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1.  BTB Protein Keap1 Targets Antioxidant Transcription Factor Nrf2 for Ubiquitination by the Cullin 3-Roc1 Ligase 
Molecular and Cellular Biology  2005;25(1):162-171.
The concentrations and functions of many eukaryotic proteins are regulated by the ubiquitin pathway, which consists of ubiquitin activation (E1), conjugation (E2), and ligation (E3). Cullins are a family of evolutionarily conserved proteins that assemble by far the largest family of E3 ligase complexes. Cullins, via a conserved C-terminal domain, bind with the RING finger protein Roc1 to recruit the catalytic function of E2. Via a distinct N-terminal domain, individual cullins bind to a protein motif present in multiple proteins to recruit specific substrates. Cullin 3 (Cul3), but not other cullins, binds directly with BTB domains to constitute a potentially large number of BTB-CUL3-ROC1 E3 ubiquitin ligases. Here we report that the human BTB-Kelch protein Keap1, a negative regulator of the antioxidative transcription factor Nrf2, binds to CUL3 and Nrf2 via its BTB and Kelch domains, respectively. The KEAP1-CUL3-ROC1 complex promoted NRF2 ubiquitination in vitro and knocking down Keap1 or CUL3 by short interfering RNA resulted in NRF2 protein accumulation in vivo. We suggest that Keap1 negatively regulates Nrf2 function in part by targeting Nrf2 for ubiquitination by the CUL3-ROC1 ligase and subsequent degradation by the proteasome. Blocking NRF2 degradation in cells expressing both KEAP1 and NRF2 by either inhibiting the proteasome activity or knocking down Cul3, resulted in NRF2 accumulation in the cytoplasm. These results may reconcile previously observed cytoplasmic sequestration of NRF2 by KEAP1 and suggest a possible regulatory step between KEAP1-NRF2 binding and NRF2 degradation.
doi:10.1128/MCB.25.1.162-171.2005
PMCID: PMC538799  PMID: 15601839
2.  CAND1-Mediated Substrate Adaptor Recycling Is Required for Efficient Repression of Nrf2 by Keap1 
Molecular and Cellular Biology  2006;26(4):1235-1244.
The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2. Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex to repress steady-state levels of Nrf2 and Nrf2-dependent transcription. Cullin-dependent ubiquitin ligase complexes have been proposed to undergo dynamic cycles of assembly and disassembly that enable substrate adaptor exchange or recycling. In this report, we have characterized the importance of substrate adaptor recycling for regulation of Keap1-mediated repression of Nrf2. Association of Keap1 with Cul3 was decreased by ectopic expression of CAND1 and was increased by small interfering RNA (siRNA)-mediated knockdown of CAND1. However, both ectopic overexpression and siRNA-mediated knockdown of CAND1 decreased the ability of Keap1 to target Nrf2 for ubiquitin-dependent degradation, resulting in stabilization of Nrf2 and activation of Nrf2-dependent gene expression. Neddylation of Cul3 on Lys 712 is required for Keap1-dependent ubiquitination of Nrf2 in vivo. However, the K712R mutant Cul3 molecule, which is not neddylated, can still assemble with Keap1 into a functional ubiquitin ligase complex in vitro. These results provide support for a model in which substrate adaptor recycling is required for efficient substrate ubiquitination by cullin-dependent E3 ubiquitin ligase complexes.
doi:10.1128/MCB.26.4.1235-1244.2006
PMCID: PMC1367193  PMID: 16449638
3.  Characterization of the role of COP9 signalosome in regulating cullin E3 ubiquitin ligase activity 
Molecular Biology of the Cell  2011;22(24):4706-4715.
Cullin RING E3 ligases require covalent modification with Nedd8 for activity. Neddylation is reversed by the COP9 signalosome (CSN). We characterize the role of CSN-dependent deneddylation in vivo and propose a model in which CSN binds to cullin ligases in their active conformation and functions to recruit important regulatory factors.
Cullin RING ligases (CRLs) are the largest family of cellular E3 ubiquitin ligases and mediate polyubiquitination of a number of cellular substrates. CRLs are activated via the covalent modification of the cullin protein with the ubiquitin-like protein Nedd8. This results in a conformational change in the cullin carboxy terminus that facilitates the ubiquitin transfer onto the substrate. COP9 signalosome (CSN)-mediated cullin deneddylation is essential for CRL activity in vivo. However, the mechanism through which CSN promotes CRL activity in vivo is currently unclear. In this paper, we provide evidence that cullin deneddylation is not intrinsically coupled to substrate polyubiquitination as part of the CRL activation cycle. Furthermore, inhibiting substrate-receptor autoubiquitination is unlikely to account for the major mechanism through which CSN regulates CRL activity. CSN also did not affect recruitment of the substrate-receptor SPOP to Cul3, suggesting it may not function to facilitate the exchange of Cul3 substrate receptors. Our results indicate that CSN binds preferentially to CRLs in the neddylation-induced, active conformation. Binding of the CSN complex to active CRLs may recruit CSN-associated proteins important for CRL regulation. The deneddylating activity of CSN would subsequently promote its own dissociation to allow progression through the CRL activation cycle.
doi:10.1091/mbc.E11-03-0251
PMCID: PMC3237615  PMID: 22013077
4.  USP15 negatively regulates Nrf2 through deubiquitination of Keap1 
Molecular cell  2013;51(1):68-79.
Summary
Nrf2 is a master regulator of the antioxidant response. Under basal conditions Nrf2 is polyubiquitinated by the Keap1-Cul3-E3 ligase and degraded by the 26S-proteasome. In response to Nrf2 inducers there is a switch in polyubiquitination from Nrf2 to Keap1. Currently, regulation of the Nrf2-Keap1 pathway by ubiquitination is largely understood. However, the mechanism responsible for removal of ubiquitin conjugated to Nrf2 or Keap1 remains unknown. Here we report that the deubiquitinating enzyme, USP15, specifically deubiquitinates Keap1, which suppresses the Nrf2 pathway. We demonstrated that deubiquitinated-Keap1 incorporates into the Keap1-Cul3-E3 ligase complex more efficiently, enhancing the complex stability and enzymatic activity. Consequently, there is an increase in Nrf2 protein degradation and a reduction in Nrf2 target gene expression. Furthermore, USP15-siRNA enhances chemoresistance of cells through upregulation of Nrf2. These findings further our understanding of how the Nrf2-Keap1 pathway is regulated, which is imperative in targeting this pathway for chemoprevention or chemotherapy.
doi:10.1016/j.molcel.2013.04.022
PMCID: PMC3732832  PMID: 23727018
Nrf2; Keap1; USP15; Cul3; ubiquitination; deubiquitination; antioxidant response; chemoresistance
5.  Oxidative Stress Sensor Keap1 Functions as an Adaptor for Cul3-Based E3 Ligase To Regulate Proteasomal Degradation of Nrf2 
Molecular and Cellular Biology  2004;24(16):7130-7139.
