Integrated mass spectrometry-based chemoproteomic and genomic technologies for studying dynamic kinase interactomes

程序摘要：基于集成质谱学的化学蛋白质组和基因组技术。 研究动态蛋白激酶相互作用的GES 蛋白质-蛋白质和蛋白质-DNA相互作用(PPI和PDI)的动态变化控制着大多数细胞亲和性。 CESS，包括细胞信号和转录；毁灭性的疾病重新连接PPI和PDI网络以驱动 疾病进展、治疗抵抗和免疫逃避。动态交互映射的新方法 因此，迫切需要网络来确定疾病机制和药物目标。蛋白激酶是 在大多数细胞PPI和PDI网络中的关键调控节点，在疾病中通常是不受调控的，并且高度 可与合成的ATP竞争性抑制剂一起下药。相应地。洞察疾病如何利用激酶来 重新布线PPI和PDI网络与抗击许多疾病极其相关。定量技术的研究进展 质谱学(MS)使蛋白质组学发生了革命性的变化，然而，对于系统、灵敏和 目前尚缺乏高通量的蛋白激酶PPI、位点特异性PDI及其动力学研究。我们将发展 将细胞透过性亲和探针与化学交联和邻近性相结合的变革性方法 原位标记以全局编码激酶相互作用，随后进行整合的LC-MS和测序分析。 细胞可塑性驱动生理和病理去分化和转分化，以及谱系转换， 对发育、组织修复、癌症转移、器官纤维化、治疗和免疫有重要贡献 在许多疾病中逃脱。为了确定对抗这些疾病表型的药物靶点，我们迫切需要 需要了解细胞可塑性不足的信号和转录网络。我们对病理学的研究- 人类约20%的蛋白激酶与细胞可塑性有关，其中许多未被研究。 激活剂。我们发现，70%的这些激酶定位于细胞核，并与转录因子和 染色质改变剂。我们还发现，细胞的可塑性动态地改变了翻译后修饰。 这些激酶的转录因子(PTM)和PPI。可塑性通路如何协调PTM、PPI和PPI的动态变化 然而，系统地改变染色质状态和转录的PDI网络在很大程度上仍然未知。 ING的关键分子机制和药物靶点尚未探索。我们将开发简化的工作流程，以供研究- 与蛋白质组学、表观基因组学、 和转录学分析，以及我们新的交互切除法平台，并应用这些工作流来揭示 在细胞去分化和转分化过程中，可塑性通路在时空上控制着蛋白激酶。 总而言之，我们的计划寻求开发新的生物分析方法和工作流程，以系统地 研究动态激酶相互作用，阐明病理性细胞可塑性的机制。追求 为了实现我们的目标，我们创建了一个雄心勃勃、严谨和富有成效的研究计划，以促进包容性和 创新，培养蛋白质组学、细胞信号和化学生物学方面的下一代科学领军人物。

PROGRAM ABSTRACT: Integrated mass spectrometry-based chemoproteomic and genomic technolo- gies for studying dynamic kinase interactomes Dynamic changes in protein-protein and protein-DNA interactions (PPIs and PDIs) control most cellular pro- cesses, including cell signaling and transcription; devastating diseases rewire PPI and PDI networks to drive disease progression, therapy resistance, and immune escape. Novel methods for mapping dynamic interaction networks are, therefore, urgently required to identify disease mechanisms and drug targets. Protein kinases are critical regulatory nodes in most cellular PPI and PDI networks, are often dysregulated in disease, and are highly druggable with synthetic, ATP-competitive inhibitors. Accordingly. insights into how diseases utilize kinases to rewire PPI and PDI networks are extremely relevant for combating many diseases. Advances in quantitative mass spectrometry (MS) have revolutionized proteomics, yet, facile methods for the systematic, sensitive, and high-throughput profiling of kinase PPIs, locus-specific PDIs, and their dynamics are lacking. We will develop transformative methods that combine cell-permeable affinity probes with chemical crosslinking and proximity labeling to globally encode kinase interactomes in situ, followed by integrated LC-MS and sequencing analyses. Cellular plasticity drives physiological and pathological de- and transdifferentiation, and lineage switching, critically contributing to development, tissue repair, cancer metastasis, organ fibrosis, and therapy and immune escape in numerous diseases. To identify drug targets for combating these disease phenotypes, we pressingly need to understand the signaling and transcriptional network that underly cellular plasticity. Our studies of patho- logical kinome rewiring linked ~20% of human kinases to cellular plasticity, among them numerous understudied kinases. We found that 70% these kinases localize to the nucleus and interact with transcription factors and chromatin remodelers. We also found that cellular plasticity dynamically alters the post-translational modifica- tions (PTMs) and PPIs of these kinases. How plasticity pathways coordinate dynamic changes in PTM, PPI and PDI networks to systematically alter chromatin states and transcription, however, remains largely unknown, leav- ing critical molecular mechanisms and drug targets unexplored. We will develop streamlined workflows for stud- ying nuclear kinase dynamics, combining kinobead/LC-MS kinome profiling with global proteomics, epigenomics, and transcriptomics analyses, and our novel interactomic platforms, and apply these workflows to unravel how plasticity pathways spatiotemporally control kinases during cellular de- and transdifferentiation. To summarize, our program seeks to develop novel bioanalytical methods and workflows to systematically study dynamic kinase interactomes, and to illuminate the mechanisms of pathological cellular plasticity. Pursuing our goals, we created an ambitious, rigorous, and productive research program that fosters inclusiveness and creativity, training the next generation of scientific leaders in proteomics, cell signaling, and chemical biology.