In mammalian cells, the enzyme HMG CoA reductase catalyzes reduction of HMG CoA to mevalonate, a rate-determining step in the synthesis of cholesterol and non-sterol isoprenoids. Reductase is integrated into the ER membrane through an N-terminal domain that contains eight membrane-spanning helices. The C-terminus of reductase projects into the cytosol and exerts catalytic activity. End-products of mevalonate metabolism accelerate ER-associated degradation (ERAD) of reductase as part of a complex feedback system that ensures cholesterol homeostasis. Excess sterols cause binding of the membrane domain of reductase to ER membrane proteins called Insig-1 and Insig-2, resulting in the poly-ubiquitination of reductase. This ubiquitination is obligatory for recognition and delivery of reductase to cytosolic 26S proteasomes for degradation. The reaction has been reconstituted in Drosophila S2 cells by overexpressing the membrane domain of mammalian reductase and Insig-1 or Insig-2. As a model system to study fundamental questions in biology, S2 cells offer a number of advantages. For example, transgenes can be easily overexpressed in S2 cells for study of their function and RNAi is simpler and much more effective in S2 cells than in mammalian cells. To gain further insight into mechanisms for Insig-mediated degradation of reductase, we propose three specific aims: 1) Determine mechanism for sterol-accelerated degradation of mammalian HMG CoA reductase in S2 cells;2) Define role of Hrd1 ubiquitin ligase complex components in sterol-accelerated degradation of reductase in mammalian cells;and 3) Identify novel genes required for degradation of reductase through a genome-wide RNAi screen in S2 cells. Collectively, these studies will provide crucial information regarding mechanisms for degradation of reductase and other polytopic proteins from the ER. In addition, these studies have significant clinical implications. Reductase is the target of statins, a family of widely prescribed drugs that lower blood LDL-cholesterol and reduce the incidence of coronary artery disease. Statins trigger responses that lead to accumulation of active reductase protein, thereby blunting their effects. Part of this increase is due to slowed degradation of reductase. Thus, elucidating mechanisms for the ERAD of reductase may lead to new therapies that increase the effectiveness of statins and ultimately reduce the incidence of heart attacks.

Public Health Relevance

The key enzyme in cholesterol synthesis is HMG CoA reductase, which is tightly controlled through multiple mechanisms that includes regulation of protein stability. Competitive inhibitors of HMG CoA reductase called statins are routinely used to lower blood cholesterol, but they trigger regulatory responses that lead to the accumulation of reductase protein. This grant will investigate the mechanism for the degradation of reductase, the elucidation of which will provide insight into development of therapies to counteract statin-induced accumulation of reductase and improve the effectiveness of the drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM090216-04
Application #
8494639
Study Section
Integrative Nutrition and Metabolic Processes Study Section (INMP)
Program Officer
Gindhart, Joseph G
Project Start
2010-07-01
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
4
Fiscal Year
2013
Total Cost
$296,207
Indirect Cost
$109,914
Name
University of Texas Sw Medical Center Dallas
Department
Genetics
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Schumacher, Marc M; Jun, Dong-Jae; Jo, Youngah et al. (2016) Geranylgeranyl-regulated transport of the prenyltransferase UBIAD1 between membranes of the ER and Golgi. J Lipid Res 57:1286-99
Morris, Lindsey L; Hartman, Isamu Z; Jun, Dong-Jae et al. (2014) Sequential actions of the AAA-ATPase valosin-containing protein (VCP)/p97 and the proteasome 19 S regulatory particle in sterol-accelerated, endoplasmic reticulum (ER)-associated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. J Biol Chem 289:19053-66
Elsabrouty, Rania; Jo, Youngah; Dinh, Tammy T et al. (2013) Sterol-induced dislocation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from membranes of permeabilized cells. Mol Biol Cell 24:3300-8
Fernandes, Nicolle V; Yeganehjoo, Hoda; Katuru, Rajasekhar et al. (2013) Geranylgeraniol suppresses the viability of human DU145 prostate carcinoma cells and the level of HMG CoA reductase. Exp Biol Med (Maywood) 238:1265-74
Jo, Youngah; Hartman, Isamu Z; DeBose-Boyd, Russell A (2013) Ancient ubiquitous protein-1 mediates sterol-induced ubiquitination of 3-hydroxy-3-methylglutaryl CoA reductase in lipid droplet-associated endoplasmic reticulum membranes. Mol Biol Cell 24:169-83
Faulkner, Rebecca A; Nguyen, Andrew D; Jo, Youngah et al. (2013) Lipid-regulated degradation of HMG-CoA reductase and Insig-1 through distinct mechanisms in insect cells. J Lipid Res 54:1011-22
Jo, Youngah; Lee, Peter C W; Sguigna, Peter V et al. (2011) Sterol-induced degradation of HMG CoA reductase depends on interplay of two Insigs and two ubiquitin ligases, gp78 and Trc8. Proc Natl Acad Sci U S A 108:20503-8
Ye, Jin; DeBose-Boyd, Russell A (2011) Regulation of cholesterol and fatty acid synthesis. Cold Spring Harb Perspect Biol 3:
Jo, Youngah; Sguigna, Peter V; DeBose-Boyd, Russell A (2011) Membrane-associated ubiquitin ligase complex containing gp78 mediates sterol-accelerated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. J Biol Chem 286:15022-31