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1.  Aldehyde dehydrogenase 2 activation and coevolution of its εPKC-mediated phosphorylation sites 
Background
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 (εPKC). 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 εPKC 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 εPKC.
Methods
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 εPKC phosphorylation in vitro. Coevolution of ALDH2 and its εPKC phosphorylation sites was delineated by multiple sequence alignments among a diverse range of species and within the ALDH multigene family.
Results
We identified S279 as a critical εPKC 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 εPKC likely also protects the enzyme against reactive aldehydes. Finally, we demonstrate that the three ALDH2 phosphorylation sites co-evolved with εPKC over a wide range of species. Alignment of 18 human ALDH isozymes, indicates that T185 and S279 are unique ALDH2, εPKC 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.
Conclusion
εPKC phosphorylation and its coevolution with ALDH2 play an important role in the regulation and protection of ALDH2 enzyme activity.
Electronic supplementary material
The online version of this article (doi:10.1186/s12929-016-0312-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s12929-016-0312-x
PMCID: PMC5217657  PMID: 28056995
ALDH2; Aldehyde dehydrogenase 2; εPKC; Phosphorylation; Coevolution; 4HNE
2.  Murine hepatic aldehyde dehydrogenase 1a1 is a major contributor to oxidation of aldehydes formed by lipid peroxidation 
Chemico-biological interactions  2011;191(0):278-287.
Reactive lipid aldehydes are implicated in the pathogenesis of various oxidative stress-mediated diseases, including non-alcoholic steatohepatitis, atherosclerosis, Alzheimer’s and cataract. In the present study, we sought to define which hepatic Aldh isoform plays a major role in detoxification of lipid-derived aldehydes, such as acrolein and HNE by enzyme kinetic and gene expression studies. The catalytic efficiencies for metabolism of acrolein by Aldh1a1 was comparable to that of Aldh3a1 (Vmax/Km = 23). However, Aldh1a1 exhibits far higher affinity for acrolein (Km = 23.2 μM) compared to Aldh3a1 (Km = 464 μM). Aldh1a1 displays a 3-fold higher catalytic efficiency for HNE than Aldh3a1 (218 vs 69 ml/min/mg). The endogenous Aldh1a1 gene was highly expressed in mouse liver and a liver-derived cell line (Hepa-1c1c7) compared to Aldh2, Aldh1b1 and Aldh3a1. Aldh1a1 mRNA levels was 34-fold and 73-fold higher than Aldh2 in mouse liver and Hepa-1c1c7 cells respectively. Aldh3a1 gene was absent in mouse liver, but moderately expressed in Hepa-1c1c7 cells compared to Aldh1a1. We demonstrated that knockdown of Aldh1a1 expression by siRNA caused Hepa-1c1c7 cells to be more sensitive to acrolein-induced cell death and resulted in increased accumulation of acrolein-protein adducts and caspase 3 activation. These results indicate that Aldh1a1 plays a major role in cellular defense against oxidative damage induced by reactive lipid aldehydes in mouse liver. We also noted that hepatic Aldh1a1 mRNA levels were significantly increased (≈ 3 fold) in acrolein-fed mice compared to control. In addition, hepatic cytosolic ALDH activity was induced by acrolein when 1 mM NAD+ was used as cofactor, suggesting an Aldh1a1-protective mechanism against acrolein toxicity in mice liver. Thus, mechanisms to induce Aldh1a1 gene expression may provide a useful rationale for therapeutic protection against oxidative stress-induced pathologies.
doi:10.1016/j.cbi.2011.01.013
PMCID: PMC4409133  PMID: 21256123
aldehyde dehydrogenase 1a1; aldehydes; 4-hydroxy-2-nonenal; acrolein; toxicity
3.  Characterization of the molecular mechanisms underlying increased ischemic damage in the aldehyde dehydrogenase 2 genetic polymorphism using a human induced pluripotent stem cell model system 
Science translational medicine  2014;6(255):255ra130.
Nearly 8% of the human population carries an inactivating point mutation in the gene that encodes the cardioprotective enzyme aldehyde dehydrogenase 2 (ALDH2). This genetic polymorphism (ALDH2*2) is linked to more severe outcomes from ischemic heart damage and an increased risk of coronary artery disease (CAD), but the underlying molecular bases are unknown. We investigated the ALDH2*2 mechanisms in a human model system of induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs) generated from individuals carrying the most common heterozygous form of the ALDH2*2 genotype. We showed that the ALDH2*2 mutation gave rise to elevated amounts of reactive oxygen species and toxic aldehydes, thereby inducing cell cycle arrest and activation of apoptotic signaling pathways, especially during ischemic injury. We established that ALDH2 controls cell survival decisions by modulating oxidative stress levels and that this regulatory circuitry was dysfunctional in the loss-of-function ALDH2*2 genotype, causing up-regulation of apoptosis in cardiomyocytes after ischemic insult. These results reveal a new function for the metabolic enzyme ALDH2 in modulation of cell survival decisions. Insight into the molecular mechanisms that mediate ALDH2*2-related increased ischemic damage is important for the development of specific diagnostic methods and improved risk management of CAD and may lead to patient-specific cardiac therapies.
doi:10.1126/scitranslmed.3009027
PMCID: PMC4215699  PMID: 25253673
4.  ALDH2 in Alcoholic Heart Diseases: Molecular Mechanism and Clinical Implications 
Pharmacology & therapeutics  2011;132(1):86-95.
Alcoholic cardiomyopathy is manifested as cardiac hypertrophy, disrupted contractile function and myofibrillary architecture. An ample amount of clinical and experimental evidence has depicted a pivotal role for alcohol metabolism especially the main alcohol metabolic product acetaldehyde, in the pathogenesis of this myopathic state. Findings from our group and others have revealed that the mitochondrial isoform of aldehyde dehydrogenase (ALDH2), which metabolizes acetaldehyde, governs the detoxification of acetaldehyde formed following alcohol consumption and the ultimate elimination of alcohol from the body. The ALDH2 enzymatic cascade may evolve as a unique detoxification mechanism for environmental alcohols and aldehydes to alleviate the undesired cardiac anomalies in ischemia-reperfusion and alcoholism. Polymorphic variants of the ALDH2 gene encode enzymes with altered pharmacokinetic properties and a significantly higher prevalence of cardiovascular diseases associated with alcoholism. The pathophysiological effects of ALDH2 polymorphism may be mediated by accumulation of acetaldehyde and other reactive aldehydes. Inheritance of the inactive ALDH2*2 gene product is associated with a decreased risk of alcoholism but an increased risk of alcoholic complications. This association is influenced by gene-environment interactions such as those associated with religion and national origin. The purpose of this review is to recapitulate the pathogenesis of alcoholic cardiomyopathy with a special focus on ALDH2 enzymatic metabolism. It will be important to dissect the links between ALDH2 polymorphism and prevalence of alcoholic cardiomyopathy, in order to determine the mechanisms underlying such associations. The therapeutic value of ALDH2 as both target and tool in the management of alcoholic tissue damage will be discussed.
doi:10.1016/j.pharmthera.2011.05.008
PMCID: PMC3144032  PMID: 21664374
Alcohol; ALDH2; enzyme; metabolism; myocardial; transgenic mice
5.  Inhibition of Aldehyde Dehydrogenase 2 by Oxidative Stress Is Associated with Cardiac Dysfunction in Diabetic Rats 
Molecular Medicine  2010;17(3-4):172-179.
