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At least 1,495 records · Page 83Linked to original sources
PubMed2000

Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases.

Ubiquitin-protein ligases (E3s) regulate diverse cellular processes by mediating protein ubiquitination. The c-Cbl proto-oncogene is a RING family E3 that recognizes activated receptor tyrosine kinases, promotes their ubiquitination by a ubiquitin-conjugating enzyme (E2) and terminates signaling. The crystal structure of c-Cbl bound to a cognate E2 and a kinase peptide shows how the RING domain recruits the E2. A comparison with a HECT family E3-E2 complex indicates that a common E2 motif is recognized by the two E3 families. The structure reveals a rigid coupling between the peptide binding and the E2 binding domains and a conserved surface channel leading from the peptide to the E2 active site, suggesting that RING E3s may function as scaffolds that position the substrate and the E2 optimally for ubiquitin transfer.

Amino Acid Sequence↗
PubMed2003

Structural basis for phosphodependent substrate selection and orientation by the SCFCdc4 ubiquitin ligase.

Cell cycle progression depends on precise elimination of cyclins and cyclin-dependent kinase (CDK) inhibitors by the ubiquitin system. Elimination of the CDK inhibitor Sic1 by the SCFCdc4 ubiquitin ligase at the onset of S phase requires phosphorylation of Sic1 on at least six of its nine Cdc4-phosphodegron (CPD) sites. A 2.7 A X-ray crystal structure of a Skp1-Cdc4 complex bound to a high-affinity CPD phosphopeptide from human cyclin E reveals a core CPD motif, Leu-Leu-pThr-Pro, bound to an eight-bladed WD40 propeller domain in Cdc4. The low affinity of each CPD motif in Sic1 reflects structural discordance with one or more elements of the Cdc4 binding site. Reengineering of Cdc4 to reduce selection against Sic1 sequences allows ubiquitination of lower phosphorylated forms of Sic1. These features account for the observed phosphorylation threshold in Sic1 recognition and suggest an equilibrium binding mode between a single receptor site in Cdc4 and multiple low-affinity CPD sites in Sic1.

Amino Acid Motifs↗
PubMed2003

Pirh2, a p53-induced ubiquitin-protein ligase, promotes p53 degradation.

The p53 tumor suppressor exerts anti-proliferative effects in response to various types of stress including DNA damage and abnormal proliferative signals. Tight regulation of p53 is essential for maintaining normal cell growth and this occurs primarily through posttranslational modifications of p53. Here, we describe Pirh2, a gene regulated by p53 that encodes a RING-H2 domain-containing protein with intrinsic ubiquitin-protein ligase activity. Pirh2 physically interacts with p53 and promotes ubiquitination of p53 independently of Mdm2. Expression of Pirh2 decreases the level of p53 protein and abrogation of endogenous Pirh2 expression increases the level of p53. Furthermore, Pirh2 represses p53 functions including p53-dependent transactivation and growth inhibition. We propose that Pirh2 is involved in the negative regulation of p53 function through physical interaction and ubiquitin-mediated proteolysis. Hence, Pirh2, like Mdm2, participates in an autoregulatory feedback loop that controls p53 function.

Amino Acid Sequence↗
PubMedDate not supplied

Identification of an E3 ubiquitin-protein ligase in the softshell clam (Mya arenaria).

Softshell clams (Mya arenaria) were exposed to dioxin in controlled laboratory experiments in order to study their molecular response to dioxin exposure. A complementary DNA (cDNA) fragment with sequence similarity to E3 ubiquitin-protein ligase appeared to be upregulated in dioxin-exposed clams compared to controls. E3 covalently ligates ubiquitin onto a protein, targeting it for degradation. Our findings suggest that the ubiquitin-mediated proteolytic pathway in the softshell clam may be activated by dioxin exposure. Because the clam E3-predicted amino acid sequence is most similar to a specific vertebrate E3 protein (E6-AP), we hypothesize that dioxin may stimulate ubiquitin-mediated degradation of cell-cycle regulatory proteins, such as the tumor suppressor p53, which promotes cell proliferation. This pathway has been observed in human cervical cancer. Partial cDNA sequence of the clam E3 has been identified using the differential display polymerase chain reaction (ddPCR) and RACE (Rapid Amplification of cDNA Ends) PCR; the full-length sequence is currently being determined. Discovering the molecular mechanism(s) stimulated by dioxin exposure in this invertebrate model may contribute to a better understanding of the effects of dioxin on marine organisms.

