Identification of the interaction site within acyl-CoA:cholesterol acyltransferase 2 for the isoform-specific inhibitor pyripyropene A.
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.
Das
Akash
A
Department of Biochemistry, Section on Lipid Science, Wake Forest University School of Medicine, Winston Salem, North Carolina 27157-1040, USA.
Davis
Matthew A
MA
Tomoda
Hiroshi
H
Omura
Satoshi
S
Rudel
Lawrence L
LL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D006224:Cricetinae; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D020033:Protein Isoforms; D011725:Pyridines / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012717:Sesquiterpenes / Q000737:chemistry*; D002785:Sterol O-Acyltransferase / Q000502:physiology*; D000097587:Sterol O-Acyltransferase 2
D020033:Protein Isoforms; D011725:Pyridines; D011993:Recombinant Fusion Proteins; D012717:Sesquiterpenes; D002785:Sterol O-Acyltransferase; D000097587:Sterol O-Acyltransferase 2; C082792:pyripyropene A