NEDD8 Deamidation Inhibits Cullin RING Ligase Dynamics.

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/αß 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.

A Destiny for Degradation: Interplay between Cullin-RING E3 Ligases and Autophagy.

Autophagy and the ubiquitin-proteasome system (UPS) are two major pathways for protein degradation. The cullin-RING E3 ligases (CRLs) are the largest E3 ligase family and have key biological functions in maintaining protein homeostasis. We provide an updated review of the interactions between CRLs and autophagy, focusing on the regulatory effects of CRLs on the core autophagy machinery that consists of several autophagy-related protein (ATG) complexes and their key upstream signaling pathways. The involvement of such functional interactions in health and disease is also discussed. Understanding the role of CRLs in autophagy is helpful for the development of therapeutic strategies for diseases in which CRLs and autophagy are dysregulated, such as cancer and neurodegenerative conditions.

Targeting Protein Neddylation to Inactivate Cullin-RING Ligases by Gossypol: A Lucky Hit or a New Start?

Cullin-RING E3 ligases (CRLs) are the largest family of E3 ubiquitin ligases, responsible for about 20% of the protein degradation by the ubiquitin-proteasome system (UPS). Given their vital roles in multiple cellular processes, and over-activation in many human cancers, CRLs are validated as promising targets for anti-cancer therapies. Activation of CRLs requires cullin neddylation, a process catalysed by three neddylation enzymes. Recently, our group established an AlphaScreen-based in vitro cullin neddylation assay and employed it for high-throughput screening to search for small-molecule inhibitors targeting cullin neddylation. During our pilot screen, gossypol, a natural product extracted from cottonseeds, was identified as one of the most potent neddylation inhibitors of cullin-1 and cullin-5. We further demonstrated that gossypol blocks cullin neddylation by binding to cullin-1/-5 to inactivate CRL1/5 ligase activity, leading to accumulation of MCL-1 and NOXA, the substrates of CRL1 and CRL5, respectively. The combination of gossypol and an MCL-1 inhibitor synergistically enhanced the anti-proliferative effect in multiple human cancer cell lines. Our study unveiled a rational combination of two previously known inhibitors of the Bcl-2 family for enhanced anti-cancer efficacy and identified a novel activity of gossypol as an inhibitor of CRL1 and CRL5 E3s, thus providing a new possibility in the development of novel CRL inhibitors for anti-cancer therapy.

A first-in-class inhibitor, MLN4924 (pevonedistat), induces cell-cycle arrest, senescence, and apoptosis in human renal cell carcinoma by suppressing UBE2M-dependent neddylation modification.

PURPOSE: MLN4924 is a second-generation inhibitor that targets ubiquitin-proteasome system by inhibiting neddylation activation enzyme (NAE), and subsequently blocking the neddylation-dependent activation of Cullin-RING E3 ligases (CRLs), which leads to the accumulation of CRLs substrates and hence, suppressing diverse tumor development. In this study, we investigated the potential application of this first-in-class inhibitor MLN4924 in the treatment of human renal cell carcinoma both in vitro and in vivo. METHODS: The impact of MLN4924 on renal cancer cells was determined by measuring viability (MTS), proliferation cell count test and clonogenic assays, cell cycle progression (flow cytometry with propidium iodide staining), apoptosis (flow cytometry with annexin V-FITC labeling) and DNA damage (immunofluorescent staining). The cell cycle regulatory molecules, apoptosis-related molecules, and cell stress-related proteins were examined by Western blotting. The influence of tumor cell migration was analyzed by wound healing assays. A well-established SCID xenograft mouse model was used to evaluate the effects of MLN4924 on tumor growth in vivo. RESULTS: The data showed that MLN4924 induced a dose-dependent cytotoxicity, anti-proliferation, anti-migration, and apoptosis in human renal cancer cells; and caused cell cycle arrested at the G2 phase. In addition, the E2 conjugating enzymes of Neddylation UBE2M played a major role in the proliferation control of renal cancer cells. Finally, we confirmed MLN4924 inhibited tumor growth in a RCC xenograft mouse model with minimal general toxicity. CONCLUSION: We concluded that MLN4924 induces apoptosis and cell cycle arrest. These findings implied that MLN4924 provides a novel strategy for the treatment of RCC.

