title: Light-induced disulfide dimerization of recoverin under ex vivo and in vivo conditions.
abstract: Despite vast knowledge of the molecular mechanisms underlying photochemical damage of photoreceptors, linked to progression of age-related macular degeneration, information on specific protein targets of the light-induced oxidative stress is scarce. Here, we demonstrate that prolonged intense illumination (halogen bulb, 1500 lx, 1-5 h) of mammalian eyes under ex vivo (cow) or in vivo (rabbit) conditions induces disulfide dimerization of recoverin, a Ca(2+)-dependent inhibitor of rhodopsin kinase. Western blotting and mass spectrometry analysis of retinal extracts reveals illumination time-dependent accumulation of disulfide homodimers of recoverin and its higher order disulfide cross-linked species, including a minor fraction of mixed disulfides with intracellular proteins (tubulins, etc.). Meanwhile, monomeric bovine recoverin remains mostly reduced. These effects are accompanied by accumulation of disulfide homodimers of visual arrestin. Histological studies demonstrate that the light-induced oxidation of recoverin and arrestin occurs in intact retina (illumination for 2 h), while illumination for 5 h is associated with damage of the photoreceptor layer. A comparison of ex vivo levels of disulfide homodimers of bovine recoverin with redox dependence of its in vitro thiol-disulfide equilibrium (glutathione redox pair) gives the lowest estimate of redox potential in rod outer segments under illumination from -160 to -155 mV. Chemical crosslinking and dynamic light scattering data demonstrate an increased propensity of disulfide dimer of bovine recoverin to multimerization/aggregation. Overall, the oxidative stress caused by the prolonged intense illumination of retina might affect rhodopsin desensitization via concerted disulfide dimerization of recoverin and arrestin. The developed herein models of eye illumination are useful for studies of the light-induced thiol oxidation of visual proteins.
authors.affiliation: Department of Cell Signaling, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992 Russia. Electronic address: zerni@belozersky.msu.ru.
authors.forename: Evgeni Yu
authors.initials: EY
authors.lastname: Zernii
authors.affiliation: Protein Research Group, Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region, 142290 Russia.
authors.forename: Aliya A
authors.initials: AA
authors.lastname: Nazipova
authors.affiliation: Department of Cell Signaling, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992 Russia.
authors.forename: Olga S
authors.initials: OS
authors.lastname: Gancharova
authors.affiliation: Protein Research Group, Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region, 142290 Russia.
authors.forename: Alexey S
authors.initials: AS
authors.lastname: Kazakov
authors.affiliation: Department of Cell Signaling, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992 Russia.
authors.forename: Marina V
authors.initials: MV
authors.lastname: Serebryakova
authors.affiliation: Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Pushchino, Moscow region, 142290 Russia.
authors.forename: Dmitry V
authors.initials: DV
authors.lastname: Zinchenko
authors.affiliation: Department of Cell Signaling, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992 Russia.
authors.forename: Natalya K
authors.initials: NK
authors.lastname: Tikhomirova
authors.affiliation: Department of Cell Signaling, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992 Russia.
authors.forename: Ivan I
authors.initials: II
authors.lastname: Senin
authors.affiliation: Department of Cell Signaling, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992 Russia.
authors.forename: Pavel P
authors.initials: PP
authors.lastname: Philippov
authors.affiliation: Protein Research Group, Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region, 142290 Russia; Department of Biomedical Engineering, Pushchino State Institute of Natural Sciences, Pushchino, Moscow region, 142290 Russia.
authors.forename: Eugene A
authors.initials: EA
authors.lastname: Permyakov
authors.affiliation: Protein Research Group, Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region, 142290 Russia; Department of Biomedical Engineering, Pushchino State Institute of Natural Sciences, Pushchino, Moscow region, 142290 Russia.
authors.forename: Sergei E
authors.initials: SE
authors.lastname: Permyakov
mesh_terms: D000818:Animals; D019390:Arrestins / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D002417:Cattle; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D005136:Eye Proteins / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D005260:Female; D008027:Light; D010084:Oxidation-Reduction; D011817:Rabbits; D051603:Recoverin / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D012160:Retina / Q000166:cytology / Q000378:metabolism* / Q000528:radiation effects
chemical_list: D019390:Arrestins; D004220:Disulfides; D005136:Eye Proteins; D051603:Recoverin
keywords: Age-related macular degeneration; Disulfide dimerization; EF-hand; NCS family; Photochemical damage; Photoreceptor; Recoverin; Visual arrestin