Function Of Aldehyde Dehydrogenase Research Articles

Neuroprotection by acrolein sequestration through exogenously applied scavengers and endogenous enzymatic enabling strategies in mouse EAE model

We have previously shown that the pro-oxidative aldehyde acrolein is a critical factor in MS pathology. In this study, we found that the acrolein scavenger hydralazine (HZ), when applied from the day of induction, can suppress acrolein and alleviate motor and sensory deficits in a mouse experimental autoimmune encephalomyelitis (EAE) model. Furthermore, we also demonstrated that HZ can alleviate motor deficits when applied after the emergence of MS symptoms, making potential anti-acrolein treatment a more clinically relevant strategy. In addition, HZ can reduce both acrolein and MPO, suggesting a connection between acrolein and inflammation. We also found that in addition to HZ, phenelzine (PZ), a structurally distinct acrolein scavenger, can mitigate motor deficits in EAE when applied from the day of induction. This suggests that the likely chief factor of neuroprotection offered by these two structurally distinct acrolein scavengers in EAE is their common feature of acrolein neutralization. Finally, up-and-down regulation of the function of aldehyde dehydrogenase 2 (ALDH2) in EAE mice using either a pharmacological or genetic strategy led to correspondent motor and sensory changes. This data indicates a potential key role of ALDH2 in influencing acrolein levels, oxidative stress, inflammation, and behavior in EAE. These findings further consolidate the critical role of aldehydes in the pathology of EAE and its mechanisms of regulation. This is expected to reinforce and expand the possible therapeutic targets of anti-aldehyde treatment to achieve neuroprotection through both endogenous and exogenous manners.

Effects of rhizobia and arbuscular mycorrhizal fungi on yield, size distribution and fatty acid of soybean seeds grown under drought stress

Soybean (Glycine max L.) is among the most economically important legumes that provide more than 1/4 of food (for man) and animal feed. However, its yield is comparatively low, most especially under drought stress. The aim of this study therefore was to assess the ability of Rhizobium spp. and mycorrhizal fungi to enhance the yield, seed size and fatty acid content of soybean grown under semi-arid environment. Rhizobium sp. strain R1 was found to possess nitrogen-fixing gene coniferyl aldehyde dehydrogenase function while Rhizobium cellulosilyticum strain R3 was found to have nitrogen-fixing genes cysteine desulfurase SufS and cysteine desulfurase IscS activity. Soybean (Glycine max L) seeds inoculated with Rhizobium spp. and mycorrhizal fungi were cultivated in soil exposed to drought stress. Rhizobium spp. inoculation and mycorrhization alleviate drought stress and increase yield, size and fat content of soybean seeds. This increase in the aboveground parameters was accompanied with an increase in belowground mycorrhizal spore number, percentage root mycorrhization and aboveground shoot relative water content (RWC) in the dually inoculated (R1 + R3MY) soybean plants. In particular, the dually inoculated (R1 + R3MY) soybean plants revealed 34.3 g fresh weight, 15.1 g dry weight and soybean plants singly inoculated with Rhizobium sp. strain R1 (R1) produced more large seeds with 12.03 g dry weight. The non-inoculated (control) seeds contained a higher percentage of moisture content compared to the microbially amended seeds while seeds co-inoculated with Rhizobium cellulosilyticum strain R3 and mycorrhizal consortium revealed the highest percent (8.4 %) of fat. Several fatty acids that are of significant health benefits to humans were observed in the soybean seeds. In order to gain insights into the bacterial communities of rhizospheric soil collected at different stages of soybean growth, class-based Heat-map analysis was performed on the Miseq sequenced data. The core bacteria that were found in the rhizospheric soil were Verrumicrobia, Proteobacteria, Gemmatimonadetes, Firmicutes, Cyanobacteria, Chloroflexi, Bacteroidetes, Actinobacteria, Acidobacteria, Planctomycetes, Deinococcus thermus and Nitrospira suggesting that the rhizobia and fungi used in this study can also improve soil microbial diversity.

