Regulation and Function of Adiponectin Oligomerization

描述(申请人提供)：脂联素是一种脂肪组织衍生的激素，具有抗糖尿病、抗动脉粥样硬化和抗炎功能。循环脂联素以三聚体、六聚体和高分子量物种的形式存在，激活不同的信号通路，并在其靶组织上发挥不同的生物学功能。脂联素多聚体高度稳定，不会自发地从一个物种转换到另一个物种。然而，目前尚不清楚不同脂联素物种的生物发生是如何在细胞中调节的。为了筛选与脂联素功能调控相关的脂联素相互作用蛋白，我们以脂联素为诱饵，从人胎脑中筛选出酵母双杂交文库。这一筛查导致了一种 25 kDa的蛋白质，以前命名为谷胱甘肽-S-转移酶-kappa。GST-kappa含有两个硫氧还蛋白结构域，C端的硫氧还蛋白结构域与细菌二硫键A(DsbA)氧化还原酶具有高度的结构和序列同源性，DsbA氧化还原酶参与革兰氏阴性细菌周质中二硫键的形成。我们的初步研究表明，GST-kappa和脂联素共同定位于不同的细胞器结构中，并且在体外和细胞内GST-kappa与脂联素相互作用。此外，我们还发现GST-kappa在脂肪组织中高表达，在肥胖的小鼠和人类以及TNF1a治疗后，GST-kappa的表达降低，并被曲格列酮诱导。此外，我们发现RNAi对GST-kappa的抑制显著且特异地降低了分化的3T3-L1脂肪细胞的脂联素水平和分泌。基于这些新的发现，我们推测GST-kappa可能作为一种蛋白质二硫键异构酶，在调节脂联素的多聚化和分泌中发挥重要作用。为了进一步验证这一假设，我们将：1)确定GST-kappa是否调节3T3-L1脂肪细胞的脂联素多聚体组装和/或分泌；2)阐明 GST-kappa调节脂联素多聚体和/或分泌；以及3)建立脂肪组织特异性GST-kappa基因敲除小鼠，以确定GST-kappa在体内的生理作用。这些研究的结果不仅有助于我们了解脂联素多聚化和分泌的调控机制，而且将为设计新的药物干预措施治疗代谢疾病，如胰岛素抵抗和2型糖尿病提供有价值的信息。公共卫生意义：脂联素是一种脂肪组织衍生的激素，具有抗糖尿病和抗炎功能。循环脂联素在细胞内经历多聚化，不同的寡聚体亚型发挥不同的生物学功能。本研究旨在阐明脂联素多聚化调控的生化和细胞机制，为胰岛素抵抗和2型糖尿病等代谢性疾病的新药干预设计提供重要信息。

DESCRIPTION (provided by applicant): Adiponectin is an adipose tissue-derived hormone with anti-diabetic, anti-atherogenic, and anti-inflammatory functions. Circulating adiponectin exists as trimer, hexamer, and high molecular weight species which activate divergent signaling pathways and exert distinct biological functions on their target tissues. Adiponectin multimers are highly stable and do not interconvert from one species to another spontaneously. However, it is currently unknown how the biogenesis of the different adiponectin species is regulated in cells. To identify adiponectin interacting proteins involved in regulating adiponectin function, we screened a yeast two-hybrid cDNA library derived from human fetal brain, using adiponectin as bait. This screening led to the identification of a 25 kDa protein, previously named glutathione-S-transferase (GST)-kappa. GST-kappa contains two thioredoxin domains and the C-terminal thioredoxin domain shares high structural and sequence homology to bacterial disulfide-bond-A (DsbA) oxidoreductase, an enzyme involved in disulfide bond formation in the periplasm of Gram-negative bacteria. Our preliminary studies showed that GST-kappa and adiponectin are co-localized in distinct cellular organelle structures and GST-kappa interacts with adiponectin in vitro and in cells. In addition, we found that GST-kappa is highly expressed in adipose tissue and the expression of GST-kappa is reduced in obese mice and human subjects and by TNF1a-treatment, and is induced by troglitazone. Furthermore, we found that suppression of GST-kappa by RNAi markedly and specifically reduced adiponectin levels and secretion in differentiated 3T3-L1 adipocytes. Based on these novel findings, we hypothesize that GST-kappa may function as a protein disulfide isomerase and play an important role in regulating adiponectin multimerization and secretion. To further test this hypothesis, we will: 1) determine whether GST-kappa regulates adiponectin multimeric assembly and/or secretion in 3T3-L1 adipocytes; 2) elucidate the mechanism by which GST-kappa regulates adiponectin multimeric multimerization and/or secretion; and 3) generate adipose tissue-specific GST-kappa knockout mice to determine the physiological roles of GST-kappa in vivo. Results from these studies will not only shed light on our understanding of the mechanism regulating adiponectin multimerization and secretion, but will also provide valuable information on the design of new pharmacological interventions for metabolic diseases such as insulin resistance and type 2 diabetes. PUBLIC HEALTH RELEVANCE: Adiponectin is an adipose tissue-derived hormone with anti-diabetic and anti- inflammatory functions. Circulating adiponectin undergoes multimerization in cells and different oligomer isoforms exert distinct biological functions. The proposed study seeks to elucidate the biochemical and cellular mechanisms by which adiponectin multimerization is regulated, which will provide important information on the design of new pharmacological interventions for metabolic diseases such as insulin resistance and type 2 diabetes.