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Analysis of Novel Genes of the Mevalonate Pathway;
Role of Peroxisomes in Cholesterol Metabolism
The isoprenoid biosynthetic pathway, is unrivaled
in nature for the chemical diversity of the compounds it produces. Products from
this pathway include all metabolically produced isoprene-containing compounds and
sterols which provide chemical signals (hormones, pheromones) as well as structural
components of enzymes (coenzyme Q and the isoprenoid moiety of heme a), and
membranes (cholesterol, ergosterol). Mevalonate is also the precursor for farnesyl
diphosphate (FPP) and geranyl-geranyl diphosphate (GGPP), both of which are required
for the isoprenylation of various G proteins, most notable of which is the product
of the ras gene, p21, a key transducer of mitogenic signals. The diversity of the
isoprenoid products and their perturbation in cellular signaling, development and
metabolic regulation suggests: 1) that regulation of isoprenoid biosynthesis has
a pervasive influence on cell function and 2) there must be an intricate balance
through which regulatory isoprenoid molecules are derived from a single biosynthetic
pathway. It is to these two major proposals that our research is directed.
In mammalian cells, 3-hydroxy-3-methylglutaryl coenzyme
A (HMG-CoA) reductase is the rate-limiting enzyme for the synthesis of mevalonic
acid, the precursor of cholesterol and other non sterol isoprenoids. Because of its
role in cholesterol biosynthesis, the regulation of HMG-CoA reductase has been intensely
studied. The levels of the endoplasmic reticulum (ER) enzyme are governed by regulation
of transcription, translation, and enzyme degradation. Another critical role for
this enzyme has emerged in recent years, due to the requirement of FPP and GGPP in
isoprenylation of proteins.
Analysis of a Novel HMG-CoA Reductase: We
and others have demonstrated that HMG-CoA reductase is localized in two distinct
intracellular compartments, ER and peroxisomes. No information is available regarding
the function of the peroxisomal reductase in cholesterol/isoprenoid metabolism and
the structure of the peroxisomal HMG-CoA reductase has yet to be determined. Accordingly,
to facilitate our studies of the function and regulation of the peroxisomal HMG-CoA
reductase and to determine its structure we have developed a mammalian cell line
that expresses only one HMG-CoA reductase protein of 90 kDa and which is localized
exclusively to peroxisomes. These cells provide a model system to study the peroxisomal
HMG-CoA reductase independent of the ER reductase. The wild type CHO cells, contain
two HMG-CoA reductase proteins, the well characterized 97 kDa protein, localized
in the endoplasmic reticulum, and a 90 kDa protein localized in peroxisomes. Thus,
our specific aims for this project are: 1) to study the regulation and function of
the peroxisomal HMG-CoA reductase in this cell line; and 2) to isolate a cDNA encoding
the peroxisomal HMG-CoA reductase.
Regulation of Cellular FPP Levels: FPP is a
key intermediate that serves as a substrate for a number of critical branch-point
enzymes, thus, the regulation and levels of FPP are important since large perturbations
in FPP could alter the flux of isoprenoid compounds down the various branches of
the pathway. We have recently made the significant finding that the entire pathway
for the synthesis of FPP from mevalonate is localized in peroxisomes. This means
that the cell's FPP is produced in the peroxisomes. Thus, FPP and/or farnesol has
to be transported out of peroxisomes for further metabolism. In addition, phosphorylated
products of mevalonate and isopentenyl are not able to cross the peroxisomal membrane.
This implies that FPP is also impermeable and has to be transported out of the peroxisome.
Therefore, we propose that regulated transport of FPP from its site of synthesis
in peroxisomes into the cytoplasm plays a fundamental regulatory role in the utilization
of FPP for sterol and non-sterol products. Thus, the major aims of this project are
designed to answer important questions regarding the cellular levels, regulation
and transport of FPP. The techniques utilized include protein biochemistry, cell
biology and molecular genetics.
The Zellweger Mouse Model: The cerebro-hepato-renal
syndrome of Zellweger is a fatal disease caused by deficient import of peroxisomal
matrix proteins. The pathogenic mechanisms leading to death are unknown. Cholesterol
levels and peroxisomal enzymes required for cholesterol biosynthesis are decreased
in tissues from patients diagnosed with peroxisomal deficiency diseases. All of these
diseases have severe neurological abnormalities whose pathophysiology is not known
but may be related to cholesterol, since recent studies have shown that cholesterol
is critical for normal brain development. However, very little information is available
regarding cholesterol biosynthesis or compartmentalization of isoprene metabolism
in the CNS.
