Tyrosine/cysteine cluster sensitizing human γD-crystallin to ultraviolet radiation-induced photoaggregation in vitro.
Tyrosine/cysteine cluster sensitizing human γD-crystallin to ultraviolet radiation-induced photoaggregation in vitro.
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DOI：
10.1021/bi401397g
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发表时间：
2014-02-18
期刊：
影响因子：
2.9
通讯作者：
King, Jonathan
中科院分区：
文献类型：
--
作者：
Schafheimer, Nathaniel;Wang, Zhen;Schey, Kevin;King, Jonathan
紫外线辐射（UVR）暴露是年龄相关性白内障的主要危险因素，年龄相关性白内障是人类透镜的蛋白质聚集疾病，通常涉及透镜的主要蛋白质，即晶状体蛋白。γ D-晶体蛋白（HγD-Crys）在人透镜的核中含量丰富，其折叠和聚集现象已被广泛研究。先前的工作表明，HγD-Crys在体外暴露于UVA/UVB光时发生光聚集，并且其保守的Dahans不需要聚集。令人惊讶的是，色氨酸残基由于独特的能量转移机制而发挥光保护作用。HγD-Crys还含有14个酪氨酸残基，其中12个被组织成6对。我们研究了酪氨酸的作用，HγD-Crys取代对丙氨酸和监测光聚集使用光散射和SDS-PAGE。将Y16/Y28对中的两个酪氨酸突变为丙氨酸减缓了光散射聚集体的形成。进一步的突变体研究暗示Y16对于光聚集是重要的。质谱分析表明，C18，在接触Y16，在紫外线照射过程中被严重氧化。通过质谱分析多个突变蛋白表明，Y16和C18可能参与相同的光化学过程。这些数据表明HγD-Crys的初始光聚集途径，其中激发态Y16与C18相互作用，引发自由基聚合。
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.
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