Transcription factor Nrf2 is a major regulator of genes encoding phase 2 detoxifying enzymes and antioxidant stress proteins in response to electrophilic agents and oxidative stress. In the absence of such stimuli, Nrf2 is inactive owing to its cytoplasmic retention by Keap1 and rapid degradation through the proteasome system. We examined the contribution of Keap1 to the rapid turnover of Nrf2 (half-life of less than 20 min) and found that a direct association between Keap1 and Nrf2 is required for Nrf2 degradation. In a series of domain function analyses of Keap1, we found that both the BTB and intervening-region (IVR) domains are crucial for Nrf2 degradation, implying that these two domains act to recruit ubiquitin-proteasome factors. Indeed, Cullin 3 (Cul3), a subunit of the E3 ligase complex, was found to interact specifically with Keap1 in vivo. Keap1 associates with the N-terminal region of Cul3 through the IVR domain and promotes the ubiquitination of Nrf2 in cooperation with the Cul3-Roc1 complex. These results thus provide solid evidence that Keap1 functions as an adaptor of Cul3-based E3 ligase. To our knowledge, Nrf2 and Keap1 are the first reported mammalian substrate and adaptor, respectively, of the Cul3-based E3 ligase system.
doi:10.1128/MCB.24.16.7130-7139.2004
PMCID: PMC479737  PMID: 15282312
6.  Regulation of Cullin-RING ubiquitin ligase 1 by Spliceosome-associated protein 130 (SAP130) 
Biology Open  2013;2(8):838-844.
Summary
Cullin-RING ubiquitin ligases (CRLs) mediate the ubiquitination of numerous protein substrates and target them for proteasomal degradation. The function of CRLs is under tight regulation by Cullin-binding proteins. It has been reported that the Spliceosome-associated protein 130 (SAP130/SF3b-3) binds to several Cullin proteins, yet it remains unknown whether SAP130 plays any role in regulating the function of CRLs. Here, we report that SAP130 overexpression reduces the binding of adaptor protein Skp1 and substrate receptor Skp2 to Cul1, whereas it has no effect on CAND1 binding to Cul1. Overexpression of SAP130 decreases the degradation rate of p27, a protein substrate of the SCFSkp2 ligase. Interestingly, silencing of SAP130 also inhibits the degradation of p27, suggesting a dual role for SAP130 in the regulation of SCF activity. We hypothesized that the regulatory role of SAP130 could extend to other CRLs; however, overexpression of SAP130 is unable to affect the protein stability of the Cul2 and Cul3 substrates, HIF-1 and NRF-2. SAP130 binds to Cul1, Cul2 and Cul4 with similar affinity, and it binds to Cul3 more strongly. SAP130 localizes in both the nucleus and the cytoplasm. Hence, the inability of SAP130 to regulate Cul2 and Cul3 CRLs cannot be explained by low binding affinity of SAP130 to these cullins or by subcellular sequestration of SAP130. We propose a novel role for SAP130 in the regulation of SCF, whereby SAP130 physically competes with the adaptor protein/F-box protein for Cul1 binding and interferes with the assembly of a functional SCF ligase.
doi:10.1242/bio.20134374
PMCID: PMC3744076  PMID: 23951410
Cullin-RING ligase 1; SAP130; p27
7.  Keap1 Controls Postinduction Repression of the Nrf2-Mediated Antioxidant Response by Escorting Nuclear Export of Nrf2▿  
Molecular and Cellular Biology  2007;27(18):6334-6349.
The transcription factor Nrf2 regulates cellular redox homeostasis. Under basal conditions, Keap1 recruits Nrf2 into the Cul3-containing E3 ubiquitin ligase complex for ubiquitin conjugation and subsequent proteasomal degradation. Oxidative stress triggers activation of Nrf2 through inhibition of E3 ubiquitin ligase activity, resulting in increased levels of Nrf2 and transcriptional activation of Nrf2-dependent genes. In this study, we identify Keap1 as a key postinduction repressor of Nrf2 and demonstrate that a nuclear export sequence (NES) in Keap1 is required for termination of Nrf2-antioxidant response element (ARE) signaling by escorting nuclear export of Nrf2. We provide evidence that ubiquitination of Nrf2 is carried out in the cytosol. Furthermore, we show that Keap1 nuclear translocation is independent of Nrf2 and the Nrf2-Keap1 complex does not bind the ARE. Collectively, our results suggest the following mechanism of postinduction repression: upon recovery of cellular redox homeostasis, Keap1 translocates into the nucleus to dissociate Nrf2 from the ARE. The Nrf2-Keap1 complex is then transported out of the nucleus by the NES in Keap1. Once in the cytoplasm, the Keap1-Nrf2 complex associates with the E3 ubiquitin ligase, resulting in degradation of Nrf2 and termination of the Nrf2 signaling pathway. Hence, postinduction repression of the Nrf2-mediated antioxidant response is controlled by the nuclear export function of Keap1 in alliance with the cytoplasmic ubiquitination and degradation machinery.
doi:10.1128/MCB.00630-07
PMCID: PMC2099624  PMID: 17636022
8.  Pathogenic Bacteria Target NEDD8-Conjugated Cullins to Hijack Host-Cell Signaling Pathways 
PLoS Pathogens  2010;6(9):e1001128.
The cycle inhibiting factors (Cif), produced by pathogenic bacteria isolated from vertebrates and invertebrates, belong to a family of molecules called cyclomodulins that interfere with the eukaryotic cell cycle. Cif blocks the cell cycle at both the G1/S and G2/M transitions by inducing the stabilization of cyclin-dependent kinase inhibitors p21waf1 and p27kip1. Using yeast two-hybrid screens, we identified the ubiquitin-like protein NEDD8 as a target of Cif. Cif co-compartmentalized with NEDD8 in the host cell nucleus and induced accumulation of NEDD8-conjugated cullins. This accumulation occurred early after cell infection and correlated with that of p21 and p27. Co-immunoprecipitation revealed that Cif interacted with cullin-RING ubiquitin ligase complexes (CRLs) through binding with the neddylated forms of cullins 1, 2, 3, 4A and 4B subunits of CRL. Using an in vitro ubiquitylation assay, we demonstrate that Cif directly inhibits the neddylated CUL1-associated ubiquitin ligase activity. Consistent with this inhibition and the interaction of Cif with several neddylated cullins, we further observed that Cif modulates the cellular half-lives of various CRL targets, which might contribute to the pathogenic potential of diverse bacteria.
