BC-box Proteins Research Articles

Cullin-RING ligases employ geometrically optimized catalytic partners for substrate targeting

Cullin-RING ligases (CRLs) ubiquitylate specific substrates selected from other cellular proteins. Substrate discrimination and ubiquitin transferase activity were thought to be strictly separated. Substrates are recognized by substrate receptors, such as Fbox or BCbox proteins. Meanwhile, CRLs employ assorted ubiquitin-carrying enzymes (UCEs, which are a collection of E2 and ARIH-family E3s) specialized for either initial substrate ubiquitylation (priming) or forging poly-ubiquitin chains. We discovered specific human CRL-UCE pairings governing substrate priming. The results reveal pairing of CUL2-based CRLs and UBE2R-family UCEs in cells, essential for efficient PROTAC-induced neo-substrate degradation. Despite UBE2R2's intrinsic programming to catalyze poly-ubiquitylation, CUL2 employs this UCE for geometrically precise PROTAC-dependent ubiquitylation of a neo-substrate and for rapid priming of substrates recruited to diverse receptors. Cryo-EM structures illuminate how CUL2-based CRLs engage UBE2R2 to activate substrate ubiquitylation. Thus, pairing with a specific UCE overcomes E2 catalytic limitations to drive substrate ubiquitylation and targeted protein degradation.

BC-Box Motif-Mediated Neuronal Differentiation of Somatic Stem Cells.

Von Hippel-Lindau tumor suppressor protein (pVHL) functions to induce neuronal differentiation of neural stem/progenitor cells (NSCs) and skin-derived precursors (SKPs). Here we identified a neuronal differentiation domain (NDD) in pVHL. Neuronal differentiation of SKPs was induced by intracellular delivery of a peptide composed of the amino-acid sequences encoded by the NDD. Neuronal differentiation mediated by the NDD was caused by the binding between it and elongin C followed by Janus kinase-2 (JAK2) ubiquitination of JAK2 and inhibition of the JAK2/the signal transducer and activator of transcription-3(STAT)3 pathway. The NDD in pVHL contained the BC-box motif ((A,P,S,T)LXXX (A,C) XXX(A,I,L,V)) corresponding to the binding site of elongin C. Therefore, we proposed that other BC-box proteins might also contain an NDD; and subsequently also identified in them an NDD containing the amino-acid sequence encoded by the BC-box motif in BC-box proteins. Furthermore, we showed that different NDD peptide-delivered cells differentiated into different kinds of neuron-like cells. That is, dopaminergic neuron-like cells, cholinergic neuron-like cells, GABAnergic neuron-like cells or rhodopsin-positive neuron-like cells were induced by different NDD peptides. These novel findings might contribute to the development of a new method for promoting neuronal differentiation and shed further light on the mechanism of neuronal differentiation of somatic stem cells.

Neuronal differentiation of skin-derived precursors by intracellular delivery of synthesized peptides derived from BC-box proteins

Neuronal differentiation of skin-derived precursors by intracellular delivery of synthesized peptides derived from BC-box proteins

BC-box protein domain-related mechanism for VHL protein degradation

The tumor suppressor VHL (von Hippel-Lindau) protein is a substrate receptor for Ubiquitin Cullin Ring Ligase complexes (CRLs), containing a BC-box domain that associates to the adaptor Elongin B/C. VHL targets hypoxia-inducible factor 1α to proteasome-dependent degradation. Gam1 is an adenoviral protein, which also possesses a BC-box domain that interacts with the host Elongin B/C, thereby acting as a viral substrate receptor. Gam1 associates with both Cullin2 and Cullin5 to form CRL complexes targeting the host protein SUMO enzyme SAE1 for proteasomal degradation. We show that Gam1 protein expression induces VHL protein degradation leading to hypoxia-inducible factor 1α stabilization and induction of its downstream targets. We also characterize the CRL-dependent mechanism that drives VHL protein degradation via proteasome. Interestingly, expression of Suppressor of Cytokine Signaling (SOCS) domain-containing viral proteins and cellular BC-box proteins leads to VHL protein degradation, in a SOCS domain-containing manner. Our work underscores the exquisite ability of viral domains to uncover new regulatory mechanisms by hijacking key cellular proteins.

