Muf1, a novel Elongin BC-interacting leucine-rich repeat protein that can assemble with Cul5 and Rbx1 to reconstitute a ubiquitin ligase

The heterodimeric Elongin BC complex has been shown to interact in vitro and in mammalian cells with a conserved BC-box motif found in a growing number of proteins including RNA polymerase II elongation factor Elongin A, SOCS-box proteins, and the von Hippel-Lindau (VHL) tumor suppressor protein. Recently, the VHL-Elongin BC complex was found to interact with a module composed of Cullin family member Cul2 and RING-H2 finger protein Rbx1 to reconstitute a novel E3 ubiquitin ligase that activates ubiquitylation by the E2 ubiquitin-conjugating enzymes Ubc5 and Cdc34. In the context of the VHL ubiquitin ligase, Elongin BC functions as an adaptor that links the VHL protein to the Cul2/Rbx1 module, raising the possibility that the Elongin BC complex could function as an integral component of a larger family of E3 ubiquitin ligases by linking alternative BC-box proteins to Cullin/Rbx1 modules. In this report, we describe identification and purification from rat liver of a novel leucine-rich repeat-containing BC-box protein, MUF1, which we demonstrate is capable of assembling with a Cullin/Rbx1 module containing the Cullin family member Cul5 to reconstitute ubiquitin ligase activity. In addition, we show that the additional BC-box proteins Elongin A, SOCS1, and WSB1 are also capable of assembling with the Cul5/Rbx1 module to reconstitute potential ubiquitin ligases. Taken together, our findings identify MUF1 as a new member of the BC-box family of proteins, and they predict the existence of a larger family of Elongin BC-based E3 ubiquitin ligases.     

Identification of Elongin C and Skp1 sequences that determine Cullin selection

The multiprotein von Hippel-Lindau (VHL) tumor suppressor and Skp1-Cul1-F-box protein (SCF) complexes belong to families of structurally related E3 ubiquitin ligases. In the VHL ubiquitin ligase, the VHL protein serves as the substrate recognition subunit, which is linked by the adaptor protein Elongin C to a heterodimeric Cul2/Rbx1 module that activates ubiquitylation of target proteins by the E2 ubiquitin-conjugating enzyme Ubc5. In SCF ubiquitin ligases, F-box proteins serve as substrate recognition subunits, which are linked by the Elongin C-like adaptor protein Skp1 to a Cul1/Rbx1 module that activates ubiquitylation of target proteins, in most cases by the E2 Cdc34. In this report, we investigate the functions of the Elongin C and Skp1 proteins in reconstitution of VHL and SCF ubiquitin ligases. We identify Elongin C and Skp1 structural elements responsible for selective interaction with their cognate Cullin/Rbx1 modules. In addition, using altered specificity Elongin C and F-box protein mutants, we investigate models for the mechanism underlying E2 selection by VHL and SCF ubiquitin ligases. Our findings provide evidence that E2 selection by VHL and SCF ubiquitin ligases is determined not solely by the Cullin/Rbx1 module, the target protein, or the integrity of the substrate recognition subunit but by yet to be elucidated features of these macromolecular complexes.     

ASB proteins interact with Cullin5 and Rbx2 to form E3 ubiquitin ligase complexes

The ankyrin repeat and SOCS box (ASB) family is composed of 18 proteins from ASB1 to ASB18 and belongs to the suppressor of cytokine signaling (SOCS) box protein superfamily. ASB2 was recently shown to interact with a certain Cul-Rbx module to form an E3 ubiquitin (Ub) ligase complex, but the functional composition of the ASB-containing E3 Ub ligase complexes remains to be characterized. Here, we show that ASB proteins interact with Cul5-Rbx2 but neither Cul2 nor Rbx1 in cells. Mutational analysis revealed that the highly conserved amino acid sequences of the BC box and Cul5 box in the SOCS box of ASB proteins were essential for the interaction with Cul5-Rbx2. Although ASB proteins show slight divergences from the consensus sequences of the BC box and Cul5 box, all five tested ASB proteins bound to Cul5-Rbx2. Furthermore, all three tested ASB complexes containing Cul5-Rbx2 were found to have E3 Ub ligase activity. These findings suggest that the ASB family proteins interact with Cul5-Rbx2 to form E3 Ub ligases and play significant roles via a ubiquitination-mediated pathway.     

