Combined use of mass spectrometry and heterologous expression for identification of membrane-interacting peptides in cytochrome P450 46A1 and NADPH-cytochrome P450 oxidoreductase.

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TitleCombined use of mass spectrometry and heterologous expression for identification of membrane-interacting peptides in cytochrome P450 46A1 and NADPH-cytochrome P450 oxidoreductase.
Publication TypeJournal Article
Year of Publication2009
AuthorsMast, N, Liao, W-L, Pikuleva, IA, Turko, IV
JournalArch Biochem Biophys
Volume483
Issue1
Pagination81-9
Date Published2009 Mar 1
ISSN1096-0384
KeywordsAmino Acid Sequence, Animals, Catalytic Domain, Cholesterol, Crystallography, X-Ray, Escherichia coli, Humans, Kinetics, Mass Spectrometry, Membranes, Models, Molecular, Molecular Sequence Data, Mutation, Missense, NADPH-Ferrihemoprotein Reductase, Peptide Fragments, Protein Structure, Tertiary, Rats, Recombinant Proteins, Steroid Hydroxylases, Substrate Specificity
Abstract

Cytochrome P450 46A1 (CYP46A1) and NADPH-cytochrome P450 oxidoreductase (CPR) are the components of the brain microsomal mixed-function monooxygenase system that catalyzes the conversion of cholesterol to 24-hydroxycholesterol. Both CYP46A1 and CPR are monotopic membrane proteins that are anchored to the endoplasmic reticulum via the N-terminal transmembrane domain. The exact mode of peripheral association of CYP46A1 and CPR with the membrane is unknown. Therefore, we studied their membrane topology by using an approach in which solution-exposed portion of heterologously expressed membrane-bound CYP46A1 or CPR was removed by digestion with either trypsin or chymotrypsin followed by extraction of the residual peptides and their identification by mass spectrometry. The identified putative membrane-interacting peptides were mapped onto available crystal structures of CYP46A1 and CPR and the proteins were positioned in the membrane considering spatial location of the missed cleavage sites located within these peptide as well as the flanking residues whose cleavage produced these peptides. Experiments were then carried out to validate the inference from our studies that the substrate, cholesterol, enters CYP46A1 from the membrane. As for CPR, its putative membrane topology indicates that the Q153R and R316W missense mutations found in patients with disordered steroidogenesis are located within the membrane-associated regions. This information may provide insight in the deleterious nature of these mutations.

DOI10.1016/j.abb.2009.01.002
Alternate JournalArch. Biochem. Biophys.
PubMed ID19161969
PubMed Central IDPMC2644731
Grant ListK02 AG024336-04 / AG / NIA NIH HHS / United States
R01 GM062882-06A2 / GM / NIGMS NIH HHS / United States