S-Nitrosation impairs activity of stress-inducible aldehyde dehydrogenases from Arabidopsis thaliana

Nitric oxide (NO) is an intracellular messenger that mediates stress responses. Several plant aldehyde dehydrogenase (ALDH) genes are expressed during abiotic stress conditions to reduce the level of cytotoxic aldehydes. We investigated a possible interference between NO and ALDHs, using the isoform ALDH3H1 of Arabidopsis thaliana as model. The physiological NO donor; S-nitrosoglutathione (GSNO), inhibits ALDH3H1 in a time- and concentration-dependent manner. Mutagenesis and ESI-MS/MS analyses show that all Cys residues of ALDH3H1 are targets of GSNO-mediated S-nitrosation. Chemical labelling indicates that the deactivation is due to the conversion of the catalytic thiol into a catalytically non-active nitrosothiol. GSNO has the same effect on the chloroplastic ALDH3I1, suggesting that susceptibility of the catalytic Cys to NO is a common feature of ALDHs. S-Nitrosation and enzymatic inhibition of ALDH were reverted by reducing agents. Our study proves that the function of ALDHs does not exclusively depend on transcriptional regulation, with stress-induced expression, but may be also susceptible to posttranslational regulation through S-nitrosation. We discuss the potential involvement of S-nitrosoglutathione reductase (GSNOR), binding specific cofactors and reducing partners in a protective system of ALDHs in vivo, which will be experimentally corroborated in our forthcoming study.

ALDH-Dependent Glycolytic Activation Mediates Stemness and Paclitaxel Resistance in Patient-Derived Spheroid Models of Uterine Endometrial Cancer.

SummaryUterine endometrial cancer is associated with poor survival outcomes in patients with advanced-stage disease. Here, we developed a three-dimensional cell cultivation method of endometrioid cancer stem-like cells with high aldehyde dehydrogenase (ALDH) activity from clinical specimens. ALDH inhibition synergized with paclitaxel to block cancer proliferation. In the clinical setting, high ALDH1A1 expression was associated with poor survival. A high level of ALDH correlated with an increase of glucose uptake, activation of the glycolytic pathway, and elevation of glucose transporter 1 (GLUT1). Blockade of GLUT1 inhibited characteristics of cancer stem cells. Similarly to ALDH inhibition, GLUT1 inhibition synergized with paclitaxel to block endometrial cancer proliferation. Our data indicated that ALDH-dependent GLUT1 activation and the resulting glycolytic activation are of clinical importance for both prognostic evaluation and therapeutic decision-making in endometrial cancer patients. In addition, the synergistic effects of taxane compounds and ALDH or GLUT1 inhibitors may serve as a new clinical treatment option for endometrial cancer.

Genome-wide identification and analysis of the aldehyde dehydrogenase gene superfamily in Chinese cabbage (Brassica rapa subsp. pekinensis)

Aldehyde dehydrogenases (ALDHs) encode a class of enzymes that dehydrooxidize aldehydes into corresponding carboxylic acids, which are involved in the growth and development of plants and in the response to various biological and abiotic stresses. In this study, we identified 27 ALDH genes in the Chinese cabbage genome and grouped them into 10 different families. Chromosomal mapping revealed that, except for one gene distributed on Scaffold, the remaining 26 genes were unevenly distributed on 10 chromosomes of Chinese cabbage. Based on a comparison of the homologous relationship between BrALDHs and ALDH genes in Arabidopsis thaliana, duplicated patterns of the ALDH gene family in Chinese cabbage were analyzed. The exon–intron structures, conserved protein motifs, and phylogenetic relationship with ALDH in six other species were also predicted and analyzed. Finally, we used available RNA-Seq data and real-time quantitative PCR to analyze the expression of ALDH genes in different tissues of Chinese cabbage including the roots, stems, leaves, flowers, and siliques. The results showed the tissue specificity and differential expression in different tissues of BrALDHs. The analysis of ALDH gene transcriptome data of Chinese cabbage under different stress conditions (cold, heat, drought, and salinity) showed that the response levels of different genes varied under different stresses, suggesting the function of some genes in these processes. Details of the ALDH gene family in Chinese cabbage has enriched studies on the ALDH gene family in plants and animals and is crucial for understanding ALDH function during plant growth and development.

Cloning of a novel gene involved in alkane biosynthesis from Klebsiella sp.

Aliphatic medium-chain alkanes, a major component of gasoline, diesel, and jet fuels, are drop-in compatible fuels. Microorganisms with the capacity to produce medium-chain alkanes are promising for the bio-production of drop-in fuel. We found that Klebsiella sp. NBRC100048 has the ability to produce medium-chain alkanes from medium-chain aldehydes. We cloned a gene involved in conversion of aldehydes to alkanes by using a genomic fosmid library derived from Klebsiella sp. NBRC100048. The gene termed orf2991 encodes 506 amino acids and shows 62% sequence homology to the aldehyde dehydrogenase of Escherichia coli, aldB. The finding of orf2991 as a novel alkane-synthesizing enzyme gene similar to E. coli aldehyde dehydrogenase family, which is generally known to catalyze a reaction oxidizing aldehydes to fatty acids, indicated a novel function of aldehyde dehydrogenase. This finding is not only significant academically but allows developing the novel manufacturing methods of alkanes fermentation.