Mechanism of cullin3 e3 ubiquitin ligase dimerization

Cullin E3 ligases are the largest family of ubiquitin ligases with diverse cellular functions. One of seven cullin proteins serves as a scaffold protein for the assembly of the multisubunit ubiquitin ligase complex. Cullin binds the RING domain protein Rbx1/Rbx2 via its C-terminus and a cullin-specific substrate adaptor protein via its N-terminus. In the Cul3 ubiquitin ligase complex, Cul3 substrate receptors contain a BTB/POZ domain. Several studies have established that Cul3-based E3 ubiquitin ligases exist in a dimeric state which is required for binding of a number of substrates and has been suggested to promote ubiquitin transfer. In two different models, Cul3 has been proposed to dimerize either via BTB/POZ domain dependent substrate receptor homodimerization or via direct interaction between two Cul3 proteins that is mediated by Nedd8 modification of one of the dimerization partners. In this study, we show that the majority of the Cul3 proteins in cells exist as dimers or multimers and that Cul3 self-association is mediated via the Cul3 N-terminus while the Cul3 C-terminus is not required. Furthermore, we show that Cul3 self-association is independent of its modification with Nedd8. Our results provide evidence for BTB substrate receptor dependent Cul3 dimerization which is likely to play an important role in promoting substrate ubiquitination.     

The role of cullin 5-containing ubiquitin ligases

The suppressor of cytokine signaling (SOCS) box consists of the BC box and the cullin 5 (Cul5) box, which interact with Elongin BC and Cul5, respectively. SOCS box-containing proteins have ubiquitin ligase activity mediated by the formation of a complex with the scaffold protein Cul5 and the RING domain protein Rbx2, and are thereby members of the cullin RING ligase superfamily. Cul5-type ubiquitin ligases have a variety of substrates that are targeted for polyubiquitination and proteasomal degradation. Here, we review the current knowledge on the identification of Cul5 and the regulation of its expression, as well as the signaling pathways regulated by Cul5 and how viruses highjack the Cul5 system to overcome antiviral responses.     

Characterization of the Cullin7 E3 ubiquitin ligase--heterodimerization of cullin substrate receptors as a novel mechanism to regulate cullin E3 ligase activity

Cul1 and Cul7 are cullin E3 ubiquitin ligase scaffold proteins. Cul1 is known to form a complex with the RING domain protein Rbx1 and one of approximately 70 different F-box proteins. F-box proteins function as substrate receptor subunits and recruit numerous substrates for poly-ubiquitination. Similarly to Cul1, Cul7 interacts with Rbx1, however, only one F-box protein, Fbxw8, has been shown to bind to Cul7. To date only few Cul7 E3 ubiquitin ligase substrates, including cyclin D1, IRS-1 and GRASP65, have been reported, and using Fbxw8 affinity purification, we were unable to identify additional substrate proteins. Here we provide evidence for a model in which Cul7-Rbx1 can promote the ubiquitination of Cul1 substrates by forming high order complexes with Cul1-Rbx1. Binding of Cul1-Rbx1 to Cul7-Rbx1 is mediated via heterodimerization of Fbxw8 with other F-box proteins which function to recruit substrates into the E3 ligase complex. The formation of this high order complex is likely to increase polyubiquitination efficiency.     

