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NEDDylation is a post-translational modification of a protein, which transfers Ubiquitin like protein NEDD8 (Neuronal Precursor Cell-expressed Developmentally Down-regulated Protein 8) to the lysine residue of the product through a three-stage enzymatic reaction, and widely regulates many biological processes, such as cell cycle signal transduction and immune recognition. In the past ten years, we have witnessed tremendous progress in the study of protein ubiquitination modification, from modification mechanisms to drug development. Which suggests that inhibition of NEDDylation is an effective way to inhibit tumor. A variety of biological detection methods have been developed during the development of the inhibitor. In this review, we briefly introduced the modification process and substrates of NEDDylation, and discussed detection methods of NEDDylation activity in detail. This review will provide an up-to-date and comprehensive review of the methods for detecting NEDDylation activity that will contribute to NEDDylation inhibitor development., (Copyright © 2020. Published by Elsevier Ltd.)

Post-translational modification plays a critical role in viral replication. Previously we reported that neddylation of PB2 of influenza A virus (IAV) can inhibit viral replication. However, we found that NEDD8 overexpression can still inhibit the replication of PB2 K699R mutant viruses, implying that other viral protein(s) can be neddylated. In this study, we revealed that M1 of IAV can also be modified by NEDD8. We found that the E3 ligase HDM2 significantly promotes M1 neddylation. Furthermore, we identified M1 K187 as the major neddylation site. We generated an IAV M1 K187R mutant (WSN-M1 K187R) and compared the growth of wild-type and mutant viruses in Madin-Darby canine kidney (MDCK) cells. The data showed that the replication of WSN-M1 K187R was more efficient than that of wild-type WSN. More importantly, we observed that overexpression of NEDD8 inhibited the replication of the wild-type WSN more effectively than that of WSN-M1 K187R. In addition, we found that the neddylation-deficient M1 mutant (M1 K187R) had a longer half-life than that of wild-type M1, indicating that the neddylation of M1 reduces stability. Then we performed a viral infection assay and found that WSN-M1 K187R exhibited greater virulence in mice than wild-type WSN, suggesting that the neddylation of M1 reduced IAV replication in vivo . In conclusion, we uncovered that neddylation of M1 by HDM2 negatively regulates the stability of M1, which in turn inhibits viral replication.

A variety of post-translational modifications of Plasmodium falciparum proteins, including phosphorylation and ubiquitination, are shown to have key regulatory roles during parasite development. NEDD8 is a ubiquitin-like modifier of cullin-RING E3 ubiquitin ligases, which regulates diverse cellular processes. Although neddylation is conserved in eukaryotes, it is yet to be characterized in Plasmodium and related apicomplexan parasites. We characterized P. falciparum NEDD8 (PfNEDD8) and identified cullins as its physiological substrates. PfNEDD8 is a 76 amino acid residue protein without the C-terminal tail, indicating that it can be readily conjugated. The wild type and mutant (Gly75Ala/Gly76Ala) PfNEDD8 were expressed in P. falciparum. Western blot of wild type PfNEDD8-expressing parasites indicated multiple high molecular weight conjugates, which were absent in the parasites expressing the mutant, indicating conjugation of NEDD8 through Gly76. Immunoprecipitation followed by mass spectrometry of wild type PfNEDD8-expressing parasites identified two putative cullins. Furthermore, we expressed PfNEDD8 in mutant S. cerevisiae strains that lacked endogenous NEDD8 (rub1Δ) or NEDD8 conjugating E2 enzyme (ubc12Δ). The PfNEDD8 immunoprecipitate also contained S. cerevisiae cullin cdc53, further substantiating cullins as physiological substrates of PfNEDD8. Our findings lay ground for investigation of specific roles and drug target potential of neddylation in malaria parasites.

Platinum is a widely used first-line chemotherapy in treating non-small cell lung cancer of adenocarcinoma. Unfortunately, platinum resistance leads to relapse and therapeutic failure, enabling the development of platinum-sensitization strategies to be of great clinical significance. Here, we report that the upregulation of the NEDD8-conjugating enzyme UBE2F is an important way for lung cancer cells to escape platinum-induced cell apoptosis, which confers to insensitivity to platinum-based chemotherapy. Mechanistically, platinum treatment impairs the complex formation for proteasome-mediated UBE2F degradation, evidenced by the weaker association between UBE2F and Ring-box protein 1 (RBX1), an essential component of Cullin-Ring E3 ligases (CRLs), thus leading to the accumulation of UBE2F. The accumulated UBE2F promotes the neddylation levels and activity of Cullin5, in accord with the lower expression of pro-apoptotic protein NOXA, a well-known substrate of Cullin-Ring E3 ligase 5 (CRL5). Additionally, knockout of UBE2F significantly sensitizes lung cancer cells to platinum treatment by enhancing the protein levels of NOXA and subsequently promoting cell apoptosis. Our observations uncover a previously unknown regulatory mechanism of UBE2F stability upon platinum chemotherapy and suggest that UBE2F might be a novel therapy target for sensitizing lung cancer cells to platinum-based chemotherapy.

Activation of sterol regulatory element-binding protein 1 (SREBP-1), a master lipogenic transcription factor, is associated with cancer metabolism and metabolic disorders. Neddylation, the process of adding NEDD8 to its substrate, contributes to diverse biological processes. Here, we identified SREBP-1 as a substrate for neddylation by UBC12 and explored its impact on tumor aggressiveness. In cell-based assays, SREBP-1 neddylation prolonged SREBP-1 stability with a decrease in ubiquitination. Consequently, NEDD8 overexpression facilitated proliferation, migration, and invasion of SK-Hep1 liver tumor cells. MLN4924 (an inhibitor of the NEDD8-activating enzyme-E1) treatment or UBC12 knockdown prevented SREBP-1 neddylation and tumor cell phenotype change. This effect was corroborated in an in vivo xenograft model. In human specimens, SREBP-1, UBC12, and NEDD8 were all upregulated in hepatocellular carcinoma (HCC) compared to nontumorous regions. Moreover, SREBP-1 levels positively correlated with UBC12. In GEO database analyses, SREBP-1 levels were greater in metastatic HCC samples accompanying UBC12 upregulation. In HCC analysis, tumoral SREBP-1 and UBC12 levels discriminated overall patient survival rates. Additionally, MLN4924 treatment destabilized SREBP-1 in MDA-MB-231 breast cancer cells and in the tumor cell xenograft. SREBP-1 and UBC12 were also highly expressed in human breast cancer tissues. Moreover, most breast cancers with lymph node metastasis displayed predominant SREBP-1 and UBC12 expressions, which compromised overall patient survival rates. In summary, SREBP-1 is neddylated by UBC12, which may contribute to HCC and breast cancer aggressiveness through SREBP-1 stabilization, and these events can be intervented by MLN4924 therapy. Our findings may also provide potential reliable prognostic markers for tumor metastasis., (© 2020 UICC.)

Neddylation is a process by which NEDD8 is covalently conjugated to target proteins by sequential enzymatic reaction. Its role in cancer cell migration has only been recently acknowledged. Previously in cancer cell migration, the epithelial to mesenchymal transition (EMT) process has been well-known to play an important role in both invasion and metastasis by promoting mesenchymal phenotype in epithelial cells. However, the role of neddylation in the EMT process and its mechanistic details are yet to be elucidated. We recently reported that neddylation plays a crucial role in cancer cell migration through the PI3K-Akt pathway. Here, we report that inhibiting neddylation activates the hypoxia-inducible factor 1α (HIF-1α) through the PI3K-Akt pathway, which eventually regulates the EMT-activator ZEB1 (zinc finger E-box binding homeobox 1) in various cancer cell lines. As induction of HIF-1α is known to deteriorate the state of cancer and EMT process is one of the hallmarks of metastasis in cancer, our findings uncover the role of neddylation between HIF-1α and ZEB1.

