In the last few years, it has been demonstrated by this group and others that rat liver peroxisomes participate in cholesterol synthesis. These unexpected findings raise interesting questions about the characteristics of the proteins involved in peroxisomal cholesterol synthesis, the fate of peroxisome-synthesized cholesterol, and the regulation and function of the peroxisomal system.
Aim I of this proposal will focus on the mechanism of the pathway, while aims 2, 3, and 4 deal with the significance and function of this pathway. 1) What parts of the cholesterol biosynthetic pathway are localized within the peroxisomes? Sections of the cholesterol synthesis pathway will be tested in an in vitro system in order to elucidate which parts of the cholesterol biosynthetic pathway are localized within the peroxisomes (in contrast to those that require the presence of cytosolic enzymes). 2) Is cholesterol synthesis defective in cultured Zellweger (a peroxisomal deficiency disease) fibroblast cell lines? A number of biochemical abnormalities all pertaining to peroxisomal metabolism have been described in this disease. The fibroblasts from these patients show the biochemical features of the disease. In studies of peroxisomal diseases significantly low plasma cholesterol levels have been reported. Experiments are designed to measure cholesterol synthesis, ER and peroxisomal HMG-CoA reductase and sterol carrier protein-2 levels in a number of cell lines belonging to different complementation groups. 3) Are peroxisomes involved in cholesterol synthesis in cultured fibroblasts from patients with mevalonate kinase deficiency? Mevalonate kinase deficient cell lines synthesize sterols at a normal rate despite the apparent absence of cytosolic mevalonate kinase activity. Studies are designed to test if these cells contain a novel (peroxisomal) cholesterol synthesis pathway. 4) Is peroxisome-synthesized cholesterol converted to bile acids in vivo? We will test if [14C]-acetyl-CoA produced from [14C]-lignoceric acid (substrate for peroxisomal beta-oxidation) is incorporated into bile acids in vivo under conditions where peroxisomal cholesterol synthesis predominates. Since peroxisomes are essential for life, studies of the peroxisomal pathway of cholesterol biosynthesis may allow us to determine the basis of the organelle defect and the reason for the hypocholesterolemia in peroxisomal disorders. In addition, the study may also provide insights about normal cholesterol and lipoprotein metabolism.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK044350-01
Application #
3245909
Study Section
Medical Biochemistry Study Section (MEDB)
Project Start
1991-09-01
Project End
1994-08-31
Budget Start
1991-09-01
Budget End
1992-08-31
Support Year
1
Fiscal Year
1991
Total Cost
Indirect Cost
Name
San Diego State University
Department
Type
Schools of Arts and Sciences
DUNS #
073371346
City
San Diego
State
CA
Country
United States
Zip Code
92182
Krisans, S K (1996) Cell compartmentalization of cholesterol biosynthesis. Ann N Y Acad Sci 804:142-64
Biardi, L; Krisans, S K (1996) Compartmentalization of cholesterol biosynthesis. Conversion of mevalonate to farnesyl diphosphate occurs in the peroxisomes. J Biol Chem 271:1784-8
Warden, C H; Krisans, S K; Purcell-Huynh, D et al. (1994) Mouse cellular nucleic acid binding proteins: a highly conserved family identified by genetic mapping and sequencing. Genomics 24:14-9
Krisans, S K; Ericsson, J; Edwards, P A et al. (1994) Farnesyl-diphosphate synthase is localized in peroxisomes. J Biol Chem 269:14165-9
Biardi, L; Sreedhar, A; Zokaei, A et al. (1994) Mevalonate kinase is predominantly localized in peroxisomes and is defective in patients with peroxisome deficiency disorders. J Biol Chem 269:1197-205
Stamellos, K D; Shackelford, J E; Shechter, I et al. (1993) Subcellular localization of squalene synthase in rat hepatic cells. Biochemical and immunochemical evidence. J Biol Chem 268:12825-36
Stamellos, K D; Shackelford, J E; Tanaka, R D et al. (1992) Mevalonate kinase is localized in rat liver peroxisomes. J Biol Chem 267:5560-8
Krisans, S K (1992) The role of peroxisomes in cholesterol metabolism. Am J Respir Cell Mol Biol 7:358-64
Hodge, V J; Gould, S J; Subramani, S et al. (1991) Normal cholesterol synthesis in human cells requires functional peroxisomes. Biochem Biophys Res Commun 181:537-41