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Sample records for acid coa ligase

  1. Contribution of CoA Ligases to Benzenoid Biosynthesis in Petunia Flowers[W

    PubMed Central

    Klempien, Antje; Kaminaga, Yasuhisa; Qualley, Anthony; Nagegowda, Dinesh A.; Widhalm, Joshua R.; Orlova, Irina; Shasany, Ajit Kumar; Taguchi, Goro; Kish, Christine M.; Cooper, Bruce R.; D’Auria, John C.; Rhodes, David; Pichersky, Eran; Dudareva, Natalia

    2012-01-01

    Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the β-oxidative or nonoxidative pathways. The first step in the β-oxidative pathway is cinnamoyl-CoA formation, likely catalyzed by a member of the 4-coumarate:CoA ligase (4CL) family that converts a range of trans-cinnamic acid derivatives into the corresponding CoA thioesters. Using a functional genomics approach, we identified two potential CoA-ligases from petunia (Petunia hybrida) petal-specific cDNA libraries. The cognate proteins share only 25% amino acid identity and are highly expressed in petunia corollas. Biochemical characterization of the recombinant proteins revealed that one of these proteins (Ph-4CL1) has broad substrate specificity and represents a bona fide 4CL, whereas the other is a cinnamate:CoA ligase (Ph-CNL). RNA interference suppression of Ph-4CL1 did not affect the petunia benzenoid scent profile, whereas downregulation of Ph-CNL resulted in a decrease in emission of benzylbenzoate, phenylethylbenzoate, and methylbenzoate. Green fluorescent protein localization studies revealed that the Ph-4CL1 protein is localized in the cytosol, whereas Ph-CNL is in peroxisomes. Our results indicate that subcellular compartmentalization of enzymes affects their involvement in the benzenoid network and provide evidence that cinnamoyl-CoA formation by Ph-CNL in the peroxisomes is the committed step in the β-oxidative pathway. PMID:22649270

  2. 4-coumarate: CoA ligase partitions metabolites for eugenol biosynthesis.

    PubMed

    Rastogi, Shubhra; Kumar, Ritesh; Chanotiya, Chandan S; Shanker, Karuna; Gupta, Madan M; Nagegowda, Dinesh A; Shasany, Ajit K

    2013-08-01

    Biosynthesis of eugenol shares its initial steps with that of lignin, involving conversion of hydroxycinnamic acids to their corresponding coenzyme A (CoA) esters by 4-coumarate:CoA ligases (4CLs). In this investigation, a 4CL (OS4CL) was identified from glandular trichome-rich tissue of Ocimum sanctum with high sequence similarity to an isoform (OB4CL_ctg4) from Ocimum basilicum. The levels of OS4CL and OB4CL_ctg4-like transcripts were highest in O. sanctum trichome, followed by leaf, stem and root. The eugenol content in leaf essential oil was positively correlated with the expression of OS4CL in the leaf at different developmental stages. Recombinant OS4CL showed the highest activity with p-coumaric acid, followed by ferulic, caffeic and trans-cinnamic acids. Transient RNA interference (RNAi) suppression of OS4CL in O. sanctum leaves caused a reduction in leaf eugenol content and trichome transcript level, with a considerable increase in endogenous p-coumaric, ferulic, trans-cinnamic and caffeic acids. A significant reduction in the expression levels was observed for OB4CL_ctg4-related transcripts in suppressed trichome compared with transcripts similar to the other four isoforms (OB4CL_ctg1, 2, 3 and 5). Sinapic acid and lignin content were also unaffected in RNAi suppressed leaf samples. Transient expression of OS4CL-green fluorescent protein fusion protein in Arabidopsis protoplasts was associated with the cytosol. These results indicate metabolite channeling of intermediates towards eugenol by a specific 4CL and is the first report demonstrating the involvement of 4CL in creation of virtual compartments through substrate utilization and committing metabolites for eugenol biosynthesis at an early stage of the pathway.

  3. 4-coumarate: CoA ligase partitions metabolites for eugenol biosynthesis.

    PubMed

    Rastogi, Shubhra; Kumar, Ritesh; Chanotiya, Chandan S; Shanker, Karuna; Gupta, Madan M; Nagegowda, Dinesh A; Shasany, Ajit K

    2013-08-01

    Biosynthesis of eugenol shares its initial steps with that of lignin, involving conversion of hydroxycinnamic acids to their corresponding coenzyme A (CoA) esters by 4-coumarate:CoA ligases (4CLs). In this investigation, a 4CL (OS4CL) was identified from glandular trichome-rich tissue of Ocimum sanctum with high sequence similarity to an isoform (OB4CL_ctg4) from Ocimum basilicum. The levels of OS4CL and OB4CL_ctg4-like transcripts were highest in O. sanctum trichome, followed by leaf, stem and root. The eugenol content in leaf essential oil was positively correlated with the expression of OS4CL in the leaf at different developmental stages. Recombinant OS4CL showed the highest activity with p-coumaric acid, followed by ferulic, caffeic and trans-cinnamic acids. Transient RNA interference (RNAi) suppression of OS4CL in O. sanctum leaves caused a reduction in leaf eugenol content and trichome transcript level, with a considerable increase in endogenous p-coumaric, ferulic, trans-cinnamic and caffeic acids. A significant reduction in the expression levels was observed for OB4CL_ctg4-related transcripts in suppressed trichome compared with transcripts similar to the other four isoforms (OB4CL_ctg1, 2, 3 and 5). Sinapic acid and lignin content were also unaffected in RNAi suppressed leaf samples. Transient expression of OS4CL-green fluorescent protein fusion protein in Arabidopsis protoplasts was associated with the cytosol. These results indicate metabolite channeling of intermediates towards eugenol by a specific 4CL and is the first report demonstrating the involvement of 4CL in creation of virtual compartments through substrate utilization and committing metabolites for eugenol biosynthesis at an early stage of the pathway. PMID:23677922

  4. Kinetically and Crystallographically Guided Mutations of a Benzoate CoA Ligase (BadA) Elucidate Mechanism and Expand Substrate Permissivity.

    PubMed

    Thornburg, Chelsea K; Wortas-Strom, Susan; Nosrati, Meisam; Geiger, James H; Walker, Kevin D

    2015-10-13

    A benzoate CoA ligase (BadA), isolated from the bacterium Rhodopseudomonas palustris, catalyzes the conversion of benzoate to benzoyl CoA on the catabolic pathway of aromatic carboxylic acids. Herein, apparent Michaelis constants K(app)cat and K(app)M were determined for an expanded array of 31 substrates chosen to systematically probe the active site architecture of the enzyme and provide a baseline for expansion of wild-type substrate specificity. Acyl CoA products were observed for 25 of the 31 substrates; in general, BadA converted ortho-substituted substrates better than the corresponding meta and para regioisomers, and the turnover number was more affected by steric rather than electronic effects. The kinetic data are interpreted in relation to six crystal structures of BadA in complex with several substrates and a benzoyl-AMP reaction intermediate. In contrast to other known natural substrate-bound benzoate ligase structures, all substrate-bound BadA structures adopted the thiolation conformation instead of the adenylation conformation. We also observed all the aryl carboxylates to be uniquely oriented within the active site, relative to other structures. Together, the kinetics and structural data suggested a mechanism that involves substrate binding in the thiolation conformation, followed by substrate rotation to an active orientation upon the transition to the adenylation conformation. On the basis of this hypothesis and the structural data, sterically demanding active site residues were mutated, and the substrate specificity was expanded substantially versus that of BadA. Novel activities were seen for substrates with larger substituents, including phenyl acetate. Additionally, the mutant Lys427Ala identified this nonconserved residue as essential for the thiolation step of BadA, but not adenylation. These variously acylated CoAs can serve as novel substrates of acyl CoA-dependent acyltransferases in coupled enzyme assays to produce analogues of

  5. Cloning and functional characterization of a 4-coumarate CoA ligase from liverwort Plagiochasma appendiculatum.

    PubMed

    Gao, Shuai; Yu, Hai-Na; Xu, Rui-Xue; Cheng, Ai-Xia; Lou, Hong-Xiang

    2015-03-01

    Plant phenylpropanoids represent a large group of secondary metabolites which have played an important role in terrestrial plant life, beginning with the evolution of land plants from primitive green algae. 4-Coumarate: coenzyme A ligase (4CL) is a provider of activated thioester substrates within the phenylpropanoid synthesis pathway. Although 4CLs have been extensively characterized in angiosperm, gymnosperm and moss species, little is known of their functions in liverworts. Here, a 4CL homolog (designated as Pa4CL1) was isolated from the liverwort species Plagiochasma appendiculatum. The full-length cDNA sequence of Pa4CL1 contains 1644bp and is predicted to encode a protein with 547amino acids. The gene products were 40-50% identical with 4CL sequences reported in public databases. The recombinant protein was heterologously expressed in Escherichia coli and exhibited a high level of 4CL activity, catalyzing formation of hydroxycinnamate-CoA thioesters by a two-step reaction mechanism from corresponding hydroxycinnamic acids. Kinetic analysis indicated that the most favorable substrate for Pa4CL1 is p-coumaric acid. The transcription of Pa4CL1 was induced when P. appendiculatum thallus was treated with either salicylic acid or methyl jasmonate.

  6. Cloning and functional characterization of a 4-coumarate CoA ligase from liverwort Plagiochasma appendiculatum.

    PubMed

    Gao, Shuai; Yu, Hai-Na; Xu, Rui-Xue; Cheng, Ai-Xia; Lou, Hong-Xiang

    2015-03-01

    Plant phenylpropanoids represent a large group of secondary metabolites which have played an important role in terrestrial plant life, beginning with the evolution of land plants from primitive green algae. 4-Coumarate: coenzyme A ligase (4CL) is a provider of activated thioester substrates within the phenylpropanoid synthesis pathway. Although 4CLs have been extensively characterized in angiosperm, gymnosperm and moss species, little is known of their functions in liverworts. Here, a 4CL homolog (designated as Pa4CL1) was isolated from the liverwort species Plagiochasma appendiculatum. The full-length cDNA sequence of Pa4CL1 contains 1644bp and is predicted to encode a protein with 547amino acids. The gene products were 40-50% identical with 4CL sequences reported in public databases. The recombinant protein was heterologously expressed in Escherichia coli and exhibited a high level of 4CL activity, catalyzing formation of hydroxycinnamate-CoA thioesters by a two-step reaction mechanism from corresponding hydroxycinnamic acids. Kinetic analysis indicated that the most favorable substrate for Pa4CL1 is p-coumaric acid. The transcription of Pa4CL1 was induced when P. appendiculatum thallus was treated with either salicylic acid or methyl jasmonate. PMID:25593011

  7. Soraphen A, an inhibitor of acetyl CoA carboxylase activity, interferes with fatty acid elongation

    PubMed Central

    Jump, Donald B.; Torres-Gonzalez, Moises; Olson, L. Karl

    2010-01-01

    Acetyl CoA carboxylase (ACC1 & ACC2) generates malonyl CoA, a substrate for de novo lipogenesis (DNL) and an inhibitor of mitochondrial fatty acid β-oxidation (FAO). Malonyl CoA is also a substrate for microsomal fatty acid elongation, an important pathway for saturated (SFA), mono- (MUFA) and polyunsaturated fatty acid (PUFA) synthesis. Despite the interest in ACC as a target for obesity and cancer therapy, little attention has been given to the role ACC plays in long chain fatty acid synthesis. This report examines the effect of pharmacological inhibition of ACC on DNL & palmitate (16:0) and linoleate (18:2,n-6) metabolism in HepG2 and LnCap cells. The ACC inhibitor, soraphen A, lowers cellular malonyl CoA, attenuates DNL and the formation of fatty acid elongation products derived from exogenous fatty acids, i.e., 16:0 & 18:2,n-6; IC50 ~ 5 nM. Elevated expression of fatty acid elongases (Elovl5, Elovl6) or desaturases (FADS1, FADS2) failed to override the soraphen A effect on SFA, MUFA or PUFA synthesis. Inhibition of fatty acid elongation leads to the accumulation of 16- and 18-carbon unsaturated fatty acids derived from 16:0 and 18:2,n-6, respectively. Pharmacological inhibition of ACC activity will not only attenuate DNL and induce FAO, but will also attenuate the synthesis of very long chain saturated, mono- and polyunsaturated fatty acids. PMID:21184748

  8. Phenolic metabolism in petunia tissues. IV. - Properties of p-coumarate : coenzyme A ligase isoenzymes.

    PubMed

    Ranjeva, R; Boudet, A M; Faggion, R

    1976-01-01

    Three p-coumarate: CoA ligases were separated from Petunia leaves. There was no interconversion from one form to another. The isoenzymes had a number of common properties: optimum pH, instability in the absence of polyols, action on p-coumaric acid as the common substrate. These enzymes differed significantly with respect to: --their substrate specificity towards the other C6-C3 units of Petunia. Form Ia (caffeate: CoA ligase) acted on caffeic acid, form Ib (sinapate: CoA ligase) on sinapic acid form II (ferulate: CoA ligase) on ferulic acid. --their thermal stability. --their sensitivity to phenolics: (a) caffeate: CoA ligase was inhibited by p-coumaroyl and caffeoyl quinic esters. It was insensitive to p-coumaroyl-glucose, on one hand and to a number of flavonoids on the other. (b) ferulate: CoA ligase was specifically inhibited by naringenin. (c) sinapate: CoA ligase was not inhibited by the selected compounds. In all cases, the inhibition was of the non competitive type and the enzymes were desensized to the modifier action by thermal treatment independently from the enzyme activity. These results suggest the occurrence of distinct sites of reception for the substrate and the inhibitor on the enzyme molecule. All these data are consistent with the hypothesis of the possible participation of each individual form in a limited number of pathways. This would be of physiological interest since the metabolic fate of the different cinnamic acids could be independently controlled at the p-coumarate: CoA ligase level.

  9. Reconciling Ligase Ribozyme Activity with Fatty Acid Vesicle Stability

    PubMed Central

    Anella, Fabrizio; Danelon, Christophe

    2014-01-01

    The “RNA world” and the “Lipid world” theories for the origin of cellular life are often considered incompatible due to the differences in the environmental conditions at which they can emerge. One obstacle resides in the conflicting requirements for divalent metal ions, in particular Mg2+, with respect to optimal ribozyme activity, fatty acid vesicle stability and protection against RNA strand cleavage. Here, we report on the activity of a short L1 ligase ribozyme in the presence of myristoleic acid (MA) vesicles at varying concentrations of Mg2+. The ligation rate is significantly lower at low-Mg2+ conditions. However, the loss of activity is overcompensated by the increased stability of RNA leading to a larger amount of intact ligated substrate after long reaction periods. Combining RNA ligation assays with fatty acid vesicles we found that MA vesicles made of 5 mM amphiphile are stable and do not impair ligase ribozyme activity in the presence of approximately 2 mM Mg2+. These results provide a scenario in which catalytic RNA and primordial membrane assembly can coexist in the same environment. PMID:25513761

  10. Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis

    PubMed Central

    Sato, Mitsunari; Yoshida, Yasuo; Nagano, Keiji; Hasegawa, Yoshiaki; Takebe, Jun; Yoshimura, Fuminobu

    2016-01-01

    Butyryl-CoA:acetate CoA transferase, which produces butyrate and acetyl-CoA from butyryl-CoA and acetate, is responsible for the final step of butyrate production in bacteria. This study demonstrates that in the periodontopathogenic bacterium Porphyromonas gingivalis this reaction is not catalyzed by PGN_1171, previously annotated as butyryl-CoA:acetate CoA transferase, but by three distinct CoA transferases, PGN_0725, PGN_1341, and PGN_1888. Gas chromatography/mass spectrometry (GC-MS) and spectrophotometric analyses were performed using crude enzyme extracts from deletion mutant strains and purified recombinant proteins. The experiments revealed that, in the presence of acetate, PGN_0725 preferentially utilized butyryl-CoA rather than propionyl-CoA. By contrast, this preference was reversed in PGN_1888. The only butyryl-CoA:acetate CoA transferase activity was observed in PGN_1341. Double reciprocal plots revealed that all the reactions catalyzed by these enzymes follow a ternary-complex mechanism, in contrast to previously characterized CoA transferases. GC-MS analysis to determine the concentrations of short chain fatty acids (SCFAs) in culture supernatants of P. gingivalis wild type and mutant strains revealed that PGN_0725 and PGN_1888 play a major role in the production of butyrate and propionate, respectively. Interestingly, a triple deletion mutant lacking PGN_0725, PGN_1341, and PGN_1888 produced low levels of SCFAs, suggesting that the microorganism contains CoA transferase(s) in addition to these three enzymes. Growth rates of the mutant strains were mostly slower than that of the wild type, indicating that many carbon compounds produced in the SCFA synthesis appear to be important for the biological activity of this microorganism. PMID:27486457

  11. Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis.

    PubMed

    Sato, Mitsunari; Yoshida, Yasuo; Nagano, Keiji; Hasegawa, Yoshiaki; Takebe, Jun; Yoshimura, Fuminobu

    2016-01-01

    Butyryl-CoA:acetate CoA transferase, which produces butyrate and acetyl-CoA from butyryl-CoA and acetate, is responsible for the final step of butyrate production in bacteria. This study demonstrates that in the periodontopathogenic bacterium Porphyromonas gingivalis this reaction is not catalyzed by PGN_1171, previously annotated as butyryl-CoA:acetate CoA transferase, but by three distinct CoA transferases, PGN_0725, PGN_1341, and PGN_1888. Gas chromatography/mass spectrometry (GC-MS) and spectrophotometric analyses were performed using crude enzyme extracts from deletion mutant strains and purified recombinant proteins. The experiments revealed that, in the presence of acetate, PGN_0725 preferentially utilized butyryl-CoA rather than propionyl-CoA. By contrast, this preference was reversed in PGN_1888. The only butyryl-CoA:acetate CoA transferase activity was observed in PGN_1341. Double reciprocal plots revealed that all the reactions catalyzed by these enzymes follow a ternary-complex mechanism, in contrast to previously characterized CoA transferases. GC-MS analysis to determine the concentrations of short chain fatty acids (SCFAs) in culture supernatants of P. gingivalis wild type and mutant strains revealed that PGN_0725 and PGN_1888 play a major role in the production of butyrate and propionate, respectively. Interestingly, a triple deletion mutant lacking PGN_0725, PGN_1341, and PGN_1888 produced low levels of SCFAs, suggesting that the microorganism contains CoA transferase(s) in addition to these three enzymes. Growth rates of the mutant strains were mostly slower than that of the wild type, indicating that many carbon compounds produced in the SCFA synthesis appear to be important for the biological activity of this microorganism. PMID:27486457

  12. 4-Coumaroyl and caffeoyl shikimic acids inhibit 4-coumaric acid:coenzyme A ligases and modulate metabolic flux for 3-hydroxylation in monolignol biosynthesis of Populus trichocarpa.

    PubMed

    Lin, Chien-Yuan; Wang, Jack P; Li, Quanzi; Chen, Hsi-Chuan; Liu, Jie; Loziuk, Philip; Song, Jina; Williams, Cranos; Muddiman, David C; Sederoff, Ronald R; Chiang, Vincent L

    2015-01-01

    Downregulation of 4-coumaric acid:coenzyme A ligase (4CL) can reduce lignin content in a number of plant species. In lignin precursor (monolignol) biosynthesis during stem wood formation in Populus trichocarpa, two enzymes, Ptr4CL3 and Ptr4CL5, catalyze the coenzyme A (CoA) ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. CoA ligation of 4-coumaric acid is essential for the 3-hydroxylation of 4-coumaroyl shikimic acid. This hydroxylation results from sequential reactions of 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) and 4-coumaric acid 3-hydroxylase 3 (PtrC3H3). Alternatively, 3-hydroxylation of 4-coumaric acid to caffeic acid may occur through an enzyme complex of cinnamic acid 4-hydroxylase 1 and 2 (PtrC4H1 and PtrC4H2) and PtrC3H3. We found that 4-coumaroyl and caffeoyl shikimic acids are inhibitors of Ptr4CL3 and Ptr4CL5. 4-Coumaroyl shikimic acid strongly inhibits the formation of 4-coumaroyl-CoA and caffeoyl-CoA. Caffeoyl shikimic acid inhibits only the formation of 4-coumaroyl-CoA. 4-Coumaroyl and caffeoyl shikimic acids both act as competitive and uncompetitive inhibitors. Metabolic flux in wild-type and PtrC3H3 downregulated P. trichocarpa transgenics has been estimated by absolute protein and metabolite quantification based on liquid chromatography-tandem mass spectrometry, mass action kinetics, and inhibition equations. Inhibition by 4-coumaroyl and caffeoyl shikimic acids may play significant regulatory roles when these inhibitors accumulate.

  13. Toxicity of Carboxylic Acid-Containing Drugs: The Role of Acyl Migration and CoA Conjugation Investigated.

    PubMed

    Lassila, Toni; Hokkanen, Juho; Aatsinki, Sanna-Mari; Mattila, Sampo; Turpeinen, Miia; Tolonen, Ari

    2015-12-21

    Many carboxylic acid-containing drugs are associated with idiosyncratic drug toxicity (IDT), which may be caused by reactive acyl glucuronide metabolites. The rate of acyl migration has been earlier suggested as a predictor of acyl glucuronide reactivity. Additionally, acyl Coenzyme A (CoA) conjugates are known to be reactive. Here, 13 drugs with a carboxylic acid moiety were incubated with human liver microsomes to produce acyl glucuronide conjugates for the determination of acyl glucuronide half-lives by acyl migration and with HepaRG cells to monitor the formation of acyl CoA conjugates, their further conjugate metabolites, and trans-acylation products with glutathione. Additionally, in vitro cytotoxicity and mitochondrial toxicity experiments were performed with HepaRG cells to compare the predictability of toxicity. Clearly, longer acyl glucuronide half-lives were observed for safe drugs compared to drugs that can cause IDT. Correlation between half-lives and toxicity classification increased when "relative half-lives," taking into account the formation of isomeric AG-forms due to acyl migration and eliminating the effect of hydrolysis, were used instead of plain disappearance of the initial 1-O-β-AG-form. Correlation was improved further when a daily dose of the drug was taken into account. CoA and related conjugates were detected primarily for the drugs that have the capability to cause IDT, although some exceptions to this were observed. Cytotoxicity and mitochondrial toxicity did not correlate to drug safety. On the basis of the results, the short relative half-life of the acyl glucuronide (high acyl migration rate), high daily dose and detection of acyl CoA conjugates, or further metabolites derived from acyl CoA together seem to indicate that carboxylic acid-containing drugs have a higher probability to cause drug-induced liver injury (DILI). PMID:26558897

  14. Acyl CoA profiles of transgenic plants that accumulate medium-chain fatty acids indicate inefficient storage lipid synthesis in developing oilseeds.

    PubMed

    Larson, Tony R; Edgell, Teresa; Byrne, James; Dehesh, Katayoon; Graham, Ian A

    2002-11-01

    Several Brassica napus lines transformed with genes responsible for the synthesis of medium- or long-chain fatty acids were examined to determine limiting factor(s) for the subsequent accumulation of these fatty acids in seed lipids. Examination of a decanoic acid (10:0) accumulating line revealed a disproportionately high concentration of 10:0 CoA during seed development compared to long-chain acyl CoAs isolated from the same tissues, suggesting that poor incorporation of 10:0 CoA into seed lipids limits 10:0 fatty acid accumulation. This relationship was also seen for dodecanoyl (12:0) CoA and fatty acid in a high 12:0 line, but not for octadecanoic (18:0) CoA and fatty acid in a high 18:0 line. Comparison of 10:0 CoA and fatty acid proportions from seeds at different developmental stages for transgenic B. napus and Cuphea hookeriana, the source plant for the medium-chain thioesterase and 3-ketoacyl-ACP synthase transgenes, revealed that C. hookeriana incorporates 10:0 CoA into seed lipids more efficiently than transgenic B. napus. Furthermore, beta-oxidation and glyoxylate cycle activities were not increased above wild type levels during seed development in the 8:0/10:0 line, suggesting that lipid catabolism was not being induced in response to the elevated 10:0 CoA concentrations. Taken together, these data suggest that transgenic plants that are engineered to synthesize medium-chain fatty acids may lack the necessary mechanisms, such as specific acyltransferases, to incorporate these fatty acids efficiently into seed lipids.

  15. Biochemical and Molecular Characterization of Phenylacetate-Coenzyme A Ligase, an Enzyme Catalyzing the First Step in Aerobic Metabolism of Phenylacetic Acid in Azoarcus evansii

    PubMed Central

    El-Said Mohamed, Magdy

    2000-01-01

    Phenylacetate-coenzyme A ligase (PA-CoA ligase; AMP forming, EC 6.2.1.30), the enzyme catalyzing the first step in the aerobic degradation of phenylacetate (PA) in Azoarcus evansii, has been purified and characterized. The gene (paaK) coding for this enzyme was cloned and sequenced. The enzyme catalyzes the reaction of PA with CoA and MgATP to yield phenylacetyl-CoA (PACoA) plus AMP plus PPi. The enzyme was specifically induced after aerobic growth in a chemically defined medium containing PA or phenylalanine (Phe) as the sole carbon source. Growth with 4-hydroxyphenylacetate, benzoate, adipate, or acetate did not induce the synthesis of this enzyme. This enzymatic activity was detected very early in the exponential phase of growth, and a maximal specific activity of 76 nmol min−1 mg of cell protein−1 was measured. After 117-fold purification to homogeneity, a specific activity of 48 μmol min−1 mg of protein−1 was achieved with a turnover number (catalytic constant) of 40 s−1. The protein is a monomer of 52 kDa and shows high specificity towards PA; other aromatic or aliphatic acids were not used as substrates. The apparent Km values for PA, ATP, and CoA were 14, 60, and 45 μM, respectively. The PA-CoA ligase has an optimum pH of 8 to 8.5 and a pI of 6.3. The enzyme is labile and requires the presence of glycerol for stabilization. The N-terminal amino acid sequence of the purified protein showed no homology with other reported PA-CoA ligases. The gene encoding this enzyme is 1,320 bp long and codes for a protein of 48.75 kDa (440 amino acids) which shows high similarity with other reported PA-CoA ligases. An amino acid consensus for an AMP binding motif (VX2SSGTTGXP) was identified. The biochemical and molecular characteristics of this enzyme are quite different from those of the isoenzyme catalyzing the same reaction under anaerobic conditions in the same bacterium. PMID:10629172

  16. Broad substrate specificity of phosphotransbutyrylase from Listeria monocytogenes: A potential participant in an alternative pathway for provision of acyl CoA precursors for fatty acid biosynthesis.

