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MLN4924/Pevonedistat, a Nedd8-activating enzyme (NAE, E1) inhibitor, has shown notable anti-cancer effect in pre-clinical trials, but it still faces tolerance resistance risk. Combination target therapy indicates a much better clinical effect than single target, and miRNAs are beneficial for easy detection in bodily fluids and tissues. Up to now, MLN4924 and miRNA-targeting combination approaching to treat breast cancer patients remains largely unknown. Here, microRNA-seq analysis showed that the expression of miR-1303 was significantly decreased after MLN4924 treatment in breast cancer cells. Moreover, miR-1303 was abnormally high in breast cancer tissues, and breast cancer patients with high miR-1303 showed poor prognosis. Functionally, excessive miR-1303 promoted the malignant phenotypes of breast cancer cells. Excessive miR-1303 accelerated cell cycle progression by promoting G2/M arrest. Furthermore, we revealed that miR-1303 targeted p27 Kip1 to release G2/M arrest. Notably, excessive miR-1303 partially disturbed the anti-cancer effect of MLN4924. These findings provide potential evidences for combined anti-cancer target therapy of breast cancer patients in the future., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The sodium-coupled bicarbonate cotransporter 1 (NBCe1) plays an essential role in the maintenance of acid-base homeostasis in the human body. However, little research has been done regarding the modification of NBCe1. Nedd4-2 is one of the most important ubiquitin E3 ligases in the kidney where it is responsible for mediating the ubiquitylation level of many important ion channel proteins; therefore, influencing their expression and membrane localization. In this study, we performed experiments based on a prediction from bioinformatics analysis that NBCe1 might be a Nedd4-2 target protein. The results of co-immunoprecipitation and glutathione S-transferase pull-down assays showed that Nedd4-2 interacted with NBCe1. An in vitro ubiquitination assay further demonstrated that Nedd4-2 is indeed the NBCe1 ubiquitin E3 ligase. The overexpression of Nedd4-2 decreased NBCe1 expression, while MG132 rescued the changes. Nedd4-2 overexpression also altered the subcellular distribution of NBCe1. Furthermore, the kidney specific Nedd4-2-knockout mice certified the alteration of NBCe1. In addition, we speculate that neddylation activates Nedd4-2. A co-immunoprecipitation analysis indicated that Nedd4-2 interacted with Nedd8. In vitro neddylation experiments further demonstrated that Nedd4-2 underwent neddylation modification. The overexpression of Nedd8 led to decreased NBCe1 expression, while Nedd4-2 inhibition rescued the changes. These findings demonstrate that Nedd4-2 acts as the ubiquitin E3 ligase of NBCe1, mediating the degradation and altering the subcellular distribution of NBCe1, and that the neddylation modification downregulated NBCe1 expression by upregulating Nedd4-2 activity., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The ubiquitin-proteasome system is involved in numerous cellular processes, such as signal transduction, autophagy, cell cycle control, embryogenesis, and regulation of immune response. Neural precursor cell expressed developmentally downregulated 8 (NEDD8) is a ubiquitin-like protein that activates Cullin-RING ligases and modifies substrates via neddylation. However, there is limited information on how neddylation regulates innate immunity in crustaceans. In the present study, we identified the evolutionarily conserved NEDD8 with the ubiquitin homologue domain in the Chinese mitten crab (Eriocheir sinensis), named it EsNEDD8. Then, we analyzed the expression patterns and cellular location of its substrate, EsCullin4. qRT-PCR showed that both EsNEDD8 and EsCullin4 were widely expressed in all the selected tissues, and EsCullin4 was significantly upregulated in hemocytes after bacterial stimulation. Moreover, silencing of EsCullin4 significantly suppressed the expression of antimicrobial peptides (AMPs) in the hemocytes after bacterial stimulation, and inhibition of EsCullin4 neddylation by treatment with the NEDD8-activating enzyme inhibitor MLN4924 significantly inhibited the expression of the AMPs. Thus, the results show that EsNEDD8-modified EsCullin4 could control antimicrobial activities via regulation of AMPs expression in the Chinese mitten crab., (Copyright © 2018 Elsevier Ltd. All rights reserved.)