Cullin-RING Ubiquitin Ligases Research Articles

CSN7B defines a variant COP9 signalosome complex with distinct function in DNA damage response

Mammalian COP9 signalosome (CSN) exists as two variant complexes containing either CSN7A or CSN7B paralogs of unknown functional specialization. Constructing knockout cells, we found that CSN7A and CSN7B have overlapping functions in the deneddylation of cullin-RING ubiquitin ligases. Nevertheless, CSNCSN7B has a unique function in DNA double-strand break (DSB) sensing, being selectively required for ataxia telangiectasia mutated (ATM)-dependent formation of NBS1S343p and γH2AX as well as DNA-damage-induced apoptosis triggered by mitomycin C and ionizing radiation. Live-cell microscopy revealed rapid recruitment of CSN7B but not CSN7A to DSBs. Resistance of CSN7B knockout cells to DNA damage is explained by the failure to deneddylate an upstream DSB signaling component, causing a switch in DNA repair pathway choice with increased utilization of non-homologous end joining over homologous recombination. In mice, CSN7B knockout tumors are resistant to DNA-damage-inducing chemotherapy, thus providing an explanation for the poor prognosis of tumors with low CSN7B expression.

Genetic ablation of Cullin-RING E3 ubiquitin ligase 7 restrains pressure overload-induced myocardial fibrosis.

Fibrosis is a pathognomonic feature of structural heart disease and counteracted by distinct cardioprotective mechanisms, e.g. activation of the phosphoinositide 3-kinase (PI3K) / AKT pro-survival pathway. The Cullin-RING E3 ubiquitin ligase 7 (CRL7) was identified as negative regulator of PI3K/AKT signalling in skeletal muscle, but its role in the heart remains to be elucidated. Here, we sought to determine whether CRL7 modulates to cardiac fibrosis following pressure overload and dissect its underlying mechanisms. For inactivation of CRL7, the Cullin 7 (Cul7) gene was deleted in cardiac myocytes (CM) by injection of adeno-associated virus subtype 9 (AAV9) vectors encoding codon improved Cre-recombinase (AAV9-CMV-iCre) in Cul7flox/flox mice. In addition, Myosin Heavy Chain 6 (Myh6; alpha-MHC)-MerCreMer transgenic mice with tamoxifen-induced CM-specific expression of iCre were used as alternate model. After transverse aortic constriction (TAC), causing chronic pressure overload and fibrosis, AAV9-CMV-iCre induced Cul7-/- mice displayed a ~50% reduction of interstitial cardiac fibrosis when compared to Cul7+/+ animals (6.7% vs. 3.4%, p<0.01). Similar results were obtained with Cul7flox/flox Myh6-Mer-Cre-MerTg(1/0) mice which displayed a ~30% reduction of cardiac fibrosis after TAC when compared to Cul7+/+ Myh6-Mer-Cre-MerTg(1/0) controls after TAC surgery (12.4% vs. 8.7%, p<0.05). No hemodynamic alterations were observed. AKTSer473 phosphorylation was increased 3-fold (p<0.01) in Cul7-/- vs. control mice, together with a ~78% (p<0.001) reduction of TUNEL-positive apoptotic cells three weeks after TAC. In addition, CM-specific expression of a dominant-negative CUL71152stop mutant resulted in a 16.3-fold decrease (p<0.001) of in situ end-labelling (ISEL) positive apoptotic cells. Collectively, our data demonstrate that CM-specific ablation of Cul7 restrains myocardial fibrosis and apoptosis upon pressure overload, and introduce CRL7 as a potential target for anti-fibrotic therapeutic strategies of the heart.

Prophylactic and therapeutic HBV vaccination by an HBs-expressing cytomegalovirus vector lacking an interferon antagonist in mice.

