Cardiovascular diseases are a major cause of global disability and death worldwide. The prevalence of heart disease is quite high in the Unites States and similar trends are becoming apparent in various Middle Eastern and Gulf region countries such as Qatar. It has been suggested that within 10 years, atherosclerosis and its complications, mainly coronary heart disease, will become the leading cause of death and loss of productive life years worldwide. The statin therapy is one of the most effective strategies for lowering plasma low-density lipoprotein, thereby preventing atherosclerosis and reducing the incidence of heart attacks. The statin group of drugs, which include Zocor, Lipitor, and Crestor, competitively inhibit the activity of the endoplasmic reticulum (ER)-localized enzyme HMG CoA reductase. The reductase catalyzes the reduction of HMG CoA to mevalonate, a rate-determining step in the synthesis of cholesterol as well as essential nonsterol isoprenoids such as dolichol, heme, and the farnesyl and geranylgeranyl groups that are found attached to many cellular proteins. Despite the successful application in curbing cholesterol levels, statin therapy is limited because they disrupt feedback regulation of reductase causing the enzyme to accumulate to high levels. One mechanism for feedback regulation of reductase involves sterol-induced ubiquitination and subsequent degradation of the enzyme from ER membranes. Thus, a complete understanding of molecular mechanisms for reductase degradation will provide targets for novel cholesterol lowering drugs that will aid statins or be a better alternative to prevent atherosclerosis and coronary heart disease. An unresolved aspect of HMG CoA reductase degradation is how the enzyme is removed from ER membranes through a reaction called dislocation and degraded in the cytosol. To answer this, we reconstituted the cytosolic dislocation of reductase in a cell-free system. Characterization of this system revealed that dislocation of reductase from membranes required the in vitro additions of the oxysterol 25-hydroxycholesterol, the nonsterol isoprenoid geranylgeraniol, an energy source, and rat liver cytosol. We were also able to show that dislocation of reductase in the cell-free system was stimulated by two compounds, Apomine and SR-12813 that mimic sterols in stimulating reductase degradation in intact cells. This finding illustrates the utility of our cell-free system in the identification of new drugs that directly stimulate degradation of reductase. Moreover, establishment of the cell-free system positions us to identify proteins in rat liver cytosol required for reductase degradation. Modulating expression of these newly identified proteins may augment reductase dislocation and subsequent degradation, rendering them new therapeutic targets for coronary heart disease.


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