James E. Metherall, Ph.D.
Department of Human Genetics
Program in Human Molecular Biology and Genetics
The Huntsman Cancer Institute

Our lab studies the cellular and molecular processes that control cholesterol metabolism and how these processess 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 synthesized 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 from 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.