APO B TRANSLOCATION AND DEGRADATION

我们的研究将集中在确定细胞内过程 负责调节载脂蛋白B的肝脏分泌， 血浆LDL apo B的来源，其与动脉粥样硬化形成密切相关。 我们将检验载脂蛋白B跨膜转运的假设， 内质网，需要一个伴侣，并决定代谢 载脂蛋白B的命运：分泌或细胞内降解。实现这一目标 我们提出以下具体目标： (1)为了确定易位是否决定载脂蛋白B的含量， 降解或降解决定了多少载脂蛋白B被易位。 我们最近的研究结果表明，蛋白水解抑制剂ALLN阻断了 非肝CHO细胞和肝癌细胞中apo B的降解。 阻断载脂蛋白B的降解将使我们能够确定 易位通常是饱和的。 (2)为了确定外源性apo B的过表达是否降低了 通过竞争易位分泌内源性载脂蛋白B。 表达载脂蛋白B，由转染到肝细胞中的质粒编码， 并在转基因小鼠的肝脏中表达， 内源性载脂蛋白B。我们将研究这是否是 竞争的限速过程，如果是这样，确定 竞争发生的细胞内位点。 (3)为了确定载脂蛋白B的过度表达是否会导致 载脂蛋白B与分子伴侣和/或其它蛋白质。 过量表达外源形式的载脂蛋白B会使蛋白饱和 参与载脂蛋白B分泌途径，并驱动其平衡 与载脂蛋白B相关。交联型载脂蛋白的分离与鉴定 B复合物将为阐明分子细节打开新的窗口 参与apo B分泌。 (4)表征负责降解载脂蛋白B形式的蛋白酶 没有完全转移到内质网上。 确定负责apo B降解的酶将提供新的 了解这个过程是如何被监管的。激活此过程 可能提供一种减少载脂蛋白B分泌、降低血浆LDL水平的方法 减少肠道脂肪吸收。

Our research will focus on defining the intracellular processes responsible for regulating hepatic secretion of apo B, the principal source of plasma LDL apo B, which is intimately linked to atherogenesis. We will examine the hypothesis that translocation of apo B across the endoplasmic reticulum, requires a chaperon and determines the metabolic fate of apo B: secretion or intracellular degradation. To attain this goal we propose the following specific aims: (1) To determine whether translocation determines how much apo B is degraded or if degradation determines how much apo B is translocated. Our recent results show that the proteolytic inhibitor ALLN blocks the degradation of apo B in both non-hepatic CHO cells and hepatoma cells. Blocking the degradation of apo B will allow us to determine if translocation is normally saturated. (2) To determine if over-expression of exogenous apo B decreases the secretion of endogenous apo B by competition for translocation. Expression of apo B, encoded by plasmids transfected into hepatic cells, and expressed in the livers of transgenic mice decreases the secretion of the endogenous forms of apo B. We will examine if this is the result of competition for a rate-limiting process and, if so, determine the intracellular site where this competition occurs. (3) To determine if over-expression of apo B leads to the association of apo B with chaperons and/or other proteins. Over-expression of exogenous forms of apo B will saturate the proteins involved in the apo B secretion pathway and drive the equilibrium of their association with apo B. Isolation and characterization of cross-linked apo B complexes will open new windows illuminating the molecular details involved in apo B secretion. (4) To characterize the protease responsible for degrading forms of apo B that are not fully translocated across the endoplasmic reticulum. Identifying the enzyme responsible for apo B degradation will provide new insights into how this process is regulated. Activation of this process may provide a means to decrease apo B secretion, lower plasma LDL levels and reduce intestinal fat absorption.