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At least 1,135 records · Page 63Linked to original sources
PubMed2005

Ubiquitin ligase activity of TFIIH and the transcriptional response to DNA damage.

Core transcription factor (TF) IIH purified from yeast possesses an E3 ubiquitin (Ub) ligase activity, which resides, at least in part, in a RING finger (RNF) domain of the Ssl1 subunit. Yeast strains mutated in the Ssl1 RNF domain are sensitive to ultraviolet (UV) light and to methyl methanesulfonate (MMS). This increased sensitivity to DNA-damaging agents does not reflect a deficiency in nucleotide excision repair. Rather, it correlates with reduced transcriptional induction of genes involved in DNA repair, suggesting that the E3 Ub ligase activity of TFIIH mediates the transcriptional response to DNA damage.

Amino Acid Sequence↗
PubMed2006

Negative regulation of the E3 ubiquitin ligase itch via Fyn-mediated tyrosine phosphorylation.

Conjugation of ubiquitin (Ub) to a protein substrate targets the substrate for degradation or functional modification, which is tightly controlled by diverse mechanisms including phosphorylation of the substrate. An emerging mechanism involves regulation of the E3 Ub ligase, for example, the JNK-dependent phosphorylation and activation of Itch E3 ligase, which controls the turnover of Jun proteins and T cell differentiation. Here we show that Itch is also modulated by an Src kinase Fyn via tyrosine phosphorylation at the Tyr371 residue. Fyn associates with Itch, and loss of Fyn results in reduced Itch phosphorylation. Importantly, tyrosine phosphorylation of Itch appears to reduce its interaction with its substrate JunB. The turnover of JunB is accelerated in Fyn-deficient T cells, which is further reconstituted by Itch Tyr371 mutation. Thus, in contrast to the activation pathway mediated by serine/threonine phosphorylation, tyrosine phosphorylation of Itch plays a negative role in modulating Itch-promoted ubiquitination.

Amino Acid Sequence↗
PubMed2006

Histone H3 and H4 ubiquitylation by the CUL4-DDB-ROC1 ubiquitin ligase facilitates cellular response to DNA damage.

Posttranslational histone modifications play important roles in transcription and other chromatin-based processes. Compared to acetylation, methylation, and phosphorylation, very little is known about the function of histone ubiquitylation. Here, we report the purification and functional characterization of a histone H3 and H4 ubiquitin ligase complex, CUL4-DDB-ROC1. We demonstrate that CUL4-DDB-ROC1-mediated H3 and H4 ubiquitylation occurs both in vitro and in vivo. Importantly, CUL4-DDB-ROC1-mediated H3 and H4 ubiquitylation is regulated by UV irradiation. Reduction of histone H3 and H4 ubiquitylation by knockdown of CUL4A impairs recruitment of the repair protein XPC to the damaged foci and inhibits the repair process. Biochemical studies indicate that CUL4-DDB-ROC1-mediated histone ubiquitylation weakens the interaction between histones and DNA and facilitates the recruitment of repair proteins to damaged DNA. Thus, our studies uncover CUL4-DDB-ROC1 as a histone ubiquitin ligase and demonstrate that histone H3 and H4 ubiquitylation participates in the cellular response to DNA damage.

Carrier Proteins↗
PubMed2006

Dual-site regulation of MDM2 E3-ubiquitin ligase activity.

The control of p53 ubiquitination by MDM2 provides a model system to define how an E3-ligase functions on a conformationally flexible substrate. The mechanism of MDM2-mediated ubiquitination of p53 has been analyzed by deconstructing, in vitro, the MDM2-dependent ubiquitination reaction. Surprisingly, ligands binding to the hydrophobic cleft of MDM2 do not inhibit its E3-ligase function. However, peptides from within the DNA binding domain of p53 that bind the acid domain of MDM2 inhibit ubiquitination of p53, localizing a motif that harbors a key ubiquitination signal. The binding of ligands to the N-terminal hydrophobic cleft of MDM2 reactivates, in vitro and in vivo, MDM2-catalyzed ubiquitination of p53F19A, a mutant p53 normally refractory to MDM2-catalyzed ubiquitination. We propose a model in which the interaction between the p53-BOX-I domain and the N terminus of MDM2 promotes conformational changes in MDM2 that stabilize acid-domain interactions with a ubiquitination signal in the DNA binding domain of the p53 tetramer.

