Substrate Protein Interactions and Methylglyoxal Modifications Reduce the Aggregation Propensity of Human Alpha-A-Crystallin G98R Mutant.
Substrate Protein Interactions and Methylglyoxal Modifications Reduce the Aggregation Propensity of Human Alpha-A-Crystallin G98R Mutant.
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DOI：
10.3389/fmolb.2022.875205
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发表时间：
2022
影响因子：
5
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α A-晶状体蛋白G98 R突变与人类早老性白内障的发生有关先前的研究表明，突变蛋白改变结构，降低稳定性，增加寡聚体大小，伴侣样活性的丧失，以及对蛋白水解的易感性可能是白内障形成的促成因素。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βB2-晶状体蛋白（βB2）进行了分子伴侣测定，并通过多角度光散射（MALS）分析来分析反应混合物。α AG 98 R蛋白在与底物蛋白相互作用后开始稳定。伴侣蛋白-客户蛋白复合物的分析显示，野生型α A-晶状体蛋白与底物蛋白相互作用形成紧密复合物，导致寡聚体质量略微增加，而α AG 98 R与底物形成不太紧密的高分子量复合物，并且所得复合物的大小随时间推移继续增加。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，α AG 98 R的稳定性和伴侣活性可以通过用低浓度（50 µM）的甲基乙二醛（MGO）修饰蛋白质来提高。在无菌条件下于37°C孵育α AG 98 R蛋白（1 mg/ml）30天，导致突变蛋白沉淀。相比之下，用50 μM MGO进行的突变蛋白孵育保持可溶和透明。SDS-PAGE分析表明，在MGO的情况下，突变蛋白的逐渐自溶。在与MGO孵育60分钟内，突变蛋白寡聚体的平均摩尔质量从7，258 ± 12 kDa变为3，950 ± 08 kDa。当在MGO处理的第7天测试时，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α AG 98 R的初始稳定可以延迟先天性白内障的出现，而突变亚基和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的MGO在稳定突变体伴侣蛋白中的潜在益处。
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 βB2-crystallin (βB2), 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).