Viral mechanisms of adipocyte dysfuntion: Role of Vpr.

描述(申请人提供)：脂肪细胞功能障碍是广泛存在的代谢性疾病的基本基础，临床表现为肥胖或脂肪营养不良。炎症过程导致脂肪细胞功能障碍，导致血脂异常、动脉粥样硬化和胰岛素抵抗。激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原因之一。我们研究了HIV-1感染导致一种复杂的脂肪萎缩、血脂异常和胰岛素抵抗综合征的机制，该综合征被称为“HIV脂肪营养不良”。我们对HIV-1辅助蛋白VPR的研究表明：a)VPR是糖皮质激素受体(GR)的协同激活物，也是PPAR3的辅助抑制因子；b)VPR通过抑制PPAR3而阻断前脂肪细胞的分化；c)VPR N末端的亲环素A结合位点包含一个非规范的核受体辅调节基序，可以进一步增强GR活性并抑制PPAR3；d)在脂肪组织和肝脏表达VPR的小鼠表现出与GR活性增加和PPAR3活性降低一致的代谢缺陷；E)VPR在血液中循环，可以独立于完整的HIV-1进入脂肪细胞。以这些发现为出发点，我们建议通过实现以下特定目的来阐明VPR介导的脂肪细胞功能障碍的分子机制：1.明确体内依赖GR和PPAR3的机制和基因靶点，这些机制和基因靶点是VPR引起小鼠生化和脂肪动力学变化的原因；2.确定VPR导致细胞周期停滞和阻止脂肪细胞发育分化的分子机制；3.确定VPR在促进前脂肪细胞和脂肪细胞周期停滞、阻断分化和诱导凋亡中的作用；4.在体内检测VPR的两种机械信息突变形式对脂质和能量代谢的影响：一种是LQQLL辅助调节结合位点的缺陷，另一种是与VPR的细胞周期停滞作用相关的富含精氨酸的C末端的缺陷。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紧密合作的团队，我们已经证明了VPR转基因小鼠概括了我们在感染HIV的人类中详细描述的关键脂质动力学缺陷，并且VPR可以在体外阻止脂肪前体分化。我们现在准备详细介绍利用突变的VPR蛋白在相关的小鼠和脂肪细胞模型中的分子机制。因此，该项目很可能发现由慢性病毒感染引起的脂肪细胞功能障碍、血脂失调和胰岛素抵抗的新途径。与公共卫生相关：脂肪细胞的炎症导致常见的代谢性疾病，如肥胖和糖尿病，病毒感染可能是脂肪细胞炎症的原因，慢性艾滋病毒感染的患者会发展成与糖尿病和心脏病的高风险相关的严重脂肪细胞退行性疾病，称为“脂肪营养不良症”。该项目的目标是确定由HIV病毒制造的一种名为VPR的蛋白质如何导致脂肪细胞的慢性炎症并导致这些疾病。

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.