Supernatant protein factor stimulates HMG-CoA reductase in cell culture and in vitro.
Supernatant protein factor (SPF) is a 46-kDa cytosolic protein that stimulates squalene monooxygenase in vitro and, unexpectedly, cholesterol synthesis in cell culture. Because squalene monooxygenase is not thought to be rate-limiting with regard to cholesterol synthesis, we investigated the possibility that SPF might stimulate other enzymes in the cholesterol biosynthetic pathway. Substitution of [(14)C]mevalonate for [(14)C]acetate in McARH7777 hepatoma cells expressing SPF reduced the 1.8-fold increase in cholesterol synthesis by half, suggesting that SPF acted on or prior to mevalonate synthesis. This conclusion was supported by the finding that substitution with [(14)C]mevalonate completely blocked an SPF-induced increase in squalene synthesis. Evaluation of 2,3-oxidosqualene synthesis from [(14)C]mevalonate demonstrated that SPF also stimulated squalene monooxygenase (1.3-fold) in hepatoma cells. Immunoblot analysis showed that SPF did not increase HMG-CoA reductase or squalene monooxygenase enzyme levels, indicating a direct effect on enzyme activity. Addition of purified recombinant SPF to rat liver microsomes stimulated HMG-CoA reductase by about 1.5-fold, and the SPF-concentration/activation curve paralleled that for the SPF-mediated stimulation of squalene monooxygenase. These results reveal that SPF directly stimulates HMG-CoA reductase, the rate-limiting step of the cholesterol biosynthetic pathway, as well as squalene monooxygenase, and suggest a new means by which cholesterol synthesis can be rapidly modulated in response to hormonal and environmental signals.
Mokashi
Vishwesh
V
Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40536, USA.
Singh
Dev K
DK
Porter
Todd D
TD
D019342:Acetic Acid / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000378:metabolism*; D000818:Animals; D002250:Carbon Radioisotopes; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival; D002784:Cholesterol / Q000096:biosynthesis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008798:Mevalonic Acid / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D008970:Molecular Weight; D010105:Oxygenases / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013185:Squalene / Q000378:metabolism; D050603:Squalene Monooxygenase; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000214:Acyl Coenzyme A; D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D008074:Lipoproteins; D011994:Recombinant Proteins; C486622:Sec14l2 protein, rat; D015534:Trans-Activators; C008047:3-hydroxy-3-methylglutaryl-coenzyme A; D013185:Squalene; D002784:Cholesterol; D010105:Oxygenases; D050603:Squalene Monooxygenase; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019342:Acetic Acid; D008798:Mevalonic Acid