Member Of Aldo-keto Reductase Superfamily Research Articles

Molecular Docking Evaluation of Some Natural Phenolic Compounds as Aldose Reductase Inhibitors for Diabetic Complications

The enzyme aldose reductase (AR) is a member of aldoketoreductase super-family which catalyzes the formation of sorbitol from glucose through polyol pathway of glucose catabolism. Reduced sorbitol production via polyol pathway due to AR inhibition is a target of choice for controlling major complications of diabetes. Epalrestat is the only commercially available inhibitor of AR till date,thus, there is a great need to search for more economical, nontoxic and safer inhibitors of AR enzyme. Flavonoids, the polyphenol compounds in plants have been reported for inhibitory effects against AR. The objective of this study is to explore the binding modes of natural phenolic compounds with AR to design safer natural drugs as alternatives to synthetic drugs. We conducted a molecular docking study on some natural phenolic compounds with AR enzyme in complex with the synthetic inhibitor. The overlay of the docked pose of the selected natural phenols with the ARreference inhibitor complex showed that the selected natural compounds have the similar binding pattern with the active site residues of the enzyme as that of co-crystallized inhibitor. The results of docking study showed the best binding affinity of AR with that of 2-(4-hydroxy-3-methoxyphenyl) ethanoic acid and butein, having the lowest binding free energy of –9.8 kcal/mol and–9.7 kcal/mol, respectively. This information can be utilized to design potent, economical and non-toxic natural AR inhibitors from natural phenols for the therapeutics of diabetic complications.

Targeting Aldose Reductase for the Treatment of Cancer

It is strongly established by numerous studies that oxidative stress-induced inflammation is one of the major causative agents in a variety of cancers. Various factors such as bacterial, viral, parasitic infections, chemical irritants, carcinogens are involved in the initiation of oxidative stress-mediated inflammation. Chronic and persistent inflammation promotes the formation of cancerous tumors. Recent investigations strongly suggest that aldose reductase [AR; AKR1B1], a member of aldo-keto reductase superfamily of proteins, is the mediator of inflammatory signals induced by growth factors, cytokines, chemokines, carcinogens etc. Further, AR reduced product(s) of lipid derived aldehydes and their metabolites such as glutathionyl 1,4-dihydroxynonanol (GS-DHN) have been shown to be involved in the activation of transcription factors such as NF-κB and AP-1 which transcribe the genes of inflammatory cytokines. The increased inflammatory cytokines and growth factors promote cell proliferation, a main feature involved in the tumorigenesis process. Inhibition of AR has been shown to prevent cancer cell growth in vitro and in vivo models. In this review, we have described the possible association between AR with oxidative stress- and inflammation- initiated carcinogenesis. A thorough understanding of the role of AR in the inflammation -associated cancers could lead to the use of AR inhibitors as novel chemotherapeutic agents against cancer.

A new approach to monitor expression of aldo–keto reductase proteins in mouse tissues

The aldo-keto reductase (AKR) proteins catalyze reduction of diverse aldehydes and play detoxification roles in many organisms. Since many substrates are shared among AKR, it is generally accepted that these enzymes can functionally compensate each other in response to oxidative stress. Their overall abundances are the important factor that partially reflects the capacity of antioxidant and detoxification in tissues. In this study, the strategy was proposed for generation of Pan-AKR antibodies to recognize most AKR proteins in mouse tissues. Derived from bioinformatic analysis, several consensus peptides with different potential antigenicities were synthesized, conjugated to hemocyanin from keyhole limpets and further delivered to rabbits to generate polyclonal antibodies. Three Pan-AKR antibodies exhibited the immune specificities and immune sensitivities, Pan-AKR-P1 for AKR1B and AKR1C, Pan-AKR-P3 for AKR1C and Pan-AKR-P4 for all the AKR proteins. Pan-AKR-P4 antibody was employed to 2-DE Western blot to examine the AKR abundances in mouse liver and kidney, resulting in seven immune-reactive spots from each tissue. Protein identification with MS revealed that most immune-positive spots were the members of AKR superfamily. Furthermore, Pan-AKR-P4 antibody was implemented to compare the different abundances of the AKR proteins in liver and kidney between normal and diabetic mice, suggesting that diabetes did cause some abnormal changes in the AKR protein abundances.