Cytomegalovirus (CMV)-based vaccines show promising effects against chronic infections in nonhuman primates. Therefore, we examined the potential of hepatitis B virus (HBV) vaccines based on mouse CMV (MCMV) vectors expressing the small HBsAg. Immunological consequences of vaccine virus attenuation were addressed by either replacing the dispensable gene m157 ("MCMV-HBsȍ) or the gene M27 ("ΔM27-HBs"), the latter encodes a potent IFN antagonist targeting the transcription factor STAT2. M27 was chosen, since human CMV encodes an analogous gene product, which also induced proteasomal STAT2 degradation by exploiting Cullin RING ubiquitin ligases. Vaccinated mice were challenged with HBV through hydrodynamic injection. MCMV-HBs and ΔM27-HBs vaccination achieved accelerated HBV clearance in serum and liver as well as robust HBV-specific CD8+ T-cell responses. When we explored the therapeutic potential of MCMV-based vaccines, especially the combination of ΔM27-HBs prime and DNA boost vaccination resulted in increased intrahepatic HBs-specific CD8+ T-cell responses and HBV clearance in persistently infected mice. Our results demonstrated that vaccines based on a replication competent MCMV attenuated through the deletion of an IFN antagonist targeting STAT2 elicit robust anti-HBV immune responses and mediate HBV clearance in mice in prophylactic and therapeutic immunization regimes.

COVID-19 Effects on Pregnancy, Neonates and Fertility

CUL4B gene is ubiquitin ligase belongs to the Cullinring ubiquitin ligase family. In recent study we have identified a series of MTNR1B, SENP3, AKAP3 and PLOD3 genes and their functional interaction to the ligand binding capacity after prediction of 3D structure to the drug like methotrexate, which plays a significant role during spermatogenesis. Although, the role of Cul4B gene mutation and its impact to the ligand binding during development and differentiation of male gonad (testis) remains unexplored in fertility.

Cdc48 cofactor Shp1 regulates signal-induced SCFMet30 disassembly

Organisms can adapt to a broad spectrum of sudden and dramatic changes in their environment. These abrupt changes are often perceived as stress and trigger responses that facilitate survival and eventual adaptation. The ubiquitin-proteasome system (UPS) is involved in most cellular processes. Unsurprisingly, components of the UPS also play crucial roles during various stress response programs. The budding yeast SCFMet30 complex is an essential cullin-RING ubiquitin ligase that connects metabolic and heavy metal stress to cell cycle regulation. Cadmium exposure results in the active dissociation of the F-box protein Met30 from the core ligase, leading to SCFMet30 inactivation. Consequently, SCFMet30 substrate ubiquitylation is blocked and triggers a downstream cascade to activate a specific transcriptional stress response program. Signal-induced dissociation is initiated by autoubiquitylation of Met30 and serves as a recruitment signal for the AAA-ATPase Cdc48/p97, which actively disassembles the complex. Here we show that the UBX cofactor Shp1/p47 is an additional key element for SCFMet30 disassembly during heavy metal stress. Although the cofactor can directly interact with the ATPase, Cdc48 and Shp1 are recruited independently to SCFMet30 during cadmium stress. An intact UBX domain is crucial for effective SCFMet30 disassembly, and a concentration threshold of Shp1 recruited to SCFMet30 needs to be exceeded to initiate Met30 dissociation. The latter is likely related to Shp1-mediated control of Cdc48 ATPase activity. This study identifies Shp1 as the crucial Cdc48 cofactor for signal-induced selective disassembly of a multisubunit protein complex to modulate activity.

The NEDD8-activating enzyme inhibition with MLN4924 sensitizes human cancer cells of different origins to apoptosis and necroptosis

