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Tyrosine/cysteine cluster sensitizing human ?D-crystallin to ultraviolet radiation-induced photoaggregation in vitro.

ABSTRACT: Ultraviolet radiation (UVR) exposure is a major risk factor for age-related cataract, a protein-aggregation disease of the human lens often involving the major proteins of the lens, the crystallins. ?D-Crystallin (H?D-Crys) is abundant in the nucleus of the human lens, and its folding and aggregation have been extensively studied. Previous work showed that H?D-Crys photoaggregates in vitro upon exposure to UVA/UVB light and that its conserved tryptophans are not required for aggregation. Surprisingly, the tryptophan residues play a photoprotective role because of a distinctive energy-transfer mechanism. H?D-Crys also contains 14 tyrosine residues, 12 of which are organized as six pairs. We investigated the role of the tyrosines of H?D-Crys by replacing pairs with alanines and monitoring photoaggregation using light scattering and SDS-PAGE. Mutating both tyrosines in the Y16/Y28 pair to alanine slowed the formation of light-scattering aggregates. Further mutant studies implicated Y16 as important for photoaggregation. Mass spectrometry revealed that C18, in contact with Y16, is heavily oxidized during UVR exposure. Analysis of multiple mutant proteins by mass spectrometry suggested that Y16 and C18 likely participate in the same photochemical process. The data suggest an initial photoaggregation pathway for H?D-Crys in which excited-state Y16 interacts with C18, initiating radical polymerization.

SUBMITTER: Schafheimer N

PROVIDER: S-EPMC3954642 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Tyrosine/cysteine cluster sensitizing human γD-crystallin to ultraviolet radiation-induced photoaggregation in vitro.

Schafheimer Nathaniel N Wang Zhen Z Schey Kevin K King Jonathan J

Biochemistry 20140205 6

Ultraviolet radiation (UVR) exposure is a major risk factor for age-related cataract, a protein-aggregation disease of the human lens often involving the major proteins of the lens, the crystallins. γD-Crystallin (HγD-Crys) is abundant in the nucleus of the human lens, and its folding and aggregation have been extensively studied. Previous work showed that HγD-Crys photoaggregates in vitro upon exposure to UVA/UVB light and that its conserved tryptophans are not required for aggregation. Surpris ...[more]

PMID: 24410332

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Project description:BackgroundHuman skin, which is constantly exposed to solar ultraviolet radiation (UVR), has a unique ability to respond by increasing its pigmentation in a protective process driven by melanogenesis in human epidermal melanocytes (HEMs). However, the molecular mechanisms used by HEMs to detect and respond to UVR remain unclear.ObjectivesTo investigate the function and potential mechanism of opsin 5 (OPN5), a photoreceptor responsive to UVR wavelengths, in melanogenesis in HEMs.MethodsMelanin content in HEMs was determined using the NaOH method, and activity of tyrosinase (TYR) (a key enzyme in melanin synthesis) was determined by the l-DOPA method. OPN5 expression in UVR-treated vs. untreated HEMs and explant tissues was detected by reverse-transcription quantitative polymerase chain reaction (RT-qPCR), Western blotting and immunofluorescence. Short interfering RNA-mediated OPN5 knockdown and a lentivirus OPN5 overexpression model were used to examine their respective effects on TYR, tyrosinase-related protein 1 (TRP1), TRP2 and microphthalmia-associated transcription factor (MITF) expression, under UVR. Changes in expression of TYR, TRP1 and TRP2 caused by changes in OPN5 expression level were detected by RT-qPCR and Western blot. Furthermore, changes in signalling pathway proteins were assayed.ResultsWe found that OPN5 is the key sensor in HEMs responsible for UVR-induced melanogenesis. OPN5-induced melanogenesis required Ca2+ -dependent G protein-coupled receptor- and protein kinase C signal transduction, thus contributing to the UVR-induced MITF response to mediate downstream cellular effects, and providing evidence of OPN5 function in mammalian phototransduction. Remarkably, OPN5 activation was necessary for UVR-induced increase in cellular melanin and has an inherent function in melanocyte melanogenesis.ConclusionsOur results provide insight into the molecular mechanisms of UVR sensing and phototransduction in melanocytes, and may reveal molecular targets for preventing pigmentation or pigment diseases.

Dataset Information

Tyrosine/cysteine cluster sensitizing human ?D-crystallin to ultraviolet radiation-induced photoaggregation in vitro.

Publications

Tyrosine/cysteine cluster sensitizing human γD-crystallin to ultraviolet radiation-induced photoaggregation in vitro.

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