Potential diagnostic and therapeutic targets and anti-tumor mechanisms of Neddylation pathway in digestive system tumors / 中华检验医学杂志

Neddylation,a novel post-translational modification of proteins, is overactivated in digestive system tumors and can be used as a potential anti-tumor molecular target. Targeting Neddylation pathway plays an anti-tumor role by inducing cell cycle arrest, apoptosis, senescence and autophagy of digestive system tumor cells, as well as enhancing the sensitivity of digestive system tumor cells to the radiotherapy and chemotherapy. Targeting Neddylation pathway and its inhibnitor MLN4924 can act as poential targets against digestive system tumors.

Emerging role of protein modification in inflammatory bowel disease / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The onset of inflammatory bowel disease (IBD) involves many factors, including environmental parameters, microorganisms, and the immune system. Although research on IBD continues to expand, the specific pathogenesis mechanism is still unclear. Protein modification refers to chemical modification after protein biosynthesis, also known as post-translational modification (PTM), which causes changes in the properties and functions of proteins. Since proteins can be modified in different ways, such as acetylation, methylation, and phosphorylation, the functions of proteins in different modified states will also be different. Transitions between different states of protein or changes in modification sites can regulate protein properties and functions. Such modifications like neddylation, sumoylation, glycosylation, and acetylation can activate or inhibit various signaling pathways (e.g., nuclear factor-‍κB (NF-‍κB), extracellular signal-regulated kinase (ERK), and protein kinase B (AKT)) by changing the intestinal flora, regulating immune cells, modulating the release of cytokines such as interleukin-1β (IL-‍‍1β), tumor necrosis factor-α (TNF‍-‍α), and interferon-‍γ (IFN-‍γ), and ultimately leading to the maintenance of the stability of the intestinal epithelial barrier. In this review, we focus on the current understanding of PTM and describe its regulatory role in the pathogenesis of IBD.

Research on the anti-lung cancer effects of targeted Neddylation modifying pathway and its inhibitor MLN4924 and its mechanism / 国际肿瘤学杂志

Neddylation is overactivated in lung cancer, which promotes the development of lung cancer by activating its downstream CRL ubiquitin ligase and promoting the CRL tumor-suppressor protein substrate degradation. MLN4924, a small molecule inhibitor of Neddylation, plays an anti-lung cancer role by inducing cell cycle arrest, apoptosis and senescence. Furthermore, targeting the key enzymes of Neddylation and their substrates, Cullin family proteins, can inhibit the development of lung cancer.

Discovery of a small molecule inhibitor of cullin neddylation that triggers ER stress to induce autophagy

Protein neddylation is catalyzed by a three-enzyme cascade, namely an E1 NEDD8-activating enzyme (NAE), one of two E2 NEDD8 conjugation enzymes and one of several E3 NEDD8 ligases. The physiological substrates of neddylation are the family members of cullin, the scaffold component of cullin RING ligases (CRLs). Currently, a potent E1 inhibitor, MLN4924, also known as pevonedistat, is in several clinical trials for anti-cancer therapy. Here we report the discovery, through virtual screening and structural modifications, of a small molecule compound HA-1141 that directly binds to NAE in both

Neddylation inhibitor MLN4924 suppresses cilia formation by modulating AKT1

The primary cilium is a microtubule-based sensory organelle. The molecular mechanism that regulates ciliary dynamics remains elusive. Here, we report an unexpected finding that MLN4924, a small molecule inhibitor of NEDD8-activating enzyme (NAE), blocks primary ciliary formation by inhibiting synthesis/assembly and promoting disassembly. This is mainly mediated by MLN4924-induced phosphorylation of AKT1 at Ser473 under serum-starved, ciliary-promoting conditions. Indeed, pharmaceutical inhibition (by MK2206) or genetic depletion (via siRNA) of AKT1 rescues MLN4924 effect, indicating its causal role. Interestingly, pAKT1-Ser activity regulates both ciliary synthesis/assembly and disassembly in a MLN4924 dependent manner, whereas pAKT-Thr determines the ciliary length in MLN4924-independent but VHL-dependent manner. Finally, MLN4924 inhibits mouse hair regrowth, a process requires ciliogenesis. Collectively, our study demonstrates an unexpected role of a neddylation inhibitor in regulation of ciliogenesis via AKT1, and provides a proof-of-concept for potential utility of MLN4924 in the treatment of human diseases associated with abnormal ciliogenesis.

The Nedd8-activating enzyme inhibitor MLN4924 suppresses colon cancer cell growth via triggering autophagy

Neddylation is a post-translational protein modification process. MLN4924 is a newly discovered pharmaceutical neddylation inhibitor that suppresses cancer growth with several cancer types. In our study, we first investigated the effect of MLN4924 on colon cancer cells (HCT116 and HT29). MLN4924 significantly inhibited the neddylation of cullin-1 and colon cancer cell growth in a time and dose-dependent manner. MLN4924 induced G2/M cell cycle arrest and apoptosis in HCT116 and HT29 cells. Moreover, MLN4924 also triggered autophagy in HCT116 and HT29 cells via suppressing the PI3K/AKT/mTOR pathway. Inhibiting autophagy by autophagy inhibitor 3-MA or ATG5 knockdown reversed the function of MLN4924 in suppressing colon cancer cell growth and cell death. Interestingly, MLN4924 suppresses colon cell growth in a xenograft model. Together, our finding revealed that blocking neddylation is an attractive colon cancer therapy strategy, and autophagy might act as a novel anti-cancer mechanism for the treatment of colon cancer by MLN4924.

