Establishing Glyoxalase 2 as a Viable Target for the Treatment of Disease [Equipment Supplement]

将乙二醛酶 2 确立为治疗疾病的可行靶点 [设备补充]

• 批准号: 10383972
• 负责人: James J Galligan
• 金额: $ 25万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-10 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10383972
• 关键词: Address; Antioxidants; Autoimmunity; Award; Biological; Biology; CRISPR/Cas technology; Carbon; Cell Line; Cell model; Cells; Cellular Metabolic Process; Chromatin; Cues; Diabetes Mellitus; Disease; Equipment; Global Change; Glutathione; Glycolysis; Goals; Health; Histones; Homeostasis; Inflammatory; Knock-out; Laboratories; Lactoylglutathione Lyase; Lysine; Malignant Neoplasms; Metabolic; Metabolic Diseases; Metabolism; Methods; Modification; Output; Oxidation-Reduction; Oxidative Stress; Plant Roots; Post-Translational Protein Processing; Proteins; Proteomics; Reader; Reduced Glutathione; Regulation; Research; Role; Sampling; Sepsis; Signal Transduction; Site; Site-Directed Mutagenesis; Therapeutic; Transcriptional Regulation; Treatment Efficacy; Xenograft Model; in vivo; novel; programs; response; sensor; treatment strategy

PROJECT SUMMARY/ABSTRACT The ability for cells to detect and respond to metabolic cues is critical to maintaining homeostasis, and perturbations in the sensing mechanisms that respond to oscillations in metabolic flux are the root cause of many diseases, including sepsis, autoimmunity, cancer, and diabetes. There is mounting evidence that protein post- translational modifications (PTMs) are the critical sensors for these metabolic fluctuations and are often dysregulated in disease. Currently, we have a fundamental gap in our understanding of the composition, abundance, and enzymatic control of PTMs and how they are altered in disease. My laboratory focuses on the identification and characterization of PTMs and how they are regulated in both health and disease. To accomplish this goal, we have developed sensitive methods to identify and quantify global changes in PTMs across a broad spectrum of biological samples. Using this approach, we have identified a novel lysine PTM that is derived from a glycolytic by-product. These PTMs are elevated when glyoxalase 2 (GLO2) is inhibited, resulting in reduced glycolytic output and disrupted one-carbon metabolism. Our primary goal is to establish the therapeutic efficacy of a GLO2 inhibition strategy for the treatment of metabolic disorders. My research program is dedicated to understanding four fundamental questions: 1) How does GLO2 control one-carbon metabolism and cellular redox? GLO2 knockout cells have reduced glutathione and increased oxidative stress. We will quantify the role of GLO2 in the regulation of de novo glutathione synthesis. In addition, the role of GLO2 in the regulation of antioxidant responses will be evaluated in a cellular model for oxidative stress and inflammatory signaling. 2) How are LactoylLys modifications regulated? We will employ quantitative proteomics using CRISPR-Cas9 knockout cell lines of candidate proteins to identify enzymatic regulators of LactoylLys modifications in cells. 3) Is GLO2 a viable target for the treatment of glycolysis- dependent disease states? A xenograft model will be employed using GLO2 knockout cell lines to quantify proliferation and metabolic regulation in vivo. This will determine the therapeutic feasibility of targeting GLO2 for the treatment of disease. 4) Are LactoylLys modifications functional histone marks? We have identified histones as targets for modification by LactoylLys modifications in unstimulated cells. The presence of these PTMs basally suggests a putative role in transcriptional regulation. We will use proteomics to identify site-specific modifications and putative `reader' domains for LactoylLys modifications in cells. Our primary goal is to establish the role of GLO2 and LactoylLys modifications in cell metabolism and chromatin biology. This project will address a fundamental gap in our basic understanding of how cell metabolism is regulated. Understanding how these PTMs regulate homeostasis is a critical first step to understanding their role in disease. Due to the far-reaching implications of this project and the broad applications for the treatment of highly glycolytic disease states, this research program is an ideal fit for the ESI MIRA Award.

项目摘要/摘要 细胞检测和响应代谢线索的能力对于维持稳态至关重要，并且 对代谢通量中振荡响应的感应机制的扰动是许多根本原因 疾病，包括败血症，自身免疫性，癌症和糖尿病。有越来越多的证据表明蛋白质后 翻译修饰（PTM）是这些代谢波动的关键传感器，通常是 疾病失调。目前，我们对构图的理解有根本的差距 PTM的丰度和酶促控制及其在疾病中的改变。 我的实验室专注于PTM的识别和表征及其在两者中的调节方式 健康与疾病。为了实现这一目标，我们开发了敏感的方法来识别和量化全局 PTM在广泛的生物样品中的变化。使用这种方法，我们已经确定了 源自糖酵解副产品的新型赖氨酸PTM。当乙二醛酶2时，这些PTM升高 （GLO2）被抑制，从而导致糖酵解输出降低并破坏一碳代谢。我们的主要 目的是确定GLO2抑制策略治疗代谢疾病的治疗功效。 我的研究计划致力于理解四个基本问题：1）GLO2控制如何 单碳代谢和细胞氧化还原？ GLO2基因敲除细胞减少了谷胱甘肽并增加了 氧化应激。我们将量化Glo2在从头谷胱甘肽合成调节中的作用。此外， GLO2在调节抗氧化剂反应中的作用将在细胞模型中评估用于氧化。 压力和炎症信号传导。 2）如何调节乳酰胺的修饰？我们将雇用 使用CRISPR-CAS9敲除候选蛋白的细胞系来鉴定酶促的定量蛋白质组学 细胞中乳酰胺修饰的调节剂。 3）GLO2是治疗糖酵解的可行靶标 依赖疾病状态？异种移植模型将使用GLO2基因敲除细胞系来量化 体内增殖和代谢调节。这将确定靶向GLO2的治疗可行性 疾病的治疗。 4）乳酰基修饰功能组蛋白标记吗？我们已经确定了 组蛋白作为未刺激细胞中乳酰胺修饰的修饰的靶标。这些的存在 PTM基本上提出了在转录调控中的推定作用。我们将使用蛋白质组学来识别特定地点 修饰和针对细胞中乳酸化修饰的假定“读取器”域。 我们的主要目标是确定glo2和lactoyllys修饰在细胞代谢和染色质中的作用 生物学。该项目将解决我们对细胞代谢的基本理解的基本差距 受监管。了解这些PTM如何调节体内平衡是了解其角色的关键第一步 在疾病中。由于该项目的深远影响以及对待的广泛应用 高糖酵解疾病指出，该研究计划非常适合ESI MIRA奖。