Chemical Approaches to Study Protein Post-Translational Modifications

总结 细胞中的大多数生物学事件在一定水平上由蛋白质翻译后修饰介导。为 例如，由激酶和磷酸酶催化的异常蛋白质磷酸化与蛋白质磷酸化有关。 各种各样的癌症类似地，由组蛋白控制的组蛋白蛋白的不受调节的乙酰化状态， 脱乙酰基酶（HDAC）蛋白可导致转录中的表观遗传变化并最终导致疾病。关键 描述健康和疾病状态的关键是对蛋白质作用的详细分子理解， 翻译后修饰，如磷酸化和乙酰化，对蛋白质功能的影响， 交互.重要的是，调节翻译后修饰的酶，包括激酶， 磷酸酶和HDAC蛋白是药物治疗的靶点。然而，将蛋白质修饰与 下游生物活动往往有限或不可用，这阻碍了疾病的进展， 表征和药物开发。 Pflum实验室的NIGMS资助项目解决了开发创新化学品的迫切需求 发现蛋白质修饰酶在细胞生物学中的意外功能的方法。在我们的工作中 在蛋白磷酸化方面，我们在过去10年中率先使用ATP类似物进行激酶- 催化标记反应。在此之前的工作的基础上，我们建议在未来5年内：1）开发一个 一套新的方法，具有独特的能力，探测激酶和磷酸酶底物对和多个， 细胞中的蛋白质复合物，2）将我们的创新工具应用于各种生物学问题， 与多位生物学家合作。在我们对蛋白质乙酰化的研究中，我们在最后一个实验中证明了 两年来，使用捕获突变体发现HDAC 1的非组蛋白底物的能力， 揭示了HDAC 1蛋白在细胞生物学中的意想不到的作用。在未来的5年里，我们将运用这一强大的 捕获策略，以额外的HDAC蛋白亚型，这将建立HDAC蛋白的作用， 超越表观遗传学和转录调控的活动。鉴于激酶、磷酸酶的关键作用， 和HDAC酶在疾病和药物治疗中的作用，但研究这些酶的工具不足， 酶在细胞系统中，使化学策略提出了这项申请将加强 细胞信号传导和药物设计的生物医学研究。

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 and phosphatase 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, phosphatase, 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 use of ATP analogs for kinase- catalyzed labeling reactions. Building on this prior work, we propose in the next 5 years to 1) develop a new suite of methods with unique abilities to probe kinase- and phosphatase-substrate pairs and multi- protein complexes in cells, and 2) apply our innovative tools to a variety of biological problems in collaboration with multiple biologists. In our work with protein acetylation, we have demonstrated in the last two 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 the next 5 years, we will apply this powerful trapping strategy to additional HDAC protein isoforms, which will establish the role of HDAC proteins in activities beyond epigenetics and transcriptional regulation. Given the critical role of kinase, phosphatase, and HDAC enzymes in disease and drug treatment, yet the inadequate tools available to study these enzymes in cellular systems, the enabling chemical strategies proposed in this application will strengthen biomedical research in cell signaling and drug design.