ObjectivesMLN4924 is an inhibitor of NEDD8-activating enzyme (NAE) that interferes with the cullin-RING ubiquitin ligase complexes formation and the nuclear factor kappa B (NF-κB) activation. Here, we investigated the cytotoxic effect of MLN4924 and its ability to sensitize a broad range of cancer cells of different origins to tumour necrosis factor-α (TNF)-induced cell death alongside unravelling its mechanism of action. Materials and methodsCell viability and caspases processing were determined after MLN4924 treatment either alone or with zVAD-fmk (pan caspase inhibitor), necrostatin-1 (nec-1, RIPK1 inhibitor) and necrosulfonamide (NSA, MLKL inhibitor). Moreover, MLN4924 ability to potentiate TNF-induced cell death was evaluated in 24 cell lines of different cancer origins. The impact of NAE inhibition with MLN4924 on TNF-induced apoptosis and necroptosis was evaluated using zVAD-fmk and nec-1, respectively. ResultsMLN4924 alone was able to induce cell death in different cell lines that was attributed to apoptosis induction. Also, MLN4924 sensitized different cancer cell lines to TNF-induced cell death. MLN4924/TNF-induced cell death was apoptosis and necroptosis dependent that may be attributed to MLN4924 inhibition of NF-κB pathway activation. ConclusionsTargeting NAE and NF-κB pathway with MLN4924 represents a substantial approach to enhance the sensitivity of diverse types of cancer cells. Moreover, the broad in vitro screening of MLN4924 anticancer activity provides a valuable guidance for elucidating the susceptible cancer types for the prospective clinical application of MLN4924.

The COP9 Signalosome: A Multi-DUB Complex.

The COP9 signalosome (CSN) is a signaling platform controlling the cellular ubiquitylation status. It determines the activity and remodeling of ~700 cullin-RING ubiquitin ligases (CRLs), which control more than 20% of all ubiquitylation events in cells and thereby influence virtually any cellular pathway. In addition, it is associated with deubiquitylating enzymes (DUBs) protecting CRLs from autoubiquitylation and rescuing ubiquitylated proteins from degradation. The coordination of ubiquitylation and deubiquitylation by the CSN is presumably important for fine-tuning the precise formation of defined ubiquitin chains. Considering its intrinsic DUB activity specific for deneddylation of CRLs and belonging to the JAMM family as well as its associated DUBs, the CSN represents a multi-DUB complex. Two CSN-associated DUBs, the ubiquitin-specific protease 15 (USP15) and USP48 are regulators in the NF-κB signaling pathway. USP15 protects CRL1β-TrCP responsible for IκBα ubiquitylation, whereas USP48 stabilizes the nuclear pool of the NF-κB transcription factor RelA upon TNF stimulation by counteracting CRL2SOCS1. Moreover, the CSN controls the neddylation status of cells by its intrinsic DUB activity and by destabilizing the associated deneddylation enzyme 1 (DEN1). Thus, the CSN is a master regulator at the intersection between ubiquitylation and neddylation.

CSN7B Defines a Variant COP9 Signalosome Complex with Distinct Function in DNA Damage Response

Mammalian COP9 signalosome (CSN) exists as two variant complexes containing either CSN7A or CSN7B paralogs, but functional specialization of these complexes remains unknown. We found that knockout of CSN7A or CSN7B is tolerated by mammalian cells, indicating redundant functions in the deneddylation of cullin-RING ubiquitin ligases. Nevertheless, CSNCSN7B has a unique function in DNA double strand break (DSB) sensing, being selectively required for ATM-dependent formation of NBS1S343p and γH2AX as well as DNA damage-induced apoptosis. Consistently, live cell confocal microscopy revealed rapid recruitment of CSN7B but not CSN7A to DSBs. Resistance of CSN7B knockout cells to DNA damage is explained by the failure to deneddylate an upstream DSB signaling pathway component. In mice, CSN7B knockout tumors are resistant to DNA damage-inducing chemotherapy. Our loss-of-function study on the role of CSNCSN7B in DNA repair provides a mechanistic underpinning for the poor prognosis of tumors with low CSN7B expression.

