Omeprazole as a potent activator of human cytosolic aldehyde dehydrogenase ALDH1A1

BackgroundAccumulation of lipid aldehydes plays a key role in the etiology of human diseases where high levels of oxidative stress are generated. In this regard, activation of aldehyde dehydrogenases (ALDHs) prevents oxidative tissue damage during ischemia-reperfusion processes. Although omeprazole is used to reduce stomach gastric acid production, in the present work this drug is described as the most potent activator of human ALDH1A1 reported yet.MethodsDocking analysis was performed to predict the interactions of omeprazole with the enzyme. Recombinant human ALDH1A1 was used to assess the effect of omeprazole on the kinetic properties. Temperature treatment and mass spectrometry were conducted to address the nature of binding of the activator to the enzyme. Finally, the effect of omeprazole was evaluated in an in vivo model of oxidative stress, using E. coli cells expressing the human ALDH1A1.ResultsOmeprazole interacted with the aldehyde binding site, increasing 4–6 fold the activity of human ALDH1A1, modified the kinetic properties, altering the order of binding of substrates and release of products, and protected the enzyme from inactivation by lipid aldehydes. Furthermore, omeprazole protected E. coli cells over-expressing ALDH1A1 from the effects of oxidative stress generated by H₂O₂ exposure, reducing the levels of lipid aldehydes and preserving ALDH activity.ConclusionOmeprazole can be repositioned as a potent activator of human ALDH1A1 and may be proposed for its use in therapeutic strategies, to attenuate the damage generated during oxidative stress events occurring in different human pathologies.     

Identification of 3-deoxyglucosone dehydrogenase as aldehyde dehydrogenase 1A1 (retinaldehyde dehydrogenase 1)

One of the metabolic fates of 3-deoxyglucosone, a product of protein deglycation and a potent glycating agent, is to be oxidized to 2-keto-3-deoxygluconate, but the enzyme that catalyzes this reaction is presently unknown. Starting from human erythrocytes, which are known to convert 3-deoxyglucosone to 2-keto-3-deoxygluconate, we have purified to near homogeneity a NAD-dependent dehydrogenase that catalyzes this last reaction at neutral pH. Sequencing of a 55 kDa band co-eluting with the enzymatic activity in the last step indicated that it corresponded to aldehyde dehydrogenase 1A1 (ALDH1A1), an enzyme known to catalyze the oxidation of retinaldehyde to retinoic acid. Overexpression of human ALDH1A1 in HEK cells led to a more than 20-fold increase in 3-deoxyglucosone dehydrogenase activity. In mouse tissues 3-deoxyglucosone dehydrogenase activity was highest in liver, intermediate in lung and testis, and negligible or undetectable in other tissues, in agreement with the tissue distribution of ALDH1A1 mRNA. 3-deoxyglucosone dehydrogenase activity was undetectable in tissues from ALDH1A1(-/-) mice. ALDH1A1 appears therefore to be the major if not the only enzyme responsible for the oxidation of 3-deoxyglucosone to 2-keto-3-deoxygluconate. The urinary excretion of 2-keto-3-deoxygluconate amounted to 16.7 micromol/g creatinine in humans, indicating that 3-deoxyglucosone may be quantitatively a more important substrate than retinaldehyde for ALDH1A1.     

Identification of isocitrate dehydrogenase 1 as a potential diagnostic and prognostic biomarker for non-small cell lung cancer by proteomic analysis