    PubMed

    Sirobhushanam, Sirisha; Galva, Charitha; Sen, Suranjana; Wilkinson, Brian J; Gatto, Craig

    2016-09-01

    Listeria monocytogenes, the causative organism of the serious food-borne disease listeriosis, has a membrane abundant in branched-chain fatty acids (BCFAs). BCFAs are normally biosynthesized from branched-chain amino acids via the activity of branched chain α-keto acid dehydrogenase (Bkd), and disruption of this pathway results in reduced BCFA content in the membrane. Short branched-chain carboxylic acids (BCCAs) added as media supplements result in incorporation of BCFAs arising from the supplemented BCCAs in the membrane of L. monocytogenes bkd mutant MOR401. High concentrations of the supplements also effect similar changes in the membrane of the wild type organism with intact bkd. Such carboxylic acids clearly act as fatty acid precursors, and there must be an alternative pathway resulting in the formation of their CoA thioester derivatives. Candidates for this are the enzymes phosphotransbutyrylase (Ptb) and butyrate kinase (Buk), the products of the first two genes of the bkd operon. Ptb from L. monocytogenes exhibited broad substrate specificity, a strong preference for branched-chain substrates, a lack of activity with acetyl CoA and hexanoyl CoA, and strict chain length preference (C3-C5). Ptb catalysis involved ternary complex formation. Additionally, Ptb could utilize unnatural branched-chain substrates such as 2-ethylbutyryl CoA, albeit with lower efficiency, consistent with a potential involvement of this enzyme in the conversion of the carboxylic acid additives into CoA primers for BCFA biosynthesis. PMID:27320015

  17. Role of CoA and acetyl-CoA in regulating cardiac fatty acid and glucose oxidation.

    PubMed

    Abo Alrob, Osama; Lopaschuk, Gary D

    2014-08-01

    CoA (coenzyme A) and its derivatives have a critical role in regulating cardiac energy metabolism. This includes a key role as a substrate and product in the energy metabolic pathways, as well as serving as an allosteric regulator of cardiac energy metabolism. In addition, the CoA ester malonyl-CoA has an important role in regulating fatty acid oxidation, secondary to inhibiting CPT (carnitine palmitoyltransferase) 1, a key enzyme involved in mitochondrial fatty acid uptake. Alterations in malonyl-CoA synthesis by ACC (acetyl-CoA carboxylase) and degradation by MCD (malonyl-CoA decarboxylase) are important contributors to the high cardiac fatty acid oxidation rates seen in ischaemic heart disease, heart failure, obesity and diabetes. Additional control of fatty acid oxidation may also occur at the level of acetyl-CoA involvement in acetylation of mitochondrial fatty acid β-oxidative enzymes. We find that acetylation of the fatty acid β-oxidative enzymes, LCAD (long-chain acyl-CoA dehydrogenase) and β-HAD (β-hydroxyacyl-CoA dehydrogenase) is associated with an increase in activity and fatty acid oxidation in heart from obese mice with heart failure. This is associated with decreased SIRT3 (sirtuin 3) activity, an important mitochondrial deacetylase. In support of this, cardiac SIRT3 deletion increases acetylation of LCAD and β-HAD, and increases cardiac fatty acid oxidation. Acetylation of MCD is also associated with increased activity, decreases malonyl-CoA levels and an increase in fatty acid oxidation. Combined, these data suggest that malonyl-CoA and acetyl-CoA have an important role in mediating the alterations in fatty acid oxidation seen in heart failure. PMID:25110000

  18. COAs: Behind the Masks.

    ERIC Educational Resources Information Center

    Birke, Szifra

    1993-01-01

    Provides information on alcoholism and codependency to help teachers identify and respond to children of alcoholics (COAs). Discusses characteristics of alcoholic homes and problems encountered by children and adult COAs. Examines survival "masks" of COAs, including hero, rebel, adjustor, clown, and caretaker. Lists organizational, print, and…

  19. Studies of Human 2,4-Dienoyl CoA Reductase Shed New Light on Peroxisomal β-Oxidation of Unsaturated Fatty Acids

    SciTech Connect

    Hua, Tian; Wu, Dong; Ding, Wei; Wang, Jiangyun; Shaw, Neil; Liu, Zhi-Jie

    2012-10-15

    Peroxisomes play an essential role in maintaining fatty acid homeostasis. Although mitochondria are also known to participate in the catabolism of fatty acids via β-oxidation, differences exist between the peroxisomal and mitochondrial β-oxidation. Only peroxisomes, but not mitochondrion, can shorten very long chain fatty acids. Here, we describe the crystal structure of a ternary complex of peroxisomal 2,4-dienoyl CoA reductases (pDCR) with hexadienoyl CoA and NADP, as a prototype for comparison with the mitochondrial 2,4-dienoyl CoA reductase (mDCR) to shed light on the differences between the enzymes from the two organelles at the molecular level. Unexpectedly, the structure of pDCR refined to 1.84 Å resolution reveals the absence of the tyrosine-serine pair seen in the active site of mDCR, which together with a lysine and an asparagine have been deemed a hallmark of the SDR family of enzymes. Instead, aspartate hydrogen-bonded to the Cα hydroxyl via a water molecule seems to perturb the water molecule for protonation of the substrate. Our studies provide the first structural evidence for participation of water in the DCR-catalyzed reactions. Biochemical studies and structural analysis suggest that pDCRs can catalyze the shortening of six-carbon-long substrates in vitro. However, the Km values of pDCR for short chain acyl CoAs are at least 6-fold higher than those for substrates with 10 or more aliphatic carbons. Unlike mDCR, hinge movements permit pDCR to process very long chain polyunsaturated fatty acids.

  20. Transient Induction of Phenylalanine Ammonia-Lyase and 4-Coumarate: CoA Ligase mRNAs in Potato Leaves Infected with Virulent or Avirulent Races of Phytophthora infestans1

    PubMed Central

    Fritzemeier, Karl-Heinrich; Cretin, Claude; Kombrink, Erich; Rohwer, Frauke; Taylor, Janet; Scheel, Dierk; Hahlbrock, Klaus

    1987-01-01

    Infection of potato leaves with the fungal pathogen Phytophthora infestans (Pi) resulted in the rapid stimulation of phenylpropanoid metabolism. Increases in the activities of several mRNAs, including those encoding phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL), were detectable within a few hours postinoculation, as demonstrated by two-dimensional gel electrophoresis of proteins synthesized in vitro. This effect was closely mimicked by application of Pi culture filtrate through cut leaf stems. PAL and 4CL mRNA activities were also rapidly and transiently induced in potato cell suspension cultures by treatments with Pi culture filtrate or arachidonic acid. This induction was exploited to generate cDNA probes complementary to PAL and 4CL mRNAs. Blot hybridizations using these probes revealed almost immediate, transient and coordinate increases in the transcription rates and subsequent changes in the amounts of PAL and 4CL mRNAs in leaves treated with Pi culture filtrate. Similar changes in the mRNA amounts were found in infected leaves of potato cultivars carrying resistance genes R1 (cv Datura) or R4 (cv Isola), independent of whether a virulent or an avirulent Pi pathotype was used for inoculation. These results are discussed in relation to recent cytological observations with the same potato cultivars and Pi pathotypes. Images Fig. 1 Fig. 4 Fig. 5 Fig. 6 PMID:16665678

  1. [The protective effect of pantothenic acid derivatives and changes in the system of acetyl CoA metabolism in acute ethanol poisoning].

    PubMed

    Moiseenok, A G; Dorofeev, B F; Omel'ianchik, S N

    1988-01-01

    Calcium pantothenate (CaP), calcium 4'-phosphopantothenate (CaPP), pantethine, panthenol, sulfopantetheine and CoA decrease acute toxicity of acetaldehyde in mice. All studied compounds diminish duration of the narcotic action of ethanol--ET (3.5 g/kg intraperitoneally) in mice and rats. In the latter this effect is realized at the expense of "long sleeping" and "middle sleeping" animals. CaP (150 mg/kg subcutaneously) and CaPP (100 mg/kg subcutaneously) prevent hypothermia and a decrease of oxygen consumption in rats induced by ET administration. Combined administration of ET, CaP and CaPP leads to a characteristic increase of acid-soluble CoA fractions in the rat liver and a relative decrease of acetyl CoA synthetase and N-acetyltransferase reactions. The antitoxic effect of preparations of pantothenic acid is not mediated by CoA-dependent reactions of detoxication, but most probably is due to intensification of ET oxidation and perhaps to its elimination from the organism. PMID:2905277

  2. Rational Redesign of the 4-Chlorobenzoate Binding Site of 4-Chlorobenzoate: Coenzyme A Ligase for Expanded Substrate Range#%

    PubMed Central

    Wu, Rui; Reger, Albert S.; Cao, Jian; Gulick, Andrew M.; Dunaway-Mariano, Debra

    2014-01-01

    Environmental aromatic acids are transformed to chemical energy in bacteria that possess the requisite secondary pathways. Some of these pathways rely on the activation of the aromatic acid by coenzyme A (CoA) thioesterification catalyzed by an aromatic acid: CoA ligase. Adaptation of such pathways to the bioremediation of man-made pollutants such as polychlorinated biphenyl (PCB) and dichlorodiphenyltrichloroethane (DDT) requires that the chlorinated benzoic acid by-product formed can be eliminated by further degradation. To take advantage of natural benzoic acid degrading pathways requiring initial ring activation by thioesterification, the pathway aromatic acid: CoA ligase must be an effective catalyst with the chlorinated benzoic acid. The present study, which focuses on the 4-chlorobenzoate: CoA ligase (CBL) of the 4-monochlorobiphenyl degrading bacterium Alcaligenes sp strain ALP83, was carried-out to determine if the 4-chlorobenzoate binding site of this enzyme can be transformed by rational design to recognize the chlorobenzoic acids formed in course of breakdown of other environmental PCB congeners. The fundamental question addressed in this study is whether it is possible to add or subtract space from the substrate-binding pocket of this ligase (so to complement the topology of the unnatural aromatic substrate) without causing disruption of the ligase catalytic machinery. Herein, we report the results of a substrate specificity analysis that, when interpreted within the context of the X-ray crystal structures, set the stage for the rational design of the ligase for thioesterification of two PCB derived chlorobenzoic acids. The ligase was first optimized to catalyze CoA thioesterification of 3,4-dichlorobenzoic acid, a poor substrate, by truncating Ile303, a large hydrophobic residue that packs against ring meta-C(H). The structural basis for the ~100-fold enhancement in the rate of 3,4-dichlorobenzoate thioesterification catalyzed by the I303A and I303G

  3. Compartmentation of acetyl CoA studied by analysis of tricarboxylic acid cycle acids and 3-hydroxybutyrate in bile of rats given [2,2,2-2H3]ethanol.

    PubMed Central

    Norsten, C; Cronholm, T

    1990-01-01

    Acetate, 3-hydroxybutyrate, pyruvate, lactate, citrate, 2-oxoglutarate, succinate, fumarate and malate were analysed in rat bile by gas chromatography and gas chromatography/mass spectrometry of their O-melthyloxime-t-butyldimethylsilyl derivatives. The concentration of acetate increased to about 1.8 mmol/l after administration of [2,2,2-2H3]ethanol. Acetate was formed from ethanol to an extent of about 82% and retained all of the 2H at C-2, whereas 15% of the 2H had been lost in the tricarboxylic acid cycle intermediates and 24% in 3-hydroxybutyrate. Thus the exchange of 2H for 1H takes place after formation of acetyl CoA. For citrate and 3-hydroxybutyrate, 41% and 11% respectively was formed from [2,2,2-2H3]ethanol. These results indicate that different pools of acetyl CoA are used for the synthesis of ketone bodies and citrate, with the latter being derived from ethanol to a much larger extent. Smaller fractions of 2-oxoglutarate (16%) and succinate (5%) were derived from [2,2,2--2H3]ethanol, indicating significant contributions from amino acids. PMID:2405844

  4. A third MRX family (MRX68) is the result of mutation in the long chain fatty acid-CoA ligase 4 (FACL4) gene: proposal of a rapid enzymatic assay for screening mentally retarded patients

    PubMed Central

    Longo, I; Frints, S; Fryns, J; Meloni, I; Pescucci, C; Ariani, F; Borghgraef, M; Raynaud, M; Marynen, P; Schwartz, C; Renieri, A; Froyen, G

    2003-01-01

    Background: The gene encoding fatty acid CoA ligase 4 (FACL4) is mutated in families with non-specific X linked mental retardation (MRX) and is responsible for cognitive impairment in the contiguous gene syndrome ATS-MR (Alport syndrome and mental retardation), mapped to Xq22.3. This finding makes this gene a good candidate for other mental retardation disorders mapping in this region. Methods: We have screened the FACL4 gene in eight families, two MRX and six syndromic X linked mental retardation (MRXS), mapping in a large interval encompassing Xq22.3. Results: We have found a missense mutation in MRX68. The mutation (c.1001C>T in the brain isoform) cosegregates with the disease and changes a highly conserved proline into a leucine (p.P375L) in the first luciferase domain, which markedly reduces the enzymatic activity. Furthermore, all heterozygous females showed completely skewed X inactivation in blood leucocytes, as happens in all reported females with other FACL4 point mutations or deletions. Conclusions: Since the FACL4 gene is highly expressed in brain, where it encodes a brain specific isoform, and is located in hippocampal and cerebellar neurones, a role for this gene in cognitive processes can be expected. Here we report the third MRX family with a FACL4 mutation and describe the development of a rapid enzymatic assay on peripheral blood that we propose as a sensitive, robust, and efficient diagnostic tool in mentally retarded males. PMID:12525535

  5. Cinnamate:CoA ligase initiates the biosynthesis of a benzoate-derived xanthone phytoalexin in Hypericum calycinum cell cultures.

    PubMed

    Gaid, Mariam M; Sircar, Debabrata; Müller, Andreas; Beuerle, Till; Liu, Benye; Ernst, Ludger; Hänsch, Robert; Beerhues, Ludger

    2012-11-01

    Although a number of plant natural products are derived from benzoic acid, the biosynthesis of this structurally simple precursor is poorly understood. Hypericum calycinum cell cultures accumulate a benzoic acid-derived xanthone phytoalexin, hyperxanthone E, in response to elicitor treatment. Using a subtracted complementary DNA (cDNA) library and sequence information about conserved coenzyme A (CoA) ligase motifs, a cDNA encoding cinnamate:CoA ligase (CNL) was isolated. This enzyme channels metabolic flux from the general phenylpropanoid pathway into benzenoid metabolism. HcCNL preferred cinnamic acid as a substrate but failed to activate benzoic acid. Enzyme activity was strictly dependent on the presence of Mg²⁺ and K⁺ at optimum concentrations of 2.5 and 100 mM, respectively. Coordinated increases in the Phe ammonia-lyase and HcCNL transcript levels preceded the accumulation of hyperxanthone E in cell cultures of H. calycinum after the addition of the elicitor. HcCNL contained a carboxyl-terminal type 1 peroxisomal targeting signal made up by the tripeptide Ser-Arg-Leu, which directed an amino-terminal reporter fusion to the peroxisomes. Masking the targeting signal by carboxyl-terminal reporter fusion led to cytoplasmic localization. A phylogenetic tree consisted of two evolutionarily distinct clusters. One cluster was formed by CoA ligases related to benzenoid metabolism, including HcCNL. The other cluster comprised 4-coumarate:CoA ligases from spermatophytes, ferns, and mosses, indicating divergence of the two clades prior to the divergence of the higher plant lineages.

  6. Changes in acetyl CoA levels during the early embryonic development of Xenopus laevis.

    PubMed

    Tsuchiya, Yugo; Pham, Uyen; Hu, Wanzhou; Ohnuma, Shin-Ichi; Gout, Ivan

    2014-01-01

    Coenzyme A (CoA) is a ubiquitous and fundamental intracellular cofactor. CoA acts as a carrier of metabolically important carboxylic acids in the form of CoA thioesters and is an obligatory component of a multitude of catabolic and anabolic reactions. Acetyl CoA is a CoA thioester derived from catabolism of all major carbon fuels. This metabolite is at a metabolic crossroads, either being further metabolised as an energy source or used as a building block for biosynthesis of lipids and cholesterol. In addition, acetyl CoA serves as the acetyl donor in protein acetylation reactions, linking metabolism to protein post-translational modifications. Recent studies in yeast and cultured mammalian cells have suggested that the intracellular level of acetyl CoA may play a role in the regulation of cell growth, proliferation and apoptosis, by affecting protein acetylation reactions. Yet, how the levels of this metabolite change in vivo during the development of a vertebrate is not known. We measured levels of acetyl CoA, free CoA and total short chain CoA esters during the early embryonic development of Xenopus laevis using HPLC. Acetyl CoA and total short chain CoA esters start to increase around midblastula transition (MBT) and continue to increase through stages of gastrulation, neurulation and early organogenesis. Pre-MBT embryos contain more free CoA relative to acetyl CoA but there is a shift in the ratio of acetyl CoA to CoA after MBT, suggesting a metabolic transition that results in net accumulation of acetyl CoA. At the whole-embryo level, there is an apparent correlation between the levels of acetyl CoA and levels of acetylation of a number of proteins including histones H3 and H2B. This suggests the level of acetyl CoA may be a factor, which determines the degree of acetylation of these proteins, hence may play a role in the regulation of embryogenesis. PMID:24831956

  7. Recent NASA Dryden COA Experience

    NASA Technical Reports Server (NTRS)

    Cobleigh, Brent

    2008-01-01

    This viewgraph presentation concerns the experience that Dryden has had with Certificate of Authorization (COA) in reference to unmanned aerial systems (UAS). It reviews recent Certificate of Authorization UAS's i.e., 2005 Altair NOAA Mission, 2006 Altair Western States Fire Mission, and 2007 Ikhana. The priorities for the safety process is reviewed, as are typical UAS hazards. Slides also review the common COA provisions, best practices and lessons learned, the 2005 NOAA/NASA Science Demonstration Flights and the use of the UAS systems during fire emergencies.

  8. The pepper E3 ubiquitin ligase RING1 gene, CaRING1, is required for cell death and the salicylic acid-dependent defense response.

    PubMed

    Lee, Dong Hyuk; Choi, Hyong Woo; Hwang, Byung Kook

    2011-08-01

    Ubiquitination is essential for ubiquitin/proteasome-mediated protein degradation in plant development and defense. Here, we identified a novel E3 ubiquitin ligase RING1 gene, CaRING1, from pepper (Capsicum annuum). In pepper, CaRING1 expression is induced by avirulent Xanthomonas campestris pv vesicatoria infection. CaRING1 contains an amino-terminal transmembrane domain and a carboxyl-terminal RING domain. In addition, it displays in vitro E3 ubiquitin ligase activity, and the RING domain is essential for E3 ubiquitin ligase activity in CaRING1. CaRING1 also localizes to the plasma membrane. In pepper plants, virus-induced gene silencing of CaRING1 confers enhanced susceptibility to avirulent X. campestris pv vesicatoria infection, which is accompanied by compromised hypersensitive cell death, reduced expression of PATHOGENESIS-RELATED1, and lowered salicylic acid levels in leaves. Transient expression of CaRING1 in pepper leaves induces cell death and the defense response that requires the E3 ubiquitin ligase activity of CaRING1. By contrast, overexpression of CaRING1 in Arabidopsis (Arabidopsis thaliana) confers enhanced resistance to hemibiotrophic Pseudomonas syringae pv tomato and biotrophic Hyaloperonospora arabidopsidis infections. Taken together, these results suggest that CaRING1 is involved in the induction of cell death and the regulation of ubiquitination during the defense response to microbial pathogens.

  9. DNA and RNA ligases: structural variations and shared mechanisms.

    PubMed

    Pascal, John M

    2008-02-01

    DNA and RNA ligases join 3' OH and 5' PO4 ends in polynucleotide substrates using a three-step reaction mechanism that involves covalent modification of both the ligase enzyme and the polynucleotide substrate with AMP. In the past three years, several polynucleotide ligases have been crystallized in complex with nucleic acid, providing the introductory views of ligase enzymes engaging their substrates. Crystal structures for two ATP-dependent DNA ligases, an NAD+-dependent DNA ligase, and an ATP-dependent RNA ligase demonstrate how ligases utilize the AMP group and their multi-domain architectures to manipulate nucleic acid structure and catalyze the end-joining reaction. Together with unliganded crystal structures of DNA and RNA ligases, a more comprehensive and dynamic understanding of the multi-step ligation reaction mechanism has emerged.

  10. Targeted degradation of abscisic acid receptors is mediated by the ubiquitin ligase substrate adaptor DDA1 in Arabidopsis.

    PubMed

    Irigoyen, María Luisa; Iniesto, Elisa; Rodriguez, Lesia; Puga, María Isabel; Yanagawa, Yuki; Pick, Elah; Strickland, Elizabeth; Paz-Ares, Javier; Wei, Ning; De Jaeger, Geert; Rodriguez, Pedro L; Deng, Xing Wang; Rubio, Vicente

    2014-02-01

    CULLIN4-RING E3 ubiquitin ligases (CRL4s) regulate key developmental and stress responses in eukaryotes. Studies in both animals and plants have led to the identification of many CRL4 targets as well as specific regulatory mechanisms that modulate their function. The latter involve COP10-DET1-DDB1 (CDD)-related complexes, which have been proposed to facilitate target recognition by CRL4, although the molecular basis for this activity remains largely unknown. Here, we provide evidence that Arabidopsis thaliana DET1-, DDB1-ASSOCIATED1 (DDA1), as part of the CDD complex, provides substrate specificity for CRL4 by interacting with ubiquitination targets. Thus, we show that DDA1 binds to the abscisic acid (ABA) receptor PYL8, as well as PYL4 and PYL9, in vivo and facilitates its proteasomal degradation. Accordingly, we found that DDA1 negatively regulates ABA-mediated developmental responses, including inhibition of seed germination, seedling establishment, and root growth. All other CDD components displayed a similar regulatory function, although they did not directly interact with PYL8. Interestingly, DDA1-mediated destabilization of PYL8 is counteracted by ABA, which protects PYL8 by limiting its polyubiquitination. Altogether, our data establish a function for DDA1 as a substrate receptor for CRL4-CDD complexes and uncover a mechanism for the desensitization of ABA signaling based on the regulation of ABA receptor stability. PMID:24563205

  11. ABD1 is an Arabidopsis DCAF substrate receptor for CUL4-DDB1-based E3 ligases that acts as a negative regulator of abscisic acid signaling.

    PubMed

    Seo, Kyoung-In; Lee, Jae-Hoon; Nezames, Cynthia D; Zhong, Shangwei; Song, Eunyoung; Byun, Myung-Ok; Deng, Xing Wang

    2014-02-01

    Members of the DDB1-CUL4-associated factors (DCAFs) family directly bind to DAMAGED DNA BINDING PROTEIN1 (DDB1) and function as the substrate receptors in CULLIN4-based E3 (CUL4) ubiquitin ligases, which regulate the selective ubiquitination of proteins. Here, we describe a DCAF protein, ABD1 (for ABA-hypersensitive DCAF1), that negatively regulates abscisic acid (ABA) signaling in Arabidopsis thaliana. ABD1 interacts with DDB1 in vitro and in vivo, indicating that it likely functions as a CUL4 E3 ligase substrate receptor. ABD1 expression is induced by ABA, and mutations in ABD1 result in ABA- and NaCl-hypersensitive phenotypes. Loss of ABD1 leads to hyperinduction of ABA-responsive genes and higher accumulation of the ABA-responsive transcription factor ABA INSENSITIVE5 (ABI5), hypersensitivity to ABA during seed germination and seedling growth, enhanced stomatal closure, reduced water loss, and, ultimately, increased drought tolerance. ABD1 directly interacts with ABI5 in yeast two-hybrid assays and associates with ABI5 in vivo by coimmunoprecipitation, and the interaction was found in the nucleus by bimolecular fluorescence complementation. Furthermore, loss of ABD1 results in a retardation of ABI5 degradation by the 26S proteasome. Taken together, these data suggest that the DCAF-CUL4 E3 ubiquitin ligase assembled with ABD1 is a negative regulator of ABA responses by directly binding to and affecting the stability of ABI5 in the nucleus. PMID:24563203

  12. [Phosphoprotein phosphatase nonspecifically hydrolyzes CoA].

    PubMed

    Reziapkin, V I; Moiseenok, A G

    1988-01-01

    CoA hydrolysis was studied by a homogenous phosphoprotein phosphatase (EC 3.1 3.16) preparation from bovine spleen nuclei at pH 5.8. Phosphoprotein phosphatase catalyzed hydrolysis of the CoA 3'-phosphoester bond to form dephospho-CoA and Pi. The Km value for phosphoprotein phosphatase with CoA as substrate was 3.7 mM, the specific activity - 0.26 mmol Pi.min-1.mg-1. Phosphoprotein phosphatase did not essentially catalyze the calcium pantothenate hydrolysis (not more than 2% as compared with the CoA hydrolysis rate). PMID:2849829

  13. Structural and mutational analysis of archaeal ATP-dependent RNA ligase identifies amino acids required for RNA binding and catalysis

    PubMed Central

    Gu, Huiqiong; Yoshinari, Shigeo; Ghosh, Raka; Ignatochkina, Anna V.; Gollnick, Paul D.; Murakami, Katsuhiko S.; Ho, C. Kiong

    2016-01-01

    An ATP-dependent RNA ligase from Methanobacterium thermoautotrophicum (MthRnl) catalyzes intramolecular ligation of single-stranded RNA to form a closed circular RNA via covalent ligase-AMP and RNA-adenylylate intermediate. Here, we report the X-ray crystal structures of an MthRnl•ATP complex as well as the covalent MthRnl–AMP intermediate. We also performed structure-guided mutational analysis to survey the functions of 36 residues in three component steps of the ligation pathway including ligase-adenylylation (step 1), RNA adenylylation (step 2) and phosphodiester bond synthesis (step 3). Kinetic analysis underscored the importance of motif 1a loop structure in promoting phosphodiester bond synthesis. Alanine substitutions of Thr117 or Arg118 favor the reverse step 2 reaction to deadenylate the 5′-AMP from the RNA-adenylate, thereby inhibiting step 3 reaction. Tyr159, Phe281 and Glu285, which are conserved among archaeal ATP-dependent RNA ligases and are situated on the surface of the enzyme, are required for RNA binding. We propose an RNA binding interface of the MthRnl based on the mutational studies and two sulfate ions that co-crystallized at the active site cleft in the MthRnl–AMP complex. PMID:26896806

  14. Feed intake is related to changes in plasma nonesterified fatty acid concentration and hepatic acetyl CoA content following feeding in lactating dairy cows.

    PubMed

    Piantoni, P; Ylioja, C M; Allen, M S

    2015-10-01

    The relationship between hepatic acetyl CoA (AcCoA) content and dry matter intake (DMI) was evaluated using 28 multiparous Holstein cows; 14 were early postpartum (PP; 12.6 ± 3.8 d in milk) and 14 were late-lactation cows (LL; 269 ± 30 d in milk). Cows were fed once daily, and DMI was determined for the first 4h after feeding. Liver and blood samples were collected before feeding and 4h after feeding. Feed intake over the 4-h period ranged from 3.7 to 9.6 kg of dry matter and was similar for the 2 stages of lactation. Before feeding, hepatic AcCoA content was greater for PP compared with LL cows (34.4 vs. 12.5 nmol/g), and decreased over the 4h after feeding for PP only (28.7 vs. 34.4 nmol/g). The range for change in AcCoA over the 4-h period was wide for both PP (-24.3 to 10.4 nmol/g) and LL (-5.7 to 16.1 nmol/g), and was related negatively to DMI at 4h for both PP (R(2) = 0.55) and LL (R(2) = 0.31). The reduction in plasma NEFA concentration over the 4-h period was greater for PP than LL cows (-681 vs. -47 µEq/L), and was related to DMI at 4h for both PP and LL (both R(2) = 0.38). Greater DMI among cows over the first 4h after feeding might have been from a sharper reduction in supply of AcCoA in the liver for oxidation during meals because of the reduction in plasma NEFA concentration. Consistent with this is that the change in AcCoA was positively related to the reduction in plasma NEFA concentration for PP cows (R(2) = 0.31). However, change in plasma NEFA concentration was not related to change in hepatic AcCoA in LL cows, indicating that the pool of AcCoA in LL cows is not as dependent on NEFA flux to the liver as that of PP cows. Further research is required to determine production and fate of AcCoA within the timeframe of meals and the effects of feeding on energy charge in hepatic tissue.

  15. Characterization of the formation of branched short-chain fatty acid:CoAs for bitter acid biosynthesis in hop glandular trichomes.