Abstract 1662: Synthesis and biologic evaluation of diethylbenzaldehyde analogues to probe functional activity of aldehyde dehydrogenase activity in cancer

Abstract The human aldehyde dehydrogenases (ALDHs) play a major role in detoxifying highly reactive aldehydes into carboxylic acids. Deregulation of ALDHs has implications in a number of cancers. They play an important role as a cancer stem cell (CSC) marker due to high activity found in CSCs, while high expression is also known to lead to resistance to chemotherapeutic drugs. Although the exact roles of ALDHs are not fully understood, emerging information indicates several isoforms from the ALDH1 family, ALDH3A1 and ALDH7A1, play key roles in several cancer types. To probe the role of ALDH functional activity, we here report on the perturbation of ALDH expression and function using diethylbenzaldehyde (DEAB) analogues. The H1299 lung cancer cell line has negligible amount of ALDH activity, and ALDH-expressing variants were used to study the novel DEAB analogues. H1299 wild-type cells (WT) and cells expressing RFP were used as controls. Expression of the proteins was confirmed by Western blots and ALDH enzymatic activity assay. We used cell culture, colorimetric MTT assay, aldefluor assay and in vitro treatment with DEAB analogues. ALDH-expressing lysates were used to obtain biochemical inhibition data of the DEAB analogues using propionaldehyde as substrate with NAD+ as a co-factor. DEAB has been shown to covalently bind to ALDH7A1, and computational modeling was employed to understand how chemical modifications to DEAB affected the binding mode. Selected ALDH-affinic probe compounds were tested in a panel of colon, lung and prostate cancer cell lines including docetaxel-resistant variant and patient samples to investigate if DEAB analogues had an effect on cell viability as single agents or in combination with docetaxel. In conclusion, the novel DEAB analogues aid us to understand ALDH functional activity and provide a platform for discovering more selective ALDH inhibitors Citation Format: Ali Ibrahim, Maria Sadiq, Daniela Presa, Ioannis Ntafoulis, Ahmad Alkawi, Fiona Frame, Norman J. Maitland, Zoe Cournia, Paul M. Loadman, Jan Moreb, Klaus Pors. Synthesis and biologic evaluation of diethylbenzaldehyde analogues to probe functional activity of aldehyde dehydrogenase activity in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1662.

Aldehyde Dehydrogenases Function in the Homeostasis of Pyridine Nucleotides in Arabidopsis thaliana

Aldehyde dehydrogenase enzymes (ALDHs) catalyze the oxidation of aliphatic and aromatic aldehydes to their corresponding carboxylic acids using NAD+ or NADP+ as cofactors and generating NADH or NADPH. Previous studies mainly focused on the ALDH role in detoxifying toxic aldehydes but their effect on the cellular NAD(P)H contents has so far been overlooked. Here, we investigated whether the ALDHs influence the cellular redox homeostasis. We used a double T-DNA insertion mutant that is defective in representative members of Arabidopsis thaliana ALDH families 3 (ALDH3I1) and 7 (ALDH7B4), and we examined the pyridine nucleotide pools, glutathione content, and the photosynthetic capacity of the aldh mutants in comparison with the wild type. The loss of function of ALDH3I1 and ALDH7B4 led to a decrease of NAD(P)H, NAD(P)H/NAD(P) ratio, and an alteration of the glutathione pools. The aldh double mutant had higher glucose-6-phosphate dehydrogenase activity than the wild type, indicating a high demand for reduced pyridine nucleotides. Moreover, the mutant had a reduced quantum yield of photosystem II and photosynthetic capacity at relatively high light intensities compared to the wild type. Altogether, our data revealed a role of ALDHs as major contributors to the homeostasis of pyridine nucleotides in plants.