Neddylation-induced conformational control regulates cullin RING ligase activity in vivo

Cullin RING ligases (CRLs) constitute the largest family of ubiquitin ligases with diverse cellular functions. Conjugation of the ubiquitin-like molecule Nedd8 to a conserved lysine residue on the cullin scaffold is essential for the activity of CRLs. Using structural studies and in vitro assays, it has been demonstrated that neddylation stimulates CRL activity through conformational rearrangement of the cullin C-terminal winged-helix B domain and Rbx1 RING subdomain from a closed architecture to an open and dynamic structure, thus promoting ubiquitin transfer onto the substrate. Here, we tested whether the proposed mechanism operates in vivo in intact cells and applies to other CRL family members. To inhibit cellular neddylation, we used a cell line with tetracycline-inducible expression of a dominant-negative form of the Nedd8 E2 enzyme or treatment of cells with the Nedd8 E1 inhibitor MLN4924. Using these cellular systems, we show that different mutants of Cul2 and Cul3 and of Rbx1 that confer increased Rbx1 flexibility mimic neddylation and rescue CRL activity in intact cells. Our findings indicate that in vivo neddylation functions by inducing conformational changes in the C-terminal domain of Cul2 and Cul3 that free the RING domain of Rbx1 and bridge the gap for ubiquitin transfer onto the substrate.     

The Cullin3 ubiquitin ligase functions as a Nedd8-bound heterodimer

Cullins are members of a family of scaffold proteins that assemble multisubunit ubiquitin ligase complexes to confer substrate specificity for the ubiquitination pathway. Cullin3 (Cul3) forms a catalytically inactive BTB-Cul3-Rbx1 (BCR) ubiquitin ligase, which becomes functional upon covalent attachment of the ubiquitin homologue neural-precursor-cell-expressed and developmentally down regulated 8 (Nedd8) near the C terminus of Cul3. Current models suggest that Nedd8 activates cullin complexes by providing a recognition site for a ubiquitin-conjugating enzyme. Based on the following evidence, we propose that Nedd8 activates the BCR ubiquitin ligase by mediating the dimerization of Cul3. First, Cul3 is found as a neddylated heterodimer bound to a BTB domain-containing protein in vivo. Second, the formation of a Cul3 heterodimer is mediated by a Nedd8 molecule, which covalently attaches itself to one Cul3 molecule and binds to the winged-helix B domain at the C terminus of the second Cul3 molecule. Third, complementation experiments revealed that coexpression of two distinct nonfunctional Cul3 mutants can rescue the ubiquitin ligase function of the BCR complex. Likewise, a substrate of the BCR complex binds heterodimeric Cul3, suggesting that the Cul3 complex is active as a dimer. These findings not only provide insight into the architecture of the active BCR complex but also suggest assembly as a regulatory mechanism for activation of all cullin-based ubiquitin ligases.     

PEST sequences mediate heat shock factor 2 turnover by interacting with the Cul3 subunit of the Cul3-RING ubiquitin ligase

Cullin-RING ubiquitin ligases promote the polyubiquitination and degradation of many important cellular proteins, which previous studies indicated can be targeted for degradation via interaction with BTB domain-containing subunits of this E3 ligase complex. PEST domains are known to promote the degradation of proteins that contain them. However, the molecular mechanism by which PEST sequences promote degradation of these proteins is not understood. Here we show that the PEST sequences of a short-lived protein called HSF2 interact with Cullin3, a subunit of a Cullin-RING E3 ubiquitin ligase, and that this interaction mediates the Cul3-dependent ubiquitination and degradation of HSF2. These results indicate how, at the molecular level, PEST sequences can promote the proteolysis of proteins that contain them. They also expand understanding of the mechanisms by which substrates can be recruited to Cullin-RING E3 ubiquitin ligases to include interactions between PEST sequences and Cul3.     

