Investigating the ACAT2/GP78/INSIG1 Tri-Protein Complex

Abstract

The ER membrane protein ACAT2 is responsible for converting free cholesterol into storable cholesteryl ester. Increased ACAT activity and the subsequent buildup of esters is a major contributor to hypercholesterolemia and cholesterol-induced diseases, including atherosclerosis and several forms of cancer. ACAT2 is a promising drug target to counter cholesterol-associated diseases because its activation is inducible and it is selectively localized in only two cell types: the enterocytes in the intestines and the hepatocytes in the liver. Recent findings have identified that the proteins INSIG1 and GP78 facilitate the ubiquitination and subsequent degradation of ACAT2 via the ubiquitin proteasome pathway. It is hypothesized that during this process ACAT2, INSIG1 and GP78 form a tri-protein complex wherein INSIG1 is directly bound to both ACAT2 and GP78, and ACAT2 and GP78 are not bound to each other. Thus far, no cryo-EM structure has been resolved for GP78 or INSIG1 alone, nor for the entire ACAT2/GP78/INSIG1 complex. GP78 and INSIG1 also facilitate the degradation of other key proteins in the cholesterol pathway, including the protein HMGCR, which catalyzes the rate-limiting step of cholesterol biosynthesis. Resolving the structure of the ACAT2/GP78/INSIG1 complex could reveal crucial information about the mechanisms of GP78 and INSIG1-mediated ubiquitination in general, and could pave the way for the development of new therapies for treating hypercholesterolemia. To lay the groundwork for the elucidation of this challenging structure, ACAT2, GP78 and INSIG1 plasmids were generated, purified and transfected into HEK293F suspension cells. Cells were harvested and proteins were collected and purified. Protein purification was optimized by changing several parameters, including the concentration of detergent and construct of protein selected to purify (full length vs dNTD). A Western Blot was performed to test for the presence of ACAT2 and GP78. Cryo-EM samples were prepared and preliminary structural data was collected. Here, we present preliminary findings including agarose gels to verify plasmid expression, SEC chromatograms of different optimization conditions, SDS-Page gels of purified proteins, Western Blots testing for ACAT2 and GP78, and Cryo-EM images. We hope that our purification troubleshooting of the three proteins and optimized methodology will lead to a resolved ACAT2/GP78/INSIG1 cryo-EM structure.