Molecular cloning and sequence analysis of cDNA encoding δ<sup>4</sup>‐3‐ketosteroid 5β‐reductase of rat liver

Onishi, Yoshiaki; Noshiro, Mitsuhide; Shimosato, Tsunehiro; Okuda, Kyuichiro

doi:10.1016/0014-5793(91)80591-p

Cited by 47 publications

(35 citation statements)

References 18 publications

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“…4, panel B). Conversely, anti-37-kd protein IgG reacted D 4 -3-ketosteroid 5b-reductase as reported by Onishi et al 20 with liver extracts from both the control and the alcohol-fed rats as well as the purified 37-kd protein ( 2 and 4). The antibody also recognized the recombinant protein (Fig.…”

supporting

confidence: 68%

“…3) was found to be identical to that of D 4 -3-ketosteroid 5b-reductase. 20 In addition to the sequence of PEP 1, from which degenerate oligonucleotide sequences were designed to screen the rat liver lgt11 cDNA library, the Table 1. However, its sequence differed from PEP 3…”

Section: Discussionmentioning

confidence: 99%

“…The phosphate/min at 25ЊC), representing a 645-fold increase over final identification of the 37-kd protein that can form crude rat liver soluble protein. For some experiments, the 37-kd liver protein was purified by chromatography through protein-AA in vivo was achieved herein by a molecular a DE52 column 20 before gel filtration chromatography (Sephcloning approach as described in this article.…”

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confidence: 99%

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Identification of the 37-kd rat liver protein that forms an acetaldehyde adductin vivo as ?4-3-ketosteroid 5?-reductase

et al. 1996

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After alcohol ingestion, ethanol is oxidized to acetalAcetaldehyde, the first product of alcohol metabolism, is highly reactive. Several proteins have been shown to dehyde in the liver mainly by the action of alcohol dehybe covalently modified by acetaldehyde in vivo. We have drogenase 1 and, on prolonged alcohol consumption, by previously reported the detection of a cytosolic 37-kd microsomal cytochrome P450IIE1. 2 Little acetaldehyde protein-acetaldehyde adduct (-AA) in the liver of alcohol-accumulates during alcohol oxidation because this infed rats. The liver extract from an alcohol-fed rat was termediate is rapidly converted to acetate by aldehyde subjected to 2-dimensional (2D) sodium dodecylsulfate dehydrogenase. Nevertheless, acetaldehyde is highly (SDS)-polyacrylamide gel electrophoresis (PAGE), trans-reactive. For many years, investigators have shown ferred to polyvinylidene difluoride (PVDF) membrane, that acetaldehyde can covalently modify proteins in and the 37-kd protein-AA spot was digested with trypsin vitro to form protein-acetaldehyde adducts (-AAs). 3 The and sequenced for amino acids. Degenerate oligonucleoformation of protein-AAs in vivo has been demontides corresponding to a peptide sequence of the proteinstrated more recently. 4-9 Our laboratory was the first AA were used as the probe to screen a lgt11 rat liver to report the detection of a liver protein-AA (apparent complementary DNA (cDNA) library. A clone that extended to a potential ATG start codon was identified. MW, 37,000) by Western immunoblot in rats fed alcohol The open reading frame was 978 nucleotides long, encod-chronically. 4 The 37-kd liver protein-AA can be deing 326 amino acid residues. The sequence matched that tected in rats within 1 week of alcohol feeding (either of rat liver D 4 -3-ketosteroid 5b-reductase. The cloned the high-fat Lieber-DeCarli or the low-fat AIN'76 liquid cDNA was expressed in Escherichia coli using pGEX-KG diet containing alcohol [Bio-Serv, Inc., Frenchtown, as the vector. The expressed protein was found to be of NJ]). 10 The bond between the 37-kd protein and acetalcorrect molecular weight. It reacted with an antibody dehyde was very stable, and its stability was not enthat recognized the unmodified liver 37-kd protein by hanced by incubating with cyanoborohydride as a reWestern blotting. Peptide profiles of tryptic-digested re-ducing agent. 4 Adding pyrazole (an inhibitor of alcohol combinant protein and the purified rat liver 37-kd pro-dehydrogenase) 11,12 to the alcohol-containing diet abro- dehydrogenase) 14 raised plasma acetaldehyde concentrations more than sixfold and greatly increased the intensity of the 37-kd protein-AA band on immunoblots. 13 These experiments therefore provide strong Abbreviations: AA, acetaldehyde adduct; KLH, keyhole limpet hemocyanin; evidence that the 37-kd band detected by immunoblot-SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; 2D, ting is truly a protein adduct of acetaldehyde. On subtwo-dimensional; Ig, immunoglobulin; PVDF, polyvinylid...