Wnt signaling plays a critical role in production and differentiation of neurons and undergoes a progressive reduction during cortical development. However, how Wnt signaling is regulated is not well understood. Here we provide evidence for an indispensable role of neddylation, a ubiquitylation-like protein modification, in inhibiting Wnt/β-catenin signaling. We show that β-catenin is neddylated; and inhibiting β-catenin neddylation increases its nuclear accumulation and Wnt/β-catenin signaling. To test this hypothesis in vivo, we mutated Nae1, an obligative subunit of the E1 for neddylation in cortical progenitors. The mutation leads to eventual reduction in radial glia progenitors (RGPs). Consequently, the production of intermediate progenitors (IPs) and neurons is reduced, and neuron migration is impaired, resulting in disorganization of the cerebral cortex. These phenotypes are similar to those of β-catenin gain-of-function mice. Finally, suppressing β-catenin expression is able to rescue deficits of Nae1 mutant mice. Together, these observations identified a mechanism to regulate Wnt/β-catenin signaling in cortical development., Competing Interests: The authors declare no competing interest.

Neddylation plays a distinct role in stabilization of a subset of ribosomal proteins. Whether the family of ribosomal proteins S27 (RPS27 and RPS27-like) is subjected to neddylation regulation with associated biological consequence is totally unknown. Here, we report that both family members are subjected to neddylation by MDM2 E3 ubiquitin ligase, and deneddylation by NEDP1. Blockage of neddylation with MLN4924, a small molecule inhibitor of neddylation-activating enzyme, destabilizes RPS27L and RPS27 by shortening their protein half-lives. Biologically, knockdown of RPS27L and RPS27 sensitizes, whereas ectopic expression of RPS27L and RPS27 desensitizes cancer cells to MLN4924-induced apoptosis. Taken together, our study demonstrates that neddylation stabilizes RPS27L and RPS27 to confer the survival of cancer cells., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Nedd8 is a ubiquitin-like protein that covalently conjugates to target proteins through neddylation. In addition to cullin-RING ligases, neddylation also modifies non-cullin proteins to regulate protein activity, stability and localization. However, the roles of NEDD8 remain largely unknown in vivo Here, we found that loss of nedd8 in female zebrafish led to defects in oogenesis, disrupted oocyte maturation and stimulated growth of the breeding tubercles (BTs) on the pectoral fins. The BTs are normally present in males, not females. However, the loss of one copy of ar can partially rescue the phenotypes displayed by nedd8 -null female zebrafish. Further assays indicated that Nedd8 conjugates to Ar and Ar is neddylated at lysine 475 and lysine 862. Moreover, Nedd8 conjugation efficiently suppressed Ar transcriptional activity. Lysine 862 (K862) of Ar is the key site modified by neddylation to modulate Ar transcriptional activity. Thus, our results not only demonstrated that Nedd8 modulates ovarian maturation and the maintenance of female secondary sexual characteristics of female zebrafish in vivo , but also indicated that androgen signaling is strictly regulated by nedd8 ., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Neddylation is an ubiquitin-like modification of proteins that affects the activity, stability and protein-protein interaction of its substrates. Apart from its role as a promoter for Cullin ring E3 ligase to positively regulate the ubiquitylation process, other functional studies about neddylation are still lacking. In this study, we developed a system to explore the impact of neddylation on changes in the subcellular localization of proteins at the omics level. By applying a method combining subcellular protein extraction and immunoprecipitation-mass spectrometry (IP-MS), 81 proteins with a tendency to shuttle between the cytoplasm and nucleus due to different neddylation levels were obtained. Among the 81 candidates, transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) and growth arrest and DNA damage protein 45a (Gadd45a) were confirmed as novel substrates of Nedd8, and neddylation promotes TAK1 nuclear import as well as Gadd45a nuclear export., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Protein neddylation, a process of conjugating neural precursor cell expressed, developmentally downregulated 8 (NEDD8) to substrates, plays a tumor-promoting role in lung carcinogenesis. Our previous study showed MLN4924, an inhibitor of NEDD8 activating enzyme (E1), significantly inhibits the growth of multiple cancer cells. However, resistance can develop to MLN4924 by mutation. Therefore, it is important to further understand how NEDD8 acts in lung cancer. In the present study, we demonstrated NEDD8 is overactivated in lung cancers and confers a worse patient overall survival. Furthermore, we report that in lung adenocarcinoma cells, NEDD8 depletion significantly suppressed lung cancer cell growth and progression both in vitro and in vivo. Mechanistic studies revealed that NEDD8 depletion induced the accumulation of a panel of tumor-suppressive cullin-RING ubiquitin ligase substrates (e.g., p21, p27, and Wee1) via blocking their degradation, triggering cell cycle arrest at G 2 phase, thus inducing apoptosis or senescence in a cell-line-dependent manner. The present study demonstrates the role of NEDD8 in regulating the malignant phenotypes of lung cancer cells and further validates NEDD8 as a potential therapeutic target in lung cancer.

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.)

To understand gene function, the encoding DNA or mRNA transcript can be manipulated and the consequences observed. However, these approaches do not have a direct effect on the protein product of the gene, which is either permanently abrogated or depleted at a rate defined by the half-life of the protein. We therefore developed a single-component system that could induce the rapid degradation of the specific endogenous protein itself. A construct combining the RING domain of ubiquitin E3 ligase RNF4 with a protein-specific camelid nanobody mediates target destruction by the ubiquitin proteasome system, a process we describe as antibody RING-mediated destruction (ARMeD). The technique is highly specific because we observed no off-target protein destruction. Furthermore, bacterially produced nanobody-RING fusion proteins electroporated into cells induce degradation of target within minutes. With increasing availability of protein-specific nanobodies, this method will allow rapid and specific degradation of a wide range of endogenous proteins., Competing Interests: Declaration of Interests The authors declare no competing financial interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the core component of DNA-PK complex in the non-homologous end-joining (NHEJ) repair of DNA double-strand breaks, and its activity is strictly controlled by DNA-PKcs phosphorylation. The ubiquitin-like protein, NEDD8 is involved in regulation of DNA damage response, but it remains mysterious whether and how NEDD8-related neddylation affects DNA-PKcs and the NHEJ process. Here, we show that DNA-PKcs is poly-neddylated at its kinase domain. The neddylation E2-conjugating enzyme UBE2M and E3 ligase HUWE1 (HECT, UBA, and WWE domain containing E3 ubiquitin protein ligase 1) are responsible for the DNA-PKcs neddylation. Moreover, inhibition of HUWE1-dependent DNA-PKcs neddylation impairs DNA-PKcs autophosphorylation at Ser2056. Finally, depletion of HUWE1-dependent DNA-PKcs neddylation reduces the efficiency of NHEJ. These studies provide insights how neddylation modulates the activity of NHEJ core complex.

