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Ligase
Kinds of Ligase Terms modified by Ligase Selected AbstractsNAD+ -Dependent DNA Ligase: A novel target waiting for the right inhibitor,MEDICINAL RESEARCH REVIEWS, Issue 4 2008Namrata Dwivedi Abstract DNA ligases (EC.6.5.1.1) are key enzymes that catalyze the formation of phosphodiester bonds at single stranded or double stranded breaks between adjacent 5, phosphoryl and 3, hydroxyl groups of DNA. These enzymes are important for survival because they are involved in major cellular processes like DNA replication/repair and recombination. DNA ligases can be classified into two groups on the basis of their cofactor specificities. NAD+ -dependent DNA ligases are present in bacteria, some entomopox viruses and mimi virus while ATP-dependent DNA ligases are ubiquitous. The former have recently been drawing a lot of attention as novel targets for antibiotics to overcome current drug resistance issues. Currently a diverse range of inhibitors have been identified. There are several issues to be addressed in the quest for optimized inhibitors of the enzyme. In the first part of the review we summarize current structural work on these enzymes. Subsequently we describe the currently available classes of inhibitors. We also address modalities to improve the specificity and potencies of new inhibitors identified using protein structure based rational approaches. In conclusion, NAD+ -dependent ligases show great promise and represent a novel drug target whose time has come. © 2007 Wiley Periodicals, Inc. Med Res Rev, 28, No. 4, 545,568, 2008 [source] A Sensitive Fluorescence Anisotropy Method for Point Mutation Detection by Using Core,Shell Fluorescent Nanoparticles and High-Fidelity DNA LigaseCHEMISTRY - A EUROPEAN JOURNAL, Issue 27 2007Ting Deng Dr. Abstract The present study reports a proof-of-principle for a sensitive genotyping assay approach that can detect single nucleotide polymorphisms (SNPs) based on fluorescence anisotropy measurements through a core,shell fluorescent nanoparticles assembly and ligase reaction. By incorporating the core,shell fluorescent nanoparticles into fluorescence anisotropy measurements, this assay provided a convenient and sensitive detection assay that enabled straightforward single-base discrimination without the need of complicated operational steps. The assay was implemented via two steps: first, the hybridization reaction that allowed two nanoparticle-tagged probes to hybridize with the target DNA strand and the ligase reaction that generated the ligation between perfectly matched probes while no ligation occurred between mismatched ones were implemented synchronously in the same solution. Then, a thermal treatment at a relatively high temperature discriminated the ligation of probes. When the reaction mixture was heated to denature the duplex formed, the fluorescence anisotropy value of the perfect-match solution does not revert to the initial value, while that of the mismatch again comes back as the assembled fluorescent nanoparticles dispart. The present approach has been demonstrated with the discrimination of a single base mutation in codon 12 of a K-ras oncogene that is of significant value for colorectal cancers diagnosis, and the wild type and mutant type were successfully scored. Due to its ease of operation and high sensitivity, it was expected that the proposed detection approach might hold great promise in practical clinical diagnosis. [source] Rnf19a, a ubiquitin protein ligase, and Psmc3, a component of the 26S proteasome, Tether to the acrosome membranes and the head,tail coupling apparatus during rat spermatid developmentDEVELOPMENTAL DYNAMICS, Issue 7 2009Eugene Rivkin Abstract We report the cDNA cloning of rat testis Rnf19a, a ubiquitin protein ligase, and show 98% and 93% protein sequence identity of testicular mouse and human Rnf19a, respectively. Rnf19a interacts with Psmc3, a protein component of the 19S regulatory cap of the 26S proteasome. During spermatid development, Rnf19a and Psmc3 are initially found in Golgi-derived proacrosomal vesicles. Later on, Rnf19a, Psmc3, and ubiquitin are seen along the cytosolic side of the acrosomal membranes and the acroplaxome, a cytoskeletal plate linking the acrosome to the spermatid nuclear envelope. Rnf19a and Psmc3 accumulate at the acroplaxome marginal ring,manchette perinuclear ring region during spermatid head shaping and in the developing sperm head,tail coupling apparatus and tail. Rnf19a and Psmc3 may interact directly or indirectly with each other, presumably pointing to the participation of the ubiquitin,proteasome system in acrosome biogenesis, spermatid head shaping, and development of the head-tail coupling apparatus and tail. Developmental Dynamics 238:1851,1861, 2009. © 2009 Wiley-Liss, Inc. [source] Identification of enzymes involved in anaerobic benzene degradation by a strictly anaerobic iron-reducing enrichment cultureENVIRONMENTAL MICROBIOLOGY, Issue 10 2010Nidal Abu Laban Summary Anaerobic benzene degradation was studied with a highly enriched iron-reducing culture (BF) composed of mainly Peptococcaceae- related Gram-positive microorganisms. The proteomes of benzene-, phenol- and benzoate-grown cells of culture BF were compared by SDS-PAGE. A specific benzene-expressed protein band of 60 kDa, which could not be observed during growth on phenol or benzoate, was subjected to N-terminal sequence analysis. The first 31 amino acids revealed that the protein was encoded by ORF 138 in the shotgun sequenced metagenome of culture BF. ORF 138 showed 43% sequence identity to phenylphosphate carboxylase subunit PpcA of Aromatoleum aromaticum strain EbN1. A LC/ESI-MS/MS-based shotgun proteomic analysis