Advancing Proteomics Technologies to Decipher the Ubiquitin-Proteasome System

蛋白质相互作用(PPI)是蛋白质复合体形成的关键，蛋白质复合体是蛋白质的活性成分 负责多种细胞功能。异常的PPI可能会对基本的 并导致各种人类疾病。阐明PPI网络及其结构特征 是理解与人类相关的基本生物学和分子变化的核心 病理，并最终促进治疗的发展。然而，蛋白质组网络的描绘 由于现有方法的局限性，在系统级别定义细胞的功能状态是具有挑战性的。 交联质谱(XL-MS)作为一种强有力的PPI研究技术，由于其独特的优势，已经成为研究PPI的有力工具。 保存和捕获天然环境中蛋白质相互作用的独特能力，以及 用结构细节揭示PPI身份。尽管目前的XL-MS技术在全球 PPI分析，仍然存在未满足的挑战，特别是在完整说明和定量评估 蜂窝网络和空间PPI映射。这就需要新的发展来提高技术水平 推进系统结构生物学的能力。泛素-蛋白酶体系统(UPS)是主要的 真核生物中的降解途径，其网络是非常动态的，由大量的 在组成和结构上都是动态的机器，可以协调蛋白质泛素化和降解。 UPS的失调与许多人类疾病有关，包括癌症和神经退行性变 精神错乱。鉴于蛋白酶体抑制剂在临床上的成功，UPS已成为治疗 药物发现。虽然UPS的基本功能已被了解，但其多层背后的分子细节 监管和机械化的行动仍然难以捉摸。因此，阐明相互作用和结构动力学 对UPS网络的生理环境的了解不仅对于促进对UPS的理解至关重要 生物，但也增强了它们在人类健康和医学上的治疗潜力。在接下来的五年， 我们计划解决PPI研究中的几个突出技术挑战，以更好地破译UPS 通过以下两个研究方向：1)发展用于交互作用的XL-MS技术 和系统水平的结构蛋白质组学；2)绘制UPS网络图以揭示分子细节 潜在的蛋白质泛素化和降解。具体地说，我们将集中精力开发新颖的XL- MS技术，以实现对蛋白质组网络的深入和定量分析，包括结构细节和 增强的空间分辨率可定义细胞的功能状态。此外，我们还将聘用新成立的 阐明蛋白酶体调节因子在蛋白质降解中的作用机制的技术，研究库尔林 环连接酶介导的蛋白质泛素化和解剖UPS网络的组织。加在一起，这些 研究将导致蛋白质组学研究的令人兴奋的技术进步，并有助于回答 与UPS生物学相关的重要但尚未解决的生物学问题。

Protein-protein interactions (PPIs) are key to the formation of protein complexes, the active components responsible for a multitude of cellular functions. Aberrant PPIs can have detrimental effects on essential biological processes and lead to various human diseases. Elucidating PPI networks and their structural features within cells is central to understanding fundamental biology and molecular alterations associated with human pathologies, and ultimately facilitating therapeutic development. However, delineation of proteome networks to define the cell’s functional states at the systems-level is challenging due to limitations in existing approaches. Cross-linking mass spectrometry (XL-MS) has emerged as a powerful technology for PPI studies, owing to its unique capability of preserving and capturing protein interactions in their native environments, as well as uncovering PPI identities with structural details. Although current XL-MS technologies are successful in global PPI analysis, unmet challenges still remain especially for complete illustration and quantitative assessment of cellular networks, and spatial PPI mapping. This necessitates new developments to enhance technical capabilities for advancing systems structural biology. The ubiquitin-proteasome system (UPS) is the major degradation pathway in eukaryotes, whose network is remarkably dynamic and made of a large number of compositionally and structurally dynamic machines that orchestrate protein ubiquitination and degradation. Dysregulation of the UPS has been linked to many human diseases including cancer and neurodegenerative disorders. Given the clinical success of proteasome inhibitors, the UPS has become an effective platform for drug discovery. Although basic functions of the UPS are understood, molecular details underlying its multi-layer regulation and mechanistic action remain elusive. Therefore, elucidating the interaction and structural dynamics of the UPS network in its physiological context is essential not only for advancing the understanding of UPS biology, but also for augmenting their therapeutic potential in human health and medicine. In the next five years, we plan to address several outstanding technological challenges in PPI studies to better decipher the UPS pathways, by pursuing the following two research directions: 1) Advancing XL-MS technologies for interactomics and structural proteomics at the systems-level; 2) Mapping the UPS network to uncover molecular details underlying protein ubiquitination and degradation. Specifically, we will center our efforts on developing novel XL- MS technologies to enable in-depth and quantitative analysis of proteome networks with structural details and enhanced spatial resolution to define cellular functional states. In addition, we will employ the newly established technologies to delineate action mechanisms of proteasome regulators in protein degradation, investigate Cullin- RING Ligase mediated protein ubiquitination and dissect the organization of the UPS network. Together, these studies will result in an exciting technological advancement in proteomics research, and facilitate answering important but unresolved biological questions associated with UPS biology.