    PubMed

    Xu, Haiyang; Zhang, Fengxia; Liu, Baoxiu; Huhman, David V; Sumner, Lloyd W; Dixon, Richard A; Wang, Guodong

    2013-07-01

    Bitter acids, known for their use as beer flavoring and for their diverse biological activities, are predominantly formed in hop (Humulus lupulus) glandular trichomes. Branched short-chain acyl-CoAs (e.g. isobutyryl-CoA, isovaleryl-CoA and 2-methylbutyryl-CoA), derived from the degradation of branched-chain amino acids (BCAAs), are essential building blocks for the biosynthesis of bitter acids in hops. However, little is known regarding what components are needed to produce and maintain the pool of branched short-chain acyl-CoAs in hop trichomes. Here, we present several lines of evidence that both CoA ligases and thioesterases are likely involved in bitter acid biosynthesis. Recombinant HlCCL2 (carboxyl CoA ligase) protein had high specific activity for isovaleric acid as a substrate (K cat /K m = 4100 s(-1) M(-1)), whereas recombinant HlCCL4 specifically utilized isobutyric acid (Kcat/K m = 1800 s(-1) M(-1)) and 2-methylbutyric acid (Kcat/K m = 6900 s(-1) M(-1)) as substrates. Both HlCCLs, like hop valerophenone synthase (HlVPS), were expressed strongly in glandular trichomes and localized to the cytoplasm. Co-expression of HlCCL2 and HlCCL4 with HlVPS in yeast led to significant production of acylphloroglucinols (the direct precursors for bitter acid biosynthesis), which further confirmed the biochemical function of these two HlCCLs in vivo. Functional identification of a thioesterase that catalyzed the reverse reaction of CCLs in mitochondria, together with the comprehensive analysis of genes involved BCAA catabolism, supported the idea that cytosolic CoA ligases are required for linking BCAA degradation and bitter acid biosynthesis in glandular trichomes. The evolution and other possible physiological roles of branched short-chain fatty acid:CoA ligases in planta are also discussed.

  16. Brain pyruvate and 2-oxoglutarate dehydrogenase complexes are mitochondrial targets of the CoA ester of the Refsum disease marker phytanic acid.

    PubMed

    Bunik, Victoria I; Raddatz, Günter; Wanders, Ronald J A; Reiser, Georg

    2006-06-12

    Pyruvate and 2-oxoglutarate dehydrogenase complexes are strongly inhibited by phytanoyl-CoA (IC(50) approximately 10(-6)-10(-7) M). Palmitoyl-CoA is 10-fold less potent. Phytanic or palmitic acids have no inhibitory effect up to 0.3 mM. At the substrate saturation, the acyl-CoA's affect the first and second enzymatic components of the 2-oxoglutarate dehydrogenase complex, while the third component is inhibited only at a low saturation with its substrate dihydrolipoamide. Thus, key regulatory branch points of mitochondrial metabolism are targets of a cellular derivative of phytanic acid. Decreased activity of the complexes might therefore contribute to neurological symptoms upon accumulation of phytanic acid in Refsum disease.

  17. The Arabidopsis F-box E3 ligase RIFP1 plays a negative role in abscisic acid signalling by facilitating ABA receptor RCAR3 degradation.

    PubMed

    Li, Ying; Zhang, Liang; Li, Dekuan; Liu, Zhibin; Wang, Jianmei; Li, Xufeng; Yang, Yi

    2016-03-01

    The phytohormone abscisic acid (ABA) plays a vital role in plant growth and development. The function of ABA is mediated by a group of newly discovered ABA receptors, named PYRABACTIN RESISTANCE 1/PYR-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORs (PYR1/PYLs/RCARs). Here, we report that an Arabidopsis thaliana F-box protein RCAR3 INTERACTING F-BOX PROTEIN 1 (RIFP1) interacts with ABA receptor (RCAR3) and SCF E3 ligase complex subunits Arabidopsis SKP1-LIKE PROTEINs (ASKs) in vitro and in vivo. The rifp1 mutant plants displayed increased ABA-mediated inhibition of seed germination and water loss of detached leaves, while the overexpression of RIFP1 in Arabidopsis led to plants being insensitive to ABA. Meanwhile, the rifp1 mutant plants showed greater tolerance to water deficit. In addition, the RCAR3 protein level was more stable in the rifp1 mutant plants than in the wild-type plants, indicating that RIFP1 facilitates the proteasome degradation of RCAR3. Accordingly, the loss of RIFP1 increased the transcript levels of several ABA-responsive genes. Taken together, these data indicate that RIFP1 plays a negative role in the RCAR3-mediated ABA signalling pathway and likely functions as an adaptor subunit of the SCF ubiquitin ligase complex to regulate ABA receptor RCAR3 stability. PMID:26386272

  18. Purification of Pseudomonas putida acyl coenzyme A ligase active with a range of aliphatic and aromatic substrates.

    PubMed Central

    Fernández-Valverde, M; Reglero, A; Martinez-Blanco, H; Luengo, J M

    1993-01-01

    Acyl coenzyme A (acyl-CoA) ligase (acyl-CoA synthetase [ACoAS]) from Pseudomonas putida U was purified to homogeneity (252-fold) after this bacterium was grown in a chemically defined medium containing octanoic acid as the sole carbon source. The enzyme, which has a mass of 67 kDa, showed maximal activity at 40 degrees C in 10 mM K2PO4H-NaPO4H2 buffer (pH 7.0) containing 20% (wt/vol) glycerol. Under these conditions, ACoAS showed hyperbolic behavior against acetate, CoA, and ATP; the Kms calculated for these substrates were 4.0, 0.7, and 5.2 mM, respectively. Acyl-CoA ligase recognizes several aliphatic molecules (acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids) as substrates, as well as some aromatic compounds (phenylacetic and phenoxyacetic acids). The broad substrate specificity of ACoAS from P. putida was confirmed by coupling it with acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum to study the formation of several penicillins. Images PMID:8476289

  19. The Ubiquitin E3 Ligase LOSS OF GDU2 Is Required for GLUTAMINE DUMPER1-Induced Amino Acid Secretion in Arabidopsis1[C][W][OA

    PubMed Central

    Pratelli, Réjane; Guerra, Damian D.; Yu, Shi; Wogulis, Mark; Kraft, Edward; Frommer, Wolf B.; Callis, Judy; Pilot, Guillaume

    2012-01-01

    Amino acids serve as transport forms for organic nitrogen in the plant, and multiple transport steps are involved in cellular import and export. While the nature of the export mechanism is unknown, overexpression of GLUTAMINE DUMPER1 (GDU1) in Arabidopsis (Arabidopsis thaliana) led to increased amino acid export. To gain insight into GDU1’s role, we searched for ethyl-methanesulfonate suppressor mutants and performed yeast-two-hybrid screens. Both methods uncovered the same gene, LOSS OF GDU2 (LOG2), which encodes a RING-type E3 ubiquitin ligase. The interaction between LOG2 and GDU1 was confirmed by glutathione S-transferase pull-down, in vitro ubiquitination, and in planta coimmunoprecipitation experiments. Confocal microscopy and subcellular fractionation indicated that LOG2 and GDU1 both localized to membranes and were enriched at the plasma membrane. LOG2 expression overlapped with GDU1 in the xylem and phloem tissues of Arabidopsis. The GDU1 protein encoded by the previously characterized intragenic suppressor mutant log1-1, with an arginine in place of a conserved glycine, failed to interact in the multiple assays, suggesting that the Gdu1D phenotype requires the interaction of GDU1 with LOG2. This hypothesis was supported by suppression of the Gdu1D phenotype after reduction of LOG2 expression using either artificial microRNAs or a LOG2 T-DNA insertion. Altogether, in accordance with the emerging bulk of data showing membrane protein regulation via ubiquitination, these data suggest that the interaction of GDU1 and the ubiquitin ligase LOG2 plays a significant role in the regulation of amino acid export from plant cells. PMID:22291198

  20. The E3 ubiquitin ligase ZNRF2 is a substrate of mTORC1 and regulates its activation by amino acids

    PubMed Central

    Hoxhaj, Gerta; Caddye, Edward; Najafov, Ayaz; Houde, Vanessa P; Johnson, Catherine; Dissanayake, Kumara; Toth, Rachel; Campbell, David G; Prescott, Alan R; MacKintosh, Carol

    2016-01-01

    The mechanistic Target of Rapamycin complex 1 (mTORC1) senses intracellular amino acid levels through an intricate machinery, which includes the Rag GTPases, Ragulator and vacuolar ATPase (V-ATPase). The membrane-associated E3 ubiquitin ligase ZNRF2 is released into the cytosol upon its phosphorylation by Akt. In this study, we show that ZNRF2 interacts with mTOR on membranes, promoting the amino acid-stimulated translocation of mTORC1 to lysosomes and its activation in human cells. ZNRF2 also interacts with the V-ATPase and preserves lysosomal acidity. Moreover, knockdown of ZNRF2 decreases cell size and cell proliferation. Upon growth factor and amino acid stimulation, mTORC1 phosphorylates ZNRF2 on Ser145, and this phosphosite is dephosphorylated by protein phosphatase 6. Ser145 phosphorylation stimulates vesicle-to-cytosol translocation of ZNRF2 and forms a novel negative feedback on mTORC1. Our findings uncover ZNRF2 as a component of the amino acid sensing machinery that acts upstream of Rag-GTPases and the V-ATPase to activate mTORC1. DOI: http://dx.doi.org/10.7554/eLife.12278.001 PMID:27244671

  1. Catalytically Important Residues of E6AP Ubiquitin Ligase Identified Using Acid-Cleavable Photo-Cross-Linkers.

    PubMed

    Krist, David T; Statsyuk, Alexander V

    2015-07-28

    Inactivation of the E6AP E3 ubiquitin ligase (UBE3A gene) causes Angelman syndrome, while aberrant degradation of p53 by E6AP is implicated in cervical cancers. Herein, we describe the development of photo-cross-linkers to discover catalytic residues of E6AP. Using these cross-linkers, we identified covalent modifications of the E6AP catalytic cysteine and two lysines: Lys(847) and Lys(799). Lys(847) is required for the formation of Lys(48)-linked polyubiquitin chains, while the K799A E6AP mutant was more active at producing Lys(48)-linked polyubiquitin chains. Thus, opposing roles of Lys(799) and Lys(847) pave the path forward to pharmacological inhibitors or activators of E6AP for therapeutic purposes. PMID:26161728

  2. Regulation of amino acid transporter trafficking by mTORC1 in primary human trophoblast cells is mediated by the ubiquitin ligase Nedd4-2.

    PubMed

    Rosario, Fredrick J; Dimasuay, Kris Genelyn; Kanai, Yoshikatsu; Powell, Theresa L; Jansson, Thomas

    2016-04-01

    Changes in placental amino acid transfer directly contribute to altered fetal growth, which increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Placental amino acid transfer is critically dependent on the expression of specific transporters in the plasma membrane of the trophoblast, the transporting epithelium of the human placenta. However, the molecular mechanisms regulating this process are largely unknown. Nedd4-2 is an ubiquitin ligase that catalyses the ubiquitination of proteins, resulting in proteasomal degradation. We hypothesized that inhibition of mechanistic target of rapamycin complex 1 (mTORC1) decreases amino acid uptake in primary human trophoblast (PHT) cells by activation of Nedd4-2, which increases transporter ubiquitination resulting in decreased transporter expression in the plasma membrane. mTORC 1 inhibition increased the expression of Nedd4-2, promoted ubiquitination and decreased the plasma membrane expression of SNAT2 (an isoform of the System A amino acid transporter) and LAT1 (a System L amino acid transporter isoform), resulting in decreased cellular amino acid uptake. Nedd4-2 silencing markedly increased the trafficking of SNAT2 and LAT1 to the plasma membrane, which stimulated cellular amino acid uptake. mTORC1 inhibition by silencing of raptor failed to decrease amino acid transport following Nedd4-2 silencing. In conclusion, we have identified a novel link between mTORC1 signalling and ubiquitination, a common posttranslational modification. Because placental mTORC1 is inhibited in fetal growth restriction and activated in fetal overgrowth, we propose that regulation of placental amino acid transporter ubiquitination by mTORC1 and Nedd4-2 constitutes a molecular mechanisms underlying abnormal fetal growth.

  3. [Ligase chain reaction (LCR)].

    PubMed

    Yamanishi, K; Yasuno, H

    1993-06-01

    Ligase chain reaction (LCR) is a ligation-mediated amplification technique of a target DNA sequence using oligonucleotides and thermostable ligase. LCR is useful for the detection of known DNA sequences and point mutations in a limited amount of DNA. We introduce the principle, development, and protocol of this simple and convenient technique for DNA analysis.

  4. DNA ligase I, the replicative DNA ligase

    PubMed Central

    Howes, Timothy R.L.; Tomkinson, Alan E.

    2013-01-01

    Multiple DNA ligation events are required to join the Okazaki fragments generated during lagging strand DNA synthesis. In eukaryotes, this is primarily carried out by members of the DNA ligase I family. The C-terminal catalytic region of these enzymes is composed of three domains: a DNA binding domain, an adenylation domain and an OB-fold domain. In the absence of DNA, these domains adopt an extended structure but transition into a compact ring structure when they engage a DNA nick, with each of the domains contacting the DNA. The non-catalytic N-terminal region of eukaryotic DNA ligase I is responsible for the specific participation of these enzymes in DNA replication. This proline-rich unstructured region contains the nuclear localization signal and a PCNA interaction motif that is critical for localization to replication foci and efficient joining of Okazaki fragments. DNA ligase I initially engages the PCNA trimer via this interaction motif which is located at the extreme N-terminus of this flexible region. It is likely that this facilitates an additional interaction between the DNA binding domain and the PCNA ring. The similar size and shape of the rings formed by the PCNA trimer and the DNA ligase I catalytic region when it engages a DNA nick suggest that these proteins interact to form a double-ring structure during the joining of Okazaki fragments. DNA ligase I also interacts with replication factor C, the factor that loads the PCNA trimeric ring onto DNA. This interaction, which is regulated by phosphorylation of the non-catalytic N-terminus of DNA ligase I, also appears to be critical for DNA replication. PMID:22918593

  5. Cloning, nucleotide sequence, and engineered expression of Thermus thermophilus DNA ligase, a homolog of Escherichia coli DNA ligase.

    PubMed Central

    Lauer, G; Rudd, E A; McKay, D L; Ally, A; Ally, D; Backman, K C

    1991-01-01

    We have cloned and sequenced the gene for DNA ligase from Thermus thermophilus. A comparison of this sequence and those of other ligases reveals significant homology only with that of Escherichia coli. The overall amino acid composition of the thermophilic ligase and the pattern of amino acid substitutions between the two proteins are consistent with compositional biases in other thermophilic enzymes. We have engineered the expression of the T. thermophilus gene in Escherichia coli, and we show that E. coli proteins may be substantially removed from the thermostable ligase by a simple heat precipitation step. Images PMID:1840584

  6. Identification of 9α-Hydroxy-17-Oxo-1,2,3,4,10,19-Hexanorandrostan-5-Oic Acid in Steroid Degradation by Comamonas testosteroni TA441 and Its Conversion to the Corresponding 6-En-5-Oyl Coenzyme A (CoA) Involving Open Reading Frame 28 (ORF28)- and ORF30-Encoded Acyl-CoA Dehydrogenases

    PubMed Central

    Hayashi, Toshiaki; Koshino, Hiroyuki; Malon, Michal; Hirota, Hiroshi; Kudo, Toshiaki

    2014-01-01

    Comamonas testosteroni TA441 degrades steroids via aromatization and meta-cleavage of the A ring, followed by hydrolysis, and produces 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid as an intermediate compound. Herein, we identify a new intermediate compound, 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid. Open reading frame 28 (ORF28)- and ORF30-encoded acyl coenzyme A (acyl-CoA) dehydrogenase was shown to convert the CoA ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid to the CoA ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid. A homology search of the deduced amino acid sequences suggested that the ORF30-encoded protein is a member of the acyl-CoA dehydrogenase_fadE6_17_26 family, whereas the deduced amino acid sequence of ORF28 showed no significant similarity to specific acyl-CoA dehydrogenase family proteins. Possible steroid degradation gene clusters similar to the cluster of TA441 appear in bacterial genome analysis data. In these clusters, ORFs similar to ORFs 28 and 30 are often found side by side and ordered in the same manner as ORFs 28 and 30. PMID:25092028

  7. SDIR1 Is a RING Finger E3 Ligase That Positively Regulates Stress-Responsive Abscisic Acid Signaling in Arabidopsis[W

    PubMed Central

    Zhang, Yiyue; Yang, Chengwei; Li, Yin; Zheng, Nuoyan; Chen, Hao; Zhao, Qingzhen; Gao, Ting; Guo, Huishan; Xie, Qi

    2007-01-01

    Ubiquitination plays important roles in plant hormone signal transduction. We show that the RING finger E3 ligase, Arabidopsis thaliana SALT- AND DROUGHT-INDUCED RING FINGER1 (SDIR1), is involved in abscisic acid (ABA)-related stress signal transduction. SDIR1 is expressed in all tissues of Arabidopsis and is upregulated by drought and salt stress, but not by ABA. Plants expressing the ProSDIR1–β-glucuronidase (GUS) reporter construct confirmed strong induction of GUS expression in stomatal guard cells and leaf mesophyll cells under drought stress. The green fluorescent protein–SDIR1 fusion protein is colocalized with intracellular membranes. We demonstrate that SDIR1 is an E3 ubiquitin ligase and that the RING finger conservation region is required for its activity. Overexpression of SDIR1 leads to ABA hypersensitivity and ABA-associated phenotypes, such as salt hypersensitivity in germination, enhanced ABA-induced stomatal closing, and enhanced drought tolerance. The expression levels of a number of key ABA and stress marker genes are altered both in SDIR1 overexpression and sdir1-1 mutant plants. Cross-complementation experiments showed that the ABA-INSENSITIVE5 (ABI5), ABRE BINDING FACTOR3 (ABF3), and ABF4 genes can rescue the ABA-insensitive phenotype of the sdir1-1 mutant, whereas SDIR1 could not rescue the abi5-1 mutant. This suggests that SDIR1 acts upstream of those basic leucine zipper family genes. Our results indicate that SDIR1 is a positive regulator of ABA signaling. PMID:17573536

  8. Systems biology of lignin biosynthesis in Populus trichocarpa: heteromeric 4-coumaric acid:coenzyme A ligase protein complex formation, regulation, and numerical modeling.

    PubMed

    Chen, Hsi-Chuan; Song, Jina; Wang, Jack P; Lin, Ying-Chung; Ducoste, Joel; Shuford, Christopher M; Liu, Jie; Li, Quanzi; Shi, Rui; Nepomuceno, Angelito; Isik, Fikret; Muddiman, David C; Williams, Cranos; Sederoff, Ronald R; Chiang, Vincent L

    2014-03-01

    As a step toward predictive modeling of flux through the pathway of monolignol biosynthesis in stem differentiating xylem of Populus trichocarpa, we discovered that the two 4-coumaric acid:CoA ligase (4CL) isoforms, 4CL3 and 4CL5, interact in vivo and in vitro to form a heterotetrameric protein complex. This conclusion is based on laser microdissection, coimmunoprecipitation, chemical cross-linking, bimolecular fluorescence complementation, and mass spectrometry. The tetramer is composed of three subunits of 4CL3 and one of 4CL5. 4CL5 appears to have a regulatory role. This protein-protein interaction affects the direction and rate of metabolic flux for monolignol biosynthesis in P. trichocarpa. A mathematical model was developed for the behavior of 4CL3 and 4CL5 individually and in mixtures that form the enzyme complex. The model incorporates effects of mixtures of multiple hydroxycinnamic acid substrates, competitive inhibition, uncompetitive inhibition, and self-inhibition, along with characteristic of the substrates, the enzyme isoforms, and the tetrameric complex. Kinetic analysis of different ratios of the enzyme isoforms shows both inhibition and activation components, which are explained by the mathematical model and provide insight into the regulation of metabolic flux for monolignol biosynthesis by protein complex formation.

  9. Alteration of the substrate specificity of l-amino acid ligase and selective synthesis of Met-Gly as a salt taste enhancer.

    PubMed

    Kino, Haruka; Kino, Kuniki

    2015-01-01

    Dipeptides have unique physiological functions. This study focused on the salt-taste-enhancing dipeptide Met-Gly. BL00235, an l-amino acid ligase from Bacillus licheniformis NBRC12200, synthesizes Met-Gly as a major product as well as Met-Met as a by-product. To alter the substrate specificity of BL00235 and synthesize Met-Gly selectively, we chose to alter Pro85 residue based on the BL00235 crystal structure. We predicted that Met might be not recognized as a C-terminal substrate by occupying the space around C-terminal substrate. Pro85 was replaced with Phe, Tyr, and Trp, which have bulky aromatic side chains, by site-directed mutagenesis. These mutants lost the capacity to synthesize Met-Met, during the synthesis of Met-Gly. Furthermore, they did not synthesize Met-Met, even when methionine was used as a substrate. These results show that the amino acid residue at position 85 has a key role in C-terminal substrate specificity. PMID:26088155

  10. Combined walking exercise and alkali therapy in patients with CKD4-5 regulates intramuscular free amino acid pools and ubiquitin E3 ligase expression.

    PubMed

    Watson, Emma L; Kosmadakis, George C; Smith, Alice C; Viana, Joao L; Brown, Jeremy R; Molyneux, Karen; Pawluczyk, Izabella Z A; Mulheran, Michael; Bishop, Nicolette C; Shirreffs, Susan; Maughan, Ronald J; Owen, Paul J; John, Stephen G; McIntyre, Christopher W; Feehally, John; Bevington, Alan

    2013-08-01

    Muscle-wasting in chronic kidney disease (CKD) arises from several factors including sedentary behaviour and metabolic acidosis. Exercise is potentially beneficial but might worsen acidosis through exercise-induced lactic acidosis. We studied the chronic effects of exercise in CKD stage 4-5 patients (brisk walking, 30 min, 5 times/week), and non-exercising controls; each group receiving standard oral bicarbonate (STD), or additional bicarbonate (XS) (Total n = 26; Exercising + STD n = 9; Exercising +XS n = 6; Control + STD n = 8; Control + XS n = 3). Blood and vastus lateralis biopsies were drawn at baseline and 6 months. The rise in blood lactate in submaximal treadmill tests was suppressed in the Exercising + XS group. After 6 months, intramuscular free amino acids (including the branched chain amino acids) in the Exercising + STD group showed a striking chronic depletion. This did not occur in the Exercising + XS group. The effect in Exercising + XS patients was accompanied by reduced transcription of ubiquitin E3-ligase MuRF1 which activates proteolysis via the ubiquitin-proteasome pathway. Other anabolic indicators (Akt activation and suppression of the 14 kDa actin catabolic marker) were unaffected in Exercising + XS patients. Possibly because of this, overall suppression of myofibrillar proteolysis (3-methylhistidine output) was not observed. It is suggested that alkali effects in exercisers arose by countering exercise-induced acidosis. Whether further anabolic effects are attainable on combining alkali with enhanced exercise (e.g. resistance exercise) merits further investigation. PMID:23591985

  11. Crystallographic trapping of the glutamyl-CoA thioester intermediate of family I CoA transferases

    SciTech Connect

    Rangarajan,E.; Li, Y.; Ajamian, E.; Iannuzzi, P.; Kernaghan, S.; Fraser, M.; Cygler, M.; Matte, A.

    2005-01-01

    Coenzyme A transferases are involved in a broad range of biochemical processes in both prokaryotes and eukaryotes, and exhibit a diverse range of substrate specificities. The YdiF protein from Escherichia coli O157:H7 is an acyl-CoA transferase of unknown physiological function, and belongs to a large sequence family of CoA transferases, present in bacteria to humans, which utilize oxoacids as acceptors. In vitro measurements showed that YdiF displays enzymatic activity with short-chain acyl-CoAs. The crystal structures of YdiF and its complex with CoA, the first co-crystal structure for any Family I CoA transferase, have been determined and refined at 1.9 and 2.0 Angstrom resolution, respectively. YdiF is organized into tetramers, with each monomer having an open {alpha}/{beta} structure characteristic of Family I CoA transferases. Co-crystallization of YdiF with a variety of CoA thioesters in the absence of acceptor carboxylic acid resulted in trapping a covalent {gamma}-glutamyl-CoA thioester intermediate. The CoA binds within a well defined pocket at the N- and C-terminal domain interface, but makes contact only with the C-terminal domain. The structure of the YdiF complex provides a basis for understanding the different catalytic steps in the reaction of Family I CoA transferases.

  12. Differences among Adult COAs and Adult Non-COAs on Levels of Self-Esteem, Depression, and Anxiety.

    ERIC Educational Resources Information Center

    Dodd, David T.; Roberts, Richard L.

    1994-01-01

    Examined self-esteem, depression, and anxiety among 60 adult children of alcoholics (COAs) and 143 adult non-COAs. Subjects completed Children of Alcoholics Screening Test, demographic questionnaire, Beck Depression Inventory, State-Trait Anxiety Inventory, and Coopersmith Self-Esteem Inventory. Found no significant differences between COAs and…

  13. The Arabidopsis RING E3 ubiquitin ligase AtAIRP2 plays combinatory roles with AtAIRP1 in abscisic acid-mediated drought stress responses.

    PubMed

    Cho, Seok Keun; Ryu, Moon Young; Seo, Dong Hye; Kang, Bin Goo; Kim, Woo Taek

    2011-12-01

    The ubiquitin (Ub)-26S proteasome pathway is implicated in various cellular processes in higher plants. AtAIRP1, a C3H2C3-type RING (for Really Interesting New Gene) E3 Ub ligase, is a positive regulator in the Arabidopsis (Arabidopsis thaliana) abscisic acid (ABA)-dependent drought response. Here, the AtAIRP2 (for Arabidopsis ABA-insensitive RING protein 2) gene was identified and characterized. AtAIRP2 encodes a cytosolic C3HC4-type RING E3 Ub ligase whose expression was markedly induced by ABA and dehydration stress. Thus, AtAIRP2 belongs to a different RING subclass than AtAIRP1 with a limited sequence identity. AtAIRP2-overexpressing transgenic (35S:AtAIRP2-sGFP) and atairp2 loss-of-function mutant plants exhibited hypersensitive and hyposensitive phenotypes, respectively, to ABA in terms of seed germination, root growth, and stomatal movement. 35S:AtAIRP2-sGFP plants were highly tolerant to severe drought stress, and atairp2 alleles were more susceptible to water stress than were wild-type plants. Higher levels of drought-induced hydrogen peroxide production were detected in 35S:AtAIRP2-sGFP as compared with atairp2 plants. ABA-inducible drought-related genes were up-regulated in 35S:AtAIRP2-sGFP and down-regulated in atairp2 progeny. The positive effects of AtAIRP2 on ABA-induced stress genes were dependent on SNF1-related protein kinases, key components of the ABA signaling pathway. Therefore, AtAIRP2 is involved in positive regulation of ABA-dependent drought stress responses. To address the functional relationship between AtAIRP1 and AtAIRP2, FLAG-AtAIRP1 and AtAIRP2-sGFP genes were ectopically expressed in atairp2-2 and atairp1 plants, respectively. Constitutive expression of FLAG-AtAIRP1 and AtAIRP2-sGFP in atairp2-2 and atairp1 plants, respectively, reciprocally rescued the loss-of-function ABA-insensitive phenotypes during germination. Additionally, atairp1/35S:AtAIRP2-sGFP and atairp2-2/35S:FLAG-AtAIRP1 complementation lines were more tolerant to

  14. Germline Deletion of Pantothenate Kinases 1 and 2 Reveals the Key Roles for CoA in Postnatal Metabolism

    PubMed Central

    Garcia, Matthew; Leonardi, Roberta; Zhang, Yong-Mei; Rehg, Jerold E.; Jackowski, Suzanne

    2012-01-01

    Pantothenate kinase (PanK) phosphorylates pantothenic acid (vitamin B5) and controls the overall rate of coenzyme A (CoA) biosynthesis. Pank1 gene deletion in mice results in a metabolic phenotype where fatty acid oxidation and gluconeogenesis are impaired in the fasted state, leading to mild hypoglycemia. Inactivating mutations in the human PANK2 gene lead to childhood neurodegeneration, but Pank2 gene inactivation in mice does not elicit a phenotype indicative of the neuromuscular symptoms or brain iron accumulation that accompany the human disease. Pank1/Pank2 double knockout (dKO) mice were derived to determine if the mild phenotypes of the single knockout mice are due to the ability of the two isoforms to compensate for each other in CoA biosynthesis. Postnatal development was severely affected in the dKO mice. The dKO pups developed progressively severe hypoglycemia and hyperketonemia by postnatal day 10 leading to death by day 17. Hyperketonemia arose from impaired whole-body ketone utilization illustrating the requirement for CoA in energy generation from ketones. dKO pups had reduced CoA and decreased fatty acid oxidation coupled with triglyceride accumulation in liver. dKO hepatocytes could not maintain the NADH levels compared to wild-type hepatocytes. These results revealed an important link between CoA and NADH levels, which was reflected by deficiencies in hepatic oleate synthesis and gluconeogenesis. The data indicate that PanK1 and PanK2 can compensate for each other to supply tissue CoA, but PanK1 is more important to CoA levels in liver whereas PanK2 contributes more to CoA synthesis in the brain. PMID:22815849

  15. Effect of elevated total CoA levels on metabolic pathways in cultured hepatocytes

    SciTech Connect

    Steffen, C.A.; Smith, C.M.

