Identification of the interaction site within acyl-CoA:cholesterol acyltransferase 2 for the isoform-specific inhibitor pyripyropene A

Abstract

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.

FIGURE 1.

PPPA-sensitive site of ACAT2 is located outside the putative active site domain of the enzyme. A, primary structure of the chimeric proteins termed A1:1–385 and A2:1–363 are indicated where sequences from ACAT1 are in open boxes and sequences from ACAT2 are in filled boxes. B, kinetic assay data for WTA1 and A1:1–385. AC29 cells were transfected with the cDNA encoding WTA1 and A1:1–385 proteins. 72 h post-transfection cells were incubated with either vehicle (Me₂SO) or 5μm PPPA for 30 min at 37 °C. There after cells were pulse-labeled with 1 μCi of [³H]oleic acid for 2 h. The incorporation of [³H]oleic acid into cellular CE pool was measured as the determinant of the enzymatic activity of the respective proteins. Background activity was obtained by a parallel kinetic assay where cells were transfected with an empty vector. All activities were corrected by background subtraction and were normalized against the control (Ctrl) WT activity. Data represent the mean ± S.E. for n = 2. This experiment was repeated three times with similar results. C, kinetic assay data for of WTA2 and A2:1–363. The assay was performed essentially as described above, and data are presented as above. D, PNS made from cells transfected with WTA1 and A1:1–385 cDNAs were subjected to immunoblot using affinity-purified ACAT1 antibody as described under “Experimental Procedures.” E, PNS, made from the cells transfected with WTA2 and A2:1–363 cDNAs, were subjected to Western blot analysis as described above.

FIGURE 2.

PPPA-sensitive site of ACAT2 is located near the C-terminal end of the enzyme. A, amino acid sequences of the chimeras A1:1–71 and A1:1–123 are represented as in Fig. 1A. B, kinetic assay data for WTA2, A1:1–71, and A1:1–123 proteins. Assays were done as described in Fig. 1B. Data represent the mean ± S.E. for n = 2, and the experiment was repeated twice with similar results. C, PNS obtained from the cells transfected with WTA2, A1:1–71, and A1:1–123 cDNAs were subject to immunoblotting as described under “Experimental Procedures.” Primary antibodies used here were as follows. Affinity-purified ACAT2 antibody was used to detect WTA2 band, whereas affinity-purified ACAT1 antibody was used for A1:1–71 and A1:1–123 proteins. D, primary sequences of the chimeras A2:1–227 and A2:1–249 are indicated as Fig. 1A. E, amino acid sequence of the chimera A2:1–428- and ACAT2-C-terminal truncated mutant CT504. F, kinetic activity of WTA2, A2:1–428, and CT504. The enzymatic assay was performed as described in Fig. 1B. Data represent mean ± S.E. for n = 2. This experiment was repeated three times with similar outcomes. Ctrl, control. G, PNS obtained from the cells transiently transfected with WTA2, A2:1–428, and CT504 cDNAs were used for Western blotting.

FIGURE 3.

PPPA-sensitive site is located within the fifth transmembrane domain of ACAT2. A, amino acid sequence representation for ACAT2 within the region 428–526 indicating the 5th transmembrane domain with amino acids 480–498 (TM5) near the C-terminal end of the enzyme. B, amino acid sequence of the chimera Ch1 represented as Fig. 1A. C, the kinetic assay data WTA2 and Ch1. The assay was performed as described in Fig. 1A. Data represent the mean ± S.E. for n = 2. This experiment was repeated twice with similar results. D, PNS obtained from WTA2- and Ch1-transfected cells were used for Western blotting using the protocol as described under “Experimental Procedures.” E, amino acid sequence of the chimera A2:1–440 indicated as Fig. 1A. F, the kinetic activity data for WTA2 and the chimera A2:1–440. The assay was performed as described for Fig. 1B. The assay was repeated twice with identical outcome. Data represent the mean ± S.E. for n = 2. G, Western blot analysis was performed using PNS obtained from WTA2- and A2:1–440-transfected AC29 cells. H, amino acid sequence representation of chimera A2:1–479 indicated as Fig. 1A. I, kinetic activity assay data for WTA2 and A2:1–479. The assay was performed as described in Fig. 1B. Data represent the mean ± S.E. for n = 4 of two independent experiments. J, PNS obtained from WTA2 and A2:1–479-transfected cells Western blotted as described under “Experimental Procedures.”

FIGURE 4.

Identification of the residues responsible for PPPA-mediated inhibition of ACAT2. A, amino acid sequence alignment of the PPPA-sensitive region of ACAT2 with its ACAT1 counterpart. This region represents the fifth transmembrane domain of both enzymes. The asterisk represents sequence identity, the colon represents conserved substitutions, the dot represents semi-conserved substitutions, and no symbol indicates no match. The alignment was performed using ClustalW program from EMBL web site. B, enzymatic assay data of each of the single amino acid mutants where the amino acid in ACAT2 is replaced with the analogous amino acid in ACAT1 as indicated for the x axis. AC29 cells were transfected with the cDNA encoding either WTA2 or the various single amino acid mutants of ACAT2. Assays were done as indicated in Fig. 1B. Data represent the mean ± S.E. for n = 2. This experiment was repeated twice with similar results. C, Western blot analysis of the WT and each of various mutant ACAT2 proteins as indicated, performed as described under “Experimental Procedures.”

FIGURE 5.

Nature of interaction of PPPA with ACAT2 and its effects on the oligomeric state of the protein. A, microsomes were prepared from ACAT2 stable cells incubated with either Me₂SO (DMSO) or 5 μm PPPA for 30 min at 37 °C. 50 μg of Me₂SO-treated microsomes and 25 μg of PPPA-treated microsomes were used to do a microsomal ACAT assay (described in detail under “Experimental Procedures”). Specific activity was calculated by normalizing activity as dpm of cholesteryl esters formed with the protein mass and the assay run time. Data represent the mean ± S.E. for n = 2. This experiment was repeated twice with similar results. B, 50 μg of ACAT2 stable cell microsomes were incubated with either Me₂SO or 5 μm PPPA at 37 °C. This was followed by reisolation of the microsomes by centrifuging at 100 K for 15 min. Microsomal ACAT assay was then performed with the reisolated microsomes. Specific activity was calculated as above. Data represent the mean ± S.E. for n = 2. This experiment was repeated three times with similar result. C, 10 μg of protein in ACAT2 stable cell microsomes and 20 μg of protein in microsomes obtained from AC29 cells transiently transfected with A2Q492L mutant were treated with either Me₂SO or 5 μm PPPA at room temperature for 30 min. Samples were then treated with vehicle or disuccinimidyl glutarate (DSG) for another 30 min at room temperature. The cross-linking reaction was quenched by adding Western loading buffer containing 1 m Tris, pH 7.5, to the reaction, and all samples were subjected to Western blotting as described under “Experimental Procedures.” As a control ACAT2 and A2Q492L was run on the gel without any treatment (1st and 4th lane on the gel). Apparent molecular weight of the bands is shown on the right side of the gel.