Left ventricular (LV) dysfunction is a common comorbidity in diabetic patients, although the molecular mechanisms underlying this cardiomyopathic feature are not completely understood. Aldehyde dehydrogenase 2 (ALDH2) has been considered a key cardioprotective enzyme susceptible to oxidative inactivation. We hypothesized that hyperglycemia-induced oxidative stress would influence ALDH2 activity, and ALDH2 inhibition would lead to cardiac functional alterations in diabetic rats. Diabetes was induced by intraperitoneal (i.p.) injection of 60 mg/kg streptozotocin. Rats were divided randomly into four groups: control, untreated diabetic, diabetic treated with N-acetylcysteine (NAC) and diabetic treated with α-lipoic acid (α-LA). Cardiac contractile function, oxidative stress markers and reactive oxygen species (ROS) levels were assessed. ALDH2 activity and expression also were determined. The role of ALDH2 activity in change in hyperglycemia-induced mitochondrial membrane potential (Δψ) was tested in cultured neonatal cardiomyocytes. Myocardial MDA content and ROS were significantly higher in diabetic rats than in controls, whereas GSH content and Mn-SOD activity were decreased in diabetic rats. Compared with controls, diabetic rats exhibited significant reduction in LV ejection fraction and fractional shortening, accompanied by decreases in ALDH2 activity and expression. NAC and α-LA attenuated these changes. Mitochondrial Δψ was decreased greatly with hyperglycemia treatment, and high glucose combined with ALDH2 inhibition with daidzin further decreased Δψ. The ALDH2 activity can be regulated by oxidative stress in the diabetic rat heart. ALDH2 inhibition may be associated with LV reduced contractility, and mitochondrial impairment aggravated by ALDH2 inhibition might reflect an underlying mechanism which causes cardiac dysfunction in diabetic rats.
doi:10.2119/molmed.2010.00114
PMCID: PMC3060979  PMID: 20957334
6.  Comparative study of the aldehyde dehydrogenase (ALDH) gene superfamily in the glycophyte Arabidopsis thaliana and Eutrema halophytes 
Annals of Botany  2014;115(3):465-479.
Background and Aims Stresses such as drought or salinity induce the generation of reactive oxygen species, which subsequently cause excessive accumulation of aldehydes in plant cells. Aldehyde dehydrogenases (ALDHs) are considered as ‘aldehyde scavengers’ to eliminate toxic aldehydes caused by oxidative stress. The completion of the genome sequencing projects of the halophytes Eutrema parvulum and E. salsugineum has paved the way to explore the relationships and the roles of ALDH genes in the glycophyte Arabidopsis thaliana and halophyte model plants.
Methods Protein sequences of all plant ALDH families were used as queries to search E. parvulum and E. salsugineum genome databases. Evolutionary analyses compared the phylogenetic relationships of ALDHs from A. thaliana and Eutrema. Expression patterns of several stress-associated ALDH genes were investigated under different salt conditions using reverse transcription–PCR. Putative cis-elements in the promoters of ALDH10A8 from A. thaliana and E. salsugineum were compared in silico.
Key Results Sixteen and 17 members of ten ALDH families were identified from E. parvulum and E. salsugineum genomes, respectively. Phylogenetic analysis of ALDH protein sequences indicated that Eutrema ALDHs are closely related to those of Arabidopsis, and members within these species possess nearly identical exon–intron structures. Gene expression analysis under different salt conditions showed that most of the ALDH genes have similar expression profiles in Arabidopsis and E. salsugineum, except for ALDH7B4 and ALDH10A8. In silico analysis of promoter regions of ALDH10A8 revealed different distributions of cis-elements in E. salsugineum and Arabidopsis.
Conclusions Genomic organization, copy number, sub-cellular localization and expression profiles of ALDH genes are conserved in Arabidopsis, E. parvulum and E. salsugineum. The different expression patterns of ALDH7B4 and ALDH10A8 in Arabidopsis and E. salsugineum suggest that E. salsugineum uses modified regulatory pathways, which may contribute to salinity tolerance.
doi:10.1093/aob/mcu152
PMCID: PMC4332599  PMID: 25085467
Aldehyde dehydrogenases; ALDH; Arabidopsis thaliana; Eutrema parvulum; Eutrema salsugineum; genome comparisons; glycophyte; halophytes; oxidative stress; ROS; salinity stress; saline adaptation
7.  Aldehyde Dehydrogenases in Cellular Responses to Oxidative/electrophilic Stress 
Reactive oxygen species (ROS) are continuously generated within living systems and the inability to manage ROS load leads to elevated oxidative stress and cell damage. Oxidative stress is coupled to the oxidative degradation of lipid membranes, also known as lipid peroxidation. This process generates over 200 types of aldehydes, many of which are highly reactive and toxic. Aldehyde dehydrogenases (ALDHs) metabolize endogenous and exogenous aldehydes and thereby mitigate oxidative/electrophilic stress in prokaryotic and eukaryotic organisms. ALDHs are found throughout the evolutionary gamut, from single celled organisms to complex multicellular species. Not surprisingly, many ALDHs in evolutionarily distant, and seemingly unrelated, species perform similar functions, including protection against a variety of environmental stressors like dehydration and ultraviolet radiation. The ability to act as an ‘aldehyde scavenger’ during lipid peroxidation is another ostensibly universal ALDH function found across species. Up-regulation of ALDHs is a stress response in bacteria (environmental and chemical stress), plants (dehydration, salinity and oxidative stress), yeast (ethanol exposure and oxidative stress), Caenorhabditis elegans (lipid peroxidation) and mammals (oxidative stress and lipid peroxidation). Recent studies have also identified ALDH activity as an important feature of cancer stem cells. In these cells, ALDH expression helps abrogate oxidative stress and imparts resistance against chemotherapeutic agents such as oxazaphosphorine, taxane and platinum drugs. The ALDH superfamily represents a fundamentally important class of enzymes that significantly contributes to the management of electrophilic/oxidative stress within living systems. Mutations in various ALDHs are associated with a variety of pathological conditions in humans, underscoring the fundamental importance of these enzymes in physiological and pathological processes.
doi:10.1016/j.freeradbiomed.2012.11.010
PMCID: PMC3631350  PMID: 23195683
Aldehyde dehydrogenase; Cancer stem cells; Chemical stress; Dehydration; Electrophilic stress; Oxidative stress
8.  Organic Nitrate Therapy, Nitrate Tolerance, and Nitrate-Induced Endothelial Dysfunction: Emphasis on Redox Biology and Oxidative Stress 
Antioxidants & Redox Signaling  2015;23(11):899-942.
