Deamidation destabilizes and triggers aggregation of a lens protein, βA3-crystallin
Deamidation destabilizes and triggers aggregation of a lens protein, βA3-crystallin
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DOI：
10.1110/ps.035410.108
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发表时间：
2008-09-01
期刊：
影响因子：
8
通讯作者：
Lampi, Kirsten J.
中科院分区：
文献类型：
--
作者：
Takata, Takumi;Oxford, Julie T.;Lampi, Kirsten J.
蛋白质聚集是多种神经退行性疾病和白内障的标志。眼睛晶状体中的主要蛋白质是晶状体蛋白，它在一生中不断积累并被广泛修饰。脱酰胺是老化和白内障过程中晶状体的主要改变。在晶状体蛋白中，β A3 亚基被发现具有与体内不溶性蛋白质相关的多个脱酰胺位点。预计有几个位点会暴露在 β A3 表面，并在本研究中进行了研究。通过 N 端结构域上的 Q42 和 N54、C 端结构域上的 N133 和 N155 以及连接结构域的肽中的 N120 处的定点诱变来模拟脱酰胺化。脱酰胺作用改变了三级结构，但不破坏二级结构或 β A3 二聚体的形成。 C 端结构域和连接肽中的脱酰胺作用比 N 端结构域中的脱酰胺作用更大程度地降低了稳定性。 N54 和 N155 的脱酰胺化也破坏了与 β B1 亚基的关联。沉降速度实验与高分辨率分析相结合，检测到所有脱酰胺蛋白中有 15%-20% 的可溶性聚集体，但野生型 β A3 中没有。这些聚集体的摩擦比升高，表明它们被拉长。体外聚集体的检测强烈表明脱酰胺可能有助于晶状体中的蛋白质聚集。潜在的机制可能包括稳定性降低和/或与其他β亚基相互作用的改变。了解脱酰胺在长寿命晶状体蛋白中的作用对于其他聚集疾病具有重要意义。
Protein aggregation is a hallmark of several neurodegenerative diseases and also of cataracts. The major proteins in the lens of the eye are crystallins, which accumulate throughout life and are extensively modified. Deamidation is the major modification in the lens during aging and cataracts. Among the crystallins, the beta A3-subunit has been found to have multiple sites of deamidation associated with the insoluble proteins in vivo. Several sites were predicted to be exposed on the surface of beta A3 and were investigated in this study. Deamidation was mimicked by site-directed mutagenesis at Q42 and N54 on the N-terminal domain, N133 and N155 on the C-terminal domain, and N120 in the peptide connecting the domains. Deamidation altered the tertiary structure without disrupting the secondary structure or the dimer formation of beta A3. Deamidations in the C-terminal domain and in the connecting peptide decreased stability to a greater extent than deamidations in the N-terminal domain. Deamidation at N54 and N155 also disrupted the association with the beta B1-subunit. Sedimentation velocity experiments integrated with high-resolution analysis detected soluble aggregates at 15%-20% in all deamidated proteins, but not in wild-type beta A3. These aggregates had elevated frictional ratios, suggesting that they were elongated. The detection of aggregates in vitro strongly suggests that deamidation may contribute to protein aggregation in the lens. A potential mechanism may include decreased stability and/or altered interactions with other beta-subunits. Understanding the role of deamidation in the long-lived crystallins has important implications in other aggregation diseases.