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At least 793 records · Page 44Linked to original sources
PubMed2005

14-3-3eta is a novel regulator of parkin ubiquitin ligase.

Mutation of the parkin gene, which encodes an E3 ubiquitin-protein ligase, is the major cause of autosomal recessive juvenile parkinsonism (ARJP). Although various substrates for parkin have been identified, the mechanisms that regulate the ubiquitin ligase activity of parkin are poorly understood. Here we report that 14-3-3eta, a chaperone-like protein present abundantly in neurons, could bind to parkin and negatively regulate its ubiquitin ligase activity. Furthermore, 14-3-3eta could bind to the linker region of parkin but not parkin with ARJP-causing R42P, K161N, and T240R mutations. Intriguingly, alpha-synuclein (alpha-SN), another familial Parkinson's disease (PD) gene product, abrogated the 14-3-3eta-induced suppression of parkin activity. alpha-SN could bind tightly to 14-3-3eta and consequently sequester it from the parkin-14-3-3eta complex. PD-causing A30P and A53T mutants of alpha-SN could not bind 14-3-3eta, and failed to activate parkin. Our findings indicate that 14-3-3eta is a regulator that functionally links parkin and alpha-SN. The alpha-SN-positive and 14-3-3eta-negative control of parkin activity sheds new light on the pathophysiological roles of parkin.

14-3-3 Proteins↗
PubMed2005

Transactivation of Schizosaccharomyces pombe cdt2+ stimulates a Pcu4-Ddb1-CSN ubiquitin ligase.

Cullin-4 forms a scaffold for multiple ubiquitin ligases. In Schizosaccharomyces pombe, the Cullin-4 homologue (Pcu4) physically associates with Ddb1 and the COP9 signalosome (CSN). One target of this complex is Spd1. Spd1 regulates ribonucleotide reductase (RNR) activity. Spd1 degradation during S phase, or following DNA damage of G2 cells, results in the nuclear export of the small RNR subunit. We demonstrate that Cdt2, an unstable WD40 protein, is a regulatory subunit of Pcu4-Ddb1-CSN ubiquitin ligase. cdt2 deletion stabilises Spd1 and prevents relocalisation of the small RNR subunit from the nucleus to the cytoplasm. cdt2+ is periodically transcribed by the Cdc10/DSC1 transcription factor during S phase and transiently transcribed following DNA damage of G2 cells, corresponding to Spd1 degradation profiles. Cdt2 co-precipitates with Spd1, and Cdt2 overexpression results in constitutive Spd1 degradation. We propose that Cdt2 incorporation into the Pcu4-Ddb1-CSN complex prompts Spd1 targeting and subsequent degradation and that Cdt2 is a WD40 repeat adaptor protein for Cullin-4-based ubiquitin ligase.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Structure and E3-ligase activity of the Ring-Ring complex of polycomb proteins Bmi1 and Ring1b.

Polycomb group proteins Ring1b and Bmi1 (B-cell-specific Moloney murine leukaemia virus integration site 1) are critical components of the chromatin modulating PRC1 complex. Histone H2A ubiquitination by the PRC1 complex strongly depends on the Ring1b protein. Here we show that the E3-ligase activity of Ring1b on histone H2A is enhanced by Bmi1 in vitro. The N-terminal Ring-domains are sufficient for this activity and Ring1a can replace Ring1b. E2 enzymes UbcH5a, b, c or UbcH6 support this activity with varying processivity and selectivity. All four E2s promote autoubiquitination of Ring1b without affecting E3-ligase activity. We solved the crystal structure of the Ring-Ring heterodimeric complex of Ring1b and Bmi1. In the structure the arrangement of the Ring-domains is similar to another H2A E3 ligase, the BRCA1/BARD1 complex, but complex formation depends on an N-terminal arm of Ring1b that embraces the Bmi1 Ring-domain. Mutation of a critical residue in the E2/E3 interface shows that catalytic activity resides in Ring1b and not in Bmi1. These data provide a foundation for understanding the critical enzymatic activity at the core of the PRC1 polycomb complex, which is implicated in stem cell maintenance and cancer.

Amino Acid Sequence↗
PubMed2006

A novel mitochondrial ubiquitin ligase plays a critical role in mitochondrial dynamics.