Abstract
Organic nitrates, such as nitroglycerin (GTN), isosorbide-5-mononitrate and isosorbide dinitrate, and pentaerithrityl tetranitrate (PETN), when given acutely, have potent vasodilator effects improving symptoms in patients with acute and chronic congestive heart failure, stable coronary artery disease, acute coronary syndromes, or arterial hypertension. The mechanisms underlying vasodilation include the release of •NO or a related compound in response to intracellular bioactivation (for GTN, the mitochondrial aldehyde dehydrogenase [ALDH-2]) and activation of the enzyme, soluble guanylyl cyclase. Increasing cyclic guanosine-3′,-5′-monophosphate (cGMP) levels lead to an activation of the cGMP-dependent kinase I, thereby causing the relaxation of the vascular smooth muscle by decreasing intracellular calcium concentrations. The hemodynamic and anti-ischemic effects of organic nitrates are rapidly lost upon long-term (low-dose) administration due to the rapid development of tolerance and endothelial dysfunction, which is in most cases linked to increased intracellular oxidative stress. Enzymatic sources of reactive oxygen species under nitrate therapy include mitochondria, NADPH oxidases, and an uncoupled •NO synthase. Acute high-dose challenges with organic nitrates cause a similar loss of potency (tachyphylaxis), but with distinct pathomechanism. The differences among organic nitrates are highlighted regarding their potency to induce oxidative stress and subsequent tolerance and endothelial dysfunction. We also address pleiotropic effects of organic nitrates, for example, their capacity to stimulate antioxidant pathways like those demonstrated for PETN, all of which may prevent adverse effects in response to long-term therapy. Based on these considerations, we will discuss and present some preclinical data on how the nitrate of the future should be designed. Antioxid. Redox Signal. 23, 899–942.
I. Introduction
A. Historical view
B. Hemodynamic effects of nitrates in patients with coronary artery disease and acute and chronic congestive heart failure
C. Cellular mechanisms of vasodilation by organic nitrates
D. No •NO from nitroglycerin?
E. Clinical uses/usefulness of organic nitrates
F. Do nitrates beneficially influence prognosis in patients with CAD?
G. Organic nitrate chemistry and pharmacokinetics
II. Organic Nitrate Bioactivation
A. High-potency (affinity) pathway involving the ALDH-2 in GTN bioactivation
1. ALDH-2 also involved in PETN bioactivation
2. Impact of gene polymorphism on GTN bioactivation by ALDH-2
3. Redox regulation of the ALDH-2
4. Dihydrolipoic acid and thioredoxin as physiological reducing factors of ALDH-2
5. Molecular mechanism of ALDH-2 bioactivation of organic nitrates via its reductase activity
6. Impact of organic nitrate-induced ROS formation on ALDH-2 activity
7. Purified ALDH-2 as a peroxynitrite synthase in the presence of GTN
B. The low-potency (affinity) pathway for bioactivation of GTN—low-molecular-weight systems and enzymatic systems of nitrate bioactivation besides ALDH-2
1. Cytochrome P450 enzymes
2. Xanthine oxidoreductase
3. Glutathione-S-transferase
4. Glyceraldehyde-3-phosphate dehydrogenase
5. Other ALDH isoforms
III. Side Effects of Chronic Nitroglycerin Therapy
A. Nitrate tolerance
1. Pseudotolerance
2. True vascular tolerance mechanisms
a. GTN increases oxidative stress and causes tolerance
b. Oxidative stress impairs GTN biotransformation
c. Desensitization of the sGC
d. Increased sensitivity to vasoconstrictors in response to GTN therapy
e. Increased sensitivity to vasoconstrictors in response to ISMN therapy
f. Epigenetic mechanisms of GTN-induced tolerance
B. Organic nitrates cause endothelial dysfunction in the clinical setting
1. Molecular mechanisms underlying nitrate-induced endothelial dysfunction
a. BH4 deficiency
b. Changes in eNOS phosphorylation and S-glutathionylation
c. Increased NADPH oxidase activity
d. Nitration of prostacyclin synthase
IV. Strategies to Prevent Development of Nitrate Tolerance and Nitrate-Induced Endothelial Dysfunction
A. Nitrate-free interval
B. Sulfhydryl group donors
C. Antioxidants
D. ACE inhibitors and angiotensin II type 1 receptor blockers
E. Hydralazine
F. Carvedilol
G. Statins
H. Endothelin receptor blockers
I. sGC stimulators and activators
J. PDE inhibitors
V. Pleiotropic Effects of Organic Nitrates
A. Ischemic preconditioning
B. Antioxidant properties of PETN: Nrf2 and HO-1
C. Epigenetic pathways, microRNA, and gene regulation
D. The effect of organic nitrates on endothelial progenitor cells
VI. Development of New Organic Nitrates Devoid of Side Effects
VII. Summary and Future Perspectives for the Class of •NO Donors
doi:10.1089/ars.2015.6376
PMCID: PMC4752190  PMID: 26261901
9.  Molecular Characterization, Expression Analysis and Role of ALDH3B1 in The Cellular Protection Against Oxidative Stress 
Free radical biology & medicine  2010;49(9):1432-1443.
Aldehyde dehydrogenase (ALDH) enzymes are critical in the detoxification of aldehydes. The human genome contains 19 ALDH genes, mutations in which are the basis of several diseases. The expression, subcellular localization, enzyme kinetics and role of ALDH3B1 against aldehyde- and oxidant-induced cytotoxicity were investigated. ALDH3B1 was purified from Sf9 cells using chromatographic methods and enzyme kinetics were determined spectrophotometrically. ALDH3B1 demonstrated high affinity for hexanal (Km 62 μM), octanal (Km 8 μM), 4-hydroxy-2-nonenal (4HNE) (Km 52 μM) and benzaldehyde (Km 46 μM). Low affinity was seen towards acetaldehyde (Km 23.3 mM), malondialdehyde (Km 152 mM) and the ester p-nitrophenylacetate (Km 3.6 mM). ALDH3B1 mRNA was abundant in testis, lung, kidney and ovary. ALDH3B1 protein was highly expressed in these tissues and the liver. Immunofluorescence microscopy of ALDH3B1-transfected human embryonic kidney (HEK293) cells and subcellular fractionation of mouse kidney and liver revealed a cytosolic protein localization. ALDH3B1-transfected HEK293 cells were significantly protected from the lipid peroxidation-derived aldehydes trans-2-octenal, 4HNE and hexanal, and the oxidants H2O2 and menadione. In addition, ALDH3B1 protein expression was up-regulated by 4HNE in ARPE-19 cells. The results detailed in this study support a pathophysiological role for ALDH3B1 in protecting cells from the damaging effects of oxidative stress.
