Functional Dissection of Metabolic-Sensing Proline Hydroxylation Pathways

项目摘要 脯氨酸羟化(Hyp)是一种基本的翻译后修饰和调节机制， 对细胞代谢条件的变化有高度的反应能力。在肿瘤进展过程中，快速的 癌细胞的增殖创造了一个低氧的微环境，抑制了羟脯氨酸介导的 HIFA蛋白降解和激活缺氧反应细胞通路促进癌细胞存活 在缺氧状态下。除氧外，修饰酶脯氨酸羟基酶也对 铁和关键线粒体代谢物包括琥珀酸、富马酸和α-酮戊二酸的浓度， 使该途径成为细胞中关键的代谢传感器。广泛的研究表明，脯氨酸 羟基化调节蛋白质结构稳定性、蛋白质-蛋白质相互作用或蛋白酶体降解 底物蛋白质。尽管它在细胞生理学中发挥着重要作用，并在靶向分析中取得了成功 该途径的单个底物、系统范围的表征和功能量化 受阻于缺乏有效的工具和策略来定点识别Pro羟化 目标。我们的总体假设和长期目标是系统地描述“脯氨酸羟基组” 通过功能蛋白质组学方法的发展，将导致对 发育和疾病中新的Hyp介导的代谢调节。向着这个目标前进，在过去 多年来，我们已经建立了HypDB，用于Hyp蛋白质组的功能注释分析 开发一种用于系统分析细胞和组织中的Hyp底物的简化工作流程。我们 在Hyp靶标的生物化学表征方面积累了丰富的经验，即特定的 脯氨酸羟基酶及其与其他PTM调节通路的串扰。为了继续努力，我们将 扩展HypDB以量化小鼠组织中的Hyp动力学并开发功能蛋白质组学策略以 确定蛋白质结构稳定性和脯氨酸羟基酶靶标的关键Hyp位点。我们将于近期申请 开发了化学和生物化学策略来研究脯氨酸羟化和 调节底物蛋白质降解和活性的其他代谢感应修饰。此外，我们 将研究一种新的Hyp介导的表观遗传修饰途径在调控中的生理意义 基因表达和染色质活性。总体而言，我们预计该技术的开发和应用 功能蛋白质组学技术用于系统分析脯氨酸羟化蛋白质组将揭示新的 代谢感知途径，并可能导致癌症领域的范式转变概念， 代谢性疾病和发育。

Project Summary Proline hydroxylation (Hyp) is a fundamental posttranslational modification and regulatory mechanism that are highly responsive to the changes in cellular metabolic conditions. During tumor progression, the rapid proliferation of cancer cells creates a hypoxic microenvironment that inhibits the hydroxyproline-mediated degradation of HIFa proteins and activates hypoxia-response cellular pathways to promote cancer cell survival in hypoxia. In addition to oxygen, the modification enzyme prolyl hydroxylases are also sensitive to the concentration of iron and key mitochondria metabolites including succinate, fumarate, and alpha-ketoglutarate, making the pathway a critical metabolic sensor in cells. Extensive studies have demonstrated that proline hydroxylation regulates protein structural stability, protein-protein interactions, or proteasomal degradation of substrate proteins. Despite its important roles in cell physiology and success in the targeted analysis of individual substrates, system-wide characterization and functional quantification of the pathway have been hindered by the lack of effective tools and strategies for site-specific identification of proline hydroxylation targets. Our overall hypothesis and long-term goal is that systematic characterization of “proline hydroxylome” through the development of functional proteomics approaches will lead to the mechanistic understanding of novel Hyp-mediated metabolic regulations in development and diseases. Moving towards this goal, in the past years, we have established HypDB for functional annotation analysis of the Hyp proteome with the development of a streamlined workflow for systematic analysis of the Hyp substrates in cells and tissues. We have gained extensive experience in biochemical characterization of Hyp targets, the interactome of specific prolyl hydroxylase as well as its crosstalk with other PTM regulatory pathways. To continue our effort, we will expand the HypDB to quantify Hyp dynamics in mouse tissues and develop functional proteomics strategies to identify key Hyp sites in protein structural stability and prolyl hydroxylase targets. We will apply recently developed chemical and biochemical strategies to investigate the crosstalk between proline hydroxylation and other metabolic-sensing modifications in regulating substrate protein degradation and activity. Furthermore, we will study the physiological significance of a new Hyp-mediated epigenetic modification pathway in regulating gene expression and chromatin activity. Overall, we anticipate that the development and application of functional proteomics technology for system-wide analysis of proline hydroxylation proteome will reveal novel metabolic-sensing pathways and potentially lead to paradigm-shifting concepts in the fields of cancer, metabolic diseases, and development.