Apolipoprotein A-V: A Functional Proteomics Study

载脂蛋白 A-V：功能蛋白质组学研究

• 批准号: 7050130
• 负责人: ROBERT O'Mara RYAN
• 金额: $ 39.08万
• 依托单位: CHILDREN'S HOSPITAL & RES CTR AT OAKLAND
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7050130
• 关键词: apolipoproteins; blood lipoprotein; chemical cleavage; circular dichroism; conformation; fluorescence resonance energy transfer; fluorescent dye /probe; gene targeting; genetically modified animals; laboratory mouse; lipid metabolism; liver cells; molecular assembly /self assembly; protein localization; protein structure function; proteomics; site directed mutagenesis; tissue /cell culture; triglycerides

The long-term goal of our research program is to understand the molecular basis whereby exchangeable apolipoproteins regulate plasma lipid homeostasis. Recent comparative studies of human and mouse genome sequences led to the discovery of a new member of the exchangeable apolipoprotein family, apolipoprotein A-V (apoA-V). Transgenic and gene disruption experiments in mice have revealed a correlation between apoA-V and plasma triacylglycerol (TG) levels. Given the strong relationship between elevated plasma TG and risk for cardiovascular disease, apoA-V is a potential target for therapeutic intervention. The hypothesis that apoA-V modulates plasma TG levels by influencing hepatic lipoprotein assembly or secretion will be tested in primary hepatocytes from apoA-V knockout and transgenic mice as well as cultured human hepatoma cells. Studies will evaluate the extent to which apoA-V expression in liver cells affects the assembly or secretion of TG-rich lipoproteins. Conditioned medium from cultured cells will be analyzed for lipoprotein content and composition, including apoA-V levels. Fluorescence studies will examine the intracellular localization or trafficking of apoA-V in HepG2 and hepatocytes. Far U.V. circular dichroism spectroscopy will be used to characterize the free energy of unfolding and domain organization of lipid-free and lipid-associated apoA-V. Chemical cleavage of apoA-V will be performed to obtain fragments for examination of the hypothesis that the protein possesses independently folded domains. N- or C-terminal truncation variants will be engineered and characterized in terms of stability, lipid binding and biological effects. ApoA-V tertiary structure and lipid-induced conformational changes will be evaluated by fluorescence resonance energy transfer studies. Site directed mutagenesis will be performed to sequentially replace Trp residues in apoA-V, thereby generating a panel of discrete single Trp apoA-V variants. Cysteine 204 will be labeled with an extrinsic fluorescent probe and distance determinations between these donor/ acceptor pairs made. Taken together, proposed research will employ a combination of functional studies and structural characterization to improve our understanding of the mechanism whereby apoA-V modulates plasma TG levels.

我们研究计划的长期目标是了解可交换载脂蛋白调节血脂稳态的分子基础。最近对人类和小鼠基因组序列的比较研究导致发现了可交换载脂蛋白家族的新成员，载脂蛋白A-V（apoA-V）。小鼠转基因和基因破坏实验揭示了载脂蛋白A-V和血浆三酰甘油（TG）水平之间的相关性。鉴于血浆TG升高与心血管疾病风险之间的密切关系，apoA-V是治疗干预的潜在靶点。将在apoA-V敲除小鼠和转基因小鼠以及培养的人肝癌细胞的原代肝细胞中检验apoA-V通过影响肝脂蛋白组装或分泌来调节血浆TG水平的假设。研究将评估肝细胞中apoA-V表达影响富含TG的脂蛋白的组装或分泌的程度。将分析来自培养细胞的条件培养基的脂蛋白含量和组成，包括apoA-V水平。荧光研究将检查apoA-V在HepG 2和肝细胞中的细胞内定位或运输。远紫外圆二色光谱将用于表征无脂质和脂质相关的apoA-V的解折叠和结构域组织的自由能。将进行apoA-V的化学裂解以获得片段，用于检查蛋白质具有独立折叠结构域的假设。将对N-或C-末端截短变体进行工程改造，并在稳定性、脂质结合和生物学效应方面进行表征。将通过荧光共振能量转移研究评价ApoA-V三级结构和脂质诱导的构象变化。将进行定点诱变以顺序替换apoA-V中的Trp残基，从而产生一组离散的单个Trp apoA-V变体。半胱氨酸204将用外来荧光探针标记，并进行这些供体/受体对之间的距离测定。总之，拟议的研究将采用功能研究和结构表征相结合，以提高我们的理解的机制，apoA-V调节血浆TG水平。