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 study explores the role of neddylation in early trophoblast development and its alteration during the pathogenesis of recurrent spontaneous abortion (RSA). Immunofluorescence and western blot were conducted to evaluate the expression pattern of NEDD8 protein in the first-trimester placentas of healthy control and RSA patients. Neddylated-cullins, especially neddylated-cullin1, were downregulated and their substrate, p21, was accumulated in RSA samples. NEDD8 cytoplasmic recruitment was observed in extravillous trophoblast (EVT) progenitors of RSA placentas. Consistent with the results of clinical samples, neddylation inhibition using MLN4924 in trophoblast cell lines caused obvious p21 accumulation and free NEDD8 cytoplasmic recruitment. Further in vitro study demonstrated neddylation inhibition attenuated proliferation of Jeg-3 cells via p21 accumulation. Moreover, when trophoblast stem (TS) cells derived from first-trimester placentas were cultured for differentiation analyses. MLN4924 impaired the differentiation of TS cells towards EVTs by downregulating HLA-G and GATA3. p21 knockdown could partly rescue MLN4924-suppressed HLA-G and GATA3 expression. In conclusion, cullin1 neddylation-mediated p21 degradation is required for trophoblast proliferation and can affect trophoblast plasticity by affecting HLA-G and GATA3 expression. The results provide insights into the pathological mechanism of RSA and the biological regulation of trophoblast development., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)

Retinoblastoma protein (Rb) is a tumor suppressor that binds and represses E2F transcription factors. In cervical cancer cells, human papilloma virus (HPV) protein E7 binds to Rb, releasing it from E2F to promote cell cycle progression, and inducing ubiquitination of Rb. E7-mediated proteasomal degradation of Rb requires action by another protease, calpain, which cleaves Rb after Lys 810. However, it is not clear why cleavage is required for Rb degradation. Here, we report that the proteasome cannot initiate degradation efficiently on full-length Rb. Calpain cleavage exposes a region that is recognized by the proteasome, leading to rapid proteolysis of Rb. These findings identify a mechanism for regulating protein stability by controlling initiation and provide a better understanding of the molecular mechanism underlying transformation by HPV.

Nonalcoholic fatty liver disease (NAFLD) is a risk factor for progression of steatohepatitis, liver cirrhosis, and liver cancer. Although pathological condition of NAFLD, which arises from an excessive accumulation of triglyceride in the liver, is accompanied by elevated sterol regulatory element-binding protein 1c (SREBP1c) level, it is largely unknown which factors are involved in the modification of SREBP1c. In this study, we discovered that neddylation of SREBP1c competes with its ubiquitination and stabilizes SREBP1c protein level, and eventually promotes hepatic steatosis. We also demonstrated that human homolog of mouse double minute 2 (HDM2) acts as an E3 neddylation ligase of SREBP1c. Further, treatment with the neddylation inhibitor, MLN4924, attenuates high-fat diet-induced hepatic steatosis by reducing the levels of SREBP1c protein and hepatic triglyceride. Our results indicate that the blockade of SREBP1c neddylation could be a novel approach in the defense against NAFLD.

NEDDylation pathway regulates multiple physiological process, unlike inhibitors, NEDDylation activators are rarely studied. Novel amide derivatives were synthesized and evaluated for antiproliferative activity against MGC803, MCF-7 and PC-3 cells. Among them, Ⅶ-31 displayed the most potent activity with an IC 50 value of 94 nmol/L against MGC803 cells. Cellular mechanisms elucidated that Ⅶ-31 inhibited the cell viability, arrested cell cycle at G2/M phase and induced apoptosis via intrinsic and extrinsic pathways against MGC803 cells. In addition, Ⅶ-31 activated NAE1-Ubc12-Cullin1 NEDDylation via interacting with NAE1 directly. Furthermore, the activation of NEDDylation resulted in the degradation of inhibitor of apoptosis proteins (IAPs). Importantly, Ⅶ-31 inhibited tumor growth in xenograft models in vivo without the apparent toxicity. In summary, it is the first time to reveal that Ⅶ-31 deserves consideration for cancer therapy as a NEDDylation activator., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest to this work., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Post-translational modifications by ubiquitin and ubiquitin-like proteins (Ubls) have known roles in a myriad of cellular processes. Ubiquitin- and Ubl-binding domains transmit the information conferred by these post-translational modifications by recognizing functional surfaces and, when present, different chain structures. Numerous domains binding to ubiquitin have been characterized and their structures solved. Analogously, motifs selectively interacting with SUMO (small ubiquitin-like modifier) have been identified in several proteins and their role in SUMO-dependent processes investigated. On the other hand, proteins that specifically recognize other Ubl modifications are known only in a few cases. The high sequence identity between NEDD8 and ubiquitin has made the identification of specific NEDD8-binding domains further complicated due to the promiscuity in the recognition by several ubiquitin-binding domains. Two evolutionarily related domains, called CUBAN (cullin-binding domain associating with NEDD8) and CoCUN (cousin of CUBAN), have been recently described. The CUBAN binds monomeric NEDD8 and neddylated cullins, but it also interacts with di-ubiquitin chains. Conversely, the CoCUN domain only binds ubiquitin. CUBAN and CoCUN provide an intriguing example of how nature solved the issue of promiscuity versus selectivity in the recognition of these two highly related molecules. The structural information available to date suggests that the ancestor of CUBAN and CoCUN was a three-helix bundle domain that diversified in KHNYN (KH and NYN domain-containing) and N4BP1 (NEDD4-binding protein-1) by acquiring different features. Indeed, these domains diverged towards two recognition modes, that recall respectively the electrostatic interaction utilized by the E3-ligase RBX1/2 in the interaction with NEDD8, and the hydrophobic features described in the recognition of ubiquitin by CUE (coupling ubiquitin conjugation to ER degradation) domains. Intriguingly, CUBAN and CoCUN domains are only found in KHNYN and N4BP1, respectively, both proteins belonging to the PRORP family whose members are characterized by the combination of protein modules involved in RNA metabolism with domains mediating ubiquitin/NEDD8 recognition. This review recapitulates the current knowledge and recent findings of CUBAN and CoCUN domains and the proteins containing them., Competing Interests: The author declares no conflict of interest.

The maintenance of muscle function is extremely important for whole body health and exercise is essential to this process. The ubiquitin-proteasome system (UPS) is required for muscle adaptation following exercise but little is known about acute posttranslational regulation and proteome remodeling during and after high-intensity exercise. Here, we used quantitative proteomics to study ubiquitin signaling dynamics in human skeletal muscle biopsies from healthy males before, during, and after a single bout of high-intensity exercise. Exercise resulted in a marked depletion of protein ubiquitylation in the vastus lateralis muscle consistent with proteasome activation. This was also associated with acute posttranslational modification of protein abundance. Integration of these data with phosphoproteomics identified co-regulated proximal modifications suggesting a cross talk between phosphorylation and ubiquitylation. We also identified additional protein modification cross talk and showed acute activation of protein NEDDylation. In vitro experiments revealed that cAMP-dependent activation of the proteasome requires NEDD8, an ubiquitin-like protein involved in protein NEDDylation, to maintain cellular protein ubiquitylation levels. Our data reveal the complexity of ubiquitin signaling and proteome remodeling in muscle during and after high-intensity exercise. We propose a model whereby exercise and the resulting cAMP signaling activate both the proteasome and ubiquitylation via NEDDylation to rapidly remove potentially damaged proteins., (© 2020 The University of Sydney. The FASEB Journal © 2020 Federation of American Societies for Experimental Biology.)

