Evaluation of spice and herb as phyto-derived selective modulators of human retinaldehyde dehydrogenases using a simple in vitro method

Abstract

Selective modulation of retinaldehyde dehydrogenases (RALDHs)-the main aldehyde dehydrogenase (ALDH) enzymes converting retinal into retinoic acid (RA), is very important not only in the RA signaling pathway but also for the potential regulatory effects on RALDH isozyme-specific processes and RALDH-related cancers. However, very few selective modulators for RALDHs have been identified, partly due to variable overexpression protocols of RALDHs and insensitive activity assay that needs to be addressed. In the present study, deletion of the N-terminal disordered regions is found to enable simple preparation of all RALDHs and their closest paralog ALDH2 using a single protocol. Fluorescence-based activity assay was employed for enzymatic activity investigation and screening for RALDH-specific modulators from extracts of various spices and herbs that are well-known for containing many phyto-derived anti-cancer constituents. Under the established conditions, spice and herb extracts exhibited differential regulatory effects on RALDHs/ALDH2 with several extracts showing potential selective inhibition of the activity of RALDHs. In addition, the presence of magnesium ions was shown to significantly increase the activity for the natural substrate retinal of RALDH3 but not the others, while His-tag cleavage considerably increased the activity of ALDH2 for the non-specific substrate retinal. Altogether we propose a readily reproducible workflow to find selective modulators for RALDHs and suggest potential sources of selective modulators from spices and herbs.

Keywords: Fluorescence activity assay; Protein preparation; Retinal; Retinaldehyde dehydrogenase; Selective modulator.

Figure 1. Features of human RALDHs and ALDH2

(A) Catalysis of acetaldehyde to acetic acid by ALDH/RALDH family proteins. (B) Phylogenetic tree of 19 human ALDH genes encoding for corresponding members of ALDH superfamily. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. Branch lengths are in the same units as those of the evolutionary distances. (C) The substrate tunnel entrances of RALDHs and ALDH2 shown as surface models and are surrounded by a black dotted line. Surface representation of RALDH1-3 and ALDH2 were created based on Protein Data Bank (PDB) code 4WB9, 6ALJ, 5FHZ (chain D) and 1O01, respectively, and depicted using the molecular graphics system PyMOL (Ver. 2.4, Schrodinger, LLC).

Figure 2. Preparation of His-tagged and untagged RALDHs and ALDH2 proteins

(A) Schematic flowchart of the expression and purification of RALDHs/ALDH2. (B) Coomassie-stained SDS/PAGE analysis of the purified His-tagged and untagged RALDHs/ALDH2.

Figure 3. Effects of Mg²⁺ and His-tag on the activity of RALDHs and ALDH2

(A) Catalysis of RAL to RA by RALDHs/ALDH2, coupled with the generation of NADH from the cofactor NAD⁺, emitting fluorescence signals. (B) Three different buffer composition without Mg²⁺ (RB1, 2) and with Mg²⁺ (RB3). HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; and MPD, 2-methyl-2,4-pentanediol. (C–F) Effects of buffers RB1–3 and the absence or presence of His-tag on initial velocity (V₀) of reactions for RALDH1 (C), RALDH2 (D), RALDH3 (E) and ALDH2 (F). Blue, yellow and gray colored graphs represent RB1–3 buffers, respectively. Error bars represent standard deviation of mean at 95% confidence (n=3). Statistical analysis including analysis of variance followed by Tukey’s post-hoc test was performed separately for each protein under two tag conditions (His-tagged, untagged) and three buffer conditions. Means that do not share a letter are significantly different (P<0.05).

Figure 4. In vitro activity assays for RALDHs/ALDH2 using spice and herb extracts

(A) Extraction scheme for spice and herb samples. (B–E) Relative activities of RALDH1 (B), RALDH2 (C), RALDH3 (D) and ALDH2 (E) mixed with the spice and herb extracts. Error bars represent standard deviation of mean at 95% confidence (n=3).

Figure 5. The major component ANE in the star anise extract show no inhibitory effect for RALDHs/ALDH2

(A) Total ion chromatograms of the star anise extract with its most abundant component ANE. (B) Effects of monoterpenes including ANE on the activity of RALDHs/ALDH2. SC: S-(+)-carvone, RC: R-(−)-carvone. Error bars indicate standard deviations of mean at 95% confidence (n=3).