Author Summary
Among the arsenal of virulence factors used by bacterial pathogens to infect and manipulate their hosts, cyclomodulins are a growing family of bacterial toxins that interfere with the eukaryotic cell-cycle. Cif is one of these cyclomodulins produced by both mammalian and invertebrate pathogenic bacteria. Cif blocks the host cell cycle by inducing the accumulation of two regulators of cell cycle progression: the cyclin-dependent kinase inhibitors p21 and p27. To decipher the mode of action of Cif, we performed yeast two-hybrid screenings. We show that Cif binds to NEDD8 and induce accumulation of neddylated cullins early after infection. Cullins are scaffold components of cullin-RING ubiquitin ligases (CRLs), which ubiquitinate proteins and target them for degradation by the 26S proteasome. We demonstrate that Cif directly inhibits the ubiquitin ligase activity of these CRLs and consequently the targeting of p21 and p27 for ubiquitin-dependent degradation. Targeting at NEDD8 represents a novel strategy for modulation of host cell functions by bacterial pathogens. By inhibiting the most prominent class of ubiquitin-ligases, Cif controls the stability of a cohort of key regulators and impinge on not only cell cycle progression but also on many cellular and biological processes such as immunity, development, transcription, and cell signaling.
doi:10.1371/journal.ppat.1001128
PMCID: PMC2947998  PMID: 20941356
9.  Cullin-RING Ligases as Attractive Anti-cancer Targets 
Current pharmaceutical design  2013;19(18):3215-3225.
The ubiquitin-proteasome system (UPS) promotes the timely degradation of short-lived proteins with key regulatory roles in a vast array of biological processes, such as cell cycle progression, oncogenesis and genome integrity. Thus, abnormal regulation of UPS disrupts the protein homeostasis and causes many human diseases, particularly cancer. Indeed, the FDA approval of bortezomib, the first class of general proteasome inhibitor, for the treatment of multiple myeloma, demonstrated that the UPS can be an attractive anti-cancer target. However, normal cell toxicity associated with bortezomib, resulting from global inhibition of protein degradation, promotes the focus of drug discovery efforts on targeting enzymes upstream of the proteasome for better specificity. E3 ubiquitin ligases, particularly those known to be activated in human cancer, become an attractive choice. Cullin-RING Ligases (CRLs) with multiple components are the largest family of E3 ubiquitin ligases and are responsible for ubiquitination of ~20% of cellular proteins degraded through UPS. Activity of CRLs is dynamically regulated and requires the RING component and cullin neddylation. In this review, we will introduce the UPS and CRL E3s and discuss the biological processes regulated by each of eight CRLs through substrate degradation. We will further discuss how cullin neddylation controls CRL activity, and how CRLs are being validated as the attractive cancer targets by abrogating the RING component through genetic means and by inhibiting cullin neddylation via MLN4924, a small molecule indirect inhibitor of CRLs, currently in several Phase I clinical trials. Finally, we will discuss current efforts and future perspectives on the development of additional inhibitors of CRLs by targeting E2 and/or E3 of cullin neddylation and CRL-mediated ubiquitination as potential anti-cancer agents.
PMCID: PMC4034125  PMID: 23151137
Anticancer targets; autophagy; cullins; CRL/SCF E3 ligase; MLN4924; NEDD8; neddylation; protein degradation; ubiquitin; UPS
10.  C. elegans CAND-1 regulates cullin neddylation, cell proliferation and morphogenesis in specific tissues 
Developmental biology  2010;346(1):113-126.
Cullin-RING ubiquitin ligases (CRLs) are critical regulators of multiple developmental and cellular processes in eukaryotes. CAND1 is a biochemical inhibitor of CRLs, yet has been shown to promote CRL activity in plant and mammalian cells. Here we analyze CAND1 function in the context of a developing metazoan organism. C. elegans CAND-1 is capable of binding to all of the cullins, and we show that it physically interacts with CUL-2 and CUL-4 in vivo. The covalent attachment of the ubiquitin-like protein Nedd8 is required for cullin activity in animals and plants. In cand-1 mutants, the levels of the neddylated isoforms of CUL-2 and CUL-4 are increased, indicating that CAND-1 is a negative regulator of cullin neddylation. cand-1 mutants are hypersensitive to the partial loss of cullin activity, suggesting that CAND-1 facilitates CRL functions. cand-1 mutants exhibit impenetrant phenotypes, including developmental arrest, morphological defects of the vulva and tail, and reduced fecundity. cand-1 mutants share with cul-1 and lin-23 mutants the phenotypes of supernumerary seam cell divisions, defective alae formation, and the accumulation of the SCFLIN-23 target the glutamate receptor GLR-1. The observation that cand-1 mutants have phenotypes associated with the loss of the SCFLIN-23 complex, but lack phenotypes associated with other specific CRL complexes, suggests that CAND-1 is differentially required for the activity of distinct CRL complexes.
doi:10.1016/j.ydbio.2010.07.020
PMCID: PMC2955628  PMID: 20659444
CAND1; CRL; cullin; Nedd8; neddylation; ubiquitin ligase; seam cells
11.  Cullin-RING ubiquitin ligases: global regulation and activation cycles 
Cell Division  2008;3:7.
Cullin-RING ubiquitin ligases (CRLs) comprise the largest known category of ubiquitin ligases. CRLs regulate an extensive number of dynamic cellular processes, including multiple aspects of the cell cycle, transcription, signal transduction, and development. CRLs are multisubunit complexes composed of a cullin, RING H2 finger protein, a variable substrate-recognition subunit (SRS), and for most CRLs, an adaptor that links the SRS to the complex. Eukaryotic species contain multiple cullins, with five major types in metazoa. Each cullin forms a distinct class of CRL complex, with distinct adaptors and/or substrate-recognition subunits. Despite this diversity, each of the classes of CRL complexes is subject to similar regulatory mechanisms. This review focuses on the global regulation of CRL complexes, encompassing: neddylation, deneddylation by the COP9 Signalosome (CSN), inhibitory binding by CAND1, and the dimerization of CRL complexes. We also address the role of cycles of activation and inactivation in regulating CRL activity and switching between substrate-recognition subunits.
doi:10.1186/1747-1028-3-7
PMCID: PMC2266742  PMID: 18282298
12.  Association of SAP130/SF3b-3 with Cullin-RING ubiquitin ligase complexes and its regulation by the COP9 signalosome 
BMC Biochemistry  2008;9:1.
Background
Cullin-RING ubiquitin E3 ligases (CRLs) are regulated by modification of an ubiquitin-like protein, Nedd8 (also known as Rub1) on the cullin subunit. Neddylation is shown to facilitate E3 complex assembly; while un-neddylated cullins are bound by CAND1 that prevents recruitment of the substrates. The level of Nedd8 modification is critically dependent on the COP9 signalosome (CSN), an eight-subunit protein complex containing Nedd8 isopeptidase activity.