doi:10.1016/j.freeradbiomed.2010.08.004
PMCID: PMC3457645  PMID: 20699116
aldehyde dehydrogenase 3B1 (ALDH3B1); 4-hydroxy-2-nonenal; lipid peroxidation; mRNA; protein expression; enzyme kinetics; aldehyde toxicity
10.  Aldehyde dehydrogenase 7A1 (ALDH7A1) attenuates reactive aldehyde and oxidative stress induced cytotoxicity 
Chemico-Biological Interactions  2011;191(1-3):269-277.
Mammalian aldehyde dehydrogenase 7A1 (ALDH7A1) is homologous to plant ALDH7B1 which protects against various forms of stress such as increased salinity, dehydration and treatment with oxidants or pesticides. Deleterious mutations in human ALDH7A1 are responsible for pyridoxine-dependent and folinic acid-responsive seizures. In previous studies, we have shown that human ALDH7A1 protects against hyperosmotic stress presumably through the generation of betaine, an important cellular osmolyte, formed from betaine aldehyde. Hyperosmotic stress is coupled to an increase in oxidative stress and lipid peroxidation (LPO). In this study, cell viability assays revealed that stable expression of mitochondrial ALDH7A1 in Chinese hamster ovary (CHO) cells provides significant protection against treatment with the LPO-derived aldehydes hexanal and 4-hydroxy-2-nonenal (4HNE) implicating a protective function for the enzyme during oxidative stress. A significant increase in cell survival was also observed in CHO cells expressing either mitochondrial or cytosolic ALDH7A1 treated with increasing concentrations of hydrogen peroxide (H2O2) or 4HNE, providing further evidence for anti-oxidant activity. In vitro enzyme activity assays indicate that human ALDH7A1 is sensitive to oxidation and that efficiency can be at least partially restored by incubating recombinant protein with the thiol reducing agent β-mercaptoethanol (BME). We also show that after reactivation with BME, recombinant ALDH7A1 is capable of metabolizing the reactive aldehyde 4HNE. In conclusion, ALDH7A1 mechanistically appears to provide cells protection through multiple pathways including the removal of toxic LPO-derived aldehydes in addition to osmolyte generation.
doi:10.1016/j.cbi.2011.02.016
PMCID: PMC3387551  PMID: 21338592
Aldehyde dehydrogenase 7A1; ALDH7A1; Antiquitin; Oxidative stress; 4HNE; 4-Hydroxynonenal
11.  Post-translational modifications of mitochondrial aldehyde dehydrogenase and biomedical implications 
Journal of proteomics  2011;74(12):2691-2702.
Aldehyde dehydrogenases (ALDHs) represent large family members of NAD(P)+-dependent dehydrogenases responsible for the irreversible metabolism of many endogenous and exogenous aldehydes to the corresponding acids. Among 19 ALDH isozymes, mitochondrial ALDH2 is a low Km enzyme responsible for the metabolism of acetaldehyde and lipid peroxides such as malondialdehyde and 4-hydroxynonenal, both of which are highly reactive and toxic. Consequently, inhibition of ALDH2 would lead to elevated levels of acetaldehyde and other reactive lipid peroxides following ethanol intake and/or exposure to toxic chemicals. In addition, many East Asian people with a dominant negative mutation in ALDH2 gene possess a decreased ALDH2 activity with increased risks for various types of cancer, myocardial infarct, alcoholic liver disease, and other pathological conditions. The aim of this review is to briefly describe the multiple post-translational modifications of mitochondrial ALDH2, as an example, after exposure to toxic chemicals or under different disease states and their pathophysiological roles in promoting alcohol/drug-mediated tissue damage. We also briefly mention exciting preclinical translational research opportunities to identify small molecule activators of ALDH2 and its isozymes as potentially therapeutic/preventive agents against various disease states where the expression or activity of ALDH enzymes is altered or inactivated.
doi:10.1016/j.jprot.2011.05.013
PMCID: PMC3177986  PMID: 21609791
Aldehyde dehydrogenases; post-translational modifications; cellular defense; drug toxicity; disease states; translational research
12.  Impaired Cardiac SIRT1 Activity by Carbonyl Stress Contributes to Aging-Related Ischemic Intolerance 
PLoS ONE  2013;8(9):e74050.
Reactive aldehydes can initiate protein oxidative damage which may contribute to heart senescence. Sirtuin 1 (SIRT1) is considered to be a potential interventional target for I/R injury management in the elderly. We hypothesized that aldehyde mediated carbonyl stress increases susceptibility of aged hearts to ischemia/reperfusion (I/R) injury, and elucidate the underlying mechanisms with a focus on SIRT1. Male C57BL/6 young (4-6 mo) and aged (22-24 mo) mice were subjected to myocardial I/R. Cardiac aldehyde dehydrogenase (ALDH2), SIRT1 activity and protein carbonyls were assessed. Our data revealed that aged heart exhibited increased endogenous aldehyde/carbonyl stress due to impaired ALDH2 activity concomitant with blunted SIRT1 activity (P<0.05). Exogenous toxic aldehydes (4-HNE) exposure in isolated cardiomyocyte verified that aldehyde-induced carbonyl modification on SIRT1 impaired SIRT1 activity leading to worse hypoxia/reoxygenation (H/R) injury, which could all be rescued by Alda-1 (ALDH2 activator) (all P<0.05). However, SIRT1 inhibitor blocked the protective effect of Alda-1 on H/R cardiomyocyte. Interestingly, myocardial I/R leads to higher carbonylation but lower activity of SIRT1 in aged hearts than that seen in young hearts (P<0.05). The application of Alda-1 significantly reduced the carbonylation on SIRT1 and markedly improved the tolerance to in vivo I/R injury in aged hearts, but failed to protect Sirt1+/− knockout mice against myocardial I/R injury. This was verified by Alda-1 treatment improved postischemic contractile function recovery in ex vivo perfused aged but not in Sirt1+/− hearts. Thus, aldehyde/carbonyl stress is accelerated in aging heart. These results provide a new insight that impaired cardiac SIRT1 activity by carbonyl stress plays a critical role in the increased susceptibility of aged heart to I/R injury. ALDH2 activation can restore this aging-related myocardial ischemic intolerance.
doi:10.1371/journal.pone.0074050
PMCID: PMC3769351  PMID: 24040162
13.  Neuroprotective effects of aldehyde dehydrogenase 2 activation in rotenone-induced cellular and animal models of parkinsonism 
Experimental neurology  2014;263:244-253.