revealed other specifically benzene-expressed proteins with encoding genes located adjacent to ORF 138 on the metagenome. The protein products of ORF 137, ORF 139 and ORF 140 showed sequence identities of 37% to phenylphosphate carboxylase PpcD of A. aromaticum strain EbN1, 56% to benzoate-CoA ligase (BamY) of Geobacter metallireducens and 67% to 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (UbiD/UbiX) of A. aromaticum strain EbN1 respectively. These genes are proposed as constituents of a putative benzene degradation gene cluster (,17 kb) composed of carboxylase-related genes. The identified gene sequences suggest that the initial activation reaction in anaerobic benzene degradation is probably a direct carboxylation of benzene to benzoate catalysed by putative anaerobic benzene carboxylase (Abc). The putative Abc probably consists of several subunits, two of which are encoded by ORFs 137 and 138, and belongs to a family of carboxylases including phenylphosphate carboxylase (Ppc) and 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (UbiD/UbiX). [source] Efficient Increase of DNA Cleavage Activity of a Diiron(III) Complex by a Conjugating Acridine GroupEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 34 2007Xiao-Qiang Chen Abstract A new diferric complex, Fe2Lb, in which a DNA intercalator (acridine) is linked to a precursor diferric complex (Fe2La), has been designed and synthesised as a hydrolytic cleaving agent of DNA. Compared with Fe2La (without the DNA intercalator) (La: 2,6-bis{[(2-hydroxybenzyl)(pyridin-2-yl)methylamino]methyl}-4-methylphenol), Fe2Lb [Lb: 5-(acridin-9-yl)- N -(3,5-bis{[(2-hydroxybenzyl)(pyridin-2-yl)methylamino]methyl}-4-hydroxybenzyl)pentanamide] leads to a 14-fold increase in the cleavage efficiency of plasmid DNA due to the binding interaction between DNA and the acridine moiety. The interaction has been demonstrated by UV/Vis absorption, CD spectroscopy, viscidity experiments and thermal denaturation studies. The hydrolytic mechanism is supported by evidence from T4 DNA ligase assay, reactive oxygen species (ROS) quenching and BNPP [bis(4-nitrophenyl) phosphate, a DNA model] cleavage experiments. The pH dependence of the BNPP cleavage by Fe2La in aqueous buffer media shows a bell-shaped pH,kobs profile with an optimum point around a pH of 7.0 which is in good agreement with the maximum point of the pH-dependent relative concentration curve of active species from the pH titration experiments. The determination of the initial rates at a pH of 7.36 as a function of substrate concentration reveals saturation kinetics with Michaelis,Menten-like behaviour and Fe2La shows a rate acceleration increase of 4.7,×,106 times in the hydrolysis of BNPP. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source] A tripartite motif protein TRIM11 binds and destabilizes Humanin, a neuroprotective peptide against Alzheimer's disease-relevant insultsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2003Takako Niikura Abstract Humanin (HN) is a newly identified neuroprotective peptide that specifically suppresses Alzheimer's disease (AD)-related neurotoxicity. HN peptide has been detected in the human AD brain as well as in mouse testis and colon by immunoblot and immunohistochemical analyses. By means of yeast two-hybrid screening, we identified TRIM11 as a novel HN-interacting protein. TRIM11, which is a member of protein family containing a tripartite motif (TRIM), is composed of a RING finger domain, which is a putative E3 ubiquitin ligase, a B-box domain, a coiled-coil domain and a B30.2 domain. Deletion of the B30.2 domain in TRIM11 abolished the interaction with HN, whereas the B30.2 domain alone did not interact with HN. For their interaction, at least the coiled-coil domain was indispensable together with the B30.2 domain. The intracellular level of glutathione S -transferase-fused or EGFP-fused HN peptides or plain HN was drastically reduced by the coexpression of TRIM11. Disruption of the RING finger domain by deleting the first consensus cysteine or proteasome inhibitor treatment significantly diminished the effect of TRIM11 on the intracellular level of HN. These results suggest that TRIM11 plays a role in the regulation of intracellular HN level through ubiquitin-mediated protein degradation pathways. [source] Ubiquitination of E3 ubiquitin ligase TRIM5, and its potential roleFEBS JOURNAL, Issue 7 2008Keiko Yamauchi HIV-1 efficiently infects susceptible cells and causes AIDS in humans. Although HIV can also enter the cells of Old World monkeys, it encounters a block before reverse transcription. Data have shown that this species-specific restriction is mediated by tripartite motif (TRIM)5,, whose molecular function is still undefined. Here, we show that TRIM5, functions as a RING-finger-type E3 ubiquitin ligase both in vitro and in vivo and ubiquitinates itself in cooperation with the E2 ubiquitin-conjugating enzyme UbcH5B. In addition to the self-ubiquitination, we show that TRIM5, is ubiquitinated by another E3 ubiquitin ligase, Ro52, and deubiquitinated by YopJ, one of the pathogenic proteins derived from Yersinia species. Thus, the ubiquitination of TRIM5, is catalyzed by itself and Ro52 and downregulated by YopJ. Unexpectedly, although TRIM5, is ubiquitinated, our results have revealed that the proteasome inhibitors MG115 and MG132 do not stabilize it in HeLa cells, suggesting that the ubiquitination of TRIM5, does not lead to proteasomal degradation. Importantly, TRIM5, is clearly conjugated by a single ubiquitin molecule (monoubiquitination). Our monoubiquitin-fusion assay suggests that monoubiquitination is a signal for TRIM5, to translocate from cytoplasmic bodies to the cytoplasm. [source] Identification