NEDDylation is involved in hepatitis B virus pregenomic RNA encapsidation / 中华实验和临床病毒学杂志

Objective@#To investigate the effect of an ubiquitin-like modification NEDDylation on HBV life cycle.@*Methods@#Cells were incubated with various concentrations of MLN4924, which is a selective inhibitor of neural precursor cell-expressed developmentally down regulated 8 (NEDD8) activating enzyme (NAE), to block cellular protein NEDDylation. And a series concentration of lamivudine (LAM), an inhibitor of HBV polymerase was set as control. Then we tested HBV DNA replication, protein expression and nucleocapsids formation. Huh-7 cells were transfected with polymerase-inactivated HBV expression plasmid, then incubated with 0.1 μmol/L MLN4924 or çvehicle-DMSO. Then we tested capsids and capsid-associated RNA.@*Results@#MLN4924 inhibited HBV replication in a dose-dependent manner (0~2 μmol/L). When the concentration of MLN4924 was 0.1 μmol/L, the amount of nucleocapsids in the cells did not change significantly, however the encapsidation of HBV pregenomic RNA (pgRNA) decreased by about 47%.@*Conclusions@#NEDDylation is involved in hepatitis B virus pregenomic RNA encapsidation.

Advances of relationship between protein Neddylation and cancer / 中国药科大学学报

Abnormal modification in protein Neddylation is closely related to the occurrence and progression of various tumors. Studies have shown that the level of enzymes participating in Neddylation is higher than that in normal neighboring tissues,so the relationship between Neddylation and cancer has aroused much concern.Ned-dylation has been considered to be a novel anticancer target. Neddylation inactivation by a first-in-class small molecular inhibitor,MLN4924,induces cell cycle arrest,apoptosis,senescence and autophagy in different tumor cells.MLN4924 also exerts significant anticancer effects by inhibiting tumor angiogenesis and regulating the func-tion of immune cells. In this review,the latest progress of protein Neddylation and tumor cell cycle,apoptosis, senescence,autophagy,angiogenesis and regulation of immunocyte were briefly introduced to provide theoretical reference for the development of antitumor drugs targeting Neddylation.

NEDDylation antagonizes ubiquitination of proliferating cell nuclear antigen and regulates the recruitment of polymerase η in response to oxidative DNA damage

NEDDylation has been shown to participate in the DNA damage pathway, but the substrates of neural precursor cell expressed developmentally downregulated 8 (NEDD8) and the roles of NEDDylation involved in the DNA damage response (DDR) are largely unknown. Translesion synthesis (TLS) is a damage-tolerance mechanism, in which RAD18/RAD6-mediated monoubiquitinated proliferating cell nuclear antigen (PCNA) promotes recruitment of polymerase η (polη) to bypass lesions. Here we identify PCNA as a substrate of NEDD8, and show that E3 ligase RAD18-catalyzed PCNA NEDDylation antagonizes its ubiquitination. In addition, NEDP1 acts as the deNEDDylase of PCNA, and NEDP1 deletion enhances PCNA NEDDylation but reduces its ubiquitination. In response to HO stimulation, NEDP1 disassociates from PCNA and RAD18-dependent PCNA NEDDylation increases markedly after its ubiquitination. Impairment of NEDDylation by Ubc12 knockout enhances PCNA ubiquitination and promotes PCNA-polη interaction, while up-regulation of NEDDylation by NEDD8 overexpression or NEDP1 deletion reduces the excessive accumulation of ubiquitinated PCNA, thus inhibits PCNA-polη interaction and blocks polη foci formation. Moreover, Ubc12 knockout decreases cell sensitivity to HO-induced oxidative stress, but NEDP1 deletion aggravates this sensitivity. Collectively, our study elucidates the important role of NEDDylation in the DDR as a modulator of PCNA monoubiquitination and polη recruitment.

Ubiquitin ligase Smurf1 promotes Smurf2 neddylation modification / 军事医学

Objective To investigate the mechanism of Smad ubiquitination-related factor 2 (Smurf2)neddylation. Methods The Smurf2 protein level was tested by overexpression of Nedd8,while the method of immunoprecipitation(IP) and Western blotting were used to analyz Smurf2-Nedd8 modification.The GST-pulldown experiment was conducted to prove protein interactions.The protein was obtained by grinding mouse tissue and Western blotting was used to test the protein expression level.Results Over expression of Nedd8 could lead to the down regulation of the Smurf2′s protein level.Smurf1 and Smurf2 could interact in the GST-pulldown experiment. Smurf1 could enhance Smurf2-Nedd8 modification.The Smurf2′s protein level was up-regulated in mouse tissue of Smurf1 C426A.Conclusion There is some relationship and also difference between Smurf1 and Smurf2.Smurf1 can enhance Smurf2-Nedd8 modification.

Progress in Function and Regulation of Protein Neddylation / 生物化学与生物物理进展

NEDD8 is a member of the ubiquitin-like proteins. The overall structure of NEDD8 is quite similar to ubiquitin. Covalent conjugation of NEDD8 to proteins at the post-translational level is called Neddylation. Neddylation occurs similarly to ubiquitination and need enzyme cascades involving E1, E2 and E3. Neddylation has been demonstrated to be essential to maintain the ubiquitin ligase activity of Cullin-Roc based E3 ligases. Compared with the ubiquitination which was widely studied in the past two decades, few substrates were identified for Neddylation and the physiological functions of Neddylation need further investigations. The current progress of function and regulation of protein Neddylation will be reviewed.