Systemwide disassembly and assembly of SCF ubiquitin ligase complexes.

Cells respond to environmental cues by remodeling their inventories of multiprotein complexes. Cellular repertoires of SCF (SKP1-CUL1-F box protein) ubiquitin ligase complexes, which mediate much protein degradation, require CAND1 to distribute the limiting CUL1 subunit across the family of ∼70 different F box proteins. Yet, how a single factor coordinately assembles numerous distinct multiprotein complexes remains unknown. We obtained cryo-EM structures of CAND1-bound SCF complexes in multiple states and correlated mutational effects on structures, biochemistry, and cellular assays. The data suggest that CAND1 clasps idling catalytic domains of an inactive SCF, rolls around, and allosterically rocks and destabilizes the SCF. New SCF production proceeds in reverse, through SKP1-F box allosterically destabilizing CAND1. The CAND1-SCF conformational ensemble recycles CUL1 from inactive complexes, fueling mixing and matching of SCF parts for E3 activation in response to substrate availability. Our data reveal biogenesis of a predominant family of E3 ligases, and the molecular basis for systemwide multiprotein complex assembly.

Cullin-RING Ubiquitin Ligase Regulatory Circuits: A Quarter Century Beyond the F-Box Hypothesis.

Cullin-RING ubiquitin ligases (CRLs) are dynamic modular platforms that regulate myriad biological processes through target-specific ubiquitylation. Our knowledge of this system emerged from the F-box hypothesis, posited a quarter century ago: Numerous interchangeable F-box proteins confer specific substrate recognition for a core CUL1-based RING E3 ubiquitin ligase. This paradigm has been expanded through the evolution of a superfamily of analogous modular CRLs, with five major families and over 200 different substrate-binding receptors in humans. Regulation is achieved by numerous factors organized in circuits that dynamically control CRL activation and substrate ubiquitylation. CRLs also serve as a vast landscape for developing small molecules that reshape interactions and promote targeted ubiquitylation-dependent turnover of proteins of interest. Here, we review molecular principles underlying CRL function, the role of allosteric and conformational mechanisms in controlling substrate timing and ubiquitylation, and how the dynamics of substrate receptor interchange drives the turnover of selected target proteins to promote cellular decision-making.

E3 ligase autoinhibition by C-degron mimicry maintains C-degron substrate fidelity.

E3 ligase recruitment of proteins containing terminal destabilizing motifs (degrons) is emerging as a major form of regulation. How those E3s discriminate bona fide substrates from other proteins with terminal degron-like sequences remains unclear. Here, we report that human KLHDC2, a CRL2 substrate receptor targeting C-terminal Gly-Gly degrons, is regulated through interconversion between two assemblies. In the self-inactivated homotetramer, KLHDC2's C-terminal Gly-Ser motif mimics a degron and engages the substrate-binding domain of another protomer. True substrates capture the monomeric CRL2KLHDC2, driving E3 activation by neddylation and subsequent substrate ubiquitylation. Non-substrates such as NEDD8 bind KLHDC2 with high affinity, but its slow on rate prevents productive association with CRL2KLHDC2. Without substrate, neddylated CRL2KLHDC2 assemblies are deactivated via distinct mechanisms: the monomer by deneddylation and the tetramer by auto-ubiquitylation. Thus, substrate specificity is amplified by KLHDC2 self-assembly acting like a molecular timer, where only bona fide substrates may bind before E3 ligase inactivation.

PIKES Analysis Reveals Response to Degraders and Key Regulatory Mechanisms of the CRL4 Network.

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.

NEDD8 enhances Hippo signaling by mediating YAP1 neddylation.

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.

Modulation of Cullin-RING E3 ubiquitin ligase-dependent ubiquitination by small molecule compounds.

