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

摘要 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-Arrestins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 结构上相关的arrestins，称为α-arrestins，已被确定，其中共享的功能，在调节 运输商走私Txnip是α-arrestins中研究最多的成员，作为一种衔接蛋白， 葡萄糖转运蛋白（GLUT）的内吞作用，并抑制葡萄糖通过其抑制蛋白结构域流入。我们 先前发现Txnip的靶向缺失导致实质性的代谢转换， 心肌细胞在严重缺血下糖酵解代谢增强。尽管有可能 确定新的分子机制，其他α-arrestins的功能，Arrestin结构域蛋白 （Arrdc）1-5，在心脏中仍然很大程度上不确定。在这里，我们提出的初步数据表明，两个α- arrestins Arrdc 4和Txnip与它们保守的arrestin结构域相关，并且共享抑制 GLUT 1。有趣的是，这种代谢抑制在Arrdc 4中比在Txnip中更有效。利用我们最近- 产生的Arrdc 4敲除小鼠模型，数据揭示了令人兴奋的发现，抑制Arrdc 4增强 心肌葡萄糖摄取在缺氧和改善心肌梗死后的结果。这些结果 定义了Arrdc 4-GLUT 1通路的轮廓，该通路可能提供心脏葡萄糖代谢之间的联系 和心肌细胞存活。该项目旨在描述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测试了三个非排他性假设， Arrdc 4调节心肌细胞中的GLUT 1功能：（a）Arrdc 4的特异性抑制蛋白结构域介导网格蛋白- （B）Arrdc 4通过E3连接酶介导的内吞作用促进GLUT 1泛素化 （c）Arrdc 4和Txnip在心肌细胞葡萄糖代谢的调节中是互补的。目的 2采用Arrdc 4基因敲除小鼠模型来测试心脏保护的总体意义， 心肌缺血通过GLUT 1介导的机制在体内。通过基因“重组”心脏 在Arrdc 4基因敲除小鼠与信息Arrdc 4突变体，这一目的也测试了特定的作用， 缺血性心脏病中Arrdc 4和GLUT 1的分子机制此外，使用组合 通过虚拟筛选和基于细胞的分析，Aim 3将寻找Arrdc 4-GLUT 1可能的相互作用 抑制剂，可以改善能量稳态和提高心肌细胞在缺氧条件下的存活。这些 研究是高度创新的，因为我们提出了一种从未被视为心脏代谢途径的途径， 调节器由此产生的知识将为理解防御提供一个新的机制基础 保护心肌细胞免受缺血下代谢挑战性环境的机制。因此，在本发明中， 我们相信Arrdc 4的作用机制将为治疗策略提供新的和相关的见解。

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.