PLASMA LIPOPROTEIN ASSEMBLY AND SECRETION

我们最近发现了脂蛋白中两个潜在的新步骤， 装配途径-apoB与蛋白质的相互作用（指定为 “蛋白X”）在内质网和蛋白质的参与 二硫键异构酶（PDI）在apoB分泌中的作用。 该补助金旨在 检验apoB与蛋白X和PDI相互作用的假设 这些相互作用影响apoB的比例， 是由肝细胞分泌的 我们将探讨蛋白X是否在小鼠apoB分泌中起作用 肝细胞 我们已经发现，共表达的一个结构域， 蛋白X与apoB的结合显著增加了apoB的分泌率， 杆状病毒感染的昆虫细胞，这意味着蛋白质-蛋白质 分泌途径内的相互作用。 此外，我们还发现 apoB和蛋白质X在这些细胞内的物理相互作用。 我们将通过以下小学实验来扩展这些研究。 小鼠肝细胞。 我们将测量apoB分泌率， 正常和蛋白X敲除小鼠的原代小鼠肝细胞。 我们 将研究几种类型的蛋白X对apoB分泌的影响。 我们将研究蛋白质二硫键异构酶 (PDI)参与apoB二硫键形成和apoB分泌。 我们已经发现，共表达一种酶失活突变体， PDI抑制apoB的分泌。 抑制作用是可逆的， 补充高生理浓度（1 mM）的油酸酯。 我们 将与apoB 48共表达PDI活性位点突变体， 发现PDI的哪些酶活性在apoB中起作用 分泌物 我们将直接评估二硫键的形成 为了验证甘油三酯 转移到分泌途径影响二硫键的形成。 我们 将尝试通过以下方法分离PDI-apoB混合二硫化物中间体： 表达缺乏氧化还原活性但具有 shufflase活性 我们会破坏细胞的氧化还原电位 用几种新的二硫醇试剂还原表达apoB 48 接近ER腔的电位，以研究潜在的作用 二硫键形成的脂质收购的新合成的 apoB.

We have recently identified two potential new steps in the lipoprotein assembly pathway-the interaction of apoB with a protein (designated "protein X") in the endoplasmic reticulum and the involvement of protein disulfide isomerase (PDI) in apoB secretion. This grant proposes to test the hypothesis that apoB interacts with protein X and with PDI within the ER and that these interactions affect the proportion of apoB that is secreted from hepatocytes. We shall ask if protein X plays a role in apoB secretion in mouse hepatocytes. We have discovered that co-expression of a domain of protein X with apoB dramatically increases the apoB secretion rate in baculovirus-infected insect cells, implying a protein-protein interaction within the secretory pathway. In addition, we have detected a physical interaction between apoB and protein X within these cells. We shall extend these studies with the following experiments in primary mouse hepatocytes. We shall measure the apoB secretion rate from primary mouse hepatocytes of normal and protein X knockout mice. We shall study the effect of several types of protein X on apoB secretion. We shall investigate the mechanism by which protein disulfide isomerase (PDI) is involved in apoB disulfide bond formation and apoB secretion. We have found that co-expression of an enzymatically inactive mutant form of PDI inhibits apoB secretion. The inhibition is reversible by oleate supplementation at high physiological concentration (1 mM). We shall co-express PDI active site mutants with apoB48 in order to discover which of PDI's enzymatic activities plays a role in apoB secretion. We shall directly assess the formation of disulfide bonds in nascent apoB in order to test the hypothesis that triglyceride transfer to the secretory pathway affects disulfide bond formation. We shall try to isolate a PDI-apoB mixed disulfide intermediate by expressing a mutant form of PDI lacking redox activity but possessing shufflase activity. We shall disrupt the redox potential of cells expressing apoB48 with several novel dithiol reagents with reduction potentials close to that of the ER lumen, to study the potential role of disulfide bond formation in lipid acquisition by newly-synthesized apoB.