    1987-05-01

    Livers from fasted rats have 30% higher total CoA levels than fed rats. To determine whether this increase of total CoA influences metabolism, the rates of gluconeogenesis, fatty acid oxidation and ketogenesis were measured in hepatocytes with cyanamide (CYM) or pantothenate (PA) deficient medium used to vary total CoA levels independently of hormonal status. Primary cultures of rat hepatocytes were incubated 14 hrs with Bt/sub 2/ cAMP, dexamethasone + theophylline in PA deficient medium or with CYM (500 ..mu..M) + PA, rinsed and preincubated 0.5 hr to remove the CYM. Hepatocytes treated with CYM had total CoA levels 10-24% higher than PA deficient cells and lower rates of glucose production from lactate + pyruvate (L/P) or from alanine (0.23 +/- 0.05 and 0.089 +/- 0.02 ..mu..m/mg protein, respectively in CYM treated cells compared to 0.33 +/- 0.06 and 0.130 +/- 0.006 in PA deficient cells). This decrease was not due to CYM per se, as the direct addition of CYM stimulated glucose production from L/P. CYM treated cells with 15-40% higher total CoA and 30% higher fatty acyl-CoA levels had the same rates of (/sup 14/C)-palmitate oxidation as PA deficient cells. However, rates of ketogenesis were lower in CYM treated cells (163 +/- 11 nm/mg compared to 217 +/- 14 nm/mg protein). These results suggest that physiological alterations of hepatic total CoA levels are not necessary for fasting rates of gluconeogenesis, fatty acid oxidation and ketogenesis.

  16. Characterization and Functional Analysis of 4-Coumarate:CoA Ligase Genes in Mulberry

    PubMed Central

    Yu, Jian; Cai, Yu-Xiang; Zhu, Pan-Pan; Liu, Chang-Ying; Zhao, Ai-Chun; Lü, Rui-Hua; Li, Meng-Jiao; Xu, Feng-Xiang; Yu, Mao-De

    2016-01-01

    A small, multigene family encodes 4-coumarate:CoA ligases (4CLs) that catalyze the ligation of CoA to hydroxycinnamic acids, a branch point directing metabolites to flavonoid or monolignol pathways. In this study, we characterized four 4CL genes from M. notabilis Genome Database, and cloned four Ma4CL genes from M. atropurpurea cv. Jialing No.40. A tissue-specific expression analysis indicated that Ma4CL3 was expressed at higher levels than the other genes, and that Ma4CL3 was strongly expressed in root bark, stem bark, and old leaves. Additionally, the expression pattern of Ma4CL3 was similar to the trend of the total flavonoid content throughout fruit development. A phylogenetic analysis suggested that Mn4CL1, Mn4CL2, and Mn4CL4 belong to class I 4CLs, and Mn4CL3 belongs to class II 4CLs. Ma4CL genes responded differently to a series of stresses. Ma4CL3 expression was higher than that of the other Ma4CL genes following wounding, salicylic acid, and ultraviolet treatments. An in vitro enzyme assay indicated that 4-coumarate acid was the best substrate among cinnamic acid, 4-coumarate acid, and caffeate acid, but no catalytic activity to sinapate acid and ferulate acid. The results of subcellular localization experiments showed that Ma4CL3 localized to the cytomembrane, where it activated transcription. We used different vectors and strategies to fuse Ma4CL3 with stilbene synthase (STS) to construct four Ma4CL-MaSTS co-expression systems to generate resveratrol. The results indicated that only a transcriptional fusion vector, pET-Ma4CL3-T-MaSTS, which utilized a T7 promoter and lac operator for the expression of MaSTS, could synthesize resveratrol. PMID:27213624

  17. Characterization and Functional Analysis of 4-Coumarate:CoA Ligase Genes in Mul-berry.

    PubMed

    Wang, Chuan-Hong; Yu, Jian; Cai, Yu-Xiang; Zhu, Pan-Pan; Liu, Chang-Ying; Zhao, Ai-Chun; Lü, Rui-Hua; Li, Meng-Jiao; Xu, Feng-Xiang; Yu, Mao-De

    2016-01-01

    A small, multigene family encodes 4-coumarate:CoA ligases (4CLs) that catalyze the ligation of CoA to hydroxycinnamic acids, a branch point directing metabolites to flavonoid or monolignol pathways. In this study, we characterized four 4CL genes from M. notabilis Genome Database, and cloned four Ma4CL genes from M. atropurpurea cv. Jialing No.40. A tissue-specific expression analysis indicated that Ma4CL3 was expressed at higher levels than the other genes, and that Ma4CL3 was strongly expressed in root bark, stem bark, and old leaves. Additionally, the expression pattern of Ma4CL3 was similar to the trend of the total flavonoid content throughout fruit development. A phylogenetic analysis suggested that Mn4CL1, Mn4CL2, and Mn4CL4 belong to class I 4CLs, and Mn4CL3 belongs to class II 4CLs. Ma4CL genes responded differently to a series of stresses. Ma4CL3 expression was higher than that of the other Ma4CL genes following wounding, salicylic acid, and ultraviolet treatments. An in vitro enzyme assay indicated that 4-coumarate acid was the best substrate among cinnamic acid, 4-coumarate acid, and caffeate acid, but no catalytic activity to sinapate acid and ferulate acid. The results of subcellular localization experiments showed that Ma4CL3 localized to the cytomembrane, where it activated transcription. We used different vectors and strategies to fuse Ma4CL3 with stilbene synthase (STS) to construct four Ma4CL-MaSTS co-expression systems to generate resveratrol. The results indicated that only a transcriptional fusion vector, pET-Ma4CL3-T-MaSTS, which utilized a T7 promoter and lac operator for the expression of MaSTS, could synthesize resveratrol. PMID:27213624

  18. Transcript profiling of jasmonate-elicited Taxus cells reveals a β-phenylalanine-CoA ligase.

    PubMed

    Ramírez-Estrada, Karla; Altabella, Teresa; Onrubia, Miriam; Moyano, Elisabeth; Notredame, Cedric; Osuna, Lidia; Vanden Bossche, Robin; Goossens, Alain; Cusido, Rosa M; Palazon, Javier

    2016-01-01

    Plant cell cultures constitute eco-friendly biotechnological platforms for the production of plant secondary metabolites with pharmacological activities, as well as a suitable system for extending our knowledge of secondary metabolism. Despite the high added value of taxol and the importance of taxanes as anticancer compounds, several aspects of their biosynthesis remain unknown. In this work, a genomewide expression analysis of jasmonate-elicited Taxus baccata cell cultures by complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) indicated a correlation between an extensive elicitor-induced genetic reprogramming and increased taxane production in the targeted cultures. Subsequent in silico analysis allowed us to identify 15 genes with a jasmonate-induced differential expression as putative candidates for genes encoding enzymes involved in five unknown steps of taxane biosynthesis. Among them, the TB768 gene showed a strong homology, including a very similar predicted 3D structure, with other genes previously reported to encode acyl-CoA ligases, thus suggesting a role in the formation of the taxol lateral chain. Functional analysis confirmed that the TB768 gene encodes an acyl-CoA ligase that localizes to the cytoplasm and is able to convert β-phenylalanine, as well as coumaric acid, into their respective derivative CoA esters. β-phenylalanyl-CoA is attached to baccatin III in one of the last steps of the taxol biosynthetic pathway. The identification of this gene will contribute to the establishment of sustainable taxol production systems through metabolic engineering or synthetic biology approaches.

  19. Tag Team Ubiquitin Ligases.

    PubMed

    Kleiger, Gary; Deshaies, Raymond

    2016-08-25

    Cullin-RING (CRL) and RING1-IBR-RING2 (RBR) are two distinct types of ubiquitin ligases. In this issue, Scott et al. show that CRLs activate the RBR enzyme ARIH1 to initiate ubiquitin chains on CRL substrates, thereby marking an unexpected and important advance in our understanding of both enzymes. PMID:27565338

  20. Effect of acetate formation pathway and long chain fatty acid CoA-ligase on the free fatty acid production in E. coli expressing acy-ACP thioesterase from Ricinus communis.

    PubMed

    Li, Mai; Zhang, Xiujun; Agrawal, Arpita; San, Ka-Yiu

    2012-07-01

    Microbial biosynthesis of fatty acid like chemicals from renewable carbon sources has attracted significant attention in recent years. Free fatty acids can be used as precursors for the production of fuels or chemicals. Wild type E. coli strains produce fatty acids mainly for the biosynthesis of lipids and cell membranes and do not accumulate free fatty acids as intermediates in lipid biosynthesis. However, free fatty acids can be produced by breaking the fatty acid elongation through the overexpression of an acyl-ACP thioesterase. Since acetyl-CoA might be an important factor for fatty acid synthesis (acetate formation pathways are the main competitive pathways in consuming acetyl-CoA or pyruvate, a precursor of acetyl-CoA), and the long chain fatty acid CoA-ligase (FadD) plays a pivotal role in the transport and activation of exogenous fatty acids prior to their subsequent degradation, we examined the composition and the secretion of the free fatty acids in four different strains including the wild type MG1655, a mutant strain with inactivation of the fatty acid beta-oxidation pathway (fadD mutant (ML103)), and mutant strains with inactivation of the two major acetate production pathways (an ack-pta (acetate kinase/phosphotransacetylase), poxB (pyruvate oxidase) double mutant (ML112)) and a fadD, ack-pta, poxB triple mutant (ML115). The engineered E. coli cells expressing acyl-ACP thioesterase with glucose yield is higher than 40% of theoretical yield. Compared to MG1655(pXZ18) and ML103(pXZ18), acetate forming pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar quantity of total free fatty acids, which indicated that acetyl-CoA availability does not appear to be limiting factor for fatty acid production in these strains. However, these strains did show significant differences in the composition of free fatty acids. Different from MG1655(pXZ18) and ML103(pXZ18), acetate formation pathway deletion strains such as ML112(pXZ18) and ML115

  1. Structural Characterization of a 140 Degree Domain Movement in the Two-Step Reaction Catalyzed By 4-Chlorobenzoate:CoA Ligase

    SciTech Connect

    Reger, A.S.; Wu, R.; Dunaway-Mariano, D.; Gulick, A.M.

    2009-05-21

    Members of the adenylate-forming family of enzymes play a role in the metabolism of halogenated aromatics and of short, medium, and long chain fatty acids, as well as in the biosynthesis of menaquinone, peptide antibiotics, and peptide siderophores. This family includes a subfamily of acyl- and aryl-CoA ligases that catalyze thioester synthesis through two half-reactions. A carboxylate substrate first reacts with ATP to form an acyl-adenylate. Subsequent to the release of the product PP{sub 1}, the enzyme binds CoA, which attacks the activated acyl group to displace AMP. Structural and functional studies on different family members suggest that these enzymes alternate between two conformations during catalysis of the two half-reactions. Specifically, after the initial adenylation step, the C-terminal domain rotates by 140{sup o} to adopt a second conformation for thioester formation. Previously, we determined the structure of 4-chlorobenzoate:CoA ligase (CBL) in the adenylate forming conformation bound to 4-chlorobenzoate. We have determined two new crystal structures. We have determined the structure of CBL in the original adenylate-forming conformation, bound to the adenylate intermediate. Additionally, we have used a novel product analogue, 4-chlorophenacyl-CoA, to trap the enzyme in the thioester-forming conformation and determined this structure in a new crystal form. This work identifies a novel binding pocket for the CoA nucleotide. The structures presented herein provide the foundation for biochemical analyses presented in the accompanying manuscript in this issue [Wu et al. (2008) Biochemistry 47, 8026-8039]. The complete characterization of this enzyme allows us to provide an explanation for the use of the domain alternation strategy by these enzymes.

  2. Structural Characterization of a 140 degrees Domain Movement in the Two-Step Reaction Catalyzed by 4-Chlorobenzoate:CoA Ligase

    SciTech Connect

    Reger,A.; Wu, R.; Dunaway-Mariano, D.; Gulick, A.

    2008-01-01

    Members of the adenylate-forming family of enzymes play a role in the metabolism of halogenated aromatics and of short, medium, and long chain fatty acids, as well as in the biosynthesis of menaquinone, peptide antibiotics, and peptide siderophores. This family includes a subfamily of acyl- and aryl-CoA ligases that catalyze thioester synthesis through two half-reactions. A carboxylate substrate first reacts with ATP to form an acyl-adenylate. Subsequent to the release of the product PPi, the enzyme binds CoA, which attacks the activated acyl group to displace AMP. Structural and functional studies on different family members suggest that these enzymes alternate between two conformations during catalysis of the two half-reactions. Specifically, after the initial adenylation step, the C-terminal domain rotates by 140 to adopt a second conformation for thioester formation. Previously, we determined the structure of 4-chlorobenzoate:CoA ligase (CBL) in the adenylate forming conformation bound to 4-chlorobenzoate. We have determined two new crystal structures. We have determined the structure of CBL in the original adenylate-forming conformation, bound to the adenylate intermediate. Additionally, we have used a novel product analogue, 4-chlorophenacyl-CoA, to trap the enzyme in the thioester-forming conformation and determined this structure in a new crystal form. This work identifies a novel binding pocket for the CoA nucleotide. The structures presented herein provide the foundation for biochemical analyses presented in the accompanying manuscript in this issue. The complete characterization of this enzyme allows us to provide an explanation for the use of the domain alternation strategy by these enzymes.

  3. A Hybrid Non-Ribosomal Peptide/Polyketide Synthetase Containing Fatty-Acyl Ligase (FAAL) Synthesizes the β-Amino Fatty Acid Lipopeptides Puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum

    PubMed Central

    Mareš, Jan; Hájek, Jan; Urajová, Petra; Kopecký, Jiří; Hrouzek, Pavel

    2014-01-01

    A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a fatty acid residue via fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-amino-2-hydroxy-4-methyl dodecanoic acid (4-methyl-Ahdoa) or 3-amino-2-hydroxy-4-methyl tetradecanoic acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the fatty acyl-AMP ligase and one of the amino acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-amino fatty acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides. PMID:25369527

  4. Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

    SciTech Connect

    Z Zhang; R Zhou; J Sauder; P Tonge; S Burley; S Swaminathan

    2011-12-31

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  5. Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

    SciTech Connect

    Zhang, Z.; Swaminathan, S.; Zhou, R.; Sauder, J. M.; Tonge, P. J.; Burley, S. K.

    2011-02-18

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  6. Discovery and characterization of a thermostable bacteriophage RNA ligase homologous to T4 RNA ligase 1.

    PubMed

    Blondal, Thorarinn; Hjorleifsdottir, Sigridur H; Fridjonsson, Olafur F; Aevarsson, Arnthor; Skirnisdottir, Sigurlaug; Hermannsdottir, Anna Gudny; Hreggvidsson, Gudmundur O; Smith, Albert Vernon; Kristjansson, Jakob K

    2003-12-15

    Thermophilic viruses represent a novel source of genetic material and enzymes with great potential for use in biotechnology. We have isolated a number of thermophilic viruses from geothermal areas in Iceland, and by combining high throughput genome sequencing and state of the art bioinformatics we have identified a number of genes with potential use in biotechnology. We have also demonstrated the existence of thermostable counterparts of previously known bacteriophage enzymes. Here we describe a thermostable RNA ligase 1 from the thermophilic bacteriophage RM378 that infects the thermophilic eubacterium Rhodothermus marinus. The RM378 RNA ligase 1 has a temperature optimum of 60-64 degrees C and it ligates both RNA and single-stranded DNA. Its thermostability and ability to work under conditions of high temperature where nucleic acid secondary structures are removed makes it an ideal enzyme for RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), and other RNA and DNA ligation applications.

  7. Four Isoforms of Arabidopsis 4-Coumarate:CoA Ligase Have Overlapping yet Distinct Roles in Phenylpropanoid Metabolism1[OPEN

    PubMed Central

    Kim, Jeong Im

    2015-01-01

    The biosynthesis of lignin, flavonoids, and hydroxycinnamoyl esters share the first three enzymatic steps of the phenylpropanoid pathway. The last shared step is catalyzed by 4-coumarate:CoA ligase (4CL), which generates p-coumaroyl CoA and caffeoyl CoA from their respective acids. Four isoforms of 4CL have been identified in Arabidopsis (Arabidopsis thaliana). Phylogenetic analysis reveals that 4CL1, 4CL2, and 4CL4 are more closely related to each other than to 4CL3, suggesting that the two groups may serve different biological functions. Promoter-GUS analysis shows that 4CL1 and 4CL2 are expressed in lignifying cells. In contrast, 4CL3 is expressed in a broad range of cell types, and 4CL3 has acquired a distinct role in flavonoid metabolism. Sinapoylmalate, the major hydroxycinnamoyl ester found in Arabidopsis, is greatly reduced in the 4cl1 4cl3 mutant, showing that 4CL1 and 4CL3 function redundantly in its biosynthesis. 4CL1 accounts for the majority of the total 4CL activity, and loss of 4CL1 leads to reduction in lignin content but no growth defect. The 4cl1 4cl2 and 4cl1 4cl2 4cl3 mutants are both dwarf but do not have further reduced lignin than the 4cl1 mutant, indicating that either 4CL1 or 4CL2 is required for normal plant growth. Although 4CL4 has a limited expression profile, it does make a modest contribution to lignin biosynthesis. Together, these data show that the four isoforms of 4CL in Arabidopsis have overlapping yet distinct roles in phenylpropanoid metabolism. PMID:26491147

  8. Genetics Home Reference: succinyl-CoA:3-ketoacid CoA transferase deficiency

    MedlinePlus

    ... CoA:3-ketoacid CoA transferase deficiency succinyl-CoA:3-ketoacid CoA transferase deficiency Enable Javascript to view ... PDF Open All Close All Description Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is an inherited ...

  9. Structural basis for a bispecific NADP+ and CoA binding site in an archaeal malonyl-coenzyme A reductase.

    PubMed

    Demmer, Ulrike; Warkentin, Eberhard; Srivastava, Ankita; Kockelkorn, Daniel; Pötter, Markus; Marx, Achim; Fuchs, Georg; Ermler, Ulrich

    2013-03-01

    Autotrophic members of the Sulfolobales (crenarchaeota) use the 3-hydroxypropionate/4-hydroxybutyrate cycle to assimilate CO2 into cell material. The product of the initial acetyl-CoA carboxylation with CO2, malonyl-CoA, is further reduced to malonic semialdehyde by an NADPH-dependent malonyl-CoA reductase (MCR); the enzyme also catalyzes the reduction of succinyl-CoA to succinic semialdehyde onwards in the cycle. Here, we present the crystal structure of Sulfolobus tokodaii malonyl-CoA reductase in the substrate-free state and in complex with NADP(+) and CoA. Structural analysis revealed an unexpected reaction cycle in which NADP(+) and CoA successively occupy identical binding sites. Both coenzymes are pressed into an S-shaped, nearly superimposable structure imposed by a fixed and preformed binding site. The template-governed cofactor shaping implicates the same binding site for the 3'- and 2'-ribose phosphate group of CoA and NADP(+), respectively, but a different one for the common ADP part: the β-phosphate of CoA aligns with the α-phosphate of NADP(+). Evolution from an NADP(+) to a bispecific NADP(+) and CoA binding site involves many amino acid exchanges within a complex process by which constraints of the CoA structure also influence NADP(+) binding. Based on the paralogous aspartate-β-semialdehyde dehydrogenase structurally characterized with a covalent Cys-aspartyl adduct, a malonyl/succinyl group can be reliably modeled into MCR and discussed regarding its binding mode, the malonyl/succinyl specificity, and the catalyzed reaction. The modified polypeptide surrounding around the absent ammonium group in malonate/succinate compared with aspartate provides the structural basis for engineering a methylmalonyl-CoA reductase applied for biotechnical polyester building block synthesis.

  10. Analysis of five rice 4-coumarate:coenzyme A ligase enzyme activity and stress response for potential roles in lignin and flavonoid biosynthesis in rice

    SciTech Connect

    Sun, Haiyan; Li, Ying; Feng, Shengqiu; Zou, Weihua; Guo, Kai; Fan, Chunfen; Si, Shengli; and others

    2013-01-18

    Highlights: ► 4CLs play important roles in both lignin and flavonoids biosynthesis. ► PA and FA are the two main substrates of 4CL (Os4CL1/3/4/5) for lignin biosynthesis. ► Os4CL2 is suggested for flavonoid formation in defense against UV radiation. -- Abstract: 4-Coumarate:coenzyme A ligase (4CL) catalyzes the conversion of hydroxycinnamates into corresponding CoA esters for biosynthesis of flavonoids and lignin. In this study, five members of the 4CL gene family from rice were cloned and analyzed. Recombinant 4CL data revealed that 4-coumaric acid and ferulic acid were the two main substrates of 4CL (Os4CL1/3/4/5) for monolignol biosynthesis in rice. Os4CL2 was specifically expressed in the anther and was strongly activated by UV irradiation, suggesting its potential involvement in flavonoid formation. Moreover, bioinformatics analysis showed that the existence of valine residue at the substrate-binding pocket may mainly affect rice 4CL activities toward sinapic acid.

  11. Coagglutination (COA) test for the rapid diagnosis of cryptococcal meningitis.

    PubMed

    Koshi, G; Anandi, V; Shastry, J C; Cheriyan, A M; Abraham, J

    1989-07-01

    Cryptococcus coagglutination (COA) test reagent was prepared locally and showed no cross reactions with different species of bacteria or yeasts or with 75 control sera including 25 that gave positive results for RA factor. We used the COA test to detect cryptococcus antigen in the CSF and we could confirm the diagnosis of 11 out of 115 suspected cases of fungal meningitis; the titre varied from 4 to 128. A four-fold rise in titre confirmed the diagnostic value and a steady fall in titre in three patients on therapy indicated the prognostic value of the test. The earliest confirmation was in a renal transplant patient on the eighth day after onset of symptoms. The COA test was negative with the CSF of 118 patients with chronic meningitis. Cryptococcal colony forming units (cfu) in CSF varied from 100 to greater than 100,000/ml and correlated well with microscopy and with the COA antigen titre in CSF. Four out of the 11 patients who had cryptococcaemia, had 50,000-100,000 cfu/ml in the CSF. Cryptococcus antigen was detected by COA in the serum of all 11 patients, even in those with only 100 cfu/ml in CSF. In the three post-renal transplant patients, who were being monitored regularly, the diagnosis was made early and all three recovered on antifungal therapy with no relapse to date (1-2 years). All the others, including the two primary CNS infections, succumbed to the disease because they presented late for diagnosis and therapy. The cryptococcus COA test is a simple and specific test that can be used as a rapid test to confirm early diagnosis and permit prompt therapy, which should improve the prognosis in CNS and other forms of systemic cryptococcosis. Moreover, it is reproducible and cost-effective, particularly in countries where the latex and other expensive test reagents are not generally available. PMID:2664182

  12. Materials and methods for the alteration of enzyme and acetyl CoA levels in plants

    DOEpatents

    Nikolau, Basil J.; Wurtele, Eve S.; Oliver, David J.; Schnable, Patrick S.; Wen, Tsui-Jung

    2009-04-28

    The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.alpha. subunit of pPDH, the E1.beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyruvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.alpha. pPDH, E1.beta. pPDH, E2 pPDH, mtPDH or ALDH.

  13. Materials and methods for the alteration of enzyme and acetyl CoA levels in plants

    DOEpatents

    Nikolau, Basil J.; Wurtele, Eve S.; Oliver, David J.; Behal, Robert; Schnable, Patrick S.; Ke, Jinshan; Johnson, Jerry L.; Allred, Carolyn C.; Fatland, Beth; Lutziger, Isabelle; Wen, Tsui-Jung

    2004-07-20

    The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.sub..alpha. subunit of pPDH, the E1.sub..beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyurvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.sub..alpha. pPDH, E1.sub..beta. pPDH, E2 pPDH, mtPDH or ALDH.

  14. Materials and methods for the alteration of enzyme and acetyl CoA levels in plants

    DOEpatents

    Nikolau, Basil J.; Wurtele, Eve S.; Oliver, David J.; Behal, Robert; Schnable, Patrick S.; Ke, Jinshan; Johnson, Jerry L.; Allred, Carolyn C.; Fatland, Beth; Lutziger, Isabelle; Wen, Tsui-Jung

    2005-09-13

    The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.alpha. subunit of pPDH, the E1.beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyruvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.alpha. pPDH, E1.beta. pPDH, E2 pPDH, mtPDH or ALDH.

  15. Pseudomonas fluorescens WCS374r-Induced Systemic Resistance in Rice against Magnaporthe oryzae Is Based on Pseudobactin-Mediated Priming for a Salicylic Acid-Repressible Multifaceted Defense Response1[C][OA

    PubMed Central

    De Vleesschauwer, David; Djavaheri, Mohammad; Bakker, Peter A.H.M.; Höfte, Monica

    2008-01-01

    Selected strains of nonpathogenic rhizobacteria can reduce disease in foliar tissues through the induction of a defense state known as induced systemic resistance (ISR). Compared with the large body of information on ISR in dicotyledonous plants, little is known about the mechanisms underlying rhizobacteria-induced resistance in cereal crops. Here, we demonstrate the ability of Pseudomonas fluorescens WCS374r to trigger ISR in rice (Oryza sativa) against the leaf blast pathogen Magnaporthe oryzae. Using salicylic acid (SA)-nonaccumulating NahG rice, an ethylene-insensitive OsEIN2 antisense line, and the jasmonate-deficient mutant hebiba, we show that this WCS374r-induced resistance is regulated by an SA-independent but jasmonic acid/ethylene-modulated signal transduction pathway. Bacterial mutant analysis uncovered a pseudobactin-type siderophore as the crucial determinant responsible for ISR elicitation. Root application of WCS374r-derived pseudobactin (Psb374) primed naive leaves for accelerated expression of a pronounced multifaceted defense response, consisting of rapid recruitment of phenolic compounds at sites of pathogen entry, concerted expression of a diverse set of structural defenses, and a timely hyperinduction of hydrogen peroxide formation putatively driving cell wall fortification. Exogenous SA application alleviated this Psb374-modulated defense priming, while Psb374 pretreatment antagonized infection-induced transcription of SA-responsive PR genes, suggesting that the Psb374- and SA-modulated signaling pathways are mutually antagonistic. Interestingly, in sharp contrast to WCS374r-mediated ISR, chemical induction of blast resistance by the SA analog benzothiadiazole was independent of jasmonic acid/ethylene signaling and involved the potentiation of SA-responsive gene expression. Together, these results offer novel insights into the signaling circuitry governing induced resistance against M. oryzae and suggest that rice is endowed with multiple

  16. Genes of succinyl-CoA ligase from Saccharomyces cerevisiae.

    PubMed

    Przybyla-Zawislak, B; Dennis, R A; Zakharkin, S O; McCammon, M T

    1998-12-01

    Succinyl-CoA ligase (succinyl-CoA synthetase) catalyzes the nucleotide-dependent conversion of succinyl-CoA to succinate. This enzyme functions in the tricarboxylic acid (TCA) cycle and is also involved in ketone-body breakdown in animals. The enzyme is composed of alpha and beta subunits that are required for catalytic activity. Two genes, LSC1 (YOR142W) and LSC2 (YGR244C), with high similarity to succinyl-CoA ligase subunits from other species were isolated from Saccharomyces cerevisiae. The expression of these genes was repressed by growth on glucose and was induced threefold to sixfold during growth on nonfermentable carbon sources. The LSC genes were deleted singly and in combination. Unlike other yeast strains with defects in TCA cycle genes, strains lacking either or both LSC genes were able to grow with acetate as a carbon source. However, growth on glycerol or pyruvate was impaired. An antiserum against both subunits of the Escherichia coli enzyme was capable of recognizing the yeast succinyl-CoA ligase alpha subunit, and this band was absent in delta lsc1 deletion strains. Succinyl-CoA ligase activity was absent in mitochondria isolated from strains deleted for one or both LSC genes, but activity was restored by the presence of the appropriate LSC gene on a plasmid. The yeast succinyl-CoA ligase was shown to utilize ATP but not GTP for succinyl-CoA synthesis.