Many studies have shown that mitochondrial aldehyde dehydrogenase 2 (ALDH2) functions as a cellular protector against oxidative stress by detoxification of cytotoxic aldehydes. Within dopaminergic neurons, dopamine is metabolized by monoamine oxidase to yield 3,4-dihydroxyphenylacetaldehyde (DOPAL) then converts to a less toxic acid product by ALDH. The highly toxic and reactive DOPAL has been hypothesized to contribute to the selective neurodegeneration in Parkinson’s disease (PD). In this study, we investigated the neuroprotective mechanism and therapeutic effect of ALDH2 in rotenone models for parkinsonism. Overexpression of wild-type ALDH2 gene, but not the enzymatically deficient mutant ALDH2*2 (E504K), reduced rotenone-induced cell death. Application of a potent activator of ALDH2, Alda-1, was effective in protecting against rotenone-induced apoptotic cell death in both SH-SY5Y cells and primary cultured substantia nigra (SN) dopaminergic neurons. In addition, intraperitoneal administration of Alda-1 significantly reduced rotenone- or MPTP-induced death of SN tyrosine hydroxylase (TH)-positive dopaminergic neurons. The attenuation of rotenone-induced apoptosis by Alda-1 resulted from decreasing ROS accumulation, reversal of mitochondrial membrane potential depolarization, and inhibition of activation of proteins related to mitochondrial apoptotic pathway. The present study demonstrates that ALDH2 plays a crucial role in maintaining normal mitochondrial function to protect against neurotoxicity and that Alda-1 is effective in ameliorating mitochondrial dysfunction and inhibiting mitochondria-mediated apoptotic pathway. These results indicate that ALDH2 activation could be a neuroprotective therapy for PD.
doi:10.1016/j.expneurol.2014.09.016
PMCID: PMC4415848  PMID: 25263579
Alda-1; aldehyde dehydrogenase 2 (ALDH2); mitochondrial dysfunction; Parkinson’s disease; rotenone; substantia nigra dopaminergic neurons
14.  ALDH3A1 Plays a Functional Role in Maintenance of Corneal Epithelial Homeostasis 
PLoS ONE  2016;11(1):e0146433.
Aldehyde dehydrogenase 1A1 (ALDH1A1) and ALDH3A1 are corneal crystallins. They protect inner ocular tissues from ultraviolet radiation (UVR)-induced oxidative damage through catalytic and non-catalytic mechanisms. Additionally, ALDH3A1 has been postulated to play a regulatory role in the corneal epithelium based on several studies that report an inverse association between ALDH3A1 expression and corneal cell proliferation. The underlying molecular mechanisms and the physiological significance of such association remain poorly understood. In the current study, we established Tet-On human corneal epithelial cell (hTCEpi) lines, which express tetracycline-inducible wild-type (wt) or catalytically-inactive (mu) ALDH3A1. Utilizing this cellular model system, we confirmed that human ALDH3A1 decreases corneal cell proliferation; importantly, this effect appears to be partially mediated by its enzymatic activity. Mechanistically, wt-ALDH3A1, but not mu-ALDH3A1, promotes sequestering of tumor suppressor p53 in the nucleus. In the mouse cornea, however, augmented cell proliferation is noted only in Aldh1a1-/-/3a1-/- double knockout (DKO) mice, indicating in vivo the anti-proliferation effect of ALDH3A1 can be rescued by the presence of ALDH1A1. Interestingly, the hyper-proliferative epithelium of the DKO corneas display nearly complete loss of p53 expression, implying that p53 may be involved in ALDH3A1/1A1-mediated effect. In hTCEpi cells grown in high calcium concentration, mRNA levels of a panel of corneal differentiation markers were altered by ALDH3A1 expression and modulated by its enzyme activity. In conclusion, we show for the first time that: (i) ALDH3A1 decreases corneal epithelial proliferation through both non-enzymatic and enzymatic properties; (ii) ALDH1A1 contributes to the regulation of corneal cellular proliferation in vivo; and (iii) ALDH3A1 modulates corneal epithelial differentiation. Collectively, our studies indicate a functional role of ALDH3A1 in the maintenance of corneal epithelial homeostasis by simultaneously modulating proliferation and differentiation through both enzymatic and non-enzymatic mechanisms.
doi:10.1371/journal.pone.0146433
PMCID: PMC4708999  PMID: 26751691
15.  Corneal Aldehyde Dehydogenases: Multiple Functions and Novel Nuclear Localization 
Brain research bulletin  2009;81(2-3):211-218.
Aldehyde dehydrogenases (ALDHs) represent a superfamily of NAD(P)+-dependent enzymes which catalyze the oxidation of a wide variety of endogenous and exogenous aldehydes to their corresponding acids. Some ALDHs have been identified as corneal crystallins and thereby contribute to the protective and refractive properties of the cornea. ALDH3A1 is highly expressed in the cornea of most mammals with the exception of rabbit, who abundantly expresses ALDH1A1 in the cornea instead of ALDH3A1. In this study, we examined the gene expression of other ALDHs and found high messenger levels of ALDH1B1, ALDH2 and ALDH7A1 in mouse cornea and lens. Substantial evidence supports a protective role for ALDH3A1 and ALDH1A1 against ultraviolet radiation (UVR)-induced oxidative damage to ocular tissues. The mechanism by which this protection occurs includes UVR filtering, detoxification of reactive aldehydes generated by UVR exposure and antioxidant activity. We recently have identified ALDH3A1 as a nuclear protein in corneal epithelium. Herein, we show that ALDH3A1 is also found in the nucleus of rabbit keratocytes. The nuclear presence of ALDH3A1 may be involved in cell cycle regulation.
doi:10.1016/j.brainresbull.2009.08.017
PMCID: PMC3025408  PMID: 19720116
16.  Mitochondrial Aldehyde Dehydrogenase 2 Plays Protective Roles in Heart Failure After Myocardial Infarction via Suppression of the Cytosolic JNK/p53 Pathway in Mice 
Background
Increasing evidence suggests a critical role for mitochondrial aldehyde dehydrogenase 2 (ALDH2) in protection against cardiac injuries; however, the downstream cytosolic actions of this enzyme are largely undefined.
