Dataset Information

Activation of Human Salivary Aldehyde Dehydrogenase by Sulforaphane: Mechanism and Significance.

ABSTRACT: Cruciferous vegetables contain the bio-active compound sulforaphane (SF) which has been reported to protect individuals against various diseases by a number of mechanisms, including activation of the phase II detoxification enzymes. In this study, we show that the extracts of five cruciferous vegetables that we commonly consume and SF activate human salivary aldehyde dehydrogenase (hsALDH), which is a very important detoxifying enzyme in the mouth. Maximum activation was observed at 1 ?g/ml of cabbage extract with 2.6 fold increase in the activity. There was a ~1.9 fold increase in the activity of hsALDH at SF concentration of ? 100 nM. The concentration of SF at half the maximum response (EC50 value) was determined to be 52 ± 2 nM. There was an increase in the Vmax and a decrease in the Km of the enzyme in the presence of SF. Hence, SF interacts with the enzyme and increases its affinity for the substrate. UV absorbance, fluorescence and CD studies revealed that SF binds to hsALDH and does not disrupt its native structure. SF binds with the enzyme with a binding constant of 1.23 x 107 M-1. There is one binding site on hsALDH for SF, and the thermodynamic parameters indicate the formation of a spontaneous strong complex between the two. Molecular docking analysis depicted that SF fits into the active site of ALDH3A1, and facilitates the catalytic mechanism of the enzyme. SF being an antioxidant, is very likely to protect the catalytic Cys 243 residue from oxidation, which leads to the increase in the catalytic efficiency and hence the activation of the enzyme. Further, hsALDH which is virtually inactive towards acetaldehyde exhibited significant activity towards it in the presence of SF. It is therefore very likely that consumption of large quantities of cruciferous vegetables or SF supplements, through their activating effect on hsALDH can protect individuals who are alcohol intolerant against acetaldehyde toxicity and also lower the risk of oral cancer development.

SUBMITTER: Alam MF

PROVIDER: S-EPMC5172892 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Activation of Human Salivary Aldehyde Dehydrogenase by Sulforaphane: Mechanism and Significance.

Alam Md Fazle MF Laskar Amaj Ahmed AA Maryam Lubna L Younus Hina H

PloS one 20161220 12

Cruciferous vegetables contain the bio-active compound sulforaphane (SF) which has been reported to protect individuals against various diseases by a number of mechanisms, including activation of the phase II detoxification enzymes. In this study, we show that the extracts of five cruciferous vegetables that we commonly consume and SF activate human salivary aldehyde dehydrogenase (hsALDH), which is a very important detoxifying enzyme in the mouth. Maximum activation was observed at 1 μg/ml of c ...[more]

PMID: 27997560

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Project description:Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a key enzyme for the metabolism of many toxic aldehydes such as acetaldehyde, derived from alcohol drinking, and 4HNE, an oxidative stress-derived lipid peroxidation aldehyde. Post-translational enhancement of ALDH2 activity can be achieved by serine/threonine phosphorylation by epsilon protein kinase C (?PKC). Elevated ALDH2 is beneficial in reducing injury following myocardial infarction, stroke and other oxidative stress and aldehyde toxicity-related diseases. We have previously identified three ?PKC phosphorylation sites, threonine 185 (T185), serine 279 (S279) and threonine 412 (T412), on ALDH2. Here we further characterized the role and contribution of each phosphorylation site to the enhancement of enzymatic activity by ?PKC.Each individual phosphorylation site was mutated to a negatively charged amino acid, glutamate, to mimic a phosphorylation, or to a non-phosphorylatable amino acid, alanine. ALDH2 enzyme activities and protection against 4HNE inactivation were measured in the presence or absence of ?PKC phosphorylation in vitro. Coevolution of ALDH2 and its ?PKC phosphorylation sites was delineated by multiple sequence alignments among a diverse range of species and within the ALDH multigene family.We identified S279 as a critical ?PKC phosphorylation site in the activation of ALDH2. The critical catalytic site, cysteine 302 (C302) of ALDH2 is susceptible to adduct formation by reactive aldehyde, 4HNE, which readily renders the enzyme inactive. We show that phosphomimetic mutations of T185E, S279E and T412E confer protection of ALDH2 against 4HNE-induced inactivation, indicating that phosphorylation on these three sites by ?PKC likely also protects the enzyme against reactive aldehydes. Finally, we demonstrate that the three ALDH2 phosphorylation sites co-evolved with ?PKC over a wide range of species. Alignment of 18 human ALDH isozymes, indicates that T185 and S279 are unique ALDH2, ?PKC specific phosphorylation sites, while T412 is found in other ALDH isozymes. We further identified three highly conserved serine/threonine residues (T384, T433 and S471) in all 18 ALDH isozymes that may play an important phosphorylation-mediated regulatory role in this important family of detoxifying enzymes.?PKC phosphorylation and its coevolution with ALDH2 play an important role in the regulation and protection of ALDH2 enzyme activity.

Project description:BackgroundCytosolic aldehyde dehydrogenase, or ALDH1A1, functions in ethanol detoxification, metabolism of neurotransmitters, and synthesis of retinoic acid. Because the promoter region of a gene can influence gene expression, the ALDH1A1 promoter regions were studied to identify polymorphism, to assess their functional significance, and to determine whether they were associated with a risk for developing alcoholism.MethodsSequence analysis was performed in the promoter region by using Asian, Caucasian, and African American subjects. The resulting polymorphisms were assessed for frequency in Asian, Caucasian, Jewish, and African American populations and tested for associations with alcohol dependence in Asian and African American populations of alcoholics and controls. The functional significance of each polymorphism was determined through in vitro expression analysis by using HeLa and HepG2 cells.ResultsTwo polymorphisms, a 17 base pair (bp) deletion (-416/-432) and a 3 bp insertion (-524), were discovered in the ALDH1A1 promoter region: ALDH1A1*2 and ALDH1A1*3, respectively. ALDH1A1*2 was observed at frequencies of 0.035, 0.023, 0.023, and 0.012 in the Asian, Caucasian, Jewish, and African American populations, respectively. ALDH1A1*3 was observed only in the African American population, at a frequency of 0.029. By using HeLa and HepG2 cells for in vitro expression, the activity of the luciferase reporter gene was significantly decreased after transient transfection of ALDH1A1*3-luciferase compared with the wild-type construct ALDH1A1*1-luciferase. In an African American population, a trend for higher frequencies of the ALDH1A1*2 and ALDH1A1*3 alleles was observed in a population of alcoholics (p = 0.03 and f = 0.12, respectively) compared with the control population.ConclusionsALDH1A1*2 and ALDH1A1*3 may influence ALDH1A1 gene expression. Both ALDH1A1*2 and ALDH1A1*3 produce a trend in an African American population that may be indicative of an association with alcoholism; however, more samples are required to validate this observation. The underlying mechanisms contributing to these trends are still unknown.

Dataset Information

Activation of Human Salivary Aldehyde Dehydrogenase by Sulforaphane: Mechanism and Significance.

Publications

Activation of Human Salivary Aldehyde Dehydrogenase by Sulforaphane: Mechanism and Significance.

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