  17. SCF ubiquitin ligase targeted therapies

    PubMed Central

    Skaar, Jeffrey R.; Pagan, Julia K.; Pagano, Michele

    2015-01-01

    Summary The recent clinical successes of inhibitors of the proteasome for the treatment of cancer have highlighted the therapeutic potential of this protein degradation system. Proteasome inhibitors prevent the degradation of numerous proteins, so increased specificity could be achieved by inhibiting the components of the ubiquitin-proteasome system that target specific subsets of proteins for degradation. F-box proteins are the substrate-targeting subunits of SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complexes. Through the degradation of a plethora of diverse substrates, SCF ubiquitin ligases control a large number of processes at the cellular and organismal levels, and their misregulation is implicated in many pathologies. SCF ligases are characterized by a high specificity for their substrates, so they represent promising drug targets. However, the potential for therapeutic manipulation of SCF complexes remains an underdeveloped area. This review will explore and discuss potential strategies to target SCF-mediated biology to treat human diseases. PMID:25394868

  18. Very long-chain acyl CoA dehydrogenase deficiency which was accepted as infanticide.

    PubMed

    Eminoglu, Tuba F; Tumer, Leyla; Okur, Ilyas; Ezgu, Fatih S; Biberoglu, Gursel; Hasanoglu, Alev

    2011-07-15

    Very-long-chain acyl-coenzyme A (CoA) dehydrogenase deficiency (VLCADD) (OMIM #201475) is an autosomal recessive disorder of fatty acid oxidation. Major phenotypic expressions are hypoketotic hypoglycemia, hepatomegaly, cardiomyopathy, myopathy, rhabdomyolysis, elevated creatinine kinase, and lipid infiltration of liver and muscle. At the same time, it is a rare cause of Sudden Infant Death Syndrome (SIDS) or unexplained death in the neonatal period [1-4]. We report a patient with VLCADD whose parents were investigated for infanticide because her three previous siblings had suddenly died after normal deliveries.

  19. Global Hawk Pacific (GloPac) COA and Mission Coordination

    NASA Technical Reports Server (NTRS)

    Dillon, Mark; Hall, Philip

    2010-01-01

    This slide presentation reviews the science objectives of the Global Hawk unmanned aircraft system (UAS) in the Pacific region, shows examp le flight tracks, the satellite under-flight requirement, the flight planning, and the agencies coordination of the airspace required for the Certificate of Authorization (COA).

  20. A Chemo-Enzymatic Road Map to the Synthesis of CoA Esters.

    PubMed

    Peter, Dominik M; Vögeli, Bastian; Cortina, Niña Socorro; Erb, Tobias J

    2016-01-01

    Coenzyme A (CoA) is a ubiquitous cofactor present in every known organism. The thioesters of CoA are core intermediates in many metabolic processes, such as the citric acid cycle, fatty acid biosynthesis and secondary metabolism, including polyketide biosynthesis. Synthesis of CoA-thioesters is vital for the study of CoA-dependent enzymes and pathways, but also as standards for metabolomics studies. In this work we systematically tested five chemo-enzymatic methods for the synthesis of the three most abundant acyl-CoA thioester classes in biology; saturated acyl-CoAs, α,β-unsaturated acyl-CoAs (i.e., enoyl-CoA derivatives), and α-carboxylated acyl-CoAs (i.e., malonyl-CoA derivatives). Additionally we report on the substrate promiscuity of three newly described acyl-CoA dehydrogenases that allow the simple conversion of acyl-CoAs into enoyl-CoAs. With these five methods, we synthesized 26 different CoA-thioesters with a yield of 40% or higher. The CoA esters produced range from short- to long-chain, include branched and α,β-unsaturated representatives as well as other functional groups. Based on our results we provide a general guideline to the optimal synthesis method of a given CoA-thioester in respect to its functional group(s) and the commercial availability of the precursor molecule. The proposed synthetic routes can be performed in small scale and do not require special chemical equipment, making them convenient also for biological laboratories. PMID:27104508

  1. α-Lipoic acid protects against the oxidative stress and cytotoxicity induced by cadmium in HepG2 cells through regenerating glutathione regulated by glutamate-cysteine ligase.

    PubMed

    Xu, Yuan; Zhou, Xue; Shi, Chunli; Wang, Jiachun; Wu, Zhigang

    2015-01-01

    Alpha-lipoic acid (α-LA) is an important antioxidant that is capable of regenerating other antioxidants, such as glutathione (GSH). In the present study, we examined the protective effects of α-LA against the oxidative stress and cytotoxicity induced by cadmium in human hepatoma cell lines (HepG2) and investigated if the process was mediated through regenerating GSH. Our results showed that after exposure to 25 μM cadmium for 16 h, there was a significant decrease in the cell viability and glutathione levels and a significant increase in lipid peroxidation (p<0.01) compared with untreated cells. The presence of α-LA significantly attenuated cadmium-induced cytotoxicity and lipid peroxidation, and reversed cellular GSH levels compared with cadmium-treated cells (p<0.05). Compared with the cells treated with cadmium, co-treatment with α-LA and cadmium significantly increased the activities of γ-glutamylcysteine ligase (γ-GCL), the rate limiting enzyme in GSH biosynthesis and the mRNA and the protein levels of γ-GCL catalytic subunit (GCLC) and a modifier subunit (GCLM). In conclusion, our results indicated that α-LA is an effective agent to reduce the oxidative stress and cytotoxicity induced by cadmium by regenerating GSH levels through increasing the activities and the expressions of γ-GCL.

  2. Human DNA ligase I cDNA: Cloning and functional expression in Saccharomyces cerevisiae

    SciTech Connect

    Barnes, D.E.; Kodama, Kenichi; Tomkinson, A.E.; Lindahl, T.; Lasko, D.D. ); Johnston, L.H. )

    1990-09-01

    Human cDNA clones encoding the major DNA ligase activity in proliferating cells, DNA ligase I, were isolated by two independent methods. In one approach, a human cDNA library was screened by hybridization with oligonucleotides deduced from partial amino acid sequence of purified bovine DNA ligase I. In an alternative approach, a human cDNA library was screened for functional expression of a polypeptide able to complement a cdc9 temperature-sensitive DNA ligase mutant of Saccharomuces cerevisiae. The sequence of an apparently full-length cDNA encodes a 102-kDa protein, indistinguishable in size from authentic human DNA ligase I. The deduced amino acid sequence of the human DNA ligase I cDNA is 40% homologous to the smaller DNA ligases of S. cerevisiae and Schizosaccharomyces pombe, homology being confined to the carboxyl-terminal regions of the respective proteins. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is transcribed from a single-copy gene on chromosome 19.

  3. Using molecular beacon to monitor activity of E. coli DNA ligase.

    PubMed

    Liu, Lingfeng; Tang, Zhiwen; Wang, Kemin; Tan, Weihong; Li, Jun; Guo, Qiuping; Meng, Xiangxian; Ma, Changbei

    2005-03-01

    NAD(+)-dependent DNA ligase has been widely used in gene diagnostics for disease-associated mutation detection and has proved to be necessary for screening bactericidal drugs targeted to DNA ligases. However, further research has been restricted since conventional ligase assay technology is limited to gel electrophoresis, which is discontinuous, time-consuming and laborious. An innovative approach is developed for monitoring the activity of E. coli DNA ligase catalyzing nucleic acid ligation in the report. This approach utilizes a molecular beacon hybridized with two single-stranded DNA (ssDNA) segments to be ligated to form a hybrid with a nick, and could therefore be recognized by the enzyme. Ligation of the two ssDNA segments would cause conformation changes of the molecular beacon, leading to significant fluorescence enhancement. Compared to gel electrophoresis, this approach can provide real time information about ligase, is more time efficient, and is easier to use. The effect of quinacrine, a drug for malaria, on the activity of the ligase is detected, thereby certifying the capability of the method for developing novel antibacterial drugs targeted at NAD(+)-dependent ligase. The fidelity of strand joining by the ligase is examined based on this approach. The effects of external factors on activity of the ligase are analyzed, and then an assay of E. coli DNA ligase is performed with a broad linear range of 4.0 x 10(-4) Weiss Unit mL(-1) to 0.4 Weiss Unit mL(-1) and the detection limit of 4.0 x 10(-4) Weiss Unit mL(-1).

  4. Inherited disorders of 3-methylcrotonyl CoA carboxylation.

    PubMed

    Leonard, J V; Seakins, J W; Bartlett, K; Hyde, J; Wilson, J; Clayton, B

    1981-01-01

    The clinical course of 4 patients who had reduced activities of 3-methylcrotonyl CoA carboxylase (also called 3-methylcrotonylglycinuria) is described. Two children presented with a metabolic acidosis, one in the neonatal period and the other with episodes of acidosis that started in the second year of life. In the other 2 children neurological symptoms were prominent, one having infantile spasms and the other developmental regression with a skin rash and alopecia. Three of the children responded well to oral biotin and dietary protein restriction but the fourth, despite a biochemical response to biotin, has a severe neurological handicap. The clinical presentation of inborn errors of 3-methylcrotonyl CoA carboxylase is variable. Metabolic acidosis may not be conspicuous and instead neurological features may predominate.

  5. Metabolic biology of 3-methylglutaconic acid-uria: a new perspective

    PubMed Central

    Su, Betty; Ryan, Robert O.

    2014-01-01

    Summary Over the past twenty-five years a growing number of distinct syndromes / mutations associated with compromised mitochondrial function have been identified that share a common feature: urinary excretion of 3-methylglutaconic acid (3MGA). In the leucine degradation pathway, carboxylation of 3-methylcrotonyl CoA leads to formation of 3-methylglutaconyl CoA while 3-methylglutaconyl CoA hydratase converts this metabolite to 3-hydroxy-3-methylglutaryl CoA (HMG CoA). In “primary” 3MGA-uria, mutations in the hydratase are directly responsible for the accumulation of 3MGA. On the other hand, in all “secondary” 3MGA-urias, no defect in leucine catabolism exists and the metabolic origin of 3MGA is unknown. Herein, a path to 3MGA from mitochondrial acetyl CoA is proposed. The pathway is initiated when syndrome-associated mutations / DNA deletions result in decreased Krebs cycle flux. When this occurs, acetoacetyl CoA thiolase condenses two acetyl CoA into acetoacetyl CoA plus CoASH. Subsequently, HMG CoA synthase 2 converts acetoacetyl CoA and acetyl CoA to HMG CoA. Under syndrome-specific metabolic conditions, 3-methylglutaconyl CoA hydratase converts HMG CoA into 3-methylglutaconyl CoA in a reverse reaction of the leucine degradation pathway. This metabolite fails to proceed further up the leucine degradation pathway owing to the kinetic properties of 3-methylcrotonyl CoA carboxylase. Instead, hydrolysis of the CoA moiety of 3-methylglutaconyl CoA generates 3MGA, which appears in urine. If experimentally confirmed, this pathway provides an explanation for the occurrence of 3MGA in multiple disorders associated with compromised mitochondrial function. PMID:24407466

  6. Metabolic biology of 3-methylglutaconic acid-uria: a new perspective.

    PubMed

    Su, Betty; Ryan, Robert O

    2014-05-01

    Over the past 25 years a growing number of distinct syndromes/mutations associated with compromised mitochondrial function have been identified that share a common feature: urinary excretion of 3-methylglutaconic acid (3MGA). In the leucine degradation pathway, carboxylation of 3-methylcrotonyl CoA leads to formation of 3-methylglutaconyl CoA while 3-methylglutaconyl CoA hydratase converts this metabolite to 3-hydroxy-3-methylglutaryl CoA (HMG CoA). In "primary" 3MGA-uria, mutations in the hydratase are directly responsible for the accumulation of 3MGA. On the other hand, in all "secondary" 3MGA-urias, no defect in leucine catabolism exists and the metabolic origin of 3MGA is unknown. Herein, a path to 3MGA from mitochondrial acetyl CoA is proposed. The pathway is initiated when syndrome-associated mutations/DNA deletions result in decreased Krebs cycle flux. When this occurs, acetoacetyl CoA thiolase condenses two acetyl CoA into acetoacetyl CoA plus CoASH. Subsequently, HMG CoA synthase 2 converts acetoacetyl CoA and acetyl CoA to HMG CoA. Under syndrome-specific metabolic conditions, 3-methylglutaconyl CoA hydratase converts HMG CoA into 3-methylglutaconyl CoA in a reverse reaction of the leucine degradation pathway. This metabolite fails to proceed further up the leucine degradation pathway owing to the kinetic properties of 3-methylcrotonyl CoA carboxylase. Instead, hydrolysis of the CoA moiety of 3-methylglutaconyl CoA generates 3MGA, which appears in urine. If experimentally confirmed, this pathway provides an explanation for the occurrence of 3MGA in multiple disorders associated with compromised mitochondrial function.

  7. Synthesis and magnetic properties of superparamagnetic CoAs nanostructures

    NASA Astrophysics Data System (ADS)

    Desai, P.; Ashokaan, N.; Masud, J.; Pariti, A.; Nath, M.

    2015-03-01

    This article provides a comprehensive guide on the synthesis and characterization of superparamagnetic CoAs nanoparticles and elongated nanostructures with high blocking temperature, (TB), via hot-injection precipitation and solvothermal methods. Cobalt arsenides constitute an important family of magnetically active solids that find a variety of applications ranging from magnetic semiconductors to biomedical imaging. While the higher temperature hot-injection precipitation technique (300 °C) yields pure CoAs nanostructures, the lower temperature solvothermal method (200 °C) yields a mixture of CoAs nanoparticles along with other Co-based impurity phases. The synthesis in all these cases involved usage of triphenylarsine ((C6H5)3As) as the As precursor which reacts with solid Co2(CO)8 by ligand displacement to yield a single source precursor. The surfactant, hexadecylamine (HDA) further assists in controlling the morphology of the nanostructures. HDA also provides a basic medium and molten flux-like conditions for the redox chemistry to occur between Co and As at elevated temperatures. The influence of the length of reaction time was investigated by studying the evolution of product morphology over time. It was observed that while spontaneous nucleation at higher temperature followed by controlled growth led to the predominant formation of short nanorods, with longer reaction time, the nanorods were further converted to nanoparticles. The size of the nanoparticles obtained, was mostly in the range of 10-15 nm. The key finding of this work is exceptionally high coercivity in CoAs nanostructures for the first time. Coercivity observed was as high as 0.1 T (1000 Oe) at 2 K. These kinds of magnetic nanostructures find multiple applications in spintronics, whereas the superparamagnetic nanoparticles are viable for use in magnetic storage, ferrofluids and as contrast enhancing agents in MRI.

  8. A structural mapping of mutations causing succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency.

    PubMed

    Shafqat, Naeem; Kavanagh, Kate L; Sass, Jörn Oliver; Christensen, Ernst; Fukao, Toshiyuki; Lee, Wen Hwa; Oppermann, Udo; Yue, Wyatt W

    2013-11-01

    Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is a rare inherited metabolic disorder of ketone metabolism, characterized by ketoacidotic episodes and often permanent ketosis. To date there are ~20 disease-associated alleles on the OXCT1 gene that encodes the mitochondrial enzyme SCOT. SCOT catalyzes the first, rate-limiting step of ketone body utilization in peripheral tissues, by transferring a CoA moiety from succinyl-CoA to form acetoacetyl-CoA, for entry into the tricarboxylic acid cycle for energy production. We have determined the crystal structure of human SCOT, providing a molecular understanding of the reported mutations based on their potential structural effects. An interactive version of this manuscript (which may contain additional mutations appended after acceptance of this manuscript) may be found on the web address: http://www.thesgc.org/jimd/SCOT . PMID:23420214

  9. Acyl CoA synthetase 5 (ACSL5) ablation in mice increases energy expenditure and insulin sensitivity and delays fat absorption

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: The family of acyl-CoA synthetase enzymes (ACSL) activates fatty acids within cells to generate long chain fatty acyl CoA (FACoA). The differing metabolic fates of FACoAs such as incorporation into neutral lipids, phospholipids, and oxidation pathways are differentially regulated by the ...

  10. Bitterness in Almonds1[C][OA

    PubMed Central

    Sánchez-Pérez, Raquel; Jørgensen, Kirsten; Olsen, Carl Erik; Dicenta, Federico; Møller, Birger Lindberg

    2008-01-01

    Bitterness in almond (Prunus dulcis) is determined by the content of the cyanogenic diglucoside amygdalin. The ability to synthesize and degrade prunasin and amygdalin in the almond kernel was studied throughout the growth season using four different genotypes for bitterness. Liquid chromatography-mass spectrometry analyses showed a specific developmentally dependent accumulation of prunasin in the tegument of the bitter genotype. The prunasin level decreased concomitant with the initiation of amygdalin accumulation in the cotyledons of the bitter genotype. By administration of radiolabeled phenylalanine, the tegument was identified as a specific site of synthesis of prunasin in all four genotypes. A major difference between sweet and bitter genotypes was observed upon staining of thin sections of teguments and cotyledons for β-glucosidase activity using Fast Blue BB salt. In the sweet genotype, the inner epidermis in the tegument facing the nucellus was rich in cytoplasmic and vacuolar localized β-glucosidase activity, whereas in the bitter cultivar, the β-glucosidase activity in this cell layer was low. These combined data show that in the bitter genotype, prunasin synthesized in the tegument is transported into the cotyledon via the transfer cells and converted into amygdalin in the developing almond seed, whereas in the sweet genotype, amygdalin formation is prevented because the prunasin is degraded upon passage of the β-glucosidase-rich cell layer in the inner epidermis of the tegument. The prunasin turnover may offer a buffer supply of ammonia, aspartic acid, and asparagine enabling the plants to balance the supply of nitrogen to the developing cotyledons. PMID:18192442

  11. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    DOE PAGES

    Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; et al

    2014-10-13

    In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of themore » intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.« less

  12. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    SciTech Connect

    Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; Drennan, Catherine L.; Baker, David; Ting, Alice Y.

    2014-10-13

    In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.

  13. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    PubMed Central

    Liu, Daniel S.; Nivón, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; Drennan, Catherine L.; Baker, David; Ting, Alice Y.

    2014-01-01

    Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies. PMID:25313043

  14. Structural Basis for Specificity and Flexibility in a Plant 4-Coumarate:CoA Ligase.

    PubMed

    Li, Zhi; Nair, Satish K

    2015-11-01

    Plant 4-coumarate:CoA ligase (4CL) serves as a central catalyst in the phenylpropanoid pathway that provides precursors for numerous metabolites and regulates carbon flow. Here, we present several high-resolution crystal structures of Nicotiana tabacum 4CL isoform 2 (Nt4CL2) in complex with Mg(2+) and ATP, with AMP and coenzyme A (CoA), and with three different hydroxycinnamate-AMP intermediates: 4-coumaroyl-AMP, caffeoyl-AMP, and feruloyl-AMP. The Nt4CL2-Mg(2+)-ATP structure is captured in the adenylate-forming conformation, whereas the other structures are in the thioester-forming conformation. These structures represent a rare example of an ANL enzyme visualized in both conformations, and also reveal the binding determinants for both CoA and the hydroxycinnamate substrate. Kinetic studies of structure-based variants were used to identify residues crucial to catalysis, ATP binding, and hydroxycinnamate specificity. Lastly, we characterize a deletion mutant of Nt4CL2 that possesses the unusual sinapinate-utilizing activity. These studies establish a molecular framework for the engineering of this versatile biocatalyst. PMID:26412334

  15. Identification of a novel CoA synthase isoform, which is primarily expressed in Brain

    SciTech Connect

    Nemazanyy, Ivan . E-mail: nemazanyy@imbg.org.ua; Panasyuk, Ganna; Breus, Oksana; Zhyvoloup, Alexander; Filonenko, Valeriy; Gout, Ivan T. . E-mail: i.gout@ucl.ac.uk

    2006-03-24

    CoA and its derivatives Acetyl-CoA and Acyl-CoA are important players in cellular metabolism and signal transduction. CoA synthase is a bifunctional enzyme which mediates the final stages of CoA biosynthesis. In previous studies, we have reported molecular cloning, biochemical characterization, and subcellular localization of CoA synthase (CoASy). Here, we describe the existence of a novel CoA synthase isoform, which is the product of alternative splicing and possesses a 29aa extension at the N-terminus. We termed it CoASy {beta} and originally identified CoA synthase, CoASy {alpha}. The transcript specific for CoASy {beta} was identified by electronic screening and by RT-PCR analysis of various rat tissues. The existence of this novel isoform was further confirmed by immunoblot analysis with antibodies directed to the N-terminal peptide of CoASy {beta}. In contrast to CoASy {alpha}, which shows ubiquitous expression, CoASy {beta} is primarily expressed in Brain. Using confocal microscopy, we demonstrated that both isoforms are localized on mitochondria. The N-terminal extension does not affect the activity of CoA synthase, but possesses a proline-rich sequence which can bring the enzyme into complexes with signalling proteins containing SH3 or WW domains. The role of this novel isoform in CoA biosynthesis, especially in Brain, requires further elucidation.

  16. The Natural Mentors of Adolescent Children of Alcoholics (COAs): Implications for Preventive Practices.

    ERIC Educational Resources Information Center

    Cavell, Timothy A.; Meehan, Barbara T.; Heffer, Robert W.; Holladay, Janice J.

    2002-01-01

    Late adolescent children of alcoholics (COAs) were interviewed about their relationship with a natural mentor. Results showed that a typical mentor was a same-sex relative who had been responsible for initiating the mentor-like relationship. Differences in the reported adjustment of COAs with and without natural mentors are considered in light of…

  17. A purple acidophilic di-ferric DNA ligase from Ferroplasma.

    PubMed

    Ferrer, Manuel; Golyshina, Olga V; Beloqui, Ana; Böttger, Lars H; Andreu, José M; Polaina, Julio; De Lacey, Antonio L; Trautwein, Alfred X; Timmis, Kenneth N; Golyshin, Peter N

    2008-07-01

    We describe here an extraordinary purple-colored DNA ligase, LigFa, from the acidophilic ferrous iron-oxidizing archaeon Ferroplasma acidiphilum, a di-ferric enzyme with an extremely low pH activity optimum. Unlike any other DNA ligase studied to date, LigFa contains two Fe(3+)-tyrosinate centers and lacks any requirement for either Mg(2+) or K(+) for activity. DNA ligases from closest phylogenetic and ecophysiological relatives have normal pH optima (6.0-7.5), lack iron, and require Mg(2+)/K(+) for activity. Ferric iron retention is pH-dependent, with release resulting in partial protein unfolding and loss of activity. Reduction of the Fe(3+) to Fe(2+) results in an 80% decrease in DNA substrate binding and an increase in the pH activity optimum to 5.0. DNA binding induces significant conformational change around the iron site(s), suggesting that the ferric irons of LigFa act both as structure organizing and stabilizing elements and as Lewis acids facilitating DNA binding at low pH.

  18. DNA ligases in the repair and replication of DNA.

    PubMed

    Timson, D J; Singleton, M R; Wigley, D B

    2000-08-30

    DNA ligases are critical enzymes of DNA metabolism. The reaction they catalyse (the joining of nicked DNA) is required in DNA replication and in DNA repair pathways that require the re-synthesis of DNA. Most organisms express DNA ligases powered by ATP, but eubacteria appear to be unique in having ligases driven by NAD(+). Interestingly, despite protein sequence and biochemical differences between the two classes of ligase, the structure of the adenylation domain is remarkably similar. Higher organisms express a variety of different ligases, which appear to be targetted to specific functions. DNA ligase I is required for Okazaki fragment joining and some repair pathways; DNA ligase II appears to be a degradation product of ligase III; DNA ligase III has several isoforms, which are involved in repair and recombination and DNA ligase IV is necessary for V(D)J recombination and non-homologous end-joining. Sequence and structural analysis of DNA ligases has shown that these enzymes are built around a common catalytic core, which is likely to be similar in three-dimensional structure to that of T7-bacteriophage ligase. The differences between the various ligases are likely to be mediated by regions outside of this common core, the structures of which are not known. Therefore, the determination of these structures, along with the structures of ligases bound to substrate DNAs and partner proteins ought to be seen as a priority.

  19. Leucine: tRNA Ligase from Cultured Cells of Nicotiana tabacum var. Xanthi

    PubMed Central

    Gore, Nigel R.; Wray, John L.

    1978-01-01

    Leucine:tRNA ligase was assayed in extracts from cultured tobacco (Nicotiana tabacum) XD cells by measuring the initial rate of aminoacylation of transfer RNA with l-[4,5-3H]leucine. Transfer RNA was purified from tobacco XD cells after the method of Vanderhoef et al. (Phytochemistry 9: 2291-2304). The buoyant density of leucine:tRNA ligase from cells grown for 100 generations in 2.5 mm [15N]nitrate and 30% deuterium oxide was 1.3397. After transfer of cells into light medium (2.5 mm [14N]nitrate and 100% H2O) the ligase activity increased and the buoyant density decreased with time to 1.3174 at 72 hours after transfer. It was concluded that leucine:tRNA ligase molecules were synthesized de novo from light amino acids during the period of activity increase. The width at half-peak height of the enzyme distribution profiles following isopycnic equilibrium centrifugation in caesium chloride remained constant at all times after transfer into light medium providing evidence for the loss of preexisting functional ligase molecules. It was concluded that during the period of activity increase the cellular level of enzyme activity was determined by a balance between de novo synthesis and the loss of functional enzyme molecules due to either inactivation or degradation. PMID:16660229

  20. Exploring Peptide Ligase Orthologs in Actinobacteria-Discovery of Pseudopeptide Natural Products, Ketomemicins.

    PubMed

    Ogasawara, Yasushi; Kawata, Junpei; Noike, Motoyoshi; Satoh, Yasuharu; Furihata, Kazuo; Dairi, Tohru

    2016-06-17

    We recently identified a novel peptide ligase (PGM1), an ATP-grasp-ligase, that catalyzes amide bond formation between (S)-2-(3,5-dihydroxy-4-methoxyphenyl)-2-guanidinoacetic acid and ribosomally supplied oligopeptides in pheganomycin biosynthesis. This was the first example of an ATP-grasp-ligase utilizing peptides as nucleophiles. To explore the potential of this type of enzyme, we performed a BLAST search and identified many orthologs. The orthologs of Streptomyces mobaraensis, Salinispora tropica, and Micromonospora sp. were found in similar gene clusters consisting of six genes. To probe the functions of these genes, we heterologously expressed each of the clusters in Streptomyces lividans and detected novel and structurally similar pseudotripeptides in the broth of all transformants. Moreover, a recombinant PGM1 ortholog of Micromonospora sp. was demonstrated to be a novel dipeptide ligase catalyzing amide bond formation between amidino-arginine and dipeptides to yield tripeptides; this is the first report of a peptide ligase utilizing dipeptides as nucleophiles.