Lung cancer is the leading cause of cancer-related death in the world. To explore tumor biomarkers for clinical application, two-dimensional fluorescence difference gel electrophoresis and subsequent MALDI-TOF/TOF mass spectrometry were performed to identify proteins differentially expressed in 12 pairs of lung squamous cell tumors and their corresponding normal tissues. A total of 28 nonredundant proteins were identified with significant alteration in lung tumors. The up-regulation of isocitrate dehydrogenase 1 (IDH1), superoxide dismutase 2, 14-3-3ε, and receptor of activated protein kinase C1 and the down-regulation of peroxiredoxin 2 in tumors were validated by RT-PCR and Western blot analysis in independent 15 pairs of samples. Increased IDH1 expression was further verified by the immunohistochemical study in extended 73 squamous cell carcinoma and 64 adenocarcinoma clinical samples. A correlation between IDH1 expression and poor overall survival of non-small cell lung cancer (NSCLC) patients was observed. Furthermore, ELISA analysis showed that the plasma level of IDH1 was significantly elevated in NSCLC patients compared with benign lung disease patients and healthy individuals. In addition, knockdown of IDH1 by RNA interference suppressed the proliferation of NSCLC cell line and decreased the growth of xenograft tumors in vivo. These observations suggested that IDH1, as a protein promoting tumor growth, could be used as a plasma biomarker for diagnosis and a histochemical biomarker for prognosis prediction of NSCLC.     

Oxidation of lactaldehyde by cytosolic aldehyde dehydrogenase and inhibition of cytosolic and mitochondrial aldehyde dehydrogenase by metabolites

An enzyme fraction which oxidizes lactaldehyde to lactic acid has been purified from goat liver. This enzyme was found to be identical with the cytosolic aldehyde dehydrogenase. Lactaldehyde was found to be primarily oxidized by this enzyme. Almost 90% of the total lactaldehyde-oxidizing activity is located in the cytosol. Methylglyoxal and glyceraldehyde 3-phosphate were found to be strong competitive inhibitors of this enzyme. Aldehyde dehydrogenase from goat liver mitochondria has also been partially purified and found to be strongly inhibited by these metabolites. The inhibitory effects of these metabolites on both these enzymes are highly pH dependent. The inhibitory effects of both the metabolites have been found to be stronger for the cytosolic enzyme at pH values higher than the physiological pH. For the mitochondrial enzyme, the inhibition with methylglyoxal was more pronounced at higher pH values, whereas stronger inhibition was observed with glyceraldehyde 3-phosphate at physiological pH.     

High expression of Cks1 in human non-small cell lung carcinomas

Enhanced degradation of cyclin-dependent kinase (CDK) inhibitor p27(Kip1) is known to be a powerful prognostic marker in many types of human cancers. Human CDK subunit 1 (Cks1) and S-phase kinase associated protein 2 (Skp2) are components of the SCF(Skp2) complex, which acts as a ubiquitin ligase for p27(Kip1). There are no reports about the involvement of Cks1 in the pathogenesis of human cancer. Here we show high expression of Cks1 in non-small cell lung cancers (NSCLCs) using Western blotting and quantitative real-time RT-PCR. The Skp2 mRNA expression level was high in squamous cell carcinomas and was inversely related with the p27(Kip1) protein level in individual clinical samples. In contrast, Cks1 mRNA expression had no such relationship with p27(Kip1), although Cks1 mRNA was significantly elevated in adenocarcinomas. These results suggest that high expression of Skp2 and Cks1 may be involved in the pathogenesis of NSCLCs via different mechanisms.     

Characterization of Xenopus cytosolic thyroid-hormone-binding protein (xCTBP) with aldehyde dehydrogenase activity

Multiple cytosolic thyroid-hormone-binding proteins (CTBPs) with varying characteristics, depending on the species and tissue, have been reported. We first purified a 59-kDa CTBP from Xenopus liver (xCTBP), and found that it is responsible for major [¹²⁵I]T₃-binding activity in Xenopus liver cytosol. Amino acid sequencing of internal peptide fragments derived from xCTBP demonstrated high identity to the corresponding sequence of mammalian aldehyde dehydrogenases 1 (ALDH1). To confirm whether or not xCTBP is identical to xALDH1, we isolated cDNAs encoding xALDH1 from an adult Xenopus hepatic cDNA library. The amino acid sequences deduced from the two isolated xALDH1 cDNAs were very similar to those of mammalian ALDH1 enzymes. The recombinant xALDH1 protein exhibited both T₃-binding activity and ALDH activity converting retinal to retinoic acid (RA), which were similar to those of xCTBP purified from liver cytosol. The T₃-binding activity was inhibited by NAD, while the ALDH activity was inhibited by thyroid hormones. Our results demonstrate that xCTBP is identical to ALDH1 and suggest that this protein might modulate RA synthesis and intracellular concentration of free T₃. Communications between thyroid hormone and retinoid pathways are discussed.     

