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

基于集成质谱的化学蛋白质组学和基因组技术，用于研究动态激酶相互作用组

• 批准号: 10714921
• 负责人: Martin Golkowski
• 金额: $ 38.5万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10714921
• 关键词: Affinity; Automobile Driving; Biological Process; Biology; Cell Nucleus; Cell physiology; Cells; Chemicals; Chromatin; Creativeness; DNA-Protein Interaction; Development; Disease; Disease Progression; Drug Targeting; Drug resistance; Fibrosis; Fostering; Genetic Transcription; Genomics; Goals; Human; Immune; In Situ; Label; Link; Maps; Mass Spectrum Analysis; Methods; Molecular; Neoplasm Metastasis; Nuclear; Organ; Pathologic; Pathologic Processes; Pathway Analysis; Pathway interactions; Permeability; Phosphotransferases; Physiological; Post-Translational Protein Processing; Productivity; Protein Kinase; Proteins; Proteomics; Research; Resistance; Signal Transduction; Technology; Training; Virus Diseases; chemoproteomics; crosslink; disease phenotype; epigenomics; human disease; inhibitor; insight; next generation; novel; programs; spatiotemporal; tissue repair; transcription factor; transcriptomics; transdifferentiation; virtual

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.

项目摘要：基于集成质谱的化学蛋白质组学和基因组技术 研究动态激酶相互作用组的基金 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 过程，包括细胞信号传导和转录；毁灭性疾病重新连接 PPI 和 PDI 网络以推动 疾病进展、治疗抵抗和免疫逃逸。映射动态交互的新方法 因此，迫切需要网络来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在疾病中常常失调，并且高度 可与合成的 ATP 竞争性抑制剂一起使用。因此。深入了解疾病如何利用激酶 重新连接 PPI 和 PDI 网络对于对抗许多疾病极为相关。定量方面的进展 质谱 (MS) 彻底改变了蛋白质组学，但系统性、灵敏性和定量分析的简便方法 缺乏激酶 PPI、位点特异性 PDI 及其动力学的高通量分析。我们将开发 将细胞渗透性亲和探针与化学交联和邻近相结合的变革方法 标记以原位全局编码激酶相互作用组，然后进行集成 LC-MS 和测序分析。 细胞可塑性驱动生理和病理的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做出重要贡献 逃避许多疾病。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背后的信号传导和转录网络。我们对病理学的研究 逻辑激酶组重连将约 20% 的人类激酶与细胞可塑性联系起来，其中许多尚未得到充分研究 激酶。我们发现 70% 这些激酶定位于细胞核并与转录因子和 染色质重塑者。我们还发现细胞可塑性动态地改变翻译后修饰 这些激酶的 PTM 和 PPI。可塑性途径如何协调 PTM、PPI 和 然而，系统地改变染色质状态和转录的 PDI 网络仍然很大程度上未知， 关键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 ying 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组学平台，并应用这些工作流程来揭示如何 可塑性途径在细胞去分化和转分化过程中时空控制激酶。 总而言之，我们的计划旨在开发新颖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组，并阐明病理细胞可塑性的机制。追求 为了实现我们的目标，我们创建了一个雄心勃勃、严谨且富有成效的研究计划，以促进包容性和 创造力，培训蛋白质组学、细胞信号传导和化学生物学领域的下一代科学领导者。