Description
TitleA functional characterization of CGI-58
Date Created2012
Other Date2012-10 (degree)
Extentxii, 122 p. : ill.
DescriptionMutations in the gene CGI-58/ABHD5 cause Chanarin-Dorfman Syndrome, a Neutral Lipid Storage Disorder (NLSD) where many cells and tissues, including human skin fibroblasts, store excessive triacylglycerol (TAG). The protein, CGI‐58, has been characterized in vitro as both a co‐activator of adipose triglyceride lipase (ATGL) and a lysophosphatidic acid acyltransferase (LPAAT). We hypothesized that CGI‐58 LPAAT activity is not necessary for co-activation of ATGL. This hypothesis was investigated through 3 specific aims: 1) to identify LPAAT active site residues, 2) to demonstrate that CGI‐58 lacking LPAAT activity can co-activate ATGL, and 3) to analyze the lipid composition of cultured NLSD fibroblasts relative to normal human skin fibroblasts. A molecular model of CGI‐58 was created to identify potential active site residues. In the model, the putative LPAAT active site residues H329 and D334 were not in close proximity, suggesting that they may not be active site residues. Recombinant H329A and D334A CGI‐58 variants, when purified from BL21(DE3) E. coli, showed higher levels of LPAAT activity than purified wild-type CGI‐58. LPAAT activity was linked to a protein contaminant, likely plsC, the endogenous E. coli LPAAT. The purification of recombinant CGI‐58 was optimized to reduce contaminant proteins. These new preparations lacked LPAAT activity, yet retained the ability to co‐activate ATGL. Additionally, extracts of Bl21(DE3) cells expressing GST-tagged CGI‐58 lacked LPAAT activity when plsC was removed by centrifugation. The previously observed LPAAT activity was due to a protein contaminant; thus, CGI‐58 lacks LPAAT activity and LPAAT activity is not necessary for the co-activation of ATGL. Additionally, using a protein‐lipid overlay, CGI‐58 bound to phosphatidylinositol 3-phosphate [PI(3)P] and phosphatidylinositol 5-phosphate [PI(5)P] but not lysophosphatidic acid [LPA], the LPAAT substrate. CGI‐58 binding of PI(3)P or PI(5)P does not alter co-activation of ATGL. Finally, CGI-58 variant H84R, found in humans with NLSDi, was expressed in cultured NLSD cells and studied in vitro. H84R CGI-58 failed to reduce accumulated TAG of NLSD fibroblasts, unlike unmodified CGI-58 or the H84A variant. Both H84 variants lacked the ability to co-activate ATGL in vitro. Thus, H84R CGI‐58 contributes to the NLSD phenotype by failing to co-activate ATGL.
NotePh.D.
NoteIncludes bibliographical references
Noteby Derek B. McMahon
Genretheses, ETD doctoral
Languageeng
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.
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