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.
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