Chemoenzymatic Protein Semisynthesis Approaches Toward Cell Signaling Enzymes

摘要 蛋白质磷酸化在许多细胞过程中起着关键作用，通过蛋白质之间的动态平衡， 蛋白激酶和磷酸酶。在许多疾病中，这种平衡倾向于激酶 磷酸酶失活和/或过度活化，这与它们的翻译后表达的变化高度相关。 修改（PTM）。能够将多个PTM引入到这样的特异性信号传导酶中， 化学定义明确的方式对于理解细胞信号传导途径既有影响力又具有变革性， PTM的功能。在以往的研究中，通过采用蛋白质半合成的方法， 同源蛋白激酶Akt作为底物形成或在Ser 473处化学计量磷酸化， 校准标准，我们提供了mTOR复合体2（mTORC 2）而不是mTORC 1的详细描述 可以选择性识别并磷酸化Akt Ser 473，从而激活这一关键信号激酶。在这一提议中， 我们继续开发和利用蛋白质半合成方法来阐明两个关键的调节， 信号传导酶，包括蛋白激酶S6 K1和异源三聚体磷酸酶PP 2A，通过PTM， 其他变构机制。S6 K1是mTORC 1的关键下游效应子，并且受到 C-末端的一簇Ser/Thr残基（Ser 411、Ser 418、Thr 421和Ser 424）的磷酸化 自身抑制结构域（CTD）。然而，CTD磷酸化如何调节S6 K1的结构和功能， 定义不好。PP 2A磷酸酶异源三聚体负责绝大多数Ser/Thr 在真核细胞中磷酸酶活性，其组装与C-末端PTM的变化有关 包括Thr 304和Tyr 307磷酸化和Leu 309甲基化。然而，函数 这些PTM的特征尚未完全确定，并且仍然是该领域的一大兴趣。也是很少的 已知PP 2A如何招募其底物，限制了我们对PP 2A调节信号的理解。我们将 产生这两种含有位点特异性PTM及其不可水解类似物的酶， 整合动力学分析、结构分析、结合测量和基于细胞的研究，以阐明 结构和催化特征。此外，我们还开发了一种新的邻近交联方法， 纳米抗体作为邻近导向剂用于分析不同细胞条件下的PP 2A相互作用组， 对各种刺激的反应。这个项目的成功完成不仅会提供详细的分子 了解这两种信号传导酶如何受特定PTM调节，但激发了新的治疗方法 战略与与其失调相关的疾病作斗争。

ABSTRACT Protein phosphorylation plays a key role in numerous cellular processes through a dynamic balance between protein kinases and phosphatases. In many disease conditions, this balance is inclined toward kinase hyperactivation and/or phosphatase inactivation, which are highly related to changes of their posttranslational modifications (PTMs). Being able to introduce multiple PTMs on such specific signaling enzymes in a chemically well-defined manner is both impactful and transformative to understand cell signaling pathways and the function of PTMs. In previous study, by employing protein semisynthesis methods to generate homogenous protein kinase Akt forms as substrates or stoichiometrically phosphorylated at Ser473 as calibration standards, we provided a detailed portrait of how mTOR Complex 2 (mTORC2) but not mTORC1 can selectively recognize and phosphorylate Akt Ser473 to activate this key signaling kinase. In this proposal, we continue to develop and utilize protein semisynthesis methods to elucidate the regulation of two key signaling enzymes including protein kinase S6K1 and the heterotrimeric phosphatase PP2A by PTMs and other allosteric mechanisms. S6K1 is a crucial downstream effector of mTORC1, and is critically regulated by the phosphorylation of a cluster of Ser/Thr residues (Ser411, Ser418, Thr421 and Ser424) in the C-terminal autoinhibitory domain (CTD). Yet how the CTD phosphorylation modulates S6K1 structure and function has been poorly defined. The PP2A phosphatase heterotrimer is responsible to the vast majority Ser/Thr phosphatase activity in eukaryotic cells, and its assembly has been linked to changes in the C-terminal PTMs of the C subunit including Thr304 and Tyr307 phosphorylations and Leu309 methylation. However, the function of these PTMs has yet to be fully characterized, and remain a great of interest in the field. It is also very little known how PP2A recruits its substrates, limiting our understanding of PP2A-regulated signaling. We will produce these two enzymes containing site-specific PTMs and their non-hydrolyzable analogs, and will integrate kinetic assays, structural analysis, binding measurements, and cell-based studies to clarify the structural and catalytic features. Moreover, we have developed a novel proximity crosslinking method using nanobodies as proximity-directing agents for analyzing PP2A interactome in different cellular conditions in response to various stimuli. Successful completion of this project will not only provide a detailed molecular understanding of how these two signaling enzymes regulated by specific PTMs, but inspire novel therapeutic strategies combat the diseases associated with their dysregulation.