小热休克蛋白(sHSPs)是ATP非依赖性伴侣,对细胞蛋白稳定至关重要,预防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-晶状体蛋白,αA-晶状体蛋白(αAc)和αB-晶状体蛋白(αBc),代表原型sHSP,表现出复杂的多分散寡聚组装和快速亚基交换动力学。然而,我们对这种可塑性如何有助于伴侣功能的理解仍然知之甚少。使用生化和生物物理分析结合单粒子电子显微镜(EM),我们检查了αAc的结构变化,αBc和天然异聚晶状体α-晶状体蛋白(αLc)处于apo状态,并处于不同程度的伴侣饱和度,导致共同聚集,使用溶菌酶和胰岛素作为模型客户。定量单粒子分析揭示了在共聚集过程中形成的寡聚态的连续谱,以显著的客户触发的扩展和sHSP寡聚支架的准有序延伸为标志,由此,本机笼状sHSP组件显示定向生长以适应客户端隔离的饱和条件。这些结构修饰最终导致伴侣-客户复合物的明显无定形崩溃,导致产生能够散射可见光的共聚集体。有趣的是,这些共聚集体保持了高度伸长的sHSP低聚物的内部形态特征,与从老化的晶状体组织中分离的聚合α-晶状体蛋白种类非常相似。这种机制在整个αAc中似乎是一致的,αBc和αLc,尽管对客户诱导的共聚集有不同程度的易感性。重要的是,我们的发现表明,客户诱导的共同聚集遵循独特的机械和准序轨迹,不同于纯粹的无定形过程。这些见解重塑了我们对α-晶状体蛋白的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的影响。
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. Using biochemical and biophysical analyses combined with single-particle electron microscopy (EM), we examined structural changes in αAc, αBc and native heteromeric lens α-
crystallins (αLc) in their apo-states and at varying degree of chaperone saturation leading to co-aggregation, using lysozyme and insulin as model clients. 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 oligomeric scaffold, whereby the native cage-like sHSP assembly displays a directional growth to accommodate saturating conditions of client sequestration. 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 oligomers with striking resemblance to polymeric α-crystallin species isolated from aged lens tissue. This mechanism appears consistent across αAc, αBc and αLc, 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 α-
crystallins, carrying potential implications for a pathway toward cataract formation.