ASSEMBLY PATHWAY OF VLDL--A THREE STEP PROCESS

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DESCRIPTION (Adapted from applicant's abstract): The investigators wish to develop a model for the assembly of VLDL by the liver. Their studies are designed to define the steps in the assembly of VLDL. It is their hypothesis that there are three steps in this process. Two steps occur in the rough endoplasmic reticulum (ER), producing triglyceride-rich lipoproteins. The third step involves addition of triglyceride and phospholipid to the maturing particle and occurs as the particle is in route to the Golgi or within the Golgi. The studies in this proposal will test the following hypotheses related to these assembly steps: 1) Acyl CoA cholesterol acyltransferase (ACAT) and diacylglycerol acyltransferase (DGAT) provide lipid for the first two assembly steps in the rough ER. 2) Triglyceride and cholesterol ester are transferred to the lumen of the rough ER by a process that does not require microsomal triglyceride transfer protein (MTP). MTP transfers lipid from this lumenal pool. to apoB. The pool is used for bulk core lipid transfer in the second assembly step. 3) MTP associates with apoB on small dense particles in the rough ER as lipid synthesis is augmented. 4) The formation of triglyceride and cholesterol ester promotes translocation of apoB from the rough ER membrane to the lumen. 5) Triglyceride and phospholipid derived from the smooth ER are added to the forming VLDL particle in a distinct third step as the particle moves from the rough ER to the Golgi. 6) ApoB-48 on small lipoprotein particles that have not yet undergone second or possibly third is phosphorylated in the Golgi, protecting it from degradation. These hypotheses will be tested with the following specific aims: 1) To define the steps in the assembly of apoB-100 and apoB-48 containing VLDL. 2) To define the roles of ACAT, DGAT, and MTP in the first two steps of VLDL assembly. 3) To determine and elucidate the role of apoB phosphorylation in the assembly and secretion of apoB-containing lipoproteins. The relevance to human disease is clear because of its relationships to VLDL and LDL metabolism. These experiments will be carried out, for specific aim 1, with rats that will be injected with 35S-methionine or 3H-glycerol. They may also use labeled palmitic acid as a precursor. They will carry out isolated liver profusion studies with these rat livers and isolate lipoproteins. Apolipoproteins will be assessed by SDS-PAGE. The role of ACAT, DGAT, and MTP in the assembly process will be investigated utilizing rough ER fractions, presumably from rat hepatocytes, although it is not stated. They will do additional studies using the MTP inhibitor BMS200150. In specific sub-aim 2.1, they will also use the rough ER fractions as they will for sub-aim 2.2 and 2.3, examining issues of whether MTP associates with apoB in a specific lipoprotein class during active lipid synthesis and lipid transfer. In specific aim 3, where they will examining the role of phosphorylation of apoB and the secretion of apoB lipoproteins will use rat hepatic Golgi apparatus-rich fractions. They will examine the questions about what are the key features of apoB phosphorylation by the Golgi and what the phosphorylation sites actually are. They will ask the question of on what lipoprotein species does this phosphorylation of apoB occur and in which compartment of the Golgi does it occur. They will ask the question of what is the 490 kD protein that is phosphorylated in the Golgi and what is the relationship of this to apoB degradation. They will assess the relationship between phosphorylation of apoB and its secretion and/or degradation. They will use MCA cells for these studies, and they will examine the effects of cAMP-dependent protein kinase inhibitor and look at bisindolylmaleimide as well as okadaic acid, which inhibits protein phosphatase 1A and 2A. Specific methods are spelled out in much greater detail in a separate section on specific methods. Both the rat and the cell culture experiments are very well defined as is the proposed time table for the studies.