Functional characterization of CGI-58

The lipase ATGL (adipose triglyceride lipase) and the protein CGI-58 (comparative gene identification 58) play very important roles in lipolysis, the mobilization of stored triacylglycerols into free fatty acids and glycerol. In adipose tissue, ATGL is the rate limiting enzyme of triacylglycerol hydrolysis and catalyzes the degradation of triacyglycerol into diacylglycerol and free fatty acid. In the presence of CGI-58, the activity of ATGL is enhanced up to 20-fold. Patients with mutations in CGI-58 or ATGL suffer from triglyceride accumulation in various tissues as expected from this known function. However, the phenotype related to mutations in CGI-58 additionally shows a severe form of ichthyosis, clearly indicating a yet unknown function of CGI-58, which is independent of the ATGL stimulating activity. Despite the acknowledged importance of CGI-58 in lipid metabolism and skin function, the underlying molecular mechanisms are completely unknown. The goals of the proposed research are to establish structure-function relationships of CGI-58. The research aims at a better understanding of the biological functions of CGI-58 by studying the mechanisms of CGI-58 mediated ATGL stimulation, LD binding, and a recently reported acyl-CoA dependent acyltransferase activity at a molecular level. To achieve these goals, we propose a multifaceted approach to further unveil the function of CGI-58: In an attempt to explain CGI-58 function by the structural features of the protein, we generated a 3D homology model based on sequence similarity with other proteins. Interestingly, the model of CGI-58 reveals that the N-terminus forms an extension compared to the otherwise compact structure of the protein. Since the dispersion of CGI-58 from the lipid droplet into the cytosol upon lipolytic stimulation seems to play a crucial role in the stimulating mechanism, we will also explore the lipid droplet binding properties of CGI-58. In our preliminary work we identified a short region within this N-terminus which is essential for localization of CGI-58 to the lipid droplet. Using NMR spectroscopy, we will gain structural information about the lipid droplet binding capacity of this peptide region on a per-residue basis. Having our 3D model in mind, we will also use a biochemical approach to determine the minimum requirements of the activator CGI-58 for stimulating ATGL activity. Furthermore we will investigate the recently identified acyl-CoA-dependent lysophosphatidic acid- specific acyltransferase activity of CGI-58 using biochemical and biophysical techniques. The outcome of this work will significantly enhance our understanding of the biological roles of CGI-58 in lipid metabolism and phospholipid metabolism at a molecular level.

Our increasingly sedentary life styles often lead to imbalances in energy metabolism. These lead to major health problems in the affected individuals and represent a global health problem. The health consequences associated with excessive energy intake include diabetes, cardiovascular disease and metabolic syndrome. Free fatty acids are harmful to the body and are associated with lipotoxicity. Therefore, mammals store excess dietary fatty acids as relatively inert triacylglycerols, in which three fatty acid molecules are esterified with one molecule of glycerol. These triacylglycerols are stored in lipid droplets mainly in adipose tissue. In the case of a low energy level of the body, stored triacylglycerols can be mobilized and supplied into the chain of events for biochemical degradation of fatty acids for energy production. This mobilization process (also referred to as lipolysis), is carried out by proteins called lipases and subject to various control mechanisms. Cleavage of the first fatty acid is carried out by adipose triglyceride lipase (ATGL), whose activity is stimulated by the protein CGI-58. In this project, the role of CGI-58 and the underlying mechanisms have been investigated in more detail. Through the use of different biochemical and biophysical techniques, regions within CGI-58 and ATGL have been identified that are essential for the interaction of CGI-58 with ATGL and the lipid droplets. The three-dimensional structure of a region of CGI-58, which induces the interaction with the lipid droplets, was determined by nuclear magnetic resonance spectroscopy (NMR). In addition, new interactions of CGI-58 with other proteins were discovered, which collectively contribute to a concerted reaction during lipolysis. An in-depth characterization of lipid catabolism is essential because only detailed understanding of the processes enable rationally based therapeutic interventions. Therefore, it is essential to understand which physiological processes are affected by the protein CGI-58.