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  • br Materials and methods br Results br Discussion


    Materials and methods
    Discussion UPP components including the proteasome, E1s, E2s, E3s and DUBs may become one the most important Schisandrol B mg of therapeutic targets for the pharmaceutical industry in the near future and supersede those which are involved in the phosphorylation system [26]. Until recently, there have been no UPP component regulators approved by the FDA except proteasomal inhibitors. An E1 inhibitor and inhibitors of E3 are now in clinical trials, whereas no E2 or DUB inhibitors have entered clinical trials [19], [27]. In recent years, some small-molecule DUB inhibitors have been identified possessing good selectivity against 19S RP-associated DUBs (USP14 and UCH-L5) such as b-AP15 [24], but most of the DUB inhibitors have poor selectivity. Additionally, the mechanisms of small chemical DUB inhibitors are elusive. The chemical structure of curcusone D is different from all of the reported DUB inhibitors. It contains cross-conjugated α, β-unsaturated ketones (Fig. 1A). A similar pharmacophore was previously described in the pan-DUB inhibitor prostaglandin lipid derivatives, which function as potent Michael receptors to inhibit the activities of DUBs [28], [29]. The alpha, beta-unsaturated carbonyl center in the cyclopentenone ring of prostaglandins could react with the DUB catalytic cysteine thiol group, resulting in a covalent adduct [30]. Our results indicated that curcusone D could induce ROS, which are responsible for the inhibition of DUBs (Fig. 5C), similar to the activity of prostaglandin. However, it has been reported that NAC is not only an ROS scavenger but can also antagonize the activity of the proteasomal inhibitor piperlongumine via direct interaction [31]. Trolox, another ROS scavenger that could not interact with proteasomal inhibitors, was used to further evaluate whether Trolox could reverse curcusone D Schisandrol B mg induced polyubiquitin level. As the Supplemental Fig. 2 showed, trolox also could significantly reverse the Curcusone D induced polyubiquitin level. So, here we showed that both NAC and Trolox could reverse the effect of Curcusone D on protein polyubiquitin, which indicated that Curcusone D might not like the proteasome inhibitor piperlongumine [31], which could only be reversed by NAC via a direct interaction with NAC. And as Supplemental Fig. 3 shown, trolox also significantly reversed the curcusone D-induced cell growth inhibition in a dose-dependent manner in both NCI-H929 and RPMI-8226 multiple myeloma cells, which confirmed that the inhibition of cell growth by curcusone D requires ROS production. There are 100 putative DUBs encoded by the human genome, which consist of five different families, of which four are cysteine proteases (ubiquitin C-terminal hydrolase (UCH), ubiquitin specific protease (USP/UBP), ovarian tumor domain (OTU), and Josephin domain (MJD) subfamilies), and one contains a metalloprotease (the JAMM subfamily) [32], [33]. Recently, it has been confirmed that not all but many DUBs including USP, UCH and OUT subfamily members could be reversibly inactivated by H2O2 treatment in vitro and in cells [33], [34], [35]. Curcusone D inhibited USP5, 7, 8, 13, 14, 15, and 22 activities, but had no effect on UCH-L1, UCH-L3, and UCH-37 (Fig. 4C), which indicates the different sensitivity of DUB members to ROS as mentioned previously [33], [34], [35]. Still, there are some differences between curcusone D and H2O2 treatment, such as that UCH-L1 and UCH-L3 could not be inhibited by curcusone D but could be actually inhibited by H2O2[34], [36]. Additionally, chalcone-based derivatives AM146, RA-9 and RA-14, which contain α,β-unsaturated carbonyl groups, directly target and suppress the activity of UCH-L1, UCH-L3, USP2, USP5 and USP8 without affecting the 20S proteasome catalytic-core activity [37]. More details of oxidative stress regulating DUBs could be uncovered as more and more small molecular probes, including curcusone D, are identified in the future. The oxidation of the sulfenic acid group (SOH) at the cysteine residue inhibits the activity of DUBs, which could be reversed in the presence of a reducing agent, but further oxidation to sulfinic acid (SO2H) or sulfonic acid (SO3H) could induce the degradation of DUBs [38]. As Fig. 3C shown, the HA labeling of DUBs decreased significantly with curcusone D treatment, implying that the activity of some DUBs, including USP5, 7, 8, 13, 14, 15 and 22, was downregulated. However, whether the related DUB protein levels were downregulated deserves specific attention in future studies, which may indirectly reveal the oxidative status of these DUBs.