Amino Acid Sequence↗
PubMed2006

The polycomb protein Ring1B generates self atypical mixed ubiquitin chains required for its in vitro histone H2A ligase activity.

Polycomb complexes mediate gene silencing, in part by modifying histones. Ring1B and Bmi1 are RING finger proteins that are members of the Polycomb repressive complex 1 (PRC1). Ring1B is an E3 that mediates its own polyubiquitination and monoubiquitination of histone H2A. In contrast, Bmi1 has no self-ubiquitinating activity. We show that unlike other RING finger proteins that are believed to mediate their own ubiquitination and degradation, Ring1B and Bmi1 are degraded by an exogenous E3, independent of their RING domain. The RING domains of both proteins mediate their association and subsequent stabilization. Consistent with the nonproteolytic self-ligase activity of Ring1B, it generates atypical mixed K6-, K27-, and K48-based polyubiquitin chains, which require the presence of all these lysine residues on the same ubiquitin molecule. The modification is required for Ring1B ability to monoubiquitinate H2A in vitro, unraveling an as yet undescribed mechanism for ligase activation via noncanonical self-ubiquitination.

Animals↗
PubMed2006

Loss of Ubr2, an E3 ubiquitin ligase, leads to chromosome fragility and impaired homologous recombinational repair.

The N-end rule pathway of protein degradation targets proteins with destabilizing N-terminal residues. Ubr2 is one of the E3 ubiquitin ligases of the mouse N-end rule pathway. We have previously shown that Ubr2-/- male mice are infertile, owing to the arrest of spermatocytes between the leptotene/zygotene and pachytene of meiosis I, the failure of chromosome pairing, and subsequent apoptosis. Here, we report that mouse fibroblast cells derived from Ubr2-/- embryos display genome instability. The frequency of chromosomal bridges and micronuclei were much higher in Ubr2-/- fibroblasts than in +/+ controls. Metaphase chromosome spreads from Ubr2-/- cells revealed a high incidence of spontaneous chromosomal gaps, indicating chromosomal fragility. These fragile sites were generally replicated late in S phase. Ubr2-/- cells were hypersensitive to mitomycin C, a DNA cross-linking agent, but displayed normal sensitivity to gamma-irradiation. A reporter assay showed that Ubr2-/- cells are significantly impaired in the homologous recombination repair of a double strand break. In contrast, Ubr2-/- cells appeared normal in an assay for non-homologous end joining. Our results therefore unveil the role of the ubiquitin ligase Ubr2 in maintaining genome integrity and in homologous recombination repair.

Animals↗
PubMed2004

Mechanism of cell-cycle control: ligating the ligase.

The F-box protein SKP2 promotes the G1-S transition by targeting key regulators for proteasomal degradation via its capacity to function as the specificity factor for the SKP1 Cullin F-box SCF(SKP2) ubiquitin ligase. SKP2 is a labile protein, the levels of which oscillate in a cell cycle-dependent manner. SKP2 accumulation is often deregulated in cancer, which indicates that temporal control of SKP2 is essential for normal cell proliferation. Two new studies now suggest that SKP2 accumulation is determined by a second ubiquitin ligase, the anaphase-promoting complex or cyclosome, APC/C(CDH1). These studies highlight a novel mechanism wherein mitotic machinery communicates with proteins that regulate G1 phase progression.

Animals↗
PubMed2006

Regulating the regulator: negative regulation of Cbl ubiquitin ligases.

Cbl proteins are regulators of signal transduction through many pathways and, consequently, regulate cell function and development. They are ubiquitin ligases that ubiquitinate and target many signaling molecules for degradation. The Cbl proteins themselves are regulated by an increasingly complex network of interactions that fine-tune the effects that Cbl proteins have on signaling. The negative regulation of Cbl protein function can occur via cis-acting structural elements that prevent inappropriate ubiquitin ligase activity, degradation of the Cbl proteins, inhibition without degradation owing to interaction with other signaling proteins, deubiquitination of Cbl substrates, and regulation of assembly of the endosomal ESCRT-I complex. Defects in the regulatory mechanisms that control Cbl function are implicated in the development of immunological and malignant diseases.

Animals↗
PubMed2006

Assembly of HIV-1 Vif-Cul5 E3 ubiquitin ligase through a novel zinc-binding domain-stabilized hydrophobic interface in Vif.

