兰州大学机构库 >生命科学学院
β/γ晶状体蛋白C端结构域遗传突变导致先天性白内障的分子机理研究
Alternative TitleMolecular mechanism of congenital hereditary cataract caused by mutations in the C-terminal domain of β/γ-crystallin
席艺博
Thesis Advisor吉尚戎
2014-05-27
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name博士
Keyword先天性白内障 晶状体蛋白 遗传突变体 聚集 蛋白质稳定性
Abstract晶状体蛋白基因突变是导致遗传性先天性白内障的一个重要因素。临床研究已经报道了50多个β/γ晶状体蛋白的致病突变,但其中大部分遗传突变导致先天性白内障的分子机制尚未得到研究。β/γ晶状体蛋白是晶状体中主要的结构蛋白,它们都包含有N端和C端两个结构域,每个结构域含有两个Greek key基序。它们的结构差异在于,β晶状体蛋白以同源或异源寡聚体形式存在,而γ晶状体蛋白以单体形式存在。本论文选取了β和γ晶状体蛋白C端结构域的几个新发现的突变体作为研究对象,利用生物物理研究手段研究这些遗传突变对β和γ晶状体蛋白的结构、稳定性和聚集机制的影响。 βB1晶状体蛋白在晶状体中可能调节了β晶状体蛋白的寡聚分布,而βB1的一个显著特点是具有长的N端和C端延伸区。前人报导位于C端延伸区的R233H突变会引发遗传性显性核性白内障。我们通过多种生物物理研究手段发现,R233H突变不影响βB1在中性条件下的稳定性,但降低了βB1在酸性条件下的稳定性。更重要的是,R233H突变极大降低了βB1/βA3的结构稳定性。结果显示C末端延伸处233位点很可能参与了β晶状体蛋白异源寡聚体的形成,同时对维持异源二聚体的结构稳定性具有重要作用。 βB2是晶状体中含量最多的一种β晶状体蛋白。我们之前的研究表明位于C端结构域最后一个β折叠片上的遗传突变V187M和R188H会导致βB2稳定性下降和易于聚集,但对于这两个位点是否直接参与了聚集核心的形成并不清楚。本论文进一步利用酸变性实验分清了突变体对蛋白质结构、稳定性、无定形聚集和规则的淀粉样纤维形成的影响。研究结果表明,V187M并不影响βB2的聚集机制,更可能是影响了蛋白质的稳定性;而R188H调节了聚集核心的形成,从而加速了无定形聚集和纤维化的速度。 我们与合作者共同鉴定了一个新的γC晶状体蛋白遗传突变G129C。该突变会引发遗传性显性核性白内障。机制研究表明该突变显著降低了蛋白的热稳定性及长时间稳定性。在去折叠过程中,该突变稳定了较低盐酸胍浓度下的折叠中间态,从而促进了蛋白的聚集和纤维化,且对细胞具有一定的毒性。 总之,本论文的研究结果表明,β/γ晶状体蛋白的C端结构域对于维持β/γ晶状体蛋白的寡聚稳定性上都有着重要作用,特别是较少研究的C末端突变可能调节了β/γ晶状体蛋白聚集核心的形成。
Other AbstractCongenital cataract is one of the leading causes of childhood blindness worldwide, and about one-third of the cases have a genetic origin. Among the familiar cataract, about one-third cases are caused by mutationsin crystallin genes. About 50 mutations have been reported in β/γ-crystallins, but only a few mutations have been investigated in detail. β/γ-Crystallinsare the predominant structural proteins in the lens, and are highly homologous in their tertiary structures composing four Greek key motif divided into two domains (the N- and C-terminal domains). The major difference between β- and γ-crystallins is their oligomeric states: β-crystallins are homomers or heteromers, whileγ-crystallins are monomers. In this research, we studied the molecular mechanisms of several cataract-linked mutations located at the C-terminal domain of β/γ-crystallins via biophysical and structural methods. βB1 has been found to have a role in regulating the size distributions of β-crystallins in the lens. The most striking feature in βB1 primary sequence is the long N- and C-terminal extensions. R233H is the only inherited mutationoccurred at the C-terminal extension of βB1. The results herein indicated that the mutation did not affect βB1 structural stability under neutral conditions, but decreased βB1 stability under mild acidic conditions. Furthermore, we found that the mutation significantly decreased the stability of βB1/βA3 hereomer. These results suggested that R233 might be involved in heteromer formation, and play a role in the structural stability of βB1/βA3 hereomer. βB2 is the most abundant among various crystallins in the lens. Our previous results have indicated that the mutations V187M and R188H decrease βB2 stability and promote βB2 aggregation. However, it remains elusive whether these two mutations are involved in the core of βB2 aggregates. In this research, acid denaturation was used to separate the effects of mutations on βB2 structure, stability, amorphousaggregation and fibrilization. Our results indicated that V187M did not affect the aggregation mechanism of βB2, but was more likely to cause cataract by decreasing protein stability. On the contrary, R188H not only decreased protein stability, but also promoted aggregation/fibrilization by regulating the core of aggregates/fibrils. Collaborated with doctors in hospitals, we have identified a novel G129C mutation in γC-crystallin, which is linked to autosomal dominant conge...
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221824
Collection生命科学学院
Recommended Citation
GB/T 7714
席艺博. β/γ晶状体蛋白C端结构域遗传突变导致先天性白内障的分子机理研究[D]. 兰州. 兰州大学,2014.
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