Inhibition of protein SUMOylation by davidiin, an ellagitannin from Davidia involucrata

Abstract

Conjugation of small ubiquitin-related modifier (SUMO) to lysine residues in target proteins is a multistep enzymatic reaction analogous to ubiquitination.1 Protein SUMOylation regulates numerous biological processes including transcription, the cell cycle, DNA repair and innate immunity.1 In the first step of the reaction, SUMO is cleaved from the SUMO precursor by SUMO-specific proteases. Next, SUMO is bound to the cysteine residue of the SUMO-activating enzyme (E1), forming a thioester linkage in an ATP-dependent manner. SUMO is then transferred from E1 to the cysteine residue of the SUMO-conjugating enzyme (E2). Finally, SUMO ligase (E3) catalyzes the SUMOylation of specific substrates via a direct interaction with E2 and the substrates. Like ubiquitination, SUMOylation is reversible; the deSUMOylation process is mediated by SUMO-specific proteases. Abnormal SUMOylation is implicated in various diseases including neurodegenerative disease,2 viral infection3 and cancer.4,5 Therefore, enzymes responsible for the SUMO conjugation pathway represent potential targets for drug discovery. To date, several natural products including ginkgolic acid,6 anacardic acid,6 kerriamycin B7 and spectomycin B18 as well as synthetic compounds,9 have been reported to inhibit protein SUMOylation. Here, we report another natural product that functions as a SUMOylation inhibitor: davidiin, purified from the plant Davidia involucrata. Although most known SUMOylation inhibitors function in the micromolar range, davidiin is particularly potent, inhibiting at sub-micromolar concentrations. Materials for this study were obtained as follows. Goat polyclonal anti-SUMO-1 (N-19) and goat polyclonal anti-p53 (FL393)-G antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). A mouse monoclonal anti-T7 antibody was from Novagen (Darmstadt, Germany). Mouse monoclonal anti-a-tubulin (B-5-1-2) and anti-FLAG (M2) antibodies were purchased from Sigma (St. Louis, MO, USA). Recombinant Hisand T7-tagged RanGAP1-C2, GST-Aos1-Uba2 fusion protein (E1), His-tagged Ubc9 (E2), and Histagged SUMO-1 proteins were purified as described previously.10 293T, H1299, MKN-45, DU-145 and NCI-H460 cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10% FBS at 37 1C under 5% CO2. The in vitro SUMOylation reaction was performed as described.6 Briefly, in vitro SUMOylation reaction was performed for 2 h at 30 1C in 20ml buffer (50 mM Tris-HCl (pH 7.4), 6 mM MgCl2, 2 mM ATP and 1 mM dithiothreitol) containing Hisand T7-tagged RanGAP1-C2, GST-Aos1/Uba2 (E1), His-tagged Ubc9 and His-tagged SUMO-1. Samples were separated by 10% SDS–PAGE followed by immunoblotting using an anti-T7 antibody and an anti-SUMO-1 antibody. The reaction for thioester bond formation between SUMO and E1 was performed as described.6 Briefly, the reaction for the thioester bond formation was performed for 20 min at 37 1C in 20ml buffer (50 mM Tris-HCl (pH 7.4), 6 mM MgCl2, 2 mM ATP) containing GSTAos1/Uba2 (E1) and biotinylated SUMO-1 in the absence of dithiothreitol. Samples were separated by 11% SDS–PAGE and the E1–biotinylated SUMO-1 intermediate was detected by avidin-conjugated horseradish peroxidase (Sigma). A screen of 750 samples of botanical and food ingredients extracts using an in situ cell-based SUMOylation assay11 revealed several samples that could inhibit protein SUMOylation, including an extract of D. involucrata (data not shown).6 The inhibitory activity of the D. involucrata extract was confirmed by in vitro SUMOylation assay using RanGAP1-C2 as substrate (Figure 1a). Compound A was isolated by activity-guided fractionation and it was identified by