The plasma level of apoprotein B (apoB) is among the strongest risk factors for coronary artery disease;thus, understanding the regulation of apoB-lipoprotein production is fundamentally interesting, clinically relevant, and may ultimately suggest new therapeutic approaches to dyslipidemias. ApoB100 is the form of apoB made by human liver and is the predominant protein component of atherogenic very low (VLDL) and low density (LDL) lipoproteins. Secretion of apoB100 from hepatic cells is controlled primarily by pre-secretory degradation. We have been at the forefront of defining apoB100 degradative pathways, and in this proposal focus on 1) endoplasmic reticulum-associated degradation (ERAD), which is mediated by the proteasome;and, 2) post- ER, presecretory proteolysis (PERPP), which is stimulated by dietary fatty acids used clinically to reduce VLDL levels, and which we believe is mediated by autophagy.
In aim 1, we propose to characterize the process that targets nascent apoB for ERAD. We have previously shown that this process requires a distinct cohort of chaperones and chaperone-like proteins. We will continue to identify the factors that control apoB biogenesis in the ER using in vitro and in vivo assays, as well as a new apoB yeast expression system.
In aim 2, we propose to determine the role of autophagy in post-ER apoB100 turnover under basal and perturbed metabolic states. Based on our recent data, we hypothesize that autophagy regulates the degradation of apoB100 under basal conditions and when hepatic cells are incubated with fish oil fatty acids (such as DHA) or other polyunsaturated fatty acids (PUFA) that lower VLDL levels. To address this hypothesis, we will study apoB100 turnover in hepatic cells and in mice in which the activities of specific autophagic factors have been manipulated. Further, based on the characteristics of insulin-mediated apoB degradation, we will also test the hypothesis that autophagy and the insulin-responsive pathways intersect to modulate apoB100 metabolism.
In aim 3, we propose to determine the oxidant(s) responsible for apoB100 degradation and the effects of reactive oxygen species (ROS) on the VLDL assembly process. When hepatic cells are incubated with DHA and other PUFA, ROS and lipid peroxidation increase and apoB100 becomes damaged, aggregated, and targeted for autophagy. Based on our preliminary data, we hypothesize that superoxide (SO) plays a central role in apoB100 aggregation/degradation. We will test this hypothesis in hepatic cells and mice with genetic alterations in oxidant pathways. Other preliminary data suggest that PUFA decrease the amount of fully matured VLDL particles in the Golgi. By combining pulse-chase studies with sub-cellular fractionation and lipid analytical techniques, we will determine whether this results from aborted VLDL assembly or from the rapid targeting of VLDL to autophagy after assembly. In summary, these studies will lead to a more detailed molecular understanding of the intracellular metabolism of apoB100 and will contribute to the development of methods to control the production of atherogenic lipoproteins in normal and pathological states.
Atherosclerosis is caused by the accumulation of particles (lipoproteins) containing cholesterol. The major source of these particles is the liver, which assembles the cholesterol with a protein, apoB, which is absolutely required for formation of and exit from the liver of the particles. We have identified ways by which the liver can destroy apoB and thereby reduce the release of the atherosclerosis-causing particles into the blood stream. The proposal's main goal is to uncover the molecules that regulate this destruction, so that ultimately, new targets for therapy to lower heart disease risk can be developed.
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