Abstract：
Congenital cataracts, a prevalent cause of blindness in children, are associated with protein aggregation. γD-crystallin, essential for sustaining lens transparency, exists as a monomer and exhibits excellent structural stability. In our cohort, we identified a nonsense mutation (c.451_452insGACT, p.Y151X) in the CRYGD gene. To explore the effect of truncation mutations on the structure of γD-crystallin, we examined the Y151X and T160RfsX8 mutations, both located in the Greek key motif 4 at the cellular and protein level in this study. Both truncation mutations induced protein misfolding and resulted in the formation of insoluble aggregates when overexpressed in HLE B3 and HEK 293T cells. Moreover, heat, UV irradiation, and oxidative stress increased the proportion of aggregates of mutants in the cells. We next purified γD-crystallin to estimate its structural changes. Truncation mutations led to conformational disruption and a concomitant decrease in protein solubility. Molecular dynamics simulations further demonstrated that partial deletion of the conserved domain within the Greek key motif 4 markedly compromised the overall stability of the protein structure. Finally, co-expression of α-crystallins facilitated the proper folding of truncated mutants and mitigated protein aggregation. In summary, the structural integrity of the Greek key motif 4 in γD-crystallin is crucial for overall structural stability.
摘要：
先天性白内障,儿童失明的普遍原因，与蛋白质聚集有关。γD-晶状体蛋白，对于维持镜头透明度至关重要，作为单体存在并表现出优异的结构稳定性。在我们的队列中，我们确定了一个无义突变(c.451_452insgact，p.Y151X)在CRYGD基因中。探讨截短突变对γD-晶状体蛋白结构的影响,我们检查了Y151X和T160RfsX8突变，在这项研究中，两者都位于细胞和蛋白质水平的希腊关键基序4中。当在HLEB3和HEK293T细胞中过表达时，两种截短突变均诱导蛋白质错误折叠并导致不溶性聚集体的形成。此外，热,紫外线照射，和氧化应激增加了细胞中突变体聚集体的比例。我们接下来纯化γD-晶状体蛋白以估计其结构变化。截断突变导致构象破坏和伴随的蛋白质溶解度降低。分子动力学模拟进一步证明，希腊关键基序4中保守结构域的部分缺失显着损害了蛋白质结构的整体稳定性。最后,α-晶状体蛋白的共表达促进了截短突变体的正确折叠并减轻了蛋白质聚集。总之,γD-晶状体蛋白中希腊关键基序4的结构完整性对于整体结构稳定性至关重要。