Abstract 24035: Human iPSC-Derived Endothelial Cells Predict Predilection to Atherogenesis by Endothelial Proinflammatory Activation

Introduction: Coronary artery disease (CAD), the direct outcome of atherosclerosis, is the leading cause of death in the United States. Previous studies demonstrated that impaired function of aldehyde dehydrogenase 2 (ALDH2), a key enzyme for alcohol metabolism, is linked to increased susceptibility to CAD. A single-nucleotide polymorphism that generates E487K mutation (ALDH2*2) reduces enzymatic activity of ALDH2 to less than 40% of the wild type (WT) and is present in ~560 million people. However, it remains unclear how ALDH2 regulates atherosclerotic progression. Hypothesis: Recent studies suggest a critical role of ALDH2 in plaque development and endothelial activation. Therefore, we hypothesize that endothelial cells of ALDH2*2 carriers possess greater susceptibility to proinflammatory activation, whereby endothelial cells recruit immune cells, leading to increased risk of atherogenesis. Methods: To study the patient-specific effects of ALDH2*2 mutation on endothelial proinflammatory activation, we generated and characterized iPSC-derived endothelial cells (iPSC-ECs) from 5 WT subjects and 5 ALDH2*2 carriers. We exposed the iPSC-ECs to pro-inflammatory conditions and assessed the level of endothelial proinflammatory activation by gene expression analysis and monocyte adhesion assay. Results: Our preliminary data show ALDH2*2-iPSC-ECs exhibit impaired ALDH2 function resulting in metabolic dysregulation compared to WT. Presence of ALDH2*2 mutation resulted in enhanced inflammatory response in the iPSC-ECs when treated with proinflammatory cytokines such as TNFα and IL-1β, as evidenced by up-regulation of cell adhesion molecules and augmented adherence to monocytes. The ALDH2*2-iPSC-ECs also exhibited an increased basal expression of vascular endothelial growth factor receptor 1 (FLT1) gene, which was further augmented upon inflammatory stimulation. FLT1 is a receptor for vascular endothelial growth factor ligands, playing a critical role in endothelial homeostasis and biology. Conclusion: Taken together, we elucidate the effects of impaired ALDH2 function on increased susceptibility to atherogenesis by endothelial proinflammatory activation using patient-derived iPSC-ECs.

Use of retinoic acid/aldehyde dehydrogenase pathway as potential targeted therapy against cancer stem cells.

A large number of studies have investigated possible drug resistance mechanisms of cancer cells and suggested strategies to overcome it. In this review, we outline the role and function of aldehyde dehydrogenase (ALDH) activity in multiple cellular functions and in cancer stem cells (CSCs) and focus on the role of retinoic acid (RA), one of the products of ALDH isozymes. We discuss our observation that ATRA and other RAs can suppress ALDH activity and decrease different ALDH isozyme proteins and result in detrimental effects on cell proliferation, invasion and chemotherapy sensitivity. We review the known uses of different RAs in the treatment of cancers. We review the use of RAs in combination with chemo-/radiotherapy and the major signaling pathways affected in different tumor types. We provide follow-up on studies that may have used our prior observation with the aim of targeting the CSCs. We conclude with summary of the findings and potential impact of published studies on future use of RAs in the targeting of CSCs and drug resistance.

Abstract 4093: The impact of the prostate cancer microenvironment on the expression and regulation of aldehyde dehydrogenases

Abstract Introduction: Aldehyde dehydrogenase (ALDH) enzymes are involved in the detoxification of specific endogenous and exogenous aldehyde substrates. High ALDH activity is used to identify stem cells and has also been associated with poor prognosis in cancer. In prostate cancer (PCa) high ALDH activity has been associated with elevated clonogenicity, migratory behaviour, tumor progression and metastasis. Despite these observations there exists a poor understanding of the role of selected ALDH isoforms in PCa. In an attempt to understand if the tumor microenvironment impacts on ALDH function, we investigated their expression under hypoxia. Methods: Gene and protein expression analysis of ALDH isoforms -1A1, -1A2, -1A3, -1B1, -2, -3A1 and -7A1 in normal prostate cell line PNT2C2, and a panel of PCa cell lines with different stages of cancer was carried out by quantitative PCR and western blot under normoxic and hypoxic conditions at 24 and 48 hours. Gene expression of the ALDHs in primary prostate cultures was assessed following retinoic acid treatment. siRNA knockdown technology was used to study functional roles of selected ALDH isoforms. Results: Gene expression of ALDH1A3 was increased in Bob cells at 24h under hypoxia, at 48h this was less apparent but the expression of most ALDHs in Bob cells including ALDH1A2, -2, -3A1 and -7A1 was reduced at 48h. In SerBob cells, the gene expression of ALDH1A1 and -1B1 was reduced at both time points, whereas the expression of ALDH1A2, -2, and -7A1 was increased under hypoxia. At protein level, the expression of ALDH1A3 appeared low under hypoxia at both time points in SerBob cells while the expression of ALDH3A1 also appeared low after 24h exposure to hypoxia. In DU145 cells, there was an increase in ALDH1A1, -1A2, 1A3, -2, and -3A1 gene expression under hypoxia at 48h. In LNCAP cells, the gene expression of ALDH2 was increased at 48h under hypoxia whereas the expression of ALDH7A1 was reduced at 48h. The protein expression of ALDH7A1 was higher in PC3 cells under hypoxia at 24h. A significant increase was observed in ALDH1A3 and ALDH3A1 expression in response to retinoic acid. ALDH1A3 knockdown showed a significant reduction of cell viability in PC3 cells. Other investigations underway that will be reported at the conference are focused on how prostate cancer stem-like cells expressing ALDHs are responding to hypoxia. Conclusion: Our data suggests that the expression of certain ALDHs is affected by hypoxia and ALDH1A3 potentially is involved in cell survival in PCa. Future work will investigate if any of these isoforms can be used as biomarkers to distinguish indolent from malignant PCa. Citation Format: Maria Sadiq, Simon J. Allison, Fiona Frame, Mark Sutherland, Roger M. Phillips, Norman J. Maitland, Klaus Pors. The impact of the prostate cancer microenvironment on the expression and regulation of aldehyde dehydrogenases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4093.

