Creation and activity of COS-1 cells stably expressing the F2 fusion of the human cholesterol side-chain cleavage enzyme system.
A fusion construct for the human cholesterol side-chain cleavage enzyme system termed F2 (H(2)N-P450scc-adrenodoxin reductase-adrenodoxin-COOH), was stably expressed in nonsteroidogenic COS-1 cells. Multiple clones were obtained and analyzed, identifying the clone COS-F2-130 as the most active in converting 22R-hydroxycholesterol (22R-OH-C) to pregnenolone. The F2 fusion construct was properly transcribed and translated in COS-F2-130 cells, indicating that these cells did not proteolytically cleave the F2 protein. Steroid analyses show that the COS-F2-130 cells do not convert appreciable quantities of pregnenolone to other steroids. Isolated COS-F2-130 mitochondria showed enhanced steroidogenesis when incubated with biosynthetic N-62 StAR protein in vitro. The cells were easily transfectable with StAR expression vectors, showing that COS-F2-130 cells exhibited both StAR-independent and StAR-dependent activity. Transient expression of either full-length or N-62 StAR stimulated steroidogenesis to approximately 45% of the maximal steroidogenic capacity, as indicated by incubation with 22R-OH-C. Single, double, and triple transfections of individual vectors expressing P450scc, adrenodoxin reductase, and adrenodoxin demonstrated that the P450 moiety of the F2 fusion protein could only receive electrons from the covalently linked adrenodoxin moiety, but that free adrenodoxin reductase could foster activity of the fusion enzyme. COS-F2-130 cells provide a useful system for studying steroidogenesis, as these are the only cells described to date that convert cholesterol to pregnenolone but lack downstream enzymes that catalyze other steroidogenic reactions.
Huang
M C
MC
Department of Pediatrics and the Metabolic Research Unit, University of California, San Francisco 94143-0978, USA.
Miller
W L
WL
D000325:Adrenodoxin / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells / Q000201:enzymology*; D002250:Carbon Radioisotopes; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics* / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D006888:Hydroxycholesterols / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D010750:Phosphoproteins / Q000235:genetics / Q000502:physiology; D011284:Pregnenolone / Q000096:biosynthesis / Q000378:metabolism; D011374:Progesterone / Q000096:biosynthesis; D012313:RNA / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013256:Steroids / Q000096:biosynthesis; D014162:Transfection; D000098682:Steroidogenic Acute Regulatory Protein
D002250:Carbon Radioisotopes; D006888:Hydroxycholesterols; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D013256:Steroids; D000098682:Steroidogenic Acute Regulatory Protein; D000325:Adrenodoxin; C003585:22-hydroxycholesterol; D011374:Progesterone; D012313:RNA; D011284:Pregnenolone; D002786:Cholesterol Side-Chain Cleavage Enzyme; D005287:Ferredoxin-NADP Reductase