In this study, we have identified a novel mitochondrial ubiquitin ligase, designated MITOL, which is localized in the mitochondrial outer membrane. MITOL possesses a Plant Homeo-Domain (PHD) motif responsible for E3 ubiquitin ligase activity and predicted four-transmembrane domains. MITOL displayed a rapid degradation by autoubiquitination activity in a PHD-dependent manner. HeLa cells stably expressing a MITOL mutant lacking ubiquitin ligase activity or MITOL-deficient cells by small interfering RNA showed an aberrant mitochondrial morphology such as fragmentation, suggesting the enhancement of mitochondrial fission by MITOL dysfunction. Indeed, a dominant-negative expression of Drp1 mutant blocked mitochondrial fragmentation induced by MITOL depletion. We found that MITOL associated with and ubiquitinated mitochondrial fission protein hFis1 and Drp1. Pulse-chase experiment showed that MITOL overexpression increased turnover of these fission proteins. In addition, overexpression phenotype of hFis1 could be reverted by MITOL co-overexpression. Our finding indicates that MITOL plays a critical role in mitochondrial dynamics through the control of mitochondrial fission proteins.

Amino Acid Sequence↗
PubMed2006

COMMD1 promotes the ubiquitination of NF-kappaB subunits through a cullin-containing ubiquitin ligase.

NF-kappaB is a pleiotropic transcription factor involved in multiple processes, including inflammation and oncogenesis. We have previously reported that COMMD1 represses kappaB-dependent transcription by negatively regulating NF-kappaB-chromatin interactions. Recently, ubiquitination of NF-kappaB subunits has been similarly implicated in the control of NF-kappaB recruitment to chromatin. We report here that COMMD1 accelerates the ubiquitination and degradation of NF-kappaB subunits through its interaction with a multimeric ubiquitin ligase containing Elongins B and C, Cul2 and SOCS1 (ECS(SOCS1)). COMMD1-deficient cells demonstrate stabilization of RelA, greater nuclear accumulation of RelA after TNF stimulation, de-repression of several kappaB-responsive genes, and enhanced NF-kappaB-mediated cellular responses. COMMD1 binds to Cul2 in a stimulus-dependent manner and serves to facilitate substrate binding to the ligase by stabilizing the interaction between SOCS1 and RelA. Our data uncover that ubiquitination and degradation of NF-kappaB subunits by this COMMD1-containing ubiquitin ligase is a novel and critical mechanism of regulation of NF-kappaB-mediated transcription.

Adaptor Proteins, Signal Transducing↗
PubMed2004

The many faces of beta-TrCP E3 ubiquitin ligases: reflections in the magic mirror of cancer.

Beta-transducin repeats-containing proteins (beta-TrCP) serve as the substrate recognition subunits for the SCFbeta-TrCP E3 ubiquitin ligases. These ligases ubiquitinate specifically phosphorylated substrates and play a pivotal role in the regulation of cell division and various signal transduction pathways, which, in turn, are essential for many aspects of tumorigenesis. We review the functions of the SCFbeta-TrCP ligases in the light of their relevance to cell growth, survival and transformation. Mechanisms underlying beta-TrCP regulation and their aberration in human and animal cancer as well as prospective of targeting beta-TrCP as a means of anticancer therapy are also discussed.

Animals↗
PubMed2006

HIF-1alpha and EPAS ubiquitination mediated by the VHL tumour suppressor involves flexibility in the ubiquitination mechanism, similar to other RING E3 ligases.

Hypoxia-inducible factor 1alpha (HIF-1alpha) degradation under normoxia is critical to modulating vascular growth. This degradation is mediated during normoxia by the von Hippel-Lindau tumour suppressor protein (VHL)-E3 ubiquitin ligase in partnership with the E2 enzyme UbcH5. In current models of the functionally similar Skp1, cullin, F-box (SCF)-E3 ligase, the E3 binds the target protein and the E2 catalyses ubiquitin transfer to lysines in an appropriately positioned domain. In the present study, we report that for efficient ubiquitination of HIF-1alpha to occur, three conserved lysines are required in both the HIF-1alpha and endothelial Per-ARNT-Sim domain protein (EPAS) sequences. The site of ubiquitin attachment via UbcH5 was mapped, and is shown to involve three HIF-1alpha lysines, K532, K538 and K547, and the same aligned lysines in EPAS. Only one of these lysines need to be intact for full ubiquitination to occur, analogous to the mechanism of Sic1 ubiquitination by the SCF/Cdc34 complex and further strengthening the functional link between the VHL and SCF-E3 ubiquitin ligases. We also report that lysines can be moved around the HIF-1alpha sequence with only minor losses in ubiquitination efficiency, thus suggesting HIF-1alpha and EPAS regulation by hypoxia depends primarily on an interaction with VHL per se, rather than the highly specific positioning of flanking lysine acceptors.

