Quantification of cholesterol-metabolizing P450s CYP27A1 and CYP46A1 in neural tissues reveals a lack of enzyme-product correlations in human retina but not human brain.

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TitleQuantification of cholesterol-metabolizing P450s CYP27A1 and CYP46A1 in neural tissues reveals a lack of enzyme-product correlations in human retina but not human brain.
Publication TypeJournal Article
Year of Publication2011
AuthorsLiao, W-L, Heo, G-Y, Dodder, NG, Reem, RE, Mast, N, Huang, S, Dipatre, PLuigi, Turko, IV, Pikuleva, IA
JournalJ Proteome Res
Volume10
Issue1
Pagination241-8
Date Published2011 Jan 7
ISSN1535-3907
KeywordsCell Membrane, Cholestanetriol 26-Monooxygenase, Cholestenes, Cholesterol, Humans, Hydroxycholesterols, Isotope Labeling, Mass Spectrometry, Nitrogen Isotopes, Reproducibility of Results, Retina, Steroid Hydroxylases, Temporal Lobe
Abstract

Cytochrome P450 enzymes (CYP or P450) 46A1 and 27A1 play important roles in cholesterol elimination from the brain and retina, respectively, yet they have not been quantified in human organs because of their low abundance and association with membrane. On the basis of our previous development of a multiple reaction monitoring (MRM) workflow for measurements of low-abundance membrane proteins, we quantified CYP46A1 and CYP27A1 in human brain and retina samples from four donors. These enzymes were quantified in the total membrane pellet, a fraction of the whole tissue homogenate, using ¹⁵N-labled recombinant P450s as internal standards. The average P450 concentrations/mg of total tissue protein were 345 fmol of CYP46A1 and 110 fmol of CYP27A1 in the temporal lobe, and 60 fmol of CYP46A1 and 490 fmol of CYP27A1 in the retina. The corresponding P450 metabolites were then measured in the same tissue samples and compared to the P450 enzyme concentrations. Investigation of the enzyme-product relationships and analysis of the P450 measurements based on different signature peptides revealed a possibility of retina-specific post-translational modification of CYP27A1. The data obtained provide important insights into the mechanisms of cholesterol elimination from different neural tissues.

DOI10.1021/pr1008898
Alternate JournalJ. Proteome Res.
PubMed ID21049985
PubMed Central IDPMC3064498
Grant ListAG024336 / AG / NIA NIH HHS / United States
EY018383 / EY / NEI NIH HHS / United States
K02 AG024336 / AG / NIA NIH HHS / United States
K02 AG024336-06 / AG / NIA NIH HHS / United States
R01 EY018383 / EY / NEI NIH HHS / United States
R01 EY018383-04 / EY / NEI NIH HHS / United States
T32 EY007157 / EY / NEI NIH HHS / United States
T32 EY007157-09 / EY / NEI NIH HHS / United States
T32 EY007157-10 / EY / NEI NIH HHS / United States
T32 EY07157 / EY / NEI NIH HHS / United States