Human Aldehyde Dehydrogenase 3A1 (ALDH3A1) Exhibits Chaperone-Like Function

Aldehyde dehydrogenase 3A1 (ALDH3A1) is a metabolic enzyme that catalyzes the oxidation of various aldehydes. Certain types of epithelial tissues in mammals, especially those continually exposed to environmental stress (e.g., corneal epithelium), express ALDH3A1 at high levels and its abundance in such tissues is perceived to help to maintain cellular homeostasis under conditions of oxidative stress. Metabolic as well as non-metabolic roles for ALDH3A1 have been associated with its mediated resistance to cellular oxidative stress. In this study, we provide evidence that ALDH3A1 exhibits molecular chaperone-like activity further supporting its multifunctional role. Specifically, we expressed and purified the human ALDH3A1 in E. coli and used the recombinant protein to investigate its in vitro ability to protect SmaI and citrate synthase (from precipitation and/or deactivation) under thermal stress conditions. Our results indicate that recombinant ALDH3A1 exhibits significant chaperone function in vitro. Furthermore, over-expression of the fused histidine-tagged ALDH3A1 confers host E. coli cells with enhanced resistance to thermal shock, while ALDH3A1 over-expression in the human corneal cell line HCE-2 was sufficient for protecting them from the cytotoxic effects of both hydrogen peroxide and tert-butyl hydroperoxide. These results further support the chaperone-like function of human ALDH3A1. Taken together, ALDH3A1, in addition to its primary metabolic role in fundamental cellular detoxification processes, appears to play an essential role in protecting cellular proteins against aggregation under stress conditions

Aldehyde dehydrogenase 3A1 promotes multi-modality resistance and alters gene expression profile in human breast adenocarcinoma MCF-7 cells

Aldehyde dehydrogenases participate in a variety of cellular homeostatic mechanisms like metabolism, proliferation, differentiation, apoptosis, whereas recently, they have been implicated in normal and cancer cell stemness. We explored roles for ALDH3A1 in conferring resistance to chemotherapeutics/radiation/oxidative stress and whether ectopic overexpression of ALDH3A1 could lead to alterations of gene expression profile associated with cancer stem cell-like phenotype. MCF-7 cells were stably transfected either with an empty vector (mock) or human aldehyde dehydrogenase 3A1 cDNA. The expression of aldehyde dehydrogenase 3A1 in MCF-7 cells was associated with altered cell proliferation rate and enhanced cell resistance against various chemotherapeutic drugs (4-hydroxyperoxycyclophosphamide, doxorubicin, etoposide, and 5-fluorouracil). Aldehyde dehydrogenase 3A1 expression also led to increased tolerance of MCF-7 cells to gamma radiation and hydrogen peroxide-induced stress. Furthermore, aldehyde dehydrogenase 3A1-expressing MCF-7 cells exhibited gene up-regulation of cyclins A, B1, B2, and down-regulation of cyclin D1 as well as transcription factors p21, CXR4, Notch1, SOX2, SOX4, OCT4, and JAG1. When compared to mock cells, no changes were observed in mRNA levels of ABCA2 and ABCB1 protein pumps with only a minor decrease of the ABCG2 pump in the aldehyde dehydrogenase 3A1-expressing cells. Also, the adhesion molecules EpCAM and CD49F were also found to be up-regulated in aldehyde dehydrogenase 3A1expressing cells. Taken together, ALDH3A1 confers a multi-modality resistance phenotype in MCF-7 cells associated with slower growth rate, increased clonogenic capacity, and altered gene expression profile, underlining its significance in cell homeostasis

Identification of a peptide ligand for human ALDH3A1 through peptide phage display: Prediction and characterization of protein interaction sites and inhibition of ALDH3A1 enzymatic activity