Amino Acid Sequence↗
PubMed1969

Effect of cholesteryl 14-methylhexadecanoate on the activity of some amino acid-transfer ribonucleic acid ligases from mammalian tissues.

1. l-Tyrosine-, l-alanine-, l-tryptophan- and l-threonine-tRNA ligases (where tRNA is transfer RNA) were purified from mammalian tissues and the relative contents of cholesteryl 14-methylhexadecanoate were determined in fractions obtained during the isolation. Purified enzymes were extracted with various organic solvents. 2. Cholesteryl 14-methylhexadecanoate contents in purified ligases were up to 210-fold that in the starting material. Different enzymes showed different contents of this cholesteryl ester. 3. Extracted enzymes lost in most cases their ability to catalyse formation of the aminoacylhydroxamate and aminoacyl-tRNA complexes. Enzymes extracted with various solvents showed a variable decreased activity. 4. The original activity could be restored to 70-100% by the addition of cholesteryl 14-methylhexadecanoate. Cholesteryl palmitate, cholesteryl margarate and cholesteryl stearate were inactive in this respect. 5. Incubation mixtures of extracted enzymes with cholesteryl 14-methylhexadecanoate added showed an initial delay in the time-course of both reactions assayed. 6. It is concluded that the effect of cholesteryl 14-methylhexadecanoate on the activity of amino acid-tRNA ligases seems to be specific and that this compound may play some role in the function of these enzymes.

Alanine↗
PubMed1990

Use of ATP, dATP and their alpha-thio derivatives to study DNA ligase adenylation.

Bacteriophage-T4 and human type I DNA ligases were found capable of self-adenylating upon exposure to both ribo- and deoxyribo-[alpha-35S]thio-ATP. However, the joining reaction does not take place in the presence of the deoxyribotriphosphates. Enzyme adenylation is reversed in all cases by an excess of PPi, but the rate of reversion is lower with thio derivatives. Therefore thio derivatives can be used to study the adenylation of DNA ligases and to search for specific inhibitors of the first step of the ligation reaction. In addition we show that thio derivatives can be used to detect DNA ligase adenylation activity covalently bound to a solid matrix.

Adenosine Triphosphate↗
PubMed1991

Correlation between anthracycline structure and human DNA ligase inhibition.

A total of 19 anthracycline derivatives were tested for their ability to interfere in vitro with the action of the human replicative DNA ligase. Only those with the sugar devoid of unmodified amino groups or with large configurational modifications were found to be inactive. Maximal inhibition of DNA-joining activity was found to require a 4'-deoxy-3'-amino sugar. Self-adenylation of DNA ligase was largely insensitive to these drugs. An important general finding is that slight modifications of the anthracycline structure have pronounced effects on DNA-ligase-inhibitory activity and might be related to the specificity of anthracycline anti-tumour activity.

Adenosine Monophosphate↗
PubMed2004

Choice of a start codon in a single transcript determines DNA ligase 1 isoform production and intracellular targeting in Arabidopsis thaliana.

DNA ligase 1 (AtLIG1) is the only essential DNA ligase activity in Arabidopsis and is implicated in the important processes of DNA replication, repair and recombination and in transgene insertion during Agrobacterium-mediated plant transformations. The mitochondrial and nuclear forms of DNA ligase 1 in Arabidopsis are translated from a single mRNA species through the control of translation initiation from either the first (M1) or second (M2) in-frame AUG codons respectively. Translation from a third in-frame AUG codon (M3) occurs on transcripts in which M1 and M2 are mutagenized to stop codons. Wild-type AtLIG1-GFP constructs (where GFP stands for green fluorescent protein) can be targeted in planta to both the nucleus and mitochondria. AtLIG1-GFP translation from M1 specifically targets the fusion protein only to mitochondria in planta, whereas translation from M2 or M3 targets the fusion protein only to the nucleus. Interestingly, the AtLIG1-GFP fusion protein in which translation is initiated from M1 contains both an N-terminal mtPS (mitochondrial targeting presequence) and a nuclear localization signal; nonetheless, this protein is only targeted to the mitochondria. This result raises intriguing questions on the translational control mechanisms that regulate how the protein products of a single transcript are targeted to more than one cellular compartment.

Amino Acid Sequence↗
PubMed2003

Kinetics and thermodynamics of nick sealing by T4 DNA ligase.

