Viral mechanisms of adipocyte dysfuntion: Role of Vpr.

描述（由申请人提供）：脂肪细胞功能障碍是临床上表现为肥胖或脂肪营养不良的广泛代谢性疾病的基础。炎症过程显著促进脂肪细胞功能障碍，导致血脂异常、动脉粥样硬化和胰岛素抵抗。引发这些过程的机制尚不清楚。持续的病毒感染是慢性组织炎症的一个原因。我们已经研究了HIV-1感染导致脂肪萎缩、血脂异常和胰岛素抵抗的复杂综合征的机制，称为“HIV脂肪营养不良”。我们对HIV-1辅助蛋白Vpr的研究表明：a) Vpr作为糖皮质激素受体（GR）的辅助激活因子和PPAR3的辅助抑制因子，通过核受体c端区的协同调节结合位点起作用；b) Vpr通过抑制PPAR3阻断前脂肪细胞分化；c) Vpr n端区域的亲环蛋白a结合位点含有非规范核受体共调节基序，可进一步增强GR活性并抑制PPAR3；d)脂肪组织和肝脏中表达Vpr的小鼠表现出代谢缺陷，与脂肪细胞中GR活性升高和PPAR3活性降低一致；e) Vpr在血液中循环，可以独立于完整的HIV-1进入脂肪细胞。以这些发现为跳板，我们建议通过实现以下具体目标来明确vpr介导的脂肪细胞功能障碍的分子机制：1。明确Vpr在小鼠体内产生生化和脂质动力学改变的GR-和ppar3依赖机制和基因靶点；2. 确定Vpr导致细胞周期阻滞和脂肪细胞发育分化的分子机制；3. 当暴露于hiv感染的T淋巴细胞时，确定Vpr在促进细胞周期阻滞、阻断分化和诱导前脂肪细胞和脂肪细胞凋亡中的作用；4. 确定Vpr在体内对脂质和能量代谢的两种机制信息突变形式的影响：一种是LQQLL共调节因子结合位点缺陷，另一种是与Vpr细胞周期阻滞作用相关的富含精氨酸的c端基序缺陷。作为一个由脂肪细胞生物化学、免疫学和蛋白质化学专家组成的密切合作团队，我们已经证明了Vpr转基因小鼠概括了我们在HIV感染人类中详细描述的关键脂质动力学缺陷，并且Vpr可以在体外阻断前脂肪型分化。我们现在正准备利用突变Vpr蛋白在相关小鼠和脂肪细胞模型中详细研究分子机制。因此，该项目有可能揭示慢性病毒感染导致的脂肪细胞功能障碍、脂质失调和胰岛素抵抗的新途径。公共卫生相关性：脂肪细胞的炎症导致常见的代谢疾病，如肥胖和糖尿病。病毒感染可能是脂肪细胞炎症的一个原因，慢性艾滋病毒感染的患者会出现严重的脂肪细胞退行性疾病，称为“脂肪营养不良”，与糖尿病和心脏病的高风险相关。该项目的目标是确定HIV病毒制造的一种名为Vpr的蛋白质是如何引起脂肪细胞的慢性炎症并导致这些疾病的。

DESCRIPTION (provided by applicant): Adipocyte dysfunction is the fundamental basis of widespread metabolic diseases that present clinically as obesity or lipodystrophy. Inflammatory processes contribute significantly to adipocyte dysfunction, leading to dyslipidemia, atherosclerosis and insulin resistance. The mechanisms that incite these processes are unclear. Persistent viral infections are a cause of chronic tissue inflammation. We have investigated mechanisms whereby HIV-1 infection contributes to a complex syndrome of fat atrophy, dyslipidemia and insulin resistance termed "HIV lipodystrophy". Our studies of the HIV-1 accessory protein Vpr have revealed that: a) Vpr functions as a coactivator of the glucocorticoid receptor (GR) and a corepressor of PPAR3 via a nuclear receptor coregulator binding site in its C-terminal region; b) Vpr blocks preadipocyte differentiation by inhibiting PPAR3; c) a cyclophilin A binding site in the N-terminal region of Vpr contains a non-canonical nuclear receptor coregulator motif that could further enhance GR activity and repress PPAR3; d) mice expressing Vpr in adipose tissue and liver display metabolic defects consistent with increased GR activity and decreased PPAR3 activity in adipocytes; e) Vpr circulates in the blood and can enter adipocytes independent of intact HIV-1. With these findings as a springboard, we propose to specify Vpr-mediated molecular mechanisms of adipocyte dysfunction by achieving the following Specific Aims: 1. Specify the in vivo GR- and PPAR3-dependent mechanisms and gene targets that are responsible for the biochemical and lipid kinetic alterations produced in mice by Vpr; 2. Determine the molecular mechanisms whereby Vpr causes cell cycle arrest and blocks differentiation in adipocyte development; 3. Determine Vpr's role in promoting cell cycle arrest, blocking differentiation and inducing apoptosis of preadipocytes and adipocytes, when exposed to HIV-infected T lymphocytes; 4. Determine in vivo effects of two mechanistically informative mutant forms of Vpr on lipid and energy metabolism: one defective in the LQQLL coregulator binding site, and the other defective in the arginine-rich C-terminus motif associated with Vpr's cell cycle arrest effects in vitro. As a closely collaborative team comprising experts in adipocyte biochemistry, immunology and protein chemistry, we have demonstrated that Vpr transgenic mice recapitulate key lipid kinetic defects we have detailed in humans with HIV infection, and that Vpr can block preadipoctye differentiation in vitro. We are now poised to detail the molecular mechanisms in relevant mouse and adipocyte models utilizing mutant Vpr proteins. Thus, this project is likely to uncover novel pathways of adipocyte dysfunction, lipid dysregulation and insulin resistance resulting from a chronic viral infection. PUBLIC HEALTH RELEVANCE: Inflammation of fat cells leads to common metabolic diseases such as obesity and diabetes Viral infections could be a cause of inflammation in fat cells and patients with chronic HIV infection develop serious fat cell degenerative condition termed "lipodystrophy" associated with high risk of diabetes and heart disease. The goal of this project is to determine how a protein made by the HIV virus, termed Vpr, can cause chronic inflammation of fat cells and lead to these disorders.