Dead enzymes in the aldehyde dehydrogenase gene family: role in drug metabolism and toxicology

<div><p><b><i>Introduction</i></b>: Dead enzymes are gene products (proteins) that lack key residues required for catalytic activity. In the pre-genome era, dead enzymes were thought to occur only rarely. However, they now have been shown to represent upwards of 10% of the total enzyme population in many families. The aldehyde dehydrogenase (ALDH) gene family encodes proteins that, depending on the isozyme, may be either catalytically-active or -inactive. Importantly, several ALDHs exhibit biological activities independent of their catalytic activity. For many of these, the physiological and pathophysiological functions remain to be established.</p><p><b><i>Areas covered</i>:</b> This article reviews the non-enzymatic functions of the ALDH superfamily. In addition, a search for additional non-catalytic ALDH records is undertaken. Our computational analyses reveal that there are currently 182 protein records (divided into 19 groups) that meet the criteria for dead enzymes.</p><p><b><i>Expert Opinion</i>:</b> Dead enzymes have the potential to exert biological actions through protein-protein interaction and allosteric modulation of the activity of an active enzyme. In addition, a dead enzyme may also influence availability of substrate for other active enzymes by sequestering substrate, and/or anchoring the substrate to a particular subcellular space. A large number of putatively non-catalytic ALDH proteins exist that warrant further study.</p></div

Overexpression of wild-type and mutant ALDH3A1 retards hTCEpi cell proliferation.

<p>The effect of ALDH3A1 on cell proliferation was investigated in Tet-On hTCEpi cell lines induced to express (<b>A</b>) no ALDH3A1 (hTCEpi-TR-Lenti), (<b>B</b>) wild-type ALDH3A1 (hTCEpi-TR-3A1wt) or (<b>C</b>) catalytically-inactive mutant ALDH3A1 (hTCEpi-TR-3A1mu). Cells were seeded in 100 mm culture dishes (2.0 x 10⁵ cells/dish) and treated with (+TET) or without (-TET) tetracycline (1μg/ml). After 4 d, cells were trypsinized and viable cells were counted using trypan blue exclusion on a hemocytometer. Consecutively, 2.0 x 10⁵ cells were transferred to a new dish and this process repeated for a total of 8 passages, i.e., for 32 d until treated cells showed decreasing cell numbers. Cumulative population doubling (PD) number at each passage was calculated as: CPD number = ln [N_E/N_B] / ln 2 + CPD_o (N_E: end cell count; N_B: cell count in the beginning; CPD: CPD_o at previous passage). Results are reported as mean + standard error from n = 3 experiments. Error bars are contained within the symbols. *<i>p</i> < 0.05, Students unpaired t-test, compared to PD numbers of untreated cells (-TET) at the same passage. ^<i>p</i> < 0.05, Students unpaired t-test, compared to the PD time (hr) of untreated cells (-TET).</p

BrDU labeling of corneal epithelial cells.

<p>(<b>A</b>) Representative H&E staining of corneas from WT and DKO mice; scale bar = 50 μm. Higher magnification images of central (C- dotted box) and peripheral (P-solid box) cells are shown in the right panels; scale bar = 5 μm. (<b>B</b>) Representative images of BrDU immunostaining of corneas from WT and DKO mice; scale bar = 100 μm. Higher magnification of central (C) and peripheral (P) corneas are shown in the right panels; scale bar = 25μm. (<b>C</b>) The numbers of total cells (left panel) in the central and peripheral regions of corneal epithelium were counted in WT and DKO mice. The numbers of BrDU-positive cells (right graph) in these regions were counted and expressed as a percentage of total cells in the same visual field. Data represent the mean ± standard deviation (N = 3). *<i>p</i> < 0.05, Students unpaired t-test, compared to WT.</p

Scheme of proposed regulatory function of ALDH3A1 in corneal epithelial cells.

<p>ALDH3A1 in corneal epithelial cells retards proliferation by transcriptional regulation of cell cycle modulators. Wild-type ALDH3A1 contributes to p53 sequestration into the nucleus, while protecting it from degradation. ALDH3A1 protein also contributes to differentiation through association with transcription factors involved in regulating mRNA levels of differentiation specific markers.</p

Subcellular levels of p53 protein in hTCEpi-TR cell lines.

<p>(<b>A</b>) Cytosolic and nuclear fractions from cell lysates of hTCEpi-TR-ALDH3A1wt and hTCEpi-TR-ALDH3A1mu cells treated with vehicle (-TET) or tetracycline (1μg/ml) (+TET) were immunoblotted for p53, ALDH3A1, GAPDH or PARP1 expression. (<b>B</b>) Relative levels of ALDH3A1 (upper panels) and p53 (lower panels) expression by densitometry analysis of immunoblots. Results are expressed as the fold of untreated cells (-TET) after correction for respective loading controls (GAPDH for cytosolic extracts; PARP-1 for nuclear extracts). Data are presented as mean + S.E.M (N = 3). *<i>p</i> <0.05, Students unpaired t-test, compared to untreated cells (-TET) of the same cell line. ^#<i>p</i> <0.05, Students unpaired t-test, compared to treated (+TET) ALDH3A1wt cells.</p

Proposed mechanism by which ALDH1B1 modulates colon tumorigenesis.

<p>ALDH1B1 play important role in cell survival and proliferation by modulating Wnt-, Notch- and PI3K/Akt signaling pathways and thereby contributes in colon tumorigenesis.</p

Evaluation of human <i>ALDH1B1</i> promoter.