Methods and Results
Proteomic analysis identified a significant downregulation of mitochondrial ALDH2 in the heart of a rat heart failure model after myocardial infarction. The mechanistic insights underlying ALDH2 action were elucidated using murine models overexpressing ALDH2 or its mutant or with the ablation of the ALDH2 gene (ALDH2 knockout) and neonatal cardiomyocytes undergoing altered expression and activity of ALDH2. Left ventricle dilation and dysfunction and cardiomyocyte death after myocardial infarction were exacerbated in ALDH2‐knockout or ALDH2 mutant‐overexpressing mice but were significantly attenuated in ALDH2‐overexpressing mice. Using an anoxia model of cardiomyocytes with deficiency in ALDH2 activities, we observed prominent cardiomyocyte apoptosis and increased accumulation of the reactive aldehyde 4‐hydroxy‐2‐nonenal (4‐HNE). We subsequently examined the impacts of mitochondrial ALDH2 and 4‐HNE on the relevant cytosolic protective pathways. Our data documented 4‐HNE‐stimulated p53 upregulation via the phosphorylation of JNK, accompanying increased cardiomyocyte apoptosis that was attenuated by inhibition of p53. Importantly, elevation of 4‐HNE also triggered a reduction of the cytosolic HSP70, further corroborating cytosolic action of the 4‐HNE instigated by downregulation of mitochondrial ALDH2.
Conclusions
Downregulation of ALDH2 in the mitochondria induced an elevation of 4‐HNE, leading to cardiomyocyte apoptosis by subsequent inhibition of HSP70, phosphorylation of JNK, and activation of p53. This chain of molecular events took place in both the mitochondria and the cytosol, contributing to the mechanism underlying heart failure.
doi:10.1161/JAHA.113.000779
PMCID: PMC4323818  PMID: 25237043
ALDH2; apoptosis; heart failure; myocardial infarction; p53
17.  Genome-Wide Identification and Functional Classification of Tomato (Solanum lycopersicum) Aldehyde Dehydrogenase (ALDH) Gene Superfamily 
PLoS ONE  2016;11(10):e0164798.
Aldehyde dehydrogenases (ALDHs) is a protein superfamily that catalyzes the oxidation of aldehyde molecules into their corresponding non-toxic carboxylic acids, and responding to different environmental stresses, offering promising genetic approaches for improving plant adaptation. The aim of the current study is the functional analysis for systematic identification of S. lycopersicum ALDH gene superfamily. We performed genome-based ALDH genes identification and functional classification, phylogenetic relationship, structure and catalytic domains analysis, and microarray based gene expression. Twenty nine unique tomato ALDH sequences encoding 11 ALDH families were identified, including a unique member of the family 19 ALDH. Phylogenetic analysis revealed 13 groups, with a conserved relationship among ALDH families. Functional structure analysis of ALDH2 showed a catalytic mechanism involving Cys-Glu couple. However, the analysis of ALDH3 showed no functional gene duplication or potential neo-functionalities. Gene expression analysis reveals that particular ALDH genes might respond to wounding stress increasing the expression as ALDH2B7. Overall, this study reveals the complexity of S. lycopersicum ALDH gene superfamily and offers new insights into the structure-functional features and evolution of ALDH gene families in vascular plants. The functional characterization of ALDHs is valuable and promoting molecular breeding in tomato for the improvement of stress tolerance and signaling.
doi:10.1371/journal.pone.0164798
PMCID: PMC5068750  PMID: 27755582
18.  Molecular Cloning, Characterization, and Potential Roles of Cytosolic and Mitochondrial Aldehyde Dehydrogenases in Ethanol Metabolism in Saccharomyces cerevisiae† 
Journal of Bacteriology  1998;180(4):822-830.
The full-length DNAs for two Saccharomyces cerevisiae aldehyde dehydrogenase (ALDH) genes were cloned and expressed in Escherichia coli. A 2,744-bp DNA fragment contained an open reading frame encoding cytosolic ALDH1, with 500 amino acids, which was located on chromosome XVI. A 2,661-bp DNA fragment contained an open reading frame encoding mitochondrial ALDH5, with 519 amino acids, of which the N-terminal 23 amino acids were identified as the putative leader sequence. The ALDH5 gene was located on chromosome V. The commercial ALDH (designated ALDH2) was partially sequenced and appears to be a mitochondrial enzyme encoded by a gene located on chromosome XV. The recombinant ALDH1 enzyme was found to be essentially NADP dependent, while the ALDH5 enzyme could utilize either NADP or NAD as a cofactor. The activity of ALDH1 was stimulated two- to fourfold by divalent cations but was unaffected by K+ ions. In contrast, the activity of ALDH5 increased in the presence of K+ ions: 15-fold with NADP and 40-fold with NAD, respectively. Activity staining of isoelectric focusing gels showed that cytosolic ALDH1 contributed 30 to 70% of the overall activity, depending on the cofactor used, while mitochondrial ALDH2 contributed the rest. Neither ALDH5 nor the other ALDH-like proteins identified from the genomic sequence contributed to the in vitro oxidation of acetaldehyde. To evaluate the physiological roles of these three ALDH isoenzymes, the genes encoding cytosolic ALDH1 and mitochondrial ALDH2 and ALDH5 were disrupted in the genome of strain TWY397 separately or simultaneously. The growth of single-disruption Δald1 and Δald2 strains on ethanol was marginally slower than that of the parent strain. The Δald1 Δald2 double-disruption strain failed to grow on glucose alone, but growth was restored by the addition of acetate, indicating that both ALDHs might catalyze the oxidation of acetaldehyde produced during fermentation. The double-disruption strain grew very slowly on ethanol. The role of mitochondrial ALDH5 in acetaldehyde metabolism has not been defined but appears to be unimportant.
PMCID: PMC106960  PMID: 9473035
19.  Genome-Wide Identification and Analysis of Grape Aldehyde Dehydrogenase (ALDH) Gene Superfamily 
PLoS ONE  2012;7(2):e32153.
Background
The completion of the grape genome sequencing project has paved the way for novel gene discovery and functional analysis. Aldehyde dehydrogenases (ALDHs) comprise a gene superfamily encoding NAD(P)+-dependent enzymes that catalyze the irreversible oxidation of a wide range of endogenous and exogenous aromatic and aliphatic aldehydes. Although ALDHs have been systematically investigated in several plant species including Arabidopsis and rice, our knowledge concerning the ALDH genes, their evolutionary relationship and expression patterns in grape has been limited.
Methodology/Principal Findings
A total of 23 ALDH genes were identified in the grape genome and grouped into ten families according to the unified nomenclature system developed by the ALDH Gene Nomenclature Committee (AGNC). Members within the same grape ALDH families possess nearly identical exon-intron structures. Evolutionary analysis indicates that both segmental and tandem duplication events have contributed significantly to the expansion of grape ALDH genes. Phylogenetic analysis of ALDH protein sequences from seven plant species indicates that grape ALDHs are more closely related to those of Arabidopsis. In addition, synteny analysis between grape and Arabidopsis shows that homologs of a number of grape ALDHs are found in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes arose before the speciation of the grape and Arabidopsis. Microarray gene expression analysis revealed large number of grape ALDH genes responsive to drought or salt stress. Furthermore, we found a number of ALDH genes showed significantly changed expressions in responses to infection with different pathogens and during grape berry development, suggesting novel roles of ALDH genes in plant-pathogen interactions and berry development.
