The applicant has trained clinically in internal medicine and completed her cardiology fellowship two years ago. Subsequently, she has worked in Dr. Lange's laboratory in the area of intracellular cholesterol metabolism, work represented by two manuscripts submitted for publication. A FIRST Award would be used to establish an independent laboratory by the applicant. The principal objective of this work is the identification and subsequent characterization of acyl-CoA-cholesterol-O- acyltransferase (ACAT), which is the principal intracellular cholesterol esterifying activity in vascular tissue. Despite the critical role of this enzyme as the final common pathway for accumulation of cholesteryl esters in vascular tissue during atherogenesis, it has never been identified and so detailed study of its regulation has proved impossible. Identification on ACAT will be accomplished through the use of a synthesized 125I-labeled photoreactive fatty acid CoA analog to photolytically label the enzyme. This compound binds to the active site of ACAT and inhibits ACAT reversibly under dark conditions with a Ki of 5 muM. Photolysis of microsomes in the presence of this compound irreversible inhibitors ACAT, and leads to covalent labeling of several polypeptides assessed by SDS-PAGE and autoradiography. The proposed experiments include differential labeling experiments using multiple active-site directed compounds including ACAT substrates, inhibitors, and selective amino acid modifying reagents, to identify the enzyme through protection from 125I- labeling. Radioactive forms of selective amino acid modifying reagents (203Hg PMB, and 3H or 14C-labeled DEP and acetic anhydride) will also be used in protein labeling experiments to identity ACAT. Confirmation of the identity of a given protein as ACAT will be obtained using polyclonal and monoclonal antibodies generated to the protein of interest. After identification of ACAT, the developed techniques will be used to study ACAT and its regulation in vascular tissue and in cell culture systems. The major focus of this work is the understanding of molecular events regulating intracellular cholesterol metabolism in vascular tissue, especially those occurring after interaction of cells with atherogentic factors such as LDL and B-VLDL.
|Kinnunen, P M; Klopf, F H; Bastiani, C A et al. (1990) 12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoic acid: biological recognition by cholesterol esterase and acyl-CoA:cholesterol O-acyltransferase. Biochemistry 29:1648-54|