The tong-term goal of this project is to utilize somatic cell mutants in cholesterol metabolism and macrophage cells to delineate various intracellular cholesterol trafficking events that are relevant to cellular cholesteryl ester accumulation and macrophage foam cell formation in human atherosclerosis. For the proposed granting period, our goals are: (1) To examine the elements involved in endosomal cholesterol trafficking in mammalian cells. (2) To investigate the mechanism by which aggregated lipoproteins cause the resident ER enzyme acyl-coenzyme A: cholesterol acyltransferase 1 (ACAT1) to become more accessible to cholesterol present in various cholesterol-rich compartments.
The specific aims are: 1. To identify the elements involved in late endosomal cholesterol trafficking. We will design experiments to address the following questions: a. Does endoCHOL play important roles in maintaining the functions of late endosome in a manner not replaceable by LDL-derived cholesterol? b. Does intracellular caveolin 1 play important roles in late endosomal cholesterol trafficking? 2. To demonstrate NPC1 as a sterol sensing protein in late endosomes. We will design experiments to address the following questions: a. Is the intact cell cross-linking between NPC1 and azocholestanol affected by the presence of NPC2 and/or caveolin 1? b. Does azocholestanol bind to the NPC1 within the sterol sensing domain of NPC1? c. Do NPC1, NPC2, and/or caveolin1 form a protein complex in vitro? Is the in vitro cross-linking between NPC1 and the azocholestanol affected by the presence of other ligands such as fatty acids or glycosphingolipids? ? 3. To test the possibility that an increase in ER fragmentation/translocation is an early response to certain atherogenic lipoproteins in macrophages. We will design experiments to address the following question: Do aggregated LDLs and latex beads share the same ability to stimulate the endoplasmic reticulum (ER) fragmentation and translocation process in macrophage cells? ? ?
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