Aldehyde dehydrogenase 3A1 (ALDH3A1) by oxidizing medium chain aldehydes to their corresponding carboxylic acids, is involved in the detoxification of toxic byproducts and is considered to play an important role in antioxidant cellular defense. ALDH3A1 has been implicated in various other functions such as cell proliferation, cell cycle regulation, and DNA damage response. Recently, it has been identified as a putative biomarker of prostate, gastric, and lung cancer stem cell phenotype. Although ALDH3A1 has multifaceted functions in both normal and cancer homeostasis, its modes of action are currently unknown. To this end, we utilized a random 12-mer peptide phage display library to identify efficiently human ALDH3A1-interacting peptides. One prevailing peptide (P1) was systematically demonstrated to interact with the protein of interest, which was further validated in vitro by peptide ELISA. Bioinformatic analysis indicated two putative P1 binding sites on the protein surface implying biomedical potential and potent inhibitory activity of the P1 peptide on hALDH3A1 activity was demonstrated by enzymatic studies. Furthermore, in search of potential hALDH3A1 interacting players, a BLASTp search demonstrated that no protein in the database includes the full-length amino acid sequence of P1, but identified a list of proteins containing parts of the P1 sequence, which may prove potential hALDH3A1 interacting partners. Among them, Protein Kinase C Binding Protein 1 and General Transcription Factor II-I are candidates of high interest due to their cellular localization and function. To conclude, this study identifies a novel peptide with potential biomedical applications and further suggests a list of protein candidates be explored as possible hALDH3A1-interacting partners in future studies

Aldehyde Dehydrogenase 1B1 Is Associated with Altered Cell Morphology, Proliferation, Migration and Chemosensitivity in Human Colorectal Adenocarcinoma Cells

Aldehyde dehydrogenases (ALDHs) are NAD(P) -dependent enzymes that catalyze the oxidation of endogenous and exogenous aldehydes to their corresponding carboxylic acids. ALDHs participate in a variety of cellular mechanisms, such as metabolism, cell proliferation and apoptosis, as well as differentiation and stemness. Over the last few years, ALDHs have emerged as cancer stem cell markers in a wide spectrum of solid tumors and hematological malignancies. In this study, the pathophysiological role of ALDH1B1 in human colorectal adenocarcinoma was investigated. Human colon cancer HT29 cells were stably transfected either with human green fluorescent protein (GFP)-tagged ALDH1B1 or with an empty lentiviral expression vector. The overexpression of ALDH1B1 was correlated with altered cell morphology, decreased proliferation rate and reduced clonogenic efficiency. Additionally, ALDH1B1 triggered a G2/M arrest at 24 h post-cell synchronization, probably through p53 and p21 upregulation. Furthermore, ALDH1B1-overexpressing HT29 cells exhibited enhanced resistance against doxorubicin, fluorouracil (5-FU) and etoposide. Finally, ALDH1B1 induced increased migratory potential and displayed epithelial-mesenchymal transition (EMT) through the upregulation of and and the consequent downregulation of Taken together, ALDH1B1 confers alterations in the cell morphology, cell cycle progression and gene expression, accompanied by significant changes in the chemosensitivity and migratory potential of HT29 cells, underlying its potential significance in cancer progression

Propolis Extracts Inhibit UV-Induced Photodamage in Human Experimental In Vitro Skin Models

The aim of this study was to assess the antioxidant, photoprotective, and antiaging effects of Greek propolis. Propolis was subjected to n-heptane or methanol extraction. Total phenolic/flavonoid content and antioxidant potential were determined in the extracts. Promising extracts were evaluated for their cytoprotective properties using human immortalized keratinocyte (HaCaT) or reconstituted human skin tissue following exposure to UVB. Assessment of cytotoxicity, DNA damage, oxidative status, and gene/protein expression levels of various matrix metalloproteinases (MMPs) were performed. The propolis methanolic fractions exhibited higher total phenolic and flavonoid contents and significant in vitro antioxidant activity. Incubation of HaCaT cells with certain methanolic extracts significantly decreased the formation of DNA strand breaks following exposure to UVB and attenuated UVB-induced decrease in cell viability. The extracts had no remarkable effect on the total antioxidant status, but significantly lowered total protein carbonyl content used as a marker for protein oxidation in HaCaT cells. MMP-1, -3, -7, and -9, monitored as endpoints of antiaging efficacy, were significantly reduced by propolis following UVB exposure in a model of reconstituted skin tissue. In conclusion, propolis protects against the oxidative and photodamaging effects of UVB and could be further explored as a promising agent for developing natural antiaging strategies