Results
We report isolation of SAP130 (SF3b-3) as a CSN1 interacting protein. SAP130 is homologous to DDB1, and is a component of SF3b RNA splicing complex and STAGA/TFTC transcription complexes, but its specific function within these complexes is unknown. We show that SAP130 can interact with a variety of cullin proteins. It forms tertiary complexes with fully assembled CRL E3 complexes such as SCFSkp2, Elongin B/C -Cul2- VHL and Cul4-DDB complex by binding to both N-terminal and C-terminal domain of cullins. SAP130 preferentially associates with neddylated cullins in vivo. However knock-down of CAND1 abolished this preference and increased association of SAP130 with Cul2. Furthermore, we provide evidence that CSN regulates SAP130-Cul2 interaction and SAP130-associated polyubiquitinating activity.
Conclusion
SAP130 is a cullin binding protein that is likely involved in the Nedd8 pathway. The association of SAP130 with various cullin member proteins such as Cul1, Cul2 and Cul4A is modulated by CAND1 and CSN. As an established component of transcription and RNA processing complexes, we hypothesis that SAP130 may link CRL mediated ubiquitination to gene expression.
doi:10.1186/1471-2091-9-1
PMCID: PMC2265268  PMID: 18173839
13.  Keap1 Is a Redox-Regulated Substrate Adaptor Protein for a Cul3-Dependent Ubiquitin Ligase Complex 
Molecular and Cellular Biology  2004;24(24):10941-10953.
The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2 and controls both the subcellular localization and steady-state levels of Nrf2. In this report, we demonstrate that Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 assembles into a functional E3 ubiquitin ligase complex with Cul3 and Rbx1 that targets multiple lysine residues located in the N-terminal Neh2 domain of Nrf2 for ubiquitin conjugation both in vivo and in vitro. Keap1-dependent ubiquitination of Nrf2 is inhibited following exposure of cells to quinone-induced oxidative stress and sulforaphane, a cancer-preventive isothiocyanate. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 is markedly resistant to inhibition by either quinone-induced oxidative stress or sulforaphane. Inhibition of Keap1-dependent ubiquitination of Nrf2 correlates with decreased association of Keap1 with Cul3. Neither quinone-induced oxidative stress nor sulforaphane disrupts association between Keap1 and Nrf2. Our results suggest that the ability of Keap1 to assemble into a functional E3 ubiquitin ligase complex is the critical determinant that controls steady-state levels of Nrf2 in response to cancer-preventive compounds and oxidative stress.
doi:10.1128/MCB.24.24.10941-10953.2004
PMCID: PMC533977  PMID: 15572695
14.  A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62▿  
Molecular and Cellular Biology  2010;30(13):3275-3285.
In response to stress, cells can utilize several cellular processes, such as autophagy, which is a bulk-lysosomal degradation pathway, to mitigate damages and increase the chances of cell survival. Deregulation of autophagy causes upregulation of p62 and the formation of p62-containing aggregates, which are associated with neurodegenerative diseases and cancer. The Nrf2-Keap1 pathway functions as a critical regulator of the cell's defense mechanism against oxidative stress by controlling the expression of many cellular protective proteins. Under basal conditions, Nrf2 is ubiquitinated by the Keap1-Cul3-E3 ubiquitin ligase complex and targeted to the 26S proteasome for degradation. Upon induction, the activity of the E3 ubiquitin ligase is inhibited through the modification of cysteine residues in Keap1, resulting in the stabilization and activation of Nrf2. In this current study, we identified the direct interaction between p62 and Keap1 and the residues required for the interaction have been mapped to 349-DPSTGE-354 in p62 and three arginines in the Kelch domain of Keap1. Accumulation of endogenous p62 or ectopic expression of p62 sequesters Keap1 into aggregates, resulting in the inhibition of Keap1-mediated Nrf2 ubiquitination and its subsequent degradation by the proteasome. In contrast, overexpression of mutated p62, which loses its ability to interact with Keap1, had no effect on Nrf2 stability, demonstrating that p62-mediated Nrf2 upregulation is Keap1 dependent. These findings demonstrate that autophagy deficiency activates the Nrf2 pathway in a noncanonical cysteine-independent mechanism.
doi:10.1128/MCB.00248-10
PMCID: PMC2897585  PMID: 20421418
15.  The cullin protein family 
Genome Biology  2011;12(4):220.
Summary
Cullin proteins are molecular scaffolds that have crucial roles in the post-translational modification of cellular proteins involving ubiquitin. The mammalian cullin protein family comprises eight members (CUL1 to CUL7 and PARC), which are characterized by a cullin homology domain. CUL1 to CUL7 assemble multi-subunit Cullin-RING E3 ubiquitin ligase (CRL) complexes, the largest family of E3 ligases with more than 200 members. Although CUL7 and PARC are present only in chordates, other members of the cullin protein family are found in Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and yeast. A cullin protein tethers both a substrate-targeting unit, often through an adaptor protein, and the RING finger component in a CRL. The cullin-organized CRL thus positions a substrate close to the RING-bound E2 ubiquitin-conjugating enzyme, which catalyzes the transfer of ubiquitin to the substrate. In addition, conjugation of cullins with the ubiquitin-like molecule Nedd8 modulates activation of the corresponding CRL complex, probably through conformational regulation of the interactions between cullin's carboxy-terminal tail and CRL's RING subunit. Genetic studies in several model organisms have helped to unravel a multitude of physiological functions associated with cullin proteins and their respective CRLs. CRLs target numerous substrates and thus have an impact on a range of biological processes, including cell growth, development, signal transduction, transcriptional control, genomic integrity and tumor suppression. Moreover, mutations in CUL7 and CUL4B genes have been linked to hereditary human diseases.
doi:10.1186/gb-2011-12-4-220
PMCID: PMC3218854  PMID: 21554755
16.  Deconjugation of Nedd8 from Cul1 Is Directly Regulated by Skp1-F-box and Substrate, and the COP9 Signalosome Inhibits Deneddylated SCF by a Noncatalytic Mechanism* 
The Journal of Biological Chemistry  2012;287(35):29679-29689.
Background: A detailed description of the kinetics of deneddylation of cullin by CSN has been lacking.
Results: Selected factors and SCF subunits are able to inhibit deneddylation to varying degrees. CSN interferes with SCF-mediated ubiquitination through a noncatalytic mechanism.
Conclusion: Deneddylation of Cul1 by CSN is regulated by F-box protein, substrate, and other factors.
Significance: Our work reported here could facilitate the development of directed therapies.