Characterization of cullin-based E3 ubiquitin ligases in intact mammalian cells--evidence for cullin dimerization

Cullin-based E3 ligases are a large family of ubiquitin ligases with diverse cellular functions. They are composed of one of six mammalian cullin homologues, the Ring finger containing protein Roc1/Rbx1 and cullin homologue-specific adapter and substrate recognition subunits. To be active, cullin-based ligases require the covalent modification of a conserved lysine residue in the cullin protein with the ubiquitin-like protein Nedd8. To characterize this family of E3 ligases in intact cells, we generated a cell line with tetracycline-inducible expression of a dominant-negative mutant of the Nedd8-conjugating enzyme Ubc12, a reported inhibitor of cullin neddylation. Using this cell line, we demonstrate that the substrate recognition subunit Skp2 and the adaptor protein Skp1 are subject to Ubc12-dependent autoubiquitination and degradation. In contrast, cullin protein stability is not regulated by neddylation in mammalian cells. We also provide evidence that Cul1 and Cul3, as well as their associated substrate recognition subunits Skp2 and Keap1, respectively, homooligomerize in intact cells, suggesting that cullin-based ligases are dimeric. Cul3, but not Cul1 homooligomerization is dependent on substrate recognition subunit dimer formation. As shown for other E3 ubiquitin ligases, dimerization may play a role in regulating the activity of cullin-based E3 ligases.     

Structural Basis for Cul3 Protein Assembly with the BTB-Kelch Family of E3 Ubiquitin Ligases

Cullin-RING ligases are multisubunit E3 ubiquitin ligases that recruit substrate-specific adaptors to catalyze protein ubiquitylation. Cul3-based Cullin-RING ligases are uniquely associated with BTB adaptors that incorporate homodimerization, Cul3 assembly, and substrate recognition into a single multidomain protein, of which the best known are BTB-BACK-Kelch domain proteins, including KEAP1. Cul3 assembly requires a BTB protein “3-box” motif, analogous to the F-box and SOCS box motifs of other Cullin-based E3s. To define the molecular basis for this assembly and the overall architecture of the E3, we determined the crystal structures of the BTB-BACK domains of KLHL11 both alone and in complex with Cul3, along with the Kelch domain structures of KLHL2 (Mayven), KLHL7, KLHL12, and KBTBD5. We show that Cul3 interaction is dependent on a unique N-terminal extension sequence that packs against the 3-box in a hydrophobic groove centrally located between the BTB and BACK domains. Deletion of this N-terminal region results in a 30-fold loss in affinity. The presented data offer a model for the quaternary assembly of this E3 class that supports the bivalent capture of Nrf2 and reveals potential new sites for E3 inhibitor design.     

The SCF ubiquitin ligase: an extended look

The SCF E3 ubiquitin ligases select specific proteins for ubiquitination (and typically destruction) by coupling variable adaptor (F box) proteins that bind protein substrates to a conserved catalytic engine containing a cullin, Cul1, and the Rbx1/Roc1 RING finger protein. A new crystal structure of the SCF(Skp2) ubiquitin ligase shows the molecular organization of this complex and raises important questions as to how substrate ubiquitination is accomplished.     

The structure and regulation of Cullin 2 based E3 ubiquitin ligases and their biological functions

Background

Cullin-RING E3 ubiquitin ligase complexes play a central role in targeting cellular proteins for ubiquitination-dependent protein turnover through 26S proteasome. Cullin-2 is a member of the Cullin family, and it serves as a scaffold protein for Elongin B and C, Rbx1 and various substrate recognition receptors to form E3 ubiquitin ligases.

Main body of the abstract

First, the composition, structure and the regulation of Cullin-2 based E3 ubiquitin ligases were introduced. Then the targets, the biological functions of complexes that use VHL, Lrr-1, Fem1b, Prame, Zyg-11, BAF250, Rack1 as substrate targeting subunits were described, and their involvement in diseases was discussed. A small molecule inhibitor of Cullins as a potential anti-cancer drug was introduced. Furthermore, proteins with VHL box that might bind to Cullin-2 were described. Finally, how different viral proteins form E3 ubiquitin ligase complexes with Cullin-2 to counter host viral defense were explained.

