Tobacco smoking is the most widely known cause of human cancer-related death worldwide. NNK is one of the proved human carcinogens contributing to the development of several types of cancer. The carcinogenic effect of NNK depends on the metabolic pathway. Reduction of NNK by carbonyl reducing enzymes leads to the formation of NNAL. This pathway is generally regarded as detoxification pathway although the conditions and circumstances are quite complicated - the process depends on a formed enantiomer of NNAL. In this study a novel method for the determination of the metabolite NNAL was developed. This makes it possible to findand characterize carbonyl reducing enzymes that are involved in NNK metabolism. This simple HPLC method uses conventional HPLC instrumentation and is designed mainly for biochemical laboratories. A new microsomal carbonyl reducing enzyme participating in the metabolism of NNK in vitro has been described. Its activity was compared with other carbonyl reducing enzymes taking part in the biotransformation of NNK.
- MeSH
- alkoholoxidoreduktasy metabolismus MeSH
- jaterní mikrozomy metabolismus MeSH
- karcinogeny metabolismus MeSH
- kouření škodlivé účinky metabolismus MeSH
- lidé MeSH
- nitrosaminy analýza metabolismus MeSH
- pyridiny analýza MeSH
- stereoizomerie MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 4-(methylnitrosamino)-1-(3-pyridyl)-1-butan-1-ol MeSH Prohlížeč
- 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone MeSH Prohlížeč
- alkoholoxidoreduktasy MeSH
- karcinogeny MeSH
- nitrosaminy MeSH
- pyridiny MeSH
Cytochrome P450 2A13 is an omitted brother of CYP2A6 that has an important role in the drug metabolism of liver. Due to extrahepatic expression, it has gained less attention than CYP2A6, despite the fact that it plays a significant role in toxicant-induced pulmonary lesions and, therefore, lung cancer. The purpose of this mini-review is to summarize the basic knowledge about this enzyme in relation to the substrates, inhibitors, genetic polymorphisms, and transcriptional regulation that are known so far (September 2021).
- Klíčová slova
- FOXA2, NNK, aflatoxin, lung cancer, polymorphism, regulation, skatole,
- MeSH
- aromatické hydroxylasy antagonisté a inhibitory genetika metabolismus MeSH
- lidé MeSH
- methoxsalen farmakologie MeSH
- nádory plic enzymologie genetika patologie MeSH
- plíce enzymologie metabolismus patologie MeSH
- polymorfismus genetický * MeSH
- regulace genové exprese enzymů * MeSH
- regulace genové exprese u nádorů * MeSH
- substrátová specifita MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- aromatické hydroxylasy MeSH
- CYP2A13 protein, human MeSH Prohlížeč
- methoxsalen MeSH
Dehydrogenase/reductase SDR family member 7 (DHRS7, SDR34C1, retSDR4) is one of the many endoplasmic reticulum bound members of the SDR superfamily. Preliminary results indicate its potential significance in human metabolism. DHRS7 containing TEV-cleavable His10 and FLAG-tag expressed in the Sf9 cell line was solubilised, purified, and reconstituted into liposomes to enable the improved characterisation of this enzyme in the future. Igepal CA-630 was determined to be the best detergent for the solubilisation process. The solubilised DHRS7 was purified using affinity chromatography, and the purified enzyme was subjected to TEV cleavage of the affinity tags and then repurified using subtractive Ni-IMAC. The cleaved and uncleaved versions of DHRS7 were successfully reconstituted into liposomes. In addition, using tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as the substrate, the cleaved liposomal DHRS7 was found to be inactive, whereas the pure and uncleaved liposomal DHRS7 were confirmed as enzymes, which reduce carbonyl group of the substrates.
- Klíčová slova
- Carbonyl reductase activity, DHRS7, Purification, Reconstitution, Short-chain dehydrogenases/reductases, Solubilisation,
- MeSH
- buněčná membrána MeSH
- lidé MeSH
- membránové proteiny chemie genetika izolace a purifikace metabolismus MeSH
- oxidoreduktasy chemie genetika izolace a purifikace metabolismus MeSH
- rekombinantní proteiny chemie genetika izolace a purifikace metabolismus MeSH
- Sf9 buňky MeSH
- Spodoptera MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- membránové proteiny MeSH
- oxidoreduktasy MeSH
- rekombinantní proteiny MeSH
Dehydrogenase/reductase (SDR family) member 7 (DHRS7, retSDR4, SDR34C1) is a previously uncharacterized member of the short-chain dehydrogenase/reductase (SDR) superfamily. While human SDR members are known to play an important role in various (patho)biochemical pathways including intermediary metabolism and biotransformation of xenobiotics, only 20% of them are considered to be well characterized. Based on phylogenetic tree and SDR sequence clusters analysis DHRS7 is a close relative to well-known SDR member 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) that participates in metabolism of endogenous and xenobiotic substances with carbonyl group. The aim of present study is to determine the basic biochemical properties of DHRS7 and its possible involvement in metabolism of substrates with carbonyl group. For the first time the computational predictions of this membrane protein and membrane topology were experimentally confirmed. DHRS7 has been demonstrated to be an integral protein facing the lumen of the endoplasmic reticulum with lack of posttranscriptional glycosylation modification. Subsequently, NADP(H) cofactor preference and enzymatic reducing activity of DHRS7 was determined towards endogenous substrates with a steroid structure (cortisone, 4-androstene-3,17-dion) and also toward relevant exogenous substances bearing a carbonyl group harmful to human health (1,2-naphtoquinone, 9,10-phenantrenequinone). In addition to 11β-HSD1, DHRS7 is another enzyme from SDR superfamily that have been proved, at least in vitro, to contribute to the metabolism of xenobiotics with carbonyl group.
