JAMES E. METHERALL, Ph.D.

LAB WEBSITE

B.A. 1981, University of Vermont
M.B.A. 1998, University of Utah
PhD. 1986, Yale University

RESEARCH:

Our lab studies the cellular and molecular processes that control cholesterol metabolism and how these processes relate to coronary heart disease and other diseases of cholesterol metabolism. Our work uses a wide range of approaches including biochemical and metabolic assays, human genetics, somatic cell genetics, and mammalian expression cloning.

Mammalian cells maintain exquisite control over the level of free cholesterol within the cell by a classic end-product feedback mechanism. Elevated sterol levels suppress transcription of the genes for the first two enzymes of the cholesterol biosynthetic pathway: HMG CoA synthase and HMG CoA reductase. In addition, sterols suppress transcription of the gene for the LDL receptor, a cell surface receptor that mediates the uptake of cholesterol-rich lipoprotein particles. A DNA binding protein (SREBP) interacts with the promoter of each of these genes to regulate transcription. The DNA binding protein is syntehsized as a transmembrane precursor anchored to the endoplasmic reticulum (ER). In the absence of sterols, the precursor is protealized releasing the DNA binding portion of the protein which enters the nucleus and activates transcription of the three sterol-regulated genes. When cholesterol levels rise, a conformational change in SREBP renders it resistant to proteolysis and transcription of the three genes decreases.

We have isolated mutant cell lines (SRD-1,2,3) with gene rearrangements that truncate the DNA binding protein, thereby eliminating the transmembrane domain. The truncated proteins are free to enter the nucleus and activate transcription even in the presence of sterols. We have also isolated a mutant cell line (SRD-6) that lacks the protease activity. In SRD-6 cells, the DNA binding protein remains trapped in the ER, and therefore fails to activate transcription even in the absence of sterols. In addition, we have isolated a mutant cell line (SRD-7) that fails to deliver cholesterol to the ER. While cholesterol is found predominantly in the plasma membrane, the triggering event for proteolysis and many other important processes involved in cholesterol metabolism occur in the ER. SRD-7 cells accumulate cholesterol-rich vesicles in the cytoplasm suggesting that vesicular trafficking normally delivers cholesterol to the ER. We have demonstrated that this transport process also requires the activity of a known protein, the multiple-drug resistance pump (MDR). We are currently investigating the mechanism by which MDR functions in this process. Another of our mutant lines lacks acyl CoA:cholesterol acyltransferase : (ACAT), an important enzyme involved in coronary heart disease.

Having identified mutants with specific defects in cholesterol metabolism, we use mammalian expression cloning approaches to isolate the genes that are defective in these cells. In addition to the mutant cell lines developed in our laboratory, we are also using expression cloning approaches to isolate the genes responsible for two diseases of cholesterol metabolism. Smith-Lemli-Opitz (SLO) disease is the most common form of inherited mental retardation. SLO is an autosomal recessive disorder resulting from a genetic defect in an enzyme required for cholesterol biosynthesis. Neimann-Pick Type C (NP-C) disease is a less common autosomal recessive disorder that results form improper intracellular cholesterol transport and cholesterol accumulation in tissues. In order to clone these disease genes and other genes involved in cholesterol metabolism, we have developed mammalian expression cloning approaches that utilize cDNA expression libraries and extensive automated robotics facilities. A number of these expression cloning projects are currently underway in the laboratory.

SELECT PUBLICATIONS:

Hoshijima, K., Metherall, J.E., Grunwald, D.J. (2002) A Protein Disulfide Isomerase Expressed in the Embryonic Midline is Required for Left/Right Asymmetries. Genes and Development 16, 2518-2529.

Simin, K., Scuderi, A., Reamey, J., Dunn, D., Weiss, R., Metherall, J.E., Letsou, A. (2002) Profiling Patterned Transcripts in Drosophila Embryos. Genome Research 12, 1040-1047. (see commentary: Oliver, B. (2002) Fly Factory. Genome Research 12, 1017-1018.)

Gao, Z.-H., Metherall, J.E., Virshup, D. M. (1999) Library Screening to Identify Casein Kinase I Substrates. Identification of Casein Kinase I Substrates by in vitro Expression Cloning (IVEC) Screening. Biochem. Biophys. Res. Com. 268, 562-566.

