title: Tyrosine/cysteine cluster sensitizing human γD-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. 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. Surprisingly, the tryptophan residues play a photoprotective role because of a distinctive energy-transfer mechanism. HγD-Crys also contains 14 tyrosine residues, 12 of which are organized as six pairs. We investigated the role of the tyrosines of HγD-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 HγD-Crys in which excited-state Y16 interacts with C18, initiating radical polymerization.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology , 68-330, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
authors.forename: Nathaniel
authors.initials: N
authors.lastname: Schafheimer
authors.forename: Zhen
authors.initials: Z
authors.lastname: Wang
authors.forename: Kevin
authors.initials: K
authors.lastname: Schey
authors.forename: Jonathan
authors.initials: J
authors.lastname: King
mesh_terms: D019943:Amino Acid Substitution; D002386:Cataract / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects*; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary / Q000528:radiation effects; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects*; D014466:Ultraviolet Rays; D038222:gamma-Crystallins / Q000235:genetics / Q000528:radiation effects*
chemical_list: C504903:CRYGD protein, human; D038222:gamma-Crystallins; D014443:Tyrosine; D014364:Tryptophan; D003545:Cysteine