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

化学蛋白质组学和基因组学的综合质谱技术 研究动态激酶相互作用组的方法 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前体， 包括细胞信号传导和转录;破坏性疾病重新连接PPI和PDI网络， 疾病进展、治疗抗性和免疫逃逸。映射动态交互的新方法 因此，迫切需要建立网络，以确定疾病机制和药物靶点。蛋白激酶是 大多数细胞PPI和PDI网络中的关键调节节点，通常在疾病中失调，并且在疾病中高度表达。 合成的ATP竞争性抑制剂相应地了解疾病如何利用激酶 重新连接PPI和PDI网络对于防治许多疾病极为重要。定量研究的进展 质谱（MS）已经彻底改变了蛋白质组学，然而，系统的，灵敏的， 缺乏激酶PPI、基因座特异性PDI及其动力学的高通量分析。我们将开发 将联合收割机可渗透细胞的亲和探针与化学交联和邻近结合起来的变革性方法 标记以原位全局编码激酶相互作用组，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、器官纤维化以及治疗和免疫有重要贡献。 逃避各种疾病。为了确定对抗这些疾病表型的药物靶点，我们迫切地 需要了解细胞可塑性的信号和转录网络。我们对病理学的研究- 逻辑激酶组重新布线将约20%的人类激酶与细胞可塑性联系起来，其中许多尚待研究 激酶。我们发现，70%的激酶定位于细胞核，并与转录因子相互作用， 染色质重塑。我们还发现，细胞可塑性动态地改变了翻译后修饰， 这些激酶的功能（PTM）和PPI。可塑性通路如何协调PTM，PPI和 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是未知的， 关键的分子机制和药物靶点尚未探索。我们将为研究开发简化的工作流程- 利用核激酶动力学，将kinobead/LC-MS激酶组分析与全局蛋白质组学、表观基因组学相结合， 和转录组学分析，以及我们新颖的相互作用平台，并应用这些工作流程来揭示 可塑性途径在细胞去分化和转分化期间时空控制激酶。 总之，我们的计划旨在开发新的生物分析方法和工作流程， 研究动态激酶相互作用组，并阐明病理细胞可塑性的机制。追求 我们的目标，我们创建了一个雄心勃勃的，严格的，富有成效的研究计划，促进包容性， 创造力，培养蛋白质组学，细胞信号和化学生物学的下一代科学领导者。

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.