Deficient DNA mismatch repair (dMMR) induces a hypermutator phenotype that can lead to tumorigenesis; however, the functional impact of the high mutation burden resulting from this phenotype remains poorly explored. Here, we demonstrate that dMMR-induced destabilizing mutations lead to proteome instability in dMMR tumors, resulting in an abundance of misfolded protein aggregates. To compensate, dMMR cells utilize a Nedd8-mediated degradation pathway to facilitate clearance of misfolded proteins. Blockade of this Nedd8 clearance pathway with MLN4924 causes accumulation of misfolded protein aggregates, ultimately inducing immunogenic cell death in dMMR cancer cells. To leverage this immunogenic cell death, we combined MLN4924 treatment with PD1 inhibition and found the combination was synergistic, significantly improving efficacy over either treatment alone., Competing Interests: Declaration of Interests G.B.M. consults with AstraZeneca, ImmunoMET, Ionis, Nuevolution, PDX bio, Signalchem, Symphogen, and Tarveda, has stock options with Catena Pharmaceuticals, ImmunoMet, SignalChem, Spindle Top Ventures, and Tarveda, sponsored research from AstraZeneca, Immunomet, Pfizer, Nanostring, and Tesaro, travel support from Chrysallis Bio, and has licensed technology to Nanostring and Myriad Genetics. The other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Co-opting Cullin4 RING ubiquitin ligases (CRL4s) to inducibly degrade pathogenic proteins is emerging as a promising therapeutic strategy. Despite intense efforts to rationally design degrader molecules that co-opt CRL4s, much about the organization and regulation of these ligases remains elusive. Here, we establish protein interaction kinetics and estimation of stoichiometries (PIKES) analysis, a systematic proteomic profiling platform that integrates cellular engineering, affinity purification, chemical stabilization, and quantitative mass spectrometry to investigate the dynamics of interchangeable multiprotein complexes. Using PIKES, we show that ligase assemblies of Cullin4 with individual substrate receptors differ in abundance by up to 200-fold and that Cand1/2 act as substrate receptor exchange factors. Furthermore, degrader molecules can induce the assembly of their cognate CRL4, and higher expression of the associated substrate receptor enhances degrader potency. Beyond the CRL4 network, we show how PIKES can reveal systems level biochemistry for cellular protein networks important to drug development., Competing Interests: Declaration of Interests K.M.R. is a Ph.D. candidate at Caltech and an employee of Genentech, Inc., through the Visiting Scientist Program; E.V., C.M.R., W.d.B., I.E.W., and D.S.K. are employees and shareholders of Genentech, Inc.,; J.M.R. is an employee and shareholder of Abbvie Inc.; R.S. is an employee and shareholder of Brystol-Myers Squibb Inc.; R.J.D. is an employee and shareholder of Amgen Inc., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Neddylation is a ubiquitination-like pathway that controls cell survival and proliferation by covalently conjugating NEDD8 to lysines in specific substrate proteins. However, the physiological role of neddylation in mammalian metabolism remains elusive, and no mitochondrial targets have been identified. Here, we report that mouse models with liver-specific deficiency of NEDD8 or ubiquitin-like modifier activating enzyme 3 (UBA3), the catalytic subunit of the NEDD8-activating enzyme, exhibit neonatal death with spontaneous fatty liver as well as hepatic cellular senescence. In particular, liver-specific UBA3 deficiency leads to systemic abnormalities similar to glutaric aciduria type II (GA-II), a rare autosomal recessive inherited fatty acid oxidation disorder resulting from defects in mitochondrial electron transfer flavoproteins (ETFs: ETFA and ETFB) or the corresponding ubiquinone oxidoreductase. Neddylation inhibition by various strategies results in decreased protein levels of ETFs in neonatal livers and embryonic hepatocytes. Hepatic neddylation also enhances ETF expression in adult mice and prevents fasting-induced steatosis and mortality. Interestingly, neddylation is active in hepatic mitochondria. ETFs are neddylation substrates, and neddylation stabilizes ETFs by inhibiting their ubiquitination and degradation. Moreover, certain mutations of ETFs found in GA-II patients hinder the neddylation of these substrates. Taken together, our results reveal substrates for neddylation and add insight into GA-II., Competing Interests: The authors declare no competing interest.

Eukaryotic cell biology depends on cullin-RING E3 ligase (CRL)-catalysed protein ubiquitylation 1 , which is tightly controlled by the modification of cullin with the ubiquitin-like protein NEDD8 2-6 . However, how CRLs catalyse ubiquitylation, and the basis of NEDD8 activation, remain unknown. Here we report the cryo-electron microscopy structure of a chemically trapped complex that represents the ubiquitylation intermediate, in which the neddylated CRL1 β-TRCP promotes the transfer of ubiquitin from the E2 ubiquitin-conjugating enzyme UBE2D to its recruited substrate, phosphorylated IκBα. NEDD8 acts as a nexus that binds disparate cullin elements and the RING-activated ubiquitin-linked UBE2D. Local structural remodelling of NEDD8 and large-scale movements of CRL domains converge to juxtapose the substrate and the ubiquitylation active site. These findings explain how a distinctive ubiquitin-like protein alters the functions of its targets, and show how numerous NEDD8-dependent interprotein interactions and conformational changes synergistically configure a catalytic CRL architecture that is both robust, to enable rapid ubiquitylation of the substrate, and fragile, to enable the subsequent functions of cullin-RING proteins.

Neddylation is the post-translational protein modification most closely related to ubiquitination. Whereas the ubiquitin-like protein NEDD8 is well studied for its role in activating cullin-RING E3 ubiquitin ligases, little is known about other substrates. We developed serial NEDD8-ubiquitin substrate profiling (sNUSP), a method that employs NEDD8 R74K knock-in HEK293 cells, allowing discrimination of endogenous NEDD8- and ubiquitin-modification sites by MS after Lys-C digestion and K-εGG-peptide enrichment. Using sNUSP, we identified 607 neddylation sites dynamically regulated by the neddylation inhibitor MLN4924 and the de-neddylating enzyme NEDP1, implying that many non-cullin proteins are neddylated. Among the candidates, we characterized lysine 112 of the actin regulator cofilin as a novel neddylation event. Global inhibition of neddylation in developing neurons leads to cytoskeletal defects, altered actin dynamics and neurite growth impairments, whereas site-specific neddylation of cofilin at K112 regulates neurite outgrowth, suggesting that cofilin neddylation contributes to the regulation of neuronal actin organization.

Gartanin, a 4-prenylated xanthone, has been identified from the purple mangosteen fruit as a potent growth inhibitor of various cancer cell lines, including prostate cancer. However, much of Gartanin's anticancer mechanism remains unknown. We have discovered that Gartanin docked onto the regulatory subunit of the precursor cell-expressed developmentally downregulated 8 (NEDD8)-activating enzyme (NAE) complex and next to the NEDD8 binding complex, which leads to inhibit NEDD8 conjugations to both Cullin1 and Ubc12 in prostate cancer cell lines and Ubc12 NEDDylation in an in vitro assay. The S phase kinase-associated protein (Skp2) and F-box and WD-repeat domain-containing 2 (FBXW2), the NEDD8 family members of E3 ubiqutin ligases, were also downregulated and upregulated by Gartainin, respectively. Knock-down of NEDD8 expression by short harpin (Sh) RNAs blocked or attenuated these effects of Gartainin. Finally, Gartanin demonstrated its ability to inhibit growth of prostate cancer lines via autophagy initiation. Our data support that Gartanin is a naturally occurring NEDDylation inhibitor and deserves further investigation for prostate cancer prevention and treatment., (© 2019 Wiley Periodicals, Inc.)

Neddylation modification is often over-expressed in a variety of human tumor cells. Therefore, targeting neddylation pathway may represent a potential approach to the treatment of human tumors. Herein, we describe the discovery of a hit scaffold from our in-house library and further structure-based optimizations. In this work, compound V11 could block the neddylation and inhibit the activity of NAE (with an EC 50 value of 3.56 µM), and a dose-dependent reduction of the Ubc12-NEDD8 conjugations was also observed. Molecular docking results suggest compound V11 could bind tightly to NAE via hydrogen bonds and hydrophobic interactions. Compound V11 showed the best antiproliferative ability with an IC 50 value of 8.22 μM against gastric cancer MGC-803 cells. Further anticancer activity studies suggested that compound V11 inhibited MGC-803 cell growth, caused a cell cycle arrestment at G2/M phase and induced apoptosis via extrinsic and intrinsic apoptosis pathways. All the findings suggest that 1,2,4-triazine scaffold might provide a novel scaffold for the further development of neddylation inhibitors and compound V11 might be a potential neddylation inhibitor with anticancer activity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Neddylation is a posttranslational modification that conjugates a ubiquitin-like protein NEDD8 to substrate proteins. The best-characterized substrates of neddylation are the cullin subunits of cullin-RING E3 ubiquitin ligase complexes (CRLs). CRLs as the largest family of E3 ubiquitin ligases control many important biological processes, including tumorigenesis, through promoting ubiquitylation and subsequent degradation of a variety of key regulatory proteins. The process of protein neddylation is overactivated in multiple types of human cancers, providing a sound rationale as an attractive anticancer therapeutic strategy, evidenced by the development of the NEDD8-activating enzyme (NAE) inhibitor MLN4924 (also known as pevonedistat). Recently, increasing evidence strongly indicates that neddylation inhibition by MLN4924 exerts anticancer effects mainly by triggering cell apoptosis, senescence, and autophagy and causing angiogenesis suppression, inflammatory responses, and chemo-/radiosensitization in a context-dependent manner. Here, we briefly summarize the latest progresses in this field, focusing on the preclinical studies to validate neddylation modification as a promising anticancer target.

Background: NEDD8 activating enzyme (NAE) plays a critical role in various cellular functions in carcinomas. The selective inhibition of NAE could mediate the rate of ubiquitination and the subsequent degradation of proteins associated with cancer so as to achieve the purpose of treatment., Objective: In this article, we decided to study the synthesis and screening of 4-amino substituted 2H-chromen-2-one derivatives against cancer cell lines, specifically the human pancreatic cancer cell line BxPC-3., Methods: After synthesis of twenty targeted compounds, we evaluated their anti-proliferative activity against six cancer cell lines, cytotoxicity against three normal cell lines through MTT assay, and hemolysis to screen out the candidate compound, which was further conducted drug-like physical property measurement, target confirmation by enzyme-based experiment, cell apoptosis, and synergistic effect research., Results: Starting from intermediates 4 and 5, several new 4-amino substituted 2H-chromen-2-one derivatives (9-28) were synthesized and evaluated for their cell activities using six cancer cell lines. We performed tests of cytotoxicity, hemolysis, ATP-dependent NAE inhibition in the enzyme- based system, apoptosis, and synergistic effect in BxPC-3 cells against the best candidate compound 21., Conclusion: Based on these results, we found that compound 21 inhibited NAE activity in an ATP-dependent manner in the enzyme-based system, induced apoptosis in BxPC-3 cells, and synergized with bortezomib on BxPC-3 cell growth inhibition. Additionally, it had low toxicity with reasonable Log P-value and water solubility., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

MLN4924, also known as pevonedistat, is a highly selective small-molecule inhibitor of NEDD8 (neuronal precursor cell-expressed developmentally downregulated protein 8)-activating enzyme (NAE) to block the entire neddylation modification cascade, leading to inactivation of cullin-RING ligases (CRLs), since activation of CRLs requires cullin neddylation. MLN4924 showed impressive anticancer activity in many preclinical studies and is currently in several Phase I/II clinical trials for anticancer therapy as a single agent or in combination with chemotherapeutic drugs.In addition to well-characterized anti-neddylation activity, recent studies showed that MLN4924 has several neddylation-independent activities. First, MLN4924 triggers EGFR dimerization to activate EGFR and its downstream RAS/MAPK and PI3K/AKT1 signals, leading to enhanced tumor sphere formation, accelerated EGF-mediated wound healing, and inhibited ciliogenesis. Second, MLN4924 induces PKM2 tetramerization to promote glycolysis, thus affecting energy metabolism. Third, MLN4924 inhibits the interaction between ACT1 (NF-κB activator 1) and TRAF6 (tumor necrosis factor receptor-associated factor 6) and attenuates IL-17A-mediated activation of NF-κB to reduce pulmonary inflammation. Fourth, MLN4924 inhibits IRF3 binding to the IFN-β promoter to inhibit IFN-β production. And finally, MLN4924 activates the JNK signaling pathway to reduce c-FLIP levels, thus enhancing TRAIL-induced apoptosis. This chapter will summarize these neddylation-independent activities of MLN4924 and discuss the underlying mechanisms and potential therapeutic applications.

Background: Amyloid Precursor Protein (APP)-Binding Protein 1 (APP-BP1) is a crucial regulator of many key signaling pathways and functions mainly as a scaffold protein to enhance molecular interactions and facilitate catalytic reactions. The interaction of APP-BP1 with Amyloid Precursor Protein (APP) plays a role in cell cycle transit control, which determines the mechanism behind the loss of cell cycle regulation in Alzheimer's Disease (AD). In contrast, neddylation, a posttranslational modification mediated by conjugation of ubiquitin-like protein neural precursor cell expressed developmentally downregulated protein 8 (NEDD8), is activated by a heterodimer composed of APP-BP1 and NEDD8-activating enzyme E1 catalytic subunit (Uba3). NEDD8 controls vital biological events, and along with APP-BP1, its levels are deregulated in AD., Objective: The present study investigated the role of melatonin in regulating the APP-BP1 pathway under both physiological and pathological conditions to develop an understanding of the underlying mechanisms., Methods: Therefore, human SH-SY5Y neuroblastoma cells were treated with various concentrations of Aβ42 to induce neurotoxic conditions comparable to AD., Results: The results are the first to demonstrate that melatonin prevents Aβ42-induced enhancement of APP-BP1 protein expression and alteration in the cellular localization of NEDD8. Moreover, using MLN4924 (APP-BP1 pathway blocker), we also verified the components of the downstream effector cascade of the APP-BP1 pathway, including tau, APP-cleaving secretases, β-catenin and p53., Conclusion: These findings indicate that melatonin regulates the interplay of molecular signaling associated with the APP-BP1 pathway and might preclude the pathogenic mechanisms occurring during disease development, thus providing a propitious therapeutic strategy for preventing AD., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Protein neddylation is one type of posttranslational modifications that regulates the activity of the substrate proteins. Neddylation modification is catalyzed by NEDD8-activating enzyme (NAE, E1), NEDD8-conjugating enzyme (E2), and NEDD8 ligase (E3) to attach NEDD8, an ubiquitin-like molecule, to a lysine residue of a substrate protein. The best known neddylation substrates are cullin family members, which are scaffold components of cullin-RING ligases (CRLs), and cullin neddylation is required for activation of CRLs. In mammalian cells, there are one E1, two E2s (UBC12/UBE2M and UBE2F), and over a dozen E3s. MLN4924, the first-in-class small-molecule inhibitor of NAE, blocks the entire neddylation modification to inactivate activity of all CRLs. MLN4924 is currently in the Phase I/II clinical trials for anticancer application.In the last few years, targeting protein-protein interactions of the neddylation complexes has been pursued as a potential strategy to selectively inhibit the activity of individual CRL. Analysis of the co-crystal structures of DCN1, a co-E3 for neddylation, and its binding partners UBC12 (a neddylation E2) suggested that it may be amenable for the design of potent, small-molecule inhibitors. In this chapter, we will review the discovery of small-molecule inhibitors that block the interactions of DCN1 with UBC12 (hereafter called DCN1 inhibitors) from a number of laboratories, including ours, leading to selective inactivation of CRL-1 and/or CRL-3. We will also discuss potential therapeutic applications of these small-molecule inhibitors.

Cullin-RING ubiquitin ligases (CRLs) represent the largest superfamily of multi-subunit E3s conserved in all eukaryotes. Soon after the discovery of these important ubiquitin ligase machineries, structural studies have made tremendous contributions to our understanding of their functions. Identification of the key components of CRLs by early studies raised immediate questions as to how these multi-subunit complexes assemble to promote the polyubiquitination of substrates. Specifically, how do the CRL subunits interact with each other to form a versatile E3 platform? How do they recognize specific substrates? How are the CRL-substrate interactions regulated in response to upstream signals? How are the CRL E3s themselves activated and deactivated, and how are substrate receptor subunits of CRLs exchanged in the cell? Even though we might not yet have complete answers to these questions, extensive structural analyses of CRL complexes in the past two decades have begun to unveil the themes and variations of CRL biology. In this chapter we will discuss both classic and emerging structures that help elucidate the overall architecture of CRLs, their substrate recognition modes, and regulatory mechanism of CRLs by NEDD8 modification.

Background: Neuroinflammation, typified by elevated levels of interleukin-1 (IL-1) α and β, and deficits in proteostasis, characterized by accumulation of polyubiquitinated proteins and other aggregates, are associated with neurodegenerative disease independently and through interactions of the two phenomena. We investigated the influence of IL-1β on ubiquitination via its impact on activation of the E3 ligase parkin by either phosphorylated ubiquitin (P-Ub) or NEDD8., Methods: Immunohistochemistry and Proximity Ligation Assay were used to assess colocalization of parkin with P-tau or NEDD8 in hippocampus from Alzheimer patients (AD) and controls. IL-1β effects on PINK1, P-Ub, parkin, P-parkin, and GSK3β-as well as phosphorylation of parkin by GSK3β-were assessed in cell cultures by western immunoblot, using two inhibitors and siRNA knockdown to suppress GSK3β. Computer modeling characterized the binding and the effects of P-Ub and NEDD8 on parkin. IL-1α, IL-1β, and parkin gene expression was assessed by RT-PCR in brains of 2- and 17-month-old PD-APP mice and wild-type littermates., Results: IL-1α, IL-1β, and parkin mRNA levels were higher in PD-APP mice compared with wild-type littermates, and IL-1α-laden glia surrounded parkin- and P-tau-laden neurons in human AD. Such neurons showed a nuclear-to-cytoplasmic translocation of NEDD8 that was mimicked in IL-1β-treated primary neuronal cultures. These cultures also showed higher parkin levels and GSK3β-induced parkin phosphorylation; PINK1 levels were suppressed. In silico simulation predicted that binding of either P-Ub or NEDD8 at a singular position on parkin opens the UBL domain, exposing Ser 65 for parkin activation., Conclusions: The promotion of parkin- and NEDD8-mediated ubiquitination by IL-1β is consistent with an acute neuroprotective role. However, accumulations of P-tau and P-Ub and other elements of proteostasis, such as translocated NEDD8, in AD and in response to IL-1β suggest either over-stimulation or a proteostatic failure that may result from chronic IL-1β elevation, easily envisioned considering its early induction in Down's syndrome and mild cognitive impairment. The findings further link autophagy and neuroinflammation, two important aspects of AD pathogenesis, which have previously been only loosely related.

Post-translational modifications, like phosphorylation, ubiquitylation, and sumoylation, have been shown to impact on synaptic neurotransmission by modifying pre- and postsynaptic proteins and therefore alter protein stability, localization, or protein-protein interactions. Previous studies showed that post-translational modifications are essential during the induction of synaptic plasticity, defined by a major reorganization of synaptic proteins. We demonstrated before that neddylation, a post-translational modification that covalently binds Nedd8 to lysine-residues, strongly affects neuronal maturation and spine stability. We now analysed the consequences of inhibiting neddylation on excitatory synaptic transmission and plasticity, which will help to narrow down possible targets, to make educated guesses, and test specific candidates. Here, we show that acute inhibition of neddylation impacts on synaptic neurotransmission before morphological changes occur. Our data indicate that pre- and postsynaptic proteins are neddylated since the inhibition of neddylation impacts on presynaptic release probability and postsynaptic receptor stabilization. In addition, blocking neddylation during the induction of long-term potentiation and long-term inhibition abolished both forms of synaptic plasticity. Therefore, this study shows the importance of identifying synaptic targets of the neddylation pathway to understand the regulation of synaptic transmission and plasticity.

Macroautophagy/autophagy is a cellular process in which cytosolic contents are degraded by lysosome in response to various stress conditions. Apart from its role in the maintenance of cellular homeostasis, autophagy also involves in regulation of cell cycle progression under nutrient-deprivation conditions. However, whether and how autophagy is regulated by the cell cycle especially during mitosis remains largely undefined. Here we show that WIPI2/ATG18B (WD repeat domain, phosphoinositide interacting 2), an autophagy-related (ATG) protein that plays a critical role in autophagosome biogenesis, is a direct substrate of CUL4-RING ubiquitin ligases (CRL4s). Upon mitosis induction, CRL4s are activated via neddylation, and recruit WIPI2 via DDB1 (damage specific DNA binding protein 1), leading to polyubiquitination and proteasomal degradation of WIPI2 and suppression of autophagy. The WIPI2 protein level and autophagy during mitosis could be rescued by knockdown of CRL4s or treatment with MLN4924/Pevonedistat, a selective inhibitor of CRLs, via suppression of NAE1 (NEDD8 activating enzyme E1 subunit 1). Moreover, restoration of WIPI2 rescues autophagy during mitosis and leads to mitotic slippage and cell senescence. Our study thus discovers a novel function of CRL4s in autophagy by targeting WIPI2 for polyubiquitination and proteasomal degradation during mitosis. Abbreviations : ACTB, actin beta; ATG, autophagy-related; AMPK, AMP-activated protein kinase; AURKB/ARK2, aurora kinase B; BafA1, bafilomycin A 1 ; CCNB1, cyclin B1; CDK1, cyclin dependent kinase 1; CHX, cycloheximide; CQ, chloroquine; CRL4s, CUL4-RING ubiquitin ligases; DDB1, damage specific DNA binding protein 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GST, glutathione S-transferase; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; STK11/LKB1,serine/threonine kinase 11; MTORC1/MTOR complex 1, mechanistic target of rapamycin kinase complex 1; NAE1, NEDD8 activating enzyme E1 subunit 1; NOC, nocodazole; RING, really interesting new gene; RBX1, ring-box 1; SA-GLB1/β-gal, senescence-associated galactosidase beta 1; TSC2, TSC complex subunit 2; TUBA, tubulin alpha; WIPI2, WD repeat domain, phosphoinositide interacting 2.

Posttranslational modifications by phosphorylation, ubiquitination, neddylation and other pathways have emerged as major regulators of cellular functions. NEDD8 ultimate buster 1, NUB1, is an adaptor protein, which negatively regulates the levels of the ubiquitin-like protein NEDD8 as well as neddylated proteins through proteasomal degradation. We previously reported that NUB1 is highly involved in the pathogenesis of synucleinopathy including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). In general, since phosphorylation is strongly related to the alteration of protein propensity, we examined if the fundamental function of NUB1 can be modulated by its phosphorylation. We created a series of phosphomimic mutants of NUB1. Among them, we found that phosphorylation of NUB1 at S46 (P-NUB46) efficiently degrades aggregates using a cell-based assay. Immunohistochemical studies have shown that specific antibodies against P-NUB46 reacted with Lewy bodies in PD and DLB but not with glial cytoplasmic inclusions in MSA. Moreover, P-NUB46 levels were significantly higher in the brains of patients with DLB than in control brains, and P-NUB46 was extracted in an insoluble fraction of DLB. These findings suggest that the phosphorylation of NUB1 is modulated during the pathological process of Lewy body disease., (© 2019 International Society of Neuropathology.)

Plasmodium parasites are the causative agents of malaria, a disease with wide public health repercussions. Increasing drug resistance and the absence of a vaccine make finding new chemotherapeutic strategies imperative. Components of the ubiquitin and ubiquitin-like pathways have garnered increased attention as novel targets given their necessity to parasite survival. Understanding how these pathways are regulated in Plasmodium and identifying differences to the host is paramount to selectively interfering with parasites. Here, we focus on Nedd8 modification in Plasmodium falciparum, given its central role to cell division and DNA repair, processes critical to Plasmodium parasites given their unusual cell cycle and requirement for refined repair mechanisms. By applying a functional chemical approach, we show that deNeddylation is controlled by a different set of enzymes in the parasite versus the human host. We elucidate the molecular determinants of the unusual dual ubiquitin/Nedd8 recognition by the essential PfUCH37 enzyme and, through parasite transgenics and drug assays, determine that only its ubiquitin activity is critical to parasite survival. Our experiments reveal interesting evolutionary differences in how neddylation is controlled in higher versus lower eukaryotes, and highlight the Nedd8 pathway as worthy of further exploration for therapeutic targeting in antimalarial drug design., Competing Interests: The authors have declared no competing interests exist.

Patients with small intestinal neuroendocrine tumors (SI-NETs) frequently develop spread disease; however, the underlying molecular mechanisms of disease progression are not known and effective preventive treatment strategies are lacking. Here, protein expression profiling was performed by HiRIEF-LC-MS in 14 primary SI-NETs from patients with and without liver metastases detected at the time of surgery and initial treatment. Among differentially expressed proteins, overexpression of the ubiquitin-like protein NEDD8 was identified in samples from patients with liver metastasis. Further, NEDD8 correlation analysis indicated co-expression with RBX1, a key component in cullin-RING ubiquitin ligases (CRLs). In vitro inhibition of neddylation with the therapeutic agent pevonedistat (MLN4924) resulted in a dramatic decrease of proliferation in SI-NET cell lines. Subsequent mass spectrometry-based proteomics analysis of pevonedistat effects and effects of the proteasome inhibitor bortezomib revealed stabilization of multiple targets of CRLs including p27, an established tumor suppressor in SI-NET. Silencing of NEDD8 and RBX1 using siRNA resulted in a stabilization of p27, suggesting that the cellular levels of NEDD8 and RBX1 affect CRL activity. Inhibition of CRL activity, by either NEDD8/RBX1 silencing or pevonedistat treatment of cells resulted in induction of apoptosis that could be partially rescued by siRNA-based silencing of p27. Differential expression of both p27 and NEDD8 was confirmed in a second cohort of SI-NET using immunohistochemistry. Collectively, these findings suggest a role for CRLs and the ubiquitin proteasome system in suppression of p27 in SI-NET, and inhibition of neddylation as a putative therapeutic strategy in SI-NET.

Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 from activated Cullins. Here we present structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single-particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (XL-MS). These structures suggest a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery. Using hydrogen-deuterium exchange (HDX)-MS we show that CRL2 activates CSN5/CSN6 in a neddylation-independent manner. The presence of NEDD8 is required to activate the CSN5 active site. Overall, by synergising cryo-EM with MS, we identify sensory regions of the CSN that mediate its stepwise activation and provide a framework for understanding the regulatory mechanism of other Cullin family members.

Serine rich splicing factor 3 (SRSF3) plays a critical role in liver function and its loss promotes chronic liver damage and regeneration. As a consequence, genetic deletion of SRSF3 in hepatocytes caused progressive liver disease and ultimately led to hepatocellular carcinoma. Here we show that SRSF3 is decreased in human liver samples with non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or cirrhosis that was associated with alterations in RNA splicing of known SRSF3 target genes. Hepatic SRSF3 expression was similarly decreased and RNA splicing dysregulated in mouse models of NAFLD and NASH. We showed that palmitic acid-induced oxidative stress caused conjugation of the ubiquitin like NEDD8 protein to SRSF3 and proteasome mediated degradation. SRSF3 was selectively neddylated at lysine11 and mutation of this residue (SRSF3-K11R) was sufficient to prevent both SRSF3 degradation and alterations in RNA splicing. Finally prevention of SRSF3 degradation in vivo partially protected mice from hepatic steatosis, fibrosis and inflammation. These results highlight a neddylation-dependent mechanism regulating gene expression in the liver that is disrupted in early metabolic liver disease and may contribute to the progression to NASH, cirrhosis and ultimately hepatocellular carcinoma.

Objective: This study aimed to investigate the function and mechanism of neddylation of HDAC1 underlying drug resistance of AML cells., Methods: Evaluation experiments of effects of HDAC1 on drug resistance of AML cells were performed with AML cell transfected with constructs overexpressing HDAC1 or multi-drug resistance AML cells transfected with siRNA for HDAC1 through observing cell viability, percentage of apoptotic cell, doxorubicin-releasing index and multidrug resistance associated protein 1 (MRP1) expression. Neddylation or ubiquitination of HDAC1 was determined by immunoprecipitation or Ni2 + pull down assay followed by western blot. The role of HDAC1 was in vivo confirmed by xenograft in mice., Results: HDAC1 was significantly upregulated in refractory AML patients, and in drug-resistant AML cells (HL-60/ADM and K562/A02). Intracellular HDAC1 expression promoted doxorubicin resistance of HL-60, K562, and primary bone marrow cells (BMCs) of remission AML patients as shown by increasing cell viability and doxorubicin-releasing index, inhibiting cell apoptosis. Moreover, HDAC1 protein level in AML cells was regulated by the Nedd8-mediated neddylation and ubiquitination, which further promoted HDAC1 degradation. In vivo, HDAC1 overexpression significantly increased doxorubicin resistance; while HDACs inhibitor Panobinostat markedly improved the inhibitory effect of doxorubicin on tumor growth. Furthermore, HDAC1 silencing by Panobinostat and/or lentivirus mediated RNA interference against HDAC1 effectively reduced doxorubicin resistance, resulting in the inhibition of tumor growth in AML bearing mice., Conclusion: Our findings suggested that HDAC1 contributed to the multidrug resistance of AML and its function turnover was regulated, at least in part, by post-translational modifications, including neddylation and ubiquitination.

Tumor-associated macrophages (TAMs) are the most abundant cancer stromal cells and play an essential role in tumor immunosuppression, providing a suitable microenvironment for cancer development and progression. However, mechanisms of regulating TAMs infiltration in tumor sites are not fully understood. Here, we show that inactivation of neddylation pathway significantly inhibits infiltration of TAMs, leading to the suppression of lung cancer metastasis. RNA-sequencing analysis revealed that neddylation inactivation suppresses the transactivation of chemotactic cytokine ligand 2 (CCL2). Mechanistically, neddylation inactivation inhibits the activity of Cullin-RING ligases (CRLs) and induces the accumulation of its substrate IκBα to block NF-κB transcriptional activity and CCL2 transactivation. As a result, neddylation inactivation exhibits lower chemotaxis of monocytes, thereby decreasing TAMs infiltration, which can be alleviated by CCL2 addition. Moreover, the expression level of NEDD8 is positively correlated with high CCL2 expression in lung adenocarcinoma, conferring a worse overall patient survival. Together, neddylation pathway promotes CCL2 transactivation and TAMs infiltration in lung cancer to provide a tumor-promoting microenvironment, which validates neddylation pathway as a promising target for anti-TAMs therapeutic strategies.

