Arrestin domain-containing protein 4 as a novel regulator of glucose metabolism in the ischemic heart

含 Arrestin 结构域的蛋白 4 作为缺血心脏葡萄糖代谢的新型调节剂

• 批准号: 10735139
• 负责人: Jun Yoshioka
• 金额: $ 39.25万
• 依托单位: CITY COLLEGE OF NEW YORK
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10735139
• 关键词: Adaptor Signaling Protein; Arrestins; Artificial Intelligence; Binding; Binding Proteins; Biochemical; Biological Assay; CRISPR/Cas technology; Cardiac; Cardiac Myocytes; Cardiovascular system; Cell Death; Cell Survival; Cell physiology; Cells; Clathrin; Complex; Computer Analysis; Cytoprotection; Data; Defense Mechanisms; Endocytosis; Environment; Family; Gene Silencing; Genes; Genetic Transcription; Genome engineering; Glucose; Glucose Transporter; Glycolysis; Heart; Homeostasis; Hypoxia; Impairment; In Vitro; Ischemia; Knock-out; Knockout Mice; Knowledge; Link; Mediating; Membrane; Metabolic; Metabolism; Molecular; Mus; Myocardial; Myocardial Infarction; Myocardial Ischemia; Nature; Nutrient; Outcome; Oxidative Phosphorylation; Pathogenesis; Pathway interactions; Physiological; Proteins; Regulation; Reporting; Role; SLC2A1 gene; Scaffolding Protein; Signal Transduction; Specificity; Starvation; Stress; Structure; TXNIP gene; Testing; Therapeutic; Therapeutic Intervention; Ubiquitination; Warburg Effect; Wild Type Mouse; aerobic glycolysis; beta-adrenergic receptor; beta-arrestin; cardioprotection; desensitization; drug discovery; experimental study; genetic manipulation; glucose metabolism; glucose transport; glucose uptake; improved; improved outcome; in vivo; inhibitor; innovation; insight; knowledge of results; member; mouse model; mutant; neoplastic cell; novel; novel therapeutics; receptor; receptor function; reconstitution; small molecule; therapeutic target; trafficking; ubiquitin-protein ligase; virtual screening

ABSTRACT The complexity of cardiomyocyte signaling requires scaffolding proteins to coordinate the cellular processes driven by receptors and transporters. β-Arrestins are prototypical intracellular scaffold proteins that negatively regulate cardiac β-adrenergic receptor function via desensitization. Recently, a larger and more ancient family of structurally related arrestins, termed α-arrestins, has been identified, which shares functions in regulating transporter trafficking. Txnip, the best-studied member of α-arrestins, serves as an adaptor protein to facilitate endocytosis of glucose transporters (GLUTs) and suppresses glucose influx through its arrestin domains. We previously found that targeted deletion of Txnip leads to a substantial metabolic switch, directing cardiomyocytes toward enhanced glycolytic metabolism under severe ischemia. Despite the potential to identify new molecular mechanisms, the functions of other α-arrestins, Arrestin domain-containing protein (Arrdc) 1-5, remain largely undefined in the heart. Here we present preliminary data demonstrating that two α- arrestins Arrdc4 and Txnip are related to their conserved arrestin domains and share the function to inhibit GLUT1. Interestingly, this metabolic inhibition was more potent in Arrdc4 than in Txnip. Using our recently- generated Arrdc4 knockout mouse model, the data reveal exciting findings that inhibition of Arrdc4 enhances myocardial glucose uptake during hypoxia and improves outcomes after myocardial infarction. These results define the outlines of an Arrdc4-GLUT1 pathway that may provide a link between cardiac glucose metabolism and cardiomyocyte survival. This project aims to delineate the molecular nature of this pathway and tests its role in the pathogenesis of ischemic heart disease. Aim 1 tests three non-exclusive hypotheses by which Arrdc4 regulates GLUT1 function in cardiomyocytes: (a) specific arrestin domains of Arrdc4 mediate clathrin- dependent endocytosis of GLUT1; (b) Arrdc4 promotes GLUT1 ubiquitination through an E3 ligase-mediated pathway; (c) Arrdc4 and Txnip are complementary in the regulation of cardiomyocyte glucose metabolism. Aim 2 employs the Arrdc4 knockout mouse model to test the overall significance of cardioprotection against myocardial ischemia through a GLUT1-mediated mechanism in vivo. By genetically “reconstituting” the hearts of Arrdc4 knockout mice with the informative Arrdc4 mutant, this aim also tests the roles of the specific molecular mechanisms linking Arrdc4 and GLUT1 in ischemic heart disease. Furthermore, using a combination of virtual screening and cell-based assays, Aim 3 will search for the possible Arrdc4-GLUT1 interaction inhibitors that may improve energy homeostasis and enhance cardiomyocyte survival under hypoxia. These studies are highly innovative as we propose a pathway that has never been entertained as a cardiac metabolic regulator. The resulting knowledge will provide a novel mechanistic basis for understanding the defense mechanism to protect cardiomyocytes against metabolically-challenging environments under ischemia. Thus, we believe that the mechanism of action of Arrdc4 will give new and relevant insights into therapeutic strategy.

