Molecular Mechanisms of Arrestin-Domain Containing Proteins in Metabolism

Arrestin超家族由两个亚家族组成：经典的视觉/β-arrestins，最初被确定为G蛋白偶联受体信号的调节因子，以及更古老的arrestin结构域包含蛋白的分支，有时被称为“α-arrestins”。最近，α-arrestin家族的一些成员在哺乳动物新陈代谢中的作用已经被揭示。α-arrestin家族中研究最多的成员是硫氧还蛋白相互作用蛋白(TXNIP)，它调节葡萄糖和果糖代谢。Arrestin结构域4(ARRDC4)是α-arrestin家族中的一个成员，尚未得到广泛的研究。ARRDC4的arrestin结构域与TXNIP的氨基酸序列有41%的相似性，因此ARRDC4可能具有与TXNIP相似的功能。然而，我们已经证明TXNIP是该家族中唯一与硫氧还蛋白共价结合的成员。与我们的生物化学研究一致，最近解算的晶体结构表明，TXNIP的半胱氨酸247残基是与硫氧还蛋白共价结合所必需的。此外，我们已经报道，TXNIP缺失的小鼠改善了胰岛素敏感性，表明氧化还原状态和葡萄糖之间存在潜在的相似 通过硫氧还蛋白代谢。在这里，我们展示了新的未发表的实验，揭示了arrdc4全局缺失的小鼠改善了胰岛素敏感性。我们还发现，由于arrdc4基因的缺失，小鼠代谢组织中的胰岛素信号得到了改善。我们的初步数据表明，在胰岛素刺激的条件下，ARRDC4蛋白直接与葡萄糖转运蛋白4(GLUT4)相互作用。此外，我们提出了一种新的带有TXNIP基因C247突变的小鼠模型，并表明TXNIP中这种单一氨基酸的变化改善了高脂饮食小鼠的胰岛素敏感性。在这个项目中，我们将利用这些新发现和小鼠模型来了解arrestin结构域蛋白如何在体内和分子机制水平上调节葡萄糖代谢。我们的具体目标是：特定目标1将检验α-arrestin蛋白ARRDC4调节体内胰岛素刺激的葡萄糖摄取的假设。具体目标2将研究含有α-arrestin结构域的蛋白与葡萄糖转运蛋白和胰岛素受体相互作用的分子机制。具体目标3将验证TXNIP可以通过硫氧还蛋白非依赖性机制调节胰岛素敏感性的假设，并确定负责的目标组织。

The arrestin superfamily is composed of two subfamilies: the classical visual/beta-arrestins that were first identified as regulators of G-protein coupled receptor signaling, and the more ancient branch of arrestin domain-containing proteins that are sometimes called the “alpha-arrestins”. Recently, the roles of some members of the alpha-arrestin family have been revealed in mammalian metabolism. The most studied member of the alpha-arrestin family is thioredoxin interacting protein (TXNIP), which regulates glucose and fructose metabolism. Arrestin domain-containing 4 (ARRDC4) is a member of the alpha-arrestin family that has not been subject to extensive investigation. The arrestin domains of ARRDC4 show 41% amino acid sequence similarity to TXNIP; therefore, ARRDC4 might be expected to have functions similar to TXNIP. However, we have shown that TXNIP is the only member of the family that binds covalently to thioredoxin. Consistent with our biochemistry studies, the recently solved crystal structure revealed that the cysteine 247 residue of TXNIP is essential for binding covalently to thioredoxin. Furthermore, we have reported that mice with deletion of TXNIP have improved insulin sensitivity, indicating a potential like between redox state and glucose metabolism through thioredoxin. Here we show new unpublished experiments revealing that mice with global deletion of arrdc4 have improved insulin sensitivity. We also show improved insulin signaling in metabolic tissues from mice with deletion of arrdc4. Our preliminary data demonstrate that the ARRDC4 protein interacts directly with glucose transporter protein 4 (GLUT4) in insulin-stimulated conditions. Furthermore, we present a new mouse model with mutation of C247 of TXNIP and show that this single amino acid change in TXNIP improves insulin sensitivity in mice fed a High Fat Diet. In this project, we will use these new discoveries and mouse models to understand how arrestin domain-containing proteins regulate glucose metabolism in vivo and at the molecular mechanism level. Our Specific Aims are: Specific Aim 1 will test the hypothesis that alpha-arrestin protein ARRDC4 regulates insulin-stimulated glucose uptake in vivo. Specific Aim 2 will investigate the molecular mechanisms of alpha arrestin domain-containing protein interactions with glucose transporters and insulin receptor. Specific Aim 3 will test the hypothesis that TXNIP can regulate insulin sensitivity through thioredoxin-independent mechanisms and identify the target tissue responsible.