Chemoenzymatic Protein Semisynthesis Approaches Toward Cell Signaling Enzymes

细胞信号酶的化学酶蛋白半合成方法

• 批准号: 10713633
• 负责人: Nam Ky Chu
• 金额: $ 39.38万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2028-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10713633
• 关键词: Binding; Biological Assay; C-terminal; Calibration; Cell physiology; Cells; Chemicals; Complex; Diabetes Mellitus; Disease; Enzymes; Equilibrium; Eukaryotic Cell; FRAP1 gene; Goals; Kinetics; Link; Malignant Neoplasms; Measurement; Methods; Methylation; Molecular; Neurodegenerative Disorders; Obesity; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Portraits; Post-Translational Protein Processing; Protein Kinase; Protein Phosphatase 2A Regulatory Subunit PR53; Protein phosphatase; Proteins; Regulation; Signal Pathway; Signal Transduction; Site; Stimulus; Structure; analog; combat; crosslink; human disease; insight; interest; nanobodies; novel; novel therapeutic intervention; recruit; response

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.

抽象的 蛋白质磷酸化通过之间的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，这与其翻译后的变化高度相关 修饰（PTM）。能够在此类特定信号酶上引入多个 PTM 化学上明确定义的方式对于理解细胞信号传导途径和 PTM 的功能。在之前的研究中，通过采用蛋白质半合成方法来生成 同质蛋白激酶 Akt 形成为底物或在 Ser473 处按化学计量磷酸化为 校准标准，我们提供了 mTOR Complex 2 (mTORC2) 而不是 mTORC1 的详细描述 可以选择性识别并磷酸化 Akt Ser473 以激活这一关键信号激酶。在这个提案中， 我们继续开发和利用蛋白质半合成方法来阐明两个关键的调控 信号酶，包括蛋白激酶 S6K1 和异源三聚磷酸酶 PP2A 通过 PTM 和 其他变构机制。 S6K1 是 mTORC1 的重要下游效应子，并受到重要调控 C 端一簇 Ser/Thr 残基（Ser411、Ser418、Thr421 和 Ser424）的磷酸化 自抑制结构域（CTD）。然而，CTD 磷酸化如何调节 S6K1 的结构和功能尚不清楚。 定义不明确。 PP2A 磷酸酶异三聚体负责绝大多数 Ser/Thr 真核细胞中的磷酸酶活性及其组装与 C 端 PTM 的变化有关 C 亚基的结构包括 Thr304 和 Tyr307 磷酸化以及 Leu309 甲基化。然而，该函数 其中一些 PTM 尚未得到充分表征，并且仍然引起该领域的极大兴趣。数量也很少 已知 PP2A 如何招募其底物，限制了我们对 PP2A 调节信号传导的理解。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，并将 整合动力学测定、结构分析、结合测量和基于细胞的研究来阐明 结构和催化特征。此外，我们还开发了一种新颖的邻近交联方法 纳米抗体作为邻近导向剂，用于分析不同细胞条件下的 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受特定 PTM 调节，但激发新的治疗方法 策略对抗与其失调相关的疾病。