  1. Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis.

    PubMed

    Nguyen, Giang K T; Wang, Shujing; Qiu, Yibo; Hemu, Xinya; Lian, Yilong; Tam, James P

    2014-09-01

    Proteases are ubiquitous in nature, whereas naturally occurring peptide ligases, enzymes catalyzing the reverse reactions of proteases, are rare occurrences. Here we describe the discovery of butelase 1, to our knowledge the first asparagine/aspartate (Asx) peptide ligase to be reported. This highly efficient enzyme was isolated from Clitoria ternatea, a cyclic peptide-producing medicinal plant. Butelase 1 shares 71% sequence identity and the same catalytic triad with legumain proteases but does not hydrolyze the protease substrate of legumain. Instead, butelase 1 cyclizes various peptides of plant and animal origin with yields greater than 95%. With Kcat values of up to 17 s(-1) and catalytic efficiencies as high as 542,000 M(-1) s(-1), butelase 1 is the fastest peptide ligase known. Notably, butelase 1 also displays broad specificity for the N-terminal amino acids of the peptide substrate, thus providing a new tool for C terminus-specific intermolecular peptide ligations. PMID:25038786

  2. Trypanosomatidae produce acetate via a mitochondrial acetate:succinate CoA transferase.

    PubMed

    Van Hellemond, J J; Opperdoes, F R; Tielens, A G

    1998-03-17

    Hydrogenosome-containing anaerobic protists, such as the trichomonads, produce large amounts of acetate by an acetate:succinate CoA transferase (ASCT)/succinyl CoA synthetase cycle. The notion that mitochondria and hydrogenosomes may have originated from the same alpha-proteobacterial endosymbiont has led us to look for the presence of a similar metabolic pathway in trypanosomatids because these are the earliest-branching mitochondriate eukaryotes and because they also are known to produce acetate. The mechanism of acetate production in these organisms, however, has remained unknown. Four different members of the trypanosomatid family: promastigotes of Leishmania mexicana mexicana, L. infantum and Phytomonas sp., and procyclics of Trypanosoma brucei were analyzed as well as the parasitic helminth Fasciola hepatica. They all use a mitochondrial ASCT for the production of acetate from acetyl CoA. The succinyl CoA that is produced during acetate formation by ASCT is recycled presumably to succinate by a mitochondrial succinyl CoA synthetase, concomitantly producing ATP from ADP. The ASCT of L. mexicana mexicana promastigotes was further characterized after partial purification of the enzyme. It has a high affinity for acetyl CoA (Km 0.26 mM) and a low affinity for succinate (Km 6.9 mM), which shows that significant acetate production can occur only when high mitochondrial succinate concentrations prevail. This study identifies a metabolic pathway common to mitochondria and hydrogenosomes, which strongly supports a common origin for these two organelles.

  3. Purification and characterization of a Galactomyces reessii hydratase that converts 3-methylcrotonic acid to 3-hydroxy-3-methylbutyric acid.

    PubMed

    Dhar, A; Dhar, K; Rosazza, J P N

    2002-02-01

    Cell free extracts of Galactomyces reessii contain a hydratase as the key enzyme for the transformation of 3-methylcrotonic acid to 3-hydroxy-3-methylbutyric acid. Highest levels of hydratase activity were obtained during growth on isovaleric acid. The enzyme, an enoyl CoA hydratase, was purified 147-fold by precipitation with ammonium sulphate and successive chromatography over columns of DE-52, Blue Sepharose CL-6B and Sephacryl S-200. During purification, hydratase activity was measured spectrophotometrically (OD change at 263 nm) for 3-methylcrotonyl CoA and crotonyl CoA as substrates. The enzyme displayed highest activity with crotonyl CoA with a Kcat of 1,050,000 min(-1). The ratio of crotonyl CoA to 3-methylcrotonyl CoA activities was constant (20:1) during all steps of purification. The Kcat for crotonyl CoA was also about 20 times greater than the Kcat for 3-methylcrotonyl CoA (51,700 min(-1). The enzyme had pH and temperature optima at 7.0 and 35 degrees C, a native Mr of 260 +/- 4.5 kDa and a subunit Mr of 65 kDa, suggesting that the enzyme was a homotetramer. The pI of the purified hydratase was 5.5, and the N-terminal amino acid sequence was VPEGYAEDLLKGKMMRFFDS. Hydratase activity for 3-methylcrotonyl CoA was competitively inhibited by acetyl CoA, propionyl CoA and acetoacetyl CoA.

  4. Conformational transitions of cinnamoyl CoA reductase 1 from Leucaena leucocephala.

    PubMed

    Sonawane, Prashant D; Khan, Bashir M; Gaikwad, Sushama M

    2014-03-01

    Conformational transitions of cinnamoyl CoA reductase, a key regulatory enzyme in lignin biosynthesis, from Leucaena leucocephala (Ll-CCRH1) were studied using fluorescence and circular dichroism spectroscopy. The native protein possesses four trp residues exposed on the surface and 66% of helical structure, undergoes rapid structural transitions at and above 45 °C and starts forming aggregates at 55 °C. Ll-CCRH1 was transformed into acid induced (pH 2.0) molten globule like structure, exhibiting altered secondary structure, diminished tertiary structure and exposed hydrophobic residues. The molten globule like structure was examined for the thermal and chemical stability. The altered secondary structure of L1-CCRH1 at pH 2.0 was stable up to 90 °C. Also, in presence of 0.25 M guanidine hydrochloride (GdnHCl), it got transformed into different structure which was stable in the vicinity of 2M GdnHCl (as compared to drastic loss of native structure in 2M GdnHCl) as seen in far UV-CD spectra. The structural transition of Ll-CCRH1 at pH 2.0 followed another transition after readjusting the pH to 8.0, forming a structure with hardly any similarity to that of native protein. PMID:24309513

  5. A 2-oxoglutarate-dependent dioxygenase from Ruta graveolens L. exhibits p-coumaroyl CoA 2'-hydroxylase activity (C2'H): a missing step in the synthesis of umbelliferone in plants.

    PubMed

    Vialart, Guilhem; Hehn, Alain; Olry, Alexandre; Ito, Kyoko; Krieger, Celia; Larbat, Romain; Paris, Cedric; Shimizu, Bun-Ichi; Sugimoto, Yukihiro; Mizutani, Masaharu; Bourgaud, Frederic

    2012-05-01

    Coumarins are important compounds that contribute to the adaptation of plants to biotic or abiotic stresses. Among coumarins, umbelliferone occupies a pivotal position in the plant phenylpropanoid network. Previous studies indicated that umbelliferone is derived from the ortho-hydroxylation of p-coumaric acid by an unknown biochemical step to yield 2,4-dihydroxycinnamic acid, which then undergoes spontaneous lactonization. Based on a recent report of a gene encoding a 2-oxoglutarate-dependent dioxygenase from Arabidopsis thaliana that exhibited feruloyl CoA 6'-hydroxylase activity (Bourgaud et al., 2006), we combined a bioinformatic approach and a cDNA library screen to identify an orthologous ORF (Genbank accession number JF799117) from Ruta graveolens L. This ORF shares 59% amino acid identity with feruloyl CoA 6'-hydroxylase, was functionally expressed in Escherichia coli, and converted feruloyl CoA into scopoletin and p-coumaroyl CoA into umbelliferone with equal activity. Its bi-functionality was further confirmed in planta: transient expression of JF799117 in Nicotiana benthamiana yielded plants with leaves containing high levels of umbelliferone and scopoletin when compared to control plants, which contained barely detectable traces of these compounds. The expression of JF799117 was also tightly correlated to the amount of umbelliferone that was found in UV-elicited R. graveolens leaves. Therefore, JF799117 encodes a p-coumaroyl CoA 2'-hydroxylase in R. graveolens, which represents a previously uncharacterized step in the synthesis of umbelliferone in plants. Psoralen, which is an important furanocoumarin in R. graveolens, was found to be a competitive inhibitor of the enzyme, and it may exert this effect through negative feedback on the enzyme at an upstream position in the pathway.

  6. Identification and characterization of two bile acid coenzyme A transferases from Clostridium scindens, a bile acid 7α-dehydroxylating intestinal bacterium

    PubMed Central

    Ridlon, Jason M.; Hylemon, Phillip B.

    2012-01-01

    The human bile acid pool composition is composed of both primary bile acids (cholic acid and chenodeoxycholic acid) and secondary bile acids (deoxycholic acid and lithocholic acid). Secondary bile acids are formed by the 7α-dehydroxylation of primary bile acids carried out by intestinal anaerobic bacteria. We have previously described a multistep biochemical pathway in Clostridium scindens that is responsible for bile acid 7α-dehydroxylation. We have identified a large (12 kb) bile acid inducible (bai) operon in this bacterium that encodes eight genes involved in bile acid 7α-dehydroxylation. However, the function of the baiF gene product in this operon has not been elucidated. In the current study, we cloned and expressed the baiF gene in E. coli and discovered it has bile acid CoA transferase activity. In addition, we discovered a second bai operon encoding three genes. The baiK gene in this operon was expressed in E. coli and found to encode a second bile acid CoA transferase. Both bile acid CoA transferases were determined to be members of the type III family by amino acid sequence comparisons. Both bile acid CoA transferases had broad substrate specificity, except the baiK gene product, which failed to use lithocholyl-CoA as a CoA donor. Primary bile acids are ligated to CoA via an ATP-dependent mechanism during the initial steps of 7α-dehydroxylation. The bile acid CoA transferases conserve the thioester bond energy, saving the cell ATP molecules during bile acid 7α-dehydroxylation. ATP-dependent CoA ligation is likely quickly supplanted by ATP-independent CoA transfer. PMID:22021638

  7. Residues in the acetyl CoA binding site of pyruvate carboxylase involved in allosteric regulation.

    PubMed

    Choosangtong, Kamonman; Sirithanakorn, Chaiyos; Adina-Zada, Abdul; Wallace, John C; Jitrapakdee, Sarawut; Attwood, Paul V

    2015-07-22

    We have examined the roles of Asp1018, Glu1027, Arg469 and Asp471 in the allosteric domain of Rhizobium etli pyruvate carboxylase. Arg469 and Asp471 interact directly with the allosteric activator acetyl coenzyme A (acetyl CoA) and the R469S and R469K mutants showed increased enzymic activity in the presence and absence of acetyl CoA, whilst the D471A mutant exhibited no acetyl CoA-activation. E1027A, E1027R and D1018A mutants had increased activity in the absence of acetyl CoA, but not in its presence. These results suggest that most of these residues impose restrictions on the structure and/or dynamics of the enzyme to affect activity. PMID:26149215

  8. Structure of succinyl-CoA:3-ketoacid CoA transferase from Drosophila melanogaster

    PubMed Central

    Zhang, Min; Xu, Han-Yang; Wang, Yi-Cui; Shi, Zhu-Bing; Zhang, Nan-Nan

    2013-01-01

    Succinyl-CoA:3-ketoacid CoA transferase (SCOT) plays a crucial role in ketone-body metabolism. SCOT from Drosophila melanogaster (DmSCOT) was purified and crystallized. The crystal structure of DmSCOT was determined at 2.64 Å resolution and belonged to space group P212121, with unit-cell parameters a = 76.638, b = 101.921, c = 122.457 Å, α = β = γ = 90°. Sequence alignment and structural analysis identified DmSCOT as a class I CoA transferase. Compared with Acetobacter aceti succinyl-CoA:acetate CoA transferase, DmSCOT has a different substrate-binding pocket, which may explain the difference in their substrate specificities. PMID:24100554

  9. RBR E3-ligases at work.

    PubMed

    Smit, Judith J; Sixma, Titia K

    2014-02-01

    The RING-in-between-RING (RBR) E3s are a curious family of ubiquitin E3-ligases, whose mechanism of action is unusual in several ways. Their activities are auto-inhibited, causing a requirement for activation by protein-protein interactions or posttranslational modifications. They catalyse ubiquitin conjugation by a concerted RING/HECT-like mechanism in which the RING1 domain facilitates E2-discharge to directly form a thioester intermediate with a cysteine in RING2. This short-lived, HECT-like intermediate then modifies the target. Uniquely, the RBR ligase HOIP makes use of this mechanism to target the ubiquitin amino-terminus, by presenting the target ubiquitin for modification using its distinctive LDD region.

  10. Cardiac-specific deletion of acetyl CoA carboxylase 2 (ACC2) prevents metabolic remodeling during pressure-overload hypertrophy

    PubMed Central

    Kolwicz, Stephen C.; Olson, David P.; Marney, Luke C.; Garcia-Menendez, Lorena; Synovec, Robert E.; Tian, Rong

    2012-01-01

    Rationale Decreased fatty acid oxidation (FAO) with increased reliance on glucose are hallmarks of metabolic remodeling that occurs in pathological cardiac hypertrophy and is associated with decreased myocardial energetics and impaired cardiac function. To date, it has not been tested whether prevention of the metabolic switch that occurs during the development of cardiac hypertrophy has unequivocal benefits on cardiac function and energetics. Objectives Since malonyl CoA production via acetyl CoA carboxylase 2 (ACC2) inhibits mitochondrial fatty acid transport, we hypothesized that mice with a cardiac-specific deletion of ACC2 (ACC2H−/−) would maintain cardiac fatty acid oxidation (FAO) and improve function and energetics during the development of pressure-overload hypertrophy. Methods and Results ACC2 deletion led to a significant reduction in cardiac malonyl CoA levels. In isolated perfused heart experiments, left ventricular (LV) function and oxygen consumption were similiar in ACC2H−/− mice despite an ~60% increase in FAO compared to controls (CON). After 8 weeks of pressure-overload via transverse aortic constriction (TAC), ACC2H−/− mice exhibited a substrate utilization profile similar to sham animals while CON-TAC hearts had decreased FAO with increased glycolysis and anaplerosis. Myocardial energetics, assessed by 31P NMR spectroscopy, and cardiac function were maintained in ACC2H−/− after 8 weeks of TAC. Furthermore, ACC2H−/−-TAC demonstrated an attenuation of cardiac hypertrophy with a significant reduction in fibrosis relative to CON-TAC. Conclusions These data suggest that reversion to the fetal metabolic profile in chronic pathological hypertrophy is associated with impaired myocardial function and energetics and maintenance of the inherent cardiac metabolic profile and mitochondrial oxidative capacity is a viable therapeutic strategy. PMID:22730442

  11. Lys-63-linked Ubiquitination of γ-Aminobutyric Acid (GABA), Type B1, at Multiple Sites by the E3 Ligase Mind Bomb-2 Targets GABAB Receptors to Lysosomal Degradation*

    PubMed Central

    Zemoura, Khaled; Trümpler, Claudia; Benke, Dietmar

    2016-01-01

    GABAB receptors are heterodimeric G protein-coupled receptors, which control neuronal excitability by mediating prolonged inhibition. The magnitude of GABAB receptor-mediated inhibition essentially depends on the amount of receptors in the plasma membrane. However, the factors regulating cell surface expression of GABAB receptors are poorly characterized. Cell surface GABAB receptors are constitutively internalized and either recycled to the plasma membrane or degraded in lysosomes. The signal that sorts GABAB receptors to lysosomes is currently unknown. Here we show that Mind bomb-2 (MIB2)-mediated Lys-63-linked ubiquitination of the GABAB1 subunit at multiple sites is the lysosomal sorting signal for GABAB receptors. We found that inhibition of lysosomal activity in cultured rat cortical neurons increased the fraction of Lys-63-linked ubiquitinated GABAB receptors and enhanced the expression of total as well as cell surface GABAB receptors. Mutational inactivation of four putative ubiquitination sites in the GABAB1 subunit significantly diminished Lys-63-linked ubiquitination of GABAB receptors and prevented their lysosomal degradation. We identified MIB2 as the E3 ligase triggering Lys-63-linked ubiquitination and lysosomal degradation of GABAB receptors. Finally, we show that sustained activation of glutamate receptors, a condition occurring in brain ischemia that down-regulates GABAB receptors, considerably increased the expression of MIB2 and Lys-63-linked ubiquitination of GABAB receptors. Interfering with Lys-63-linked ubiquitination by overexpressing ubiquitin mutants or GABAB1 mutants deficient in Lys-63-linked ubiquitination prevented glutamate-induced down-regulation of the receptors. These findings indicate that Lys-63-linked ubiquitination of GABAB1 at multiple sites by MIB2 controls sorting of GABAB receptors to lysosomes for degradation under physiological and pathological conditions. PMID:27573246

  12. Autoactivation of the MDM2 E3 Ligase by Intramolecular Interaction

    PubMed Central

    Cheng, Qian; Song, Tanjing; Chen, Lihong

    2014-01-01

    The RING domain ubiquitin E3 ligase MDM2 is a key regulator of p53 degradation and a mediator of signals that stabilize p53. The current understanding of the mechanisms by which MDM2 posttranslational modifications and protein binding cause p53 stabilization remains incomplete. Here we present evidence that the MDM2 central acidic region is critical for activating RING domain E3 ligase activity. A 30-amino-acid minimal region of the acidic domain binds to the RING domain through intramolecular interactions and stimulates the catalytic function of the RING domain in promoting ubiquitin release from charged E2. The minimal activation sequence is also the binding site for the ARF tumor suppressor, which inhibits ubiquitination of p53. The acidic domain-RING domain intramolecular interaction is modulated by ATM-mediated phosphorylation near the RING domain or by binding of ARF. These results suggest that MDM2 phosphorylation and association with protein regulators share a mechanism in inhibiting the E3 ligase function and stabilizing p53 and suggest that targeting the MDM2 autoactivation mechanism may be useful for therapeutic modulation of p53 levels. PMID:24842904

  13. Abolition of mitochondrial substrate-level phosphorylation by itaconic acid produced by LPS-induced Irg1 expression in cells of murine macrophage lineage.

    PubMed

    Németh, Beáta; Doczi, Judit; Csete, Dániel; Kacso, Gergely; Ravasz, Dora; Adams, Daniel; Kiss, Gergely; Nagy, Adam M; Horvath, Gergo; Tretter, Laszlo; Mócsai, Attila; Csépányi-Kömi, Roland; Iordanov, Iordan; Adam-Vizi, Vera; Chinopoulos, Christos

    2016-01-01

    Itaconate is a nonamino organic acid exhibiting antimicrobial effects. It has been recently identified in cells of macrophage lineage as a product of an enzyme encoded by immunoresponsive gene 1 (Irg1), acting on the citric acid cycle intermediate cis-aconitate. In mitochondria, itaconate can be converted by succinate-coenzyme A (CoA) ligase to itaconyl-CoA at the expense of ATP (or GTP), and is also a weak competitive inhibitor of complex II. Here, we investigated specific bioenergetic effects of increased itaconate production mediated by LPS-induced stimulation of Irg1 in murine bone marrow-derived macrophages (BMDM) and RAW-264.7 cells. In rotenone-treated macrophage cells, stimulation by LPS led to impairment in substrate-level phosphorylation (SLP) of in situ mitochondria, deduced by a reversal in the directionality of the adenine nucleotide translocase operation. In RAW-264.7 cells, the LPS-induced impairment in SLP was reversed by short-interfering RNA(siRNA)-but not scrambled siRNA-treatment directed against Irg1. LPS dose-dependently inhibited oxygen consumption rates (61-91%) and elevated glycolysis rates (>21%) in BMDM but not RAW-264.7 cells, studied under various metabolic conditions. In isolated mouse liver mitochondria treated with rotenone, itaconate dose-dependently (0.5-2 mM) reversed the operation of adenine nucleotide translocase, implying impairment in SLP, an effect that was partially mimicked by malonate. However, malonate yielded greater ADP-induced depolarizations (3-19%) than itaconate. We postulate that itaconate abolishes SLP due to 1) a "CoA trap" in the form of itaconyl-CoA that negatively affects the upstream supply of succinyl-CoA from the α-ketoglutarate dehydrogenase complex; 2) depletion of ATP (or GTP), which are required for the thioesterification by succinate-CoA ligase; and 3) inhibition of complex II leading to a buildup of succinate which shifts succinate-CoA ligase equilibrium toward ATP (or GTP) utilization. Our results

  14. Structure and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases

    PubMed Central

    Riley, B.E.; Lougheed, J.C.; Callaway, K.; Velasquez, M.; Brecht, E.; Nguyen, L.; Shaler, T.; Walker, D.; Yang, Y.; Regnstrom, K.; Diep, L.; Zhang, Z.; Chiou, S.; Bova, M.; Artis, D.R.; Yao, N.; Baker, J.; Yednock, T.; Johnston, J.A.

    2013-01-01

    Parkin is a RING-between-RING E3 ligase that functions in the covalent attachment of ubiquitin to specific substrates, and mutations in Parkin are linked to Parkinson’s disease, cancer and mycobacterial infection. The RING-between-RING family of E3 ligases are suggested to function with a canonical RING domain and a catalytic cysteine residue usually restricted to HECT E3 ligases, thus termed ‘RING/HECT hybrid’ enzymes. Here we present the 1.58 Å structure of Parkin-R0RBR, revealing the fold architecture for the four RING domains, and several unpredicted interfaces. Examination of the Parkin active site suggests a catalytic network consisting of C431 and H433. In cells, mutation of C431 eliminates Parkin-catalysed degradation of mitochondria, and capture of an ubiquitin oxyester confirms C431 as Parkin’s cellular active site. Our data confirm that Parkin is a RING/HECT hybrid, and provide the first crystal structure of an RING-between-RING E3 ligase at atomic resolution, providing insight into this disease-related protein. PMID:23770887

  15. Overexpression of a Soybean Ariadne-Like Ubiquitin Ligase Gene GmARI1 Enhances Aluminum Tolerance in Arabidopsis

    PubMed Central

    Zhang, Xiaolian; Wang, Ning; Chen, Pei; Gao, Mengmeng; Liu, Juge; Wang, Yufeng; Zhao, Tuanjie; Li, Yan; Gai, Junyi

    2014-01-01

    Ariadne (ARI) subfamily of RBR (Ring Between Ring fingers) proteins have been found as a group of putative E3 ubiquitin ligases containing RING (Really Interesting New Gene) finger domains in fruitfly, mouse, human and Arabidopsis. Recent studies showed several RING-type E3 ubiquitin ligases play important roles in plant response to abiotic stresses, but the function of ARI in plants is largely unknown. In this study, an ariadne-like E3 ubiquitin ligase gene was isolated from soybean, Glycine max (L.) Merr., and designated as GmARI1. It encodes a predicted protein of 586 amino acids with a RBR supra-domain. Subcellular localization studies using Arabidopsis protoplast cells indicated GmARI protein was located in nucleus. The expression of GmARI1 in soybean roots was induced as early as 2–4 h after simulated stress treatments such as aluminum, which coincided with the fact of aluminum toxicity firstly and mainly acting on plant roots. In vitro ubiquitination assay showed GmARI1 protein has E3 ligase activity. Overexpression of GmARI1 significantly enhanced the aluminum tolerance of transgenic Arabidopsis. These findings suggest that GmARI1 encodes a RBR type E3 ligase, which may play important roles in plant tolerance to aluminum stress. PMID:25364908

  16. Chromosome demise in the wake of ligase-deficient replication

    PubMed Central

    Kouzminova, Elena A.; Kuzminov, Andrei

    2012-01-01

    Summary Bacterial DNA ligases, NAD+-dependent enzymes, are distinct from eukaryotic ATP-dependent ligases, representing promising targets for broad-spectrum antimicrobials. Yet, the chromosomal consequences of ligase-deficient DNA replication, during which Okazaki fragments accumulate, are still unclear. Using ligA251(Ts), the strongest ligase mutant of Escherichia coli, we studied ligase-deficient DNA replication by genetic and physical approaches. Here we show that replication without ligase kills after a short resistance period. We found that double-strand break repair via RecA, RecBCD, RuvABC and RecG explains the transient resistance, whereas irreparable chromosomal fragmentation explains subsequent cell death. Remarkably, death is mostly prevented by elimination of linear DNA degradation activity of ExoV, suggesting that non-allelic double-strand breaks behind replication forks precipitate DNA degradation that enlarge them into allelic double-strand gaps. Marker frequency profiling of synchronized replication reveals stalling of ligase-deficient forks with subsequent degradation of the DNA synthesized without ligase. The mechanism that converts unsealed nicks behind replication forks first into repairable double-strand breaks and then into irreparable double-strand gaps may be behind lethality of any DNA damaging treatment. PMID:22582878

  17. KF-1 Ubiquitin Ligase: An Anxiety Suppressor

    PubMed Central

    Hashimoto-Gotoh, Tamotsu; Iwabe, Naoyuki; Tsujimura, Atsushi; Takao, Keizo; Miyakawa, Tsuyoshi

    2009-01-01

    Anxiety is an instinct that may have developed to promote adaptive survival by evading unnecessary danger. However, excessive anxiety is disruptive and can be a basic disorder of other psychiatric diseases such as depression. The KF-1, a ubiquitin ligase located on the endoplasmic reticulum (ER), may prevent excessive anxiety; kf-1−/− mice exhibit selectively elevated anxiety-like behavior against light or heights. It is surmised that KF-1 degrades some target proteins, responsible for promoting anxiety, through the ER-associated degradation pathway, similar to Parkin in Parkinson's disease (PD). Parkin, another ER-ubiquitin ligase, prevents the degeneration of dopaminergic neurons by degrading the target proteins responsible for PD. Molecular phylogenetic studies have revealed that the prototype of kf-1 appeared in the very early phase of animal evolution but was lost, unlike parkin, in the lineage leading up to Drosophila. Therefore, kf-1−/− mice may be a powerful tool for elucidating the molecular mechanisms involved in emotional regulation, and for screening novel anxiolytic/antidepressant compounds. PMID:19753093

  18. Isolation of ubiquitinated substrates by tandem affinity purification of E3 ligase-polyubiquitin-binding domain fusions (ligase traps).

    PubMed

    Mark, Kevin G; Loveless, Theresa B; Toczyski, David P

    2016-02-01

    Ubiquitination is an essential protein modification that influences eukaryotic processes ranging from substrate degradation to nonproteolytic pathway alterations, including DNA repair and endocytosis. Previous attempts to analyze substrates via physical association with their respective ubiquitin ligases have had some success. However, because of the transient nature of enzyme-substrate interactions and rapid protein degradation, detection of substrates remains a challenge. Ligase trapping is an affinity purification approach in which ubiquitin ligases are fused to a polyubiquitin-binding domain, which allows the isolation of ubiquitinated substrates. Immunoprecipitation is first used to enrich for proteins that are bound to the ligase trap. Subsequently, affinity purification is used under denaturing conditions to capture proteins conjugated with hexahistidine-tagged ubiquitin. By using this protocol, ubiquitinated substrates that are specific for a given ligase can be isolated for mass spectrometry or western blot analysis. After cells have been collected, the described protocol can be completed in 2-3 d.

  19. Bisubstrate Adenylation Inhibitors of Biotin Protein Ligase from Mycobacterium tuberculosis

    PubMed Central

    Duckworth, Benjamin P.; Geders, Todd W.; Tiwari, Divya; Boshoff, Helena I.; Sibbald, Paul A.; Barry, Clifton E.; Schnappinger, Dirk; Finzel, Barry C.; Aldrich, Courtney C.