Elsea, S. H., Mykytyn, K., Ferrell, K., Das, P., Dubiel, W., Patel, P. I., Metherall, J. E. (1999) The COP9 Signalosome Subunit Gene, SGN3, Maps within the Smith-Magenis Syndrome Critical Interval. Am. J. Med. Gen. 87, 342-348.

Neklason, D. W., Kelley, R., Metherall, J. E. (1999) Biochemical Variants of 7-Dehydrocholesterol Reductase Deficiency. Am. J. Med. Genetics 85, 517-523.

DeBry, P., Nash, E. A., Neklason, D., Metherall, J. E. (1997) Role of Multidrug Resistance (MDR) P-Glycoproteins in Cholesterol Esterification. J. Biol. Chem. 272, 1026-1031.

Jackson, S. M., Ericsson, J., Goto, A., Metherall, J. E., and Edwards, P. A. (1996) Sterol Regulatory Element Binding Protein is Involved in the Regulation of Farnesyl Diphosphate Synthase and Cholesterol and Fatty Acid Synthesis: Evidence from Sterol Regulation-Defective Cell Lines. J. Lipid Research 37, 1712-1721.

Metherall, J. E., Waugh, K., Li, H. (1996) Progesterone Inhibits Cholesterol Biosynthesis: Accumulation of Sterol Precursors J. Biol. Chem. 271, 2627-2633.

Metherall, J. E., Li, H., Waugh, K. (1996) Role of Multidrug Resistance (MDR) P-Glycoproteins in Cholesterol Biosynthesis. J. Biol. Chem. 271, 2634-2640.

Evans, M. J., Metherall, J. E. (1993) Loss of Transcriptional Repression of Three Sterol regulated Genes in Mutant Hamster Cell Lines. Mol. Cell. Biol. 13, 5175-5185.

Naglich, J., Metherall, J. E., Russell, D. W., Eidels, L. (1992) Expression Cloning of a Diphtheria Toxin Receptor : Identity with a Heparin-Binding EGF-Like Growth Factor Precursor. Cell 69, 1051-1061.

Metherall, J. E., Ridgway, N. D., Dawson, P. A., Goldstein, J. L., Brown, M. S. (1991) A 25-Hydroxycholesterol Resistant Cell Line Deficient in Acyl CoA:Cholesterol Acyltransferase J. Biol. Chem. 266, 12734-12740.
Dawson, P. A., Metherall, J. E., Ridgway, N. D., Brown, M. S., Goldstein, J. L. (1991) Separation of Transcriptional and Post transcriptional Regulation of 3 hydroxy 3 methylglutaryl Coenzyme A Reductase in Mutant Hamster Cells. J. Biol.Chem. 266, 9128-9134.

Metherall, J. E., Goldstein, J. L., Luskey, K. L., Brown, M. S. (1989) Loss of Transcriptional Repression of Three Sterol regulated Genes in Mutant Hamster Cell Lines. J. Biol. Chem. 264, 15634 15641.

Glazer, P. M., Greggio, N. A., Metherall, J. E., Summers, W. C. (1989) UV induced DNA binding Proteins in Human Cells. Proc. Natl. Acad. Sci. 86, 1163 1167.

Metherall, J. E., Gillespie, F. P. and Forget, B.G. (1988) Analysis of Linked ?- Globin Genes Suggests that Non deletion forms of Hereditary Persistence of Fetal Hemoglobin are bona fide Switching Mutants. Am. J. of Human Genetics 42, 476 481.

Stoeckert, C. J., Metherall, J. E., Yamakawa. M., Forget, B. G., Eisenstadt, J. M. and Weissman, S. M. (1987) Elevated Expression of a Greek Hereditary Persistence of Fetal Hemoglobin A? Gene in a Human Erythroid Cell Line. Mol. Cell. Bio. 7, 2999 3003.

Metherall, J. E., Collins, F. S., Pan, J., Weissman, S. M. and Forget, B. G. (1986) ?0 Thalassemia Caused by a Base Substitution that Creates an Alternate Splice Acceptor Site. EMBO J. 5, 2551 2557.

Collins, F. S., Metherall, J. E., Yamakawa, M., Pan, J., Weissman, S. M. and Forget, B. G. (1985) A Point Mutation in the A?-Globin Gene Promoter in Greek Hereditary Persistence of Fetal Hemoglobin. Nature 313, 325 326.