Alda-1 Constrains TLR-Induced Cytokine Overproduction By Fanconi Anemia Macrophages By Enhancing ALDH1A1 Activity

Replication and survival of FA-deficient hematopoietic stem and progenitor cells (HSPC) are highly suppressed by exposure to inflammatory cytokines including TNFα, IL-1β, IFNγ, and MIP-1α. Additionally, FA macrophages exposed to specific toll-like receptor (TLR) ligands overproduce the very inflammatory cytokines that exhaust the stem cell pool. Recent evidence using double knockout mice suggests that aldehyde dehydrogenases (ALDH) may play a role in protecting FA HSPC but the mechanisms involved are unclear. We tested the hypothesis that the TLR-dependent overproduction of inflammatory cytokines in FA macrophages results from a loss of FA protein-dependent ALDH function. Control (T-shNT) or FANCC-deficient (T-shFC) THP-1 human monocytic leukemia cells were treated with Alda-1 (a small molecule ALDH agonist known to enhance the activity of both ALDH1A1 and ALDH2), before exposing them to the TLR-7/8 agonist R848 and quantifying the production of TNFα (ELISA of 24- culture supernatants). While R848 alone induced a 6-fold increase in TNFα production by the FANCC-deficient cells, Alda-1 suppressed TLR-induced TNFα production in a dose-dependent manner, by both T-shNT and T-shFC cells (Fig. 1). To identify the ALDH isoform that played a role in the suppression of TNFα production, we ectopically expressed ALDH1A1 or ALDH2 in T-shNT and T-shFC cells. Overexpression of ALDH2 had little effect on R848-induced TNFα production, but ALDH1A1 overexpression completely suppressed TNFα production by FANCC-deficient cells and suppressed (by approximately 50%) the production of TNFα by control cells (Fig. 2). Likewise, in loss-of-function studies, siRNA knockdown of ALDH1A1 (but not ALDH2) enhanced R848-induced production of TNFα (1.8-fold) in T-shNT cells but did not enhance TNFα overproduction in either FANCC-deficient (T-shFC) or FANCA-deficient (T-shFA [FANCA knockdown]) cells. Additionally, ALDH1A1 (but not ALDH2) mRNA was highly inducible in both THP-1 cells (by R848) and in Lin- Sca-1+ Kit+ murine marrow cells (by TNFα). While ALDH1A1 mRNA is expressed similarly in both T-shNT and T-shFC cells, our results indicate that in normal macrophages ALDH1A1 plays a role in the constraining TLR-induced TNF production but is significantly less functional in FANCC-deficient macrophages. In summary, (1) either pharmacological enhancement of ALDH function with Alda-1 or overexpression of ALDH1A1 is sufficient to restore the TLR-suppressive function of ALDH1A1 in FANCC-deficient macrophages, and (2) specific suppression of ALDH1A1, (but not ALDH2) induces an FA-like phenotype in control macrophages. We conclude that (1) optimal function of ALDH1A1 is FANCC- and FANCA-dependent in normal macrophages, (2) TLR-dependent overproduction of inflammatory cytokines by FA-deficient macrophages may result either from an increase in aldehyde load or the loss of a non-canonical signal-suppressive function of ALDH1A1, and (3) enhancement of ALDH activity using small molecule agonists such as Alda-1 may alleviate the FA macrophage/cytokine phenotype and thereby protect HSC from inflammation-induced exhaustion. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract IA16: Targeting cancer stem cells to overcome chemotherapy resistance