Placenta Defects and Embryonic Lethality Resulting from Disruption of Mouse Hydroxysteroid (17-β) Dehydrogenase 2 Gene

Hydroxysteroid (17-beta) dehydrogenase 2 (HSD17B2) is a member of aldo-keto reductase superfamily, known to catalyze the inactivation of 17beta-hydroxysteroids to less active 17-keto forms and catalyze the conversion of 20alpha-hydroxyprogesterone to progesterone in vitro. To examine the role of HSD17B2 in vivo, we generated mice deficient in Hsd17b2 [HSD17B2 knockout (KO)] by a targeted gene disruption in embryonic stem cells. From the homozygous mice carrying the disrupted Hsd17b2, 70% showed embryonic lethality appearing at the age of embryonic d 11.5 onward. The embryonic lethality was associated with reduced placental size measured at embryonic d 17.5. The HSD17B2KO mice placentas presented with structural abnormalities in all three major layers: the decidua, spongiotrophoblast, and labyrinth. Most notable was the disruption of the spongiotrophoblast and labyrinthine layers, together with liquid-filled cysts in the junctional region and the basal layer. Treatments with an antiestrogen or progesterone did not rescue the embryonic lethality or the placenta defect in the homozygous mice. In hybrid background used, 24% of HSD17B2KO mice survived through the fetal period but were born growth retarded and displayed a phenotype in the brain with enlargement of ventricles, abnormal laminar organization, and increased cellular density in the cortex. Furthermore, the HSD17B2KO mice had unilateral renal degeneration, the affected kidney frequently appearing as a fluid-filled sac. Our results provide evidence for a role for HSD17B2 enzyme in the cellular organization of the mouse placenta.

Aldo–keto reductase family 1 B10 gene silencing results in growth inhibition of colorectal cancer cells: Implication for cancer intervention

Aldo-keto reductase family 1 B10 (AKR1B10), a member of aldo-keto reductase superfamily, is overexpressed in human hepatocellular carcinoma, lung squamous cell carcinoma and lung adenocarcinoma. Our previous study had demonstrated that the ectopic expression of AKR1B10 in 293T cells promotes cell proliferation. To evaluate its potential as a target for cancer intervention, in the current study we knocked down AKR1B10 expression in HCT-8 cells derived from a colorectal carcinoma, using chemically synthesized small interfering RNA (siRNA). The siRNA 1, targeted to encoding region, downregulated AKR1B10 expression by more than 60%, and siRNA 2, targeted to 3' untranslational region, reduced AKR1B10 expression by more than 95%. AKR1B10 silencing resulted in approximately a 50% decrease in cell growth rate and nearly 40% suppression of DNA synthesis. More importantly, AKR1B10 downregulation significantly reduced focus formation rate and colony size in semisolid culture, indicating the critical role of AKR1B10 in HCT-8 cell proliferation. Recombinant AKR1B10 protein showed strong enzymatic activity to acrolein and crotonaldehyde, with K(m) = 110.1 +/- 12.2 microM and V(max) = 3,122.0 +/- 64.7 nmol/mg protein/min for acrolein and K(m) = 86.7 +/- 14.3 microM and V(max) = 2,647.5 +/- 132.2 nmol/mg protein/min for crotonaldehyde. AKR1B10 downregulation enhanced the susceptibility of HCT-8 cells to acrolein (25 microM) and crotonaldehyde (50 microM), resulting in rapid oncotic cell death characterized with lactate dehydrogenase efflux and annexin-V staining. These results suggest that AKR1B10 may regulate cell proliferation and cellular response to additional carbonyl stress, thus being a potential target for cancer intervention.

Homology modeling of ρ-crystallin from bullfrog ( Rana catesbeiana) lens

ρ-Crystallins are major protein component found in the eye lenses of frogs of the genus Rana. Structural analysis has indicated that frog ρ-crystallins belong to aldo–keto reductase superfamily (AKRs) which include aldehyde and aldose reductases, prostaglandin F synthase and several detoxification enzymes. Members of AKRs catalyze the oxidation–reduction reaction over a range of substrates using NAD(P)(H) as a cofactor. In spite of higher structural similarity with AKRs and cofactor binding affinity, the ρ-crystallins were found to be catalytically inactive. This study presents comparative or homology modeling of ρ-crystallin from bullfrog ( Rana catesbeiana) in presence and absence of cofactor NADP and a competitive inhibitor, testosterone. The predicted models are explored to examine the catalytic cleft, cofactor binding affinity characteristics and substrate binding pocket.

Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases.

