Neddylation is a posttranslational modification that attaches ubiquitin-like protein Nedd8 to protein targets via Nedd8-specific E1-E2-E3 enzymes and modulates many important biological processes. Nedd8 attaches to a lysine residue of a substrate, not for degradation, but for modulation of substrate activity. We previously identified the HECT-type ubiquitin ligase Smurf1, which controls diverse cellular processes, is activated by Nedd8 through covalent neddylation. Smurf1 functions as a thioester bond-type Nedd8 ligase to catalyze its own neddylation. Numerous ubiquitination substrates of Smurf1 have been identified, but the neddylation substrates of Smurf1 remain unknown. Here, we show that Smurf1 interacts with RRP9, a core component of the U3 snoRNP complex, which is involved in pre-rRNA processing. Our in vivo and in vitro neddylation modification assays show that RRP9 is conjugated with Nedd8. RRP9 neddylation is catalyzed by Smurf1 and removed by the NEDP1 deneddylase. We identified Lys221 as a major neddylation site on RRP9. Deficiency of RRP9 neddylation inhibits pre-rRNA processing and leads to downregulation of ribosomal biogenesis. Consequently, functional studies suggest that ectopic expression of RRP9 promotes tumor cell proliferation, colony formation, and cell migration, whereas unneddylated RRP9, K221R mutant has no such effect. Furthermore, in human colorectal cancer, elevated expression of RRP9 and Smurf1 correlates with cancer progression. These results reveal that Smurf1 plays a multifaceted role in pre-rRNA processing by catalyzing RRP9 neddylation and shed new light on the oncogenic role of RRP9.

Background: Numerous studies have indicated that the neddylation pathway is closely associated with tumor development. MLN4924 (Pevonedistat), an inhibitor of the NEDD8-activating E1 enzyme, is considered a promising chemotherapeutic agent. Recently, we demonstrated that neddylation of the tumor suppressor PTEN occurs under high glucose conditions and promotes breast cancer development. It has been shown, however, that PTEN protein levels are reduced by 30–40% in breast cancer. Whether this PTEN deficiency affects the anti-tumor function of MLN4924 is unknown.Methods: In the present study, cell counting kit-8 and colony formation assays were used to detect cell proliferation, and a transwell system was used to quantify cell migration. A tumor growth assay was performed in BALB/c nude mice. The subcellular location of PTEN was detected by fluorescence microscopy. The CpG island of the UBA3 gene was predicted by the Database of CpG Islands and UCSC database. Western blotting and qRT-PCR were used to measure the expression of indicated proteins. The Human Protein Atlas database, the Cancer Genome Atlas and Gene Expression Omnibus datasets were used to validate the expression levels of UBA3 in breast cancer.Results: Our data show that the anti-tumor efficacy of MLN4924 in breast cancer cells was markedly reduced with the deletion of PTEN. PI3K/Akt signaling pathway activity correlated positively with UBA3 expression. Pathway activity correlated negatively with NEDP1 expression in PTEN-positive breast cancer patients, but not in PTEN-negative patients. We also demonstrate that high glucose conditions upregulate UBA3 mRNA by inhibiting UBA3 promoter methylation, and this upregulation results in the overactivation of PTEN neddylation in breast cancer cells.Conclusion: These data suggest a mechanism by which high glucose activates neddylation. PTEN is critical, if not indispensable, for MLN4924 suppression of tumor growth; PTEN status thus may help to identify MLN4924-responsive breast cancer patients.

Non‐alcoholic fatty liver disease (NAFLD) is one of the most common liver disorders which are characterized by the accumulation of excessive lipid in hepatocytes. The precise pathogenesis of NAFLD is very complicated and remains largely unknown. Smad ubiquitination regulatory factor 1 (Smurf1) is crucial for numerous processes including bone homeostasis, embryogenesis, and pathogenic autophagy. In this study, we found that liver steatosis was alleviated in Smurf1 ‐deficient mice fed with high‐fat diet (HFD) for 19 weeks. The deletion of Smurf1 reduced the accumulation of lipid droplets and triglycerides in hepatocytes. The stability of sterol regulatory element‐binding protein‐1c (SREBP‐1c), a key transcription factor that mediates de novo lipogenesis, was markedly reduced in Smurf1 ‐deficient mice. The mechanistic study showed that Smurf1 interacts with SREBP‐1c and protects SREBP‐1c from ubiquitination and degradation by preventing the binding of SREBP‐1c to its ubiquitin E3 ligase Fbw7a. Thus, our study presented an E3 ligase catalytic activity‐independent function of Smurf1 in the fatty liver development.

Background: CD4⁺ T cells play multiple roles in controlling tumor growth and increasing IFN-γ⁺ T-helper 1 cell population could promote cell-mediated anti-tumor immune response. We have previously showed that low-dose DNA demethylating agent decitabine therapy promotes CD3⁺ T-cell proliferation and cytotoxicity; however, direct regulation of purified CD4⁺ T cells and the underlying mechanisms remain unclear.Methods: The effects of low-dose decitabine on sorted CD4⁺ T cells were detected both in vitro and in vivo. The activation, proliferation, intracellular cytokine production and cytolysis activity of CD4⁺ T cells were analyzed by FACS and DELFIA time-resolved fluorescence assays. In vivo ubiquitination assay was performed to assess protein degradation. Moreover, phosphor-p65 and IκBα levels were detected in sorted CD4⁺ T cells from solid tumor patients with decitabine-based therapy.Results: Low-dose decitabine treatment promoted the proliferation and activation of sorted CD4⁺ T cells, with increased frequency of IFN-γ⁺ Th1 subset and enhanced cytolytic activity in vitro and in vivo. NF-κB inhibitor, BAY 11-7082, suppressed decitabine-induced CD4⁺ T cell proliferation and IFN-γ production. In terms of mechanism, low-dose decitabine augmented the expression of E3 ligase β-TrCP, promoted the ubiquitination and degradation of IκBα and resulted in NF-κB activation. Notably, we observed that in vitro low-dose decitabine treatment induced NF-κB activation in CD4⁺ T cells from patients with a response to decitabine-primed chemotherapy rather than those without a response.Conclusion: These data suggest that low-dose decitabine potentiates CD4⁺ T cell anti-tumor immunity through enhancing IκBα degradation and therefore NF-κB activation and IFN-γ production.