摘要 心肌细胞信号的复杂性需要支架蛋白来协调细胞过程 由受体和转运体驱动。β-arrestins是典型的细胞内支架蛋白，它对 通过脱敏调节心脏β-肾上腺素能受体功能。最近，一个更大更古老的家庭 在结构上相关的拦阻蛋白，称为α-拦阻蛋白，已经被鉴定出来，它们分享了调控功能 运送者贩卖。TXNIP是α-arrestins中研究最深入的成员，它是一种接头蛋白，可以促进 葡萄糖转运体(GLUT)的内吞作用，并通过其arrestin结构域抑制葡萄糖内流。我们 先前发现，TXNIP的靶向缺失会导致实质性的代谢转换，从而引导 心肌细胞在严重缺血状态下糖酵解代谢增强。尽管有可能 确定新的分子机制、其他α-arrestins、arrestin结构域包含蛋白的功能 (Arrdc)1-5，在心脏中大部分仍未定义。在这里，我们提供的初步数据表明，两个α- 抑制素Arrdc4和TXNIP与其保守的arrestin结构域有关，具有共同的抑制功能 Glut1.有趣的是，这种代谢抑制在Arrdc4中比在TXNIP中更有效。利用我们最近的- 产生了Arrdc4基因敲除小鼠模型，数据揭示了令人兴奋的发现，抑制Arrdc4增强了 低氧时的心肌葡萄糖摄取和改善心肌梗死后的预后。这些结果 确定Arrdc4-GLUT1途径的轮廓，该途径可能在心脏葡萄糖代谢之间提供联系 和心肌细胞存活。该项目旨在描绘这一途径的分子性质并测试其 在缺血性心脏病发病机制中的作用。目标1测试三个非排他性假设 Arrdc4调节心肌细胞GLUT1功能：(A)Arrdc4的特定arrestin结构域介导clathrin- 依赖的GLUT1内吞作用；(B)Arrdc4通过E3连接酶介导促进GLUT1泛素化 (C)Arrdc4和TXNIP在调节心肌细胞葡萄糖代谢方面具有互补性。目标 2采用Arrdc4基因敲除小鼠模型来测试心脏保护的整体意义。 GLUT1介导的在体心肌缺血机制。通过基因“重组”心脏 在具有信息丰富的Arrdc4突变体的Arrdc4基因敲除小鼠中，这一目的也测试了特定的 Arrdc4和GLUT1在缺血性心脏病中的分子机制。此外，使用组合 在虚拟筛选和基于细胞的分析中，Aim 3将搜索可能的Arrdc4-GLUT1相互作用 可以改善能量动态平衡和提高心肌细胞在低氧下的存活率的抑制剂。这些 研究具有很高的创新性，因为我们提出了一种从未被认为是心脏代谢的途径 调整器。由此产生的知识将为理解辩护提供新的机制基础。 缺血条件下保护心肌细胞免受代谢挑战环境的机制。因此， 我们相信，Arrdc4的作用机制将为治疗策略提供新的和相关的见解。