Proteomics of the Proteasome Interacting Networks

亨廷顿病（HD）是目前最具破坏性的神经退行性疾病（ND）之一， 缺乏有效的治疗方法。由突变亨廷顿蛋白（Htt）的毒性聚集引起， 长聚谷氨酰胺（polyQ）重复序列，它们通过蛋白酶体降解的及时去除对于 延缓疾病的发作。鉴于其作为负责退化的中央机器的关键作用， 受损和错误折叠的蛋白质，如Htt和其他易于聚集的蛋白质，26 S蛋白酶体 损伤已被认为是与神经病理学相关的ND的标志之一。虽然它有 有人认为蛋白质聚集体可以诱导26 S的构象变化以降低其功能， 通过磷酸化激活蛋白酶体似乎增强了Htt突变体的去除。然而，在这方面， HD中蛋白酶体抑制和激活的分子细节仍不清楚。解决这些 由于未知因素，必须定量评估Htt聚集和磷酸化依赖性 细胞中26 S的构象，以获得对26 S的结构-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，这种调查以前一直没有进行过。 战略布局在当前的资助周期中，我们已经证明了交联质谱法（XL-100） MS）对于研究蛋白酶体复合物的体内结构动力学是有效的。尽管制订 特异性残基靶向MS可切割交联剂进一步提高了我们绘制蛋白质-蛋白质图谱的能力 由于缺乏靶向相互作用（PPI），疏水区域的相互作用仍然难以表征， 残基因此，有必要探索用于捕获那些结构细节的替代化学。 区域，以便全面剖析细胞中蛋白酶体构象动力学。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绘图 和体外培养。此外，我们打算开发集成的QXL-MS平台，以定义 26 S蛋白酶体对Htt聚集和磷酸化的影响，产生分子细节来描绘 HD受损蛋白酶体结构-功能关系这个项目不仅代表了一个巨大的飞跃， XL-MS技术，而且还有助于解决与HD相关的重要但尚未解决的生物学问题 这些对于未来的治疗开发具有巨大的潜力。

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.