T4 DNA ligase is an Mg2+-dependent and ATP-dependent enzyme that seals DNA nicks in three steps: it covalently binds AMP, transadenylates the nick phosphate, and catalyses formation of the phosphodiester bond releasing AMP. In this kinetic study, we further detail the reaction mechanism, showing that the overall ligation reaction is a superimposition of two parallel processes: a 'processive' ligation, in which the enzyme transadenylates and seals the nick without dissociating from dsDNA, and a 'nonprocessive' ligation, in which the enzyme takes part in the abortive adenylation cycle (covalent binding of AMP, transadenylation of the nick, and dissociation). At low concentrations of ATP (<10 microM) and when the DNA nick is sealed with mismatching base pairs (e.g. five adjacent), this superimposition resolves into two kinetic phases, a burst ligation (approximately 0.2 min(-1)) and a subsequent slow ligation (approximately 2x10(-3) min(-1)). The relative rate and extent of each phase depend on the concentrations of ATP and Mg2+. The activation energies of self-adenylation (16.2 kcal.mol(-1)), transadenylation of the nick (0.9 kcal.mol(-1)), and nick-sealing (16.3-18.8 kcal.mol(-1)) were determined for several DNA substrates. The low activation energy of transadenylation implies that the transfer of AMP to the terminal DNA phosphate is a spontaneous reaction, and that the T4 DNA ligase-AMP complex is a high-energy intermediate. To summarize current findings in the DNA ligation field, we delineate a kinetic mechanism of T4 DNA ligase catalysis.

Adenosine Monophosphate↗
PubMed2004

Nitrosative stress linked to sporadic Parkinson's disease: S-nitrosylation of parkin regulates its E3 ubiquitin ligase activity.

Many hereditary and sporadic neurodegenerative disorders are characterized by the accumulation of aberrant proteins. In sporadic Parkinson's disease, representing the most prevalent movement disorder, oxidative and nitrosative stress are believed to contribute to disease pathogenesis, but the exact molecular basis for protein aggregation remains unclear. In the case of autosomal recessive-juvenile Parkinsonism, mutation in the E3 ubiquitin ligase protein parkin is linked to death of dopaminergic neurons. Here we show both in vitro and in vivo that nitrosative stress leads to S-nitrosylation of wild-type parkin and, initially, to a dramatic increase followed by a decrease in the E3 ligase-ubiquitin-proteasome degradative pathway. The initial increase in parkin's E3 ubiquitin ligase activity leads to autoubiquitination of parkin and subsequent inhibition of its activity, which would impair ubiquitination and clearance of parkin substrates. These findings may thus provide a molecular link between free radical toxicity and protein accumulation in sporadic Parkinson's disease.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2005

The Arabidopsis SUMO E3 ligase SIZ1 controls phosphate deficiency responses.

Plants sense phosphate (Pi) deficiency and initiate signaling that controls adaptive responses necessary for Pi acquisition. Herein, evidence establishes that AtSIZ1 is a plant small ubiquitin-like modifier (SUMO) E3 ligase and is a focal controller of Pi starvation-dependent responses. T-DNA insertional mutated alleles of AtSIZ1 (At5g60410) cause Arabidopsis to exhibit exaggerated prototypical Pi starvation responses, including cessation of primary root growth, extensive lateral root and root hair development, increase in root/shoot mass ratio, and greater anthocyanin accumulation, even though intracellular Pi levels in siz1 plants were similar to wild type. AtSIZ1 has SUMO E3 ligase activity in vitro, and immunoblot analysis revealed that the protein sumoylation profile is impaired in siz1 plants. AtSIZ1-GFP was localized to nuclear foci. Steadystate transcript abundances of Pi starvation-responsive genes AtPT2, AtPS2, and AtPS3 were moderate but clearly greater in siz1 seedlings than in wild type, where Pi is sufficient. Pi starvation induced the expression of these genes to the same extent in siz1 and wild-type seedlings. However, two other Pi starvation-responsive genes, AtIPS1 and AtRNS1, are induced more slowly in siz1 seedlings by Pi limitation. PHR1, a MYB transcriptional activator of AtIPS1 and AtRNS1, is an AtSIZ1 sumoylation target. These results indicate that AtSIZ1 is a SUMO E3 ligase and that sumoylation is a control mechanism that acts both negatively and positively on different Pi deficiency responses.

Anthocyanins↗
PubMed2006

Activation of the E3 ubiquitin ligase Itch through a phosphorylation-induced conformational change.