We have recently demonstrated that in peroxisome assembly
deficient Chinese Hamster Ovary cells (PEX2 mutants) the levels of cholesterol and
the rates of cholesterol and dolichol biosynthesis are significantly reduced. Now,
a peroxisomal PEX2 knockout mouse (Zellweger syndrome) has become available. These
PEX2 deficient mice will provide an excellent model for studying the role of peroxisomal
function in isoprenoid metabolism. Analysis of the PEX2 deficient animals may render
insight into the mechanism(s) responsible for the neurological phenotype seen in
the knockout mice.
We propose to: 1) Undertake a detailed analysis of
the isoprenoid/cholesterol biosynthesis pathway in neonatal brain tissue; 2) Test
for defects in lipid composition and cholesterol/dolichol metabolism in neurological
tissues obtained from PEX2 deficient mice; and 3) Investigate if decreased cholesterol
levels in PEX2 deficient mice impairs the sonic hedgehog signaling pathway.
Representative Publications
Westfall, D. Aboushadi, N. Shackelford, J. E., and
Krisans, S. K. Metabolism of Farnesol: Phosphorylation of Farnesol by Rat
Liver Microsomal and Peroxisomal Fractions. Biochem. Biophys. Res. Commun.
230:562-568. 1997.
Paton, V. G., Shackelford, J. E., and Krisans,
S. K. Cloning and Subcellular Localization of Hamster and Rat Isopentenyl Diphosphate
Dimethallyl Diphosphate Isomerase: A PTS1 Motif Targets the Enzyme to Peroxisomes.
J. Biol. Chem. 272:18945-18950. 1997.
Engfelt, W. H., Shackelford J. E., Aboushadi, N.,
Jessani, N., Masuda, K., Paton, V. G., Keller, G. A. and Krisans, S. K. Characterization
of UT2 Cells: The Induction of Peroxisomal 3-Hydroxy-3-Methyl- Glutaryl-Coenzyme
A Reductase. J. Biol. Chem. 272: 24579-24587. 1997.
Hinson, D. D., Chambliss, K. L., Hoffmann, G. F.,
Krisans, S. K., Keller, R. K., and Gibson, K. M. Identification of an Active
Site Alanine in Mevalonate Kinase Through Characterization of a Novel Mutation in
Mevalonate Kinase Deficiency. J. Biol. Chem. 272:26756-26760. 1997.
Aboushadi, N. and Krisans, S.K. Analysis of
Isoprenoid Biosynthesis in Peroxisomal Deficient CHO Cell Lines. J. Lipid Res.
39:1781-1791. 1998.
Engfelt, W.H., Masuda, K.R., Paton, V.G. and Krisans,
S.K. Splice Donor Site Mutations in the 3-Hydroxy-3-Methylglutaryl Coenzyme A
Reductase Gene Causes a Deficiency of the Endoplasmic Reticulum 3-Hydroxy-3-Methylglutaryl
Coenzyme A Reductase Protein in UT2 Cells. J. Lipid. Res. 39:2182-2191.
1998.
Olivier, L. M., Chamblis, K. L., Gibson, K. M., and
Krisans, S. K. Characterization of Phosphomevalonate Kinase: Chromosomal Localization,
Regulation, and Subcellular Targeting. J. Lipid. Res. 40:672-679. 1999.
Aboushadi, N., Engfelt, W. H., Paton, V., G. and Krisans,
S. K. Role of Peroxisomes in Isoprenoid Biosynthesis. J. Histochem. Cytochem.
47:1127-1132. 1999.
Hinson, D. D., Ross, R. M., Krisans. S. K.,
Shaw, J. L., Kozich, V., Rolland, M. O., Divry, P., Mancicni, J., Hoffmann, G. F.,
and Gibson, K. M. 1999. Itentification of a Mutation in Mevalonate kinase Deficiency
Including a New Mutation in a Patient of Memmonite Ancestry. Am J. Human. Genet.
65:327-335. 1999.
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