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.

程序摘要：基于质谱的综合化学蛋白质组和基因组技术 用于研究动态激酶相互作用的基因 蛋白质 - 蛋白质和蛋白-DNA相互作用（PPI和PDI）的动态变化控制了大多数细胞促进 塞斯，包括细胞信号传导和转录；破坏性疾病雷波威PPI和PDI网络驱动 疾病进展，耐药性和免疫逃生。映射动态互动的新方法 因此，迫切需要网络来识别疾病机制和药物靶标。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，并且高度高 可用于合成，竞争抑制剂的药物。因此。了解疾病如何利用激酶 REWIRE PPI和PDI网络对于打击许多疾病至关重要。定量的进步 质谱法（MS）彻底改变了蛋白质组学，但对于系统，敏感和 缺乏激酶PPI，基因座特异性PDI及其动态的高通量分析。我们将发展 结合细胞可渗透亲和力探针与化学交联和接近性的变革性方法 将其标记到全球编码激酶相互作用的原位，然后进行集成的LC-MS和测序分析。 细胞塑性驱动生理和病理学的脱节以及谱系转换，以及谱系切换， 严重促进发育，组织修复，癌症转移，器官纤维化以及治疗和免疫 在许多疾病中逃脱。为了识别与这些疾病表型打击的药物目标，我们紧迫 需要了解基础细胞塑性的信号传导和转录网络。我们对病理的研究 逻辑激素重新布线将〜20％的人类激酶与细胞可塑性联系起来，其中许多研究了 激酶。我们发现70％的这些激酶本地定位于细胞核，并与转录因子相互作用， 染色质重塑。我们还发现，细胞可塑性在动态上改变了翻译后的修饰 - 这些激酶的tions（PTMS）和PPI。塑性途径如何协调PTM，PPI和PPI的动态变化 但是，PDI网络系统地改变染色质状态和转录，但在很大程度上未知，leav- 未经探索的关键分子机制和药物靶标。我们将开发简化的工作流程以进行研究 Ying核激酶动力学，将Kinobead/LC-MS Kinome分析与全球蛋白质组学，表观基因组学， 和转录组学分析以及我们的新颖的相互作用平台，并应用这些工作流程来解开如何 可塑性途径在细胞脱离和转分化过程中的时空控制激酶。 总而言之，我们的计划试图开发新颖的生物分析方法和工作流程 研究动态激酶相互作用，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，严格且富有成效的研究计划，促进了包容性和 创造力，培训蛋白质组学，细胞信号和化学生物学的下一代科学领导者。