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: 6774551
• 负责人: ROBERT O'Mara RYAN
• 金额: $ 40.03万
• 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/6774551
• 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浆脂质稳态的分子基础。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）。小鼠的转基因和基因破坏实验揭示了apoA-V与血浆甘油三酯（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。apoA-V通过影响肝脏脂蛋白的组装或分泌来调节血浆TG水平的假说将在敲除apoA-V和转基因小鼠的原代肝细胞以及培养的人肝癌细胞中进行测试。研究将评估apoA-V在肝细胞中的表达对富含tg的脂蛋白的组装或分泌的影响程度。从培养细胞的条件培养基中分析脂蛋白含量和组成，包括apoA-V水平。荧光研究将检查apoA-V在HepG2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和脂相关apoA-V的展开和结构域组织的自由能。将对apoA-V进行化学切割以获得片段，以检验该蛋白质具有独立折叠结构域的假设。N端或c端截断变异将在稳定性、脂质结合和生物效应方面进行设计和表征。ApoA-V的三级结构和脂质诱导的构象变化将通过荧光共振能量转移研究来评估。位点定向诱变将依次替换apoA-V中的Trp残基，从而产生一组离散的单Trp apoA-V变体。半胱氨酸204将用外部荧光探针标记，并确定这些供体/受体对之间的距离。综上所述，拟议的研究将采用功能研究和结构表征相结合的方法来提高我们对apoA-V调节血浆TG水平的机制的理解。