CHIP functions an E3 ubiquitin ligase of Runx1

Runx1 is a key factor in the generation and maintenance of hematopoietic stem cells. Improper expression and mutations in Runx1 are frequently implicated in human leukemia. Here, we report that CHIP, the carboxyl terminus of Hsc70-interacting protein, also named Stub1, physically interacts with Runx1 through the TPR and Charged domains in the nucleus. Over-expression of CHIP directly induced Runx1 ubiquitination and degradation through the ubiquitin-proteasome pathway. Interestingly, we found that CHIP-mediated degradation of Runx1 is independent of the molecular chaperone Hsp70/90. Taken together, we propose that CHIP serves as an E3 ubiquitin ligase that regulates Runx1 protein stability via an ubiquitination and degradation mechanism that is independent of Hsp70/90.     

Chaperone functions of the E3 ubiquitin ligase CHIP

The carboxyl terminus of the Hsc70-interacting protein (CHIP) is an Hsp70 co-chaperone as well as an E3 ubiquitin ligase that protects cells from proteotoxic stress. The abilities of CHIP to interact with Hsp70 and function as a ubiquitin ligase place CHIP at a pivotal position in the protein quality control system, where its entrance into Hsp70-substrate complexes partitions nonnative proteins toward degradation. However, the manner by which Hsp70 substrates are selected for ubiquitination by CHIP is not well understood. We discovered that CHIP possesses an intrinsic chaperone activity that enables it to selectively recognize and bind nonnative proteins. Interestingly, the chaperone function of CHIP is temperature-sensitive and is dramatically enhanced by heat stress. The ability of CHIP to recognize nonnative protein structure may aid in selection of slow folding or misfolded polypeptides for ubiquitination.     

Neuralized functions as an E3 ubiquitin ligase during Drosophila development.

The Notch pathway is a widely studied means of intercellular signaling responsible for the determination of cell fate, cell differentiation, and boundary formation (reviewed in ). The main effectors of this pathway, Notch (N) and Delta (Dl), have been shown to function as a receptor and ligand, respectively. Genetic and phenotypic studies suggest that Neuralized (Neu), a RING finger protein, also plays a role within the N-Dl pathway, although its biochemical function is unknown. Here, we show that Neu is required at the plasma membrane for functional activity and that its RING finger domain acts as an E3 ubiquitin ligase. These data suggest that the role of Neu is to target components of the N-Dl pathway for ubiquitination, allowing for propagation and/or regulation of the signal.     

TRIP12 functions as an E3 ubiquitin ligase of APP-BP1

The NEDD8 pathway plays an essential role in various physiological processes, such as cell cycle progression and signal transduction. The conjugation of NEDD8 to target proteins is initiated by the NEDD8-activating enzyme composed of APP-BP1 and Uba3. In the present study, we show that APP-BP1 is degraded by ubiquitin-dependent proteolysis. To study biological functions of TRIP12, a HECT domain-containing E3 ubiquitin ligase, we used the yeast two-hybrid system and identified APP-BP1 as its binding partner. Immunoprecipitation analysis showed that TRIP12 specifically interacts with the APP-BP1 monomer but not with the APP-BP1/Uba3 heterodimer. Overexpression of TRIP12 enhanced the degradation of APP-BP1, whereas knockdown of TRIP12 stabilized it. In vitro ubiquitination assays revealed that TRIP12 functions as an E3 enzyme of APP-BP1 and additionally requires an E4 activity for polyubiquitination of APP-BP1. Moreover, neddylation of endogenous CUL1 was increased in TRIP12 knockdown cells, while complementation of the knockdown cells with TRIP12 lowered neddylated CUL1. Our data suggest that that TRIP12 promotes degradation of APP-BP1 by catalyzing its ubiquitination, which in turn modulates the neddylation pathway.     