COP9 signalosome (CSN) mediates deconjugation of the ubiquitin-like protein Nedd8 from the cullin subunits of SCF and other cullin-RING ubiquitin ligases (CRLs). This process is essential to maintain the proper activity of CRLs in cells. Here, we report a detailed kinetic characterization of CSN-mediated deconjugation of Nedd8 from SCF. CSN is an efficient enzyme, with a kcat of ∼1 s−1 and Kmfor neddylated Cul1-Rbx1 of ∼200 nm, yielding a kcat/Km near the anticipated diffusion-controlled limit. Assembly with an F-box-Skp1 complex markedly inhibited deneddylation, although the magnitude varied considerably, with Fbw7-Skp1 inhibiting by ∼5-fold but Skp2-Cks1-Skp1 by only ∼15%. Deneddylation of both SCFFbw7 and SCFSkp2-Cks1 was further inhibited ∼2.5-fold by the addition of substrate. Combined, the inhibition by Fbw7-Skp1 plus its substrate cyclin E was greater than 10-fold. Unexpectedly, our results also uncover significant product inhibition by deconjugated Cul1, which results from the ability of Cul1 to bind tightly to CSN. Reciprocally, CSN inhibits the ubiquitin ligase activity of deneddylated Cul1. We propose a model in which assembled CRL complexes engaged with substrate are normally refractory to deneddylation. Upon consumption of substrate and subsequent deneddylation, CSN can remain stably bound to the CRL and hold it in low state of reduced activity.
doi:10.1074/jbc.M112.352484
PMCID: PMC3436198  PMID: 22767593
Analytical Biochemistry; Enzyme Kinetics; Protein Degradation; Protein-Protein Interactions; Ubiquitin Ligase; CSN; Cop9; Cul1; Nedd8; Deneddylation
17.  Structural and biochemical characterization of the KLHL3–WNK kinase interaction important in blood pressure regulation 
Biochemical Journal  2014;460(Pt 2):237-246.
WNK1 [with no lysine (K)] and WNK4 regulate blood pressure by controlling the activity of ion co-transporters in the kidney. Groundbreaking work has revealed that the ubiquitylation and hence levels of WNK isoforms are controlled by a Cullin-RING E3 ubiquitin ligase complex (CRL3KLHL3) that utilizes CUL3 (Cullin3) and its substrate adaptor, KLHL3 (Kelch-like protein 3). Loss-of-function mutations in either CUL3 or KLHL3 cause the hereditary high blood pressure disease Gordon's syndrome by stabilizing WNK isoforms. KLHL3 binds to a highly conserved degron motif located within the C-terminal non-catalytic domain of WNK isoforms. This interaction is essential for ubiquitylation by CRL3KLHL3 and disease-causing mutations in WNK4 and KLHL3 exert their effects on blood pressure by disrupting this interaction. In the present study, we report on the crystal structure of the KLHL3 Kelch domain in complex with the WNK4 degron motif. This reveals an intricate web of interactions between conserved residues on the surface of the Kelch domain β-propeller and the WNK4 degron motif. Importantly, many of the disease-causing mutations inhibit binding by disrupting critical interface contacts. We also present the structure of the WNK4 degron motif in complex with KLHL2 that has also been reported to bind WNK4. This confirms that KLHL2 interacts with WNK kinases in a similar manner to KLHL3, but strikingly different to how another KLHL protein, KEAP1 (Kelch-like enoyl-CoA hydratase-associated protein 1), binds to its substrate NRF2 (nuclear factor-erythroid 2-related factor 2). The present study provides further insights into how Kelch-like adaptor proteins recognize their substrates and provides a structural basis for how mutations in WNK4 and KLHL3 lead to hypertension.
WNK kinases regulate mammalian blood pressure. The level of WNK protein in a cell is regulated by the KLHL3–CUL3 ubiquitin ligase. We define the interaction between KLHL3 and WNK, identifying the WNK degron, and present the crystal structure of the KLHL3–WNK degron complex.
doi:10.1042/BJ20140153
PMCID: PMC4019986  PMID: 24641320
Bric-a-brac; Tramtrack; and Broad complex (BTB domain); Cullin; hypertension; Kelch-like protein (KLHL); Kelch-like protein 2 (KLHL2); ubiquitin; BTB, Bric-a-brac, Tramtrack, and Broad complex; CRL3KLHL3, Cullin3-RING ligase in complex with KLHL; CUL3, Cullin3; KEAP1, Kelch-like enoyl-CoA hydratase-associated protein 1; KLHL, Kelch-like protein; NCC, Na+/Cl− ion co-transporter; NKCC2, Na+/K+/2Cl− co-transporter 2; NRF2, nuclear factor-erythroid 2-related factor 2; OSR1, oxidative stress-responsive kinase 1; rTEV, recombinant tobacco etch virus; RT-PCR, reverse transcription–PCR; SPAK, SPS1-related proline/alanine-rich kinase; TCEP, tris-(2-carboxyethyl)phosphine; TEV, tobacco etch virus; WNK, with no lysine (K)
18.  Neurospora COP9 Signalosome Integrity Plays Major Roles for Hyphal Growth, Conidial Development, and Circadian Function 
PLoS Genetics  2012;8(5):e1002712.
The COP9 signalosome (CSN) is a highly conserved multifunctional complex that has two major biochemical roles: cleaving NEDD8 from cullin proteins and maintaining the stability of CRL components. We used mutation analysis to confirm that the JAMM domain of the CSN-5 subunit is responsible for NEDD8 cleavage from cullin proteins in Neurospora crassa. Point mutations of key residues in the metal-binding motif (EXnHXHX10D) of the CSN-5 JAMM domain disrupted CSN deneddylation activity without interfering with assembly of the CSN complex or interactions between CSN and cullin proteins. Surprisingly, CSN-5 with a mutated JAMM domain partially rescued the phenotypic defects observed in a csn-5 mutant. We found that, even without its deneddylation activity, the CSN can partially maintain the stability of the SCFFWD-1 complex and partially restore the degradation of the circadian clock protein FREQUENCY (FRQ) in vivo. Furthermore, we showed that CSN containing mutant CSN-5 efficiently prevents degradation of the substrate receptors of CRLs. Finally, we found that deletion of the CAND1 ortholog in N. crassa had little effect on the conidiation circadian rhythm. Our results suggest that CSN integrity plays major roles in hyphal growth, conidial development, and circadian function in N. crassa.
