Department of Ophthalmology, School of Medicine, University of Missouri-Columbia, Columbia, MO 65212, USA.
Department of Biochemistry, School of Medicine, University of Missouri-Columbia, Columbia, MO 65211, USA.
Int J Mol Sci. 2021 Oct 5;22(19):10771. doi: 10.3390/ijms221910771.
Previously, we showed that the removal of the 54-61 residues from αB-crystallin (αBΔ54-61) results in a fifty percent reduction in the oligomeric mass and a ten-fold increase in chaperone-like activity. In this study, we investigated the oligomeric organization changes in the deletion mutant contributing to the increased chaperone activity and evaluated the cytoprotection properties of the mutant protein using ARPE-19 cells. Trypsin digestion studies revealed that additional tryptic cleavage sites become susceptible in the deletion mutant than in the wild-type protein, suggesting a different subunit organization in the oligomer of the mutant protein. Static and dynamic light scattering analyses of chaperone-substrate complexes showed that the deletion mutant has more significant interaction with the substrates than wild-type protein, resulting in increased binding of the unfolding proteins. Cytotoxicity studies carried out with ARPE-19 cells showed an enhancement in anti-apoptotic activity in αBΔ54-61 as compared with the wild-type protein. The improved anti-apoptotic activity of the mutant is also supported by reduced caspase activation and normalization of the apoptotic cascade components level in cells treated with the deletion mutant. Our study suggests that altered oligomeric assembly with increased substrate affinity could be the basis for the enhanced chaperone function of the αBΔ54-61 protein.
先前，我们已经证明从 αB- 晶体蛋白（αBΔ54-61）中去除 54-61 个残基会导致寡聚体质量减少一半，同时伴侣样活性增加十倍。在这项研究中，我们研究了导致伴侣活性增加的缺失突变体的寡聚体组织变化，并使用 ARPE-19 细胞评估了突变蛋白的细胞保护特性。胰蛋白酶消化研究表明，与野生型蛋白相比，缺失突变体中出现了更多的潜在胰蛋白酶切割位点，这表明突变体蛋白的寡聚体中存在不同的亚基组织。伴侣-底物复合物的静态和动态光散射分析表明，与野生型蛋白相比，缺失突变体与底物的相互作用更显著，导致未折叠蛋白的结合增加。用 ARPE-19 细胞进行的细胞毒性研究表明，与野生型蛋白相比，αBΔ54-61 的抗凋亡活性增强。在用缺失突变体处理的细胞中，半胱天冬酶的激活减少，凋亡级联成分的水平恢复正常，这也支持了突变体的抗凋亡活性增强。我们的研究表明，与增加的底物亲和力相关的寡聚体组装的改变可能是 αBΔ54-61 蛋白增强伴侣功能的基础。