The EBAX-type Cullin-RING E3 Ligase and Hsp90 Guard the Protein Quality of the SAX-3/Robo Receptor in Developing Neurons

Although protein quality control (PQC) is generally perceived as important for the development of thenervous system, the specific mechanisms of neuronal PQC have remained poorly understood. Here, we report that C.elegans Elongin BC-binding axon regulator (EBAX-1), a conserved BC-box protein, regulates axon guidance through PQC of the SAX-3/Robo receptor. EBAX-1 buffers guidance errors against temperature variations. As a substrate-recognition subunit in the Elongin BC-containing Cullin-RING ubiquitin ligase (CRL), EBAX-1 also binds to DAF-21, a cytosolic Hsp90 chaperone. The EBAX-type CRL and DAF-21 collaboratively regulate SAX-3-mediated axon pathfinding. Biochemical and imaging assays indicate that EBAX-1 specifically recognizes misfolded SAX-3 and promotes its degradation invitro and invivo. Importantly, vertebrate EBAX also shows substrate preference toward aberrant Robo3 implicated in horizontal gaze palsy with progressive scoliosis (HGPPS). Together, our findings demonstrate a triage PQC mechanism mediated by the EBAX-type CRL and DAF-21/Hsp90 that maintains the accuracy of neuronal wiring.

Cul8/Rtt101 Forms a Variety of Protein Complexes That Regulate DNA Damage Response and Transcriptional Silencing

The budding yeast, Saccharomyces cerevisiae, has three cullin proteins, which act as platforms for Cullin-based E3 ubiquitin ligases. Genetic evidence indicates that Cul8, together with Mms1, Mms22, and Esc4, is involved in the repair of DNA damage that can occur during DNA replication. Cul8 is thought to form a complex with these proteins, but the composition and the function of Cul8-based E3 ubiquitin ligases remain largely uncharacterized. Herein, we report a comprehensive biochemical analysis of Cul8 complexes. Cul8 was found to form a Cul8-Mms1-Mms22-Esc4 complex under physiological conditions, with Mms1 bridging Cul8 and Mms22 and Mms22 bridging Mms1 and Esc4. Domain analysis demonstrated that the N-terminal region of Mms1 and the C-terminal region of Mms22 are required for the Mms1-Mms22 interaction, whereas the N-terminal region of Mms22 is required for the Mms22-Esc4 interaction. We also found other Cul8-Mms1-binding proteins Ctf4, Esc2, and Orc5 using yeast two-hybrid screening. Esc4 and Ctf4 bound to Mms22 directly and bound to Cul8-Mms1 in the presence of Mms22, whereas Esc2 and Orc5 interacted with both Cul8 and Mms1, independently. We found that Cul8, Mms1, and Mms22 participated in the regulation of transcriptional silencing of yeast telomeres. These results suggest that Cul8-Mms1, as part of various protein complexes, is involved in the regulation of chromatin metabolism.

Structural Insight into the Human Immunodeficiency Virus Vif SOCS Box and Its Role in Human E3 Ubiquitin Ligase Assembly

Human immunodeficiency virus (HIV) virion infectivity factor (Vif) causes the proteasome-mediated destruction of human antiviral protein APOBEC3G by tethering it to a cellular E3 ubiquitin ligase composed of ElonginB, ElonginC, Cullin5, and Rbx2. It has been proposed that HIV Vif hijacks the E3 ligase through two regions within its C-terminal domain: a BC box region that interacts with ElonginC and a novel zinc finger motif that interacts with Cullin5. We have determined the crystal structure of the HIV Vif BC box in complex with human ElonginB and ElonginC. This complex presents direct structural evidence of the recruitment of a human ubiquitin ligase by a viral BC box protein that mimics the conserved interactions of cellular ubiquitin ligases. We further mutated conserved hydrophobic residues in a region downstream of the Vif BC box. These mutations demonstrate that this region, the Vif Cullin box, composes a third E3-ligase recruiting site critical for interaction between Vif and Cullin5. Furthermore, our homology modeling reveals that the Vif Cullin box and zinc finger motif may be positioned adjacent to the N terminus of Cullin5 for interaction with loop regions in the first cullin repeat of Cullin5.