Author Summary
Cullin-RING E3 ubiquitin ligases (CRLs) play important roles in regulating a wide range of processes, such as signal transduction, transcription, cell cycle progression, circadian rhythm, and development, via the ubiquitin-proteasome pathway. The activity and stability of CRLs is precisely controlled by the COP9 signalosome (CSN), an evolutionarily conserved multisubunit protein complex. Under the control of the CSN, CRL activity can be either downregulated via cleavage of NEDD8 (an ubiquitin-like protein) from cullin proteins (deneddylation) or preserved by maintaining the stability of CRL components. We generated point mutations of key residues in the JAMM domain of the CSN-5 subunit to disrupt CSN deneddylation activity, thereby creating a series of mutants containing the intact CSN complex but lacking deneddylation activity. Surprisingly, hyphal growth, conidial development, circadian rhythm, and stability of the SCFFWD-1 complex in these CSN-5 point mutants were comparable to that observed in wild-type N. crassa. Furthermore, we showed that CSN containing mutant CSN-5 efficiently prevents degradation of the substrate receptors of CRLs. Finally, deletion of the N. crassa ortholog of CAND1 (cullin-associated NEDD8-dissociated protein 1) had little effect on conidial development and the circadian clock. Our results suggest that the integrity of the CSN is important for growth and development in N. crassa.
doi:10.1371/journal.pgen.1002712
PMCID: PMC3349749  PMID: 22589747
19.  Cross-Regulations among NRFs and KEAP1 and Effects of their Silencing on Arsenic-Induced Antioxidant Response and Cytotoxicity in Human Keratinocytes 
Environmental Health Perspectives  2012;120(4):583-589.
Background: Nuclear factor E2-related factors (NRFs), including NRF2 and NRF1, play critical roles in mediating the cellular adaptive response to oxidative stress. Human exposure to inorganic arsenic, a potent oxidative stressor, causes various dermal disorders, including hyperkeratosis and skin cancer.
Objective: We investigated the cross-regulations among NRF2, NRF1, and KEAP1, a cullin-3–adapter protein that allows NRF2 to be ubiquinated and degraded by the proteasome complex, in arsenic-induced antioxidant responses.
Results: In human keratinocyte HaCaT cells, selective knockdown (KD) of NRF2 by lentiviral short hairpin RNAs (shRNAs) significantly reduced the expression of many antioxidant enzymes and sensitized the cells to acute cytotoxicity of inorganic arsenite (iAs3+). In contrast, silencing KEAP1 led to a dramatic resistance to iAs3+-induced apoptosis. Pretreatment of HaCaT cells with NRF2 activators, such as tert-butylhydroquinone, protects the cells against acute iAs3+ toxicity in an NRF2-dependent fashion. Consistent with the negative regulatory role of KEAP1 in NRF2 activation, KEAP1-KD cells exhibited enhanced transcriptional activity of NRF2 under nonstressed conditions. However, deficiency in KEAP1 did not facilitate induction of NRF2-target genes by iAs3+. In addition, NRF2 silencing reduced the expression of KEAP1 at transcription and protein levels but increased the protein expression of NRF1 under the iAs3+-exposed condition. In contrast, silencing KEAP1 augmented protein accumulation of NRF2 under basal and iAs3+-exposed conditions, whereas the iAs3+-induced protein accumulation of NRF1 was attenuated in KEAP1-KD cells.
Conclusions: Our studies suggest that NRF2, KEAP1, and NRF1 are coordinately involved in the regulation of the cellular adaptive response to iAs3+-induced oxidative stress.
doi:10.1289/ehp.1104580
PMCID: PMC3339469  PMID: 22476201
antioxidant response; arsenic; cytotoxicity; KEAP1; keratinocyte; NRF1; NRF2
20.  Cullin4A and Cullin4B Are Interchangeable for HIV Vpr and Vpx Action through the CRL4 Ubiquitin Ligase Complex 
Journal of Virology  2014;88(12):6944-6958.
ABSTRACT
Human immunodeficiency virus (HIV) seizes control of cellular cullin-RING E3 ubiquitin ligases (CRLs) to promote viral replication. HIV-1 Vpr and HIV-2/simian immunodeficiency virus (SIV) Vpr and Vpx engage the cullin4 (CUL4)-containing ubiquitin ligase complex (CRL4) to cause polyubiquitination and proteasomal degradation of host proteins, including ones that block infection. HIV-1 Vpr engages CRL4 to trigger the degradation of uracil-N-glycosylase 2 (UNG2). Both HIV-1 Vpr and HIV-2/SIV Vpr tap CRL4 to initiate G2 cell cycle arrest. HIV-2/SIV Vpx secures CRL4 to degrade the antiviral protein SAMHD1. CRL4 includes either cullin4A (CUL4A) or cullin4B (CUL4B) among its components. Whether Vpr or Vpx relies on CUL4A, CUL4B, or both to act through CRL4 is not known. Reported structural, phenotypic, and intracellular distribution differences between the two CUL4 types led us to hypothesize that Vpr and Vpx employ these in a function-specific manner. Here we determined CUL4 requirements for HIV-1 and HIV-2/SIV Vpr-mediated G2 cell cycle arrest, HIV-1 Vpr-mediated UNG2 degradation, and HIV-2 Vpx-mediated SAMHD1 degradation. Surprisingly, CUL4A and CUL4B are exchangeable for CRL4-dependent Vpr and Vpx action, except in primary macrophages, where Vpx relies on both CUL4A and CUL4B for maximal SAMHD1 depletion. This work highlights the need to consider both CUL4 types for Vpr and Vpx functions and also shows that the intracellular distribution of CUL4A and CUL4B can vary by cell type.
IMPORTANCE The work presented here shows for the first time that HIV Vpr and Vpx do not rely exclusively on CUL4A to cause ubiquitination through the CRL4 ubiquitin ligase complex. Furthermore, our finding that intracellular CUL4 and SAMHD1 distributions can vary with cell type provides the basis for reconciling previous disparate findings regarding the site of SAMHD1 depletion. Finally, our observations with primary immune cells provide insight into the cell biology of CUL4A and CUL4B that will help differentiate the functions of these similar proteins.
doi:10.1128/JVI.00241-14
PMCID: PMC4054339  PMID: 24719410
21.  Dysfunctional KEAP1–NRF2 Interaction in Non-Small-Cell Lung Cancer 
PLoS Medicine  2006;3(10):e420.
Background
Nuclear factor erythroid-2 related factor 2 (NRF2) is a redox-sensitive transcription factor that positively regulates the expression of genes encoding antioxidants, xenobiotic detoxification enzymes, and drug efflux pumps, and confers cytoprotection against oxidative stress and xenobiotics in normal cells. Kelch-like ECH-associated protein 1 (KEAP1) negatively regulates NRF2 activity by targeting it to proteasomal degradation. Increased expression of cellular antioxidants and xenobiotic detoxification enzymes has been implicated in resistance of tumor cells against chemotherapeutic drugs.