The E3 ubiquitin (Ub) ligase Itch is a critical regulator of T helper 2 (Th2) cytokine production through its ability to induce Ub-dependent JunB degradation. After T cell receptor engagement, Itch undergoes JNK1-mediated phosphorylation that greatly enhances its enzymatic activity. To investigate how phosphorylation activates an E3 Ub ligase we have identified the JNK1 phosphorylation sites within Itch as S199, S232, and T222, which are located within a Pro-rich region. Phosphorylation of these sites is necessary and sufficient for disrupting an inhibitory interaction between the WW domain of Itch and its catalytic HECT (Homologous to E6-AP C Terminus) domain and induces a conformational change that greatly enhances the catalytic activity of Itch, a HECT E3 ligase found to be directly activated upon its phosphorylation.

Cell Line↗
PubMed2006

HERC5 is an IFN-induced HECT-type E3 protein ligase that mediates type I IFN-induced ISGylation of protein targets.

Type I IFNs induce the expression of IFN-stimulated gene 15 (ISG15) and its conjugation to cellular targets. ISGylation is a multistep process involving IFN-inducible Ube1L, UbcH8, and a yet-to-be identified E3 ligase. Here we report the identification of an IFN-induced HECT-type E3 protein ligase, HERC5/Ceb1, which mediates ISGylation. We also defined a number of proteins modified by ISG15 after IFN triggering or HERC5 overexpression. A reduction in endogenous HERC5 by small interfering RNA inhibition blocks the IFN-induced ISG15 conjugation. Conversely, HERC5 coexpression with Ube1L and UbcH8 induces the ISG15 conjugation in vivo independent of IFN stimulation. A targeted substitution of Cys-994 to Ala in the HECT domain of HERC5 completely abrogates its E3 protein ligase activity. Therefore, this study demonstrates that HERC5/Ceb1 is involved in the conjugation of ISG15 to cellular proteins.

Cell Line↗
PubMed2006

Crystal structure of the SOCS2-elongin C-elongin B complex defines a prototypical SOCS box ubiquitin ligase.

Growth hormone (GH) signaling is tightly controlled by ubiquitination of GH receptors, phosphorylation levels, and accessibility of binding sites for downstream signaling partners. Members of the suppressors of cytokine signaling (SOCS) family function as key regulators at all levels of this pathway, and mouse knockout studies implicate SOCS2 as the primary suppressor. To elucidate the structural basis for SOCS2 function, we determined the 1.9-A crystal structure of the ternary complex of SOCS2 with elongin C and elongin B. The structure defines a prototypical SOCS box ubiquitin ligase with a Src homology 2 (SH2) domain as a substrate recognition motif. Overall, the SOCS box and SH2 domain show a conserved spatial domain arrangement with the BC box and substrate recognition domain of the von Hippel-Lindau (VHL) tumor suppressor protein, suggesting a common mechanism of ubiquitination in these cullin-dependent E3 ligases. The SOCS box binds elongin BC in a similar fashion to the VHL BC box and shows extended structural conservation with the F box of the Skp2 ubiquitin ligase. A previously unrecognized feature of the SOCS box is revealed with the burial of the C terminus, which packs together with the N-terminal extended SH2 subdomain to create a stable interface between the SOCS box and SH2 domain. This domain organization is conserved in SOCS1-3 and CIS1, which share a strictly conserved length of their C termini, but not in SOCS4, 5, and 7, which have extended C termini defining two distinct classes of inter- and intramolecular SOCS box interactions.

Amino Acid Sequence↗
PubMed2006

Human SHPRH is a ubiquitin ligase for Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen.

Human SHPRH gene is located at the 6q24 chromosomal region, and loss of heterozygosity in this region is seen in a wide variety of cancers. SHPRH is a member of the SWI/SNF family of ATPases/helicases, and it possesses a C(3)HC(4) RING motif characteristic of ubiquitin ligase proteins. In both of these features, SHPRH resembles the yeast Rad5 protein, which, together with Mms2-Ubc13, promotes replication through DNA lesions via an error-free postreplicational repair pathway. Genetic evidence in yeast has indicated a role for Rad5 as a ubiquitin ligase in mediating the Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Here we show that SHPRH is a functional homolog of Rad5. Similar to Rad5, SHPRH physically interacts with the Rad6-Rad18 and Mms2-Ubc13 complexes, and we show that SHPRH protein is a ubiquitin ligase indispensable for Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Based on these observations, we predict a role for SHPRH in promoting error-free replication through DNA lesions. Such a role for SHPRH is consistent with the observation that this gene is mutated in a number of cancer cell lines, including those from melanomas and ovarian cancers, which raises the strong possibility that SHPRH function is an important deterrent to mutagenesis and carcinogenesis in humans.

Adenosine Triphosphatases↗