    2011-01-01

    SUMMARY The mycobacterial biotin protein ligase (MtBPL) globally regulates lipid metabolism in Mtb through the posttranslational biotinylation of acyl coenzyme A carboxylases involved in lipid biosynthesis that catalyze the first step in fatty acid biosynthesis and pyruvate coenzyme A carboxylase, a gluconeogenic enzyme vital for lipid catabolism. Here we describe the design, development and evaluation of a rationally designed bisubstrate inhibitor of MtBPL. This inhibitor displays potent sub-nanomolar enzyme inhibition and antitubercular activity against multi- and extensively drug resistant Mtb strains. We show that the inhibitor decreases in vivo protein biotinylation of key enzymes involved in fatty acid biosynthesis and that the anti-bacterial activity is MtBPL-dependent. Additionally, the gene encoding BPL was found to be essential in M. smegmatis. Finally, the X-ray co-crystal structure of inhibitor bound MtBPL was solved providing detailed insight for further structure-activity analysis. Collectively, these data suggest that MtBPL is a promising target for further antitubercular therapeutic development. PMID:22118677

  20. Characterization of Agrobacterium tumefaciens DNA ligases C and D.

    PubMed

    Zhu, Hui; Shuman, Stewart

    2007-01-01

    Agrobacterium tumefaciens encodes a single NAD+-dependent DNA ligase and six putative ATP-dependent ligases. Two of the ligases are homologs of LigD, a bacterial enzyme that catalyzes end-healing and end-sealing steps during nonhomologous end joining (NHEJ). Agrobacterium LigD1 and AtuLigD2 are composed of a central ligase domain fused to a C-terminal polymerase-like (POL) domain and an N-terminal 3'-phosphoesterase (PE) module. Both LigD proteins seal DNA nicks, albeit inefficiently. The LigD2 POL domain adds ribonucleotides or deoxyribonucleotides to a DNA primer-template, with rNTPs being the preferred substrates. The LigD1 POL domain has no detectable polymerase activity. The PE domains catalyze metal-dependent phosphodiesterase and phosphomonoesterase reactions at a primer-template with a 3'-terminal diribonucleotide to yield a primer-template with a monoribonucleotide 3'-OH end. The PE domains also have a 3'-phosphatase activity on an all-DNA primer-template that yields a 3'-OH DNA end. Agrobacterium ligases C2 and C3 are composed of a minimal ligase core domain, analogous to Mycobacterium LigC (another NHEJ ligase), and they display feeble nick-sealing activity. Ligation at DNA double-strand breaks in vitro by LigD2, LigC2 and LigC3 is stimulated by bacterial Ku, consistent with their proposed function in NHEJ.

  1. Fluorogenic DNA ligase and base excision repair enzyme assays using substrates labeled with single fluorophores.

    PubMed

    Nikiforov, Theo T; Roman, Steven

    2015-05-15

    Continuing our work on fluorogenic substrates labeled with single fluorophores for nucleic acid modifying enzymes, here we describe the development of such substrates for DNA ligases and some base excision repair enzymes. These substrates are hairpin-type synthetic DNA molecules with a single fluorophore located on a base close to the 3' ends, an arrangement that results in strong fluorescence quenching. When such substrates are subjected to an enzymatic reaction, the position of the dyes relative to that end of the molecules is altered, resulting in significant fluorescence intensity changes. The ligase substrates described here were 5' phosphorylated and either blunt-ended or carrying short, self-complementary single-stranded 5' extensions. The ligation reactions resulted in the covalent joining of the ends of the molecules, decreasing the quenching effect of the terminal bases on the dyes. To generate fluorogenic substrates for the base excision repair enzymes formamido-pyrimidine-DNA glycosylase (FPG), human 8-oxo-G DNA glycosylase/AP lyase (hOGG1), endonuclease IV (EndoIV), and apurinic/apyrimidinic endonuclease (APE1), we introduced abasic sites or a modified nucleotide, 8-oxo-dG, at such positions that their enzymatic excision would result in the release of a short fluorescent fragment. This was also accompanied by strong fluorescence increases. Overall fluorescence changes ranged from approximately 4-fold (ligase reactions) to more than 20-fold (base excision repair reactions). PMID:25728944

  2. Probing the active site of cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala.

    PubMed

    Sonawane, Prashant; Patel, Krunal; Vishwakarma, Rishi Kishore; Srivastava, Sameer; Singh, Somesh; Gaikwad, Sushama; Khan, Bashir M

    2013-09-01

    Lack of three dimensional crystal structure of cinnamoyl CoA reductase (CCR) limits its detailed active site characterization studies. Putative active site residues involved in the substrate/NADPH binding and catalysis for Leucaena leucocephala CCR (Ll-CCRH1; GenBank: DQ986907) were identified by amino acid sequence alignment and homology modeling. Putative active site residues and proximal H215 were subjected for site directed mutagenesis, and mutated enzymes were expressed, purified and assayed to confirm their functional roles. Mutagenesis of S136, Y170 and K174 showed complete loss of activity, indicating their pivotal roles in catalysis. Mutant S212G exhibited the catalytic efficiencies less than 10% of wild type, showing its indirect involvement in substrate binding or catalysis. R51G, D77G, F30V and I31N double mutants showed significant changes in Km values, specifying their roles in substrate binding. Finally, chemical modification and substrate protection studies corroborated the presence Ser, Tyr, Lys, Arg and carboxylate group at the active site of Ll-CCRH1. PMID:23688416

  3. Omega-3 fatty acid deficiency in major depressive disorder is caused by the interaction between diet and a genetically determined abnormality in phospholipid metabolism.

    PubMed

    Ross, Brian M

    2007-01-01

    Omega-3 fatty acids are a type of polyunsaturated fatty acid (PUFA). A growing body of evidence suggests that this form PUFA is a useful and well tolerated treatment for major depressive disorder, a common and serious mental illness. The efficacy of omega-3 PUFA is routinely explained as being due to a deficiency caused by inadequate dietary intake of this class of fatty acid. The hypothesis considered states that low omega-3 PUFA abundance in patients with major depressive and related disorders is due to an underlying genetically determined abnormality. The hypothesis can explain why although a specific and consistent deficit in omega-3, but not omega-6, PUFA occurs in major depressive and related disorders, the literature does not consistently support the notion that this is due to deficient dietary intake. Specifically it is hypothesized that having genetically determined low activity of fatty acid CoA ligase 4 and/or Type IV phospholipase A(2) combined with the low dietary availability of omega-3 PUFA results in reduced cellular uptake of omega-3 PUFA and constitutes a risk factor for depression. The hypothesis also has important consequences for the pharmacological treatment of depression in that it predicts that administering agents which enhance phospholipid synthesis, particularly those containing ethanolamine such as CDP-ethanolamine, should be effective antidepressants especially when co-administered with omega-3 PUFA.

  4. Alternative Okazaki Fragment Ligation Pathway by DNA Ligase III.

    PubMed

    Arakawa, Hiroshi; Iliakis, George

    2015-01-01

    Higher eukaryotes have three types of DNA ligases: DNA ligase 1 (Lig1), DNA ligase 3 (Lig3) and DNA ligase 4 (Lig4). While Lig1 and Lig4 are present in all eukaryotes from yeast to human, Lig3 appears sporadically in evolution and is uniformly present only in vertebrates. In the classical, textbook view, Lig1 catalyzes Okazaki-fragment ligation at the DNA replication fork and the ligation steps of long-patch base-excision repair (BER), homologous recombination repair (HRR) and nucleotide excision repair (NER). Lig4 is responsible for DNA ligation at DNA double strand breaks (DSBs) by the classical, DNA-PKcs-dependent pathway of non-homologous end joining (C-NHEJ). Lig3 is implicated in a short-patch base excision repair (BER) pathway, in single strand break repair in the nucleus, and in all ligation requirements of the DNA metabolism in mitochondria. In this scenario, Lig1 and Lig4 feature as the major DNA ligases serving the most essential ligation needs of the cell, while Lig3 serves in the cell nucleus only minor repair roles. Notably, recent systematic studies in the chicken B cell line, DT40, involving constitutive and conditional knockouts of all three DNA ligases individually, as well as of combinations thereof, demonstrate that the current view must be revised. Results demonstrate that Lig1 deficient cells proliferate efficiently. Even Lig1/Lig4 double knockout cells show long-term viability and proliferate actively, demonstrating that, at least in DT40, Lig3 can perform all ligation reactions of the cellular DNA metabolism as sole DNA ligase. Indeed, in the absence of Lig1, Lig3 can efficiently support semi-conservative DNA replication via an alternative Okazaki-fragment ligation pathway. In addition, Lig3 can back up NHEJ in the absence of Lig4, and can support NER and HRR in the absence of Lig1. Supporting observations are available in less elaborate genetic models in mouse cells. Collectively, these observations raise Lig3 from a niche-ligase to a

  5. Suramin inhibits cullin-RING E3 ubiquitin ligases

    PubMed Central

    Wu, Kenneth; Chong, Robert A.; Yu, Qing; Bai, Jin; Spratt, Donald E.; Ching, Kevin; Lee, Chan; Miao, Haibin; Tappin, Inger; Hurwitz, Jerard; Zheng, Ning; Shaw, Gary S.; Sun, Yi; Felsenfeld, Dan P.; Sanchez, Roberto; Zheng, Jun-nian; Pan, Zhen-Qiang

    2016-01-01

    Cullin-RING E3 ubiquitin ligases (CRL) control a myriad of biological processes by directing numerous protein substrates for proteasomal degradation. Key to CRL activity is the recruitment of the E2 ubiquitin-conjugating enzyme Cdc34 through electrostatic interactions between E3′s cullin conserved basic canyon and the acidic C terminus of the E2 enzyme. This report demonstrates that a small-molecule compound, suramin, can inhibit CRL activity by disrupting its ability to recruit Cdc34. Suramin, an antitrypansomal drug that also possesses antitumor activity, was identified here through a fluorescence-based high-throughput screen as an inhibitor of ubiquitination. Suramin was shown to target cullin 1’s conserved basic canyon and to block its binding to Cdc34. Suramin inhibits the activity of a variety of CRL complexes containing cullin 2, 3, and 4A. When introduced into cells, suramin induced accumulation of CRL substrates. These observations help develop a strategy of regulating ubiquitination by targeting an E2–E3 interface through small-molecule modulators. PMID:27001857

  6. 3-Hydroxy-3-methylglutaryl CoA lyase (HL): Mouse and human HL gene (HMGCL) cloning and detection of large gene deletions in two unrelated HL-deficient patients

    SciTech Connect

    Wang, S.P.; Robert, M.F.; Mitchell, G.A.

    1996-04-01

    3-hydroxy-3-methylglutaryl CoA lyase (HL, EC 4.1.3.4) catalyzes the cleavage of 3-hydroxy-3-methylglutaryl CoA to acetoacetic acid and acetyl CoA, the final reaction of both ketogenesis and leucine catabolism. Autosomal-recessive HL deficiency in humans results in episodes of hypoketotic hypoglycemia and coma. Using a mouse HL cDNA as a probe, we isolated a clone containing the full-length mouse HL gene that spans about 15 kb of mouse chromosome 4 and contains nine exons. The promoter region of the mouse HL gene contains elements characteristic of a housekeeping gene: a CpG island containing multiple Sp1 binding sites surrounds exon 1, and neither a TATA nor a CAAT box are present. We identified multiple transcription start sites in the mouse HL gene, 35 to 9 bases upstream of the translation start codon. We also isolated two human HL genomic clones that include HL exons 2 to 9 within 18 kb. The mouse and human HL genes (HGMW-approved symbol HMGCL) are highly homologous, with identical locations of intron-exon junctions. By genomic Southern blot analysis and exonic PCR, was found 2 of 33 HL-deficient probands to be homozygous for large deletions in the HL gene. 26 refs., 4 figs., 2 tabs.

  7. The Structure of LiuC, a 3-Hydroxy-3-Methylglutaconyl CoA Dehydratase Involved in Isovaleryl-CoA Biosynthesis in Myxococcus xanthus, Reveals Insights into Specificity and Catalysis.

    PubMed

    Bock, Tobias; Reichelt, Joachim; Müller, Rolf; Blankenfeldt, Wulf

    2016-09-01

    Myxobacteria are able to produce the important metabolite isovaleryl coenzyme A by a route other than leucine degradation. The first step into this pathway is mediated by LiuC, a member of the 3-methylglutaconyl CoA hydratases (MGCH). Here we present crystal structures refined to 2.05 and 1.1 Å of LiuC in the apo form and bound to coenzyme A, respectively. By using simulated annealing we modeled the enzyme substrate complex and identified residues potentially involved in substrate binding, specificity, and catalysis. The dehydration of 3-hydroxy-3-methylglutaconyl CoA to 3-methylglutaconyl CoA catalyzed by LiuC involves Glu112 and Glu132 and likely employs the typical crotonase acid-base mechanism. In this, Tyr231 and Arg69 are key players in positioning the substrate to enable catalysis. Surprisingly, LiuC shows higher sequence and structural similarity to human MGCH than to bacterial forms, although they convert the same substrate. This study provides structural insights into the alternative isovaleryl coenzyme A biosynthesis pathway and might open a path for biofuel research, as isovaleryl-CoA is a source for isobutene, a precursor for renewable fuels and chemicals. PMID:27271456

  8. Dysregulation of ubiquitin ligases in cancer

    PubMed Central

    Ronai, Ze’ev A.

    2015-01-01

    Ubiquitin ligases are critical components of the ubiquitin proteasome system (UPS), which governs fundamental processes regulating normal cellular homeostasis, metabolism, and cell cycle in response to external stress signals and DNA damage. Among multiple steps of the UPS system required to regulate protein ubiquitination and stability, UBLs define specificity, as they recognize and interact with substrates in a temporally- and spatially-regulated manner. Such interactions are required for substrate modification by ubiquitin chains, which marks proteins for recognition and degradation by the proteasome, or alters their subcellular localization or assembly into functional complexes. UBLs are often deregulated in cancer, altering substrate availability or activity in a manner that can promote cellular transformation. Such deregulation can occur at the epigenetic, genomic, or post-translational levels. Alterations in UBL can be used to predict their contributions, affecting tumor suppressors or oncogenes in select tumors. Better understanding of mechanisms underlying UBL expression and activities is expected to drive the development of next generation modulators that can serve as novel therapeutic modalities. This review summarizes our current understanding of UBL deregulation in cancer and highlights novel opportunities for therapeutic interventions. PMID:26690337

  9. Structural basis for the design of potent and species-specific inhibitors of 3-hydroxy-3-methylglutaryl CoA synthases.

    PubMed

    Pojer, Florence; Ferrer, Jean-Luc; Richard, Stéphane B; Nagegowda, Dinesh A; Chye, Mee-Len; Bach, Thomas J; Noel, Joseph P

    2006-08-01

    3-Hydroxy-3-methylglutaryl CoA synthase (HMGS) catalyzes the first committed step in the mevalonate metabolic pathway for isoprenoid biosynthesis and serves as an alternative target for cholesterol-lowering and antibiotic drugs. We have determined a previously undescribed crystal structure of a eukaryotic HMGS bound covalently to a potent and specific inhibitor F-244 [(E,E)-11-[3-(hydroxymethyl)-4-oxo-2-oxytanyl]-3,5,7-trimethyl-2,4-undecadienenoic acid]. Given the accessibility of synthetic analogs of the F-244 natural product, this inhibited eukaryotic HMGS structure serves as a necessary starting point for structure-based methods that may improve the potency and species-specific selectivity of the next generation of F-244 analogs designed to target particular eukaryotic and prokaryotic HMGS.

  10. Structural Basis for the Design of Potent and Species-specific Inhibitors of 3-hydroxy-3-methylglutaryl CoA Synthases

    SciTech Connect

    Pojer,F.; Ferrer, J.; Richard, S.; Nagegowda, D.; Chye, M.; Bach, T.; Noel, J.

    2006-01-01

    3-Hydroxy-3-methylglutaryl CoA synthase (HMGS) catalyzes the first committed step in the mevalonate metabolic pathway for isoprenoid biosynthesis and serves as an alternative target for cholesterol-lowering and antibiotic drugs. We have determined a previously undescribed crystal structure of a eukaryotic HMGS bound covalently to a potent and specific inhibitor F-244 [(E,E)-11-[3-(hydroxymethyl)-4-oxo-2-oxytanyl]-3,5,7-trimethyl-2,4-undecadienenoic acid]. Given the accessibility of synthetic analogs of the F-244 natural product, this inhibited eukaryotic HMGS structure serves as a necessary starting point for structure-based methods that may improve the potency and species-specific selectivity of the next generation of F-244 analogs designed to target particular eukaryotic and prokaryotic HMGS.

  11. Substrates of IAP ubiquitin ligases identified with a designed orthogonal E3 ligase, the NEDDylator

    PubMed Central

    Zhuang, Min; Guan, Shenheng; Wang, Haopeng; Burlingame, Alma L.; Wells, James A.

    2012-01-01

    SUMMARY Inhibitors of Apoptosis Proteins (IAPs) are guardian ubiquitin ligases that keep classic pro-apoptotic proteins in check. Systematic identification of additional IAP substrates is challenged by the heterogeneity and sheer number of ubiquitinated proteins (>5000). Here we report a powerful catalytic tagging tool, the NEDDylator, which fuses a NEDD8 E2 conjugating enzyme, Ubc12, to the ubiquitin ligase, XIAP or cIAP1. This permits transfer of the rare ubiquitin homolog NEDD8 to the ubiquitin E3 substrates allowing them to be efficiently purified for LC/MS/MS identification. We have identified >50 potential IAP substrates of both cytosolic and mitochondrial origin that bear hallmark N-terminal IAP binding motifs. These substrates include the recently discovered protein phosphatase, PGAM5, which we show is proteolytically processed, accumulates in cytosol during apoptosis, and sensitizes cells to death. These studies reveal mechanisms and antagonistic partners for specific IAPs, and provide a powerful technology for labeling binding partners in transient protein-protein complexes. PMID:23201124

  12. Characterization of Agrobacterium tumefaciens DNA ligases C and D

    PubMed Central

    Zhu, Hui; Shuman, Stewart

    2007-01-01

    Agrobacterium tumefaciens encodes a single NAD+-dependent DNA ligase and six putative ATP-dependent ligases. Two of the ligases are homologs of LigD, a bacterial enzyme that catalyzes end-healing and end-sealing steps during nonhomologous end joining (NHEJ). Agrobacterium LigD1 and AtuLigD2 are composed of a central ligase domain fused to a C-terminal polymerase-like (POL) domain and an N-terminal 3′-phosphoesterase (PE) module. Both LigD proteins seal DNA nicks, albeit inefficiently. The LigD2 POL domain adds ribonucleotides or deoxyribonucleotides to a DNA primer-template, with rNTPs being the preferred substrates. The LigD1 POL domain has no detectable polymerase activity. The PE domains catalyze metal-dependent phosphodiesterase and phosphomonoesterase reactions at a primer-template with a 3′-terminal diribonucleotide to yield a primer-template with a monoribonucleotide 3′-OH end. The PE domains also have a 3′-phosphatase activity on an all-DNA primer-template that yields a 3′-OH DNA end. Agrobacterium ligases C2 and C3 are composed of a minimal ligase core domain, analogous to Mycobacterium LigC (another NHEJ ligase), and they display feeble nick-sealing activity. Ligation at DNA double-strand breaks in vitro by LigD2, LigC2 and LigC3 is stimulated by bacterial Ku, consistent with their proposed function in NHEJ. PMID:17488851

  13. [Cloning and tissue expression of 4-coumarate coenzyme A ligase gene in Angelica sinensis].

    PubMed

    Wen, Sui-chao; Wang, Yin-quan; Luo, Jun; Xia, Qi; Fan, Qin; Li, Shu-nan; Wang, Zhen-heng

    2015-12-01

    4-coumarate coenzyme A ligase is a key enzyme of phenylpropanoid metabolic pathway in higher plant and may regulate the biosynthesis of ferulic acid in Angelica sinensis. In this study, the homology-based cloning and rapid amplification of cDNA ends (RACE) technique were used to clone a full length cDNA encoding 4-coumarate coenzyme A ligase gene (4CL), and then qRT-PCR was taken for analyzing 4CL gene expression levels in the root, stem and root tissue at different growth stages of seedlings of A. sinensis. The results showed that a full-length 4CL cDNA (1,815 bp) was obtained (GenBank accession number: KT880508) which shares an open reading frame (ORF) of 1 632 bp, encodes 544 amino acid polypeptides. We found 4CL gene was expressed in all tissues including leaf, stem and root of seedlings of A. sinensis. The expressions in the leave and stem were increased significantly with the growth of seedlings of A. sinensis (P < 0.05), while it in the root showed little change. It indicates a time-space pattern of 4CL gene expression in seedlings of A. sinensis. These findings will be useful for establishing an experiment basis for studying the structure and function of 4CL gene and elucidating mechanism of ferulic acid biosynthesis and space-time regulation in A. sinensis. PMID:27245029

  14. Flexible DAQ card for detector systems utilizing the CoaXPress communication standard

    NASA Astrophysics Data System (ADS)

    Neue, G.; Hejtmánek, M.; Marčišovský, M.; Voleš, P.

    2015-04-01

    This work concerns the design and construction of a flexible FPGA based data acquisition system aimed for particle detectors. The interface card as presented was designed for large area detectors with millions of individual readout channels. Flexibility was achieved by partitioning the design into multiple PCBs, creating a set of modular blocks, allowing the creation of a wide variety of configurations by simply stacking functional PCBs together. This way the user can easily toggle the polarity of the high voltage bias supply or switch the downstream interface from CoaXPress to PCIe or stream directly HDMI. We addressed the issues of data throughput, data buffering, bias voltage generation, trigger timing and fine tuning of the whole readout chain enabling a smooth data transmission. On the current prototype, we have wire-bonded a MediPix2 MXR quad and connected it to a XILINX FPGA. For the downstream interface, we implemented the CoaXPress communication protocol, which enables us to stream data at 3.125 Gbps to a standard PC.

  15. Mapping L1 Ligase ribozyme conformational switch

    PubMed Central

    Giambaşu, George M.; Lee, Tai-Sung; Scott, William G.; York, Darrin M.

    2012-01-01

    L1 Ligase (L1L)molecular switch is an in vitro optimized synthetic allosteric ribozyme that catalyzes the regioselective formation of a 5’-to-3’ phosphodiester bond, a reaction for which there is no known naturally occurring RNA catalyst. L1L serves as a proof of principle that RNA can catalyze a critical reaction for prebiotic RNA self-replication according to the RNA World hypothesis. L1L crystal structure captures two distinct conformations that differ by a re-orientation of one of the stems by around 80 Å and are presumed to correspond to the active and inactive state, respectively. It is of great interest to understand the nature of these two states in solution, and the pathway for their interconversion. In this study, we use explicit solvent molecular simulation together with a novel enhanced sampling method that utilizes concepts from network theory to map out the conformational transition between active and inactive states of L1L. We find that the overall switching mechanism can be described as a 3-state/2-step process. The first step involves a large-amplitude swing that re-orients stem C. The second step involves the allosteric activation of the catalytic site through distant contacts with stem C. Using a conformational space network representation of the L1L switch transition, it is shown that the connection between the three states follows different topographical patterns: the stem C swing step passes through a narrow region of the conformational space network, whereas the allosteric activation step covers a much wider region and a more diverse set of pathways through the network. PMID:22771572

  16. Association between the enterotoxin production and presence of Coa, Nuc genes among Staphylococcus aureus isolated from various sources, in Shiraz.

    PubMed

    Moghassem Hamidi, R; Hosseinzadeh, S; Shekarforoush, S S; Poormontaseri, M; Derakhshandeh, A

    2015-01-01

    The present study was aimed to identify the frequency of coagulase (Coa) and thermonuclease (Nuc) genes and Staphylococcal enterotoxin A (Sea) production among Staphylococcus aureus isolated from various sources in Shiraz. Moreover, the correlation between the Sea gene and coagulase and thermonuclease enzymes is also considered. A total of 100 S. aureus were isolated from various sources including 40 humans, 30 animals and 30 food samples by the routine biochemical tests. The frequency of Coa, Nuc and Sea genes was evaluated by PCR assay. Correlation among those genes was finally evaluated by statistical analysis. The PCR results showed that the prevalence of Coa, Nuc and Sea genes was 91%, 100% and 14%, respectively. The evaluation of the enterotoxin production indicated that 78.6% of the Sea gene was expressed. The presence of enterotoxin A was not necessarily correlated to the production of toxin. As a final conclusion to detect the enterotoxigenic strains, both genotypic and phenotypic methods are highly recommended.

  17. Purification, gene cloning, and characterization of γ-butyrobetainyl CoA synthetase from Agrobacterium sp. 525a.

    PubMed

    Fujimitsu, Hiroshi; Matsumoto, Akira; Takubo, Sayaka; Fukui, Akiko; Okada, Kazuma; Mohamed Ahmed, Isam A; Arima, Jiro; Mori, Nobuhiro

    2016-08-01

    The report is the first of purification, overproduction, and characterization of a unique γ-butyrobetainyl CoA synthetase from soil-isolated Agrobacterium sp. 525a. The primary structure of the enzyme shares 70-95% identity with those of ATP-dependent microbial acyl-CoA synthetases of the Rhizobiaceae family. As distinctive characteristics of the enzyme of this study, ADP was released in the catalytic reaction process, whereas many acyl CoA synthetases are annotated as an AMP-forming enzyme. The apparent Km values for γ-butyrobetaine, CoA, and ATP were, respectively, 0.69, 0.02, and 0.24 mM. PMID:27125317

  18. Association between the enterotoxin production and presence of Coa, Nuc genes among Staphylococcus aureus isolated from various sources, in Shiraz

    PubMed Central

    Moghassem Hamidi, R; Hosseinzadeh, S; Shekarforoush, S. S.; Poormontaseri, M; Derakhshandeh, A

    2015-01-01

    The present study was aimed to identify the frequency of coagulase (Coa) and thermonuclease (Nuc) genes and Staphylococcal enterotoxin A (Sea) production among Staphylococcus aureus isolated from various sources in Shiraz. Moreover, the correlation between the Sea gene and coagulase and thermonuclease enzymes is also considered. A total of 100 S. aureus were isolated from various sources including 40 humans, 30 animals and 30 food samples by the routine biochemical tests. The frequency of Coa, Nuc and Sea genes was evaluated by PCR assay. Correlation among those genes was finally evaluated by statistical analysis. The PCR results showed that the prevalence of Coa, Nuc and Sea genes was 91%, 100% and 14%, respectively. The evaluation of the enterotoxin production indicated that 78.6% of the Sea gene was expressed. The presence of enterotoxin A was not necessarily correlated to the production of toxin. As a final conclusion to detect the enterotoxigenic strains, both genotypic and phenotypic methods are highly recommended. PMID:27175208

  19. A key role of PGC-1α transcriptional coactivator in production of VEGF by a novel angiogenic agent COA-Cl in cultured human fibroblasts.