RNF220, an E3 ubiquitin ligase that targets Sin3B for ubiquitination

Modification of proteins by ubiquitination plays important roles in various cellular processes. During this process, the target specificity is determined by ubiquitin ligases. Here we identify RNF220 (RING finger protein 220) as a novel ubiquitin ligase for Sin3B. As a conserved RING protein, RNF220 can bind E2 and mediate auto-ubiquitination of itself. Through a yeast two-hybrid screen, we isolated Sin3B as one of its targets, which is a scaffold protein of the Sin3/HDAC (histone deacetylase) corepressor complex. RNF220 specifically interacts with Sin3B both in vitro and in vivo. Sin3B can be regulated by the ubiquitin-proteasome system. Co-expression of RNF220 promotes the ubiquitination and proteasomal degradation of Sin3B. Taken together, these results reveal a new mechanism for regulating the Sin3/HDAC complex.     

Autoantigen Ro52 is an E3 ubiquitin ligase

Anti-Ro/SSA antibodies are classic autoantibodies commonly found in patients with Sj?gren's syndrome, a chronic autoimmune disease characterized by dryness of the eyes and mouth. The autoantibodies recognize a RING-finger protein, Ro52, whose function is still unknown. Since many RING-finger proteins have been identified as E3 ubiquitin ligases, this study was designed to determine whether Ro52 functions as an E3 ubiquitin ligase. For this purpose, recombinant Ro52 was purified from bacterial lysate and used to investigate its activity of E3 ubiquitin ligase in vitro. Its enzymatic activity was also tested in HEK293T cells using wild-type Ro52 and its RING-finger mutant. Our results indicated that Ro52 ubiquitinates itself in cooperation with E2 ubiquitin-conjugating enzyme UbcH5B, thereby validating that Ro52 is a RING-finger-type E3 ubiquitin ligase. Importantly, this ubiquitin modification is predominantly monoubiquitination, which does not target Ro52 to the proteasome for degradation.     

A viral ubiquitin ligase has substrate preferential SUMO targeted ubiquitin ligase activity that counteracts intrinsic antiviral defence

Intrinsic antiviral resistance represents the first line of intracellular defence against virus infection. During herpes simplex virus type-1 (HSV-1) infection this response can lead to the repression of viral gene expression but is counteracted by the viral ubiquitin ligase ICP0. Here we address the mechanisms by which ICP0 overcomes this antiviral response. We report that ICP0 induces the widespread proteasome-dependent degradation of SUMO-conjugated proteins during infection and has properties related to those of cellular SUMO-targeted ubiquitin ligases (STUbLs). Mutation of putative SUMO interaction motifs within ICP0 not only affects its ability to degrade SUMO conjugates, but also its capacity to stimulate HSV-1 lytic infection and reactivation from quiescence. We demonstrate that in the absence of this viral countermeasure the SUMO conjugation pathway plays an important role in mediating intrinsic antiviral resistance and the repression of HSV-1 infection. Using PML as a model substrate, we found that whilst ICP0 preferentially targets SUMO-modified isoforms of PML for degradation, it also induces the degradation of PML isoform I in a SUMO modification-independent manner. PML was degraded by ICP0 more rapidly than the bulk of SUMO-modified proteins in general, implying that the identity of a SUMO-modified protein, as well as the presence of SUMO modification, is involved in ICP0 targeting. We conclude that ICP0 has dual targeting mechanisms involving both SUMO- and substrate-dependent targeting specificities in order to counteract intrinsic antiviral resistance to HSV-1 infection.     

Selective recruitment of an E2~ubiquitin complex by an E3 ubiquitin ligase

RING E3 ligases are proteins that must selectively recruit an E2-conjugating enzyme and facilitate ubiquitin transfer to a substrate. It is not clear how a RING E3 ligase differentiates a naked E2 enzyme from the E2∼ubiquitin-conjugated form or how this is altered upon ubiquitin transfer. RING-box protein 1 (Rbx1/ROC1) is a key protein found in the Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligase complex that functions with the E2 ubiquitin conjugating enzyme CDC34. The solution structure of Rbx1/ROC1 revealed a globular RING domain (residues 40–108) stabilized by three structural zinc ions (root mean square deviation 0.30 ± 0.04 Å) along with a disordered N terminus (residues 12–39). Titration data showed that Rbx1/ROC1 preferentially recruits CDC34 in its ubiquitin-conjugated form and favors this interaction by 50-fold compared with unconjugated CDC34. Furthermore, NMR and biochemical assays identified residues in helix α2 of Rbx1/ROC1 that are essential for binding and activating CDC34∼ubiquitin for ubiquitylation. Taken together, this work provides the first direct structural and biochemical evidence showing that polyubiquitylation by the RING E3 ligase Rbx1/ROC1 requires the preferential recruitment of an E2∼ubiquitin complex and subsequent release of the unconjugated E2 protein upon ubiquitin transfer to a substrate or ubiquitin chain.     