Characterization of Xenopus cytosolic thyroid-hormone-binding protein (xCTBP) with aldehyde dehydrogenase activity

Multiple cytosolic thyroid-hormone-binding proteins (CTBPs) with varying characteristics, depending on the species and tissue, have been reported. We first purified a 59-kDa CTBP from Xenopus liver (xCTBP), and found that it is responsible for major [125I]T(3)-binding activity in Xenopus liver cytosol. Amino acid sequencing of internal peptide fragments derived from xCTBP demonstrated high identity to the corresponding sequence of mammalian aldehyde dehydrogenases 1 (ALDH1). To confirm whether or not xCTBP is identical to xALDH1, we isolated cDNAs encoding xALDH1 from an adult Xenopus hepatic cDNA library. The amino acid sequences deduced from the two isolated xALDH1 cDNAs were very similar to those of mammalian ALDH1 enzymes. The recombinant xALDH1 protein exhibited both T(3)-binding activity and ALDH activity converting retinal to retinoic acid (RA), which were similar to those of xCTBP purified from liver cytosol. The T(3)-binding activity was inhibited by NAD, while the ALDH activity was inhibited by thyroid hormones. Our results demonstrate that xCTBP is identical to ALDH1 and suggest that this protein might modulate RA synthesis and intracellular concentration of free T(3). Communications between thyroid hormone and retinoid pathways are discussed.     

Studies on the mechanism of sheep liver cytosolic aldehyde dehydrogenase.

The dissociation of the aldehyde dehydrogenase X NADH complex was studied by displacement with NAD+. The association reaction of enzyme and NADH was also studied. These processes are biphasic, as shown by McGibbon, Buckley & Blackwell [(1977) Biochem. J. 165, 455-462], but the details of the dissociation reaction are significantly different from those given by those authors. Spectral and kinetic experiments provide evidence for the formation of abortive complexes of the type enzyme X NADH X aldehyde. Kinetic studies at different wavelengths with transcinnamaldehyde as substrate provide evidence for the formation of an enzyme X NADH X cinnamoyl complex. Hydrolysis of the thioester relieves a severe quenching effect on the fluorescence of enzyme-bound NADH.     

Retinal oxidation activity and biological role of human cytosolic aldehyde dehydrogenase.

The major cytosolic aldehyde dehydrogenase isozyme (ALDH1) exhibits strong activity for oxidation of retinal to retinoic acid, while the major mitochondrial ALDH2 and the stomach cytosolic ALDH3 have no such activity. The Km of ALDH1 for retinal is about 0.06 mumol/l at pH 7.5, and the catalytic efficiency (Vmax/Km) for retinal is about 600 times higher than that for acetaldehyde. Thus, ALDH1 can efficiently produce retinoic acid from retinal in tissues with low retinal concentrations (< 0.01 mumol/l). The gene for ALDH1 has hormone response elements. These findings suggest that the major physiological substrate of human ALDH1 is retinal, and that its primary biological role is generation of retinoic acid resulting in modulation of cell differentiation including hormone-mediated development.     