Characterization of Cullin-box Sequences That Direct Recruitment of Cul2-Rbx1 and Cul5-Rbx2 Modules to Elongin BC-based Ubiquitin Ligases

The Elongin BC-box protein family includes the von Hippel-Lindau tumor suppressor and suppressor of cytokine signaling proteins, which are substrate recognition subunits of structurally related classes of E3 ubiquitin ligases composed of Elongin C-Elongin B-Cullin 2-Rbx1 (Cul2 ubiquitin ligases) or of Elongin C-Elongin B-Cullin 5-Rbx2 (Cul5 ubiquitin ligases). The Elongin BC complex acts as an adaptor that links a substrate recognition subunit to heterodimers of either Cullin 2 (Cul2) and RING finger protein Rbx1 or Cullin 5 (Cul5) and Rbx2. It has been shown ( Kamura, T., Maenaka, K., Kotoshiba, S., Matsumoto, M., Kohda, D., Conaway, R. C., Conaway, J. W., and Nakayama, K. I. (2004) Genes Dev. 18, 3055-3065 ) that interaction of BC-box proteins with their cognate Cul-Rbx module is determined by specific regions, called Cul2- or Cul5-boxes, located immediately downstream of their BC-boxes. Here, we investigate further the mechanisms governing assembly of BC-box proteins with their specific Cul-Rbx modules. Through purification and characterization of a larger collection of BC-box proteins that serve as substrate recognition subunits of Cul2 and Cul5 ubiquitin ligases and through structure-function studies, we define Cul2- and Cul5-boxes in greater detail. Although it previously appeared that there was little sequence similarity between Cul5- and Cul2-box motifs, analyses of newly identified BC-box proteins reveal that residues conserved in the Cul2-box represent a subset of those conserved in the Cul5-box. The sequence motif LPPhiP, which is conserved in most Cul5-boxes and has been suggested to specify assembly of Cul5 ligases, is compatible with Cul2 interaction. Finally, the spacing between BC- and Cullin-boxes is much more flexible than has been appreciated and can vary from as few as 3 and as many as approximately 80 amino acids. Taken together, our findings shed new light on the mechanisms by which BC-box proteins direct recruitment of Cullin-Rbx modules during reconstitution of ubiquitin ligases.

Adenovirus E4orf6 assembles with Cullin5‐ElonginB‐ElonginC E3 ubiquitin ligase through an HIV/SIV Vif‐like BC‐box to regulate p53

The adenovirus protein E4orf6 targets p53 for polyubiquitination and proteasomal degradation and is known to form a complex with the Cul5-ElonginB-ElonginC E3 ubiquitin ligase. However, whether Cul5 is directly responsible for the E4orf6-mediated degradation of p53 remains unclear. By using a dominant-negative mutant of Cul5 and silencing Cul5 expression through RNA interference, we have now demonstrated that E4orf6-mediated p53 degradation requires Cul5. Furthermore, we have identified a lentiviral Vif-like BC-box motif in E4orf6 that is highly conserved among adenoviruses from multiple species. More importantly, we have shown that this Vif-like BC-box is essential for the recruitment of Cul5-ElonginB-ElonginC E3 ubiquitin ligase by E4orf6 and is also required for E4orf6-mediated p53 degradation. E4orf6 selectively recruited Cul5 despite the lack of either a Cul5-box, which is used by cellular substrate receptors to recruit Cul5, or a newly identified HCCH zinc-binding motif, which is used by primate lentiviral Vif to recruit Cul5. Therefore, adenovirus E4orf6 molecules represent a novel family of viral BC-box proteins the cellular ancestor of which is as yet unknown.

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.

Mammalian mediator subunit mMED8 is an Elongin BC-interacting protein that can assemble with Cul2 and Rbx1 to reconstitute a ubiquitin ligase.

The heterodimeric Elongin BC complex has been shown to interact in vitro and in cells with a conserved BC-box motif found in an increasing number of proteins including RNA polymerase II elongation factor Elongin A, suppressor of cytokine signaling (SOCS)-box proteins, and the von Hippel-Lindau tumor suppressor protein. Recently, the Elongin BC complex was found to function as an adaptor that links these BC-box proteins to a module composed of Cullin family members Cul2 or Cul5 and RING-H2 finger protein Rbx1 to reconstitute a family of E3 ubiquitin ligases that activate ubiquitylation by the E2 ubiquitin-conjugating enzyme Ubc5. As part of our effort to understand the functions of Elongin BC-based ubiquitin ligases, we exploited a modified yeast two-hybrid screen to identify a mammalian BC-box protein similar in sequence to Saccharomyces cerevisiae Mediator subunit Med8p. In this report we demonstrate (i) that mammalian MED8 is a subunit of the mammalian Mediator complex and (ii) that MED8 can assemble with Elongins B and C, Cul2, and Rbx1 to reconstitute a ubiquitin ligase. Taken together, our findings are consistent with the model that MED8 could function to recruit ubiquitin ligase activity directly to the RNA polymerase II transcriptional machinery.

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.