Isolevuglandins and mitochondrial enzymes in the retina: mass spectrometry detection of post-translational modification of sterol-metabolizing CYP27A1.

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TitleIsolevuglandins and mitochondrial enzymes in the retina: mass spectrometry detection of post-translational modification of sterol-metabolizing CYP27A1.
Publication TypeJournal Article
Year of Publication2011
AuthorsCharvet, C, Liao, W-L, Heo, G-Y, Laird, J, Salomon, RG, Turko, IV, Pikuleva, IA
JournalJ Biol Chem
Volume286
Issue23
Pagination20413-22
Date Published2011 Jun 10
ISSN1083-351X
KeywordsCardiovascular Diseases, Cholestanetriol 26-Monooxygenase, Eye Proteins, Humans, Lipoylation, Macular Degeneration, Mass Spectrometry, Mitochondrial Proteins, Peptide Mapping, Protein Processing, Post-Translational, Retina
Abstract

We report the first peptide mapping and sequencing of an in vivo isolevuglandin-modified protein. Mitochondrial cytochrome P450 27A1 (CYP27A1) is a ubiquitous multifunctional sterol C27-hydroxylase that eliminates cholesterol and likely 7-ketocholesterol from the retina and many other tissues. We investigated the post-translational modification of this protein with isolevuglandins, arachidonate oxidation products. Treatment of purified recombinant CYP27A1 with authentic iso[4]levuglandin E(2) (iso[4]LGE(2)) in vitro diminished enzyme activity in a time- and phospholipid-dependent manner. A multiple reaction monitoring protocol was then developed to identify the sites and extent of iso[4]LGE(2) adduction. CYP27A1 exhibited only three Lys residues, Lys(134), Lys(358), and Lys(476), that readily interact with iso[4]LGE(2) in vitro. Such selective modification enabled the generation of an internal standard, (15)N-labeled CYP27A1 modified with iso[4]LGE(2), for the subsequent analysis of a human retinal sample. Two multiple reaction monitoring transitions arising from the peptide AVLK(358)(-C(20)H(26)O(3))ETLR in the retinal sample were observed that co-eluted with the corresponding two (15)N transitions from the supplemented standard. These data demonstrate that modified CYP27A1 is present in the retina. We suggest that such protein modification impairs sterol elimination and likely has other pathological sequelae. We also propose that the post-translational modifications identified in CYP27A1 exemplify a general mechanism whereby oxidative stress and inflammation deleteriously affect protein function, contributing, for example, to cholesterol-rich lesions associated with age-related macular degeneration and cardiovascular disease. The proteomic protocols developed in this study are generally applicable to characterization of lipid-derived oxidative protein modifications occurring in vivo, including proteins bound to membranes.

DOI10.1074/jbc.M111.232546
Alternate JournalJ. Biol. Chem.
PubMed ID21498512
PubMed Central IDPMC3121529
Grant ListAG024336 / AG / NIA NIH HHS / United States
EY018383 / EY / NEI NIH HHS / United States
GM21249 / GM / NIGMS NIH HHS / United States
P30 EY11373 / EY / NEI NIH HHS / United States
R01 EY018383 / EY / NEI NIH HHS / United States
T32 EY007157 / 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 EY007157-11 / EY / NEI NIH HHS / United States