TY - JOUR
T1 - Selective inhibition of sterol O-acyltransferase 1 isozyme by beauveriolide III in intact cells
AU - Ohshiro, Taichi
AU - Kobayashi, Keisuke
AU - Ohba, Mio
AU - Matsuda, Daisuke
AU - Rudel, Lawrence L.
AU - Takahashi, Takashi
AU - Doi, Takayuki
AU - Tomoda, Hiroshi
N1 - Funding Information:
This work was supported by JSPS KAKENHI Grant numbers JP26253009 (HT), JP15H05837 in Middle Molecular Strategy (TD) and JP16K18900 (TO), the grant of Research Fellow of Japan Society for the Promotion of Science (KK) and a Kitasato University Research Grant for Young Researchers (TO).
Publisher Copyright:
© The Author(s) 2017.
PY - 2017/12/1
Y1 - 2017/12/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41598-017-04177-8
DO - 10.1038/s41598-017-04177-8
M3 - Article
C2 - 28646165
AN - SCOPUS:85021298218
VL - 7
JO - Scientific Reports
JF - Scientific Reports
SN - 2045-2322
IS - 1
M1 - 4163
ER -