RESUMO
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.
Assuntos
Colestanotriol 26-Mono-Oxigenase/metabolismo , Proteínas do Olho/metabolismo , Lipoilação , Proteínas Mitocondriais/metabolismo , Processamento de Proteína Pós-Traducional , Retina/enzimologia , Doenças Cardiovasculares/metabolismo , Humanos , Degeneração Macular/metabolismo , Espectrometria de Massas/métodos , Mapeamento de PeptídeosRESUMO
Of the different oxygenated cholesterol metabolites, 7-ketocholesterol (7KCh) is considered a noxious oxy-sterol implicated in the development of certain pathologies, including those found in the eye. Here we elucidated whether sterol 27-hydroxylase cytochrome P450 27A1 (CYP27A1) is involved in elimination of 7KCh from the posterior part of the eye: the neural retina and underlying retinal pigment epithelium (RPE). We first established that the affinities of purified recombinant CYP27A1 for 7KCh and its endogenous substrate cholesterol are similar, yet 7KCh is metabolized at a 4-fold higher rate than cholesterol in the reconstituted system in vitro. Lipid extracts from bovine neural retina and RPE were then analyzed by isotope dilution GC-MS for the presence of the 7KCh-derived oxysterols. Two metabolites, 3ß,27-dihydroxy-5-cholesten-7-one (7KCh-27OH) and 3ß-hydroxy-5-cholesten-7-one-26-oic acid (7KCh-27COOH), were detected in the RPE but not in the neural retina. 7KCh-27OH was also formed when RPE homogenates were supplemented with NADPH and the mitochondrial redox system. Quantifications in human RPE showed that CYP27A1 is indeed expressed in the RPE at 2-4-fold higher levels than in the neural retina. The data obtained represent evidence for the role of CYP27A1 in retinal metabolism of 7KCh and suggest that, in addition to cholesterol removal, the functions of this enzyme could also include elimination of toxic endogenous compounds.