Miller Adam P, O'Neill Susan E, Lampi Kirsten J, Reichow Steve L
Department of Chemical Physiology and Biochemistry, Oregon Health & Science University, Portland, Oregon 97239, USA.
Vollum Institute, Oregon Health & Science University, Portland, Oregon 97239, USA.
bioRxiv. 2023 Aug 17:2023.08.15.553435. doi: 10.1101/2023.08.15.553435.
Small heat shock proteins (sHSPs) are ATP-independent chaperones vital to cellular proteostasis, preventing protein aggregation events linked to various human diseases including cataract. The α-crystallins, αA-crystallin (αAc) and αB-crystallin (αBc), represent archetypal sHSPs that exhibit complex polydispersed oligomeric assemblies and rapid subunit exchange dynamics. Yet, our understanding of how this plasticity contributes to chaperone function remains poorly understood. This study investigates structural changes in αAc and αBc during client sequestration under varying degree of chaperone saturation. Using biochemical and biophysical analyses combined with single-particle electron microscopy (EM), we examined αAc and αBc in their apo-states and at various stages of client-induced co-aggregation, using lysozyme as a model client. Quantitative single-particle analysis unveiled a continuous spectrum of oligomeric states formed during the co-aggregation process, marked by significant client-triggered expansion and quasi-ordered elongation of the sHSP scaffold. These structural modifications culminated in an apparent amorphous collapse of chaperone-client complexes, resulting in the creation of co-aggregates capable of scattering visible light. Intriguingly, these co-aggregates maintain internal morphological features of highly elongated sHSP scaffolding with striking resemblance to polymeric α-crystallin species isolated from aged lens tissue. This mechanism appears consistent across both αAc and αBc, albeit with varying degrees of susceptibility to client-induced co-aggregation. Importantly, our findings suggest that client-induced co-aggregation follows a distinctive mechanistic and quasi-ordered trajectory, distinct from a purely amorphous process. These insights reshape our understanding of the physiological and pathophysiological co-aggregation processes of sHSPs, carrying potential implications for a pathway toward cataract formation.
小热休克蛋白（sHSPs）是对细胞蛋白质稳态至关重要的不依赖ATP的分子伴侣，可防止与包括白内障在内的各种人类疾病相关的蛋白质聚集事件。α-晶体蛋白，即αA-晶体蛋白（αAc）和αB-晶体蛋白（αBc），是典型的sHSPs，表现出复杂的多分散寡聚体组装和快速的亚基交换动力学。然而，我们对这种可塑性如何有助于分子伴侣功能的理解仍然很有限。本研究调查了在不同程度的分子伴侣饱和状态下，客户蛋白隔离过程中αAc和αBc的结构变化。结合生化和生物物理分析以及单颗粒电子显微镜（EM），我们以溶菌酶作为模型客户蛋白，研究了处于无配体状态以及客户蛋白诱导的共聚集不同阶段的αAc和αBc。定量单颗粒分析揭示了共聚集过程中形成的连续寡聚体状态谱，其特征是客户蛋白触发的sHSP支架的显著扩张和准有序伸长。这些结构修饰最终导致分子伴侣-客户蛋白复合物明显的无定形塌陷，从而形成能够散射可见光的共聚物。有趣的是，这些共聚物保留了高度伸长的sHSP支架的内部形态特征，与从老化晶状体组织中分离出的聚合α-晶体蛋白种类惊人地相似。尽管对客户蛋白诱导的共聚集的敏感性程度不同，但这种机制在αAc和αBc中似乎是一致的。重要的是，我们的研究结果表明，客户蛋白诱导的共聚集遵循独特的机制和准有序轨迹，不同于纯粹的无定形过程。这些见解重塑了我们对sHSPs生理和病理生理共聚集过程的理解，对白内障形成途径具有潜在意义。