Abstract While most ovarian cancer patients will have a complete response with surgery and chemotherapy, the majority will relapse and die of their disease. The high relapse rate in ovarian cancer after complete clinical response is highly consistent with a cancer stem-like cell (CSC) model. The CSC hypothesis suggests that only rare, inherently chemoresistant cancer cells with stem cell like characteristics are capable of proliferating and differentiating to regenerate the various cell types within a tumor, thereby causing relapse of the disease. In theory and in experimental models, the elimination of CSC can overcome chemotherapy resistance. We are developing novel therapies to specifically target ovarian CSC (OvCSC). Aldehyde Dehydrogenase (ALDH) enzymatic activity was one of the first, and remains one of the best supported CSC markers. ALDH enzymes perform numerous critical functions including the biosynthesis of retinoic acid. Our group and others have found that ALDH identifies a population cancer cells with ovarian CSC (OvCSC) activity. Primary human ALDH+ cells initiate and propagate tumors in mice while ALDH-cells do not. ALDH used in combination with the stem cell marker CD133 enriches CSC isolation. ALDH1A+ or ALDH1A1+CD133+ cells in primary tumor samples predict poor prognosis for ovarian cancer patients [9, 11]. ALDH and CD133 are enriched in tumor specimens immediately following chemotherapy [12]. ALDH1A1 is 100 fold upregulated in chemoresistant ovarian cancer cells and ALDH1A knockdown restores chemosensitivity. Finally, normal ovary epithelial stem cells also express ALDH and CD133, and when mutated for p53 and Rb, these cells gain CSC capacity and initiate serous tumors. Taken together these data strongly support a role for ALDH in serous ovarian CSC. Based on the CSC specific function of ALDH and the function of we generated and screened. We have therefore identified and screened dozens of ALDH inhibitors and molecular analogs of known ALDH inhibitors. Using cell lines with well-defined CSC populations, we have identified several compounds which kill cells expressing CSC markers in vitro. CSC die via necrosis. Lead compounds prevent tumor sphere formation in vitro and restrict tumor initiation in vivo. The novel ALDH inhibitors are highly synergistic with chemotherapy and reverse chemotherapy resistance in primary human tumor xenografts. Taken together our data support ALDH as a CSC specific target and identifies novel compounds as potential new therapeutics. Citation Format: Ronald J. Buckanovich. Targeting cancer stem cells to overcome chemotherapy resistance. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr IA16.

Structural Determinants of Oligomerization of Δ1-Pyrroline-5-Carboxylate Dehydrogenase: Identification of a Hexamerization Hot Spot

The aldehyde dehydrogenase (ALDH) superfamily member Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) catalyzes the NAD+-dependent oxidation of glutamate semialdehyde to glutamate, which is the final step of proline catabolism. Defects in P5CDH activity lead to the metabolic disorder type II hyperprolinemia, P5CDH is essential for virulence of the fungal pathogen Cryptococcus neoformans, and bacterial P5CDHs have been targeted for vaccine development. Although the enzyme oligomeric state is known to be important for ALDH function, the oligomerization of P5CDH has remained relatively unstudied. Here we determine the oligomeric states and quaternary structures of four bacterial P5CDHs using a combination of small-angle X-ray scattering, X-ray crystallography, and dynamic light scattering. The P5CDHs from Thermus thermophilus and Deinococcus radiodurans form trimer-of-dimers hexamers in solution, which is the first observation of a hexameric ALDH in solution. In contrast, two Bacillus P5CDHs form dimers in solution but do not assemble into a higher-order oligomer. Site-directed mutagenesis was used to identify a hexamerization hot spot that is centered on an arginine residue in the NAD+-binding domain. Mutation of this critical Arg residue to Ala in either of the hexameric enzymes prevents hexamer formation in solution. Paradoxically, the dimeric Arg-to-Ala T. thermophilus mutant enzyme packs as a hexamer in the crystal state, which illustrates the challenges associated with predicting the biological assembly in solution from crystal structures. The observation of different oligomeric states among P5CDHs suggests potential differences in cooperativity and protein–protein interactions.