Conclusions

Cullin-2 based E3 ubiquitin ligases, using many different substrate recognition receptors, recognize a number of substrates and regulate their protein stability. These complexes play critical roles in biological processes and diseases such as cancer, germline differentiation and viral defense. Through the better understanding of their biology, we can devise and develop new therapeutic strategies to treat cancers, inherited diseases and viral infections.

     

Flexible cullins in cullin-RING E3 ligases allosterically regulate ubiquitination

How do the cullins, with conserved structures, accommodate substrate-binding proteins with distinct shapes and sizes? Cullin-RING E3 ubiquitin ligases facilitate ubiquitin transfer from E2 to the substrate, tagging the substrate for degradation. They contain substrate-binding, adaptor, cullin, and Rbx proteins. Previously, we showed that substrate-binding and Rbx proteins are flexible. This allows shortening of the E2-substrate distance for initiation of ubiquitination or increasing the distance to accommodate the polyubiquitin chain. However, the role of the cullin remained unclear. Is cullin a rigid scaffold, or is it flexible and actively assists in the ubiquitin transfer reaction? Why are there different cullins, and how do these cullins specifically facilitate ubiquitination for different substrates? To answer these questions, we performed structural analysis and molecular dynamics simulations based on Cul1, Cul4A, and Cul5 crystal structures. Our results show that these three cullins are not rigid scaffolds but are flexible with conserved hinges in the N-terminal domain. However, the degrees of flexibilities are distinct among the different cullins. Of interest, Cul1 flexibility can also be changed by deletion of the long loop (which is absent in Cul4A) in the N-terminal domain, suggesting that the loop may have an allosteric functional role. In all three cases, these conformational changes increase the E2-substrate distance to a specific range to facilitate polyubiquitination, suggesting that rather than being inert scaffold proteins, cullins allosterically regulate ubiquitination.     

A zinc-binding region in Vif binds Cul5 and determines cullin selection

Human immunodeficiency virus-1 (HIV-1) Vif overcomes the anti-viral activity of APOBEC3G by targeting it for ubiquitination via a Cullin 5-ElonginB-ElonginC (Cul5-EloBC) E3 ligase. Vif associates with Cul5-EloBC through a BC-box motif that binds EloC, but the mechanism by which Vif selectively recruits Cul5 is poorly understood. Here we report that a region of Vif (residues 100-142) upstream of the BC-box binds selectively to Cul5 in the absence of EloC. This region contains a zinc coordination site HX5CX17-18CX3-5H (HCCH), with His/Cys residues at positions 108, 114, 133, and 139 coordinating one zinc ion. The HCCH zinc coordination site, which is conserved among primate lentivirus Vif proteins, does not correspond to any known class of zinc-binding motif. Mutations of His/Cys residues in the HCCH motif impair zinc coordination, Cul5 binding, and APOBEC3G degradation. Mutations of conserved hydrophobic residues (Ile-120, Ala-123, and Leu-124) located between the two Cys residues in the HCCH motif disrupt binding of the zinc-coordinating region to Cul5 and inhibit APOBEC3G degradation. The Vif binding site maps to the first cullin repeat in the N terminus of Cul5. These data suggest that the zinc-binding region in Vif is a novel cullin interaction domain that mediates selective binding to Cul5. We propose that the HCCH zinc-binding motif facilitates Vif-Cul5 binding by playing a structural role in positioning hydrophobic residues for direct contact with Cul5.     

