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J. Biol. Chem., Vol. 283, Issue 16, 10453-10460, April 18, 2008

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Identification of the Interaction Site within Acyl-CoA:Cholesterol Acyltransferase 2 for the Isoform-specific Inhibitor Pyripyropene A^*

Akash Das, Matthew A. Davis, Hiroshi Tomoda^¶, Satoshi Ômura^||, and Lawrence L. Rudel¹

From the Department of Biochemistry and Department of Pathology, Section on Lipid Science, Wake Forest University School of Medicine, Winston Salem, North Carolina 27157-1040 and ^¶School of Pharmacy and ^||Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, Tokyo 108-8641, Japan

Targeted deletion of acyl-CoA:cholesterol acyltransferase 2 (ACAT2) (A2), especially in the liver, protects hyperlipidemic mice from diet-induced hypercholesterolemia and atherosclerosis, whereas the deletion of ACAT1 (A1) is not as effective, suggesting ACAT2 may be the more appropriate target for treatment of atherosclerosis. Among the numerous ACAT inhibitors known, pyripyropene A (PPPA) is the only compound that has high selectivity (>2000-fold) for inhibition of ACAT2 compared with ACAT1. In the present study we sought to determine the PPPA interaction site of ACAT2. To achieve this goal we made several chimeric proteins where parts of ACAT2 were replaced by the analogous region of ACAT1. Differences in the amino acid sequence and the membrane topology were utilized to design the chimeras. Among chimeras, A2:1–428/A1:444–550 had 50% reduced PPPA selectivity, whereas C-terminal-truncated ACAT2 mutant A2:1–504 (C-terminal last 22 amino acids were deleted) remained selectively inhibited, indicating the PPPA-sensitive site is located within a region between amino acids 440 and 504. Three additional chimeras within this region helped narrow down the PPPA-sensitive site to a region containing amino acids 480–504, representing the fifth putative transmembrane domain of ACAT2. Subsequently, for this region we made single amino acid mutants where each amino acid in ACAT2 was individually changed to its ACAT1 counterpart. Mutation of Q492L, V493L, S494A resulted in only 30, 50, and 70% inhibition of the activity by PPPA, respectively (as opposed to greater than 95% with the wild type enzyme), suggesting these three residues are responsible for the selective inhibition by PPPA of ACAT2. Additionally, we found that PPPA non-covalently interacts with ACAT2 apparently without altering the oligomeric structure of the protein. The present study provides the first evidence for a unique motif in ACAT2 that can be utilized for making an ACAT2-specific drug.

Received for publication, November 19, 2007 , and in revised form, February 15, 2008.

^* This work was supported by National Institutes of Health Grant NIH-P01-HL49373 (to L. L. R.) and Kakenhi Grant 16073215 from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to H. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. A–C.

¹ To whom correspondence should be addressed: Dept. of Pathology, Section on Lipid Sciences, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1040. Tel.: 336-716-2823; Fax: 336-716-6279; E-mail: lrudel{at}wfubmc.edu.

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