and functional characterization of an Src homology domain 3 domain-binding site on CblFEBS JOURNAL, Issue 23 2006Archana Sanjay§ Cbl is an adaptor protein and ubiquitin ligase that binds and is phosphorylated by the nonreceptor tyrosine kinase Src. We previously showed that the primary interaction between Src and Cbl is mediated by the Src homology domain 3 (SH3) of Src binding to proline-rich sequences of Cbl. The peptide Cbl RDLPPPPPPDRP(540,551), which corresponds to residues 540,551 of Cbl, inhibited the binding of a GST,Src SH3 fusion protein to Cbl, whereas RDLAPPAPPPDR(540,551) did not, suggesting that Src binds to this site on Cbl in a class I orientation. Mutating prolines 543,548 reduced Src binding to the Cbl 479,636 fragment significantly more than mutating the prolines in the PPVPPR(494,499) motif, which was previously reported to bind Src SH3. Mutating Cbl prolines 543,548 to alanines substantially reduced Src binding to Cbl, Src-induced phosphorylation of Cbl, and the inhibition of Src kinase activity by Cbl. Expressing the mutated Cbl in osteoclasts induced a moderate reduction in bone-resorbing activity and increased amounts of Src protein. In contrast, disabling the tyrosine kinase-binding domain of full-length Cbl by mutating glycine 306 to glutamic acid, and thereby preventing the previously described binding of the tyrosine kinase-binding domain to the Src phosphotyrosine 416, had no effect on Cbl phosphorylation, the inhibition of Src activity by full-length Cbl, or bone resorption. These data indicate that the Cbl RDLPPPP(540,546) sequence is a functionally important binding site for Src. [source] Inhibition of the D -alanine:D -alanyl carrier protein ligase from Bacillus subtilis increases the bacterium's susceptibility to antibiotics that target the cell wallFEBS JOURNAL, Issue 12 2005Juergen J. May The surface charge as well as the electrochemical properties and ligand binding abilities of the Gram-positive cell wall is controlled by the d -alanylation of the lipoteichoic acid. The incorporation of d -Ala into lipoteichoic acid requires the d -alanine:d -alanyl carrier protein ligase (DltA) and the carrier protein (DltC). We have heterologously expressed, purified, and assayed the substrate selectivity of the recombinant proteins DltA with its substrate DltC. We found that apo-DltC is recognized by both endogenous 4,-phosphopantetheinyl transferases AcpS and Sfp. After the biochemical characterization of DltA and DltC, we designed an inhibitor (d -alanylacyl-sulfamoyl-adenosine), which is able to block the d -Ala adenylation by DltA at a Ki value of 232 nmin vitro. We also performed in vivo studies and determined a significant inhibition of growth for different Bacillus subtilis strains when the inhibitor is used in combination with vancomycin. [source] Kinetics and thermodynamics of nick sealing by T4 DNA ligaseFEBS JOURNAL, Issue 21 2003Alexey V. Cherepanov 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 µm) 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 (, 0.2 min,1) and a subsequent slow ligation (, 2 × 10,3 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. [source] Biotinylation in the hyperthermophile Aquifex aeolicusFEBS JOURNAL, Issue 6 2003Isolation of a cross-linked BPL:BCCP complex Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl CoA carboxylase and this post-translational modification of a single lysine residue is exceptionally specific. The exact details of the protein,protein interactions involved are unclear as a BPL:BCCP complex has not yet been isolated. Moreover, detailed information is lacking on the composition, biosynthesis and role of fatty acids in hyperthermophilic organisms. We have cloned, overexpressed and purified recombinant BPL and the biotinyl domain of BCCP (BCCP,67) from the extreme hyperthermophile Aquifex aeolicus. In vitro assays have demonstrated that BPL catalyses biotinylation of lysine 117 on BCCP,67 at temperatures of up to 70 °C. Limited proteolysis of BPL with trypsin and chymotrypsin revealed a single protease-sensitive site located 44 residues from the N-terminus. This site is adjacent to the predicted substrate-binding site and proteolysis of BPL is significantly reduced in the presence of MgATP and biotin. Chemical crosslinking with 1-ethyl-3-(dimethylamino-propyl)-carbodiimide (EDC) allowed the isolation of a BPL:apo-BCCP,67 complex. Furthermore, this complex was also formed between BPL and a BCCP,67 mutant lacking the lysine residue (BCCP,67 K117L) however, complex formation was considerably reduced using holo-BCCP,67. These observations provide evidence that addition of the biotin prosthetic group reduces the ability of BCCP,67 to heterodimerize with BPL, and emphasizes that a network of interactions between residues on both proteins mediates protein recognition. [source] Cdc20 protein contains a destruction-box but, unlike Clb2, its proteolysisis not acutely dependent on the activity of anaphase-promoting complexFEBS JOURNAL, Issue 2 2000Phuay-Yee Goh Both chromosome segregation and the final exit from mitosis require a ubiquitin-protein ligase called anaphase-promoting complex (APC) or cyclosome. This multiprotein complex ubiquitinates various substrates, such as the anaphase inhibitor Pds1 and mitotic cyclins, and thus targets them for proteolysis by the 26S proteasome. The ubiquitination by APC is dependent on the presence of a destruction-box sequence in the N-terminus of target proteins. Recent reports have strongly suggested that Cdc20, a WD40 repeat-containing protein required for nuclear division in the budding yeast Saccharomyces cerevisiae, is essential for the APC-mediated proteolysis. To understand the function of CDC20, we have studied its