APOBEC3G (A3G) and related cytidine deaminases are potent inhibitors of retroviruses. HIV-1 Vif hijacks the cellular Cul5-E3 ubiquitin ligase to degrade APOBEC3 proteins and render them ineffective against these viruses. Here, we report that HIV-1 Vif is a novel zinc-binding protein containing an H-x(5)-C-x(17-18)-C-x(3-5)-H motif that is distinct from other recognized classes of zinc fingers. Zinc-binding stabilized a conserved hydrophobic interface within the HCCH motif that is critical for Vif-Cul5 E3 assembly and Vif function. An N-terminal region in the first Cullin repeat of Cul5, which is dispensable for adaptor ElonginC binding, was required for interaction with Vif. This region is the most divergent sequence between Cul2 and Cul5, a factor that may contribute to the selection of Cul5 and not Cul2 by Vif. This is the first example of a zinc-binding substrate receptor responsible for the assembly of a Cullin-RING ligase, representing a new target for antiviral development.

Amino Acid Motifs↗
PubMed2004

Ubiquitin protein ligase activity of the anti-apoptotic baculovirus protein Op-IAP3.

The baculovirus inhibitor of apoptosis protein (IAP) Op-IAP3 is required to prevent apoptosis during infection of insect cells by Orgyia pseudotsugata M nucleopolyhedrovirus (OpMNPV) and inhibits apoptosis when overexpressed in insect and mammalian cells. Although previous reports have demonstrated that the RING domain is important for the anti-apoptotic function of Op-IAP3, the function of this domain in Op-IAP3 has not been studied. Here, the ability of Op-IAP3 to function as an E3 ubiquitin protein ligase was examined. Op-IAP3 expressed in the insect cell line Spodoptera frugiperda (Sf21) was ubiquitinated, but only if the RING domain was intact. In addition, co-expression of Op-IAP3 and the pro-apoptotic Drosophila protein HID resulted in the ubiquitination of HID. Recombinant Op-IAP3 protein also promoted the ubiquitination of both itself and recombinant HID protein in vitro, and the ubiquitination of HID required both the RING and BIR2 of Op-IAP3. Thus, we conclude that Op-IAP3 is a functional E3 ubiquitin ligase, and the ability to ubiquitinate pro-apoptotic cellular proteins such as HID may play an important role in the anti-apoptotic function of Op-IAP3.

Animals↗
PubMed2005

TRIM37 defective in mulibrey nanism is a novel RING finger ubiquitin E3 ligase.

Mulibrey nanism is an autosomal recessive prenatal-onset growth disorder characterized by dysmorphic features, cardiomyopathy, and hepatomegaly. Mutations in TRIM37 encoding a tripartite motif (TRIM, RING-B-box-coiled-coil)-family protein underlie mulibrey nanism. We investigated the ubiquitin ligase activity predicted for the RING domain of TRIM37 by analyzing its autoubiquitination. Full-length TRIM37 and its TRIM domain were highly polyubiquitinated when co-expressed with ubiquitin. Polyubiquitination was decreased in a mutant protein with disrupted RING domain (Cys35Ser;Cys36Ser) and in the Leu76Pro mutant protein, a disease-associated missense mutation affecting the TRIM domain of TRIM37. Bacterially produced GST-TRIM domain fusion protein, but not its Cys35Ser;Cys36Ser or Leu76Pro mutants, were polyubiquitinated in cell-free conditions, implying RING-dependent modification. Ubiquitin was also identified as an interaction partner for TRIM37 in a yeast two-hybrid screen. Ectopically expressed TRIM37 rapidly formed aggregates that were ubiquitin-, proteasome subunit-, and chaperone-positive in immunofluorescence analysis, defining them as aggresomes. The Cys35Ser;Cys36Ser mutant and the Leu76Pro and Gly322Val patient mutant proteins were markedly less prone to aggregation, implying that aggresomal targeting reflects a physiological function of TRIM37. These findings suggest that TRIM37 acts as a TRIM domain-dependent E3 ubiquitin ligase and imply defective ubiquitin-dependent degradation of an as-yet-unidentified target protein in the pathogenesis of mulibrey nanism.

