Integration of Inflammatory Signaling and the Unfolded Protein Response by Nitrosylation Signaling in Obesity

肥胖中炎症信号传导与亚硝基化信号传导的未折叠蛋白反应的整合

基本信息

批准号：
10062953
负责人：
Ling Yang
金额：
$ 38.13万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-12-01 至 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10062953
关键词：
Address Affect Animal Model Attenuated Binding Proteins Biochemical Biological Butyric Acids Cells Chemicals Chronic Data Development Diabetes Mellitus Dyslipidemias Endoplasmic Reticulum Enhancers Enzymes Excision Failure Functional disorder Glutathione Goals Hepatic Homeostasis Human Immunoprecipitation Impairment Inflammation Inflammatory Inositol Insulin Insulin Resistance Knockout Mice Knowledge Laboratory Research Liver Maps Mediating Mediator of activation protein Metabolic Metabolic Diseases Metabolism Mission Modification Molecular Mus NOS2A gene Nicotinamide adenine dinucleotide Nitric Oxide Nitric Oxide Synthase Non-Insulin-Dependent Diabetes Mellitus Obese Mice Obesity Organelles Outcome Output Oxidoreductase Pancreatic ribonuclease Pathology Pathway interactions Post-Translational Protein Processing Production Protein S Proteins Proteome Proteomics Public Health RNA Processing RNA Splicing RNA-Protein Interaction Regulation Research Resistance Ribonucleases Role S-Nitrosoglutathione Signal Transduction Site Speed Stress Supplementation Testing Therapeutic Thinness Tissues United States National Institutes of Health Up-Regulation base biological adaptation to stress blood glucose regulation crosslink diabetic patient diet-induced obesity disability endoplasmic reticulum stress improved in vivo innovation insight insulin sensitivity mouse S-nitrosoglutathione reductase mouse model new therapeutic target nitrosative stress novel obesity development overexpression protein complex response

项目摘要

PROJECT SUMMARY In the setting of obesity, endoplasmic reticulum (ER) stress has been identified as a prominent feature in metabolic tissues in both animal models and in humans. To cope with ER stress, cells activate the unfolded protein response (UPR) to mitigate stress. However, failure of the UPR results in chronic unresolved stress, contributing to the development of obesity-induced insulin resistance. Nitric oxide (NO) is a key mediator of obesity-associated inflammation. We recently demonstrated that NO-mediated protein modification (S- nitrosylation) impairs the RNase activity of inositol-requiring enzyme-α (IRE1α), resulting in unresolved ER stress and insulin resistance. Although nitric oxide synthase (NOS) provides the general intracellular NO pools for protein S-nitrosylation, the rate of these modifications is also affected by the targeted removal of NO groups by protein denitrosylation. Our strong preliminary data demonstrate that obesity impairs the activity of S- nitrosoglutathione reductase (GSNOR, a major denitrosylase), leading to elevated nitrosative stress in the liver. Furthermore, deletion or overexpression of GSNOR directly regulates the S-nitrosylation state of IRE1α and ER function in mice with diet-induced obesity (DIO). Notably, liver-specific GSNOR overexpression ameliorated obesity-associated insulin resistance. However, the molecular mechanism that underlies the regulation of ER homeostasis by GSNOR-mediated denitrosylation signaling is unknown. We propose to test the central hypothesis that obesity attenuates GSNOR-dependent protein denitrosylation, resulting in elevated nitrosative stress in the ER that contributes to obesity-associated hepatic insulin resistance. To test this hypothesis, we will undertake 2 specific aims. In Aim 1, we will: 1) determine whether liver-specific GSNOR deletion impacts hepatic insulin sensitivity and whole body glucose homeostasis using DIO mouse model; 2) establish whether GSNOR regulates hepatic insulin action directly via modulation of UPR; and 3) assess the therapeutic potential of enhancing hepatic GSNOR activity in obese mice by glutathione supplementation and enhancing cellular nicotinamide adenine dinucleotide (NAD) metabolism. In Aim 2, we will: 1) profile the ER S-nitrosylation proteome and characterize the endogenous S-nitrosylation sites on IRE1α; 2) address how GSNOR-mediated denitrosylation signaling modulates the IRE1α RNase activity; and 3) establish how GSNOR-mediated denitrosylation signaling affects IRE1α interactome formation. The approaches used here are innovative, combining the use of cellular and molecular biological analysis, biochemical analysis for S-nitrosylated proteins, elucidating the ER S-nitrosylation proteome, protein-RNA interaction analysis, and in vivo mouse metabolic profiling. The mechanisms elucidated in this project will provide further understanding of how inflammatory and ER stress pathways are integrated in the context of obesity-associated hepatic insulin resistance, dyslipidemia and ultimately type 2 diabetes. Insight into the mechanisms by which maladaptive organelle stress responses drive these pathologies should speed development of novel therapeutic targets for metabolic diseases.

