Establishing Glyoxalase 2 as a Viable Target for the Treatment of Disease

项目摘要/摘要 细胞检测并对代谢信号做出反应的能力对维持体内平衡至关重要，而且 对新陈代谢流量波动做出反应的感知机制的扰动是许多 疾病，包括败血症、自身免疫、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 在疾病中调节失调。目前，我们对构图的理解存在着根本性的差距， PTM的丰度和酶控制，以及它们在疾病中如何改变。 我的实验室专注于PTM的鉴定和表征，以及它们是如何在 无论是健康还是疾病。为了实现这一目标，我们开发了敏感的方法来识别和量化 在广泛的生物样本中的PTMS的全球变化。使用这种方法，我们已经确定了 一种由糖酵解副产物衍生的新型赖氨酸PTM。当乙二醛酶2时，这些PTM升高 (GLO2)被抑制，导致糖酵解产量减少，并扰乱一碳代谢。我们的初选 目的是确定GLO2抑制策略在治疗代谢紊乱方面的疗效。 我的研究计划致力于理解四个基本问题：1)GLO2如何控制 一碳代谢和细胞氧化还原？GLO2基因敲除细胞减少了谷胱甘肽，增加了 氧化应激。我们将量化GLO2在调节从头合成谷胱甘肽中的作用。此外, GLO2在调节抗氧化反应中的作用将在氧化的细胞模型中进行评估 压力和炎症信号。2)LactoylLys的修饰是如何监管的？我们将聘用 应用CRISPR-Cas9基因敲除细胞系鉴定酶的定量蛋白质组学 细胞中乳酸赖氨酸修饰的调节剂。3)GLO2是治疗糖酵解的可行靶点吗？ 依赖疾病状态？将采用异种移植模型，使用GLO2基因敲除细胞系来量化 体内的增殖和代谢调节。这将决定靶向GLO2治疗的可行性 疾病的治疗。4)LactoylLys修饰是否是功能性组蛋白标记？我们已经确定了 未刺激细胞中组蛋白作为乳酸赖氨酸修饰的靶点。这些东西的存在 PTMS基本提示在转录调控中可能起作用。我们将使用蛋白质组学来识别特定的位点 细胞中LactoylLys修饰的修饰和推测的‘阅读器’结构域。 我们的主要目标是确定GLO2和LactoylLys修饰在细胞代谢和 染色质生物学。这个项目将解决我们对细胞新陈代谢的基本理解中的一个根本差距 是受监管的。了解这些PTM如何调节动态平衡是了解它们的 在疾病中的作用。由于该项目的深远影响和广泛的治疗应用 对于高度糖酵解疾病状态，这一研究计划是ESI Mira奖的理想选择。

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.