Psh1 is an E3 ubiquitin ligase that targets the centromeric histone variant Cse4

Cse4 is a variant of histone H3 that is incorporated into a single nucleosome at each centromere in budding yeast. We have discovered an E3 ubiquitin ligase, called Psh1, which controls the cellular level of Cse4 via ubiquitylation and proteolysis. The activity of Psh1 is dependent on both its RING and zinc finger domains. We demonstrate the specificity of the ubiquitylation activity of Psh1 toward Cse4 in vitro and map the sites of ubiquitylation. Mutation of key lysines prevents ubiquitylation of Cse4 by Psh1 in vitro and stabilizes Cse4 in vivo. While deletion of Psh1 stabilizes Cse4, elimination of the Cse4-specific chaperone Scm3 destabilizes Cse4, and the addition of Scm3 to the Psh1-Cse4 ubiquitylation reaction prevents Cse4 ubiquitylation, together suggesting Scm3 may protect Cse4 from ubiquitylation. Without Psh1, Cse4 overexpression is toxic and Cse4 is found at ectopic locations. Our results suggest Psh1 functions to prevent the mislocalization of Cse4.     

The EHEC type III effector NleL is an E3 ubiquitin ligase that modulates pedestal formation

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and may result in potentially fatal hemolytic uremia syndrome in humans. EHEC colonize the intestinal mucosa and promote the formation of actin-rich pedestals via translocated type III effectors. Two EHEC type III secreted effectors, Tir and EspFu/TccP, are key players for pedestal formation. We discovered that an EHEC effector protein called Non-LEE-encoded Ligase (NleL) is an E3 ubiquitin ligase. In vitro, we showed that the NleL C753 residue is critical for its E3 ligase activity. Functionally, we demonstrated that NleL E3 ubiquitin ligase activity is involved in modulating Tir-mediated pedestal formation. Surprisingly, EHEC mutant strain deficient in the E3 ligase activity induced more pedestals than the wild-type strain. The canonical EPEC strain E2348/69 normally lacks the nleL gene, and the ectopic expression of the wild-type EHEC nleL, but not the catalytically-deficient nleL(C753A) mutant, in this strain resulted in fewer actin-rich pedestals. Furthermore, we showed that the C. rodentium NleL homolog is a E3 ubiquitin ligase and is required for efficient infection of murine colonic epithelial cells in vivo. In summary, our study demonstrated that EHEC utilizes NleL E3 ubiquitin ligase activity to modulate Tir-mediated pedestal formation.     

Essential role of E3 ubiquitin ligase activity in Cbl-b-regulated T cell functions

E3 ubiquitin ligases have been placed among the essential molecules involved in the regulation of T cell functions and T cell tolerance. However, it has never been experimentally proven in vivo whether these functions indeed depend on the catalytic E3 ligase activity. The Casitas B-cell lymphoma (Cbl) family protein Cbl-b was the first E3 ubiquitin ligase directly implicated in the activation and tolerance of the peripheral T cell. In this study, we report that selective genetic inactivation of Cbl-b E3 ligase activity phenocopies the T cell responses observed when total Cbl-b is ablated, resulting in T cell hyperactivation, spontaneous autoimmunity, and impaired induction of T cell anergy in vivo. Moreover, mice carrying a Cbl-b E3 ligase-defective mutation spontaneously reject tumor cells that express human papilloma virus Ags. These data demonstrate for the first time, to our knowledge, that the catalytic function of an E3 ligase, Cbl-b, is essential for negative regulation of T cells in vivo. Thus, modulation of the E3 ligase activity of Cbl-b might be a novel modality to control T cell immunity in vaccination, cancer biology, or autoimmunity.     

