Santhoshkumar Puttur, Sharma Krishna K
Department of Ophthalmology, University of Missouri, Columbia, MO, United States.
Department of Biochemistry, University of Missouri, Columbia, MO, United States.
Front Mol Biosci. 2022 Apr 6;9:875205. doi: 10.3389/fmolb.2022.875205. eCollection 2022.
The G98R mutation in αA-crystallin is associated with presenile cataract development in humans. Previous studies have indicated that mutant proteins altered structure, decreased stability, increased oligomeric size, loss of chaperone-like activity, and susceptibility to proteolysis could be contributing factors to cataract formation. To evaluate the effect of substrate protein interactions with the mutant protein on cataract formation, we have performed chaperone assays with alcohol dehydrogenase (ADH), citrate synthase (CS), and βB-crystallin (βB), and analyzed the reaction mixtures by multi-angle light scattering (MALS) analysis. It appears that αAG98R protein initially gets stabilized upon interaction with substrate proteins. Analysis of the chaperone-client protein complexes revealed that wild-type αA-crystallin interacts with substrate proteins to form compact complexes leading to a slight increase in oligomeric mass, whereas αAG98R forms less compact and high molecular weight complexes with the substrate, and the resulting complexes continue to increase in size over time. As a result, the soluble complexes formed initially by the mutant protein begin to scatter light and precipitate. We found that the stability and chaperone activity of the αAG98R can be improved by modifying the protein with low concentrations (50 µM) of methylglyoxal (MGO). Incubation of αAG98R protein (1 mg/ml) under aseptic conditions for 30 days at 37°C resulted in precipitation of the mutant protein. In contrast, mutant protein incubations carried out with 50 µM MGO remained soluble and transparent. SDS-PAGE analysis showed gradual autolysis of the mutant protein in the absence of MGO. The average molar mass of the mutant protein oligomers changed from 7,258 ± 12 kDa to 3,950 ± 08 kDa within 60 min of incubation with MGO. There was no further significant change in the molar mass of mutant protein when tested on day 7 of MGO treatment. Our data suggest that the initial stabilization of αAG98R by substrate proteins could delay congenital cataracts' appearance, and the uncontrolled long-term interaction amongst mutant subunits and substrate proteins could be the rationale behind presenile cataracts formation. The results also demonstrate the potential benefit of low concentrations of MGO in stabilizing mutant chaperone protein(s).
αA-晶状体蛋白中的G98R突变与人类早老性白内障的发生有关。先前的研究表明，突变蛋白结构改变、稳定性降低、寡聚体尺寸增加、伴侣样活性丧失以及对蛋白水解的敏感性可能是导致白内障形成的因素。为了评估底物蛋白与突变蛋白的相互作用对白内障形成的影响，我们用乙醇脱氢酶（ADH）、柠檬酸合酶（CS）和βB-晶状体蛋白（βB）进行了伴侣蛋白分析，并通过多角度光散射（MALS）分析对反应混合物进行了分析。似乎αAG98R蛋白在与底物蛋白相互作用时最初会得到稳定。对伴侣蛋白-客户蛋白复合物的分析表明，野生型αA-晶状体蛋白与底物蛋白相互作用形成紧密复合物，导致寡聚体质量略有增加，而αAG98R与底物形成的复合物较松散且分子量较高，并且随着时间的推移，形成的复合物尺寸会持续增加。结果，突变蛋白最初形成的可溶性复合物开始散射光并沉淀。我们发现，用低浓度（50µM）的甲基乙二醛（MGO）修饰该蛋白可以提高αAG98R的稳定性和伴侣蛋白活性。在无菌条件下，将αAG98R蛋白（1mg/ml）在37°C孵育30天会导致突变蛋白沉淀。相比之下，用50µM MGO进行的突变蛋白孵育仍保持可溶且透明。SDS-PAGE分析表明，在没有MGO的情况下，突变蛋白会逐渐自溶。与MGO孵育60分钟内，突变蛋白寡聚体的平均摩尔质量从7258±12kDa变为3950±08kDa。在MGO处理第7天进行测试时，突变蛋白的摩尔质量没有进一步的显著变化。我们的数据表明，底物蛋白对αAG98R的初始稳定作用可能会延迟先天性白内障的出现，而突变亚基与底物蛋白之间不受控制的长期相互作用可能是早老性白内障形成的原因。结果还证明了低浓度MGO在稳定突变伴侣蛋白方面的潜在益处。