Cullin (CUL)-RING (Really Interesting New Gene) E3 ubiquitin (Ub) ligases (CRLs) are the largest E3 family. The E3 CRL core ligase is a subcomplex formed by the CUL C-terminal domain bound with the ROC1/RBX1 RING finger protein, which acts as a hub that mediates and organizes multiple interactions with E2, Ub, Nedd8, and the ARIH family protein, thereby resulting in Ub transfer to the E3-bound substrate. This report describes the modulation of CRL-dependent ubiquitination by small molecule compounds including KH-4-43, #33, and suramin, which target the CRL core ligases. We show that both KH-4-43 and #33 inhibit the ubiquitination of CK1α by CRL4CRBN. However, either compound's inhibitory effect on this reaction is significantly reduced when a neddylated form of CRL4CRBN is used. On the other hand, both #33 and KH-4-43 inhibit the ubiquitination of ß-catenin by CRL1ß-TrCP and Nedd8-CRL1ß-TrCP almost equally. Thus, neddylation of CRL1ß-TrCP does not negatively impact the sensitivity to inhibition by #33 and KH-4-43. These findings suggest that the effects of neddylation to alter the sensitivity of CRL inhibition by KH-4-43/#33 is dependent upon the specific CRL type. Suramin, a compound that targets CUL's basic canyon, can effectively inhibit CRL1/4-dependent ubiquitination regardless of neddylation status, in contrast to the results observed with KH-4-43/#33. This observed differential drug sensitivity of KH-4-43/#33 appears to echo CUL-specific Nedd8 effects on CRLs as revealed by recent high-resolution structural biology efforts. The highly diversified CRL core ligase structures may provide opportunities for specific targeting by small molecule modulators.

Mixed in chains: NEDD8 polymers in the Protein Quality Control system.

Post-translational modification of proteins with the Ubiquitin-like molecule NEDD8 is a critical regulatory mechanism for several biological processes and a potential target for therapeutic intervention. The role of NEDD8 has been mainly characterised through its modification as single moiety on the cullin family of proteins and control of Cullin-Ring-Ligases, but also on non-cullin substrates. In addition to monoNEDDylation, recent studies have now revealed that NEDD8 can also generate diverse polymers. This is either through modification of the 9 available lysines in NEDD8 and the formation of polyNEDD8 chains, or NEDDylation of Ubiquitin and SUMO-2 for the generation of hybrid NEDD8 chains. Here, we review recent findings that characterise the formation of NEDD8 polymers under distinct modes of protein NEDDylation (canonical/atypical) and their potential role as regulatory signals of the proteotoxic stress response and the Protein Quality Control system.

DYRK2 maintains genome stability via neddylation of cullins in response to DNA damage.

Neural precursor cell-expressed developmentally down-regulated 8 (NEDD8), an ubiquitin-like protein, is an essential regulator of the DNA damage response. Numerous studies have shown that neddylation (conjugation of NEDD8 to target proteins) dysfunction causes several human diseases, such as cancer. Hence clarifying the regulatory mechanism of neddylation could provide insight into the mechanism of genome stability underlying the DNA damage response (DDR) and carcinogenesis. Here, we demonstrate that dual-specificity tyrosine-regulated kinase 2 (DYRK2) is a novel regulator of neddylation and maintains genome stability. Deletion of DYRK2 leads to persistent DNA double-strand breaks (DSBs) and subsequent genome instability. Mechanistically, DYRK2 promotes neddylation through forming a complex with NAE1, which is a component of NEDD8-activating enzyme E1, and maintaining its protein level by suppressing polyubiquitylation. The present study is the first to demonstrate that DYRK2 controls neddylation and is necessary for maintaining genome stability. This article has an associated First Person interview with the first author of the paper.

Biochemical and Structural Consequences of NEDD8 Acetylation.

Similar to ubiquitin, the ubiquitin-like protein NEDD8 is not only conjugated to other proteins but is itself subject to posttranslational modifications including lysine acetylation. Yet, compared to ubiquitin, only little is known about the biochemical and structural consequences of site-specific NEDD8 acetylation. Here, we generated site-specifically mono-acetylated NEDD8 variants for each known acetylation site by genetic code expansion. We show that, in particular, acetylation of K11 has a negative impact on the usage of NEDD8 by the NEDD8-conjugating enzymes UBE2M and UBE2F and that this is likely due to electrostatic and steric effects resulting in conformational changes of NEDD8. Finally, we provide evidence that p300 acts as a position-specific NEDD8 acetyltransferase.

Identification of novel benzothiazole derivatives as inhibitors of NEDDylation pathway to inhibit the progression of gastric cancer.

The overexpression of neddylation modification is frequently observed in human tumor cells. Targeting the neddylation pathway has been recognized as a promising anticancer therapeutic strategy, thus discovering potent and selective neddylation inhibitors is of great importance. In this study, we designed and synthesized a series of novel neddylation inhibitors bearing benzothiazole scaffolds by molecular hybridization strategy and all compounds were evaluated for antiproliferative activity against MGC-803, MCF-7, A549 and KYSE-30 cell lines. In vitro anti-tumor studies showed that the most promising compound X-10, effectively suppressed MGC-803 cells growth and migration, induced apoptosis and arrested cell cycle at G2/M phase. Importantly, by directly interacting with NAE1, X-10 blocked NAE1 activity, specifically preventing neddylation of Cullin 3 and Cullin 1, and produced a dose-response decline in the level of UBC12-NEDD8 complex. Overall, our data indicate that X-10 inhibits the process of neddylation, making it a potentially agent for both cancer prevention and therapy purposes.