Ubiquitin-dependent proteasomal degradation of AMPK gamma subunit by Cereblon inhibits AMPK activity

Cereblon (CRBN), a substrate receptor for Cullin-ring E3 ubiquitin ligase (CRL), is a major target protein of immunomodulatory drugs. An earlier study demonstrated that CRBN directly interacts with the catalytic α subunit of AMP-activated protein kinase (AMPK), a master regulator of energy homeostasis, down-regulating the enzymatic activity of AMPK. However, it is not clear how CRBN modulates AMPK activity. To investigate the mechanism of CRBN-dependent AMPK inhibition, we measured protein levels of each AMPK subunit in brains, livers, lungs, hearts, spleens, skeletal muscles, testes, kidneys, and embryonic fibroblasts from wild-type and Crbn−/− mice. Protein levels and stability of the regulatory AMPKγ subunit were increased in Crbn−/− mice. Increased stability of AMPKγ in Crbn−/− MEFs was dramatically reduced by exogenous expression of Crbn. In wild-type MEFs, the proteasomal inhibitor MG132 blocked degradation of AMPKγ. We also found that CRL4CRBN directly ubiquitinated AMPKγ. Taken together, these findings suggest that CRL4CRBN regulates AMPK through ubiquitin-dependent proteasomal degradation of AMPKγ.

The SCFSkp2 ubiquitin ligase complex modulates TRAIL-R2-induced apoptosis by regulating FLIP(L)

TRAIL-R2 (DR5) is a clinically-relevant therapeutic target and a key target for immune effector cells. Herein, we identify a novel interaction between TRAIL-R2 and the Skp1-Cullin-1-F-box (SCF) Cullin-Ring E3 Ubiquitin Ligase complex containing Skp2 (SCFSkp2). We find that SCFSkp2 can interact with both TRAIL-R2’s pre-ligand association complex (PLAC) and ligand-activated death-inducing signalling complex (DISC). Moreover, Cullin-1 interacts with TRAIL-R2 in its active NEDDylated form. Inhibiting Cullin-1’s DISC recruitment using the NEDDylation inhibitor MLN4924 (Pevonedistat) or siRNA increased apoptosis induction in response to TRAIL. This correlated with enhanced levels of the caspase-8 regulator FLIP at the TRAIL-R2 DISC, particularly the long splice form, FLIP(L). We subsequently found that FLIP(L) (but not FLIP(S), caspase-8, nor the other core DISC component FADD) interacts with Cullin-1 and Skp2. Importantly, this interaction is enhanced when FLIP(L) is in its DISC-associated, C-terminally truncated p43-form. Prevention of FLIP(L) processing to its p43-form stabilises the protein, suggesting that by enhancing its interaction with SCFSkp2, cleavage to the p43-form is a critical step in FLIP(L) turnover. In support of this, we found that silencing any of the components of the SCFSkp2 complex inhibits FLIP ubiquitination, while overexpressing Cullin-1/Skp2 enhances its ubiquitination in a NEDDylation-dependent manner. DISC recruitment of TRAF2, previously identified as an E3 ligase for caspase-8 at the DISC, was also enhanced when Cullin-1’s recruitment was inhibited, although its interaction with Cullin-1 was found to be mediated indirectly via FLIP(L). Notably, the interaction of p43-FLIP(L) with Cullin-1 disrupts its ability to interact with FADD, caspase-8 and TRAF2. Collectively, our results suggest that processing of FLIP(L) to p43-FLIP(L) at the TRAIL-R2 DISC enhances its interaction with co-localised SCFSkp2, leading to disruption of p43-FLIP(L)’s interactions with other DISC components and promoting its ubiquitination and degradation, thereby modulating TRAIL-R2-mediated apoptosis.

A cullin-RING ubiquitin ligase promotes thermotolerance as part of the intracellular pathogen response in Caenorhabditis elegans