Amino Acid Sequence↗
PubMed1998

Characterization of the monovalent and divalent cation requirements for the xenobiotic carboxylic acid: CoA ligases of bovine liver mitochondria.

The XL-I, XL-II and XL-III forms of xenobiotic/medium-chain fatty acid: CoA ligase were found to be inactive toward benzoate in the absence of either monovalent or divalent cations. The absolute requirement for monovalent cation was satisfied by either K+, Rb+, or NH4+. Na+ only supported a very low rate. Varying the nature of the anion had only a minor effect. For XL-I and XI-II, the optimum concentration of K+ was 50 mM; higher (physiologic) concentrations led to a decrease in activity. K+ did not inhibit XL-III. The absolute requirement for divalent cation was satisfied by Mg2+ or Mn2+, or to a lesser extent by Co2+ or Fe2+. For the XL-I and XL-II, excess uncomplexed Mg2+ or Mn2+ decreased the rate; the optimum concentration of Mn2+ was approximately the same as the concentration of ATP in the assay, and the optimum concentration of Mg2+ was approximately double the concentration of ATP in the assay. This is consistent with the concept that the divalent cation is required to complex with ATP and with the known stability constants for the ATP complexes of these two divalent cations. XL-III was not inhibited by uncomplexed divalent cations. Uncomplexed ATP was a moderate inhibitor of XL-I and XL-II, and a weak inhibitor of XL-III. The data indicate that in vivo benzoate conjugation is K+ and Mg2+ dependent, and that the cation effects are complex and differ for XL-I and XL-II as compared with XL-III.

Adenosine Triphosphate↗
PubMed1998

Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation.

The E6-AP ubiquitin ligase (human/mouse gene UBE3A/Ube3a) promotes the degradation of p53 in association with papilloma E6 protein, and maternal deficiency causes human Angelman syndrome (AS). Ube3a is imprinted with silencing of the paternal allele in hippocampus and cerebellum in mice. We found that the phenotype of mice with maternal deficiency (m-/p+) for Ube3a resembles human AS with motor dysfunction, inducible seizures, and a context-dependent learning deficit. Long-term potentiation (LTP) was severely impaired in m-/p+ mice despite normal baseline synaptic transmission and neuroanatomy, indicating that ubiquitination may play a role in mammalian LTP and that LTP may be abnormal in AS. The cytoplasmic abundance of p53 was increased in postmitotic neurons in m-/p+ mice and in AS, providing a potential biochemical basis for the phenotype through failure to ubiquitinate and degrade various effectors.

Acoustic Stimulation↗
PubMed1999

Mutation of the E6-AP ubiquitin ligase reduces nuclear inclusion frequency while accelerating polyglutamine-induced pathology in SCA1 mice.

Mutant ataxin-1, the expanded polyglutamine protein causing spinocerebellar ataxia type 1 (SCA1), aggregates in ubiquitin-positive nuclear inclusions (NI) that alter proteasome distribution in affected SCA1 patient neurons. Here, we observed that ataxin-1 is degraded by the ubiquitin-proteasome pathway. While ataxin-1 [2Q] and mutant ataxin-1 [92Q] are polyubiquitinated equally well in vitro, the mutant form is three times more resistant to degradation. Inhibiting proteasomal degradation promotes ataxin-1 aggregation in transfected cells. And in mice, Purkinje cells that express mutant ataxin-1 but not a ubiquitin-protein ligase have significantly fewer NIs. Nonetheless, the Purkinje cell pathology is markedly worse than that of SCA1 mice. Taken together, NIs are not necessary to induce neurodegeneration, but impaired proteasomal degradation of mutant ataxin-1 may contribute to SCA1 pathogenesis.

Animals↗
PubMed2002

The ret finger protein-like 4 gene, Rfpl4, encodes a putative E3 ubiquitin-protein ligase expressed in adult germ cells.