The co-ordinates reported have been submitted to the Protein Data Bank under accession number 1MI3. Xylose reductase (XR; AKR2B5) is an unusual member of aldo-keto reductase superfamily, because it is one of the few able to efficiently utilize both NADPH and NADH as co-substrates in converting xylose into xylitol. In order to better understand the basis for this dual specificity, we have determined the crystal structure of XR from the yeast Candida tenuis in complex with NAD(+) to 1.80 A resolution (where 1 A=0.1 nm) with a crystallographic R -factor of 18.3%. A comparison of the NAD(+)- and the previously determined NADP(+)-bound forms of XR reveals that XR has the ability to change the conformation of two loops. To accommodate both the presence and absence of the 2'-phosphate, the enzyme is able to adopt different conformations for several different side chains on these loops, including Asn(276), which makes alternative hydrogen-bonding interactions with the adenosine ribose. Also critical is the presence of Glu(227) on a short rigid helix, which makes hydrogen bonds to both the 2'- and 3'-hydroxy groups of the adenosine ribose. In addition to changes in hydrogen-bonding of the adenosine, the ribose unmistakably adopts a 3'- endo conformation rather than the 2'- endo conformation seen in the NADP(+)-bound form. These results underscore the importance of tight adenosine binding for efficient use of either NADH or NADPH as a co-substrate in aldo-keto reductases. The dual specificity found in XR is also an important consideration in designing a high-flux xylose metabolic pathway, which may be improved with an enzyme specific for NADH.

Fate of 4-hydroxynonenal in vivo: disposition and metabolic pathways

Due to the cytotoxicity of 4-hydroxynonenal (HNE), and to the fact that this major product of lipid peroxidation is a rather long-living compound compared with reactive oxygen species, the capability of organisms to inactivate and eliminate HNE has received increasing attention during the last decade. Several recent in vivo studies have addressed the issue of the diffusion, kinetics, biotransformation and excretion of HNE. Part of these studies are primarily concerned with the toxicological significance of HNE biotransformation and more precisely with the metabolic pathways by which HNE is inactivated and eliminated. The other aim of in vivo metabolic study is the characterisation of end-metabolites, especially in urine, in order to develop specific and non-invasive biomarkers of lipid peroxidation. When HNE is administered intravenously or intraperitoneally, it is mainly excreted into urine and bile as conjugated metabolites, in a proportion that is dependent on the administration route. However, biliary metabolites undergo an enterohepatic cycle that limits the final excretion of faecal metabolites. Only a very low amount of metabolites is found to be bound to macromolecules. The main urinary metabolites are represented by two groups of compounds. One comes from the mercapturic acid formation from (i) 1,4 dihydroxynonene-glutathione (DHN-GSH) which originates from the conjugation of HNE with GSH by glutathione-S-transferases and the subsequent reduction of the aldehyde by a member of aldo-keto reductase superfamily; (ii) the lactone of 4-hydroxynonanoic-GSH (HNA-lactone-GSH) which originates from the conjugation of HNE followed by the oxidation of the aldehyde by aldehyde dehydrogenase; (iii) HNA-GSH which originates from the hydrolysis of the corresponding lactone. The other one is a group of metabolites issuing from the ω-hydroxylation of HNA or HNA-lactone by cytochromes P450 4A, followed eventually, in the case of ω-oxidized-HNA-lactone, by conjugation with GSH and subsequent mercapturic acid formation. Biliary metabolites are GSH or mercapturic acid conjugates of DHN, HNE and HNA. Stereochemical aspects of HNE metabolism are also discussed.

Human testis specific protein: a new member of aldo-keto reductase superfamily

Human testis specific protein, HTSP, was identified initially by the search of the Expressed Sequence Tag database, followed by the screening of human testis cDNA library. Among various organs examined, the HTSP transcripts were detected only in the testis, not in other reproductive organs such as vas deferens and prostate. No cross-hybridizing signal was detected in the testis of mouse or rat, indicating that this gene is specifically expressed in the human testis. We isolated four isoforms, HTSP1, 2, 3 and 4. Screening of the high throughput genomic sequence database indicated the localization of the HTSP gene in chromosome 10. Thus, HTSP isoforms were generated by alternative splicing of a single gene. HTSP4, the longest gene product, was composed of 307 amino acids and shared 56% identity to mouse vas deferens protein as well as human aldose reductase in amino acid levels. Bacterially expressed recombinant HTSP protein showed small but significant activity towards 9,10-phenanthrenequinone among the putative substrates so far tested. Accordingly, HTSP is a new member of the aldo-keto reductase superfamily with as yet unidentified function.

Androgen-regulated expression of a novel member of the aldo-keto reductase superfamily in regrowing rat prostate.