Intracellular pathogen infection leads to proteotoxic stress in host organisms. Previously we described a physiological program in the nematode Caenorhabditis elegans called the intracellular pathogen response (IPR), which promotes resistance to proteotoxic stress and appears to be distinct from canonical proteostasis pathways. The IPR is controlled by PALS-22 and PALS-25, proteins of unknown biochemical function, which regulate expression of genes induced by natural intracellular pathogens. We previously showed that PALS-22 and PALS-25 regulate the mRNA expression of the predicted ubiquitin ligase component cullin cul-6, which promotes thermotolerance in pals-22 mutants. However, it was unclear whether CUL-6 acted alone, or together with other cullin-ring ubiquitin ligase components, which comprise a greatly expanded gene family in C. elegans Here we use coimmunoprecipitation studies paired with genetic analysis to define the cullin-RING ligase components that act together with CUL-6 to promote thermotolerance. First, we identify a previously uncharacterized RING domain protein in the TRIM family we named RCS-1, which acts as a core component with CUL-6 to promote thermotolerance. Next, we show that the Skp-related proteins SKR-3, SKR-4, and SKR-5 act redundantly to promote thermotolerance with CUL-6. Finally, we screened F-box proteins that coimmunoprecipitate with CUL-6 and find that FBXA-158 and FBXA-75 promote thermotolerance. In summary, we have defined the three core components and two F-box adaptors of a cullin-RING ligase complex that promotes thermotolerance as part of the IPR in C. elegans, which adds to our understanding of how organisms cope with proteotoxic stress.

NRBP1-Containing CRL2/CRL4A Regulates Amyloid β Production by Targeting BRI2 and BRI3 for Degradation

Alzheimer's disease (AD) is a progressive neurodegenerative disease caused by accumulations of Aβ peptides. Production and fibrillation of Aβ are downregulated by BRI2 and BRI3, which are physiological inhibitors of amyloid precursor protein (APP) processing and Aβ oligomerization. Here, we identify nuclear receptor binding protein 1 (NRBP1) as a substrate receptor of a Cullin-RING ubiquitin ligase (CRL) that targets BRI2 and BRI3 for degradation. Moreover, we demonstrate that (1) dimerized NRBP1 assembles into a functional Cul2- and Cul4A-containing heterodimeric CRL through its BC-box and an overlapping cryptic H-box, (2) both Cul2 and Cul4A contribute to NRBP1 CRL function, and (3) formation of the NRBP1 heterodimeric CRL is strongly enhanced by chaperone-like function of TSC22D3 and TSC22D4. NRBP1 knockdown in neuronal cells results in an increase in the abundance of BRI2 and BRI3 and significantly reduces Aβ production. Thus, disrupting interactions between NRBP1 and its substrates BRI2 and BRI3 may provide a useful therapeutic strategy for AD.

PPARγ and RhoBTB1 in hypertension.

This review provides an up-to-date understanding of how peroxisome proliferator activated receptor γ (PPARγ) exerts its cardioprotective effect in the vasculature through its activation of novel PPARγ target genes in endothelium and vascular smooth muscle. In vascular endothelial cells, PPARγ plays a protective role by increasing nitric oxide bioavailability and preventing oxidative stress. RBP7 is a PPARγ target gene enriched in vascular endothelial cells, which is likely to form a positive feedback loop with PPARγ. In vascular smooth muscle cells, PPARγ antagonizes the renin-angiotensin system, maintains vascular integrity, suppresses vasoconstriction, and promotes vasodilation through distinct pathways. Rho-related BTB domain containing protein 1 (RhoBTB1) is a novel PPARγ gene target in vascular smooth muscle cells that mediates the protective effect of PPARγ by serving as a substrate adaptor between the Cullin-3 RING ubiquitin ligase and phosphodiesterase 5, thus restraining its activity through ubiquitination and proteasomal degradation. In the vasculature, PPARγ exerts its cardioprotective effect through its transcriptional activity in endothelium and vascular smooth muscle. From the understanding of PPARγ's transcription targets in those pathways, novel hypertension therapy target(s) will emerge.

The Impact of Cand1 in Prostate Cancer.

