Unmasking Crucial Residues in Adipose Triglyceride Lipase (ATGL) for Co-Activation with Comparative Gene Identification-58 (CGI-58)

Kulminskaya, N.; Rodriguez Gamez, C. F.; Hofer, P.; Cerk, I. K.; Dubey, N.; Viertlmayr, R.; Sagmeister, T.; Pavkov-Keller, T.; Zechner, R.; Oberer, M.

doi:10.1101/2023.06.26.546551

bioRxiv · 10.1101/2023.06.26.546551

Unmasking Crucial Residues in Adipose Triglyceride Lipase (ATGL) for Co-Activation with Comparative Gene Identification-58 (CGI-58)

Kulminskaya, N.·Rodriguez Gamez, C. F.·Hofer, P.·Cerk, I. K.·Dubey, N.·Viertlmayr, R.·Sagmeister, T.·Pavkov-Keller, T.·Zechner, R.·Oberer, M.

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Abstract

Lipolysis is an essential metabolic process that releases unesterified fatty acids from neutral lipid stores to maintain energy homeostasis in living organisms. Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis and can be co-activated upon interaction with the protein comparative gene identification-58 (CGI-58). The underlying molecular mechanism of ATGL stimulation by CGI-58 is incompletely understood. Based on analysis of evolutionary conservation, we used site directed mutagenesis to study a C-terminally truncated variant and full-length mouse ATGL providing insights in the protein co-activation on a per-residue level. We identified the region from residues N209-N215 in mouse ATGL as essential for co-activation by mouse CGI-58. ATGL variants with amino-acids exchanges in this region were still able to hydrolyze triacylglycerol at the basal level and to interact with CGI-58, yet could not be activated by CGI-58. Our studies also demonstrate that full-length mouse ATGL showed higher tolerance to specific single amino acid exchanges in the N209-N215 region upon CGI-58 co-activation compared to C-terminally truncated ATGL variants. The region is either directly involved in protein-protein interaction or essential for conformational changes required in the co-activation process. Three-dimensional models of the ATGL/CGI-58 complex with the artificial intelligence software AlphaFold demonstrated that a large surface area is involved in the protein-protein interaction. Mapping important amino acids for co-activation of both proteins, ATGL and CGI-58, onto the 3D model of the complex locates these essential amino acids at the predicted ATGL/CGI-58 interface thus strongly corroborating the significance of these residues in CGI-58 mediated co-activation of ATGL.

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Kulminskaya, N., Rodriguez Gamez, C. F., Hofer, P., Cerk, I. K., Dubey, N., Viertlmayr, R., Sagmeister, T., Pavkov-Keller, T., Zechner, R., Oberer, M.. 2023-06-26. Unmasking Crucial Residues in Adipose Triglyceride Lipase (ATGL) for Co-Activation with Comparative Gene Identification-58 (CGI-58). https://doi.org/10.1101/2023.06.26.546551

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