Chen Yuwen, An Yuxin, Pan Hui, Gong Zhou, Li Zhiying, Chen Jing, Liang Zhen, Zhang Yukui, Liu Yu, Zhao Qun, Zhang Lihua
State Key Laboratory of Medical Proteomics, National Chromatographic R. & A. Center, CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Anal Chem. 2024 Dec 10;96(49):19778-19786. doi: 10.1021/acs.analchem.4c05071. Epub 2024 Nov 27.
Protein aggregation is a hallmark of numerous degenerative diseases, yet its underlying mechanisms remain poorly understood due to the challenges in identifying the composition and interaction networks of these aggregates. To address this issue, we developed TAggiXL, a novel method that combines fluorescence-traceable aggregate isolation with cross-linking proteomics, significantly enhancing the efficiency and precision of isolating protein aggregates. This method facilitates unbiased profiling of aggregated proteomes and their interactomes in live cells. The TAggiXL approach leverages advanced cross-linking proteomics, density gradient centrifugation, and fluorescence tracking to provide detailed characterization of protein aggregation under various stress conditions including HSP90 and proteasome inhibition. Using TAggiXL, we identified key components and interactions within the aggregates, particularly highlighting E3 ubiquitin ligase TRIM26, which plays a crucial role in aggregate formation and autophagic clearance under stress and pathogenic conditions. Moreover, TAggiXL revealed that HSPA1B functions as a central interaction hub within the aggregated proteome. It preferentially interacts with intrinsically disordered regions (IDRs) of aggregate components and demonstrates dynamic behavior within the aggregate. In summary, TAggiXL offers a powerful tool for dissecting the complex composition and interaction networks of protein aggregates, with a significant potential to advance our understanding of protein aggregation in degenerative diseases. It also holds promise for the development of future therapeutic interventions.
蛋白质聚集是许多退行性疾病的一个标志，然而由于在识别这些聚集体的组成和相互作用网络方面存在挑战，其潜在机制仍知之甚少。为了解决这个问题，我们开发了TAggiXL，这是一种将荧光可追踪聚集体分离与交联蛋白质组学相结合的新方法，显著提高了分离蛋白质聚集体的效率和精度。该方法有助于在活细胞中对聚集蛋白质组及其相互作用组进行无偏分析。TAggiXL方法利用先进的交联蛋白质组学、密度梯度离心和荧光追踪，对包括HSP90和蛋白酶体抑制在内的各种应激条件下的蛋白质聚集进行详细表征。使用TAggiXL，我们确定了聚集体中的关键成分和相互作用，特别突出了E3泛素连接酶TRIM26，它在应激和致病条件下的聚集体形成和自噬清除中起关键作用。此外，TAggiXL揭示HSPA1B在聚集蛋白质组中作为一个中心相互作用枢纽发挥作用。它优先与聚集成分的内在无序区域（IDR）相互作用，并在聚集体中表现出动态行为。总之，TAggiXL为剖析蛋白质聚集体的复杂组成和相互作用网络提供了一个强大的工具，在推进我们对退行性疾病中蛋白质聚集的理解方面具有巨大潜力。它也为未来治疗干预的发展带来了希望。
