title: Isolevuglandins and mitochondrial enzymes in the retina: mass spectrometry detection of post-translational modification of sterol-metabolizing CYP27A1.
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.
authors.affiliation: Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland,Ohio 44106, USA .
authors.forename: Casey
authors.initials: C
authors.lastname: Charvet
authors.forename: Wei-Li
authors.initials: WL
authors.lastname: Liao
authors.forename: Gun-Young
authors.initials: GY
authors.lastname: Heo
authors.forename: James
authors.initials: J
authors.lastname: Laird
authors.forename: Robert G
authors.initials: RG
authors.lastname: Salomon
authors.forename: Illarion V
authors.initials: IV
authors.lastname: Turko
authors.forename: Irina A
authors.initials: IA
authors.lastname: Pikuleva
mesh_terms: D002318:Cardiovascular Diseases / Q000378:metabolism; D053493:Cholestanetriol 26-Monooxygenase / Q000378:metabolism*; D005136:Eye Proteins / Q000378:metabolism*; D006801:Humans; D054878:Lipoylation; D008268:Macular Degeneration / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D024101:Mitochondrial Proteins / Q000378:metabolism*; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D012160:Retina / Q000201:enzymology*
chemical_list: D005136:Eye Proteins; D024101:Mitochondrial Proteins; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase