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This study aimed to explore the underlying mechanism of neddylation in macrophage polarization during sepsis. A mouse model of sepsis was established by cecal ligation and puncture (CLP). ELISA and Flow cytometry were performed to analyze the generation of pro-inflammatory factors and M1/M2 macrophage polarization, respectively. Western blotting was applied to detect NEDD8-mediated neddylation and glycolysis-related proteins. ECAR method was used to analyze the glycolysis level. HE staining was applied to detect the lung injury. The bacterial load in peritoneal cavity and peripheral blood was determined by counting the colony-forming units. The results showed the upregulated neddylation, M1 polarization and glycolysis of macrophage in patients with sepsis and CLP-challenged mice. NEDD8-mediated Cullin1 neddylation promoted M1 polarization and glycolysis to accelerate inflammation via NF-κB p65 pathway in E.coli-treated Raw264.7 cells. MLN4924 treatment alleviated sepsis by inhibiting neddylation to prevent M1 polarization in CLP-challenged mice. In summary, this study demonstrated that upregulation of NEDD8-mediated Cullin1 neddylation promotes glycolysis and M1 polarization of macrophage via NF-κB p65 pathway, accelerating inflammation in sepsis., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The ongoing obesity epidemic has raised awareness of the complex physiology of adipose tissue. Abnormal adipocyte differentiation results in the development of systemic metabolic disorders such as insulin resistance and diabetes. The conjugation of NEDD8 (neural precursor cell expressed, developmentally downregulated 8) to target protein, termed neddylation, has been shown to mediate adipogenesis. However, much remains unknown about its role in adipogenesis. Here, we demonstrated that neddylation and its targets, the cullin (CUL) family members, are differentially regulated during mouse and human adipogenesis. Inhibition of neddylation by MLN4924 significantly reduced adipogenesis of 3T3-L1 and human stromal vascular cells. Deletion of NAE1, a subunit of the only NEDD8 E1 enzyme, suppressed neddylation and impaired adipogenesis. Neddylation deficiency did not affect mitotic cell expansion. Instead, it disrupted CREB/CEBPβ/PPARγ signaling, essential for adipogenesis. Interestingly, among the neddylation-targeted CUL family members, deletion of CUL3, but not CUL1, CUL2, or CUL4A, largely replicated the adipogenic defects observed with neddylation deficiency. A PPARγ agonist minimally rescued the adipogenic defects caused by the deletion of NAE1 and CUL3. In conclusion, our study demonstrates that neddylation and its targeted CUL3 are crucial for adipogenesis. These findings provide potential targets for therapeutic intervention in obesity and metabolic disorders.

Cullin-based RING ligases (CRLs) comprise the largest family of ubiquitin E3 ligases. CRL activity is tightly regulated by cullin neddylation, which has been associated with various diseases. Although inhibitors of CRLs neddylation have been reported, there is a lack of small molecules that can selectively target individual cullins. Here, we identified a natural product, liquidambaric acid (LDA), with relatively selective inhibition properties against cullin (Cul) 2 neddylation, and found that its target, Tumor Necrosis Factor receptor-associated factor 2 (TRAF2) was required for the activity. TRAF2 associates with the Cul2 neddylation complex and regulates the machinery assembly, especially that of E2 (UBC12) and E3 (RBX1) enzymes. In addition, we demonstrated that by intervention of the associations between TRAF2 and the neddylation machinery, LDA disturbed NEDD8 transfer from E1 to E2, therefore blocking Cul2 neddylation. Taken together, we show that TRAF2 plays a positive role in neddylation cascades, and we have identified a small molecule capable of selective modulation of cullin neddylation., (© 2024 Federation of European Biochemical Societies.)

Cancer affects millions of people and understanding the molecular mechanisms related to disease development and progression is essential to manage the disease. Post-translational modification (PTM) processes such as ubiquitination and neddylation have a significant role in cancer development and progression by regulating protein stability, function, and interaction with other biomolecules. Both ubiquitination and neddylation are analogous processes that involves a series of enzymatic steps leading to the covalent attachment of ubiquitin or NEDD8 to target proteins. Neddylation modifies the CRL family of E3 ligase and regulates target proteins' function and stability. The DCUN1D1 protein is a regulator of protein neddylation and ubiquitination and acts promoting the neddylation of the cullin family components of E3-CRL complexes and is known to be upregulated in several types of cancers. In this review we compare the PTM ubiquitination and neddylation. Our discussion is focused on the neddylation process and the role of DCUN1D1 protein in cancer development. Furthermore, we provide describe DCUN1D1 protein and discuss its role in pathogenesis and signalling pathway in six different types of cancer. Additionally, we explore both the neddylation and DCUN1D1 pathways as potential druggable targets for therapeutic interventions. We focus our analysis on the development of compounds that target specifically neddylation or DCUN1D1. Finally, we provide a critical analysis about the challenges and perspectives in the field of DCUN1D1 and neddylation in cancer research. KEY POINTS: Neddylation is a post-translational modification that regulates target proteins' function and stability. One regulator of the neddylation process is a protein named DCUN1D1 and it is known to have its expression deregulated in several types of cancers. Here, we provide a detailed description of DCUN1D1 structure and its consequence for the development of cancer. We discuss both the neddylation and DCUN1D1 pathways as potential druggable targets for therapeutic interventions and provide a critical analysis about the challenges and perspectives in the field of DCUN1D1 and neddylation in cancer research., 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 © 2024 Elsevier B.V. All rights reserved.)

Plasmodium parasites, the causative agents of malaria, rely on sophisticated cellular mechanisms to survive and proliferate within their hosts. Plasmodium complex life cycle requires posttranslational modifications (PTMs) to control cellular activities. Neddylation is a type of PTM in which NEDD8 is covalently attached to target proteins and plays an important role in cell cycle control and metabolism. Covalent attachment to its substrates requires the Nedd8-activating enzyme, E1; the NEDD8-conjugating enzyme, E2; and the ligase, E3. In Plasmodium , protein neddylation is essential for parasite development during the stage I-II transition from zygote to ookinete differentiation and malaria transmission. Here, we discuss the current understanding of protein neddylation in Plasmodium, which is involved in malaria transmission.

The Hippo-YAP signaling pathway plays a central role in many biological processes such as regulating cell fate, organ size, and tissue growth, and its key components are spatiotemporally expressed and posttranslationally modified during these processes. Neddylation is a posttranslational modification that involves the covalent attachment of NEDD8 to target proteins by NEDD8-specific E1-E2-E3 enzymes. Whether neddylation is involved in Hippo-YAP signaling remains poorly understood. Here, we provide evidence supporting the critical role of NEDD8 in facilitating the Hippo-YAP signaling pathway by mediating neddylation of the transcriptional coactivator yes-associated protein 1 (YAP1). Overexpression of NEDD8 induces YAP1 neddylation and enhances YAP1 transactivity, but inhibition of neddylation suppresses YAP1 transactivity and attenuates YAP1 nuclear accumulation. Furthermore, inhibition of YAP1 signaling promotes MLN4924-induced ovarian granulosa cells apoptosis and disruption of nedd8 in zebrafish results in downregulation of yap1-activated genes and upregulation of yap1-repressed genes. Further assays show that the xiap ligase promotes nedd8 conjugates to yap1 and that yap1 neddylation. In addition, we identify lysine 159 as a major neddylation site on YAP1. These findings reveal a novel mechanism for neddylation in the regulation of Hippo-YAP signaling., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Drug-induced liver injury (DILI) is a significant cause of acute liver failure (ALF) and liver transplantation in the Western world. Acetaminophen (APAP) overdose is a main contributor of DILI, leading to hepatocyte cell death through necrosis. Here, we identified that neddylation, an essential post-translational modification involved in the mitochondria function, was upregulated in liver biopsies from patients with APAP-induced liver injury (AILI) and in mice treated with an APAP overdose. MLN4924, an inhibitor of the neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8)-activating enzyme (NAE-1), ameliorated necrosis and boosted liver regeneration in AILI. To understand how neddylation interferes in AILI, whole-body biotinylated NEDD8 ( bio NEDD8) and ubiquitin ( bio UB) transgenic mice were investigated under APAP overdose with and without MLN4924. The cytidine diphosphate diacylglycerol (CDP-DAG) synthase TAM41, responsible for producing cardiolipin essential for mitochondrial activity, was found modulated under AILI and restored its levels by inhibiting neddylation. Understanding this ubiquitin-like crosstalk in AILI is essential for developing promising targeted inhibitors for DILI treatment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Diabetic retinopathy (DR) is a highly hazardous and widespread complication of diabetes mellitus (DM). The accumulated reactive oxygen species (ROS) play a central role in DR development. The aim of this research was to examine the impact and mechanisms of mesenchymal stem cell (MSC)-derived small extracellular vesicles (sEV) on regulating ROS and retinal damage in DR. Intravitreal injection of sEV inhibited Cullin3 neddylation, stabilized Nrf2, decreased ROS, reduced retinal inflammation, suppressed Müller gliosis, and mitigated DR. Based on MSC-sEV miRNA sequencing, bioinformatics software, and dual-luciferase reporter assay, miR-143-3p was identified to be the key effector for MSC-sEV's role in regulating neural precursor cell expressed developmentally down-regulated 8 (NEDD8)-mediated neddylation. sEV were able to be internalized by Müller cells. Compared to advanced glycation end-products (AGEs)-induced Müller cells, sEV coculture decreased Cullin3 neddylation, activated Nrf2 signal pathway to combat ROS-induced inflammation. The barrier function of endothelial cells was impaired when endothelial cells were treated with the supernatant of AGEs-induced Müller cells, but was restored when treated with supernatant of AGEs-induced Müller cells cocultured with sEV. The protective effect of sEV was, however, compromised when miR-143-3p was inhibited in sEV. Moreover, the protective efficacy of sEV was diminished when NEDD8 was overexpressed in Müller cells. These findings showed MSC-sEV delivered miR-143-3p to inhibit Cullin3 neddylation, stabilizing Nrf2 to counteract ROS-induced inflammation and reducing vascular leakage. Our findings suggest that MSC-sEV may be a potential nanotherapeutic agent for DR, and that Cullin3 neddylation could be a new target for DR therapy., 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 © 2024 Elsevier Inc. All rights reserved.)

Immune checkpoint blockade therapy aims to activate the immune system to eliminate cancer cells. However, clinical benefits are only recorded in a subset of patients. Here, we leverage genome-wide CRISPR/Cas9 screens in a Tumor-Immune co-Culture System focusing on triple-negative breast cancer (TNBC). We reveal that NEDD8 loss in cancer cells causes a vulnerability to nivolumab (anti-PD-1). Genetic deletion of NEDD8 only delays cell division initially but cell proliferation is unaffected after recovery. Since the NEDD8 gene is commonly essential, we validate this observation with additional CRISPR screens and uncover enhanced immunogenicity in NEDD8 deficient cells using proteomics. In female immunocompetent mice, PD-1 blockade lacks efficacy against established EO771 breast cancer tumors. In contrast, we observe tumor regression mediated by CD8+ T cells against Nedd8 deficient EO771 tumors after PD-1 blockade. In essence, we provide evidence that NEDD8 is conditionally essential in TNBC and presents as a synergistic drug target for PD-1/L1 blockade therapy., (© 2024. The Author(s).)

The E3 ligase-degron interaction determines the specificity of the ubiquitin‒proteasome system. We recently discovered that FEM1B, a substrate receptor of Cullin 2-RING ligase (CRL2), recognizes C-degrons containing a C-terminal proline. By solving several cryo-EM structures of CRL2 FEM1B bound to different C-degrons, we elucidate the dimeric assembly of the complex. Furthermore, we reveal distinct dimerization states of unmodified and neddylated CRL2 FEM1B to uncover the NEDD8-mediated activation mechanism of CRL2 FEM1B . Our research also indicates that, FEM1B utilizes a bipartite mechanism to recognize both the C-terminal proline and an upstream aromatic residue within the substrate. These structural findings, complemented by in vitro ubiquitination and in vivo cell-based assays, demonstrate that CRL2 FEM1B -mediated polyubiquitination and subsequent protein turnover depend on both FEM1B-degron interactions and the dimerization state of the E3 ligase complex. Overall, this study deepens our molecular understanding of how Cullin-RING E3 ligase substrate selection mediates protein turnover., (© 2024. The Author(s).)

The clinical efficacy of VSVΔ51 oncolytic virotherapy has been limited by tumor resistance to viral infection, so strategies to transiently repress antiviral defenses are warranted. Pevonedistat is a first-in-class NEDD8-activating enzyme (NAE) inhibitor currently being tested in clinical trials for its antitumor potential. In this study, we demonstrate that pevonedistat sensitizes human and murine cancer cells to increase oncolytic VSVΔ51 infection, increase tumor cell death, and improve therapeutic outcomes in resistant syngeneic murine cancer models. Increased VSVΔ51 infectivity was also observed in clinical human tumor samples. We further identify the mechanism of this effect to operate via blockade of the type 1 interferon (IFN-1) response through neddylation-dependent interferon-stimulated growth factor 3 (ISGF3) repression and neddylation-independent inhibition of NF-κB nuclear translocation. Together, our results identify a role for neddylation in regulating the innate immune response and demonstrate that pevonedistat can improve the therapeutic outcomes of strategies using oncolytic virotherapy., Competing Interests: Declaration of interests B.W., A.B., R.A., and J.-S.D. are co-inventors on a patent covering the use of NAE inhibitors as an enhancer of RNA viruses., (Copyright © 2023. Published by Elsevier Inc.)

Background: Hepatitis B Virus (HBV) is the most common cause of chronic liver disease worldwide. The mechanisms that regulate HBV viral replication remain poorly defined. Here, we show that blocking of the neddylation elicits antiviral effect against HBV replication, indicating that NEDD8 supports viral production., Methods and Results: To explore role of neddylation, HBV-replicating HepG2.2.15.7 cells and HBV-infected HepG2-hNTCP-30 cells were treated with siNEDD8 and MLN4924, a potent and selective NEDD8-activating enzyme inhibitor. Cell viability, intracellular and extracellular HBV DNA, covalently closed circular DNA (cccDNA), HBsAg, HBeAg, and HBcrAg were measured to assess the consequences of the various treatments on viral replication. Our data showed that HBV infection increased NEDD8 expression in human liver cell lines. Symmetrically, NEDD8 knockdown by siRNA or MLN4924 treatments decreased HBV replication in HepG2.2.15.7 and HepG2-hNTCP-30 cells. Notably, HBsAg, and HBeAg secretions were strongly suppressed in the culture supernatants, but not the HBcrAg. These results indicate that the suppression of NEDD8 decreases HBV replication. However, cccDNA steady level confirms once again its persistence and longevity in chronic infection., Conclusion: The manipulation of the neddylation pathway can thus provide new tools interfering with HBV persistence as well as novel therapeutic strategies against chronic hepatitis B., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Through catalyzing the ubiquitination of key regulatory proteins, cullin-RING ubiquitin ligases (CRLs) play essential biological roles and their activities are controlled by multiple mechanisms including neddylation, the conjugation of NEDD8 to cullins. Upon neddylation, a CRL, such as the CUL1-based CRL1, undergoes conformational changes that accelerate substrate ubiquitination. Given the structural diversity across subfamilies of CRLs and their substrates, to what extent neddylation modulates the activity of individual CRLs remains to be evaluated. Here, through reconstituting the CRL2 ubiquitination reaction in vitro, we showed that neddylation promotes CRL2 VHL -dependent degradation of both full-length HIF1α and the degron peptide of HIF1α, resulting in more than 10-fold increase in the rate of substrate ubiquitination. Consistently, pevonedistat (also known as MLN4924), an inhibitor of neddylation, inhibits the degradation of HIF1α in RCC4 cells stably expressing VHL in cycloheximide chase assays. However, such inhibitory effect of pevonedistat on HIF1α degradation was not observed in HEK293 cells, which was further found to be due to CRL2 VHL -independent degradation that was active in HEK293 but not RCC4 cells. After truncating HIF1α to its Carboxy-terminal Oxygen-Dependent Degradation (CODD) domain, we showed that pevonedistat inhibited the degradation of CODD and increased its half-life by six-fold in HEK293 cells. Our results demonstrate that neddylation plays a significant role in activating CRL2, and the cellular activity of CRL2 VHL is better reflected by the degradation of CODD than that of HIF1α, especially under conditions where CRL2-independent degradation of HIF1α is active., (© 2021 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Knocking down the oncogene ROC1 with siRNA inhibits the proliferation of cancer cells by suppressing the Neddylation pathway. However, methods for delivering siRNA in vivo to induce this high anticancer activity with low potential side effects are urgently needed. Herein, a folic acid (FA)-modified polydopamine (PDA) nanomedicine used in photothermal therapy was designed for siRNA delivery. The designed nanovector can undergo photothermal conversion with good biocompatibility. Importantly, this genetic nanomedicine was selectively delivered to liver cancer cells by FA through receptor-mediated endocytosis. Subsequently, the siRNA cargo was released from the PDA nanomedicine into the tumor microenvironment by controlled release triggered by pH. More importantly, the genetic nanomedicine not only inhibited liver cancer cell proliferation but also promoted liver cell apoptosis by slowing ROC1 activity, suppressing the Neddylation pathway, enabling the accumulation of apototic factor ATF4 and DNA damage factor P-H2AX. Combined with photothermal therapy, this genetic nanomedicine showed superior inhibition of the growth of liver cancer in vitro and in vivo. Taken together, the results indicate that this biodegradable nanomedicine exhibits good target recognition, an effective pH response, application potential for genetic therapy, photothermal imaging and treatment of liver cancer. Therefore, this work contributes to the design of a multifunctional nanoplatform that combines genetic therapy and photothermal therapy for the treatment of liver cancer., (© 2021. The Author(s).)

Ubiquitin (Ub) and Ub-like proteins (Ubls) such as NEDD8 are best known for their function as covalent modifiers of other proteins but they are also themselves subject to post-translational modifications including phosphorylation. While functions of phosphorylated Ub (pUb) have been characterized, the consequences of Ubl phosphorylation remain unclear. Here we report that NEDD8 can be phosphorylated at S65 - the same site as Ub - and that S65 phosphorylation affects the structural dynamics of NEDD8 and Ub in a similar manner. While both pUb and phosphorylated NEDD8 (pNEDD8) can allosterically activate the Ub ligase Parkin, they have different protein interactomes that in turn are distinct from those of unmodified Ub and NEDD8. Among the preferential pNEDD8 interactors are HSP70 family members and we show that pNEDD8 stimulates HSP70 ATPase activity more pronouncedly than unmodified NEDD8. Our findings highlight the general importance of Ub/NEDD8 phosphorylation and support the notion that the function of pUb/pNEDD8 does not require their covalent attachment to other proteins., (© 2021. The Author(s).)

An emerging mechanism of ubiquitylation involves partnering of two distinct E3 ligases. In the best-characterized E3-E3 pathways, ARIH-family RING-between-RING (RBR) E3s ligate ubiquitin to substrates of neddylated cullin-RING E3s. The E3 ARIH2 has been implicated in ubiquitylation of substrates of neddylated CUL5-RBX2-based E3s, including APOBEC3-family substrates of the host E3 hijacked by HIV-1 virion infectivity factor (Vif). However, the structural mechanisms remained elusive. Here structural and biochemical analyses reveal distinctive ARIH2 autoinhibition, and activation on assembly with neddylated CUL5-RBX2. Comparison to structures of E3-E3 assemblies comprising ARIH1 and neddylated CUL1-RBX1-based E3s shows cullin-specific regulation by NEDD8. Whereas CUL1-linked NEDD8 directly recruits ARIH1, CUL5-linked NEDD8 does not bind ARIH2. Instead, the data reveal an allosteric mechanism. NEDD8 uniquely contacts covalently linked CUL5, and elicits structural rearrangements that unveil cryptic ARIH2-binding sites. The data reveal how a ubiquitin-like protein induces protein-protein interactions indirectly, through allostery. Allosteric specificity of ubiquitin-like protein modifications may offer opportunities for therapeutic targeting., (© 2021. The Author(s).)

As the largest tissue in the body, skeletal muscle has multiple functions in movement and energy metabolism. Skeletal myogenesis is controlled by a transcriptional cascade including a set of muscle regulatory factors (MRFs) that includes Myogenic Differentiation 1 (MYOD1), Myocyte Enhancer Factor 2 (MEF2), and Myogenin (MYOG), which direct the fusion of myogenic myoblasts into multinucleated myotubes. Neddylation is a posttranslational modification that covalently conjugates ubiquitin-like NEDD8 (neural precursor cell expressed, developmentally downregulated 8) to protein targets. Inhibition of neddylation impairs muscle differentiation; however, the underlying molecular mechanisms remain less explored. Here, we report that neddylation is temporally regulated during myoblast differentiation. Inhibition of neddylation through pharmacological blockade using MLN4924 (Pevonedistat) or genetic deletion of NEDD8 Activating Enzyme E1 Subunit 1 (NAE1), a subunit of the E1 neddylation-activating enzyme, blocks terminal myoblast differentiation partially through repressing MYOG expression. Mechanistically, we found that neddylation deficiency enhances the mRNA and protein expressions of class IIa histone deacetylases 4 and 5 (HDAC4 and 5) and prevents the downregulation and nuclear export of class III HDAC (NAD-Dependent Protein Deacetylase Sirtuin-1, SIRT1), all of which have been shown to repress MYOD1-mediated MYOG transcriptional activation. Together, our findings for the first time identify the crucial role of neddylation in mediating class IIa and III HDAC co-repressors to control myogenic program and provide new insights into the mechanisms of muscle disease and regeneration.

Cullin-RING ligases (CRLs) are a significant subset of Ubiquitin E3 ligases that regulate multiple cellular substrates involved in innate immunity, cytoskeleton modeling, and cell cycle. The glutamine deamidase Cycle inhibitory factor (Cif) from enteric bacteria inactivates CRLs to modulate these processes in the host cell. The covalent attachment of a Ubiquitin-like protein NEDD8 catalytically activates CRLs by driving conformational changes in the Cullin C-terminal domain (CTD). NEDDylation results in a shift from a compact to an open CTD conformation through non-covalent interactions between NEDD8 and the WHB subdomain of CTD, eliminating the latter's inhibitory interactions with the RING E3 ligase-Rbx1/2. It is unknown whether the non-covalent interactions are sufficient to stabilize Cullin CTD's catalytic conformation. We studied the dynamics of Cullin-CTD in the presence and absence of NEDD8 using atomistic molecular dynamics (MD) simulations. We uncovered that NEDD8 engages in non-covalent interactions with 4HB/αβ subdomains in Cullin-CTD to promote open conformations. Cif deamidates glutamine 40 in NEDD8 to inhibit the conformational change in CRLs by an unknown mechanism. We investigated the effect of glutamine deamidation on NEDD8 and its interaction with the WHB subdomain post-NEDDylation using MD simulations and NMR spectroscopy. Our results suggest that deamidation creates a new intramolecular salt bridge in NEDD8 to destabilize the NEDD8/WHB complex and reduce CRL activity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mohanty, Chatterjee and Das.)

Small cell lung carcinoma (SCLC) is among the most lethal of all solid tumor malignancies. In an effort to identify novel therapeutic approaches for this recalcitrant cancer type, we applied genome-scale CRISPR/Cas9 inactivation screens to cell lines that we derived from a murine model of SCLC. SCLC cells were particularly sensitive to the deletion of NEDD8 and other neddylation pathway genes. Genetic suppression or pharmacological inhibition of this pathway using MLN4924 caused cell death not only in mouse SCLC cell lines but also in patient-derived xenograft (PDX) models of pulmonary and extrapulmonary small cell carcinoma treated ex vivo or in vivo. A subset of PDX models were exceptionally sensitive to neddylation inhibition. Neddylation inhibition suppressed expression of major regulators of neuroendocrine cell state such as INSM1 and ASCL1, which a subset of SCLC rely upon for cell proliferation and survival. To identify potential mechanisms of resistance to neddylation inhibition, we performed a genome-scale CRISPR/Cas9 suppressor screen. Deletion of components of the COP9 signalosome strongly mitigated the effects of neddylation inhibition in small cell carcinoma, including the ability of MLN4924 to suppress neuroendocrine transcriptional program expression. This work identifies neddylation as a regulator of neuroendocrine cell state and potential therapeutic target for small cell carcinomas., (© 2021 Norton et al.; Published by Cold Spring Harbor Laboratory Press.)

Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men in Western countries, and there is still an urgent need for a better understanding of PCa progression to inspire new treatment strategies. Skp2 is a substrate-recruiting component of the E3 ubiquitin ligase complex, whose activity is regulated through neddylation. Slug is a transcriptional repressor involved in the epithelial-to-mesenchymal transition, which may contribute to therapy resistance. Although Skp2 has previously been associated with a mesenchymal phenotype and prostate cancer progression, the relationship with Slug deserves further elucidation. We have previously shown that a high Gleason score (≥8) is associated with higher Skp2 and lower E-cadherin expression. In this study, significantly increased expression of Skp2, AR, and Slug, along with E-cadherin downregulation, was observed in primary prostate cancer in patients who already had lymph node metastases. Skp2 was slightly correlated with Slug and AR in the whole cohort (Rs 0.32 and 0.37, respectively), which was enhanced for both proteins in patients with high Gleason scores (Rs 0.56 and 0.53, respectively) and, in the case of Slug, also in patients with metastasis to lymph nodes (Rs 0.56). Coexpression of Skp2 and Slug was confirmed in prostate cancer tissues by multiplex immunohistochemistry and confocal microscopy. The same relationship between these two proteins was observed in three sets of prostate epithelial cell lines (PC3, DU145, and E2) and their mesenchymal counterparts. Chemical inhibition of Skp2, but not RNA interference, modestly decreased Slug protein in PC3 and its docetaxel-resistant subline PC3 DR12. Importantly, chemical inhibition of Skp2 by MLN4924 upregulated p27 and decreased Slug expression in PC3, PC3 DR12, and LAPC4 cells. Novel treatment strategies targeting Skp2 and Slug by the neddylation blockade may be promising in advanced prostate cancer, as recently documented for other aggressive solid tumors.

Virtually all aspects of cell biology are regulated by a ubiquitin code where distinct ubiquitin chain architectures guide the binding events and itineraries of modified substrates. Various combinations of E2 and E3 enzymes accomplish chain formation by forging isopeptide bonds between the C terminus of their transiently linked donor ubiquitin and a specific nucleophilic amino acid on the acceptor ubiquitin, yet it is unknown whether the fundamental feature of most acceptors-the lysine side chain-affects catalysis. Here, use of synthetic ubiquitins with non-natural acceptor site replacements reveals that the aliphatic side chain specifying reactive amine geometry is a determinant of the ubiquitin code, through unanticipated and complex reliance of many distinct ubiquitin-carrying enzymes on a canonical acceptor lysine.

PTEN tumor suppressor opposes the PI3K/Akt signaling pathway in the cytoplasm and maintains chromosomal integrity in the nucleus. Nucleus-cytoplasm shuttling of PTEN is regulated by ubiquitylation, SUMOylation and phosphorylation, and nuclear PTEN has been proposed to exhibit tumor-suppressive functions. Here we show that PTEN is conjugated by Nedd8 under high glucose conditions, which induces PTEN nuclear import without effects on PTEN stability. PTEN neddylation is promoted by the XIAP ligase and removed by the NEDP1 deneddylase. We identify Lys197 and Lys402 as major neddylation sites on PTEN. Neddylated PTEN accumulates predominantly in the nucleus and promotes rather than suppresses cell proliferation and metabolism. The nuclear neddylated PTEN dephosphorylates the fatty acid synthase (FASN) protein, inhibits the TRIM21-mediated ubiquitylation and degradation of FASN, and then promotes de novo fatty acid synthesis. In human breast cancer tissues, neddylated PTEN correlates with tumor progression and poor prognosis. Therefore, we demonstrate a previously unidentified pool of nuclear PTEN in the Nedd8-conjugated form and an unexpected tumor-promoting role of neddylated PTEN.

TAS4464, a potent, selective small molecule NEDD8-activating enzyme (NAE) inhibitor, leads to inactivation of cullin-RING E3 ubiquitin ligases (CRLs) and consequent accumulations of its substrate proteins. Here, we investigated the antitumor properties and action mechanism of TAS4464 in acute myeloid leukemia (AML). TAS4464 induced apoptotic cell death in various AML cell lines. TAS4464 treatments resulted in the activation of both the caspase-9-mediated intrinsic apoptotic pathway and caspase-8-mediated extrinsic apoptotic pathway in AML cells; combined treatment with inhibitors of these caspases markedly diminished TAS4464-induced apoptosis. In each apoptotic pathway, TAS4464 induced the mRNA transcription of the intrinsic proapoptotic factor NOXA and decreased that of the extrinsic antiapoptotic factor c-FLIP. RNA-sequencing analysis showed that the signaling pathway of the CRL substrate c-Myc was enriched after TAS4464 treatment. Chromatin immunoprecipitation (ChIP) assay revealed that TAS4464-induced c-Myc bound to the PMAIP1 (encoding NOXA) and CFLAR (encoding c-FLIP) promoter regions, and siRNA-mediated c-Myc knockdown neutralized both TAS4464-mediated NOXA induction and c-FLIP downregulation. TAS4464 activated both caspase-8 and caspase-9 along with an increase in NOXA and a decrease in c-FLIP, resulting in complete tumor remission in a human AML xenograft model. These findings suggest that NAE inhibition leads to anti-AML activity via a novel c-Myc-dependent apoptosis induction mechanism.

The ubiquitin-like molecule NEDD8 controls several biological processes and is a promising target for therapeutic intervention. NEDDylation occurs through specific NEDD8 enzymes (canonical) or enzymes of the ubiquitin system (atypical). Identification of NEDD8 sites on substrates is critical for delineating the processes controlled by NEDDylation. By combining the use of the NEDD8 R74K mutant with anti-di-glycine (anti-diGly) antibodies, we identified 1,101 unique NEDDylation sites in 620 proteins. Bioinformatics analysis reveals that canonical and atypical NEDDylation have distinct proteomes; the spliceosome/mRNA surveillance/DNA replication and ribosome/proteasome, respectively. The data also reveal the formation of poly-NEDD8, hybrid NEDD8-ubiquitin, and NEDD8-SUMO-2 chains as potential molecular signals. In particular, NEDD8-SUMO-2 chains are induced upon proteotoxic stress (atypical) through NEDDylation of K11 in SUMO-2, and conjugates accumulate in previously described nucleolus-related inclusions. The study uncovers a diverse proteome for NEDDylation and is consistent with the concept of extensive cross-talk between ubiquitin and Ubls under proteotoxic stress conditions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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 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.)

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 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.)

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.

Heterologous sensitization of adenylyl cyclase (AC) is revealed as enhanced or exaggerated AC/cAMP signaling that occurs following persistent activation of G α i/o -coupled receptors. This paradoxical phenomenon was discovered more than 40 years ago and was proposed as a cellular mechanism to explain the adaptive changes that occur following chronic exposure to drugs of abuse. However, the underlying molecular mechanisms of heterologous sensitization of AC remain largely unknown. In the present study, we performed a genome-wide cell-based RNA interference screen as an unbiased approach to identify genes associated with heterologous sensitization of AC. Following a series of validation and confirmation assays, three genes that form an E3 ligase complex, cullin3 ( CUL3 ), neural precursor-cell-expressed and developmentally downregulated 8 ( NEDD8 ), and really interesting new gene (RING)-box protein 1 ( RBX1 ), were identified as specific modulators of heterologous sensitization of AC. Furthermore, based on the downstream actions of these genes, we evaluated the activity of proteasome inhibitors as well as the specific NEDD8-activating enzyme inhibitor, MLN4924 (Pevonedistat), in AC sensitization. We demonstrate that MG-132 and bortezomib treatments could mimic the inhibitory effects observed with gene knockdown, and MLN4924 was potent and efficacious in blocking the development of heterologous sensitization of endogenous and recombinant AC isoforms, including AC1, AC2, AC5, and AC6. Together, by using genetic and pharmacological approaches, we identified, for the first time, cullin3-RING ligases and the protein degradation pathway as essential modulators for heterologous sensitization of AC. SIGNIFICANCE STATEMENT: Through a genome-wide cell-based RNA interference screening, we identified three genes that form an E3 ligase complex, cullin3, neural precursor-cell-expressed and developmentally downregulated 8 ( NEDD8 ), and really interesting new gene-box protein 1, as specific modulators of heterologous sensitization of AC. The effect of cullin3 , NEDD8, or really interesting new gene-box protein 1 small interfering RNAs on heterologous sensitization was recapitulated by proteasome inhibitors, MG132 and bortezomib, and the specific NEDD8-activating enzyme inhibitor, MLN4924. These results suggest a novel hypothesis in which protein degradation is involved in the sensitization of AC signaling that occurs following chronic activation of Gα i/o -coupled receptors., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

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.

As an important regulator of neddylation, neural precursor cell expressed developmentally downregulated 8 (Nedd8)‑conjugating enzyme E2M (UBE2M) mediates cullin neddylation. Upregulation of the neddylation pathway is associated with tumor progression in intrahepatic cholangiocarcinoma (ICC). The present study was designed to assess the effects of Nedd8‑conjugating enzyme UBE2M knockdown on intrahepatic cholangiocarcinoma cells, and to determine the potential underlying mechanisms. UBE2M and associated protein expression levels were determined via immunohistochemistry and western blotting. ICC cells were transfected with short hairpin RNA to knockdown UBE2M expression. Cell Counting Kit‑8 and colony formation assays, and xenograft experiments were used to examine cell viability and colony survival in vitro, and tumor formation in vivo. Survival was evaluated using Kaplan‑Meier analysis and log‑rank tests. Patients with ICC presenting high expression of UBE2M exhibited worse accumulative recurrence and overall survival compared with patients with low expression. Knockdown of UBE2M expression led to a decrease in the viability and clonogenic survival of QBC939 and HUCCT1 cells, and suppressed tumor formation in vivo. UBE2M silencing caused accumulation of cullin‑RING ligase substrates (chromatin‑licensing and DNA replication factor 1 and origin recognition complex subunit 1), inducing DNA damage responses and apoptosis. The present findings suggested that UBE2M serves an important role in ICC progression and may present as a novel target for the treatment of ICC.

Ubiquitin and ubiquitin-like chains are finely balanced by conjugating and de-conjugating enzymes. Alterations in this balance trigger the response to stress conditions and are often observed in pathologies. How such changes are detected is not well understood. We identify the HSP70 chaperone as a sensor of changes in the balance between mono- and poly-NEDDylation. Upon DNA damage, the induction of the de-NEDDylating enzyme NEDP1 restricts the formation of NEDD8 chains, mainly through lysines K11/K48. This promotes APAF1 oligomerization and apoptosis induction, a step that requires the HSP70 ATPase activity. HSP70 binds to NEDD8, and, in vitro, the conversion of NEDD8 chains into mono-NEDD8 stimulates HSP70 ATPase activity. This effect is independent of NEDD8 conjugation onto substrates. The study indicates that the NEDD8 cycle is a regulatory module of HSP70 function. These findings may be important in tumorigenesis, as we find decreased NEDP1 levels in hepatocellular carcinoma with concomitant accumulation of NEDD8 conjugates., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

NEDD8-activating enzyme (NAE) is an essential E1 enzyme of the NEDD8 conjugation (neddylation) pathway, which controls cancer cell growth and survival through activation of cullin-RING ubiquitin ligase complexes (CRL). In this study, we describe the preclinical profile of a novel, highly potent, and selective NAE inhibitor, TAS4464. TAS4464 selectively inhibited NAE relative to the other E1s UAE and SAE. TAS4464 treatment inhibited cullin neddylation and subsequently induced the accumulation of CRL substrates such as CDT1, p27, and phosphorylated IκBα in human cancer cell lines. TAS4464 showed greater inhibitory effects than those of the known NAE inhibitor MLN4924 both in enzyme assay and in cells. Cytotoxicity profiling revealed that TAS4464 is highly potent with widespread antiproliferative activity not only for cancer cell lines, but also patient-derived tumor cells. TAS4464 showed prolonged target inhibition in human tumor xenograft mouse models; weekly or twice a week TAS4464 administration led to prominent antitumor activity in multiple human tumor xenograft mouse models including both hematologic and solid tumors without marked weight loss. As a conclusion, TAS4464 is the most potent and highly selective NAE inhibitor reported to date, showing superior antitumor activity with prolonged target inhibition. It is, therefore, a promising agent for the treatment of a variety of tumors including both hematologic and solid tumors. These results support the clinical evaluation of TAS4464 in hematologic and solid tumors., (©2019 American Association for Cancer Research.)

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.

Background: The neddylation pathway is overactivated in human cancers. Inhibition of neddylation pathway has emerged as an attractive anticancer strategy. The mechanisms underlying neddylation overactivation in cancer remain elusive. MLN4924/Pevonedistat, a first-in-class NEDD8-activating enzyme (NAE, E1) inhibitor, exerts significant anti-tumor effects, but its mutagenic resistance remains unresolved., Methods: The expression of NEDD8-conjugating enzyme UBC12/UBE2M (E2) and NEDD8 were estimated by bioinformatics analysis and western blot in human lung cancer cell lines. The malignant phenotypes of lung cancer cells were evaluated both in vitro and in vivo upon UBC12 knockdown. Cell-cycle arrest was evaluated by quantitative proteomic analysis and propidium iodide stain and fluorescence - activated cell sorting (FACS). The growth of MLN4924 - resistant H1299 cells was also evaluated upon UBC12 knockdown., Findings: The mRNA level of UBC12 in lung cancer tissues was much higher than that in normal lung tissues, increased with disease deterioration, and positively correlated with NEDD8 expression. Moreover, the overexpression of UBC12 significantly enhanced protein neddylation modification whereas the downregulation of UBC12 reduced neddylation modification of target proteins. Functionally, neddylation inactivation by UBC12 knockdown suppressed the malignant phenotypes of lung cancer cells both in vitro and in vivo. The quantitative proteomic analysis and cell cycle profiling showed that UBC12 knockdown disturbed cell cycle progression by triggering G 2 phase cell-cycle arrest. Further mechanistical studies revealed that UBC12 knockdown inhibited Cullin neddylation, led to the inactivation of CRL E3 ligases and induced the accumulation of tumor-suppressive CRL substrates (p21, p27 and Wee1) to induce cell cycle arrest and suppress the malignant phenotypes of lung cancer cells. Finally, UBC12 knockdown effectively inhibited the growth of MLN4924-resistant lung cancer cells., Interpretation: These findings highlight a crucial role of UBC12 in fine-tuned regulation of neddylation activation status and validate UBC12 as an attractive alternative anticancer target against neddylation pathway. FUND: Chinese Minister of Science and Technology grant (2016YFA0501800), National Natural Science Foundation of China (Grant Nos. 81401893, 81625018, 81820108022, 81772470, 81572340 and 81602072), Innovation Program of Shanghai Municipal Education Commission (2019-01-07-00-10-E00056), Program of Shanghai Academic/Technology Research Leader (18XD1403800), National Thirteenth Five-Year Science and Technology Major Special Project for New Drug and Development (2017ZX09304001). The funders had no role in study design, data collection, data analysis, interpretation, writing of the report., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

Sorafenib remains the standard care for patients with hepatocellular carcinoma (HCC) even though it has low antitumor efficacy. Protein neddylation is abnormally activated in many types of human cancer. However, whether dysregulation of neddylation is involved in HCC progression and whether targeting neddylation sensitizes HCC cells to sorafenib need to be ascertained. In the present study, it was demonstrated that high expression of neddylation components, neural precursor cell expressed, developmentally downregulated 8 (NEDD8) and NEDD8‑activating enzyme 1 (NAE1), were associated with poor survival of patients with HCC. Inhibition of neddylation by MLN4924, a small‑molecule inhibitor of NAE1, significantly inhibited HCC growth, reduced clonogenic survival, increased apoptosis, and decreased migration capacity. Sorafenib alone exhibited minimal anticancer efficacy. However, a combination of sorafenib with MLN4924 at a low concentration significantly enhanced the inhibition of cell proliferation and migration as well as the induction of apoptosis induced by sorafenib. In vivo HCC xenograft mouse models also showed that MLN4924 increased the antitumor efficacy of sorafenib. Mechanistically, MLN4924 enhanced the antitumor activity of sorafenib in HCC cells via upregulation of cullin‑RING E3 ubiquitin ligase (CRL)/Skp1‑Cullin1‑F box (SCF) E3 ubiquitin ligase substrates p21, p27, Deptor and IκBɑ. Taken together, these findings suggest that combination therapy of MLN4924 with sorafenib appears to present an additive effect with a maximal in the treatment of HCC.

NEDD8 is a ubiquitin-like protein that activates cullin-RING E3 ubiquitin ligases (CRLs). Here, we identify a novel role for NEDD8 in regulating the activity of poly(ADP-ribose) polymerase 1 (PARP-1) in response to oxidative stress. We show that treatment of cells with H 2 O 2 results in the accumulation of NEDD8 chains, likely by directly inhibiting the deneddylase NEDP1. One chain type, an unanchored NEDD8 trimer, specifically bound to the second zinc finger domain of PARP-1 and attenuated its activation. In cells in which Nedp1 is deleted, large amounts of tri-NEDD8 constitutively form, resulting in inhibition of PARP-1 and protection from PARP-1-dependent cell death. Surprisingly, these NEDD8 trimers are additionally acetylated, as shown by mass spectrometry analysis, and their binding to PARP-1 is reduced by the overexpression of histone de-acetylases, which rescues PARP-1 activation. Our data suggest that trimeric, acetylated NEDD8 attenuates PARP-1 activation after oxidative stress, likely to delay the initiation of PARP-1-dependent cell death., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Cullin-RING E3 ubiquitin ligases (CRLs) are large and diverse multisubunit protein complexes that contribute to about one-fifth of ubiquitin-dependent protein turnover in cells. CRLs are activated by the attachment of the ubiquitin-like protein neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) to the cullin subunits. This cullin neddylation is essential for a plethora of CRL-regulated cellular processes and is vital for life. In mammals, neddylation is promoted by the five co-E3 ligases, defective in cullin neddylation 1 domain-containing 1-5 (DCNL1-5); however, their functional regulation within the CRL complex remains elusive. We found here that the ubiquitin-associated (UBA) domain-containing DCNL1 is monoubiquitylated when bound to CRLs and that this monoubiquitylation depends on the CRL-associated Ariadne RBR ligases TRIAD1 (ARIH2) and HHARI (ARIH1) and strictly requires the DCNL1's UBA domain. Reconstitution of DCNL1 monoubiquitylation in vitro revealed that autoubiquitylated TRIAD1 mediates binding to the UBA domain and subsequently promotes a single ubiquitin attachment to DCNL1 in a mechanism previously dubbed coupled monoubiquitylation. Moreover, we provide evidence that DCNL1 monoubiquitylation is required for efficient CRL activity, most likely by remodeling CRLs and their substrate receptors. Collectively, this work identifies DCNL1 as a critical target of Ariadne RBR ligases and coupled monoubiquitylation of DCNL1 as an integrated mechanism that affects CRL activity and client-substrate ubiquitylation at multiple levels., (© 2019 Kelsall et al.)

Neddylation has been researched in many different human carcinomas. However, the roles of neural precursor cell expressed, developmentally downregulated 8 (NEDD8) in bladder cancer are still unknown. Our study was the first study which systematically investigated the possible functions of NEDD8 in bladder cancer (BC) progression. We carried out immunohistochemistry to explore associations between the expression of NEDD8 in tumor tissues and clinical outcomes of patients. RT-qPCR and western blot were used to detect the expressional levels of genes. The biological abilities of cell proliferation, migration and invasion were researched by in vitro and in vivo experiments. Results were as follows: Data from The Cancer Genome Atlas (TCGA) database showed that NEDD8 was overexpressed in BC tissues and was associated with poor patient survival. Results of immunohistochemistry found that NEDD8 was significantly associated with poor clinical outcomes of BC patients. Suppression of NEDD8 could inhibit the proliferation, migration and invasion of tumor cells. Knocking down NEDD8 could induce apoptosis and G2 phase arrest of cell cycle progression. In vivo, suppression of NEDD8 restricted growth and metastasis of tumors in mice. In conclusion, NEDD8 has important roles in regulating the progression of BC cells and was associated with poor prognosis of patients; hence, it may become a potential therapeutic target of BC., (© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Spatial management of stress-induced protein aggregation is an integral part of the proteostasis network. Protein modification by the ubiquitin-like molecule NEDD8 increases upon proteotoxic stress and it is characterised by the formation of hybrid NEDD8/ubiquitin conjugates. However, the biological significance of this response is unclear. Combination of quantitative proteomics with biological analysis shows that, during proteotoxic stress, NEDDylation promotes nuclear protein aggregation, including ribosomal proteins as a major group. This correlates with protection of the nuclear Ubiquitin Proteasome System from stress-induced dysfunction. Correspondingly, we show that NEDD8 compromises ubiquitination and prevents targeting and processing of substrates by the proteasome. Moreover, we identify HUWE1 as a key E3-ligase that is specifically required for NEDDylation during proteotoxic stress. The study reveals a specific role for NEDD8 in nuclear protein aggregation upon stress and is consistent with the concept that transient aggregate formation is part of a defence mechanism against proteotoxicity.

The ubiquitin-related protein NEDD8 is conjugated and deconjugated to and from proteins in processes related to ubiquitin conjugation and deconjugation. Neddylation is a well-studied posttranslational modification of Cullin-RING E3 ligases (CRLs). Biochemical and structural studies aiming at understanding the role of NEDD8 in CRL function have now resulted in a convincing model of how neddylation and deneddylation antagonistically regulate CRL stability, conformation, activity as well as degradation substrate receptor exchange. Studies of the Arabidopsis thaliana deneddylation-deficient den1 mutant led to the identification of many low abundant, non-Cullin NEDD8 conjugates. Examination of neddylated AUXIN RESISTANT1 (AXR1), a prominent neddylated protein in den1, suggests, however, that AXR1 neddylation may be an auto-catalytic side-reaction of Cullin-targeted neddylation and that DEN1 may serve to antagonize non-productive, auto-neddylation from substrates to provide free NEDD8 for CRL regulation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Neddylation is a posttranslational modification that regulates protein stability, activity, and subcellular localization. Here, we describe a new tool for exploring the neddylation cycle of cullin1 (Cul1) directly in a cellular context. This assay utilizes the NanoLuc ® Binary Technology (NanoBiT) to monitor the covalent neddylation status of Cul1. A stable clonal cell line derived from HEK293 was developed that expressed a C-terminus LgBiT tagged-Cul1 and N-terminus SmBiT tagged-Nedd8. Using this cell line, we screened inhibitors that are known to disrupt Nedd8 biology and demonstrated that both inhibitors of Nedd8-activating enzyme (NAE) and Constitutive photomorphogenesis 9 signalosome (CSN) complex produce concentration and time dependent signal decreases and increases, respectively. The kinetics of both responses could be monitored in real time and demonstrated that modulation of the Nedd8 pathway occurs rapidly. Further characterization of the cellular components of this cell line was performed in order to quantify the various levels of Cul1, Nedd8 and NAE and determined to be near endogenous levels. There was no difference between control and stably transfected cell lines in viability studies of NAE and CSN inhibitors. Taken together, these results suggest that the NanoBiT assay can be used to monitor Cul1 neddylation specifically and in real time., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Despite the similar enzyme cascade in the Ubiquitin and Ubiquitin-like peptide(Ubl) conjugation, the involvement of single or heterodimer E1 activating enzyme has been a mystery. Here, by using a quantitative Förster Resonance Energy Transfer (FRET) technology, aided with Analysis of Electrostatic Similarities Of Proteins (AESOP) computational framework, we elucidate in detail the functional properties of each subunit of the E1 heterodimer activating-enzyme for NEDD8, UBA3 and APPBP1. In contrast to SUMO activation, which requires both subunits of its E1 heterodimer AOS1-Uba2 for its activation, NEDD8 activation requires only one of two E1 subunits, UBA3. The other subunit, APPBP1, only contributes by accelerating the activation reaction rate. This discovery implies that APPBP1 functions mainly as a scaffold protein to enhance molecular interactions and facilitate catalytic reaction. These findings for the first time reveal critical new mechanisms and a potential evolutionary pathway for Ubl activations. Furthermore, this quantitative FRET approach can be used for other general biochemical pathway analysis in a dynamic mode.

During development, ventricular chamber maturation is a crucial step in the formation of a functionally competent postnatal heart. Defects in this process can lead to left ventricular noncompaction cardiomyopathy and heart failure. However, molecular mechanisms underlying ventricular chamber development remain incompletely understood. Neddylation is a posttranslational modification that attaches ubiquitin-like protein NEDD8 to protein targets via NEDD8-specific E1-E2-E3 enzymes. Here, we report that neddylation is temporally regulated in the heart and plays a key role in cardiac development. Cardiomyocyte-specific knockout of NAE1, a subunit of the E1 neddylation activating enzyme, significantly decreased neddylated proteins in the heart. Mice lacking NAE1 developed myocardial hypoplasia, ventricular noncompaction, and heart failure at late gestation, which led to perinatal lethality. NAE1 deletion resulted in dysregulation of cell cycle-regulatory genes and blockade of cardiomyocyte proliferation in vivo and in vitro, which was accompanied by the accumulation of the Hippo kinases Mst1 and LATS1/2 and the inactivation of the YAP pathway. Furthermore, reactivation of YAP signaling in NAE1-inactivated cardiomyocytes restored cell proliferation, and YAP-deficient hearts displayed a noncompaction phenotype, supporting an important role of Hippo-YAP signaling in NAE1-depleted hearts. Mechanistically, we found that neddylation regulates Mst1 and LATS2 degradation and that Cullin 7, a NEDD8 substrate, acts as the ubiquitin ligase of Mst1 to enable YAP signaling and cardiomyocyte proliferation. Together, these findings demonstrate a role for neddylation in heart development and, more specifically, in the maturation of ventricular chambers and also identify the NEDD8 substrate Cullin 7 as a regulator of Hippo-YAP signaling., Competing Interests: The authors declare no conflict of interest.