FBXL2 is a ubiquitin E3 ligase subunit that triggers mitotic arrest

Mitotic progression is regulated by ubiquitin E3 ligase complexes to carefully orchestrate eukaryotic cell division. Here, we show that a relatively new E3 ligase component belonging to the SCF (Skip-Cullin1-F-box protein) E3 ligase family, SCF^FBXL2, impairs cell proliferation by mediating cyclin D3 polyubiquitination and degradation. Both cyclin D3 and FBXL2 colocalize within the centrosome. FBXL2 overexpression led to G₂/M-phase arrest in transformed epithelia, resulting in the appearance of supernumerary centrosomes, tetraploidy and nuclei where condensed chromosomes are arranged on circular monopolar spindles typical of mitotic arrest. RNAi-mediated knockdown of cyclin D3 recapitulated effects of SCF^FBXL2 expression. SCF^FBXL2 impaired the ability of cyclin D3 to associate with centrosomal assembly proteins [Aurora A, polo-like kinase 4 (Plk4), CDK11]. Thus, these results suggest a role for SCF^FBXL2 in regulating the fidelity of cellular division.Key words: F-box protein, centrosome, mitosis, cyclin D3, Aurora A     

FBXL2 is a ubiquitin E3 ligase subunit that triggers mitotic arrest

Mitotic progression is regulated by ubiquitin E3 ligase complexes to carefully orchestrate eukaryotic cell division. Here, we show that a relatively new E3 ligase component belonging to the SCF (Skip-Cullin1-F-box protein) E3 ligase family, SCF^FBXL2, impairs cell proliferation by mediating cyclin D3 polyubiquitination and degradation. Both cyclin D3 and FBXL2 colocalize within the centrosome. FBXL2 overexpression led to G₂/M-phase arrest in transformed epithelia, resulting in the appearance of supernumerary centrosomes, tetraploidy and nuclei where condensed chromosomes are arranged on circular monopolar spindles typical of mitotic arrest. RNAi-mediated knockdown of cyclin D3 recapitulated effects of SCF^FBXL2 expression. SCF^FBXL2 impaired the ability of cyclin D3 to associate with centrosomal assembly proteins [Aurora A, polo-like kinase 4 (Plk4), CDK11]. Thus, these results suggest a role for SCF^FBXL2 in regulating the fidelity of cellular division.     

MEX is a testis-specific E3 ubiquitin ligase that promotes death receptor-induced apoptosis

In the present study, we report the identification and characterization of MEX (MEKK1-related protein X), a protein with homology to MEKK1 that is expressed uniquely in the testis. MEX is comprises four putative zinc-binding domains including an N-terminal SWIM (SWI2/SNF2 and MuDR) domain of unknown function and two RING (really interesting new gene) fingers separated by a ZZ zinc finger domain. Biochemical analyses revealed that MEX is self-ubiquitinated and targeted for degradation through the proteasome pathway. MEX can act as an E3, Ub (ubiquitin) ligase, through the E2, Ub-conjugating enzymes UbcH5a, UbcH5c or UbcH6. A region of MEX that contains the RING fingers and the ZZ zinc finger was required for interaction with UbcH5a and MEX self-association, whereas the SWIM domain was critical for MEX ubiquitination. The expression of MEX promoted apoptosis that was induced through Fas, DR (death receptor) 3 and DR4 signalling, but not that mediated by the BH3 (Bcl-2 homology 3)-only protein BimEL or the chemotherapeutic drug adriamycin. The enhancement of apoptosis by MEX required a functional SWIM domain, suggesting that MEX ubiquitination is critical for the enhancement of apoptosis. These results indicate that MEX acts as an E3 Ub ligase, an activity that is dependent on the SWIM domain and suggest a role for MEX in the regulation of death receptor-induced apoptosis in the testes.     

