Chemical Approaches to Characterizing Protein Post-translational Modifications by Mass Spectrometry

通过质谱表征蛋白质翻译后修饰的化学方法

• 批准号: 10410612
• 负责人: Mary Kay H Pflum
• 金额: $ 5.64万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10410612
• 关键词: Acetylation; Address; Biochemistry; Biological; Biological Models; Cells; Cellular biology; Chemicals; Collaborations; Complex; Disease; Enzymes; Epigenetic Process; Event; Exposure to; Funding; Genetic Transcription; HDAC1 gene; Histone Deacetylase; Histones; Label; Lead; Link; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Methods; Molecular; Multiprotein Complexes; National Institute of General Medical Sciences; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Protein Acetylation; Proteins; Proteomics; Reaction; Role; Scientist; Sickle Cell; Sickle Cell Anemia; Training; Work; analog; base; drug development; innovation; link protein; mutant; next generation; novel therapeutics; pre-doctoral; protein function; targeted treatment; tool

Summary Most biological events in the cell are mediated at some level by protein post-translational modifications. For example, aberrant protein phosphorylation catalyzed by kinase enzymes is linked to a wide variety of cancers. Similarly, the unregulated acetylation state of histone proteins, controlled by histone deacetylase (HDAC) proteins, can lead to epigenetic changes in transcription and ultimately disease. Key to characterizing both healthy and disease states is a detailed molecular understanding of the role of protein post-translational modifications, such as phosphorylation and acetylation, on protein function and interactions. Importantly, enzymes regulating post-translational modifications, including kinase and HDAC proteins, are targets of drug treatment. Yet, tools linking protein modifications to downstream biological activities are often limited or unavailable, which has stalled progress in disease characterization and drug development. The NIGMS-funded projects in the Pflum lab address the critical need to develop innovative chemical approaches to discover unanticipated functions of protein modifying enzymes in cell biology. In our work with protein phosphorylation, we have pioneered in the last 10 years application of ATP analogs in kinase- catalyzed labeling reactions to develop a suite of methods to probe kinase-substrate pairs and multi-protein complexes in cells. This supplemental funding application will support this project by applying our innovative tools to complex biological problems in collaboration with biologists. In our work with protein acetylation, we have demonstrated in the last several years the power of using trapping mutants to discover non-histone substrates of HDAC1, which has revealed unexpected roles of HDAC1 proteins in cell biology. In this supplemental funding application, we will apply this powerful trapping strategy to discover the substrates of HDAC1 in sickle cell anemia model systems, which will reveal unanticipated molecular mechanisms in sickle cell and possible new therapeutics. Importantly, these studies will provide an ideal training ground to expose the pre-doctoral candidate to proteomics-based mass spectrometry, biochemistry, and cell biology to prepare for entry into the biomedical workforce.

概括 细胞中的大多数生物事件在某种程度上是由蛋白质翻译后修饰介导的。为了 例如，激酶催化的异常蛋白质磷酸化与多种 癌症。同样，组蛋白的乙酰化状态不受调控，由组蛋白脱乙酰酶控制 (HDAC) 蛋白可导致转录的表观遗传变化并最终导致疾病。关键 表征健康和疾病状态是对蛋白质作用的详细分子理解 翻译后修饰，例如磷酸化和乙酰化，对蛋白质功能和 互动。重要的是，调节翻译后修饰的酶，包括激酶和 HDAC 蛋白质，是药物治疗的目标。然而，将蛋白质修饰与下游生物联系起来的工具 活动往往受到限制或无法进行，这阻碍了疾病特征和药物方面的进展 发展。 Pflum 实验室 NIGMS 资助的项目解决了开发创新化学品的迫切需求 发现细胞生物学中蛋白质修饰酶的意外功能的方法。在我们的工作中 通过蛋白质磷酸化，我们在过去 10 年中率先将 ATP 类似物应用于激酶- 催化标记反应开发一套探测激酶底物对和多蛋白的方法 细胞内的复合物。该补充资金申请将通过应用我们的创新技术来支持该项目 与生物学家合作解决复杂生物学问题的工具。在我们的蛋白质乙酰化工作中，我们 在过去的几年中已经证明了使用捕获突变体来发现非组蛋白的力量 HDAC1 的底物，揭示了 HDAC1 蛋白在细胞生物学中意想不到的作用。在这个 补充资金申请中，我们将应用这种强大的捕获策略来发现 镰状细胞贫血模型系统中的 HDAC1，这将揭示镰状细胞贫血症中意想不到的分子机制 细胞和可能的新疗法。重要的是，这些研究将为揭露 基于蛋白质组学的质谱、生物化学和细胞生物学的博士前候选人 为进入生物医学队伍做好准备。