The Arabidopsis ALF4 protein is a regulator of SCF E3 ligases.

The cullin-RING E3 ligases (CRLs) regulate diverse cellular processes in all eukaryotes. CRL activity is controlled by several proteins or protein complexes, including NEDD8, CAND1, and the CSN Recently, a mammalian protein called Glomulin (GLMN) was shown to inhibit CRLs by binding to the RING BOX (RBX1) subunit and preventing binding to the ubiquitin-conjugating enzyme. Here, we show that Arabidopsis ABERRANT LATERAL ROOT FORMATION4 (ALF4) is an ortholog of GLMN The alf4 mutant exhibits a phenotype that suggests defects in plant hormone response. We show that ALF4 binds to RBX1 and inhibits the activity of SCFTIR1, an E3 ligase responsible for degradation of the Aux/IAA transcriptional repressors. In vivo, the alf4 mutation destabilizes the CUL1 subunit of the SCF Reduced CUL1 levels are associated with increased levels of the Aux/IAA proteins as well as the DELLA repressors, substrate of SCFSLY1 We propose that the alf4 phenotype is partly due to increased levels of the Aux/IAA and DELLA proteins.

Structure-Guided Design of Peptides as Tools to Probe the Protein-Protein Interaction between Cullin-2 and Elongin†BC Substrate Adaptor in Cullin RING E3 Ubiquitin Ligases.

Cullin RING E3 ubiquitin ligases (CRLs) are large dynamic multi-subunit complexes that control the fate of many proteins in cells. CRLs are attractive drug targets for the development of small-molecule inhibitors and chemical inducers of protein degradation. Herein we describe a structure-guided biophysical approach to probe the protein-protein interaction (PPI) between the Cullin-2 scaffold protein and the adaptor subunits Elongin BC within the context of the von Hippel-Lindau complex (CRL2VHL ) using peptides. Two peptides were shown to bind at the targeted binding site on Elongin C, named the "EloC site", with micromolar dissociation constants, providing a starting point for future optimization. Our results suggest ligandability of the EloC binding site to short linear peptides, unveiling the opportunity and challenges to develop small molecules that have the potential to target selectively the Cul2-adaptor PPI within CRLs.

RNAi-Based Suppressor Screens Reveal Genetic Interactions Between the CRL2LRR-1 E3-Ligase and the DNA Replication Machinery in Caenorhabditis elegans.

Cullin-RING E3-Ligases (CRLs), the largest family of E3 ubiquitin-Ligases, regulate diverse cellular processes by promoting ubiquitination of target proteins. The evolutionarily conserved Leucine Rich Repeat protein 1 (LRR-1) is a substrate-recognition subunit of a CRL2LRR-1 E3-ligase. Here we provide genetic evidence supporting a role of this E3-enzyme in the maintenance of DNA replication integrity in Caenorhabditis elegans Through RNAi-based suppressor screens of lrr-1(0) and cul-2(or209ts) mutants, we identified two genes encoding components of the GINS complex, which is part of the Cdc45-MCM-GINS (CMG) replicative helicase, as well as CDC-7 and MUS-101, which drives the assembly of the CMG helicase during DNA replication. In addition, we identified the core components of the ATR/ATL-1 DNA replication checkpoint pathway (MUS-101, ATL-1, CLSP-1, CHK-1). These results suggest that the CRL2LRR-1 E3-ligase acts to modify or degrade factor(s) that would otherwise misregulate the replisome, eventually leading to the activation of the DNA replication checkpoint.

HIV-1 Vpr increases HCV replication through VprBP in cell culture.

Coinfection of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) occurs at a high frequency, in which HIV shows a promotion of HCV-derived liver diseases. However, the mechanism of how this occurs is not well understood. Our previous work has demonstrated that the HIV-1 accessory protein Vpr enhances HCV RNA replication in cell culture. Because Vpr performs most of its functions through host protein VprBP (DCAF1), the role of VprBP in the regulation of HCV by Vpr was investigated in this study. We found that the Vpr mutant Q65R, which is deficient in VprBP binding, could not enhance HCV replication. Furthermore, Vpr-mediated enhancement of HCV replication was severely diminished in VprBP knockdown cells. In addition, an inhibitor of Cullin RING E3 ligases, MLN4924, impaired the function of Vpr during HCV replication. Together, these results suggest that Vpr promotes HCV replication in a VprBP-dependent manner, and that the activity of Cullin RING E3 ligases is essential to this process. In conclusion, our findings demonstrate that HIV-1 Vpr makes the cellular environment more suitable for HCV replication, which might relate with the host ubiquitination system.

Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function.

The family of cullin-RING E3 Ligases (CRLs) and the constitutive photomorphogenesis 9 (COP9) signalosome (CSN) form dynamic complexes that mediate ubiquitylation of 20% of the proteome, yet regulation of their assembly/disassembly remains poorly understood. Inositol polyphosphates are highly conserved signaling molecules implicated in diverse cellular processes. We now report that inositol hexakisphosphate (IP6) is a major physiologic determinant of the CRL-CSN interface, which includes a hitherto unidentified electrostatic interaction between the N-terminal acidic tail of CSN subunit 2 (CSN2) and a conserved basic canyon on cullins. IP6, with an EC50 of 20 nM, acts as an intermolecular "glue," increasing cullin-CSN2 binding affinity by 30-fold, thereby promoting assembly of the inactive CRL-CSN complexes. The IP6 synthase, Ins(1,3,4,5,6)P5 2-kinase (IPPK/IP5K) binds to cullins. Depleting IP5K increases the percentage of neddylated, active Cul1 and Cul4A, and decreases levels of the Cul1/4A substrates p27 and p21. Besides dysregulating CRL-mediated cell proliferation and UV-induced apoptosis, IP5K depletion potentiates by 28-fold the cytotoxic effect of the neddylation inhibitor MLN4924. Thus, IP5K and IP6 are evolutionarily conserved components of the CRL-CSN system and are potential targets for cancer therapy in conjunction with MLN4924.

A lysine-to-arginine mutation on NEDD8 markedly reduces the activity of cullin RING E3 ligase through the impairment of neddylation cascades.

Neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8) is a ubiquitin-like modifier, which forms covalent conjugates on lysines of its substrates. This post-translational modification, neddylation, plays important roles in tumor cell proliferation and viability. Ubiquitin can form diverse polyubiquitin chains, on its seven lysines, which play important functions in various biological processes. However, the roles of lysines in NEDD8 have not been explored. Here, we generated nine NEDD8 point mutants, each with one lysine replaced by an arginine, to study the putative function of lysines in NEDD8. Our experiments discover that Lys27 in NEDD8 is a critical residue for protein neddylation. Replacement of this residue with arginine almost completely eliminates the conjugation of NEDD8 to its substrates. Furthermore, we find that the K27R mutant impairs NEDD8 conjugation to the E2 enzyme, which normally forms thioester bonds for further transferring NEDD8 to its ligases and substrates. Therefore, this mutation completely inhibits global protein neddylation, including neddylation of cullin family proteins, resulting in decreased activity of cullin-RING E3 ligases. This work sheds new light on the roles of NEDD8 lysines on neddylation cascades and provides a dominant negative mutant for the study of neddylation and its biological functions.

Inositol hexakisphosphate kinase-1 mediates assembly/disassembly of the CRL4-signalosome complex to regulate DNA repair and cell death.

Inositol polyphosphates containing an energetic pyrophosphate bond are formed primarily by a family of three inositol hexakisphosphate (IP6) kinases (IP6K1-3). The Cullin-RING ubiquitin ligases (CRLs) regulate diverse biological processes through substrate ubiquitylation. CRL4, comprising the scaffold Cullin 4A/B, the E2-interacting Roc1/2, and the adaptor protein damage-specific DNA-binding protein 1, is activated by DNA damage. Basal CRL4 activity is inhibited by binding to the COP9 signalosome (CSN). UV radiation and other stressors dissociate the complex, leading to E3 ligase activation, but signaling events that trigger signalosome dissociation from CRL4 have been unclear. In the present study, we show that, under basal conditions, IP6K1 forms a ternary complex with CSN and CRL4 in which IP6K1 and CRL4 are inactive. UV dissociates IP6K1 to generate IP7, which then dissociates CSN-CRL4 to activate CRL4. Thus, IP6K1 is a novel CRL4 subunit that transduces UV signals to mediate disassembly of the CRL4-CSN complex, thereby regulating nucleotide excision repair and cell death.