Methods and Findings
Here we report a systematic analysis of the KEAP1 genomic locus in lung cancer patients and cell lines that revealed deletion, insertion, and missense mutations in functionally important domains of KEAP1 and a very high percentage of loss of heterozygosity at 19p13.2, suggesting that biallelic inactivation of KEAP1 in lung cancer is a common event. Sequencing of KEAP1 in 12 cell lines and 54 non-small-cell lung cancer (NSCLC) samples revealed somatic mutations in KEAP1 in a total of six cell lines and ten tumors at a frequency of 50% and 19%, respectively. All the mutations were within highly conserved amino acid residues located in the Kelch or intervening region domain of the KEAP1 protein, suggesting that these mutations would likely abolish KEAP1 repressor activity. Evaluation of loss of heterozygosity at 19p13.2 revealed allelic losses in 61% of the NSCLC cell lines and 41% of the tumor samples. Decreased KEAP1 activity in cancer cells induced greater nuclear accumulation of NRF2, causing enhanced transcriptional induction of antioxidants, xenobiotic metabolism enzymes, and drug efflux pumps.
Conclusions
This is the first study to our knowledge to demonstrate that biallelic inactivation of KEAP1 is a frequent genetic alteration in NSCLC. Loss of KEAP1 function leading to constitutive activation of NRF2-mediated gene expression in cancer suggests that tumor cells manipulate the NRF2 pathway for their survival against chemotherapeutic agents.
Biallelic inactivation ofKEAP1, a frequent genetic alteration in NSCLC, is associated with activation of the NRF2 pathway which leads to expression of genes that contribute to resistance against chemotherapeutic drugs.
Editors' Summary
Background.
Lung cancer is the most common cause of cancer-related death worldwide. More than 150,000 people in the US alone die every year from this disease, which can be split into two basic types—small cell lung cancer and non-small-cell lung cancer (NSCLC). Four out of five lung cancers are NSCLCs, but both types are mainly caused by smoking. Exposure to chemicals in smoke produces changes (or mutations) in the genetic material of the cells lining the lungs that cause the cells to grow uncontrollably and to move around the body. In more than half the people who develop NSCLC, the cancer has spread out of the lungs before it is diagnosed, and therefore can't be removed surgically. Stage IV NSCLC, as this is known, is usually treated with chemotherapy—toxic chemicals that kill the fast-growing cancer cells. However, only 2% of people with stage IV NSCLC are still alive two years after their diagnosis, mainly because their cancer cells become resistant to chemotherapy. They do this by making proteins that destroy cancer drugs (detoxification enzymes) or that pump them out of cells (efflux pumps) and by making antioxidants, chemicals that protect cells against the oxidative damage caused by many chemotherapy agents.
Why Was This Study Done?
To improve the outlook for patients with lung cancer, researchers need to discover exactly how cancer cells become resistant to chemotherapy drugs. Detoxification enzymes, efflux pumps, and antioxidants normally protect cells from environmental toxins and from oxidants produced by the chemical processes of life. Their production is regulated by nuclear factor erythroid-2 related factor 2 (NRF2). The activity of this transcription factor (a protein that controls the expression of other proteins) is controlled by the protein Kelch-like ECH-associated protein 1 (KEAP1). KEAP1 holds NRF2 in the cytoplasm of the cell (the cytoplasm surrounds the cell's nucleus, where the genetic material is stored) when no oxidants are present and targets it for destruction. When oxidants are present, KEAP1 no longer interacts with NRF2, which moves into the nucleus and induces the expression of the proteins that protect the cell against oxidants and toxins. In this study, the researchers investigated whether changes in KEAP1 might underlie the drug resistance seen in lung cancer.
What Did the Researchers Do and Find?
The researchers looked carefully at the gene encoding KEAP1 in tissue taken from lung tumors and in several lung cancer cell lines—tumor cells that have been grown in a laboratory. They found mutations in parts of KEAP1 known to be important for its function in half the cell lines and a fifth of the tumor samples. They also found that about half of the samples had lost part of one copy of the KEAP1 gene—cells usually have two copies of each gene. Five of the six tumors with KEAP1 mutations had also lost one copy of KEAP1—geneticists call this biallelic inactivation. This means that these tumors should have no functional KEAP1. When the researchers checked this by staining the tumors for NRF2, they found that the tumor cells had more NRF2 than normal cells and that it accumulated in the nucleus. In addition, the tumor cells made more detoxification enzymes, efflux proteins, and antioxidants than normal cells. Finally, the researchers showed that lung cancer cells with KEAP1 mutations were more resistant to chemotherapy drugs than normal lung cells were.
What Do These Findings Mean?
These results indicate that biallelic inactivation of KEAP1 is a frequent genetic alteration in NSCLC and suggest that the loss of KEAP1 activity is one way that lung tumors can increase their NRF2 activity and develop resistance to chemotherapeutic drugs. More lung cancer samples need to be examined to confirm this result, and similar studies need to be done in other cancers to see whether loss of KEAP1 activity is a common mechanism by which tumors become resistant to chemotherapy. If such studies confirm that high NRF2 activity (either through mutation or by some other route) is often associated with a poor tumor response to chemotherapy, then the development of NRF2 inhibitors might help to improve treatment outcomes in patients with chemotherapy-resistant tumors.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030420.
US National Cancer Institute information on lung cancer and on cancer treatment
MedlinePlus entries on small cell lung cancer and NSCLC Cancer Research UK information on lung cancer
Wikipedia entries on lung cancer and chemotherapy (note that Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030420
PMCID: PMC1584412  PMID: 17020408
22.  New strategies to inhibit KEAP1 and the Cul3-based E3 ubiquitin ligases 
Biochemical Society Transactions  2014;42(Pt 1):103-107.
E3 ubiquitin ligases that direct substrate proteins to the ubiquitin–proteasome system are promising, though largely unexplored drug targets both because of their function and their remarkable specificity. CRLs [Cullin–RING (really interesting new gene) ligases] are the largest group of E3 ligases and function as modular multisubunit complexes constructed around a Cullin-family scaffold protein. The Cul3-based CRLs uniquely assemble with BTB (broad complex/tramtrack/bric-à-brac) proteins that also homodimerize and perform the role of both the Cullin adapter and the substrate-recognition component of the E3. The most prominent member is the BTB–BACK (BTB and C-terminal Kelch)–Kelch protein KEAP1 (Kelch-like ECH-associated protein 1), a master regulator of the oxidative stress response and a potential drug target for common conditions such as diabetes, Alzheimer's disease and Parkinson's disease. Structural characterization of BTB–Cul3 complexes has revealed a number of critical assembly mechanisms, including the binding of an N-terminal Cullin extension to a bihelical ‘3-box’ at the C-terminus of the BTB domain. Improved understanding of the structure of these complexes should contribute significantly to the effort to develop novel therapeutics targeted to CRL3-regulated pathways.
