Abstract:
Congenital cataract is the leading cause of childhood blindness, which primarily results from genetic factors. γD-crystallin is the most abundant γ-crystallin and is essential for maintaining lens transparency and refractivity. Numerous mutations in γD-crystallin have been reported with unclear pathogenic mechanism. Two different cataract-causing mutations Ser78Phe and Ser78Pro in γD-crystallin were previously identified at the same conserved Ser78 residue. In this work, firstly, we purified the mutants and characterized for the structural change using fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and size-exclusion chromatography (SEC). Both mutants were prone to form insoluble precipitates when expressed in Escherichia coli strain BL21 (DE3) cells. Compared with wild-type (WT), both mutations caused structural disruption, increased hydrophobic exposure, decreased solubility, and reduced thermal stability. Next, we investigated the aggregation of the mutants at the cellular level. Overexpression the mutants in HLE-B3 and HEK 293T cells could induce aggresome formations. The environmental stresses (including heat, ultraviolet irradiation and oxidative stress) promoted the formation of aggregates. Moreover, the intracellular S78F and S78P aggregates could be reversed by lanosterol. Molecular dynamic simulation indicated that both mutations disrupted the structural integrity of Greek-key motif 2. Hence, our results reveal the vital role of conserved Ser78 in maintaining the structural stability, which can offer new insights into the mechanism of cataract formation.
摘要:
先天性白内障是导致儿童失明的主要原因,这主要是遗传因素造成的。γD-晶状体蛋白是最丰富的γ-晶状体蛋白,对于保持晶状体的透明度和折射率至关重要。已经报道了γD-晶状体蛋白中的许多突变,其致病机制尚不清楚。先前在相同保守的Ser78残基处鉴定了γD-晶状体蛋白中的两个不同的引起白内障的突变Ser78Phe和Ser78Pro。在这项工作中,首先,我们纯化了突变体,并使用荧光光谱法表征了结构变化,圆二色性(CD)光谱,和尺寸排阻色谱(SEC)。当在大肠杆菌菌株BL21(ED3)细胞中表达时,两种突变体都倾向于形成不溶性沉淀物。与野生型(WT)相比,两种突变都导致结构破坏,增加疏水性暴露,溶解度降低,和降低热稳定性。接下来,我们在细胞水平上研究了突变体的聚集。HLE-B3和HEK293T细胞中突变体的过表达可诱导攻击体形成。环境压力(包括热量,紫外线照射和氧化应激)促进了聚集体的形成。此外,细胞内S78F和S78P聚集体可被羊毛甾醇逆转。分子动力学模拟表明,两种突变都破坏了希腊关键基序2的结构完整性。因此,我们的结果揭示了保守的Ser78在维持结构稳定性方面的重要作用,这可以为白内障形成机制提供新的见解。