Abstract
Beauveriolide III (BeauIII) inhibited sterol O-acyltransferases 1 and 2 (SOAT1 and SOAT2), which are endoplasmic reticulum (ER) membrane proteins, in an enzyme-based assay, and selectively inhibited SOAT1 in a cell-based assay using SOAT1-/SOAT2-CHO cells. This discrepancy in SOAT inhibition by BeauIII was investigated. In the enzyme-based assay, BeauIII inhibited SOAT1 and SOAT2 to a similar extent using microsomes prepared from cells disrupted under the strongest sonication condition. In semi-intact SOAT1-/SOAT2-CHO cells prepared by a treatment with digitonin (plasma membrane permeabilized), BeauIII selectively inhibited SOAT1 (IC50; 5.0 µM (SOAT1) vs >90 µM (SOAT2)), while in those treated with saponin (plasma membrane and ER membrane permeabilized), BeauIII inhibited SOAT1 (IC50, 1.8 µM) and SOAT2 (5.9 µM). SOAT1-selective inhibition by BeauIII was reproduced in intact ER fractions prepared from SOAT1/SOAT2-CHO cells. A Western blotting analysis revealed that biotin-labeled beauveriolide bound to the SOAT1 protein prepared from SOAT1-CHO cells. We concluded that BeauIII binds to a putative active site responsible for SOAT1 that is located on the cytosolic side of the ER, while BeauIII is not accessible to the corresponding active site for SOAT2 located on the luminal side.
Highlights
Beauveriolides, fungal metabolites produced by Beauveria sp
We previously reported that Beauveriolide I (BeauI) and Beauveriolide III (BeauIII) selectively inhibited SOAT1 in an intact cell-based assay, and inhibited SOAT1 and SOAT2 in a microsome assay as an enzyme source using SOAT1-/SOAT2-Chinese hamster ovary (CHO) cells
SOAT1 activity was inhibited by BeauIII with similar IC50s (2.7, 1.3, and 1.2 μM, respectively), while SOAT2 activity was disruption time-dependently inhibited by BeauIII with IC50s of >51, 31, and 6.6 μM, respectively. These results suggest that the strongest condition for cell disruption perturbed the endoplasmic reticulum (ER) membrane integrity, thereby allowing BeauIII to access the active site of the SOAT2 isozyme in the damaged ER membrane
Summary
Beauveriolides, fungal metabolites produced by Beauveria sp. FO-6979, were identified as inhibitors of lipid droplet formation in mouse peritoneal macrophages[1,2,3]. A study of their mechanism of action revealed that beauveriolides inhibit the synthesis of cholesteryl ester (CE), one of the main constituents of lipid droplets in macrophages, by blocking sterol O-acyltransferase (SOAT, known as acyl-CoA:cholesterol acyltransferase (ACAT)) activity[4]. SOAT1 is ubiquitously expressed in tissues and cells, while SOAT2 is predominantly expressed in the liver (hepatocytes) and intestines[13] Both SOAT isozymes are membrane proteins localized in the endoplasmic reticulum (ER), and are assumed to possess several hydrophobic domains that penetrate the ER membrane[15, 16]. The reason for this discrepancy in SOAT inhibition by BeauI and BeauIII between the enzyme- and intact cell-based assays is unclear. We investigated SOAT isozyme inhibition by BeauIII in semi-intact (perforated) CHO cells and an intact ER fraction prepared from CHO cells in an attempt to explain this discrepancy
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