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confidence: 68%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Identification of the 37-kd rat liver protein that forms an acetaldehyde adductin vivo as ?4-3-ketosteroid 5?-reductase

et al. 1996

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“…15, 27, 28 and 35) as previously reported; 162 these were enriched 5-12x in autophagosomal membranes (Table 1). A second steroid-metabolizing enzyme, 3-oxo-5b-steroid 4-dehydrogenase, important in bile acid synthesis, 183,184 was present as a single spot (no. 42; 3x enriched).…”

Section: Autophagosomal Membrane Proteinsmentioning

confidence: 99%

Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

Øverbye¹,

Fengsrud²,

Seglen³

2007

Autophagy

140

117

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“…22 The enzyme is liver-specific and is localized in liver cytosol. 23 It catalyzes reduced nicotinamide adenine dinucleotide phosphate dependent reduction of 7␣-hydroxy-4-cholesten-3-one (HCO) during bile acid biosynthesis. 24 Inborn error of bile acid synthesis with ⌬ 4 -3-ketosteroid-5␤ reductase deficiency has been reported.…”

mentioning

confidence: 99%

Cytotoxic effect of 7?-hydroxy-4-cholesten-3-one on HepG2 cells: Hypothetical role of acetaldehyde-modified ?4-3-ketosteroid-5?-reductase (the 37-kd-liver protein) in the pathogenesis of alcoholic liver injury in the rat

Lin¹,

Fillenwarth²,

Du³

1998

Hepatology

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We recently identified ⌬ 4 -3-ketosteroid-5␤-reductase as the 37 kd liver protein which is highly susceptible to acetaldehyde modification in rats continuously fed alcohol. The 5␤-reductase is a key enzyme involved in bile acid synthesis. We report here that the ability to degrade 7␣-hydroxy-4-cholesten-3-one (HCO) was lower in the liver cytosol of alcohol-fed rats than in control animals, suggesting an inhibition of the 5␤-reductase enzyme activity by acetaldehyde modification. We also showed that HCO exhibited a time-and concentration-dependent cytotoxicity to HepG2 cells. HCO cytotoxicity was noticeable at a concentration of 2.5 g/mL. When 10 g/mL of HCO was added to confluent cell monolayers, 57% and 37% of cells remained viable after 24 and 48 hours of treatment. The decrease in cell viability was accompanied by an increased lactic dehydrogenase activity in the culture medium. DNA extracted from HCO-treated cells showed no evidence of DNA fragmentation when analyzed by agarose gel electrophoresis. Staining with propidium iodide showed no nuclear condensation in cells. Thus, cell death by HCO treatment was caused by necrosis and not by apoptosis. Various agents, including, serum proteins, hormones, bile acids, antioxidants, Ca ϩϩ -chelators, Fe ϩϩ -chelator, CYP450 inhibitor, adenylate cyclase inhibitor, protease inhibitors, and nitric oxide synthase inhibitor, did not protect against HCO cytotoxicity. We speculate that HCO concentrations may be elevated around the pericentral area in the liver after chronic alcohol ingestion, causing local cell necrosis. The release of cellular contents and protein-acetaldehyde adducts (PAAs) may activate nonparenchymal cells and provoke autoimmune reaction. Thus, the formation of the 37 kd-PAA may play an important role in the initiation of alcoholic liver injury. (HEPATOLOGY 1998;27:100-107.)Alcohol is metabolized in the liver mainly by alcohol dehydrogenase (ADH). 1 With prolonged alcohol consumption, the metabolic pathway through cytochrome P450IIE1 becomes important. 2 Both ADH and CYPIIE1 generate acetaldehyde from ethanol.

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Molecular cloning and sequence analysis of cDNA encoding δ⁴‐3‐ketosteroid 5β‐reductase of rat liver

Cited by 47 publications

References 18 publications

Identification of the 37-kd rat liver protein that forms an acetaldehyde adductin vivo as ?4-3-ketosteroid 5?-reductase

Identification of the 37-kd rat liver protein that forms an acetaldehyde adductin vivo as ?4-3-ketosteroid 5?-reductase

Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

Cytotoxic effect of 7?-hydroxy-4-cholesten-3-one on HepG2 cells: Hypothetical role of acetaldehyde-modified ?4-3-ketosteroid-5?-reductase (the 37-kd-liver protein) in the pathogenesis of alcoholic liver injury in the rat

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Molecular cloning and sequence analysis of cDNA encoding δ4‐3‐ketosteroid 5β‐reductase of rat liver

Cited by 47 publications

References 18 publications

Identification of the 37-kd rat liver protein that forms an acetaldehyde adductin vivo as ?4-3-ketosteroid 5?-reductase

Identification of the 37-kd rat liver protein that forms an acetaldehyde adductin vivo as ?4-3-ketosteroid 5?-reductase

Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

Cytotoxic effect of 7?-hydroxy-4-cholesten-3-one on HepG2 cells: Hypothetical role of acetaldehyde-modified ?4-3-ketosteroid-5?-reductase (the 37-kd-liver protein) in the pathogenesis of alcoholic liver injury in the rat

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Molecular cloning and sequence analysis of cDNA encoding δ⁴‐3‐ketosteroid 5β‐reductase of rat liver