Identification of aldehyde dehydrogenase 1A1 modulators using virtual screening

The highly similar aldehyde dehydrogenase isozymes (ALDH1A1 and ALDH2) have been implicated in the metabolism of toxic biogenic aldehydes such as 3,4-dihydroxyphenylacetaldehyde (DOPAL) and 4-hydroxy-2E-nonenal. We report the down-regulation of ALDH1A1 mRNA found in substantia nigra tissue of human Parkinson’s disease (PD) samples using the Genome-Wide SpliceArray^? (GWSA^?) technology. Since DOPAL can rapidly inactivate ALDH1A1 in vitro, we set up a DOPAL-induced ALDH1A1 inactivation assay and used this assay to demonstrate that Alda-1, a compound originally identified as an activator of ALDH2, can also activate ALDH1A1. We carried out a virtual screening of 19,943 compounds and the top 21 hits from this screen were tested in the DOPAL inactivation assay with ALDH1A1 which led to identification of an activator as well as two inhibitors among these hits. These findings represent an attractive starting point for developing higher potency activator compounds that may have utility in restoring the metabolism of DOPAL in PD.     

p27~(Kip1)和CyclinD1在非小细胞肺癌中表达及意义

为了探讨p27Kip1蛋白和CyclinD1蛋白在非小细胞肺癌(NSCLC)中的表达及意义,收集临床手术切除的非小细胞肺癌组织蜡块64例及正常肺组织10例,应用免疫组化(S-P法)检测组织中p27Kip1蛋白和CyclinD1蛋白的表达,结合临床病理资料和随访资料进行回顾性研究。实验发现NSCLC组织中p27Kip1蛋白表达和CyclinD1蛋白表达均明显不同于正常肺组织(P<0.01)。p27Kip1蛋白表达降低与NSCLC肿瘤大小、病理分级、分期增加、淋巴结转移之间有相关性(P<0.05),但与肿瘤组织学分型无相关性(P>0.05)。CyclinD1蛋白过表达与组织学分型、肿瘤大小、病理分级、临床分期、淋巴结转移无相关性(P>0.05)。p27Kip1蛋白表达与CyclinD1蛋白表达之间呈显著负相关(P<0.01)。cox单因素及多因素分析,p27Kip1蛋白低表达及CyclinD1过表达是影响NSCLC患者预后的主要因素。实验结果显示,NSCLC组织中,p27Kip1蛋白表达降低,而CyclinD1过表达,二者与NSCLC的发生发展机制有关,可作为预后指标,有利于NSCLC患者预后判断及个体化治疗。     

Detection and partial characterization of a variant form of cytosolic aldehyde dehydrogenase isozyme

Summary A rare case of human liver cytosolic aldehyde dehydrogenase (isozyme II) variation discovered in a Chinese autopsy liver specimen is reported. While the major isozyme band was nearly absent, several additional minor bands were observed on isoelectric focusing gel. Rabbit antibodies to purified human liver ALDH II showed immunological cross-reactivity for the variant enzyme bands. The existence of additional minor bands indicates the presence of tetramer hybrid forms made up of normal and variant monomers. The observed abnormality may represent the heterozygous form of ALDH II variation. A similar variant was also detected in erythrocytes of a male Thai student.This paper is dedicated to Professor Dr. Karl Decker on his 60th birthday     

High frequency of p16 promoter methylation in non-small cell lung carcinomas from Chile

The inactivation of tumour suppressor genes by aberrant methylation of promoter regions has been described as a frequent event in neoplasia development, including lung cancer. The p16 gene is a tumour suppressor gene involved in the regulation of cell cycle progression that has been reported to be inactivated by promoter methylation in lung carcinomas at variable frequencies around the world in a smoking habit dependent manner. The purpose of this study was to investigate the methylation status of the promoter region of the p16 gene in 74 non-small cell lung carcinomas from Chile. The frequency of p16 gene inactivation by promoter methylation was determined as 79.7% (59/74). When we considered histological type, we observed that p16 promoter methylation was significantly higher in squamous cell carcinomas (30/33, 91%) compared with adenocarcinomas (21/30, 70%) (p=0.029). In addition, no association between p16 promoter methylation and gender, age or smoking habit was found (p=0.202, 0.202 and 0.147 respectively). Our results suggest that p16 promoter hypermethylation is a very frequent event in non-small cell lung carcinomas from Chile and could be smoking habit-independent.     