regulation in some detail. The expression of the CDC20 gene is cell-cycle regulated such that it is transcribed only during late S phase and mitosis. Although the protein is unstable to some extent through out the cell cycle, its degradation is particularly enhanced in G1. Cdc20 contains a destruction box sequence which, when mutated or deleted, stabilizes it considerably in G1. Surprisingly, we find that while the inactivation of APC subunits Cdc16, Cdc23 or Cdc27 results in stabilization of the mitotic cyclin Clb2 in G1, the proteolytic destruction of Cdc20 remains largely unaffected. This suggests the existence of proteolytic mechanisms in G1 that can degrade destruction-box containing proteins, such as Cdc20, in an APC-independent manner. [source] Reprogramming Hansenula polymorpha for penicillin production: expression of the Penicillium chrysogenum pcl geneFEMS YEAST RESEARCH, Issue 7 2007Loknath Gidijala Abstract We aim to introduce the penicillin biosynthetic pathway into the methylotrophic yeast Hansenula polymorpha. To allow simultaneous expression of the multiple genes of the penicillin biosynthetic pathway, additional markers were required. To this end, we constructed a novel host,vector system based on methionine auxotrophy and the H. polymorpha MET6 gene, which encodes a putative cystathionine ,-lyase. With this new host,vector system, the Penicillium chrysogenum pcl gene, encoding peroxisomal phenylacetyl-CoA ligase (PCL), was expressed in H. polymorpha. PCL has a potential C-terminal peroxisomal targeting signal type 1 (PTS1). Our data demonstrate that a green fluorescent protein,PCL fusion protein has a dual location in the heterologous host in the cytosol and in peroxisomes. Mutation of the PTS1 of PCL (SKI-COOH) to SKL-COOH restored sorting of the fusion protein to peroxisomes only. Additionally, we demonstrate that peroxisomal PCL,SKL produced in H. polymorpha displays normal enzymatic activities. [source] Rsp5 is required for the nuclear export of mRNA of HSF1 and MSN2/4 under stress conditions in Saccharomyces cerevisiaeGENES TO CELLS, Issue 2 2008Yutaka Haitani Rsp5 is an essential and multi-functional E3 ubiquitin ligase in Saccharomyces cerevisiae. We previously isolated the Ala401Glu rsp5 mutant that is hypersensitive to various stresses. In rsp5A401E cells, the transcription of the stress protein genes was defective. To understand the mechanism by which Rsp5 regulates the expression of stress proteins, we analyzed the expression and localization of two major transcription factors, Hsf1 and Msn2/4, required for stress protein gene expression in S. cerevisiae. The mRNA levels of HSF1 and MSN2/4 in rsp5A401E cells were slightly lower than those of wild-type cells. An interesting finding is that the protein levels of HSF1 and Msn2/4 were remarkably defective in rsp5A401E cells after exposure to temperature up-shift and ethanol, although these proteins are mainly localized in the nucleus under these stress conditions. We also showed that the mRNAs of HSF1 and MSN2/4 were accumulated in the nucleus of rsp5A401E cells after exposure to temperature up-shift and ethanol, and even under non-stress conditions, suggesting that Rsp5 is required for the nuclear export of these mRNAs. These results indicate that, in response to environmental stresses, Rsp5 primarily regulates the expression of Hsf1 and Msn2/4 at the post-transcriptional level and is involved in the repair system of stress-induced abnormal proteins. [source] Lafora disease in the Indian population: EPM2A and NHLRC1 gene mutations and their impact on subcellular localization of laforin and malin,HUMAN MUTATION, Issue 6 2008Shweta Singh Abstract Lafora disease (LD) is a fatal form of teenage-onset autosomal recessive progressive myoclonus epilepsy. LD is more common among geographic isolates and in populations with a higher rate of consanguinity. Mutations in two genes, EPM2A encoding laforin phosphatase, and NHLRC1 encoding malin ubiquitin ligase, have been shown to cause the LD. We describe here a systematic analysis of the EPM2A and the NHLRC1 gene sequences in 20,LD families from the Indian population. We identified 12 distinct mutations in 15,LD families. The identified novel mutations include 4 missense mutations (K140N, L310W, N148Y, and E210,K) and a deletion of exon 3 for EPM2A, and 4 missense mutations (S22R, L279P, L279P, and L126P) and a single base-pair insertional mutation (612insT) for NHLRC1. The EPM2A gene is known to encode two laforin isoforms having distinct carboxyl termini; a major isoform localized in the cytoplasm, and a minor isoform that targeted the nucleus. We show here that the effect of the EPM2A gene mutation L310W was limited to the cytoplasmic isoform of laforin, and altered its subcellular localization. We have also analyzed the impact of NHLRC1 mutations on the subcellular localization of malin. Of the 6 distinct mutants tested, three targeted the nucleus, one formed perinuclear aggregates, and two did not show any significant difference in the subcellular localization as compared to the wild-type malin. Our results suggest that the altered subcellular localization of mutant proteins of the EPM2A and NHLRC1 genes could be one of the molecular bases of the LD phenotype. © 2008 Wiley-Liss, Inc. [source] The degradation of cell cycle regulators by SKP2/CKS1 ubiquitin ligase is genetically controlled in rodent liver cancer and contributes to determine the susceptibility to the diseaseINTERNATIONAL JOURNAL OF CANCER, Issue 5 2010Diego F. Calvisi Abstract Previous work showed a genetic control of cell cycle deregulation during hepatocarcinogenesis. We now evaluated in preneoplastic lesions, dysplastic nodules and hepatocellular carcinoma (HCC), chemically