Conclusion
The genome-wide identification, evolutionary and expression analysis of grape ALDH genes should facilitate research in this gene family and provide new insights regarding their evolution history and functional roles in plant stress tolerance.
doi:10.1371/journal.pone.0032153
PMCID: PMC3280228  PMID: 22355416
20.  Discovery of a series of aromatic lactones as ALDH1/2-directed inhibitors 
In humans, the aldehyde dehydrogenase superfamily consists of 19 isoenzymes which mostly catalyze the NAD(P)+-dependent oxidation of aldehydes. Many of these isoenzymes have overlapping substrate specificities and therefore their potential physiological functions may overlap. Thus the development of new isoenyzme-selective probes would be able to better delineate the function of a single isoenyzme and its individual contribution to the metabolism of a particular substrate. This specific study was designed to find a novel modulator of ALDH2, a mitochondrial ALDH isoenzyme most well-known for its role in acetaldehyde oxidation. 53 compounds were initially identified to modulate the activity of ALDH2 by a high-throughput esterase screen from a library of 63,000 compounds. Of these initial 53 compounds, 12 were found to also modulate the oxidation of propionaldehyde by ALDH2. Single concentration measurements at 10 μM compound were performed using ALDH1A1, ALDH1A2, ALDH1A3, ALDH2, ALDH1B1, ALDH3A1, ALDH4A1, and/or ALDH5A1 to determine the selectivity of these 12 compounds towards ALDH2. Four of the twelve compounds shared an aromatic lactone structure and were found to be potent inhibitors of the ALDH1/2 isoenzymes, but have no inhibitory effect on ALDH3A1, ALDH4A1 or ALDH5A1. Two of the aromatic lactones show selectivity within the ALDH1/2 class, and one appears to be selective for ALDH2 compared to all other isoenzymes tested.
doi:10.1016/j.cbi.2014.12.038
PMCID: PMC4414788  PMID: 25641190
aldehyde dehydrogenase; high-throughput screening
21.  Development of a high-throughput in vitro assay to identify selective inhibitors for human ALDH1A1 
The human aldehyde dehydrogenase (ALDH) superfamily consists of at least 19 enzymes that metabolize endogenous and exogenous aldehydes. Currently, there are no commercially available inhibitors that target ALDH1A1 but have little to no effect on the structurally and functionally similar ALDH2. Here we present the first human ALDH1A1 structure, as the apoenzyme and in complex with its cofactor NADH to a resolution of 1.75 Å and 2.1 Å, respectfully. Structural comparisons of the cofactor binding sites in ALDH1A1 with other closely related ALDH enzymes illustrate a high degree of similarity. In order to minimize discovery of compounds that inhibit both isoenzymes by interfering with their conserved cofactor binding sites, this study reports the use of an in vitro, NAD+-independent, esterase-based high-throughput screen (HTS) of 64,000 compounds to discover novel, selective inhibitors of ALDH1A1. We describe 256 hits that alter the esterase activity of ALDH1A1. The effects on aldehyde oxidation of 67 compounds were further analyzed, with 30 selectively inhibiting ALDH1A1 compared to ALDH2 and ALDH3A1. One compound inhibited ALDH1A1 and ALDH2, while another inhibited ALDH1A1, ALDH2, and the more distantly related ALDH3A1. The results presented here indicate that this in vitro enzyme activity screening protocol successfully identified ALDH1A1 inhibitors with a high degree of isoenzyme selectivity. The compounds identified via this screen plus the screening methodology itself represent a starting point for the development of highly potent and selective inhibitors of ALDH1A1 that may be utilized to better understand the role of this enzyme in both normal and disease states.
doi:10.1016/j.cbi.2014.10.028
PMCID: PMC4414680  PMID: 25450233
aldehyde dehydrogenase; retinaldehyde dehydrogenase; high-throughput screen; small molecule inhibitors; enzyme inhibition
22.  Association between aldehyde dehydrogenase 2 polymorphisms and the incidence of diabetic retinopathy among Japanese subjects with type 2 diabetes mellitus 
Background
Mitochondrial aldehyde dehydrogenase 2 (ALDH2) detoxifies reactive aldehydes in the micro- and macrovasculature. These substrates, including methylglyoxal and 4-hydroxynonenal formed from glucose and lipids, cause protein carbonylation and mitochondrial dysfunction, forming advanced glycation end products (AGEs). The present study aimed to confirm the association between the inactive ALDH2*2 allele and diabetic retinopathy (DR).
Methods
A retrospective longitudinal analysis was conducted, among 234 Japanese patients with type 2 diabetes mellitus (DM) (156 males and 78 females) who had no DR signs at baseline and were treated for more than half a year. The ALDH2*1/*2 alleles were determined using a real-time TaqMan allelic discrimination assay. Multivariate-adjusted hazard ratios (HRs) and 95% confidential intervals (CIs) for the cumulative incidence of the development of DR were examined using a Cox proportional hazard model, taking drinking habits and the serum γ-glutamyltransferase (GGT) level into consideration.
Results
The frequency of the ALDH2*2 allele was 22.3%. Fifty-two subjects cumulatively developed DR during the follow-up period of 5.5 ± 2.5 years. The ALDH2*2 allele carriers had a significantly higher incidence of DR than the non-carriers (HR: 1.92; P = 0.02). The incidence of DR was significantly higher in the drinkers with the ALDH2*2 allele than in those with the ALDH2*1/*1 genotype (HR: 2.61; P = 0.03), while the incidence of DR in the non-drinkers did not differ significantly between the ALDH2 genotype groups (P > 0.05). The incidence of DR was significantly higher in the ALDH2*2 allele carriers with a high GGT level than in the non-carriers with a high or low GGT level (HR: 2.45; P = 0.03; and HR: 2.63; P = 0.03, respectively).
Conclusions
To the best of our knowledge, this is the first report of a significant association between the ALDH2*2 allele and the incidence of DR. These findings provide additional evidence that ALDH2 protects both microvasculature and macrovasculature against reactive aldehydes generated under conditions of sustained oxidative stress, although further investigations in larger cohorts are needed to verify the results.
doi:10.1186/1475-2840-12-132
PMCID: PMC3847457  PMID: 24028448
Advanced glycation end products; Alcohol drinking; Aldehyde dehydrogenase 2; Diabetic retinopathy; Diabetic angiopathy; γ-glutamyltransferase; Oxidative stress; Type 2 diabetes mellitus
23.  The rarity of ALDH + cells is the key to separation of normal versus leukemia stem cells by ALDH activity in AML patients 
International Journal of Cancer  2015;137(3):525-536.