Alleles↗
PubMed2006

Differential response between the p53 ubiquitin-protein ligases Pirh2 and MdM2 following DNA damage in human cancer cells.

Pirh2, a recently identified ubiquitin-protein ligase, has been reported to promote p53 degradation. Pirh2 physically interacts with p53 and promotes ubiquitination of p53 independently of MDM2. Like MDM2, Pirh2 is thought to participate in an autoregulatory feedback loop that controls p53 function. We have previously reported that Pirh2 was overexpressed in human and murine lung cancers as compared to uninvolved lung tissue. Pirh2 increase could potentially cause degradation of wildtype p53 and reduce its tumor suppression function in the lung tumor cells. Since Pirh2 has been reported to be transactivated by p53, however, the mechanisms by which a high level of Pirh2 expression is maintained in tumor cells despite low level of wildtype p53 protein are unclear. In order to evaluate p53 involvement in the transactivation of Pirh2, we evaluated Pirh2, MDM2, p53 and p21 expression with Western blot analysis and real time PCR after gamma irradiation or cisplatin DNA damage treatment using human cancer cell lines containing wildtype (A549, MCF-7), mutant (H719) and null (H1299) p53. Surprisingly, Pirh2 expression was not affected by the presence of wildtype p53 in the cancer cells. In contrast, MDM2 was upregulated by wildtype p53 in A549 and MCF-7 cells and was absent from the H1299 and the H719 cells. We conclude that Pirh2 operates in a distinct manner from MDM2 in response to DNA damage in cancer cells. Pirh2 elevation in p53 null cells indicates the existence of additional molecular mechanisms for Pirh2 upregulation and suggests that p53 is not the sole target of Pirh2 ubiquitin ligase activity.

Animals↗
PubMed2006

SanJ, an ATP-dependent picolinate-CoA ligase, catalyzes the conversion of picolinate to picolinate-CoA during nikkomycin biosynthesis in Streptomyces ansochromogenes.

Nikkomycins, a group of peptidyl nucleoside antibiotics, are competitive inhibitors of chitin synthase. The nikkomycin biosynthetic gene cluster has been cloned previously from Streptomyces ansochromogenes. The cluster contains 25 complete ORFs including sanJ. The sanJ gene was inactivated by the insertion of a kanamycin resistance gene and the resulting disruption mutants failed to produce nikkomycins. Moreover, the nikkomycin production was restored by complementation with a single copy of sanJ. The deduced product of sanJ bears striking sequence similarity with enzymes belonging to the adenylate-forming superfamily. sanJ was overexpressed as a His6-tagged fusion protein in Escherichia coli and purified to apparent homogeneity by affinity chromatography. The purified SanJ demonstrated adenylate ligase activity in the presence of picolinate or its analogs (benzoate, nicotinate, 4-methoxybenzoate, 4-hydroxybenzoate), ATP and Mg2+. SanJ was also found to catalyze the conversion of picolinate, benzoate, nicotinate to their corresponding CoA esters and 4-methoxybenzoate, 4-hydroxybenzoate to their respective AMP derivatives in vitro. This was unambiguously shown by using HPLC and electrospray ionization mass spectrometry (ESI-MS) or by comparing the reaction product with an authentic standard of benzoyl-CoA. These results indicated that sanJ encodes an ATP-dependent picolinate-CoA ligase which is essential for nikkomycin biosynthesis.

Adenosine Triphosphate↗
PubMed2005

Skeletrophin, a novel ubiquitin ligase to the intracellular region of Jagged-2, is aberrantly expressed in multiple myeloma.

Recent research has indicated that ligand-dependent activation of the Notch receptor in stromal cells plays a crucial role in the tumorigenesis of multiple myeloma. Ubiquitination of intracellular regions of Notch receptor and its ligands is important for Notch signal transduction. In vitro autoubiquitination analysis using recombinant proteins identified skeletrophin as a novel RING finger-dependent ubiquitin ligase. Skeletrophin bound the intracellular regions of the Notch ligand Jagged-2, but not to those of Delta-1, -3, -4, or Jagged-1. Skeletrophin, but not its RING-mutated form, ubiquitinized the intracellular region of Jagged-2. In malignant plasma cells from 23 of 40 multiple myeloma specimens, strong skeletrophin expression was observed, especially from patients with osteolytic bone lesions. Cytoplasmic localization, which may indicate Jagged-2 internalization, was found in many skeletrophin-positive myeloma cells. In contrast, skeletrophin was only weakly expressed in a few nonneoplasmic plasma cells in chronically inflamed tissues. Interestingly, exogenous expression of skeletrophin, but not the RING-mutated form, in Jagged-2-positive P3U1 myeloma cells induced Hes-1 (Hairy and Enhancer of Split homolog-1) gene expression in Notch receptor-positive bone marrow stromal cells through direct cell-cell contact. Thus, skeletrophin is a novel ubiquitin ligase that targets the intracellular region of Jagged-2 and is aberrantly overexpressed in multiple myeloma cells, possibly activating Hes-1 on stromal cells through ligand-dependent Notch signaling.

