Chemoenzymatic Protein Semisynthesis Approaches Toward Cell Signaling Enzymes

摘要 蛋白质磷酸化通过动态平衡在许多细胞过程中发挥关键作用 蛋白激酶和磷酸酶。在许多疾病情况下，这种平衡倾向于激酶。 过度激活和/或磷酸酶失活与其翻译后的变化高度相关 修改(PTM)。能够在这样的特定信号酶上引入多个PTM 化学上定义明确的方式对理解细胞信号通路和 PTMS的功能。在以前的研究中，通过使用蛋白质半合成方法来生成 均一蛋白激酶Akt在Ser473上形成底物或按化学计量比磷酸化 校准标准，我们提供了mTOR复合体2(MTORC2)的详细描述，但没有提供mTORC1 可以选择性地识别和磷酸化Akt Ser473来激活这个关键的信号通路。在这份提案中， 我们继续开发和利用蛋白质半合成方法来阐明两个关键蛋白的调节 包括蛋白激酶S6K1和异三聚体磷酸酶PP2A在内的信号转导酶 其他变构机制。S6K1是mTORC1的关键下游效应器，受 C末端一簇Ser/Thr残基(Ser411、Ser418、Thr421和Ser424)的磷酸化 自身抑制域(CTD)。然而，CTD磷酸化如何调节S6K1的结构和功能 定义不明确。PP2A磷酸酶异三聚体与绝大多数丝氨酸/苏氨酸有关 真核细胞中的磷酸酶活性及其组装与C-末端PTMS的变化有关 C亚基包括Thr304和Tyr307的磷酸化和Leu309的甲基化。但是，该函数 其中的PTM尚未得到充分的表征，仍是该领域的一大研究热点。它也是非常少的 已知PP2A如何招募其底物，限制了我们对PP2A调节的信号转导的理解。我们会 产生这两种含有位点特异的PTM及其不可水解类似物的酶，并将 整合动力学分析、结构分析、结合测量和基于细胞的研究，以澄清 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，该方法使用 纳米体作邻近导向剂分析不同细胞条件下的PP2A相互作用组 对各种刺激的反应。该项目的成功完成不仅将提供详细的分子 了解这两种信号酶如何由特定的PTM调节，但启发新的治疗方法 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.