induced in genetically susceptible F344 and resistant Brown Norway (BN) rats, the role of cell cycle regulating proteins in the determination of a phenotype susceptible to HCC development. p21WAF1, p27KIP1, p57KIP2 and p130 mRNA levels increased in fast growing lesions of F344 rats. Lower/no increases occurred in slowly growing lesions of BN rats. A similar behavior of RassF1A mRNA was previously found in the 2 rat strains. However, p21WAF1, p27KIP1, p57KIP, p130 and RassF1A proteins exhibited no change/low increase in the lesions of F344 rats and consistent rise in dysplastic nodules and HCC of BN rats. Increase in Cks1-Skp2 ligase and ubiquitination of cell cycle regulators occurred in F344 but not in BN rat lesions, indicating that posttranslational modifications of cell cycle regulators are under genetic control and contribute to determine a phenotype susceptible to HCC. Moreover, proliferation index of 60 human HCCs was inversely correlated with protein levels but not with mRNA levels of P21WAF1, P27KIP1, P57KIP2 and P130, indicating a control of human HCC proliferation by posttranslational modifications of cell cycle regulators. [source] Phosphorylation by COP9 Signalosome-Associated CK2 Promotes Degradation of p27 during the G1 Cell Cycle PhaseISRAEL JOURNAL OF CHEMISTRY, Issue 2 2006Xiaohua Huang The cell cycle regulator p27Kip1 (p27) is controlled by 26S proteasome-mediated proteolysis by two different pathways. From the S till the G2 phase of the cell cycle, degradation of p27 takes place in the nucleus and is initiated by CDK2-dependent phosphorylation of threonine 187 with subsequent ubiquitination by the SCFSkp2 ubiquitin ligase. During the G1 cell cycle phase (G1), p27 breakdown is cytosolic and is initiated by nuclear export with subsequent ubiquitination by a RING finger ligase called kip1 ubiquitination complex. Here we show that the COP9 signalosome (CSN) is a regulator of p27 proteolysis during G1. The CSN interacts with p27 and the CSN-associated kinase CK2 phosphorylates p27 at two regions. One is central to the protein (amino acids 101,113), and the other was mapped near to the C-terminus (amino acids 170,189). Elimination of the putative C-terminal phosphorylation sites stabilizes ectopic p27 towards proteasomal degradation and abolishes CSN,p27 binding. Inhibition of CSN-associated kinase activity by curcumin attenuates loss of p27 upon cell cycle re-entry. Similar but not additive effects of the phosphoinositol-3-kinase blocker LY 290042 may point to a common pathway of CSN-associated CK2 and protein kinase B/Akt (Akt) in regulating p27 abundance. Akt is found in Flag pulldowns of lysates obtained from cells permanently expressing Flag-tagged CSN2, indicating that Akt is a novel kinase associated with the CSN. Thus, the CSN seems to regulate p27 proteolysis at G1 downstream of Ras-mediated signal pathways. [source] ,4 phosphoprotein interacts with EDD E3 ubiquitin ligase and poly(A)-binding proteinJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2010William J. McDonald Abstract Mammalian ,4 phosphoprotein, the homolog of yeast Tap42, is a component of the mammalian target-of-rapamycin (mTOR) pathway that regulates ribogenesis, the initiation of translation, and cell-cycle progression. ,4 is known to interact with the catalytic subunit of protein phosphatase 2A (PP2Ac) and to regulate PP2A activity. Using ,4 as bait in yeast two-hybrid screening of a human K562 erythroleukemia cDNA library, EDD (E3 isolated by differential display) E3 ubiquitin ligase was identified as a new protein partner of ,4. EDD is the mammalian ortholog of Drosophila hyperplastic discs gene (hyd) that controls cell proliferation during development. The EDD protein contains a PABC domain that is present in poly(A)-binding protein (PABP), suggesting that PABP may also interact with ,4. PABP recruits translation factors to the poly(A)-tails of mRNAs. In the present study, immunoprecipitation/immunoblotting (IP/IB) analyses showed a physical interaction between ,4 and EDD in rat Nb2 T-lymphoma and human MCF-7 breast cancer cell lines. ,4 also interacted with PABP in Nb2, MCF-7 and the human Jurkat T-leukemic and K562 myeloma cell lines. COS-1 cells, transfected with Flag-tagged-pSG5-EDD, gave a (Flag)-EDD,,4 immunocomplex. Furthermore, deletion mutants of ,4 were constructed to determine the binding site for EDD. IP/IB analysis showed that EDD bound to the C-terminal region of ,4, independent of the ,4-PP2Ac binding site. Therefore, in addition to PP2Ac, ,4 interacts with EDD and PABP, suggesting its involvement in multiple steps in the mTOR pathway that leads to translation initiation and cell-cycle progression. J. Cell. Biochem. 110: 1123,1129, 2010. Published 2010 Wiley-Liss, Inc. [source] Regulation of Sprouty2 stability by mammalian Seven-in-Absentia homolog 2,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2007Robert J. Nadeau Abstract Mammalian Sprouty (Spry) gene expression is rapidly induced upon activation of the FGF receptor signaling pathway in multiple cell types including cells of mesenchymal and epithelial origin. Spry2 inhibits FGF-dependent ERK activation and thus Spry acts as a feedback inhibitor of FGF-mediated proliferation. In addition, Spry2 interacts with the ring-finger-containing E3 ubiquitin ligase, c-Cbl, in a manner that is dependent upon phosphorylation of Tyr55 of Spry2. This interaction results in the poly-ubiquitination and subsequent degradation of Spry2 by the proteasome. Here, we describe the identification of another E3 ubiquitin ligase, human Seven-in-Absentia homolog-2 (SIAH2), as a Spry2 interacting protein. We show by yeast two-hybrid analysis that the N-terminal domain of Spry2 and the ring finger domain of SIAH2 mediated this interaction. Co-expression of SIAH2 resulted in proteasomal degradation of Spry1, 2, and to a lesser extent Spry4. The related E3 ubiquitin-ligase, SIAH1, had little effect on Spry2 protein stability when co-expressed. Unlike c-Cbl-mediated degradation of Spry2, SIAH2-mediated degradation was independent of phosphorylation of Spry2 on Tyr55. Spry2 was also phosphorylated on Tyr227, and phosphorylation of this residue was also dispensable for SIAH2-mediated degradation of Spry2. Finally, co-expression of SIAH2 with Spry2 resulted in a rescue of FGF2-mediated ERK phosphorylation. These data suggest a novel mechanism whereby Spry2 stability is regulated in a manner that is independent of tyrosine phosphorylation, and provides an addition level of control of Spry2 protein levels. J. Cell. Biochem. 