Identification of the Most Impactful Asparagine Residues for γS-Crystallin Aggregation by Deamidation
Identification of the Most Impactful Asparagine Residues for γS-Crystallin Aggregation by Deamidation
复制标题
通过脱酰胺作用鉴定对 γS-晶状体蛋白聚集影响最大的天冬酰胺残基
DOI：
10.1021/acs.biochem.3c00097
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发表时间：
2023
期刊：
影响因子：
2.9
通讯作者：
Ishikawa Yoshinobu
中科院分区：
文献类型：
--
作者：
Kato Koichi;Nakayoshi Tomoki;Kitamura Yuki;Kurimoto Eiji;Oda Akifumi;Ishikawa Yoshinobu
晶状体中晶体蛋白的聚集参与了白内障的发病机制。这种聚集被认为是由非酶翻译后修饰促进的，例如氨基酸残基的脱酰胺化和立体转化。虽然在先前的研究中，在体内检测到了γS-晶体蛋白中的去酰胺化天冬酰胺残基，但目前还不清楚在生理条件下，哪些去酰胺化残基对聚集的影响最大。在这项研究中，我们利用脱酰胺模拟突变体(N14D、N37D、N53D、N76D和N143D)研究了γS晶体蛋白中所有天冬氨酸残基对结构和聚集性质的影响。用圆二色谱和分子动力学模拟研究了结构的影响，用凝胶过滤层析和分光光度方法分析了聚集态的性质。没有检测到所有突变对结构的重大影响。然而，N37D突变降低了热稳定性，并改变了一些分子间氢键的形成。聚集性分析表明，各突变体的聚集率优势随温度不同而不同。Asn残基的脱酰胺作用促进了γS-晶状体蛋白的聚集，Asn 37、Asn 53和Asn 76残基的脱酰胺作用对不溶聚集体的形成影响最大。
Crystallin aggregation in the eye lens is involved in the pathogenesis of cataracts. The aggregation is considered to be promoted by non-enzymatic post-translational modifications, such as the deamidation and stereoinversion of amino acid residues. Although in a previous study, the deamidated asparagine residues were detected in γS-crystallin in vivo, it is unclear which deamidated residues have the most impact on the aggregation under physiological conditions. In this study, we investigated the deamidation impacts of all Asn residues in γS-crystallin for the structural and aggregation properties utilizing deamidation mimetic mutants (N14D, N37D, N53D, N76D, and N143D). The structural impacts were investigated using circular dichroism analysis and molecular dynamics simulations, and the aggregation properties were analyzed by gel filtration chromatography and spectrophotometric methods. No significant structural impacts of all mutations were detected. However, the N37D mutation decreased thermal stability and changed some intermolecular hydrogen-bond formations. Aggregation analysis indicated that the superiority of the aggregation rate in each mutant varied with temperature. Deamidation at any Asn residues promoted γS-crystallin aggregation, and the deamidation at Asn37, Asn53, and Asn76 were suggested to be the most impactful in the formation of insoluble aggregations.