Deneddylating enzyme SENP8 regulates neuronal development.

Neddylation is a cellular process in which the neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) is conjugated to the lysine residue of target proteins via serial enzymatic cascades. Recently, it has been demonstrated that neddylation is required for synaptic clustering of metabotropic glutamate receptor 7 (mGlu7) and postsynaptic density protein 95 (PSD-95), and the inhibition of neddylation impairs neurite outgrowth and excitatory synaptic maturation. Similar to the balanced role of deubiquitylating enzymes (DUBs) in the ubiquitination process, we hypothesized that deneddylating enzymes can regulate neuronal development by counteracting the process of neddylation. We find that the SUMO peptidase family member, NEDD8 specific (SENP8) acts as a key neuronal deneddylase targeting the global neuronal substrates in primary rat cultured neurons. We demonstrate that SENP8 expression levels are developmentally regulated, peaking around the first postnatal week and gradually diminishing in mature brain and neurons. We find that SENP8 negatively regulates neurite outgrowth through multiple pathways, including actin dynamics, Wnt/ß-catenin signaling, and autophagic processes. Alterations in neurite outgrowth by SENP8 subsequently result in the impairment of excitatory synapse maturation. Our data indicate that SENP8 plays an essential role in neuronal development and is a promising therapeutic target for neurodevelopmental disorders.

Unanchored tri-NEDD8 inhibits PARP-1 to protect from oxidative stress-induced cell death.

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 H2O2 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.

Zebrafish Nedd8 facilitates ovarian development and the maintenance of female secondary sexual characteristics via suppression of androgen receptor activity.

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.

FOXO1-regulated lncRNA CYP1B1-AS1 suppresses breast cancer cell proliferation by inhibiting neddylation.

PURPOSE: Overactivated neddylation is considered to be a common event in cancer. Long non-coding RNAs (lncRNAs) can regulate cancer development by mediating post-translational modifications. However, the role of lncRNA in neddylation modification remains unclear. METHODS: LncRNA cytochrome P450 family 1 subfamily B member 1 antisense RNA 1 (CYP1B1-AS1) expression in breast cancer tissues was evaluated by RT-PCR and TCGA BRCA data. Gain and loss of function experiments were performed to explore the role of CYP1B1-AS1 in breast cancer cell proliferation and apoptosis in vitro and in vivo. Luciferase assay, CHIP-qPCR assay, transcriptome sequencing, RNA-pulldown assay, mass spectrometry, RIP-PCR and Western blot were used to investigate the regulatory factors of CYP1B1-AS1 expression and the molecular mechanism of CYP1B1-AS1 involved in neddylation modification. RESULTS: We found that CYP1B1-AS1 was down-regulated in breast cancer tissues and correlated with prognosis. In vivo and in vitro functional experiments confirmed that CYP1B1-AS1 inhibited cell proliferation and induced apoptosis. Mechanistically, CYP1B1-AS1 was regulated by the transcription factor, forkhead box O1 (FOXO1), and could be upregulated by inhibiting the PI3K/FOXO1 pathway. Moreover, CYP1B1-AS1 bound directly to NEDD8 activating enzyme E1 subunit 1 (NAE1) to regulate protein neddylation. CONCLUSION: This study reports for the first time that CYP1B1-AS1 inhibits protein neddylation to affect breast cancer cell proliferation, which provides a new strategy for the treatment of breast cancer by lncRNA targeting neddylation modification.

USP48 and A20 synergistically promote cell survival in Helicobacter pylori infection.

The human pathogen Helicobacter pylori represents a risk factor for the development of gastric diseases including cancer. The H. pylori-induced transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is involved in the pro-inflammatory response and cell survival in the gastric mucosa, and represents a trailblazer of gastric pathophysiology. Termination of nuclear NF-κB heterodimer RelA/p50 activity is regulated by the ubiquitin-RING-ligase complex elongin-cullin-suppressor of cytokine signalling 1 (ECSSOCS1), which leads to K48-ubiquitinylation and degradation of RelA. We found that deubiquitinylase (DUB) ubiquitin specific protease 48 (USP48), which interacts with the COP9 signalosome (CSN) subunit CSN1, stabilises RelA by deubiquitinylation and thereby promotes the transcriptional activity of RelA to prolong de novo synthesis of DUB A20 in H. pylori infection. An important role of A20 is the suppression of caspase-8 activity and apoptotic cell death. USP48 thus enhances the activity of A20 to reduce apoptotic cell death in cells infected with H. pylori. Our results, therefore, define a synergistic mechanism by which USP48 and A20 regulate RelA and apoptotic cell death in H. pylori infection.