Pam (Protein associated with Myc) functions as an E3 ubiquitin ligase and regulates TSC/mTOR signaling

The tumor suppressor tuberin, encoded by the Tuberous Sclerosis Complex (TSC) gene TSC2, negatively regulates the mammalian target of rapamycin (mTOR) pathway, which plays a key role in the control of cell growth and proliferation. In addition to naturally occurring mutations, several kinases including Akt, RSK1, and ERK are known to phosphorylate and inactivate tuberin. We demonstrate a novel mechanism of tuberin inactivation through ubiquitination by Pam, a putative RING finger-containing E3 ubiquitin (Ub) ligase in mammalian cells. We show that Pam associates with E2 ubiquitin-conjugating enzymes, and tuberin can be ubiquitinated by Pam through its RING finger domain. Tuberin ubiquitination is independent of its phosphorylation by Akt, RSK1, and ERK kinases. Pam is also self-ubiquitinated through its RING finger domain. Moreover, the TSC1 protein hamartin, which forms a heterodimer with tuberin, protects tuberin from ubiquitination by Pam. However, TSC1 fails to protect a disease-associated missense mutant of TSC2 from ubiquitination by Pam. Furthermore, Pam knockdown by RNA interference (RNAi) in rat primary neurons elevates the level of tuberin, and subsequently inhibits the mTOR pathway. Our results provide novel evidence that Pam can function as an E3 Ub ligase toward tuberin and regulate mTOR signaling, suggesting that Pam can in turn regulate cell growth and proliferation as well as neuronal function through the TSC/mTOR pathway in mammalian cells.     

The poxvirus p28 virulence factor is an E3 ubiquitin ligase

A majority of the orthopoxviruses, including the variola virus that causes the dreaded smallpox disease, encode a highly conserved 28-kDa protein with a classic RING finger sequence motif (C(3)HC(4)) at their carboxyl-terminal domains. The RING domain of p28 has been shown to be a critical determinant of viral virulence for the ectromelia virus (mousepox virus) in a murine infection model (Senkevich, T. G., Koonin, E. V., and Buller, R. M. (1994) Virology 198, 118-128). Here, we demonstrate that the p28 proteins encoded by the ectromelia virus and the variola virus possess E3 ubiquitin ligase activity in biochemical assays as well as in cultured mammalian cells. Point mutations disrupting the RING finger domain of p28 completely abolish its E3 ligase activity. In addition, p28 functions cooperatively with Ubc4 and UbcH5c, the E2 conjugating enzymes involved in 26 S proteasome degradation of protein targets. Moreover, p28 catalyzes the formation of Lys-63-linked polyubiquitin chains in the presence of Ubc13/Uev1A, a heterodimeric E2 conjugating enzyme, indicating that p28 may regulate the biological activity of its cognate viral and/or host cell target(s) by Lys-63-linked ubiquitin multimers. We thus conclude that the poxvirus p28 virulence factor is a new member of the RING finger E3 ubiquitin ligase family and has a unique polyubiquitylation activity. We propose that the E3 ligase activity of the p28 virulence factor may be targeted for therapeutic intervention against infections by the variola virus and other poxviruses.     

GIDE is a mitochondrial E3 ubiquitin ligase that induces apoptosis and slows growth

Here, we report the identification of GIDE, a mitochondrially located E3 ubiquitin ligase. GIDE contains a C-terminal RING finger domain, which is mostly conserved with those of the lAP family members and is required for GIDE＇s E3 ligase activity. Overexpression of GIDE induces apoptosis via a pathway involving activation of caspases, since caspase inhibitors, XIAP and an inactive mutant of caspase-9 block GIDE-induced apoptosis. GIDE also activates JNK, and blockage of JNK activation inhibits GIDE-induced release of cytochrome c and Smac as well as apoptosis, suggesting that JNK activation precedes release of cytochrome c and Smac and is required for GIDE- induced apoptosis. These pro-apoptotic properties of GIDE require its E3 ligase activity. When somewhat over-or underexpressed, GIDE slows or accelerates cell growth, respectively. These pro-apoptotic or growth inhibition effects of GIDE may account for its absence in tumor cells.