Chemical Approaches to Study Protein Post-Translational Modifications

摘要 细胞中的大多数生物事件在一定程度上是由蛋白质翻译后修饰介导的。为 例如，由激酶和磷酸酶催化的异常蛋白磷酸化与一种 癌症种类繁多。同样，由组蛋白控制的组蛋白未受调控的乙酰化状态 脱乙酰酶(HDAC)蛋白，可导致转录的表观遗传变化，最终导致疾病。钥匙 对健康和疾病状态的描述是对蛋白质作用的详细的分子理解 翻译后修饰，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， 磷酸酶和HDAC蛋白是药物治疗的靶点。然而，将蛋白质修饰与 下游的生物活动往往有限或无法获得，这阻碍了疾病的进展。 表征和药物开发。 NIGMS资助的Pflum实验室项目满足了开发创新化学品的迫切需要 在细胞生物学中发现蛋白质修饰酶未预料到的功能的方法。在工作中 通过蛋白质磷酸化，我们在过去10年中率先使用ATP类似物来代替激酶- 催化标记反应。在之前工作的基础上，我们建议在未来5年内1)开发一种 一套具有独特能力的新方法来探测激酶-和磷酸酶-底物对和多个 细胞中的蛋白质复合体，以及2)将我们的创新工具应用于各种生物问题 与多位生物学家合作。在我们对蛋白质乙酰化的研究中，我们在上一篇文章中展示了 两年来，使用诱捕突变体发现HDAC1的非组蛋白底物的能力，它已经 揭示了HDAC1蛋白在细胞生物学中意想不到的作用。在未来5年，我们将应用这一强大的 其他HDAC蛋白亚型的捕获策略，这将确定HDAC蛋白在 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，但可用于研究这些的工具不足 细胞系统中的酶，本申请中提出的使能化学策略将加强 细胞信号和药物设计方面的生物医学研究。

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.