ASB2 is an Elongin BC-interacting protein that can assemble with Cullin 5 and Rbx1 to reconstitute an E3 ubiquitin ligase complex

The ankyrin repeat-containing protein with a suppressor of cytokine signaling box-2 (ASB2) gene was identified as a retinoic acid-response gene and a target of the promyelocytic leukemia-retinoic acid receptor-alpha oncogenic protein characteristic of acute promyelocytic leukemia. Expression of ASB2 in myeloid leukemia cells inhibits growth and promotes commitment, recapitulating an early step known to be critical for differentiation. Here we show that ASB2, by interacting with the Elongin BC complex, can assemble with Cullin5.Rbx1 to form an E3 ubiquitin ligase complex that stimulates polyubiquitination by the E2 ubiquitin-conjugating enzyme Ubc5. This is a first indication that a member of the ASB protein family, ASB2, is a subunit of an ECS (Elongin C-Cullin-SOCS box)-type E3 ubiquitin ligase complex. Altogether, our results strongly suggest that ASB2 targets specific proteins to destruction by the proteasome in leukemia cells that have been induced to differentiate.     

Essential role for the d-Asb11 cul5 Box domain for proper notch signaling and neural cell fate decisions in vivo

ECS (Elongin BC-Cul2/Cul5-SOCS-box protein) ubiquitin ligases recruit substrates to E2 ubiquitin-conjugating enzymes through a SOCS-box protein substrate receptor, an Elongin BC adaptor and a cullin (Cul2 or Cul5) scaffold which interacts with the RING protein. In vitro studies have shown that the conserved amino acid sequence of the cullin box in SOCS-box proteins is required for complex formation and function. However, the in vivo importance of cullin boxes has not been addressed. To explore the biological functions of the cullin box domain of ankyrin repeat and SOCS-box containing protein 11 (d-Asb11), a key mediator of canonical Delta-Notch signaling, we isolated a zebrafish mutant lacking the Cul5 box (Asb11(Cul)). We found that homozygous zebrafish mutants for this allele were defective in Notch signaling as indicated by the impaired expression of Notch target genes. Importantly, asb11(Cul) fish were not capable to degrade the Notch ligand DeltaA during embryogenesis, a process essential for the initiation of Notch signaling during neurogenesis. Accordingly, proper cell fate specification within the neurogenic regions of the zebrafish embryo was impaired. In addition, Asb11(Cul) mRNA was defective in the ability to transactivate a her4::gfp reporter DNA when injected in embryos. Thus, our study reporting the generation and the characterization of a metazoan organism mutant in the conserved cullin binding domain of the SOCS-box demonstrates a hitherto unrecognized importance of the SOCS-box domain for the function of this class of cullin-RING ubiquitin ligases and establishes that the d-Asb11 cullin box is required for both canonical Notch signaling and proper neurogenesis.     

Identification of a Cullin5-ElonginB-ElonginC E3 complex in degradation of feline immunodeficiency virus Vif-mediated feline APOBEC3 proteins

Various feline APOBEC3 (fA3) proteins exhibit broad antiviral activities against a wide range of viruses, such as feline immunodeficiency virus (FIV), feline foamy virus (FFV), and feline leukemia virus (FeLV), as well as those of other species. This activity can be counteracted by the FIV Vif protein, but the mechanism by which FIV Vif suppresses fA3s is unknown. In the present study, we demonstrated that FIV Vif could act via a proteasome-dependent pathway to overcome fA3s. FIV Vif interacted with feline cellular proteins Cullin5 (Cul5), ElonginB, and ElonginC to form an E3 complex to induce degradation of fA3s. Both the dominant-negative Cul5 mutant and a C-terminal hydrophilic replacement ElonginC mutant potently disrupted the FIV Vif activity against fA3s. Furthermore, we identified a BC-box motif in FIV Vif that was essential for the recruitment of E3 ubiquitin ligase and also required for FIV Vif-mediated degradation of fA3s. Moreover, despite the lack of either a Cul5-box or a HCCH zinc-binding motif, FIV Vif specifically selected Cul5. Therefore, FIV Vif may interact with Cul5 via a novel mechanism. These finding imply that SOCS proteins may possess distinct mechanisms to bind Cul5 during formation of the Elongin-Cullin-SOCS box complex.