Proteomics of the Proteasome Interacting Networks

亨廷顿病(HD)是最具破坏性的神经退行性疾病(NDS)之一 缺乏有效的治疗方法。由携带异常的亨廷顿(Htt)突变蛋白的毒性聚集引起 长多聚谷氨酰胺(PolyQ)重复，它们通过蛋白酶体降解及时移除是至关重要的 延缓疾病的发病。鉴于其作为负责环境退化的中央机器的关键角色 受损和错误折叠的蛋白质，如Htt和其他易于聚集的蛋白质、26S蛋白酶体 损伤已被认为是与神经病理相关的NDS的特征之一。虽然它已经 有人认为蛋白质聚集体可以诱导26S的构象变化，从而降低其功能， 通过磷酸化激活蛋白酶体似乎可以增强Htt突变体的去除。然而， HD中蛋白酶体抑制和激活的分子细节仍不清楚。要解决这些问题 未知因素，有必要定量评估Htt聚集和磷酸化依赖 26S在细胞中的构象，以获得对结构-功能关系的机械理解 HD相关蛋白酶体。由于缺乏适当的调查，这类调查以前一直没有被探索过 战略。在当前的资金周期中，我们已经证明了交联质谱学(XL- MS)是研究蛋白酶体复合体体内结构动力学的有效方法。在发展的同时， 针对特定残基的MS可裂解交联剂进一步提高了我们绘制蛋白质-蛋白质图谱的能力 相互作用(PPI)，由于缺乏针对性，疏水区域的相互作用仍然难以表征 残留物。因此，有必要探索替代化学方法来捕捉这些物质的结构细节。 区域，以便全面剖析细胞中蛋白酶体的构象动力学。在这里，我们的目标是 开发基于光化学的XL-MS平台，使其能够应用于体内复杂的PPI图谱 而且是在体外。此外，我们打算开发集成的QXL-MS平台来定义 26S蛋白酶体对Htt聚集和磷酸化，产生分子细节来描述 HD受损蛋白酶体的结构-功能关系。这个项目不仅代表着 XL-MS技术，但也有助于解决与HD相关的重要但尚未解决的生物学问题 在未来的治疗开发中有很大的潜力。

Huntington’s disease (HD) is one of the most devastating neurodegenerative disorders (NDs) that currently lacks effective therapies. Caused by toxic aggregation of mutant Huntingtin (Htt) proteins carrying abnormally long polyglutamine (polyQ) repeats, their timely removal through proteasomal degradation is critical for delaying onset of the disease. Given its crucial role as the central machine responsible for degradation of damaged and misfolded proteins such as Htt and other aggregation-prone proteins, 26S proteasome impairment has been recognized as one of the hallmarks of NDs associated with neuropathology. While it has been suggested that protein aggregates can induce conformational changes in the 26S to reduce its function, activation of proteasomes through phosphorylation appears to enhance the removal of Htt mutants. However, the molecular details underlying proteasome inhibition and activation in HD remain unclear. To address these unknowns, it is essential to quantitatively assess Htt aggregation and phosphorylation-dependent conformations of the 26S in cells to obtain a mechanistic understanding of the structure-function relationship of HD-associated proteasomes. Such investigations have remained previously unexplored due to lack of proper strategies. During the current funding cycle, we have demonstrated that cross-linking mass spectrometry (XL- MS) is effective for studying in vivo structural dynamics of proteasome complexes. While the development of specific residue-targeting MS-cleavable cross-linkers has further improved our capability to map protein-protein interactions (PPIs), interactions at hydrophobic regions remain difficult to characterize due to lack of targetable residues. Therefore, it is necessary to explore alternative chemistries for capturing structural details in those regions in order to comprehensively dissect proteasome conformational dynamics in cells. Here, we aim to develop photochemistry-based XL-MS platforms to enable their application for complex PPI mapping in vivo and in vitro. In addition, we intend to develop integrated QXL-MS platforms to define the temporal dynamics of the 26S proteasome upon Htt aggregation and phosphorylation, yielding molecular details to delineate the structure-function relationship of HD-impaired proteasomes. This project not only represents a great leap in XL-MS technology, but also helps address important yet unresolved biological questions associated with HD that have great potential for future therapeutic exploitation.