100: 151,160, 2007. © 2006 Wiley-Liss, Inc. [source] Centrosome function in normal and tumor cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2006Satish Sankaran Abstract Centrosomes nucleate microtubules that form the mitotic spindle and regulate the equal division of chromosomes during cell division. In cancer, centrosomes are often found amplified to greater than two per cell, and these tumor cells frequently have aneuploid genomes. In this review, we will discuss the cellular factors that regulate the proper duplication of the centrosome and how these regulatory steps can lead to abnormal centrosome numbers and abnormal mitoses. In particular, we highlight the newly emerging role of the Breast Cancer 1 (BRCA1) ubiquitin ligase in this process. J. Cell. Biochem. 99: 1240,1250, 2006. © 2006 Wiley-Liss, Inc. [source] Association of polymorphisms of glutamate-cystein ligase and microsomal triglyceride transfer protein genes in non-alcoholic fatty liver diseaseJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 2 2010Claudia Pinto Marques Souza Oliveira Abstract Background and Aims:, Although the metabolic risk factors for non-alcoholic fatty liver disease (NAFLD) progression have been recognized, the role of genetic susceptibility remains a field to be explored. The aim of this study was to examine the frequency of two polymorphisms in Brazilian patients with biopsy-proven simple steatosis or non-alcoholic steatohepatitis (NASH): ,493 G/T in the MTP gene, which codes the protein responsible for transferring triglycerides to nascent apolipoprotein B, and ,129 C/T in the GCLC gene, which codes the catalytic subunit of glutamate-cystein ligase in the formation of glutathione. Methods:, One hundred and thirty-one biopsy-proven NAFLD patients (n = 45, simple steatosis; n = 86, NASH) and 141 unrelated healthy volunteers were evaluated. Genomic DNA was extracted from peripheral blood cells, and the ,129 C/T polymorphism of the GCLC gene was determined by restriction fragment length polymorphism (RFLP). The ,493 G/T polymorphism of the MTP gene was determined by direct sequencing of the polymerase chain reaction products. Results:, The presence of at least one T allele in the ,129 C/T polymorphism of the GCLC gene was independently associated with NASH (odds ratio 12.14, 95% confidence interval 2.01,73.35; P = 0.007), whereas, the presence of at least one G allele in the ,493 G/T polymorphism of the MTP gene differed slightly between biopsy-proven NASH and simple steatosis. Conclusion:, This difference clearly warrants further investigation in larger samples. These two polymorphisms could represent an additional factor for consideration in evaluating the risk of NAFLD progression. Further studies involving a larger population are necessary to confirm this notion. [source] Synthesis of deuterium-labelled 5,- O -[N -(Salicyl)sulfamoyl]adenosine (Sal-AMS-d4) as an internal standard for quantitation of Sal-AMSJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 2 2008Amol Gupte Abstract 5,- O -[N -(Salicyl)sulfamoyl]adenosine (Sal-AMS, 1) is a potent inhibitor of the bifunctional enzyme salicyl-AMP ligase in Mycobacterium tuberculosis. This inhibitor acts by disrupting the biosynthesis of the mycobactin siderophores that are essential for the process of iron acquisition. To aid with in vitro metabolism and in vivo pharmacokinetic studies of Sal-AMS, a stable deuterium-labelled Sal-AMS analog (Sal-AMS-d4) was synthesized. This deuterium-labelled analog was used as an internal standard to conduct in vitro plasma and microsomal stability studies. Sal-AMS was found to be stable for 24,h in human plasma and 1,h in human liver microsomes at 37°C. Copyright © 2008 John Wiley & Sons, Ltd. [source] Interaction of p53 with Mdm2 and azurin as studied by atomic force spectroscopyJOURNAL OF MOLECULAR RECOGNITION, Issue 4 2010Gloria Funari Abstract Azurin, a bacterial protein, can be internalized in cancer cells and induce apoptosis. Such anticancer effect is coupled to the formation of a complex with the tumour-suppressor p53. The mechanism by which azurin stabilizes p53 and the binding sites of their complex are still under investigation. It is also known that the predominant mechanism for p53 down-regulation implies its association to Mdm2, the main ubiquitin ligase affecting its stability. However, the p53/Mdm2 interaction, occurring at the level of both their N-terminal domains, has been characterized so far by experiments involving only partial domains of these proteins. The relevance of the p53/Mdm2 complex as a possible target of the anticancer therapies requires a deeper study of this complex as made up of the two entire proteins. Moreover, the apparent antagonist action of azurin against Mdm2, with respect of p53 regulation, might suggest the possibility that azurin binds p53 at the same site of Mdm2, preventing in such a way p53 and Mdm2 from association