Autocrine Function of Aldehyde Dehydrogenase 1 as a Determinant of Diet- and Sex-Specific Differences in Visceral Adiposity

Mechanisms for sex- and depot-specific fat formation are unclear. We investigated the role of retinoic acid (RA) production by aldehyde dehydrogenase 1 (Aldh1a1, -a2, and -a3), the major RA-producing enzymes, on sex-specific fat depot formation. Female Aldh1a1−/− mice, but not males, were resistant to high-fat (HF) diet–induced visceral adipose formation, whereas subcutaneous fat was reduced similarly in both groups. Sexual dimorphism in visceral fat (VF) was attributable to elevated adipose triglyceride lipase (Atgl) protein expression localized in clusters of multilocular uncoupling protein 1 (Ucp1)-positive cells in female Aldh1a1−/− mice compared with males. Estrogen decreased Aldh1a3 expression, limiting conversion of retinaldehyde (Rald) to RA. Rald effectively induced Atgl levels via nongenomic mechanisms, demonstrating indirect regulation by estrogen. Experiments in transgenic mice expressing an RA receptor response element (RARE-lacZ) revealed HF diet–induced RARE activation in VF of females but not males. In humans, stromal cells isolated from VF of obese subjects also expressed higher levels of Aldh1 enzymes compared with lean subjects. Our data suggest that an HF diet mediates VF formation through a sex-specific autocrine Aldh1 switch, in which Rald-mediated lipolysis in Ucp1-positive visceral adipocytes is replaced by RA-mediated lipid accumulation. Our data suggest that Aldh1 is a potential target for sex-specific antiobesity therapy.

Studying fatty aldehyde metabolism in living cells with pyrene-labeled compounds

The lack of fatty aldehyde dehydrogenase function in Sjögren Larsson Syndrome (SLS) patient cells not only impairs the conversion of fatty aldehydes into their corresponding fatty acid but also has an effect on connected pathways. Alteration of the lipid profile in these cells is thought to be responsible for severe symptoms such as ichtyosis, mental retardation, and spasticity. Here we present a novel approach to examine fatty aldehyde metabolism in a time-dependent manner by measuring pyrene-labeled fatty aldehyde, fatty alcohol, fatty acid, and alkylglycerol in the culture medium of living cells using HPLC separation and fluorescence detection. Our results show that in fibroblasts from SLS patients, fatty aldehyde is not accumulating but is converted readily into fatty alcohol. In control cells, in contrast, exclusively the corresponding fatty acid is formed. SLS patient cells did not display a hypersensitivity toward hexadecanal or hexadecanol, but 3-fold lower concentrations of the fatty alcohol than the corresponding fatty aldehyde were needed to induce toxicity in SLS patient and in control cells.

Inhibition of Aldehyde Dehydrogenase Expands Hematopoietic Stem Cells with Radioprotective Capacity

Hematopoietic stem cells (HSCs) are enriched for aldehyde dehydrogenase (ALDH) activity and ALDH is a selectable marker for human HSCs. However, the function of ALDH in HSC biology is not well understood. We sought to determine the function of ALDH in regulating HSC fate. Pharmacologic inhibition of ALDH with diethylaminobenzaldehyde (DEAB) impeded the differentiation of murine CD34(-)c-kit(+)Sca-1(+)lineage(-) (34(-)KSL) HSCs in culture and facilitated a ninefold expansion of cells capable of radioprotecting lethally irradiated mice compared to input 34(-)KSL cells. Treatment of bone marrow (BM) 34(-)KSL cells with DEAB caused a fourfold increase in 4-week competitive repopulating units, verifying the amplification of short-term HSCs (ST-HSCs) in response to ALDH inhibition. Targeted siRNA of ALDH1a1 in BM HSCs caused a comparable expansion of radioprotective progenitor cells in culture compared to DEAB treatment, confirming that ALDH1a1 was the target of DEAB inhibition. The addition of all trans retinoic acid blocked DEAB-mediated expansion of ST-HSCs in culture, suggesting that ALDH1a1 regulates HSC differentiation via augmentation of retinoid signaling. Pharmacologic inhibition of ALDH has therapeutic potential as a means to amplify ST-HSCs for transplantation purposes.