Evidence has accumulated asserting the importance of cullin-RING (really interesting new gene) ubiquitin ligases (CRLs) and their regulator Cullin-associated neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8) dissociated protein 1 (Cand1) in various cancer entities. However, the role of Cand1 in prostate cancer (PCa) has not been intensively investigated so far. Thus, in the present study, we aimed to assess the relevance of Cand1 in the clinical and preclinical setting. Immunohistochemical analyses of radical prostatectomy specimens of PCa patients showed that Cand1 protein levels are elevated in PCa compared to benign areas. In addition, high Cand1 levels were associated with higher Gleason Scores, as well as higher tumor recurrence and decreased overall survival. In line with clinical findings, in vitro experiments in different PCa cell lines revealed that knockdown of Cand1 reduced cell viability and proliferation and increased apoptosis, therefore underlining its role in tumor progression. We also found that the cyclin-dependent kinase inhibitor p21 is significantly upregulated upon downregulation of Cand1. Using bioinformatic tools, we detected genes encoding for proteins linked to mRNA turnover, protein polyubiquitination, and proteasomal degradation to be significantly upregulated in Cand1high tumors. Next generation sequencing of PCa cell lines resistant to the anti-androgen enzalutamide revealed that Cand1 is mutated in enzalutamide-resistant cells, however, with little functional and clinically relevant impact in the process of resistance development. To summarize the present study, we found that high Cand1 levels correlate with PCa aggressiveness.

Cullin-5 Adaptor SPSB1 Controls NF-κB Activation Downstream of Multiple Signaling Pathways.

The initiation of innate immune responses against pathogens relies on the activation of pattern-recognition receptors (PRRs) and corresponding intracellular signaling cascades. To avoid inappropriate or excessive activation of PRRs, these responses are tightly controlled. Cullin-RING E3 ubiquitin ligases (CRLs) have emerged as critical regulators of many cellular functions including innate immune activation and inflammation. CRLs form multiprotein complexes in which a Cullin protein acts as a scaffold and recruits specific adaptor proteins, which in turn recognize specific substrate proteins for ubiquitylation, hence providing selectivity. CRLs are divided into 5 main groups, each of which uses a specific group of adaptor proteins. Here, we systematically depleted all predicted substrate adaptors for the CRL5 family (the so-called SOCS-box proteins) and assessed the impact on the activation of the inflammatory transcription factor NF-κB. Depletion of SPSB1 resulted in a significant increase in NF-κB activation, indicating the importance of SPSB1 as an NF-κB negative regulator. In agreement, overexpression of SPSB1 suppressed NF-κB activity in a potent, dose-dependent manner in response to various agonists. Inhibition by SPSB1 was specific to NF-κB, because other transcription factors related to innate immunity and interferon (IFN) responses such as IRF-3, AP-1, and STATs remained unaffected by SPSB1. SPSB1 suppressed NF-κB activation induced via multiple pathways including Toll-like receptors and RNA and DNA sensing adaptors, and required the presence of its SOCS-box domain. To provide mechanistic insight, we examined phosphorylation and degradation of the inhibitor of κB (IκBα) and p65 translocation into the nucleus. Both remained unaffected by SPSB1, indicating that SPSB1 exerts its inhibitory activity downstream, or at the level, of the NF-κB heterodimer. In agreement with this, SPSB1 was found to co-precipitate with p65 after over-expression and at endogenous levels. Additionally, A549 cells stably expressing SPSB1 presented lower cytokine levels including type I IFN in response to cytokine stimulation and virus infection. Taken together, our results reveal novel regulatory mechanisms in innate immune signaling and identify the prominent role of SPSB1 in limiting NF-κB activation. Our work thus provides insights into inflammation and inflammatory diseases and new opportunities for the therapeutic targeting of NF-κB transcriptional activity.

Effective targeting of the ubiquitin-like modifier NEDD8 for lung adenocarcinoma treatment.