The rat prostate is dependent on androgen for normal growth and differentiation. In addition, the organ undergoes rapid cell death upon withdrawal of androgen on castration, and the atrophied tissue is capable of regrowth after androgen replacement in adult animals. In our search for novel factor(s) that participate in this androgen-induced proliferation of adult rat prostate cells, we have generated a complementary DNA (cDNA) library enriched in cDNAs transiently up-regulated after androgen stimulation in castrated rat ventral prostate using a PCR-based subtractive hybridization technique. Sequence analysis of about one hundred clones in the library showed that approximately 70% of them are identical or closely related to genes of known function, the remaining ones showing no or very low similarity to any genes characterized previously. Among the former a new member of the rat aldo-keto reductase superfamily that is closely related to aflatoxin, B1 aldehyde reductase has been identified. The newly identified protein (androgen-inducible aldehyde reductase, AIAR) and rat aflatoxin B1 aldehyde reductase (AFAR) exhibit 80% amino acid sequence homology. The enzymatic activity toward 4-nitrobenzaldehyde of recombinant AIAR expressed in Escherichia coli was about 16% of that of rat AFAR. Northern blot analysis revealed AIAR expression in various adult rat tissues in addition to the ventral and dorsolateral prostates, which differs from the highly restricted expression of AFAR in the kidney and liver. The AIAR messenger RNA (mRNA) content of the ventral prostate was low in normal and castrated rats, transiently increased after androgen administration to castrated rats, attaining a peak 12-24 h after the treatment. Although the physiological substrate(s) of AIAR has not been identified, the current results suggest that AIAR expression is associated with some growth-related processes in regrowing rat prostate.

Cloning of Mouse 20.ALPHA.-Hydroxysteroid Dehydrogenase cDNA and Its mRNA Localization during Pregnancy.

20α-Hydroxysteroid dehydrogenase (20α-HSD) converts progesterone to a biologically inactive steroid, 20α-dihydroprogesterone. To examine a potential role of 20α-HSD in tissues other than corpus luteum (CL) during pregnancy, mouse 20α-HSD cDNA was cloned and was used for the examination of its mRNA localization in the conceptus and uterus at different stages of gestation. Using probes based on the rat sequence, mouse 20α-HSD cDNA clone was isolated from a cDNA library prepared from ovaries of day 12 pseudopregnant mice. The mouse 20α-HSD cDNA was 1193 bp in length and encoded 323 amino acids with an estimated molecular weight of 37 kD. The mouse 20α-HSD was homologous to the rat 20α-HSD in nucleotide and amino acid sequences (both in 93% homology), and to other members of aldo-keto reductase superfamily at the cofactor (NADP(H)) binding site. By in situ hybridization, 20α-HSD mRNA was localized in endometrial epithelial cells on days 10, 15 and 18 of pregnancy, maternal placental endothelial cells on day 10, and fetal epidermal cells on day 15. During pseudopregnancy as well as early pregnancy, however, 20α-HSD mRNA was not detected in endometrial cells. Together, 20α-HSD mRNA was localized in cells that surround fetuses during the mid and late stages of gestation.

Purification and characterization of two major forms of naloxone reductase from rabbit liver cytosol, new members of aldo-keto reductase superfamily.

Rabbit liver cytosol produced approximately equal amounts of 6alpha-naloxol and 6beta-naloxol from naloxone in the presence of NADPH at pH 7.4, and contained at least four forms of naloxone reductase. The two major forms, NR1 and NR2, which catalyze the stereospecific reduction of naloxone to 6alpha-naloxol and 6beta-naloxol, respectively, were purified to apparent homogeneity by various chromatographic techniques. Both enzymes are monomeric proteins with similar molecular weights of 35000-36000, but NR1 is a basic protein with an isoelectric point (pI) of 9.3, while NR2 is an acidic protein (pI of 5.9). NR1 and NR2 gave the maximal activities at pH 8.0 and 6.1, respectively. NR1 exhibited considerable activity with NADH as well as with NADPH, whereas NR2 showed highly restricted specificity for NADPH. The Km and Vmax values of NR1 and NR2 for naloxone were 1.0 and 0.06 mM, and 76 and 162 munits/mg, respectively. In addition to naloxone, naltrexone and dihydromorphinone served as good substrates for NR2 but were poor substrates for NR1. Both enzymes reduced aliphatic and aromatic aldehydes, cyclic and aromatic ketones, and quinones at higher rates. The two enzymes catalyzed the dehydrogenation of 17beta-hydroxysteroids with low Km values, and NR2 showed an additional 3alpha-hydroxysteroid dehydrogenase activity. Amino acid sequence data of NRI (99% of whole protein) and NR2 (66%) showed that both enzymes belong to the aldo-keto reductase (AKR) superfamily and can be classified into the AKR1C subfamily. These findings therefore indicate that they are new members of the AKR superfamily and may be involved physiologically in the steroid metabolism as well as in the detoxification of xenobiotic carbonyl compounds.