Proteomics of the Proteasome Interacting Networks

 描述（由申请人提供）：蛋白质-蛋白质相互作用（PPI）在定义生物系统中的蛋白质功能方面发挥关键作用。异常PPI可对基本细胞过程产生剧烈影响，从而导致各种人类疾病。PPI接口最近已成为治疗学的新范例。因此，在活细胞中绘制PPI及其相互作用界面不仅是理解蛋白质功能和调控的基础，而且对于确定更好治疗的潜在靶点也至关重要。以前，我们已经开发了一种称为QTAX的集成方法，以有效地捕获和识别静态和动态蛋白质相互作用，以便提供更真实的蛋白质相互作用网络快照，因为它们存在于活细胞中。为了推进体内PPI的研究超越相互作用身份，我们成功开发了一种基于新型膜渗透性、可富集和MS可裂解交联剂（即叠氮化物-A-DSBSO）和多级串联质谱（MSn）的体内交联质谱（XL-MS）平台。该策略已成功应用于蛋白质组规模和蛋白质复合物水平的PPI图谱。这一技术进步标志着研究体内PPI的一个飞跃，并为我们进一步开发新技术提供了一个必要的框架，以实现在系统水平上定义蛋白质复合物体内动力学的最终目标。26 S蛋白酶体是负责泛素/ATP依赖性蛋白质降解的蛋白质复合物。蛋白酶体降解的失调与许多疾病有关。到目前为止，26 S蛋白酶体的高分辨率结构仍然没有得到解决，其调控机制也很不清楚。因此，对人26 S蛋白酶体复合物的详细结构分析对于揭示其功能和调控的分子细节至关重要。为此，我们建议：1）开发下一代多功能交联剂，用于定义活细胞中蛋白质复合物的PPI; 2）开发定量XL-MS策略，用于绘制H2 O2诱导的氧化应激后人26 S蛋白酶体的体内动力学; 3）剖析人26 S蛋白酶体及其相关去泛素化酶的相互作用动力学。拟议的实验不仅将导致蛋白质组学研究的令人兴奋的技术进步，而且还有助于解决与蛋白酶体生物学及其与人类疾病相关的重要但尚未解决的生物学问题。

 DESCRIPTION (provided by applicant): Protein-protein interactions (PPIs) play a key role in defining protein functions in biological systems. Aberrant PPIs can have drastic impacts on basic cellular processes, and thus lead to various human diseases. PPI interfaces have recently become a new paradigm for therapeutics. Therefore, mapping PPIs and their interaction interfaces in living cells is not only fundamental to understanding protein function and regulation but also vital to identifying potential targets for better therapeutics. Previously we have developed an integrated approach termed QTAX to effectively capture and identify static and dynamic protein interactions in order to provide a more authentic snapshot of protein interaction networks as they exist in living cells. To advance the study of in vivo PPIs beyond interaction identities, we have successfully developed an in vivo cross-linking mass spectrometry (XL-MS) platform based on a novel membrane permeable, enrichable and MS-cleavable cross-linker, i.e. Azide-A-DSBSO, and multistage tandem mass spectrometry (MSn). This strategy has been successfully applied to map PPIs at the proteome scale and the protein complex level. This technological advancement signifies a leap forward in studying in vivo PPIs and offers an essential framework for us to further develop novel technologies towards the ultimate goal of defining in vivo dynamics of protein complexes at the systems level. The 26S proteasome is the protein complex responsible for ubiquitin/ATP dependent protein degradation. Dysregulation of proteasomal degradation has been implicated in many diseases. Up to now, the high-resolution structure of the 26S proteasome remains unresolved and its regulation is poorly defined. Therefore, detailed structural analysis of the human 26S proteasome complex will be critical to uncover molecular details underlying its function and regulation. To this end, we propose 1) to develop the next generation of multifunctional cross-linkers for defining PPIs of protein complexes in living cells, 2) to develop a quantitative XL-MS strategy for charting in vivo dynamics of the human 26S proteasome upon H2O2-induced oxidative stress, 3) to dissect the interaction dynamics of the human 26S proteasome and its associated deubiquitinases. The proposed experiments will not only result in an exciting technological advancement in proteomics research, but also help address important yet unresolved biological questions associated with proteasomal biology and its relevance to human diseases.