Functional Dissection of Metabolic-Sensing Proline Hydroxylation Pathways

项目摘要 越来越多的证据表明，脯氨酸羟化(Hyp)是一种基本的翻译后 对细胞代谢环境的变化高度敏感的修饰。在癌症期间 实体瘤中癌细胞的发展、快速增殖受制于有限的氧气供应。缺氧症 微环境阻止其羟脯氨酸依赖的低氧诱导因子α蛋白的降解，并激活缺氧- 促进癌细胞在低氧中存活的反应细胞通路。除了氧气，监管机构 酶对铁和关键线粒体代谢物的浓度也很敏感。 包括琥珀酸、富马酸和α-酮戊二酸，使该途径成为细胞内关键的代谢传感器。 广泛的研究表明，底物蛋白质的脯氨酸羟基化调节蛋白质-蛋白质。 相互作用或底物蛋白质降解。尽管它在细胞生理学中扮演着重要的角色，并在 对单个底物进行有针对性的分析，全系统表征和功能量化 由于缺乏有效的工具和战略来确定全球特定地点的 脯氨酸羟化靶标。我们的总体假设和长期目标是系统地描述 通过功能蛋白质组学方法的发展将导致“脯氨酸羟基组”的机械化 理解在发育和疾病中新的Hyp介导的代谢调节。要做到这一点 目标，我们开发并应用了一种免疫沉淀辅助的策略来进行全球鉴定 脯氨酸羟化靶标。通过这一战略，我们将应对系统发现和 脯氨酸羟化蛋白质组的定量。我们将开发新的定量蛋白质组学工作流程和 应用这些策略来鉴定和验证内源性脯氨酸羟基酶靶标。vbl.使用 时间动力学分析，我们还将揭示受Hyp依赖蛋白影响的靶蛋白 退化。综合数据分析将揭示新型Hyp底物的调节酶和 因此，能够实现可信的验证以及功能表征。除了特定于目标的 降解，我们的初步蛋白质组学分析表明，Pro羟化可能调节全球蛋白质 通过调节蛋白酶体活性实现的动态平衡。我们将发展内生模式体系和 用新的定量质谱学技术确定脯氨酸的功能意义 蛋白酶体亚基上的羟化以及这种调节如何影响全球蛋白质动态平衡。总体而言，我们 展望全系统分析功能蛋白质组学技术的发展和应用 将揭示新的新陈代谢感知途径，并可能导致范式- 癌症、代谢性疾病和老龄化领域的概念转变。

Project Summary Mounting evidence have demonstrated proline hydroxylation (Hyp) as a fundamental posttranslational modification that are highly responsive to the changes in cellular metabolic environment. During cancer development, rapid proliferation of cancer cells in solid tumors suffers from limited oxygen supply. The hypoxia microenvironment prevents its hydroxyproline-dependent degradation of HIFα proteins and activates hypoxia- response cellular pathways that promote cancer cell survival in hypoxia. In addition to oxygen, the regulatory 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 of substrate proteins regulates protein-protein interactions or substrate protein degradation. Despite of its important roles in cell physiology and success in targeted analysis of individual substrates, system-wide characterization and functional quantification of the pathway have been hindered by the lack effective tools and strategies for global 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 mechanistic understanding of the novel Hyp-mediated metabolic regulations in development and diseases. To achieve this goal, we have developed and applied an immunoprecipitation-assisted strategy for global identification of proline hydroxylation targets. With this strategy, we will tackle the challenge of systematic discovery and quantification of proline hydroxylation proteome. We will develop new quantitative proteomics workflows and apply the strategies for the identification and validation of endogenous prolyl hydroxylase targets. Using temporal dynamics analysis, we will also reveal the target proteins that are subject to Hyp-dependent protein degradation. Integrated data analysis will reveal the regulatory enzyme of the novel Hyp substrates and therefore enable confident validation as well as functional characterization. In addition to the target-specific degradation, our preliminary proteomics analysis showed that proline hydroxylation may regulate global protein homeostasis through the regulation of proteasome activities. We will develop endogenous model systems and novel quantitative mass spectrometry technology to determine the functional significance of proline hydroxylation on proteasome subunits and how such regulation affect global protein homeostasis. Overall, we anticipate that the development and application of functional proteomics technology for system-wide analysis of proline hydroxylation targets will reveal novel metabolic-sensing pathways and potentially lead to paradigm- shifting concepts in the fields of cancer, metabolic diseases and aging.