Using an in silico (electronic database) subtraction, we identified a new member of the Ret Finger Protein-Like gene family, Rfpl4. Rfpl4 encodes a 287 amino acid putative E3 ubiquitin-protein ligase with a RING finger-like domain and a B30.2 motif. Reverse transcriptase polymerase chain reaction and Northern blot analyses reveal that Rfpl4 encodes a 1.7kb mRNA detectable exclusively in the gonads of adult mice. In situ hybridization localizes Rfpl4 transcripts within the ovary to oocytes of primary and later stage follicles and in the testis to elongating spermatids. The Rfpl4 gene comprises three exons and maps to mouse chromosome 7. We have identified the human ortholog, which maps to 19q13.4. These studies suggest that RFPL4 mediates protein degradation pathways important for gametogenesis or early embryonic development.

Amino Acid Sequence↗
PubMed2002

The BRCA1/BARD1 heterodimer, a tumor suppressor complex with ubiquitin E3 ligase activity.

Although the protein product of the BRCA1 tumor suppressor gene has been implicated in a surprisingly diverse array of biological processes, the molecular mechanism by which BRCA1 loss promotes tumor formation remains unclear. Nonetheless, a pivotal advance has been achieved by recent studies that establish BRCA1 and its partner polypeptide BARD1 as enzymatic mediators of protein ubiquitination. The potent ubiquitin E3 ligase activity of the BRCA1/BARD1 heterodimer may be responsible for many of the biological properties attributed to BRCA1, including its ability to suppress tumor formation in normal cells.

Amino Acid Sequence↗
PubMed2000

The SCF ubiquitin ligase protein slimb regulates centrosome duplication in Drosophila.

The duplication of the centrosome is a key event in the cell-division cycle. Although defects in centrosome duplication are thought to contribute to genomic instability [1-3] and are a hallmark of certain transformed cells and human cancer [4-6], the mechanism responsible for centrosome duplication is not understood. Recent experiments have established that centrosome duplication requires the activity of cyclin-dependent kinase 2 (Cdk2) and cyclins E and A [7-9]. The stability of cyclin E is regulated by the ubiquitin ligase SCF, which is a protein complex composed of Skp1, Cdc53 (Cullin) and F-box proteins [10-12]. The Skp1 and Cullin components have been detected on mammalian centrosomes, and shown to be essential for centrosome duplication and separation in Xenopus [13]. Here, we report that Slimb, an F-box protein that targets proteins to the SCFcomplex [14,15], plays a role in limiting centrosome replication. We found that, in the fruit fly Drosophila, the hypomorphic mutation slimb(crd) causes the appearance of additional centrosomes and mitotic defects in mutant larval neuroblasts.

Animals↗
PubMed2001

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.

Animals↗
PubMed2000

The lore of the RINGs: substrate recognition and catalysis by ubiquitin ligases.

Recently, many new examples of E3 ubiquitin ligases or E3 enzymes have been found to regulate a host of cellular processes. These E3 enzymes direct the formation of multiubiquitin chains on specific protein substrates, and - typically - the subsequent destruction of those proteins. We discuss how the modular architecture of E3 enzymes connects one of two distinct classes of catalytic domains to a wide range of substrate-binding domains. In one catalytic class, a HECT domain transfers ubiquitin directly to substrate bound to a non-catalytic domain. Members of the other catalytic class, found in the SCF, VBC and APC complexes, use a RING finger domain to facilitate ubiquitylation. The separable substrate-recognition domains of E3 enzymes provides a flexible means of linking a conserved ubiquitylation function to potentially thousands of ubiquitylated substrates in eukaryotic cells.

Animals↗
PubMed2003

PHD domains and E3 ubiquitin ligases: viruses make the connection.

PHD domains constitute a widely distributed subfamily of zinc fingers whose biochemical functions have been unclear until now. Recently, several PHD-containing viral proteins have been identified that promote immune evasion by downregulating proteins that govern immune recognition. Studies show that these viral regulators lead to ubiquitination of their targets by functioning as E3 ubiquitin ligases -- an activity that requires the PHD motif. These are the first examples linking the PHD domain to E3 activity, but the recent discovery of PHD-dependent E3 activity in the cellular kinase MEKK1 and the close structural relation of PHD domains to RING fingers hint that many other PHD proteins might share this activity.

Amino Acid Sequence↗
PubMed2002

Protein quality control: U-box-containing E3 ubiquitin ligases join the fold.

