Studies of membrane topology of mitochondrial cholesterol hydroxylases CYPs 27A1 and 11A1.

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TitleStudies of membrane topology of mitochondrial cholesterol hydroxylases CYPs 27A1 and 11A1.
Publication TypeJournal Article
Year of Publication2008
AuthorsPikuleva, IA, Mast, N, Liao, W-L, Turko, IV
JournalLipids
Volume43
Issue12
Pagination1127-32
Date Published2008 Dec
ISSN0024-4201
KeywordsAmino Acid Sequence, Animals, Cattle, Cholestanetriol 26-Monooxygenase, Cholesterol Side-Chain Cleavage Enzyme, Humans, Intracellular Membranes, Mitochondria, Molecular Sequence Data, Peptides, Trypsin
Abstract

Mitochondrial cytochrome P450 enzymes (CYP or P450, EC 1.14.13.15) play an important role in metabolism of cholesterol. CYP27A1 hydroxylates cholesterol at position 27 and, thus, initiates cholesterol removal from many extrahepatic tissues. CYP11A1 is a steroidogenic P450 that converts cholesterol to pregnenolone, the first step in the biosynthesis of all steroid hormones. We utilized a new approach to study membrane topology of CYPs 27A1 and 11A1. This approach involves heterologous expression of membrane-bound P450 in E. coli, isolation of the P450-containing E. coli membranes, treatment of the membranes with protease, removal of the digested soluble portion and extraction of the membrane-associated peptides, which are then identified by mass spectrometry. By using this approach, we found four membrane-interacting peptides in CYP27A1, and two peptides in CYP11A1. Peptides in CYP27A1 represent a contiguous portion of the polypeptide chain (residues 210-251) corresponding to the putative F-G loop and adjacent portions of the F and G helices. Peptides in CYP11A1 are from the putative F-G loop (residues 218-225) and the C-terminal portion of the G helix (residues 238-250). This data is consistent with those obtained previously by us and others and provide new information about the membrane topology of CYPs 27A1 and 11A1.

DOI10.1007/s11745-008-3234-x
Alternate JournalLipids
PubMed ID18791760
PubMed Central IDPMC2953644
Grant ListAG024336 / AG / NIA NIH HHS / United States
GM062882 / GM / NIGMS NIH HHS / United States
K02 AG024336-04 / AG / NIA NIH HHS / United States
R01 GM062882-07 / GM / NIGMS NIH HHS / United States