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.

抽象的 心肌细胞信号传导的复杂性需要支架蛋白来协调细胞过程 由受体和转运蛋白驱动。 β-抑制蛋白是典型的细胞内支架蛋白， 通过脱敏调节心脏β-肾上腺素能受体功能。最近，一个更大、更古老的家族 已鉴定出结构相关的抑制蛋白，称为 α-抑制蛋白，其在调节中具有共同功能 运输者贩运。 Txnip 是 α-arrestins 中研究最透彻的成员，可作为衔接蛋白来促进 葡萄糖转运蛋白（GLUT）的内吞作用并通过其抑制蛋白结构域抑制葡萄糖流入。我们 先前发现，Txnip 的靶向删除会导致显着的代谢转变，从而指导 心肌细胞在严重缺血下糖酵解代谢增强。尽管有潜力 识别新的分子机制、其他 α-arrestin 的功能、含 Arrestin 结构域的蛋白 (Arrdc) 1-5，在心里很大程度上仍然没有定义。在这里，我们提供初步数据，证明两个 α- 抑制蛋白 Arrdc4 和 Txnip 与其保守的抑制蛋白结构域相关，并共享抑制蛋白的功能 过剩1。有趣的是，这种代谢抑制在 Arrdc4 中比在 Txnip 中更有效。使用我们最近的- 生成 Arrdc4 敲除小鼠模型，数据揭示了令人兴奋的发现，即 Arrdc4 的抑制增强了 缺氧期间心肌葡萄糖摄取并改善心肌梗死后的结局。这些结果 定义了 Arrdc4-GLUT1 通路的轮廓，该通路可能提供心脏葡萄糖代谢之间的联系 和心肌细胞的存活率。该项目旨在描述该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 调节心肌细胞中的 GLUT1 功能：(a) Arrdc4 的特定抑制蛋白结构域介导网格蛋白- GLUT1 的依赖性内吞作用； (b) Arrdc4 通过 E3 连接酶介导促进 GLUT1 泛素化 途径； (c) Arrdc4 和 Txnip 在心肌细胞葡萄糖代谢的调节中互补。目的 2 采用 Arrdc4 敲除小鼠模型来测试心脏保护作用的整体意义 体内通过 GLUT1 介导的机制引起心肌缺血。通过基因“重建”心脏 具有信息丰富的 Arrdc4 突变体的 Arrdc4 敲除小鼠，这个目的还测试了特定的作用 缺血性心脏病中连接 Arrdc4 和 GLUT1 的分子机制。此外，使用组合 通过虚拟筛选和基于细胞的检测，Aim 3 将寻找可能的 Arrdc4-GLUT1 相互作用 可以改善能量稳态并增强缺氧下心肌细胞存活的抑制剂。这些 研究具有高度创新性，因为我们提出了一条从未被认为是心脏代谢的途径 调节器。由此产生的知识将为理解防御提供新的机制基础 保护心肌细胞免受缺血下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相关见解。