doi:10.1042/BST20130215
PMCID: PMC3935762  PMID: 24450635
antioxidant response; cancer; cell signalling; degradation; drug design; ubiquitylation; ARE, antioxidant-response element; BACK, BTB and C-terminal Kelch; BTB, broad complex/tramtrack/bric-à-brac; CRL, Cullin–RING ligase; HECT, homologous with E6-associated protein C-terminus; KEAP1, Kelch-like ECH-associated protein 1; KLHL, Kelch-like; MATH, meprin and TRAF (tumour-necrosis-factor-receptor-associated factor) homology; Nrf2, nuclear factor erythroid 2-related factor 2; POZ, pox virus and zinc finger; Rbx, RING box protein; RING, really interesting new gene; SPOP, speckle-type POZ protein
23.  UBXN7 docks on neddylated cullin complexes using its UIM motif and causes HIF1α accumulation 
BMC Biology  2012;10:36.
Background
The proteins from the UBA-UBX family interact with ubiquitylated proteins via their UBA domain and with p97 via their UBX domain, thereby acting as substrate-binding adaptors for the p97 ATPase. In particular, human UBXN7 (also known as UBXD7) mediates p97 interaction with the transcription factor HIF1α that is actively ubiquitylated in normoxic cells by a CUL2-based E3 ligase, CRL2. Mass spectrometry analysis of UBA-UBX protein immunoprecipitates showed that they interact with a multitude of E3 ubiquitin-ligases. Conspicuously, UBXN7 was most proficient in interacting with cullin-RING ligase subunits. We therefore set out to determine whether UBXN7 interaction with cullins was direct or mediated by its ubiquitylated targets bound to the UBA domain.
Results
We show that UBXN7 interaction with cullins is independent of ubiquitin- and substrate-binding. Instead, it relies on the UIM motif in UBXN7 that directly engages the NEDD8 modification on cullins. To understand the functional consequences of UBXN7 interaction with neddylated cullins, we focused on HIF1α, a CUL2 substrate that uses UBXD7/p97 as a ubiquitin-receptor on its way to proteasome-mediated degradation. We find that UBXN7 over-expression converts CUL2 to its neddylated form and causes the accumulation of non-ubiquitylated HIF1α. Both of these effects are strictly UIM-dependent and occur only when UBXN7 contains an intact UIM motif. We also show that HIF1α carrying long ubiquitin-chains can recruit alternative ubiquitin-receptors, lacking p97's ATP-dependent segregase activity.
Conclusions
Our study shows that independently of its function as a ubiquitin-binding adaptor for p97, UBXN7 directly interacts with neddylated cullins and causes the accumulation of the CUL2 substrate HIF1α. We propose that by sequestering CUL2 in its neddylated form, UBXN7 negatively regulates the ubiquitin-ligase activity of CRL2 and this might prevent recruitment of ubiquitin-receptors other than p97 to nuclear HIF1α.
doi:10.1186/1741-7007-10-36
PMCID: PMC3349548  PMID: 22537386
cullin; NEDD8; p97; ubiquitin-dependent degradation; UBXD7
24.  The Keap1-BTB Protein Is an Adaptor That Bridges Nrf2 to a Cul3-Based E3 Ligase: Oxidative Stress Sensing by a Cul3-Keap1 Ligase 
Molecular and Cellular Biology  2004;24(19):8477-8486.
The Nrf2 transcription factor promotes survival following cellular insults that trigger oxidative damage. Nrf2 activity is opposed by the BTB/POZ domain protein Keap1. Keap1 is proposed to regulate Nrf2 activity strictly through its capacity to inhibit Nrf2 nuclear import. Recent work suggests that inhibition of Nrf2 may also depend upon ubiquitin-mediated proteolysis. To address the contribution of Keap1-dependent sequestration versus Nrf2 proteolysis, we identified the E3 ligase that regulates Nrf2 ubiquitination. We demonstrate that Keap1 is not solely a cytosolic anchor; rather, Keap1 is an adaptor that bridges Nrf2 to Cul3. We demonstrate that Cul3-Keap1 complexes regulate Nrf2 polyubiquitination both in vitro and in vivo. Inhibition of either Keap1 or Cul3 increases Nrf2 nuclear accumulation, leading to promiscuous activation of Nrf2-dependent gene expression. Our data demonstrate that Keap1 restrains Nrf2 activity via its capacity to target Nrf2 to a cytoplasmic Cul3-based E3 ligase and suggest a model in which Keap1 coordinately regulates both Nrf2 accumulation and access to target genes.
doi:10.1128/MCB.24.19.8477-8486.2004
PMCID: PMC516753  PMID: 15367669
25.  Insight into the Intermolecular Recognition Mechanism between Keap1 and IKKβ Combining Homology Modelling, Protein-Protein Docking, Molecular Dynamics Simulations and Virtual Alanine Mutation 
PLoS ONE  2013;8(9):e75076.
Degradation of certain proteins through the ubiquitin-proteasome pathway is a common strategy taken by the key modulators responsible for stress responses. Kelch-like ECH-associated protein-1(Keap1), a substrate adaptor component of the Cullin3 (Cul3)-based ubiquitin E3 ligase complex, mediates the ubiquitination of two key modulators, NF-E2-related factor 2 (Nrf2) and IκB kinase β (IKKβ), which are involved in the redox control of gene transcription. However, compared to the Keap1-Nrf2 protein-protein interaction (PPI), the intermolecular recognition mechanism of Keap1 and IKKβ has been poorly investigated. In order to explore the binding pattern between Keap1 and IKKβ, the PPI model of Keap1 and IKKβ was investigated. The structure of human IKKβ was constructed by means of the homology modeling method and using reported crystal structure of Xenopus laevis IKKβ as the template. A protein-protein docking method was applied to develop the Keap1-IKKβ complex model. After the refinement and visual analysis of docked proteins, the chosen pose was further optimized through molecular dynamics simulations. The resulting structure was utilized to conduct the virtual alanine mutation for the exploration of hot-spots significant for the intermolecular interaction. Overall, our results provided structural insights into the PPI model of Keap1-IKKβ and suggest that the substrate specificity of Keap1 depend on the interaction with the key tyrosines, namely Tyr525, Tyr574 and Tyr334. The study presented in the current project may be useful to design molecules that selectively modulate Keap1. The selective recognition mechanism of Keap1 with IKKβ or Nrf2 will be helpful to further know the crosstalk between NF-κB and Nrf2 signaling.
doi:10.1371/journal.pone.0075076
PMCID: PMC3774807  PMID: 24066166

Results 1-25 (667178)