Basic Helix-Loop-Helix Proteins↗
PubMed1988

Regulation of hepatic cholesterol biosynthesis by fatty acids: effect of feeding olive oil on cytoplasmic acetoacetyl-coenzyme A thiolase, beta-hydroxy-beta-methylglutaryl-CoA synthase, and acetoacetyl-coenzyme A ligase.

We reported previously that, in the perfused rat liver, oleic acid increased the specific activity of cytosolic enzymes of cholesterol biosynthesis. In this study, we examined the effects of oral administration of olive oil on the activities of HMG-CoA synthase, AcAc-CoA thiolase, AcAc-CoA ligase and HMG-CoA reductase. Olive oil feeding increased the specific activity of hepatic HMG-CoA synthase by 50%, AcAc-CoA thiolase by 2-fold, and AcAc-CoA ligase by 3-fold. Olive oil had no effect on HMG-CoA reductase activity. These data suggest that the enzymes that supply the HMG-CoA required for hepatic cholesterogenesis are regulated in parallel by a physiological substrate, fatty acid, independent of HMG-CoA reductase under these conditions.

Acetyl-CoA C-Acetyltransferase↗
PubMed2004

NIRF is a ubiquitin ligase that is capable of ubiquitinating PCNP, a PEST-containing nuclear protein.

We previously reported the association of a novel Np95/ICBP90-like RING finger protein (NIRF) with a novel PEST-containing nuclear protein (PCNP). NIRF is a nuclear protein with a ubiquitin-like domain, a PHD finger, a YDG/SRA domain, Rb-binding motifs and a RING finger. In this study, we showed that NIRF has auto-ubiquitination activity, the hallmark of a ubiquitin ligase. PCNP was readily ubiquitinated in 293 and COS-7 cells, and NIRF ubiquitinated PCNP in vitro as well as in vivo. Considering that NIRF is implicated in cell cycle regulation, these findings suggest that NIRF and PCNP are a ubiquitin ligase and its substrate, respectively, and may constitute a novel signaling pathway with some relation to cell proliferation.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53.

The tumor suppressor p53 is degraded by the ubiquitin-proteasome system. p53 was polyubiquitinated in the presence of E1, UbcH5 as E2 and MDM2 oncoprotein. A ubiquitin molecule bound MDM2 through sulfhydroxy bond which is characteristic of ubiquitin ligase (E3)-ubiquitin binding. The cysteine residue in the carboxyl terminus of MDM2 was essential for the activity. These data suggest that the MDM2 protein, which is induced by p53, functions as a ubiquitin ligase, E3, in human papillomavirus-uninfected cells which do not have E6 protein.

Amino Acid Sequence↗
PubMed2005

High-level expression and purification of recombinant SCF ubiquitin ligases.

The SCF complexes are the prototype of a superfamily of cullin-dependent ubiquitin ligases, which regulate diverse cellular functions by promoting the ubiquitination of a large number of regulatory and signaling proteins. The SCF complexes are organized by the elongated scaffold protein subunit Cul1, which interacts with the Rbx1 RING finger protein at one end and the Skp1 adaptor protein at the other. By binding to Skp1, members of the F-box protein family are responsible for recruiting specific substrates to the ligase machine. This chapter describes methods that we have developed to achieve high-level expression and purification of two recombinant SCF complexes from both insect cells and bacteria. We emphasize the power of protein coexpression and a novel "Split-n-Coexpress" method in producing soluble and functional recombinant proteins and protein complexes. We propose that similar approaches can be used to obtain large quantities of other SCF and SCF-like complexes for biochemical and structural investigations.

Animals↗