- Klíčová slova
- 11β-HSD1, 11β-hydroxysteroid dehydrogenase, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, DHRS7, ER, Membrane topology, NNAL, NNK, Oxidoreductase activity, SDR, SDR34C1, Short-chain dehydrogenases/reductases, dehydrogenase/reductase (SDR family) member 7, endoplasmic reticulum, short-chain dehydrogenases/reductases,
- MeSH
- 11-beta-hydroxysteroiddehydrogenasa typ 1 chemie metabolismus MeSH
- benzaldehydy metabolismus MeSH
- fluorescenční protilátková technika MeSH
- intracelulární membrány metabolismus MeSH
- izoenzymy chemie metabolismus MeSH
- jaterní mikrozomy enzymologie MeSH
- kinetika MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- NAD metabolismus MeSH
- NADP metabolismus MeSH
- nitrosaminy chemie metabolismus MeSH
- oxidoreduktasy chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- Sf9 buňky MeSH
- spektrofotometrie MeSH
- substrátová specifita MeSH
- ultracentrifugace MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 11-beta-hydroxysteroiddehydrogenasa typ 1 MeSH
- 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone MeSH Prohlížeč
- 4-nitrobenzaldehyde MeSH Prohlížeč
- benzaldehydy MeSH
- DHRS7 protein, human MeSH Prohlížeč
- DHRS7c protein, human MeSH Prohlížeč
- izoenzymy MeSH
- NAD MeSH
- NADP MeSH
- nitrosaminy MeSH
- oxidoreduktasy MeSH
Dehydrogenase/reductase (SDR family) member 3 (DHRS3), also known as retinal short-chain dehydrogenase/reductase (retSDR1) is a member of SDR16C family. This family is thought to be NADP(H) dependent and to have multiple substrates; however, to date, only all-trans-retinal has been identified as a DHRS3 substrate. The reductive reaction catalysed by DHRS3 seems to be physiological, and recent studies proved the importance of DHRS3 for maintaining suitable retinoic acid levels during embryonic development in vivo. Although it seems that DHRS3 is an important protein, knowledge of the protein and its properties is quite limited, with the majority of information being more than 15 years old. This study aimed to generate a more comprehensive characterisation of the DHRS3 protein. Recombinant enzyme was prepared and demonstrated to be a microsomal, integral-membrane protein with the C-terminus oriented towards the cytosol, consistent with its preference of NADPH as a cofactor. It was determined that DHRS3 also participates in the metabolism of other endogenous compounds, such as androstenedione, estrone, and DL-glyceraldehyde, and in the biotransformation of xenobiotics (e.g., NNK and acetohexamide) in addition to all-trans-retinal. Purified and reconstituted enzyme was prepared for the first time and will be used for further studies. Expression of DHRS3 was shown at the level of both mRNA and protein in the human liver, testis and small intestine. This new information could open other areas of DHRS3 protein research.
- Klíčová slova
- DHRS3, Expression, Membrane topology, Reductase activity, retSDR1,
- MeSH
- alkoholoxidoreduktasy metabolismus MeSH
- cytosol metabolismus MeSH
- jaterní mikrozomy enzymologie metabolismus MeSH
- játra enzymologie metabolismus MeSH
- lidé MeSH
- membránové proteiny metabolismus MeSH
- NADH, NADPH oxidoreduktasy metabolismus MeSH
- NADP metabolismus MeSH
- Sf9 buňky MeSH
- Spodoptera metabolismus MeSH
- syntázy mastných kyselin metabolismus MeSH
- tenké střevo enzymologie metabolismus MeSH
- testis enzymologie metabolismus MeSH
- tretinoin metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- alkoholoxidoreduktasy MeSH
- membránové proteiny MeSH
- NADH, NADPH oxidoreduktasy MeSH
- NADP MeSH
- short chain trans-2-enoyl-CoA reductase MeSH Prohlížeč
- syntázy mastných kyselin MeSH
- tretinoin MeSH