Molecular chaperones act with folding co-chaperones to suppress protein aggregation and refold stress damaged proteins. However, it is not clear how slowly folding or misfolded polypeptides are targeted for proteasomal degradation. Generally, selection of proteins for degradation is mediated by E3 ubiquitin ligases of the mechanistically distinct HECT and RING domain sub-types. Recent studies suggest that the U-box protein family represents a third class of E3 enzymes. CHIP, a U-box-containing protein, is a degradatory co-chaperone of heat-shock protein 70 (Hsp70) and Hsp90 that facilitates the polyubiquitination of chaperone substrates. These data indicate a model for protein quality control in which the interaction of Hsp70 and Hsp90 with co-chaperones that have either folding or degradatory activity helps to determine the fate of non-native cellular proteins.

Cysteine Endopeptidases↗
PubMed2003

RING finger ubiquitin protein ligases: implications for tumorigenesis, metastasis and for molecular targets in cancer.

Covalent modification of proteins with ubiquitin regulates almost all aspects of eukaryotic cellular function. Ubiquitin protein ligases (E3s) play central regulatory roles in that they provide substrate specificity to this process and therefore, represent attractive molecular targets for disease therapy. We summarize recent advances in our understanding of RING finger and RING finger-related E3s with emphasis on BRCA1 and the tumor autocrine motility factor receptor (gp78), as well as discuss the potential for components of the ubiquitin pathway for proteasomal degradation as molecular targets.

Amino Acid Motifs↗
PubMed2000

DNA ligase IV deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway.

DNA ligase IV (LIG4) is a nonhomologous end-joining (NHEJ) protein used for V(D)J recombination and DNA repair. In mice, Lig4 deficiency causes embryonic lethality, massive neuronal apoptosis, arrested lymphogenesis, and various cellular defects. Herein, we assess potential roles in this phenotype for INK4a/ARF and p53, two proteins implicated in apoptosis and senescence. INK4a/ARF deficiency rescued proliferation/senescence defects of Lig4-deficient fibroblasts but not other phenotypic aspects. In contrast, p53 deficiency rescued embryonic lethality, neuronal apoptosis, and fibroblast proliferation/senescence defects but not lymphocyte development or radiosensitivity. Young Lig4/p53 double null mice routinely died from pro-B lymphomas. Thus, in the context of Lig4 deficiency, embryonic lethality and neuronal apoptosis likely result from a p53-dependent response to unrepaired DNA damage, and neuronal apoptosis and lymphocyte developmental defects can be mechanistically dissociated.

Animals↗
PubMed2002

CHIP is associated with Parkin, a gene responsible for familial Parkinson's disease, and enhances its ubiquitin ligase activity.

Unfolded Pael receptor (Pael-R) is a substrate of the E3 ubiquitin ligase Parkin. Accumulation of Pael-R in the endoplasmic reticulum (ER) of dopaminergic neurons induces ER stress leading to neurodegeneration. Here, we show that CHIP, Hsp70, Parkin, and Pael-R formed a complex in vitro and in vivo. The amount of CHIP in the complex was increased during ER stress. CHIP promoted the dissociation of Hsp70 from Parkin and Pael-R, thus facilitating Parkin-mediated Pael-R ubiquitination. Moreover, CHIP enhanced Parkin-mediated in vitro ubiquitination of Pael-R in the absence of Hsp70. Furthermore, CHIP enhanced the ability of Parkin to inhibit cell death induced by Pael-R. Taken together, these results indicate that CHIP is a mammalian E4-like molecule that positively regulates Parkin E3 activity.

Animals↗
PubMed2004

The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription factors.

Skeletal muscle size depends upon a dynamic balance between anabolic (or hypertrophic) and catabolic (or atrophic) processes. Previously, no link between the molecular mediators of atrophy and hypertrophy had been reported. We demonstrate a hierarchy between the signals which mediate hypertrophy and those which mediate atrophy: the IGF-1/PI3K/Akt pathway, which has been shown to induce hypertrophy, prevents induction of requisite atrophy mediators, namely the muscle-specific ubiquitin ligases MAFbx and MuRF1. Moreover, the mechanism for this inhibition involves Akt-mediated inhibition of the FoxO family of transcription factors; a mutant form of FOXO1, which prevents Akt phosphorylation, thereby prevents Akt-mediated inhibition of MuRF1 and MAFbx upregulation. Our study thus defines a previously uncharacterized function for Akt, which has important therapeutic relevance: Akt is not only capable of activating prosynthetic pathways, as previously demonstrated, but is simultaneously and dominantly able to suppress catabolic pathways, allowing it to prevent glucocorticoid and denervation-induced muscle atrophy.

Animals↗