Identification of xanthine dehydrogenase/xanthine oxidase as a rat Paneth cell zinc-binding protein

Paneth cells are zinc-containing cells localized in small intestinal crypts, but their function has not been fully elucidated. Previously, we showed that an intravenous injection of diphenylthiocarbazone (dithizone), a zinc chelator, induced selective killing of Paneth cells, and purified a zinc-binding protein in Paneth cells. In the present study, we further characterized one of these proteins, named zinc-binding protein of Paneth cells (ZBPP)-1. Partial amino acid sequences of ZBPP-1 showed identity with rat xanthine dehydrogenase (XD)/xanthine oxidase (XO). Anti-rat XD antibody (Ab) recognized ZBPP-1, and conversely anti ZBPP-1 Ab recognized 85 kDa fragment of rat XD in Western blotting. Messenger RNA and protein levels of XD were consistent with our previous data on the fluctuation of Paneth cell population after dithizone injection. Thus, ZBPP-1 is an 85 kDa fragment of XD/XO in Paneth cells. XD/XO in Paneth cells may play important roles in intestinal function.     

Genetics of alcohol dehydrogenase and aldehyde dehydrogenase from Monodelphis domestica cornea: further evidence for identity of corneal aldehyde dehydrogenase with a major soluble protein

A didelphid marsupial, the gray short-tailed opossum (Monodelphis domestica), was used as a model species to study the biochemical genetics of alcohol dehydrogenases (ADHs) and aldehyde dehydrogenase (ALDH) in corneal tissue. Isoelectric point variants of corneal ALDH (designated ALDH3) and a major soluble protein in corneal extracts were observed among eight families of animals used in studying the genetics of these proteins. Both phenotypes exhibited identical patterns following PAGE-IEF and were inherited in a normal Mendelian fashion, with two alleles at a single locus (ALDH3) showing codominant expression. The data provided evidence for genetic identity of corneal ALDH with this major soluble protein, and supported biochemical evidence, recently reported for purified bovine corneal ALDH, that this enzyme constitutes a major portion of soluble corneal protein (Abedinia et al. 1990). Isoelectric point variants for corneal ADH were also observed, with patterns for the two major forms (ADH3 and ADH4) and one minor form (ADH5) being consistent with the presence of two ADH subunits (designated gamma and delta), and variant phenotypes existing for the gamma subunit. The genetics of this enzyme was studied in the eight families, and the results were consistent with codominant expression of two alleles at a single locus (designated ADH3). It is relevant that a major detoxification function has been proposed for corneal ADH and ALDH, in the oxidoreduction of peroxidic aldehydes induced by available oxygen and UV-B light (Holmes & VandeBerg, 1986a). In addition, a direct role for corneal ALDH as a UV-B photoreceptor in this anterior eye tissue has also been proposed (Abedinia et al. 1990).     

Genetic basis for the polymorphism of rat liver cytosolic aldehyde dehydrogenase

Liver aldehyde dehydrogenase (ALDH), the enzyme involved in the oxidation of aldehydes such as acetaldehyde derived from ethanol, exists in multiple forms in most mammals. Up to five separable forms have been identified from the cytosolic fraction of Wistar rat liver. We investigated the genetic basis of a particular set of three enzyme forms by selective breeding and analysis of electrophoretic patterns of liver ALDH by isoelectric focusing. The forms of liver ALDH investigated were at pI 5.8 or 6.2, or a triple form with enzymes at pI 5.8, 6.0, and 6.2. There are two alleles found at the ALDH locus which encode in homozygotes for one of two electrophoretically separable ALDH forms. A rat heterozygous at the locus forms both ALDH types plus a hybrid. The alleles are expressed codominantly, found at an autosomal locus, and remain constant postpartum. The activities associated with the triplet enzyme form were statistically indistinguishable from a 1:2:1 ratio. This suggests that the enzymes hybridize to form a set of dimers or tetramers of the form A₂, AB, B₂ or A₄, A₂B₂, B₄, respectively.