Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University of Gdansk, University of Gdansk, Gdansk 80-307, Poland.
Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University of Gdansk, University of Gdansk, Gdansk 80-307, Poland
Proc Natl Acad Sci U S A. 2021 Dec 7;118(49). doi: 10.1073/pnas.2108163118.
Protein homeostasis is constantly being challenged with protein misfolding that leads to aggregation. Hsp70 is one of the versatile chaperones that interact with misfolded proteins and actively support their folding. Multifunctional Hsp70s are harnessed to specific roles by J-domain proteins (JDPs, also known as Hsp40s). Interaction with the J-domain of these cochaperones stimulates ATP hydrolysis in Hsp70, which stabilizes substrate binding. In eukaryotes, two classes of JDPs, Class A and Class B, engage Hsp70 in the reactivation of aggregated proteins. In most species, excluding metazoans, protein recovery also relies on an Hsp100 disaggregase. Although intensely studied, many mechanistic details of how the two JDP classes regulate protein disaggregation are still unknown. Here, we explore functional differences between the yeast Class A (Ydj1) and Class B (Sis1) JDPs at the individual stages of protein disaggregation. With real-time biochemical tools, we show that Ydj1 alone is superior to Sis1 in aggregate binding, yet it is Sis1 that recruits more Ssa1 molecules to the substrate. This advantage of Sis1 depends on its ability to bind to the EEVD motif of Hsp70, a quality specific to most of Class B JDPs. This second interaction also conditions the Hsp70-induced aggregate modification that boosts its subsequent dissolution by the Hsp104 disaggregase. Our results suggest that the Sis1-mediated chaperone assembly at the aggregate surface potentiates the entropic pulling, driven polypeptide disentanglement, while Ydj1 binding favors the refolding of the solubilized proteins. Such subspecialization of the JDPs across protein reactivation improves the robustness and efficiency of the disaggregation machinery.
蛋白质的结构稳态经常受到蛋白质错误折叠的挑战，从而导致聚集。Hsp70 是一种多功能伴侣蛋白，可与错误折叠的蛋白质相互作用，并积极支持其折叠。多功能 Hsp70 被 J 结构域蛋白（JDPs，也称为 Hsp40s）赋予特定的功能。这些共伴侣蛋白与 J 结构域的相互作用刺激 Hsp70 中的 ATP 水解，从而稳定底物结合。在真核生物中，有两类 JDPs，A 类和 B 类，参与 Hsp70 对聚集蛋白的再激活。在大多数物种中，除了后生动物外，蛋白质的恢复还依赖于 Hsp100 解聚酶。尽管已经进行了深入研究，但关于这两类 JDP 如何调节蛋白解聚的许多机制细节仍不清楚。在这里，我们在蛋白解聚的各个阶段探索了酵母 A 类（Ydj1）和 B 类（Sis1）JDP 之间的功能差异。通过实时生化工具，我们表明 Ydj1 单独在聚集物结合方面优于 Sis1，但 Sis1 能招募更多的 Ssa1 分子到底物上。Sis1 的这种优势依赖于其与 Hsp70 的 EEVD 基序结合的能力，这是大多数 B 类 JDP 的特有质量。这种第二个相互作用还调节了 Hsp70 诱导的聚集修饰，从而增强了随后由 Hsp104 解聚酶溶解聚集物的能力。我们的研究结果表明，Sis1 在聚集物表面介导的伴侣组装增强了熵拉力，驱动多肽解缠结，而 Ydj1 的结合则有利于溶解蛋白的重折叠。JDPs 在蛋白再激活过程中的这种专业化提高了解聚机制的稳健性和效率。
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