Apolipoprotein A-V: A Functional Proteomics Study

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导致了可交换载脂蛋白家族的一个新成员-载脂蛋白A-V(apoA-V)的发现。小鼠的转基因和基因阻断实验表明，载脂蛋白A-V和血浆三酰甘油(TG)水平之间存在相关性。鉴于血浆甘油三酯升高与心血管疾病风险之间的密切关系，载脂蛋白A-V是一个潜在的治疗干预靶点。ApoA-V通过影响肝脂蛋白组装或分泌来调节血浆甘油三酯水平的假设将在apoA-V基因敲除和转基因小鼠的原代肝细胞以及培养的人肝癌细胞中得到验证。研究将评估载脂蛋白A-V在肝细胞中的表达对富含甘油三酯的脂蛋白的组装或分泌的影响程度。来自培养细胞的条件培养液将被分析脂蛋白的含量和组成，包括载脂蛋白A-V水平。荧光研究将检测apoA-V在细胞内的定位或在HepG2和肝细胞中的运输。远紫外圆二色谱将被用来表征无脂和脂联apoA-V的展开自由能和结构域组织。将进行apoA-V的化学切割以获得片段，以检验蛋白质具有独立折叠结构域的假设。将设计N-末端或C-末端截断变体，并从稳定性、脂质结合和生物学效应方面对其进行表征。载脂蛋白A-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。将进行定点突变以顺序替换apoA-V中的Trp残基，从而产生一组离散的单一Trp apoA-V变体。半胱氨酸204将用外在荧光探针标记，并确定这些供体/受体对之间的距离。总之，拟议的研究将采用功能研究和结构表征相结合的方式，以提高我们对载脂蛋白A-V调节血浆甘油三酯水平的机制的理解。

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.