and thus p53 from degradation. By following the interaction of the two entire proteins by atomic force spectroscopy, we have assessed the formation of a specific complex between p53 and Mdm2. We found for it a binding strength and a dissociation rate constant typical of dynamical protein,protein interactions and we observed that azurin, even if capable to bind p53, does not compete with Mdm2 for the same binding site on p53. The formation of the p53/Mdm2/azurin ternary complex might suggest an alternative anti-cancer mechanism adopted by azurin. Copyright © 2009 John Wiley & Sons, Ltd. [source] Identification and characterization of a novel endogenous murine parkin mutationJOURNAL OF NEUROCHEMISTRY, Issue 2 2010Chenere P. Ramsey J. Neurochem. (2010) 113, 402,417. Abstract Various mutations in the PARK2 gene which encodes the protein, parkin, are causal of a disease entity-termed autosomal recessive juvenile parkinsonism. Parkin can function as an E3 ubiquitin-protein ligase, mediating the ubiquitination of specific targeted proteins and resulting in proteasomal degradation. Parkin is thought to lead to parkinsonism as a consequence of a loss in its function. In this study, immunoblot analyses of brain extracts from Balb/c, C57BL/6, C3H, and 129S mouse strains demonstrated significant variations in immunoreactivity with anti-parkin monoclonal antibodies (PRK8, PRK28, and PRK109). This resulted partly from differences in the steady-state levels of parkin protein across mouse strains. There was also a complete loss of immunoreactivity for PRK8 and PRK28 antibodies in C3H mice due to was because of a homologous nucleotide mutation resulting in an E398Q amino acid substitution. In cultured cells, parkin harboring this mutation had a greater tendency to aggregate, exhibited reduced interaction with the E2 ubiquitin-conjugating enzymes, UbcH7 and UbcH8, and demonstrated loss-of-function in promoting the proteosomal degradation of a specific putative substrate, synphilin-1. In situ, C3H mice displayed age-dependent increased levels of brain cortical synphilin-1 compared with C57BL/6, suggesting that E398Q parkin in these mice is functionally impaired and that C3H mice may be a suitable model of parkin loss-of-function similar to patients with missense mutations. [source] Neuroprotective actions of noradrenaline: effects on glutathione synthesis and activation of peroxisome proliferator activated receptor deltaJOURNAL OF NEUROCHEMISTRY, Issue 5 2007Jose L. M. Madrigal Abstract The endogenous neurotransmitter noradrenaline (NA) can protect neurons from the toxic consequences of various inflammatory stimuli, however the exact mechanisms of neuroprotection are not well known. In the current study, we examined neuroprotective effects of NA in primary cultures of rat cortical neurons. Exposure to oligomeric amyloid beta (A,) 1-42 peptide induced neuronal damage revealed by increased staining with fluorojade, and toxicity assessed by LDH release. A,-dependent neuronal death did not involve neuronal expression of the inducible nitric oxide synthase 2 (NOS2), since A, did not induce nitrite production from neurons, LDH release was not reduced by co-incubation with NOS2 inhibitors, and neurotoxicity was similar in wildtype and NOS2 deficient neurons. Co-incubation with NA partially reduced A,-induced neuronal LDH release, and completely abrogated the increase in fluorojade staining. Treatment of neurons with NA increased expression of ,-glutamylcysteine ligase, reduced levels of GSH peroxidase, and increased neuronal GSH levels. The neuroprotective effects of NA were partially blocked by co-treatment with an antagonist of peroxisome proliferator activated receptors (PPARs), and replicated by incubation with a selective PPARdelta (PPAR,) agonist. NA also increased expression and activation of PPAR,. Together these data demonstrate that NA can protect neurons from A,-induced damage, and suggest that its actions may involve activation of PPAR, and increases in GSH production. [source] Rotenone selectively kills serotonergic neurons through a microtubule-dependent mechanismJOURNAL OF NEUROCHEMISTRY, Issue 1 2007Yong Ren Abstract As a major co-morbidity of Parkinson's disease (PD), depression is associated with the loss of serotonergic neurons. Our recent study has shown that midbrain dopaminergic neurons are particularly vulnerable to microtubule-depolymerizing agents including rotenone, an environmental toxin linked to PD. Here we show that rotenone also selectively killed serotonergic neurons in midbrain neuronal cultures. Its selective toxicity was significantly decreased by the microtubule-stabilizing drug taxol and mimicked by microtubule-depolymerizing agents such as colchicine and nocodazole. Microtubule depolymerization induced by rotenone or colchicine caused vesicle accumulation in the soma and killed serotonergic neurons through a mechanism dependent on serotonin metabolism in the cytosol. Blocking serotonin synthesis or degradation, as well as application of antioxidants, significantly reduced the selective toxicity of rotenone or colchicine. Inhibition of vesicular sequestration of serotonin exerted a selective toxicity on serotonergic neurons that was mitigated by blocking serotonin metabolism. Over-expression of parkin, a protein-ubiquitin E3 ligase that strongly binds to microtubules, greatly attenuated the selective toxicity of rotenone or colchicine. The protective effects of parkin were abrogated by its PD-linked mutations. Together, our results suggest that rotenone and parkin affect the survival of serotonergic neurons by impacting on microtubules in opposing manners. [source] A ubiquitin ligase HRD1 promotes the degradation of Pael receptor, a