Blocking neddylation elicits antiviral effect against hepatitis B virus replication.

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.

Involvement of the neddylation modification system in Bombyx mori nucleopolyhedrovirus replication.

Neddylation is a posttranslational modification that is similar to ubiquitination, and involved in some critical biological processes, such as DNA repair, transcription regulation, and ubiquitin-proteasome pathway. Recently, it was found that neddylation inhibitor MLN4924 has potent antiviral activity against human viruses including herpes simplex virus (HSV)-1, HSV-2, and influenza viruses. Here, we report that MLN4924 could dramatically and dose-dependently inhibits the propagation, formation of budding virus (BV) and occlusion body (OB) of a lepidopteran virus-Bombyx mori nucleopolyhedrovirus (BmNPV), impaired OB assembly. In addition, the neddylation modification protein NEDD8 is colocalized with aggresome and autophagosome. Our findings suggest that inhibiting neddylation could be an antibaculovirus strategy and MLN4924 may be used as candidate drug for that purpose.

The HSP70 chaperone as sensor of the NEDD8 cycle upon DNA damage.

Molecular chaperones are essential components of the protein quality control system and maintenance of homeostasis. Heat Shock Protein 70 (HSP70), a highly evolutionarily conserved family of chaperones is a key regulator of protein folding, oligomerisation and prevents the aggregation of misfolded proteins. HSP70 chaperone function depends on the so-called 'HSP70-cycle', where HSP70 interacts with and is released from substrates via ATP hydrolysis and the assistance of HSP70 co-factors/co-chaperones, which also provide substrate specificity. The identification of regulatory modules for HSP70 allows the elucidation of HSP70 specificity and target selectivity. Here, we discuss how the HSP70 cycle is functionally linked with the cycle of the Ubiquitin-like molecule NEDD8. Using as an example the DNA damage response, we present a model where HSP70 acts as a sensor of the NEDD8 cycle. The NEDD8 cycle acts as a regulatory module of HSP70 activity, where conversion of poly-NEDD8 chains into mono-NEDD8 upon DNA damage activates HSP70, facilitating the formation of the apoptosome and apoptosis execution.

A first-in-human, phase 1 study of the NEDD8 activating enzyme E1 inhibitor TAS4464 in patients with advanced solid tumors.

Background This open-label, phase 1 study investigated TAS4464, a potent NEDD8-activating enzyme inhibitor, in patients with advanced/metastatic solid tumors (JapicCTI-173,488; registered 13/01/2017). The primary objective was dose-limiting toxicities (DLTs). Maximum-tolerated dose (MTD) was investigated using an accelerated titration design. Methods The starting 10-mg/m2 dose was followed by an initial accelerated stage (weekly dosing; n = 11). Based on liver function test (LFT) results, a 14-day, 20-mg/m2 dose lead-in period was implemented (weekly dosing with lead-in; n = 6). Results Abnormal LFT changes and gastrointestinal effects were the most common treatment-related adverse events (AEs). DLTs with 56-mg/m2 weekly dosing occurred in 1/5 patients; five patients had grade ≥ 2 abnormal LFT changes at 40- and 56-mg/m2 weekly doses. Further dose escalation ceased because of the possibility of severe abnormal LFT changes occurring. DLTs with weekly dosing with lead-in occurred in 1/5 patients at a 56-mg/m2 dose; MTD could not be determined because discontinuation criteria for additional enrollment at that particular dose level were met. As no further enrollment at lower doses occurred, dose escalation assessment was discontinued. Serious treatment-related AEs, AEs leading to treatment discontinuation, and DLTs were all related to abnormal LFT changes, suggesting that TAS4464 administration could affect liver function. This effect was dose-dependent but considered reversible. Complete or partial responses to TAS4464 were not observed; one patient achieved prolonged stable disease. Conclusions MTD could not be determined due to TAS4464 effects on liver function. Further evaluation of the mechanism of NEDD8-activating enzyme inhibitor-induced abnormal liver function is required. Trial registration number JapicCTI-173,488 (registered with Japan Pharmaceutical Information Center). Registration date 13 January 2017.

Quantification of exercise-regulated ubiquitin signaling in human skeletal muscle identifies protein modification cross talk via NEDDylation.

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.