[PMC free article] [PubMed] [Google Scholar] 38. These results suggest that emodin potentiates the antiproliferative effect of IFN- by activation of JAK/STAT pathway signaling through inhibition of 26S proteasome-stimulated IFNAR1 degradation. Therefore, emodin warrants further investigation as a new means to enhance the efficacy of IFN-/. and 0.05, (**) 0.01, (***) 0.001 in comparison with the DMSO control. Inhibitory effect of emodin on 26S proteasome activity Bendazac L-lysine Purified human 26S proteasome was used to examine whether emodin directly inhibited the 26S proteasome. As shown in Physique ?Physique3A,3A, emodin inhibited the Bendazac L-lysine chymotrypsin-like activity of the 26S proteasome, with an IC50 value of 1 1.22 M. Emodin also inhibited the trypsin-like and caspase-like activities of the 26S proteasome, with IC50 values of 20.85 M Bendazac L-lysine and 0.24 M, respectively (Physique 3B and 3C). To examine the effect of emodin on endogenous protein ubiquitination, HEK293A cells were treated with emodin and the cell lysates were probed with Rabbit polyclonal to beta defensin131 anti-ubiquitin antibodies. As shown in Physique ?Physique3D,3D, proteasome inhibitor MG132 significantly increased ubiquitinated protein accumulation in comparison with that of the untreated cells. In addition, emodin also increased endogenous protein ubiquitination in a concentration-dependent manner. Furthermore, emodin treatment also significantly increased endogenous protein ubiquitination in a time-dependent manner (Physique ?(Figure3E).3E). These results indicate that emodin is usually a potent inhibitor of the 26S proteasome. Open in a separate window Physique 3 Inhibitory effect of emodin on 26S proteasome activityThe purified human 26S proteasome (0.1 g) was treated with or without different concentrations of emodin and A. 40 M Suc-Leu-Leu-Val-Tyr-AMC (for measurement of chymotrypsin-like activity), B. 40 M Ac-Arg-Leu-Arg-AMC (for measurement of trypsin-like activity), or C. 40 M Z-Nle-Pro-Nle-Asp-aminoluciferin (for measurement of caspase-like activity) for 2 h at 37C. HeLa cells were treated with the indicated concentrations of emodin or 10 M MG132 for 3 h D., or with 20 M emodin or 10 M MG132 for the indicated durations E., and the cell lysates were probed with anti-ubiquitin antibodies. GAPDH was used as an internal control. Molecular docking of emodin with proteasome subunits The proteolytic activities of the proteasome are dependent on the N-terminal threonine (Thr1) residue hydroxyl group of the subunits, which are responsible for catalyzing the Bendazac L-lysine cleavage of peptides through nucleophilic attack. An docking study was performed to aid the understanding of possible binding modes of emodin with the active pocket of proteasome subunits and subsequent proteasome inhibition. Emodin was docked to the active site of the proteasome 1, 2, and 5 subunits, which are responsible for caspase-like, trypsin-like, and chymotrypsin-like activities of the proteasome, respectively. As shown in Physique ?Physique4A,4A, emodin adopted a conformation favorable for nucleophilic attack at the active site of the 1 subunit with energy of ?6.22 kcal/mol. To identify favorable binding modes of emodin to the proteasomal chymotrypsin-like active site, we analyzed hydrogen-bond (H-bond) formation and hydrophobic interactions between emodin and the 1 subunit. There are 3 polar hydrogens and 1 carbonyl-oxygen in emodin that are available for H-bonding and participate in H-bonding with the Thr1, Thr21, and Ser129 residues of the 1 subunit (Physique ?(Physique4B).4B). Emodin was also favorably inserted within the S1 hydrophobic pocket of the 1 subunit through hydrophobic interactions (Physique ?(Physique4C).4C). Similarly, emodin adopted a conformation favorable for nucleophilic attack at the active site of the 2 2 Bendazac L-lysine subunit with energy of ?6.95 kcal/mol (Figure ?(Figure4D).4D). There are 2 polar hydrogens and 1 carbonyl-oxygen on emodin that are available for H-bonding and which participate in H-bonding with the Thr21, His35, Gly45, and Gly47 residues of the 2 2 subunit (Physique ?(Figure4E).4E). Emodin was favorably inserted within the S1 hydrophobic pocket of the 2 2 subunit through hydrophobic interactions (Physique ?(Figure4F).4F). Emodin adopted a conformation favorable for nuleophilic attack at the active site of the 5 subunit with energy of ?6.22 kcal/mol (Physique ?(Physique4G).4G). There is 1 polar hydrogen on emodin that is available for H-bonding, which participates in H-bonding with the Gly47 residue of the 5 subunit (Physique ?(Physique4H).4H). Emodin was favorably inserted within the S1 hydrophobic pocket of the 2 2 subunit through hydrophobic interactions (Physique ?(Figure4I).4I). These results suggest that emodin can exhibit an orientation/conformation in proximity to the N-terminal Thr1 of the 1, 2, and 5 subunits and is thus subject to nucleophilic attack. Open in a separate window Figure 4 Molecular docking of emodin on the proteasome subunitsACC. Emodin.