br Introduction O Methylguanine DNA methyltransferase MGMT i
Introduction O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA-repair enzyme that specifically transfers alkyl adducts from the O6 position of guanine to the cysteine residue (Cys145) in its active site. In cancer cells, this ability of MGMT disrupts the cytotoxic actions of alkylating anticancer agents. Hence, MGMT is expressed at high levels in patients with various cancers  and is associated with resistance to -alkylating chemotherapeutic drugs . Inhibition of MGMT through promoter methylation ,  or shRNA  leads to increased drug sensitivity in multiple human cancers. However, several lines of evidence have revealed that treatment with the alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) leads to decreased MGMT proteins levels in various cancer types , , suggesting that BCNU mediates MGMT turnover. However, the underlying mechanism is unclear. The irreversible binding of the alkyl group to MGMT protein functionally inhibits its enzyme activity and leads to protein degradation . Proteasomal and lysosomal degradation are the two major processes for cellular protein turnover . Protein ubiquitination is a chemical process in which ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3) sequentially covalently attach ubiquitin to the lysine of a target protein . First, the ATP-dependent E1 enzyme activates ubiquitin, which is then transferred to one of approximately 40 of E2 enzymes (in mammals) and forms a thiol ester intermediate E2-ubiquitin (E2-ub). E2-ub then interacts with the E3 enzyme, which precisely binds with the substrate to achieve specific ubiquitin modification. The nature of the ubiquitin linkages generally determines the fate of the modified protein . By contrast, lysosomal protein degradation is usually initiated non-selectively and occurs during autophagy. Autophagy is a dynamic process with multiple steps, including the formation of autophagosomes, the fusion of autophagosomes with lysosomes to form autolysosomes, and the degradation of autolysosome contents . We and others have demonstrated that BCNU, cisplatin, and tamoxifen induce ubiquitin-dependent proteolysis of MGMT in numerous types of tumor SAG , , , suggesting that the proteasomal degradation process mediates MGMT turnover. We used online prediction programs to screen for human E2 enzymes that have the potential to bind with MGMT protein. UBE2B exhibited the strongest interaction with MGMT. In the present study, we aimed to elucidate the role of UBE2B in modulating MGMT, and the possible application potential of UBE2B inhibitors in cancer therapeutics.
Materials and methods
Discussion In this study, we uncovered a critical role of the ubiquitin-proteasome system in cancer cells in response to chemotherapeutics, such as the alkylating agent BCNU. Previous studies indicated the possible importance of the isocyanate moiety of nitrosoureas, such as BCNU and lomustine (CCNU), in impacting protein turnover by attacking nucleophilic amino acids , . The isocyanates generated from decomposition of BCNU have been shown to inhibit glutathione reductase ,  and DNA ligase , . We found that MGMT becomes inactive when exposed to BCNU. UBE2B and RAD18 regulate the ubiquitination of inactive MGMT. Ubiquitinated MGMT is then degraded by the proteasome. UBE2B depletion significantly decreases MGMT ubiquitination and activity, leading to increased BCNU cytotoxicity. Thus, our study identified the critical function of UBE2B in the turnover of deactivated MGMT (Fig. 6). Ubiquitination has received special attention owing to its recently assigned role as a modulator of carcinogenesis, cancer pathogenesis, and metastasis , . Certain E2s and E3s have been reported as targets in cancer therapy . UBE2B has been reported as a critical regulator of DNA repair , , ,  and the cell cycle ,  in cancer cells. Notably, a previous study indicated that yeast Rad6 (the ortholog of human UBE2B) interacts with the RING finger-containing E3 ubiquitin ligase Ubr1  and stimulates the ubiquitination of Mgt1, which is the yeast ortholog of human MGMT , , . Our study provided substantial evidence that human UBE2B governs the ubiquitination of MGMT in NPC cells. However, since we chose UBE2B for analysis because it has the strongest binding affinity for MGMT based on the binding affinity prediction analysis, the possibility of the involvement of other E2 enzymes in MGMT ubiquitination cannot be ruled out. Further analysis with other E2 enzymes should be performed to fully understand the mechanism of regulation of MGMT.