Suppressing Glioblastoma Stem Cell Function by Aldehyde Dehydrogenase Inhibition with Chloramphenicol or Disulfiram as a New Treatment Adjunct: A Hypothesis

Strong expression of aldehyde dehydrogenase is a prominent feature of both normal and cancer stem cells, including the stem cell sub-population of glioblastoma. Aldehyde dehydrogenase function is used by cancer stem cells to repopulate a tumor mass after chemotherapy cytoreduction. Cancer stem cells tend to be chemotherapy compared to the non-stem cell majority cell population in several common human cancers. Such has been demonstrated specifically in glioblastoma. In normal hematopoietic stem cells with unimpaired high levels of aldehyde dehydrogenase, stem cells divide rarely and then asymmetrically to a daughter stem cell and a daughter cell on a path of differentiation or symmetrically with both daughter cells on a differentiated path. If a parallel situation obtains in glioblastoma stem cells, the migrating, far flung paucicellular extensions will be stem cell rich and use aldehyde dehydrogenase to generate the characteristic multiple metastases made up of mostly non-stem cells. With inhibition of aldehyde dehydrogenase, stem cell division to non-stem daughter cells tends to become blocked. We have three old yet potent aldehyde dehydrogenase inhibitors on the market- chloral hydrate, chloramphenicol, and disulfiram- they should be investigated as adjuncts in glioblastoma chemotherapy. If GBM stem cell function can be thwarted by potent aldehyde dehydrogenase inhibition, they will be less able to regenerate a stem cell derived tumor mass after primary resection or chemotherapy.

Neurotoxicity and Metabolism of the Catecholamine-Derived 3,4-Dihydroxyphenylacetaldehyde and 3,4-Dihydroxyphenylglycolaldehyde: The Role of Aldehyde Dehydrogenase

Aldehydes are highly reactive molecules formed during the biotransformation of numerous endogenous and exogenous compounds, including biogenic amines. 3,4-Dihydroxyphenylacetaldehyde is the aldehyde metabolite of dopamine, and 3,4-dihydroxyphenylglycolaldehyde is the aldehyde metabolite of both norepinephrine and epinephrine. There is an increasing body of evidence suggesting that these compounds are neurotoxic, and it has been recently hypothesized that neurodegenerative disorders may be associated with increased levels of these biogenic aldehydes. Aldehyde dehydrogenases are a group of NAD(P)+ -dependent enzymes that catalyze the oxidation of aldehydes, such as those derived from catecholamines, to their corresponding carboxylic acids. To date, 19 aldehyde dehydrogenase genes have been identified in the human genome. Mutations in these genes and subsequent inborn errors in aldehyde metabolism are the molecular basis of several diseases, including Sjögren-Larsson syndrome, type II hyperprolinemia, gamma-hydroxybutyric aciduria, and pyridoxine-dependent seizures, most of which are characterized by neurological abnormalities. Several pharmaceutical agents and environmental toxins are also known to disrupt or inhibit aldehyde dehydrogenase function. It is, therefore, possible to speculate that reduced detoxification of 3,4-dihydroxyphenylacetaldehyde and 3,4-dihydroxyphenylglycolaldehyde from impaired or deficient aldehyde dehydrogenase function may be a contributing factor in the suggested neurotoxicity of these compounds. This article presents a comprehensive review of what is currently known of both the neurotoxicity and respective metabolism pathways of 3,4-dihydroxyphenylacetaldehyde and 3,4-dihydroxyphenylglycolaldehyde with an emphasis on the role that aldehyde dehydrogenase enzymes play in the detoxification of these two aldehydes.

Structural and functional studies of a NADP(+)-specific aldehyde dehydrogenase from the luminescent marine bacterium Vibrio harveyi.

Aldehyde dehydrogenase, isolated from the luminescent bacterium, Vibrio harveyi, catalyzes the oxidation of fatty aldehydes (Meighen et al.,1976). This enzyme has a preference for long chain aldehydes as reflected by a large decrease in the Km for aldehydes on increasing the chain length from acetaldehyde to tetradecanal (Bognar & Meighen, 1978; Vedadi et al., 1995). As one of the substrates for luciferase is tetradecanal, the potential involvement of aldehyde dehydrogenase in the luminescent system has been proposed. Tetradecanal is produced in luminescent bacteria by a lux-specific fatty acid reductase complex which activates (plus ATP) tetradecanoic acid to fatty acyl-AMP; the acyl group is then transferred between the fatty acid reductase subunits before being reduced with NADPH (Riendeau et al., 1982; Rodriguez et al., 1983a, 1983b). The V. harveyi aldehyde dehydrogenase(Vh ALDH) may be present so as to maintain aldehyde at nontoxic levels under conditions where excess levels of aldehyde are produced by the fatty acid reductase complex. However, the possibility that the function of aldehyde dehydrogenase is unrelated to luminescence can certainly not be excluded as Vh ALDH is under different regulation than the lux-specific proteins and has not been detected in other luminescent bacteria (Byers et al., 1988; Vedadi, M. unpublished data).KeywordsAldehyde DehydrogenaseLuminescent BacteriumFatty AldehydeVinyl KetoneSite Specific MutagenesisThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.