项目摘要在肥胖的情况下，内质网（ER）应力已被确定为一个突出特征动物模型和人类中的代谢组织。为了应对质网应力，细胞激活展开的蛋白质反应（UPR）减轻压力。但是，UPR的失败导致慢性未解决的压力，有助于肥胖引起的胰岛素抵抗的发展。一氧化氮（NO）是肥胖相关炎症。我们最近证明了无介导的蛋白质修饰（s- 硝基化）会损害肌醇酶的RNase活性，从而导致未解决的ER 压力和胰岛素抵抗。尽管一氧化氮合酶（NOS）提供了一般的细胞内无池对于蛋白质S-亚硝基化，这些修饰的速率也受到靶向去除的影响通过蛋白质硝化基化。我们强大的初步数据表明，肥胖会损害S-的活性硝基谷胱甘肽还原（GSNOR，一种主要的脱硝基酶），导致肝脏的硝化应激升高。此外，GSNOR的缺失或过表达直接调节IRE1α和饮食诱导的肥胖症（DIO）的小鼠的ER功能。值得注意的是，肝特异性GSNOR过表达得到了改善肥胖相关胰岛素抵抗。但是，基于ER调节的分子机制 GSNOR介导的非硝基化信号传导的稳态尚不清楚。我们建议测试中央肥胖症减弱GSNOR依赖性蛋白硝基糖基化的假设，导致硝化剂升高 ER中有助于肥胖相关的肝胰岛素抵抗的压力。为了检验这一假设，我们将实现2个具体目标。在AIM 1中，我们将：1）确定肝脏特异性GSNOR缺失是否影响使用DIO小鼠模型，肝胰岛素敏感性和全身葡萄糖稳态； 2）确定是否 GSNOR直接通过调节UPR调节肝胰岛素作用； 3）评估治疗潜力通过补充谷胱甘肽增强肥胖小鼠的肝脏GSNOR活性并增强细胞烟酰胺腺嘌呤二核苷酸（NAD）代谢。在AIM 2中，我们将：1）介绍ER S-硝基化蛋白质组并表征IRE1α上的内源性S-亚硝基化位点； 2）解决GSNOR介导的如何非硝基化信号传导调节IRE1αRNase活性； 3）建立GSNOR介导的如何非硝基化信号会影响IRE1α相互作用组的形成。这里使用的方法是创新的，结合了细胞和分子生物学分析的使用，生化分析用于S-亚硝基化蛋白，阐明ER S-亚硝化蛋白质组，蛋白-RNA相互作用分析和体内小鼠代谢分析。该项目中阐明的机制将进一步了解炎症和 ER应力途径在与肥胖相关的肝胰岛素抵抗，血脂异常的背景下整合最终是2型糖尿病。深入了解适应不良细胞器应力反应的机制驱动这些病理应加快代谢疾病的新型治疗靶标的发展。