To understand the precise disease driving mechanisms in acute myeloid leukemia (AML), comparison of patient matched hematopoietic stem cells (HSC) and leukemia stem cells (LSC) is essential. In this analysis, we have examined the value of aldehyde dehydrogenase (ALDH) activity in combination with CD34 expression for the separation of HSC from LSC in 104 patients with de novo AML. The majority of AML patients (80 out of 104) had low percentages of cells with high ALDH activity (ALDH+ cells; <1.9%; ALDH‐rare AML), whereas 24 patients had relatively numerous ALDH+ cells (≥1.9%; ALDH‐numerous AML). In patients with ALDH‐rare AML, normal HSC could be separated by their CD34+ALDH+ phenotype, whereas LSC were exclusively detected among CD34+ALDH− cells. For patients with ALDH‐numerous AML, the CD34+ALDH+ subset consisted mainly of LSC and separation from HSC was not feasible. Functional analyses further showed that ALDH+ cells from ALDH‐numerous AML were quiescent, refractory to ARA‐C treatment and capable of leukemic engraftment in a xenogenic mouse transplantation model. Clinically, resistance to chemotherapy and poor long‐term outcome were also characteristic for patients with ALDH‐numerous AML providing an additional risk‐stratification tool. The difference in spectrum and relevance of ALDH activity in the putative LSC populations demonstrates, in addition to phenotypic and genetic, also functional heterogeneity of leukemic cells and suggests divergent roles for ALDH activity in normal HSC versus LSC. By acknowledging these differences our study provides a new and useful tool for prospective identification of AML cases in which separation of HSC from LSC is possible.
What's new?
To understand the precise disease‐driving mechanisms in acute myeloid leukemia (AML), comparison of patient‐matched hematopoietic stem cells (HSC) and leukemia stem cells (LSC) is essential. This study demonstrates the relevance of aldehyde dehydrogenase (ALDH) for the prospective identification of AML cases in which separation of functionally normal HSC from LSC is possible. Increased activity of this biomarker also characterizes a subgroup of patients with adverse outcome, which might be helpful in risk stratification prior to therapy. Overall, this study demonstrates functional heterogeneity of leukemia cells and suggests divergent roles for ALDH activity in normal HSC versus leukemia‐initiating cells.
doi:10.1002/ijc.29410
PMCID: PMC4755039  PMID: 25545165
acute myeloid leukemia; leukemia stem cell; hematopoietic stem cell; aldehyde dehydrogenase; high risk factor
24.  Aldehyde dehydrogenase 2 activation in heart failure restores mitochondrial function and improves ventricular function and remodelling 
Cardiovascular Research  2014;103(4):498-508.
Aims
We previously demonstrated that pharmacological activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) protects the heart against acute ischaemia/reperfusion injury. Here, we determined the benefits of chronic activation of ALDH2 on the progression of heart failure (HF) using a post-myocardial infarction model.
Methods and results
We showed that a 6-week treatment of myocardial infarction-induced HF rats with a selective ALDH2 activator (Alda-1), starting 4 weeks after myocardial infarction at a time when ventricular remodelling and cardiac dysfunction were present, improved cardiomyocyte shortening, cardiac function, left ventricular compliance and diastolic function under basal conditions, and after isoproterenol stimulation. Importantly, sustained Alda-1 treatment showed no toxicity and promoted a cardiac anti-remodelling effect by suppressing myocardial hypertrophy and fibrosis. Moreover, accumulation of 4-hydroxynonenal (4-HNE)-protein adducts and protein carbonyls seen in HF was not observed in Alda-1-treated rats, suggesting that increasing the activity of ALDH2 contributes to the reduction of aldehydic load in failing hearts. ALDH2 activation was associated with improved mitochondrial function, including elevated mitochondrial respiratory control ratios and reduced H2O2 release. Importantly, selective ALDH2 activation decreased mitochondrial Ca2+-induced permeability transition and cytochrome c release in failing hearts. Further supporting a mitochondrial mechanism for ALDH2, Alda-1 treatment preserved mitochondrial function upon in vitro aldehydic load.
Conclusions
Selective activation of mitochondrial ALDH2 is sufficient to improve the HF outcome by reducing the toxic effects of aldehydic overload on mitochondrial bioenergetics and reactive oxygen species generation, suggesting that ALDH2 activators, such as Alda-1, have a potential therapeutic value for treating HF patients.
doi:10.1093/cvr/cvu125
PMCID: PMC4155470  PMID: 24817685
Oxidant stress; Heart disease; Mitochondria; Pharmacological therapy; Bioenergetics
25.  Role of the General Base Glu-268 in Nitroglycerin Bioactivation and Superoxide Formation by Aldehyde Dehydrogenase-2* 
The Journal of Biological Chemistry  2009;284(30):19878-19886.
Mitochondrial aldehyde dehydrogenase-2 (ALDH2) plays an essential role in nitroglycerin (GTN) bioactivation, resulting in formation of NO or a related activator of soluble guanylate cyclase. ALDH2 denitrates GTN to 1,2-glyceryl dinitrate and nitrite but also catalyzes reduction of GTN to NO. To elucidate the relationship between ALDH2-catalyzed GTN bioconversion and established ALDH2 activities (dehydrogenase, esterase), we compared the function of the wild type (WT) enzyme with mutants lacking either the reactive Cys-302 (C302S) or the general base Glu-268 (E268Q). Although the C302S mutation led to >90% loss of all enzyme activities, the E268Q mutant exhibited virtually unaffected rates of GTN denitration despite low dehydrogenase and esterase activities. The nucleotide co-factor NAD caused a pronounced increase in the rates of 1,2-glyceryl dinitrate formation by WT-ALDH2 but inhibited the reaction catalyzed by the E268Q mutant. GTN bioactivation measured as activation of purified soluble guanylate cyclase or release of NO in the presence of WT- or E268Q-ALDH2 was markedly potentiated by superoxide dismutase, suggesting that bioavailability of GTN-derived NO is limited by co-generation of superoxide. Formation of superoxide was confirmed by determination of hydroethidine oxidation that was inhibited by superoxide dismutase and the ALDH2 inhibitor chloral hydrate. E268Q-ALDH2 exhibited ∼50% lower rates of superoxide formation than the WT enzyme. Our results suggest that Glu-268 is involved in the structural organization of the NAD-binding pocket but is not required for GTN denitration. ALDH2-catalyzed superoxide formation may essentially contribute to oxidative stress in GTN-exposed blood vessels.
doi:10.1074/jbc.M109.005652
PMCID: PMC2740413  PMID: 19506075

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