Toxicity Profiling of Biosurfactants Produced by Novel Marine Bacterial Strains

Surface active agents (SAAs), currently used in modern industry, are synthetic chemicals produced from non-renewable sources, with potential toxic impacts on humans and the environment. Thus, there is an increased interest for the identification and utilization of natural derived SAAs. As such, the marine environment is considered a promising source of biosurfactants with low toxicity, environmental compatibility, and biodegradation compared to their synthetic counterparts. MARISURF is a Horizon 2020 EU-funded project aiming to identify and functionally characterize SAAs, derived from a unique marine bacterial collection, towards commercial exploitation. Specifically, rhamnolipids produced by Marinobacter MCTG107b and Pseudomonas MCTG214(3b1) strains were previously identified and characterized while currently their toxicity profile was assessed by utilizing well-established methodologies. Our results showed a lack of cytotoxicity in in vitro models of human skin and liver as indicated by alamar blue and propidium iodide assays. Additionally, the use of the single gel electrophoresis assay, under oxidative stress conditions, revealed absence of any significant mutagenic/anti-mutagenic potential. Finally, both 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonicacid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) cell-free assays, revealed no significant anti-oxidant capacity for neither of the tested compounds. Consequently, the absence of significant cytotoxicity and/or mutagenicity justifies their commercial exploitation and potential development into industrial end-user applications as natural and environmentally friendly biosurfactants

The Concept of Cancer Stem Cells: Elaborating on ALDH1B1 as an Emerging Marker of Cancer Progression

Cancer is a multifactorial, complex disease exhibiting extraordinary phenotypic plasticity and diversity. One of the greatest challenges in cancer treatment is intratumoral heterogeneity, which obstructs the efficient eradication of the tumor. Tumor heterogeneity is often associated with the presence of cancer stem cells (CSCs), a cancer cell sub-population possessing a panel of stem-like properties, such as a self-renewal ability and multipotency potential. CSCs are associated with enhanced chemoresistance due to the enhanced efflux of chemotherapeutic agents and the existence of powerful antioxidant and DNA damage repair mechanisms. The distinctive characteristics of CSCs make them ideal targets for clinical therapeutic approaches, and the identification of efficient and specific CSCs biomarkers is of utmost importance. Aldehyde dehydrogenases (ALDHs) comprise a wide superfamily of metabolic enzymes that, over the last years, have gained increasing attention due to their association with stem-related features in a wide panel of hematopoietic malignancies and solid cancers. Aldehyde dehydrogenase 1B1 (ALDH1B1) is an isoform that has been characterized as a marker of colon cancer progression, while various studies suggest its importance in additional malignancies. Here, we review the basic concepts related to CSCs and discuss the potential role of ALDH1B1 in cancer development and its contribution to the CSC phenotype

Efficient E.coli expression strategies for production of soluble human crystallin ALDH3A1

Aldehyde dehydrogenase 3A1 (ALDH3A1) is a recently characterized corneal crystallin with its exact functions still being unclear. Expressing recombinant human ALDH3A1 has been difficult in Escherichia coli (E. coli) because of low solubility, yield and insufficient purity issues. In this report, we compared different E. coli expression strategies (namely the maltose binding protein; MBP- and the 6-his-tagged expression systems) under conditions of auto-induction and co-expression with E. coli's molecular chaperones where appropriate. Thus, we aimed to screen the efficiency of these expression strategies in order to improve solubility of recombinant ALDH3A1 when expressed in E. coli. We showed that the MBP- tagged expression in combination with lower-temperature culture conditions resulted in active soluble recombinant ALDH3A1. Expression of the fused 6-his tagged-ALDH3A1 protein resulted in poor solubility and neither lowering temperature culture conditions nor the auto-induction strategy improved its solubility. Furthermore, higher yield of soluble, active native form of 6-his tagged-ALDH3A1 was facilitated through co-expression of the two groups of E. coli's molecular chaperones, GroES/GroEL and DnaK/DnaJ/GrpE. Convenient one step immobilized affinity chromatography methods were utilized to purify the fused ALDH3A1 hybrids. Both fusion proteins retained their biological activity and could be used directly without removing the fusion tags. Taken together, our results provide a rational option for producing sufficient amounts of soluble and active recombinant ALDH3A1 using the E. coli expression system for conducting functional studies towards elucidating the biological role(s) of this interesting corneal crystallin