Functional Dissection of Metabolic-Sensing Proline Hydroxylation Pathways

代谢传感脯氨酸羟基化途径的功能剖析

• 批准号: 10241993
• 负责人: Yue Chen
• 金额: $ 35.14万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10241993
• 关键词: Affect; Aging; Biological Models; Cell Survival; Cell physiology; Cells; Data Analyses; Development; Disease; Dissection; Environment; Enzymes; Fumarates; Goals; Hydroxylation; Hydroxyproline; Hypoxia; Immunoprecipitation; Individual; Inflammation; Iron; Lead; Link; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Metabolic; Metabolic Diseases; Mitochondria; Oxygen; Pathway interactions; Post-Translational Protein Processing; Procollagen-Proline Dioxygenase; Proline; Proteins; Proteome; Proteomics; Regulation; Research; Role; Site; Solid Neoplasm; Succinates; System; Technology; Validation; alpha ketoglutarate; cancer cell; multicatalytic endopeptidase complex; novel; prevent; protein degradation; protein protein interaction; proteostasis; response; sensor; success; tool

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.

项目摘要 越来越多的证据表明脯氨酸羟基化（HYP）是翻译后的基本 对细胞代谢环境的变化有很高响应的修饰。癌症期间 发育，实体瘤中癌细胞的快速增殖受到有限的氧气供应。缺氧 微环境可防止其HIFα蛋白的羟基依赖性降解，并激活缺氧 - 促进缺氧癌细胞存活的反应细胞途径。除氧气外，调节 酶脯氨酰羟化酶也对铁和关键线粒体代谢产物的浓度敏感 包括琥珀酸酯，富马酸酯和α-酮戊二酸，使该途径成为细胞中的关键代谢传感器。 广泛的研究表明，底物蛋白的脯氨酸羟基化调节蛋白质蛋白 相互作用或底物蛋白降解。尽管在细胞生理和成功中具有重要作用 针对单个底物的有针对性分析，全系统范围的表征和功能定量 缺乏有效的工具和策略来阻碍途径 脯氨酸羟基化靶标。我们的总体假设和长期目标是系统的表征 通过开发功能蛋白质组学方法的“脯氨酸羟基”将导致机械 了解新型催眠介导的发展和疾病中的代谢法规。实现这一目标 目标，我们已经制定并应用了一种免疫沉淀的策略，以全球识别 脯氨酸羟基化靶标。通过此策略，我们将应对系统发现的挑战和 脯氨酸羟基化蛋白质组的定量。我们将开发新的定量蛋白质组学工作流程和 将策略应用于内源性丙酰羟化酶靶标的鉴定和验证。使用 临时动力学分析，我们还将揭示受催眠蛋白的靶蛋白 降解。综合数据分析将揭示新型催眠底物的调节酶和 因此，实现自信验证和功能表征。除了特定目标 退化，我们的初步蛋白质组学分析表明，脯氨酸羟基化可能调节全局蛋白 通过调节蛋白酶体活动的稳态。我们将开发内源模型系统和 新的定量质谱技术来确定脯氨酸的功能意义 对蛋白酶体亚基的羟基化以及这种调节如何影响全球蛋白质稳态。总体而言，我们 预计功能蛋白质组学技术在全系统分析中的开发和应用 脯氨酸羟基化靶标的将揭示新的代谢途径，并有可能导致范式 在癌症，代谢疾病和衰老领域的转移概念。