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代谢障碍的广泛代谢性疾病的根本基础。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原因。我们研究了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：Vpr是糖皮质激素受体（GR）的共激活子和PPAR 3的共抑制子，Vpr的C-末端是核受体共调节子的结合位点，Vpr通过抑制PPAR 3而阻断前脂肪细胞的分化，Vpr的C-末端是核受体共调节子的结合位点，Vpr的C-末端是核受体共调节子的结合位点。c）Vpr的N-末端区域中的亲环素A结合位点含有可进一步增强GR活性并抑制PPAR 3的非典型核受体辅助调节基序; d）在脂肪组织和肝脏中表达Vpr的小鼠表现出与脂肪细胞中GR活性增加和PPAR 3活性降低一致的代谢缺陷; e）Vpr在血液中循环，并且可以独立于完整的HIV-1进入脂肪细胞。以这些发现为出发点，我们提出通过实现以下具体目标来详细说明Vpr介导的脂肪细胞功能障碍的分子机制：1.详细说明体内GR和PPAR 3依赖性机制和基因靶点，这些机制和基因靶点负责Vpr在小鼠中产生的生化和脂质动力学改变; 2.确定Vpr导致细胞周期停滞和阻断脂肪细胞发育分化的分子机制; 3.观察Vpr在HIV感染的T淋巴细胞作用下，对前脂肪细胞和脂肪细胞的细胞周期阻滞、分化阻滞和凋亡诱导的作用。确定Vpr的两种机械信息突变形式对脂质和能量代谢的体内影响：一种在LQQLL辅助调节剂结合位点有缺陷，另一种在与Vpr体外细胞周期阻滞效应相关的富含精氨酸的C-末端基序有缺陷。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Vpr转基因小鼠重现了我们在HIV感染者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细胞分化。我们现在准备详细的分子机制，在相关的小鼠和脂肪细胞模型，利用突变Vpr蛋白。因此，该项目有可能揭示由慢性病毒感染引起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，如肥胖症和糖尿病病毒感染可能是脂肪细胞炎症的原因，慢性HIV感染的患者发展为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由HIV病毒产生的蛋白质（称为Vpr）如何引起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.