Protein neddylation, a process of conjugating neural precursor cell expressed, developmentally downregulated 8 (NEDD8) to substrates, plays a tumor-promoting role in lung carcinogenesis. Our previous study showed MLN4924, an inhibitor of NEDD8 activating enzyme (E1), significantly inhibits the growth of multiple cancer cells. However, resistance can develop to MLN4924 by mutation. Therefore, it is important to further understand how NEDD8 acts in lung cancer. In the present study, we demonstrated NEDD8 is overactivated in lung cancers and confers a worse patient overall survival. Furthermore, we report that in lung adenocarcinoma cells, NEDD8 depletion significantly suppressed lung cancer cell growth and progression both in vitro and in vivo. Mechanistic studies revealed that NEDD8 depletion induced the accumulation of a panel of tumor-suppressive cullin-RING ubiquitin ligase substrates (e.g., p21, p27, and Wee1) via blocking their degradation, triggering cell cycle arrest at G2 phase, thus inducing apoptosis or senescence in a cell-line-dependent manner. The present study demonstrates the role of NEDD8 in regulating the malignant phenotypes of lung cancer cells and further validates NEDD8 as a potential therapeutic target in lung cancer.

Structural Biology of CRL Ubiquitin Ligases.

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

Coordinated Actions Between p97 and Cullin-RING Ubiquitin Ligases for Protein Degradation.

The cullin-RING ubiquitin ligases comprise the largest subfamily of ubiquitin ligases. They control ubiquitylation and degradation of a large number of protein substrates in eukaryotes. p97 is an ATPase domain-containing protein segregase. It plays essential roles in post-ubiquitylational events in the ubiquitin-proteasome pathway. Together with its cofactors, p97 collaborates with ubiquitin ligases to extract ubiquitylated substrates and deliver them to the proteasome for proteolysis. Here we review the structure, functions, and mechanisms of p97 in cellular protein degradation in coordination with its cofactors and the cullin-RING ubiquitin ligases.

Assembly and Regulation of CRL Ubiquitin Ligases.

Cullin-RING ubiquitin ligases (CRLs) determine the substrate specificity of ubiquitination reactions, and substrates are recruited to the cullin core through binding to their cognate substrate receptor modules. Because a family of substrate receptors compete for the same cullin core, the assembly and activity of CRLs are dynamically regulated to fulfill the needs of the cell to adapt to the changing pool of proteins demanding ubiquitination. Cullins are modified by NEDD8, a ubiquitin-like protein. This process, referred to as neddylation, promotes the E3 activity of CRLs by inducing conformational rearrangement in the Cullin-RING catalytic core. Cand1 is a cullin-associated protein whose binding is excluded by cullin neddylation. Although early biochemical studies suggested that Cand1 inhibits CRL activity, genetic studies revealed its positive role in ubiquitination. Emerging evidence from kinetic and quantitative proteomic studies demonstrated that Cand1 stimulates assembly of new Skp1-Cul1-F-box protein (SCF) complexes by exchanging the Skp1-F-box protein substrate receptor modules. Furthermore, aided by refined experimental design as well as computational simulation, an attractive model has been developed in which substrate, neddylation cycle and Cand1-mediated "adaptive exchange" collaborate to maintain the dynamics of the cellular SCF repertoire. Here, we review and discuss recent advances that have deepened our understanding of CRL regulation.

CRL3s: The BTB-CUL3-RING E3 Ubiquitin Ligases.

The ubiquitin proteasome pathway is one of the major regulatory tools used by eukaryotic cells. The evolutionarily conserved cullin family proteins can assemble as many as >600 distinct E3 ubiquitin ligase complexes that regulate diverse cellular pathways. In most of Cullin-RING ubiquitin ligase (CRL) complexes, separate linker and adaptor proteins build the substrate recognition module. Differently, a single BTB-containing adaptor molecule utilizing two protein interaction sites can link the CUL3 scaffold to the substrate, forming as many as 188 CUL3-BTB E3 ligase complexes in mammals. Here, we review the most recent studies on CRL3 complexes, with a focus on the model for CUL3 assembly with its BTB-containing substrate receptors. Also, we summarize the current knowledge of CRL3 substrates and their relevant biological functions. Next, we discuss the mutual exclusivity of somatic mutations in KEAP1, NRF2, and CUL3 in human lung cancer. Finally, we highlight new strategies to expand CUL3 substrates and discuss outstanding questions remaining in the field.