<p>(<b>A</b>) Putative TCF/LEF binding elements (TBEs) in the human ALDH1B1 3-Kb promoter region. TBE6, TBE5, TBE4, TBE3 and TBE2 are in sense orientation whereas TBE1 is in antisense orientation. Numbers given below TBEs represent the distance from transcription start site (TSS) in base pairs. (<b>B</b>) Activity of the 3kb ALDH1B1-pGL3 and cMyc-pBV promoter reporter constructs expressed relative to control (the empty pGL3-enhancer and pBV vectors, respectively) in COLO320, SW480 and HT29 cells. The Firefly luciferase activity is normalized to the internal control Renilla luciferase. Data are presented as mean ± SEM (representative from three individual experiments done in triplicate). (<b>C</b>) Activity of the entire 3kb ALDH1B1 pGL3-promoter construct and truncations of the construct in which putative TCF/LEF binding elements (TBEs) have been sequentially deleted. Luciferase promoter activity was measured 24h after transfection and the cells transfected with empty plasmid were used as control. The Firefly luciferase activity is normalized to the internal control Renilla luciferase. Luciferase activity was calculated as fold of empty pGL3 enhancer vector and data are expressed as per cent luciferase activity of TBE1-6 construct and presented as mean ± SEM (representative from three individual experiments done in triplicates). *<i>P</i> < 0.05, one-way ANOVA.</p

ALDH1B1 knock down depletes ALDH^bright cells.

<p>Flow cytometry was conducted in SW480 cells transfected with either ALDH1B1 shRNA or scramble shRNA. Cells were incubated with Aldefluor substrate in the presence or absence of the ALDH inhibitor diethylaminobenzaldehyde (DEAB). Cells with high fluorescence (reflecting high ALDH activity, i.e., ALDH^bright cells) were determined and arbitrarily defined as being in the oval gate in the bottom right quadrant of the graph. (<b>A</b>) Flow cytometric analysis of scramble shRNA control cells in the absence (-DEAB) or presence (+DEAB) of DEAB. (<b>B</b>) Flow cytometric analysis of ALDH1B1 shRNA cells in the absence (-DEAB) or presence (+DEAB) of DEAB; (<b>C</b>) Proportion of ALDH^bright cells in ALDH1B1 shRNA (black bar) and scramble shRNA (white bar) cell populations. Data are presented as the mean ± SEM from 3 experiments. * <i>P</i> < 0.05, Student’s unpaired t-test, compared with scramble shRNA cells.</p

Decreased ALDH1B1 expression inhibits spheroid formation and tumor growth.

<p>SW480 cells were transfected with ALDH1B1 shRNA or scramble shRNA to develop stable cell colonies. (<b>A</b>) ALDH1B1 expression was examined by Western blotting and quantified by densitometric analysis. Numbers at the bottom of ALDH1B1 Western blot bands are the average densitometry of three replicates expressed relative to scramble shRNA-transfected (control) cells. Rec, recombinant ALDH1B1; CT, Control SW480 cells without transfection. (<b>B</b>) Spheroid formation was measured 10 d after plating of cells. Representative phase contrast micrographs (left) and histograms (right) showing the number and volume of spheroids developing from SW480 cells transfected with ALDH1B1 shRNA (open bars) or with scramble shRNA (control) (closed bars) vectors. Data are presented as mean ± SEM (n = 3). # <i>P</i> < 0.01, Student’s unpaired t-test, compared with scramble shRNA. (C) Growth of tumors induced by injection of SW480 cells transfected with ALDH1B1 shRNA (circles) or scramble shRNA (squares)) into the left and right flanks of female athymic (nu+/nu+) mice. Tumor volume was calculated as (length × width² × 0.52). Data are presented as mean ± SEM (n = 10 tumors per group). * <i>P</i> < 0.05, Student’s unpaired t-test, compared with tumors in scramble shRNA group at same time point.</p

ALDH1B1 modulates Wnt/β-catenin, Notch and PI3K/Akt signaling pathways.

<p>(<b>A</b>) For the Wnt/β-catenin reporter assay, scramble or ALDH1B1 shRNA transfected SW480 cells were transiently transfected with either TOPflash, or FOPflash reporter plasmid along with pRL-TK vector as an internal control. Data are expressed as TOP/FOPflash ratio and presented as mean ± SEM (representative from three individual experiments done in triplicate). * <i>P</i> < 0.05, Student’s unpaired t-test. (<b>B</b>) RT-PCR analysis of RNA isolated from SW480 cells transfected with scramble shRNA or ALDH1B1 shRNA. mRNA levels were expressed as a ratio of the levels in scramble shRNA-transfected cells. <i>GAPDH</i> gene expression was used to normalize data. Results are presented as the mean ± SEM from three experiments. * <i>P < 0</i>.<i>05</i>, Student’s unpaired t-test, compared with results in scramble shRNA cells. Western blot analysis of expression of members of Wnt/β-catenin and Notch pathways (<b>C</b>) and of FABP5, PPARβ/δ and PI3K/Akt pathway members (<b>D</b>) were conducted in ALDH1B1 shRNA and scramble shRNA cells. Western blots were quantified by densitometry, numbers represent mean ± SEM of three independent experiments. * <i>P</i> < 0.05, compared to results in scramble shRNA cells.</p