substrate of ParkinJOURNAL OF NEUROCHEMISTRY, Issue 6 2006Tomohiro Omura Abstract It has been proposed that in autosomal recessive juvenile parkinsonism (AR-JP), a ubiquitin ligase (E3) Parkin, which is involved in endoplasmic reticulum-associated degradation (ERAD), lacks E3 activity. The resulting accumulation of Parkin-associated endothelin receptor-like receptor (Pael-R), a substrate of Parkin, leads to endoplasmic reticulum stress, causing neuronal death. We previously reported that human E3 HRD1 in the endoplasmic reticulum protects against endoplasmic reticulum stress-induced apoptosis. This study shows that HRD1 was expressed in substantia nigra pars compacta (SNC) dopaminergic neurons and interacted with Pael-R through the HRD1 proline-rich region, promoting the ubiquitylation and degradation of Pael-R. Furthermore, the disruption of endogenous HRD1 by small interfering RNA (siRNA) induced Pael-R accumulation and caspase-3 activation. We also found that ATF6 overexpression, which induced HRD1, accelerated and caused Pael-R degradation; the suppression of HRD1 expression by siRNA partially prevents this degradation. These results suggest that in addition to Parkin, HRD1 is also involved in the degradation of Pael-R. [source] Sequestosome 1/p62 shuttles polyubiquitinated tau for proteasomal degradationJOURNAL OF NEUROCHEMISTRY, Issue 1 2005Jeganathan Ramesh Babu Abstract Inclusions isolated from several neurodegenerative diseases, including Alzheimer's disease (AD), are characterized by ubiquitin-positive proteinaceous aggregates. Employing confocal and immunoelectron microscopy, we find that the ubiquitin-associating protein sequestosome1/p62, co-localizes to aggregates isolated from AD but not control brain, along with the E3 ubiquitin ligase, TRAF6. This interaction could be recapitulated by co-transfection in HEK293 cells. Employing both in vitro and in vivo approaches, tau was found to be a substrate of the TRAF6, possessing lysine 63 polyubiquitin chains. Moreover, tau recovered from brain of TRAF6 knockout mice, compared with wild type, was not ubiquitinated. Tau degradation took place through the ubiquitin,proteasome pathway and was dependent upon either the K63-polyubiquitin chains or upon p62. In brain lysates of p62 knockout mice, tau fails to co-interact with Rpt1, a proteasomal subunit, thereby indicating a requirement for p62 shuttling of tau to the proteasome. Our results demonstrate that p62 interacts with K63-polyubiquitinated tau through its UBA domain and serves a novel role in regulating tau proteasomal degradation. We propose a model whereby either a decline in p62 expression or a decrease in proteasome activity may contribute to accumulation of insoluble/aggregated K63-polyubiquitinated tau. [source] 2,,3,-Cyclic nucleotide 3,-phosphodiesterase: A novel RNA-binding protein that inhibits protein synthesisJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2009Michel Gravel Abstract 2,,3,-Cyclic nucleotide 3,-phosphodiesterase (CNP) is one of the earliest myelin-related proteins to be specifically expressed in differentiating oligodendrocytes (ODCs) in the central nervous system (CNS) and is implicated in myelin biogenesis. CNP possesses an in vitro enzymatic activity, whose in vivo relevance remains to be defined, because substrates with 2,,3,-cyclic termini have not yet been identified. To characterize CNP function better, we previously determined the structure of the CNP catalytic domain by NMR. Interestingly, the structure is remarkably similar to the plant cyclic nucleotide phosphodiesterase (CPDase) from A. thaliana and the bacterial 2,-5, RNA ligase from T. thermophilus, which are known to play roles in RNA metabolism. Here we show that CNP is an RNA-binding protein. Furthermore, by using precipitation analyses, we demonstrate that CNP associates with poly(A)+ mRNAs in vivo and suppresses translation in vitro in a dose-dependent manner. With SELEX, we isolated RNA aptamers that can suppress the inhibitory effect of CNP on translation. We also demonstrate that CNP1 can bridge an association between tubulin and RNA. These results suggest that CNP1 may regulate expression of mRNAs in ODCs of the CNS. © 2008 Wiley-Liss, Inc. [source] Polyhydroxylated fullerene derivative C60(OH)24 prevents mitochondrial dysfunction and oxidative damage in an MPP+ -induced cellular model of Parkinson's diseaseJOURNAL OF NEUROSCIENCE RESEARCH, Issue 16 2008Xiaoqing Cai Abstract To find effective agents for Parkinson's disease (PD) prevention and therapy, we examined the protective effects of the polyhydroxylated fullerene derivative C60(OH)24 in a 1-methyl-4-phenylpyridinium (MPP+)-induced acute cellular PD model in human neuroblastoma cells and the free radical scavenging effects in this model with an electron spin resonance (ESR) spectrometer. Pretreatment with C60(OH)24 at concentrations greater than 20 ,M showed significant protective effects on MPP+ -induced loss in cell viability, decreases in mitochondrial function (including mitochondrial membrane potential and activities of complex I and II), and increases in the levels of reactive oxygen species and oxidative damage to DNA and proteins. In addition, C60(OH)24 acts as a phase 2 enzyme inducer to protect cells from MPP+ -induced decreases in expression of nuclear factor-E2-related factor 2, expression and activity of ,-glutamyl cysteine ligase and level of glutathione. The ESR study showed that C60(OH)24 is a powerful radical scavenger for superoxide, hydroxyl, and lipid radicals. These data suggest that C60(OH)24 is a mitochondrial protective antioxidant with direct radical scavenging activity and indirect antioxidant inducing activity. © 2008 Wiley-Liss, Inc. [source] | ||||||||||||||||