Purpose: How the neddylation pathway functions in breast tumor and regulation of estrogen receptor (ER) expression is rarely reported. The purpose of this study was to identify the role of neddylation in breast cancer and ER expression, and further explore the underlying mechanisms., Experimental Design: Expression patterns of nedd8-activating enzyme (NAE) and nedd8, two key proteins in the neddylation pathway, were examined in human breast specimens. ER-α expression was investigated using animal 18F-FES-PET/CT and immunoblotting upon NAE inhibitor MLN4924 treatment. Chromatin immunoprecipitation assay, luciferase reporter promoter assay, and the CRISPR-Cas9 system were used to elucidate the mechanism of ER-α regulation by MLN4924. The ER-positive breast cancer mouse model was used to determine the synergetic effect of MLN4924 and fulvestrant on tumor growth. All statistical tests were two-sided., Results: Both NAE1 and nedd8 expressions were higher in the ER-positive subgroup. Higher expressions of NAE1 and nedd8 indicated poorer prognosis. Importantly, ER-α expression was significantly downregulated upon MLN4924 treatment in vitro and in vivo . Mechanistically, MLN4924 treatment delayed serum and glucocorticoid-induced protein kinase (SGK) degradation and induced Forkhead box O3a (FOXO3a) nuclear export as well as decreased binding to the ESR1 promoter. Importantly, MLN4924 single or synergized with fulvestrant significantly suppressed the growth of ER-positive breast cancer in vitro and in vivo ., Conclusions: Our proof-of-principle study determines the activation of neddylation in breast tumor tissues for the first time and reveals a new ER-α regulatory mechanism, as well as further explores an effective approach to improve fulvestrant sensitivity through a neddylation inactivation combination., (©2019 American Association for Cancer Research.)

Metastasis is the leading cause of tumor-related death from lung cancer. However, limited success has been achieved in the treatment of lung cancer metastasis due to the lack of understanding of the mechanisms that underlie the metastatic process. In this study, Lewis lung carcinoma (LLC) cells which expressed green fluorescent protein in the nucleus and red fluorescent protein in the cytoplasm were used to record metastatic process in real-time via a whole-mouse imaging system. Using this system, we show the neddylation inhibitor MLN4924 inhibits multiple steps of the metastatic process, including intravascular survival, extravasation, and formation of metastatic colonies, thus finally suppressing tumor metastasis. Mechanistically, MLN4924 efficiently inhibits the expression of MMP2, MMP9, and vimentin and disrupts the actin cytoskeleton at an early stage to impair invasive potential and subsequently causes a DNA damage response, cell cycle arrest, and apoptosis upon long exposure to MLN4924. Furthermore, MMP2 and MMP9 are overexpressed in patient lung adenocarcinoma, which conferred a worse overall survival. Together, targeting the neddylation pathway via MLN4924 suppresses multiple steps of the metastatic process, highlighting the potential therapeutic value of MLN4924 for the treatment of metastatic lung cancer.

Mammalian oocyte meiosis is a special form of cell division that provides haploid gametes for fertilization. Unlike in mitosis, post-translational modifications (PTMs) are more crucial during meiosis because of the absence of de novo mRNA transcription. As a classic PTM, protein neddylation is a biological process that mediates protein degradation by modifying cullin proteins and activating the Cullin-Ring E3 ligases. This process plays important roles in various biological processes such as autophagy and tumorigenesis. However, the function of neddylation in germ cells is unknown. In this study, we observed that the inhibition of neddylation by its specific inhibitor MLN4924 significantly arrests mouse oocyte at the stage of metaphase during meiosis. The arrested oocytes display impaired spindles with over-activation of spindle assembly checkpoint (SAC). Accordingly, we identified early mitosis inhibitor 1 (Emi1), a key inhibitor of anaphase-promoting complex/cyclosome (APC/C Fzr1 ), as a substrate of neddylation-mediated protein degradation. Thus, our study uncovered an unknown role of neddylation in female germ cells and suggests that proper neddylation is essential for oocyte maturation.

Alterations in perinatal conditions (such as preterm birth) is linked to adult health and disease, in particular, the cardiovascular system. Neddylation, a novel posttranslational modification through which the ubiquitin-like protein NEDD8 is conjugated to protein substrates, has emerged as an important mechanism regulating embryonic cardiac chamber maturation. However, the importance of neddylation in postpartum cardiac development has not been investigated. Here, we aimed to determine whether transient, postnatal inhibition of neddylation has immediate and prolonged impact on the structure and function of the neonatal and adult hearts. Sprague-Dawley pups were given three intraperitoneal injections of MLN4924 (MLN), a specific neddylation inhibitor, at postnatal days (P)1, 3, and 5. Cardiac structure and function were temporally assessed during aging and after 2 wk of isoproterenol (ISO) infusion in adulthood. MLN treatment resulted in modest reduction of neddylated proteins in neonatal hearts. The MLN-treated rats developed cardiac hypertrophy and dysfunction by P7, which was accompanied by significantly reduced cardiomyocyte proliferation. At 3 mo of age, cardiac contractile function was restored in MLN-treated rats, but MLN-treated hearts displayed hypertrophic phenotype. Whereas ISO infusion triggered compensatory cardiac hypertrophy without impairing cardiac contractility in the control rats, the MLN-treated rats displayed a similar degree of hypertrophy, which quickly progressed to decompensation with ventricular wall thinning, chamber dilatation, and reduced ejection fraction as well as exacerbated pathological cardiac remodeling. Our findings suggest that neddylation is required for postnatal cardiac development and that perturbation of neddylation during development predisposes adult hearts to cardiac failure under stress conditions. NEW & NOTEWORTHY Our study demonstrates that perinatal perturbation of neddylation induces cardiomyopathy, impairs postnatal cardiac development, and increases susceptibility to catecholamine-induced cardiac dysfunction. The results reveal a previously unappreciated role of neddylation in postnatal cardiac maturation and call for close monitoring for the potential cardiotoxicity of MLN4924 (pevonedistat) and other agents that modify neddylation, especially in pregnant women and preadolescents.

The cullin-RING E3 ubiquitin ligases (CRLs) play crucial roles in modulating the stability of proteins in the cell and are, in turn, regulated by post-translational modification by the ubiquitin-like (Ubl) protein NEDD8. This process, termed neddylation, is reversible through the action of the COP9 signalosome (CSN); a multi-subunit metalloprotease conserved among eukaryotes that plays direct or indirect roles in DNA repair, cell signaling and cell cycle regulation in part through modulating the activity of the CRLs. Previously, inhibition of CRL neddylation by MLN4924, a small molecule inhibitor of the NEDD8-activating enzyme 1 (NAE1), was shown to induce interphase cell cycle arrest and cell death. Using fixed and living cell microscopy, we re-evaluated the cell cycle effects of inhibition of neddylation by MLN4924 in both asynchronous and mitotic cell populations. Consistent with previous studies, treatment of asynchronous cells with MLN4924 increased CDT1 expression levels, induced G2 arrest and increased nuclear size. However, in synchronized cells treated in mitosis, mitotic defects were observed including lagging chromosomes and binucleated daughter cells. Consistent with neddylation and deneddylation playing a role in cytokinesis, NEDD8, as well as subunits of the CSN, could be localized at the midbody and cleavage furrow. Finally, treatment of mitotic cells with MLN4924 induced the premature accumulation of MKLP1 at the cleavage furrow, a key regulator of cytokinesis, which was concomitant with increased abscission delay and failure. Thus, these studies uncover an uncharacterized mitotic effect of MLN4924 on MKLP1 accumulation at the midbody and support a role for neddylation during cytokinesis. Abbreviations : CSN, COP9 Signalosome; MKLP1, mitotic kinesin-like protein 1; NEDD8, Neural precursor cell Expressed, Developmentally Down-regulated 8.