Kurokawa Nina, Ogawa Mima, Midorikawa Rio, Kanno Arisa, Naka Wakaba, Noguchi Keiichi, Morishima Ken, Inoue Rintaro, Sugiyama Masaaki, Yohda Masafumi
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan.
Instrumentation Analysis Center, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan.
Int J Mol Sci. 2025 Jun 16;26(12):5748. doi: 10.3390/ijms26125748.
Small heat shock proteins (sHsps) are ubiquitous low-molecular-weight chaperones that prevent protein aggregation under cellular stress conditions. In the absence of stress, they assemble into large oligomers. In response to stress, such as elevated temperatures, they undergo conformational changes that expose hydrophobic surfaces, allowing them to interact with denatured proteins. At heat shock temperatures in bacteria, large sHsp oligomers disassemble into smaller oligomeric forms. Methanogens are a diverse group of microorganisms, ranging from thermophilic to psychrophilic and halophilic species. Accordingly, their sHsps exhibit markedly different temperature dependencies based on their optimal growth temperatures. In this study, we characterized sHsps from both hyperthermophilic and mesophilic methanogens to investigate the mechanisms underlying their temperature-dependent behavior. Using analytical ultracentrifugation, we observed the dissociation of sHsps from a mesophilic methanogen into dimers. The dissociation equilibrium of these oligomers was found to be dependent not only on temperature but also on protein concentration. Furthermore, by generating various mutants, we identified the specific amino acid residues responsible for the temperature dependency observed. The C-terminal region containing the IXI/V motif and the α-crystallin domain were found to be the primary determinants of oligomer stability and its temperature dependence.
小分子热休克蛋白（sHsps）是普遍存在的低分子量伴侣蛋白，可在细胞应激条件下防止蛋白质聚集。在无应激状态下，它们组装成大的寡聚体。在应激反应中，如温度升高时，它们会发生构象变化，暴露出疏水表面，使其能够与变性蛋白相互作用。在细菌的热休克温度下，大的sHsp寡聚体会分解成较小的寡聚体形式。产甲烷菌是一类多样的微生物，包括嗜热、嗜冷和嗜盐物种。因此，它们的sHsps根据其最佳生长温度表现出明显不同的温度依赖性。在本研究中，我们对嗜热产甲烷菌和嗜温产甲烷菌的sHsps进行了表征，以研究其温度依赖性行为的潜在机制。使用分析超速离心法，我们观察到嗜温产甲烷菌的sHsps解离成二聚体。发现这些寡聚体的解离平衡不仅取决于温度，还取决于蛋白质浓度。此外，通过产生各种突变体，我们确定了导致观察到的温度依赖性的特定氨基酸残基。发现含有IXI/V基序和α-晶状体蛋白结构域的C末端区域是寡聚体稳定性及其温度依赖性的主要决定因素。
