Molecular Mechanisms of Arrestin-Domain Containing Proteins in Metabolism

抑制蛋白超家族由两个亚家族组成：经典的视觉/β-抑制蛋白，其首先被鉴定为G-蛋白偶联受体信号传导的调节剂，以及含有抑制蛋白结构域的蛋白质的更古老的分支，其有时被称为“α-抑制蛋白”。近年来，α-arrestin家族的一些成员在哺乳动物代谢中的作用已被揭示。研究最多的α-抑制蛋白家族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。含有抑制蛋白结构域的4（ARRDC 4）是α-抑制蛋白家族的成员，尚未受到广泛的研究。ARRDC 4的arrestin结构域与TXNIP显示41%的氨基酸序列相似性，因此，ARRDC 4可能具有与TXNIP相似的功能。然而，我们已经表明，TXNIP是共价结合硫氧还蛋白家族的唯一成员。与我们的生物化学研究一致，最近解决的晶体结构显示，TXNIP的半胱氨酸247残基是共价结合硫氧还蛋白所必需的。此外，我们已经报道了TXNIP缺失的小鼠具有改善的胰岛素敏感性，表明氧化还原状态和葡萄糖之间的电位类似 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arrdc 4整体缺失的小鼠具有改善的胰岛素敏感性。我们还显示了arrdc 4缺失小鼠代谢组织中胰岛素信号的改善。我们的初步数据表明，ARRDC 4蛋白直接与葡萄糖转运蛋白4（GLUT 4）在胰岛素刺激的条件下相互作用。此外，我们提出了一种新的小鼠模型与TXNIP的C247突变，并表明TXNIP中的这种单一氨基酸的变化提高了高脂饮食小鼠的胰岛素敏感性。在本项目中，我们将利用这些新发现和小鼠模型来了解含arrestin结构域的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：具体目标1将检验α-抑制蛋白ARRDC 4在体内调节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抑制蛋白结构域的蛋白质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将测试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，并确定负责的靶组织。

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 Here we show new unpublished experiments revealing that mice with global deletion of arrdc4 have improved insulin sensitivity.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.