inhibited by G0S2.A. Mouse ATGL (mATGL) contained in COS-7 cell lysates were subjected to in vitro TG hydrolase activity assay in the presence of CGI-58 (*) and without or with addition of bacterially expressed mouse G0S2 (mG0S2), using radiolabeled triolein as artificial substrate. B. E. coli expressed mATGL was assayed in in vitro TG hydrolase activity assay without or with mG0S2 as above. Gb1, the fusion tag of mATGL, does not exhibit TG hydrolase activity. Representative assays (performed in triplicates) of three independent experiments are shown. Data are presented as mean+SD. *** indicate statistical significant differences as determined by unpaired Student's t-test (two-tailed), p>0.001. As control, the corresponding fusion tag (Trigger factor –TF for mG0S2) was added to the reaction. C. Western blots confirming expression of mATGL in COS-7 cells and in E.coli. SDS-PAGE showing purified CGI-58 (∼54 kDa) and bacterial lysates of TF-mG0S2 (64 kDa), and TF alone (54 kDa). As a control, lysates of non induced cells were used.
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pone-0026349-g002: TG hydrolase activity of full length ATGL is inhibited by G0S2.A. Mouse ATGL (mATGL) contained in COS-7 cell lysates were subjected to in vitro TG hydrolase activity assay in the presence of CGI-58 (*) and without or with addition of bacterially expressed mouse G0S2 (mG0S2), using radiolabeled triolein as artificial substrate. B. E. coli expressed mATGL was assayed in in vitro TG hydrolase activity assay without or with mG0S2 as above. Gb1, the fusion tag of mATGL, does not exhibit TG hydrolase activity. Representative assays (performed in triplicates) of three independent experiments are shown. Data are presented as mean+SD. *** indicate statistical significant differences as determined by unpaired Student's t-test (two-tailed), p>0.001. As control, the corresponding fusion tag (Trigger factor –TF for mG0S2) was added to the reaction. C. Western blots confirming expression of mATGL in COS-7 cells and in E.coli. SDS-PAGE showing purified CGI-58 (∼54 kDa) and bacterial lysates of TF-mG0S2 (64 kDa), and TF alone (54 kDa). As a control, lysates of non induced cells were used.

Mentions: Previously we had shown that the activity of mATGL can also be stimulated by mouse CGI-58 (mCGI-58) expressed in E. coli [27]. We first wanted to verify that bacterially expressed mouse G0S2 (mG0S2) is functional at inhibiting mATGL expressed in COS-7 cells after activation by CGI-58. Indeed, inhibition of mATGL expressed in COS-7 and also in E.coli was observed upon addition of mG0S2 from E.coli lysates in a similar manner (Figure 2A and B). This indicates, that the underlying protein-protein interaction is independent from post-translational modifications and can be studied at the level of heterologously expressed proteins.


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)

TG hydrolase activity of full length ATGL is inhibited by G0S2.A. Mouse ATGL (mATGL) contained in COS-7 cell lysates were subjected to in vitro TG hydrolase activity assay in the presence of CGI-58 (*) and without or with addition of bacterially expressed mouse G0S2 (mG0S2), using radiolabeled triolein as artificial substrate. B. E. coli expressed mATGL was assayed in in vitro TG hydrolase activity assay without or with mG0S2 as above. Gb1, the fusion tag of mATGL, does not exhibit TG hydrolase activity. Representative assays (performed in triplicates) of three independent experiments are shown. Data are presented as mean+SD. *** indicate statistical significant differences as determined by unpaired Student's t-test (two-tailed), p>0.001. As control, the corresponding fusion tag (Trigger factor –TF for mG0S2) was added to the reaction. C. Western blots confirming expression of mATGL in COS-7 cells and in E.coli. SDS-PAGE showing purified CGI-58 (∼54 kDa) and bacterial lysates of TF-mG0S2 (64 kDa), and TF alone (54 kDa). As a control, lysates of non induced cells were used.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026349-g002: TG hydrolase activity of full length ATGL is inhibited by G0S2.A. Mouse ATGL (mATGL) contained in COS-7 cell lysates were subjected to in vitro TG hydrolase activity assay in the presence of CGI-58 (*) and without or with addition of bacterially expressed mouse G0S2 (mG0S2), using radiolabeled triolein as artificial substrate. B. E. coli expressed mATGL was assayed in in vitro TG hydrolase activity assay without or with mG0S2 as above. Gb1, the fusion tag of mATGL, does not exhibit TG hydrolase activity. Representative assays (performed in triplicates) of three independent experiments are shown. Data are presented as mean+SD. *** indicate statistical significant differences as determined by unpaired Student's t-test (two-tailed), p>0.001. As control, the corresponding fusion tag (Trigger factor –TF for mG0S2) was added to the reaction. C. Western blots confirming expression of mATGL in COS-7 cells and in E.coli. SDS-PAGE showing purified CGI-58 (∼54 kDa) and bacterial lysates of TF-mG0S2 (64 kDa), and TF alone (54 kDa). As a control, lysates of non induced cells were used.
Mentions: Previously we had shown that the activity of mATGL can also be stimulated by mouse CGI-58 (mCGI-58) expressed in E. coli [27]. We first wanted to verify that bacterially expressed mouse G0S2 (mG0S2) is functional at inhibiting mATGL expressed in COS-7 cells after activation by CGI-58. Indeed, inhibition of mATGL expressed in COS-7 and also in E.coli was observed upon addition of mG0S2 from E.coli lysates in a similar manner (Figure 2A and B). This indicates, that the underlying protein-protein interaction is independent from post-translational modifications and can be studied at the level of heterologously expressed proteins.

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