    PubMed

    Igarashi, Junsuke; Okamoto, Ryuji; Yamashita, Tetsuo; Hashimoto, Takeshi; Karita, Sakiko; Nakai, Kozo; Kubota, Yasuo; Takata, Maki; Yamaguchi, Fuminori; Tokuda, Masaaki; Sakakibara, Norikazu; Tsukamoto, Ikuko; Konishi, Ryoji; Hirano, Katsuya

    2016-03-01

    We previously demonstrated a potent angiogenic effect of a newly developed adenosine-like agent namedCOA-Cl.COA-Cl exerted tube forming activity in human umbilical vein endothelial cells in the presence of normal human dermal fibroblasts (NHDF). We therefore explored whether and howCOA-Cl modulates gene expression and protein secretion ofVEGF, a master regulator of angiogenesis, inNHDFRT-PCRandELISArevealed thatCOA-Cl upregulatedVEGF mRNAexpression and protein secretion inNHDFHIF1α(hypoxia-inducible factor 1α), a transcription factor, andPGC-1α(peroxisome proliferator-activated receptor-γcoactivator-1α), a transcriptional coactivator, are known to positively regulate theVEGFgene. Immunoblot andRT-PCRanalyses revealed thatCOA-Cl markedly upregulated the expression ofPGC-1αprotein andmRNACOA-Cl had no effect on the expression ofHIF1αprotein andmRNAin both hypoxia and normoxia. SilencingPGC-1αgene, but notHIF1αgene, by small interferingRNAattenuated the ability ofCOA-Cl to promoteVEGFsecretion. When an N-terminal fragment ofPGC-1αwas cotransfected with its partner transcription factorERRα(estrogen-related receptor-α) inCOS-7 cells,COA-Cl upregulated the expression of the endogenousVEGF mRNA However,COA-Cl had no effect on the expression ofVEGF, whenHIF1αwas transfected.COA-Cl inducesVEGFgene expression and protein secretion in fibroblasts. The transcriptional coactivatorPGC-1α, in concert withERRα, plays a key role in theCOA-Cl-inducedVEGFproduction.COA-Cl-induced activation ofPGC-1α-ERRα-VEGFpathway has a potential as a novel means for therapeutic angiogenesis.

  20. PEX2 is the E3 ubiquitin ligase required for pexophagy during starvation.

    PubMed

    Sargent, Graeme; van Zutphen, Tim; Shatseva, Tatiana; Zhang, Ling; Di Giovanni, Valeria; Bandsma, Robert; Kim, Peter Kijun

    2016-09-12

    Peroxisomes are metabolic organelles necessary for anabolic and catabolic lipid reactions whose numbers are highly dynamic based on the metabolic need of the cells. One mechanism to regulate peroxisome numbers is through an autophagic process called pexophagy. In mammalian cells, ubiquitination of peroxisomal membrane proteins signals pexophagy; however, the E3 ligase responsible for mediating ubiquitination is not known. Here, we report that the peroxisomal E3 ubiquitin ligase peroxin 2 (PEX2) is the causative agent for mammalian pexophagy. Expression of PEX2 leads to gross ubiquitination of peroxisomes and degradation of peroxisomes in an NBR1-dependent autophagic process. We identify PEX5 and PMP70 as substrates of PEX2 that are ubiquitinated during amino acid starvation. We also find that PEX2 expression is up-regulated during both amino acid starvation and rapamycin treatment, suggesting that the mTORC1 pathway regulates pexophagy by regulating PEX2 expression levels. Finally, we validate our findings in vivo using an animal model. PMID:27597759

  1. Generation of poly-β-hydroxybutyrate from acetate in higher plants: Detection of acetoacetyl CoA reductase- and PHB synthase- activities in rice.

    PubMed

    Tsuda, Hirohisa; Shiraki, Mari; Inoue, Eri; Saito, Terumi

    2016-08-20

    It has been reported that Poly-β-hydroxybutyrate (PHB) is generated from acetate in the rice root. However, no information is available about the biosynthetic pathway of PHB from acetate in plant cells. In the bacterium Ralstonia eutropha H16 (R. eutropha), PHB is synthesized from acetyl CoA by the consecutive reaction of three enzymes: β-ketothiolase (EC: 2.3.1.9), acetoacetyl CoA reductase (EC: 1.1.1.36) and PHB synthase (EC: 2.3.1.-). Thus, in this study, we examined whether the above three enzymatic activities were also detected in rice seedlings. The results clearly showed that the activities of the above three enzymes were all detected in rice. In particular, the PHB synthase activity was detected specifically in the sonicated particulate fractions (2000g 10min precipitate (ppt) and the 8000g 30min ppt) of rice roots and leaves. In addition to these enzyme activities, several new experimental results were obtained on PHB synthesis in higher plants: (a) (14)C-PHB generated from 2-(14)C-acetate was mainly localized in the 2000g 10min ppt and the 8000g 30min ppt of rice root. (b) Addition of acetate (0.1-10mM) to culture medium of rice seedlings did not increase the content of PHB in the rice root or leaf. (c) In addition to C3 plants, PHB was generated from acetate in a C4 plant (corn) and in a CAM plant (Bryophyllum pinnatum). d) Washing with ethylenediaminetetraacetic acid (EDTA) strongly suggested that the PHB synthesized from acetate was of plant origin and was not bacterial contamination. PMID:27372278

  2. Metabolic annotation of 2-ethylhydracrylic acid.

    PubMed

    Ryan, Robert O

    2015-08-25

    Increased levels of the organic acid, 2-ethylhydracrylic acid (2-EHA) occur in urine of subjects with impaired L(+)-isoleucine metabolism. Chiral intermediates formed during isoleucine degradation are (S) enantiomers. Blockage of (S) pathway flux drives racemization of (2S, 3S) L(+)-isoleucine and its (2S, 3R) stereoisomer, L(+)-alloisoleucine. This non-protein amino acid is metabolized to (R)-2-methylbutyryl CoA via enzymes common to branched chain amino acid degradation. Subsequently, (R) intermediates serve as alternate substrates for three valine metabolic enzymes, generating 2-EHA. Once formed, 2-EHA accumulates because it is poorly recognized by distal valine pathway enzymes. Thus, urinary 2-EHA represents a biomarker of isoleucine pathway defects. 2-EHA levels are also increased in rats exposed to the industrial solvent, ethylene glycol monomethyl ether or the neurotoxin precursor, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. In these cases, a block in (S) pathway isoleucine catabolism occurs at the level of (S)-2-methylbutyryl CoA conversion to tiglyl CoA via inhibition of electron transferring flavoprotein/ubiquinone oxidoreductase dependent reactions. Elevated urinary 2-EHA in propionyl CoA carboxylase deficiency and methylmalonic aciduria results from a buildup of distal intermediates in the (S) pathway of isoleucine degradation. In Barth syndrome and dilated cardiomyopathy with ataxia syndrome, 2-EHA is a byproduct of impeded propionyl CoA entry into the Krebs cycle.

  3. Metabolic annotation of 2-ethylhydracrylic acid

    PubMed Central

    Ryan, Robert O.

    2015-01-01

    Summary Increased levels of the organic acid, 2-ethylhydracrylic acid (2-EHA) occur in urine of subjects with impaired L(+)-isoleucine metabolism. Chiral intermediates formed during isoleucine degradation are (S) enantiomers. Blockage of (S) pathway flux drives racemization of (2S, 3S) L(+)-isoleucine and its (2S, 3R) stereoisomer, L(+)-alloisoleucine. This non-protein amino acid is metabolized to (R)-2-methylbutyryl CoA via enzymes common to branched chain amino acid degradation. Subsequently, (R) intermediates serve as alternate substrates for three valine metabolic enzymes, generating 2-EHA. Once formed, 2-EHA accumulates because it is poorly recognized by distal valine pathway enzymes. Thus, urinary 2-EHA represents a biomarker of isoleucine pathway defects. 2-EHA levels are also increased in rats exposed to the industrial solvent, ethylene glycol monomethyl ether or the neurotoxin precursor, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. In these cases, a block in (S) pathway isoleucine catabolism occurs at the level of (S)-2-methylbutyryl CoA conversion to tiglyl CoA via inhibition of electron transferring flavoprotein / ubiquinone oxidoreductase dependent reactions. Elevated urinary 2-EHA in propionyl CoA carboxylase deficiency and methylmalonic aciduria results from a buildup of distal intermediates in the (S) pathway of isoleucine degradation. In Barth syndrome and dilated cardiomyopathy with ataxia syndrome, 2-EHA is a byproduct of impeded propionyl CoA entry into the Krebs cycle. PMID:26115894

  4. Differential dependence on DNA ligase of type II restriction enzymes: a practical way toward ligase-free DNA automaton.

    PubMed

    Chen, Peng; Li, Jing; Zhao, Jian; He, Lin; Zhang, Zhizhou

    2007-02-16

    DNA computing study is a new paradigm in computer science and biological computing fields. As one of DNA computing approaches, DNA automaton is composed of the hardware, input DNA molecule and state transition molecules. By now restriction enzymes are key hardware for DNA computing automaton. It has been found that DNA computing efficiency may be independent on DNA ligases when type IIS restriction enzymes like FokI are used as hardware. In this study, we compared FokI with four other distinct enzymes HgaI, BsmFI, BbsI, and BseMII, and found their differential independence on T4 DNA ligase when performing automaton reactions. Since DNA automaton is a potential powerful tool to tackle gene relationship in genomic network scale, the feasible ligase-free DNA automaton may set an initial base to develop functional DNA automata for various DNA technology development and implications in genetics study in the near future.

  5. Structure of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase in complex with the feedback inhibitor CoA reveals only one active-site conformation

    SciTech Connect

    Wubben, T.; Mesecar, A.D.

    2014-10-02

    Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway, reversibly transferring an adenylyl group from ATP to 4'-phosphopantetheine to form dephosphocoenzyme A (dPCoA). To complement recent biochemical and structural studies on Mycobacterium tuberculosis PPAT (MtPPAT) and to provide further insight into the feedback regulation of MtPPAT by CoA, the X-ray crystal structure of the MtPPAT enzyme in complex with CoA was determined to 2.11 {angstrom} resolution. Unlike previous X-ray crystal structures of PPAT-CoA complexes from other bacteria, which showed two distinct CoA conformations bound to the active site, only one conformation of CoA is observed in the MtPPAT-CoA complex.

  6. Discovery of Tumor-Specific Irreversible Inhibitors of Stearoyl CoA Desaturase

    PubMed Central

    Theodoropoulos, Panayotis C.; Gonzales, Stephen S.; Winterton, Sarah E.; Rodriguez-Navas, Carlos; McKnight, John S.; Morlock, Lorraine K.; Hanson, Jordan M.; Cross, Bethany; Owen, Amy E.; Duan, Yingli; Moreno, Jose R.; Lemoff, Andrew; Mirzaei, Hamid; Posner, Bruce A.; Williams, Noelle S.

    2016-01-01

    A hallmark of targeted cancer therapies is selective toxicity among cancer cell lines. We evaluated results from a viability screen of over 200,000 small molecules to identify two chemical series, oxalamides and benzothiazoles, that were selectively toxic to the same four of 12 human lung cancer cell lines at low nanomolar concentrations. Sensitive cell lines expressed cytochrome P450 (CYP) 4F11, which metabolized the compounds into irreversible stearoyl CoA desaturase (SCD) inhibitors. SCD is recognized as a promising biological target in cancer and metabolic disease. However, SCD is essential to sebocytes, and accordingly SCD inhibitors cause skin toxicity. Mouse sebocytes were unable to activate the benzothiazoles or oxalamides into SCD inhibitors, providing a therapeutic window for inhibiting SCD in vivo. We thus offer a strategy to target SCD in cancer by taking advantage of high CYP expression in a subset of tumors. PMID:26829472

  7. OUTCROP-BASED HIGH RESOLUTION GAMMA-RAY CHARACTERIZATION OF ARSENIC-BEARING LITHOFACIES IN THE PERMIAN GARBER SANDSTONE AND WELLINGTON FORMATION, CENTRAL OKLAHOMA AQUIFER (COA). CLEVELAND COUNTY, OKLAHOMA

    EPA Science Inventory

    The COA supplies drinking water to a number of municipalities in central Oklahoma. Two major stratigraphic units in the COA, the Garber Sandstone and Wellington Formation, contain naturally occurring arsenic that exceeds government mandated drinking-water standards (EPA, 2001). ...

  8. Role of ubiquitin ligases in neural stem and progenitor cells.

    PubMed

    Naujokat, Cord

    2009-01-01

    Ubiquitin ligases are central components of the ubiquitin-proteasome system (UPS), the major machinery for regulated proteolysis in eukaryotic cells. Proteins essential for regulating development, differentiation, proliferation, cell cycling, apoptosis, gene transcription, and signal transduction undergo posttranslational processing via selection by ubiquitin ligases and subsequent controlled proteolysis by the 26S proteasome, the proteolytic unit of the UPS. Neural stem cells (NSCs) are self-renewing multipotent cells of the embryonic and adult mammalian central nervous system. In the last few years, NSCs have generated considerable interest because of their potential to repair neurological damage in preclinical models of stroke, spinal cord injury, and neurodegenerative disease. Recent evidence reveals a central role of ubiquitin ligases in controlling the development, survival, differentiation, and programming of neural stem and progenitor cells. Here the current knowledge of the role and function of ubiquitin ligases in neural stem and progenitor cells is reviewed and insight into an important mechanism of NSC homeostasis by regulated proteolysis is provided. PMID:19479207

  9. The Role of Ubiquitin Ligases in Cardiac Disease

    PubMed Central

    Willis, Monte S.; Bevilacqua, Ariana; Pulinilkunnil, Thomas; Kienesberger, Petra; Tannu, Manasi; Patterson, Cam

    2014-01-01

    Rigorous surveillance of protein quality control is essential for the maintenance of normal cardiac function, while the dysregulation of protein turnover is present in a diverse array of common cardiac diseases. Central to the protein quality control found in all cells is the ubiquitin proteasome system (UPS). The UPS plays a critical role in protein trafficking, cellular signaling, and most prominently, protein degradation. As ubiquitin ligases (E3s) control the specificity of the UPS, their description in the cardiomyocyte has highlighted how ubiquitin ligases are critical to the turnover and function of the sarcomere complex, responsible for the heart’s required continuous contraction. In this review, we provide an overview of the UPS, highlighting a comprehensive overview of the cardiac ubiquitin ligases identified to date. We then focus on recent studies of new cardiac ubiquitin ligases outlining their novel roles in protein turnover, cellular signaling, and the regulation of mitochondrial dynamics and receptor turnover in the pathophysiology of cardiac hypertrophy, cardiac atrophy, myocardial infarction, and heart failure. PMID:24262338

  10. Amelioration of adverse effects of valproic acid on ketogenesis and liver coenzyme A metabolism by cotreatment with pantothenate and carnitine in developing mice: possible clinical significance.

    PubMed

    Thurston, J H; Hauhart, R E

    1992-04-01

    Very young children with organic brain damage, intractable seizures, and developmental retardation are at particular risk of developing fatal hepatic dysfunction coincident with valproate therapy, especially if the children are also receiving other anticonvulsant drugs. The mechanism of valproate-associated hepatic failure in these children is unclear. There are two major theories of etiology. The first concerns the manyfold consequences of depletion of CoA due to sequestration into poorly metabolized valproyl CoA and valproyl CoA metabolites. The other theory proposes that the unsaturated valproate derivative 2-n-propyl-4-pentenoic acid and/or metabolically activated intermediates are toxic and directly cause irreversible inhibition of enzymes of beta-oxidation. The present study shows for the first time that in developing mice, when panthothenic acid and carnitine are administered with valproate, at least some of the effects of valproate are mitigated. Perhaps most importantly, the beta-hydroxybutyrate concentration in plasma and the free CoA and acetyl CoA levels in liver do not fall so low. Cotreatment with carnitine alone was without effect. Findings support the CoA depletion mechanism of valproate inhibition of beta-oxidation and other CoA- and acetyl CoA-requiring enzymic reactions and stress the role of carnitine in the regulation of CoA synthesis at the site of action of pantothenate kinase. PMID:1570210

  11. On the origin of 3-methylglutaconic acid in disorders of mitochondrial energy metabolism.

    PubMed

    Ikon, Nikita; Ryan, Robert O

    2016-09-01

    3-methylglutaconic acid (3MGA)-uria occurs in numerous inborn errors of metabolism (IEM) associated with compromised mitochondrial energy metabolism. This organic acid arises from thioester cleavage of 3-methylglutaconyl CoA (3MG CoA), an intermediate in leucine catabolism. In individuals harboring mutations in 3MG CoA hydratase (i.e., primary 3MGA-uria), dietary leucine is the source of 3MGA. In secondary 3MGA-uria, however, no leucine metabolism defects have been reported. While others have suggested 3MGA arises from aberrant isoprenoid shunting from cytosol to mitochondria, an alternative route posits that 3MG CoA arises in three steps from mitochondrial acetyl CoA. Support for this biosynthetic route in IEMs is seen by its regulated occurrence in microorganisms. The fungus, Ustilago maydis, the myxobacterium, Myxococcus xanthus and the marine cyanobacterium, Lyngbya majuscule, generate 3MG CoA (or acyl carrier protein derivative) in the biosynthesis of iron chelating siderophores, iso-odd chain fatty acids and polyketide/nonribosomal peptide products, respectively. The existence of this biosynthetic machinery in these organisms supports a model wherein, under conditions of mitochondrial dysfunction, accumulation of acetyl CoA in the inner mitochondrial space as a result of inefficient fuel utilization drives de novo synthesis of 3MG CoA. Since humans lack the downstream biosynthetic capability of the organisms mentioned above, as 3MG CoA levels rise, thioester hydrolysis yields 3MGA, which is excreted in urine as unspent fuel. Understanding the metabolic origins of 3MGA may increase its utility as a biomarker.

  12. On the origin of 3-methylglutaconic acid in disorders of mitochondrial energy metabolism.

    PubMed

    Ikon, Nikita; Ryan, Robert O

    2016-09-01

    3-methylglutaconic acid (3MGA)-uria occurs in numerous inborn errors of metabolism (IEM) associated with compromised mitochondrial energy metabolism. This organic acid arises from thioester cleavage of 3-methylglutaconyl CoA (3MG CoA), an intermediate in leucine catabolism. In individuals harboring mutations in 3MG CoA hydratase (i.e., primary 3MGA-uria), dietary leucine is the source of 3MGA. In secondary 3MGA-uria, however, no leucine metabolism defects have been reported. While others have suggested 3MGA arises from aberrant isoprenoid shunting from cytosol to mitochondria, an alternative route posits that 3MG CoA arises in three steps from mitochondrial acetyl CoA. Support for this biosynthetic route in IEMs is seen by its regulated occurrence in microorganisms. The fungus, Ustilago maydis, the myxobacterium, Myxococcus xanthus and the marine cyanobacterium, Lyngbya majuscule, generate 3MG CoA (or acyl carrier protein derivative) in the biosynthesis of iron chelating siderophores, iso-odd chain fatty acids and polyketide/nonribosomal peptide products, respectively. The existence of this biosynthetic machinery in these organisms supports a model wherein, under conditions of mitochondrial dysfunction, accumulation of acetyl CoA in the inner mitochondrial space as a result of inefficient fuel utilization drives de novo synthesis of 3MG CoA. Since humans lack the downstream biosynthetic capability of the organisms mentioned above, as 3MG CoA levels rise, thioester hydrolysis yields 3MGA, which is excreted in urine as unspent fuel. Understanding the metabolic origins of 3MGA may increase its utility as a biomarker. PMID:27091556

  13. A wild-type DNA ligase I gene is expressed in Bloom's syndrome cells

    SciTech Connect

    Petrini, J.H.J.; Huwiler, K.G.; Weaver, D.T. )

    1991-09-01

    Alteration of DNA ligase I activity is a consistent biochemical feature of Bloom's syndrome (BS) cells. DNA ligase I activity in BS cells either is reduced and abnormally thermolabile or is present in an anomalously dimeric form. To assess the role of DNA ligase function in the etiology of BS, the authors have cloned the DNA ligase I cDNA from normal human cells by a PCR strategy using degenerate oligonucleotide primers based on conserved regions of the Saccharomyces cerevisiae and Schizosaccharomyces pombe DNA ligase genes. Human DNA ligase I cDNAs from normal and BS cells complemented a S. cerevisiae DNA ligase mutation, and protein extracts prepared from S. cerevisiae transformants expressing normal and BS cDNA contained comparable levels of DNA ligase I activity. DNA sequencing and Northern blot analysis of DNA ligase I expression in two BS human fibroblast lines representing each of the two aberrant DNA ligase I molecular phenotypes demonstrated that this gene was unchanged in BS cells. Thus, another factor may be responsible for the observed reduction in DNA ligase I activity associated with this chromosomal breakage syndrome.

  14. Reversible phosphorylation controls the activity of cyclosome-associated cyclin-ubiquitin ligase.

    PubMed Central

    Lahav-Baratz, S; Sudakin, V; Ruderman, J V; Hershko, A

    1995-01-01

    Cyclin B/cdc2 is responsible both for driving cells into mitosis and for activating the ubiquitin-dependent degradation of mitotic cyclins near the end of mitosis, an event required for the completion of mitosis and entry into interphase of the next cell cycle. Previous work with cell-free extracts of rapidly dividing clam embryos has identified two specific components required for the ubiquitination of mitotic cyclins: E2-C, a cyclin-selective ubiquitin carrier protein that is constitutively active during the cell cycle, and E3-C, a cyclin-selective ubiquitin ligase that purifies as part of a approximately 1500-kDa complex, termed the cyclosome, and which is active only near the end of mitosis. Here, we have separated the cyclosome from its ultimate upstream activator, cdc2. The mitotic, active form of the cyclosome can be inactivated by incubation with a partially purified, endogenous okadaic acid-sensitive phosphatase; addition of cdc2 restores activity to the cyclosome after a lag that reproduces that seen previously in intact cells and in crude extracts. These results demonstrate that activity of cyclin-ubiquitin ligase is controlled by reversible phosphorylation of the cyclosome complex. Images Fig. 3 PMID:7568122

  15. Degradation of host ubiquitin E3 ligase Itch by human cytomegalovirus UL42.

    PubMed

    Koshizuka, Tetsuo; Tanaka, Keiichiro; Suzutani, Tatsuo

    2016-01-01

    Human cytomegalovirus (HCMV) UL42 is classified as a CMV-specific but function-unknown gene. According to its amino acid sequence, UL42 has a C-terminal hydrophobic domain predicted to be a transmembrane domain and two PPxY (PY) motifs in its N terminus, but no N-terminal signal peptide. These features resemble those of herpes simplex virus (HSV) UL56 and varicella-zoster virus ORF0. HCMV UL42 interacts with Itch, a member of the Nedd4 family of ubiquitin E3 ligases, through its PY motifs as observed in HSV UL56. HCMV UL42 was partially colocalized with the trans-Golgi network and cytoplasmic vesicles in transfected fibroblasts. Itch was colocalized with HCMV UL42 and accumulated in a fine-speckled pattern in the cytoplasm. UL42 induced the ubiquitination and degradation of Itch in HCMV-infected fibroblasts, and was partially colocalized with p62, a ubiquitin-binding protein, and CD63, a marker of lysosome and multivesicular bodies. The electrophoretic pattern of Itch was altered by infection with HCMV and the amount of Itch was increased by the deletion of UL42. Our findings suggest that the regulatory function of the Nedd4 E3 ligase family and the structural features of HCMV UL42 are conserved characteristics in herpesviruses. PMID:26555021

  16. Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site.

    PubMed

    Sayer, Christopher; Finnigan, William; Isupov, Michail N; Levisson, Mark; Kengen, Servé W M; van der Oost, John; Harmer, Nicholas J; Littlechild, Jennifer A

    2016-01-01

    A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions. PMID:27160974

  17. Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site

    PubMed Central

    Sayer, Christopher; Finnigan, William; Isupov, Michail N.; Levisson, Mark; Kengen, Servé W. M.; van der Oost, John; Harmer, Nicholas J.; Littlechild, Jennifer A.

    2016-01-01

    A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions. PMID:27160974

  18. Acetate/acetyl-CoA metabolism associated with cancer fatty acid synthesis: overview and application.

    PubMed

    Yoshii, Yukie; Furukawa, Takako; Saga, Tsuneo; Fujibayashi, Yasuhisa

    2015-01-28

    Understanding cancer-specific metabolism is important for identifying novel targets for cancer diagnosis and therapy. Induced acetate/acetyl CoA metabolism is a notable feature that is related to fatty acid synthesis supporting tumor growth. In this review, we focused on the recent findings related to cancer acetate/acetyl CoA metabolism. We also introduce [1-¹¹C]acetate positron emission tomography (PET), which is a useful tool to visualize up-regulation of acetate/acetyl CoA metabolism in cancer, and discuss the utility of [1-¹¹C]acetate PET in cancer diagnosis and its application to personalized medicine.

  19. Investigation of the Roles of Allosteric Domain Arginine, Aspartate, and Glutamate Residues of Rhizobium etli Pyruvate Carboxylase in Relation to Its Activation by Acetyl CoA.

    PubMed

    Sirithanakorn, Chaiyos; Jitrapakdee, Sarawut; Attwood, Paul V

    2016-08-01

    The mechanism of allosteric activation of pyruvate carboxylase by acetyl CoA is not fully understood. Here we have examined the roles of residues near the acetyl CoA binding site in the allosteric activation of Rhizobium etli pyruvate carboxylase using site-directed mutagenesis. Arg429 was found to be especially important for acetyl CoA binding as substitution with serine resulted in a 100-fold increase in the Ka of acetyl CoA activation and a large decrease in the cooperativity of this activation. Asp420 and Arg424, which do not make direct contact with bound acetyl CoA, were nonetheless found to affect acetyl CoA binding when mutated, probably through changed interactions with another acetyl CoA binding residue, Arg427. Thermodynamic activation parameters for the pyruvate carboxylation reaction were determined from modified Arrhenius plots and showed that acetyl CoA acts to decrease the activation free energy of the reaction by both increasing the activation entropy and decreasing the activation enthalpy. Most importantly, mutations of Asp420, Arg424, and Arg429 enhanced the activity of the enzyme in the absence of acetyl CoA. A main focus of this work was the detailed investigation of how this increase in activity occurred in the R424S mutant. This mutation decreased the activation enthalpy of the pyruvate carboxylation reaction by an amount consistent with removal of a single hydrogen bond. It is postulated that Arg424 forms a hydrogen bonding interaction with another residue that stabilizes the asymmetrical conformation of the R. etli pyruvate carboxylase tetramer, constraining its interconversion to the symmetrical conformer that is required for catalysis. PMID:27379711

  20. The Kaposi's sarcoma-associated herpesvirus K5 E3 ubiquitin ligase modulates targets by multiple molecular mechanisms.

    PubMed

    Means, Robert E; Lang, Sabine M; Jung, Jae U

    2007-06-01

    Kaposi's sarcoma-associated herpesvirus encodes two highly related membrane-associated, RING-CH-containing (MARCH) family E3 ubiquitin ligases, K3 and K5, that can down regulate a variety of cell surface proteins through enhancement of their endocytosis and degradation. In this report we present data that while K5 modulation of major histocompatibility complex class I (MHC-I) closely mirrors the mechanisms used by K3, alternative molecular pathways are utilized by this E3 ligase in the down regulation of intercellular adhesion molecule 1 (ICAM-1) and B7.2. Internalization assays demonstrate that down regulation of each target can occur through increased endocytosis from the cell surface. However, mutation of a conserved tyrosine-based endocytosis motif in K5 resulted in a protein lacking the ability to direct an increased rate of MHC-I or ICAM-1 internalization but still able to down regulate B7.2 in a ubiquitin-dependent but endocytosis-independent manner. Further, mutation of two acidic clusters abolished K5-mediated MHC-I degradation while only slightly decreasing ICAM-1 or B7.2 protein destruction. This same mutant abolished detectable ubiquitylation of all targets. These data indicate that while K5 can act as an E3 ubiquitin ligase to directly mediate cell surface molecule destruction, regulation of its targets occurs through multiple pathways, including ubiquitin-independent mechanisms.

  1. The Kaposi's Sarcoma-Associated Herpesvirus K5 E3 Ubiquitin Ligase Modulates Targets by Multiple Molecular Mechanisms▿

    PubMed Central

    Means, Robert E.; Lang, Sabine M.; Jung, Jae U.

    2007-01-01

    Kaposi's sarcoma-associated herpesvirus encodes two highly related membrane-associated, RING-CH-containing (MARCH) family E3 ubiquitin ligases, K3 and K5, that can down regulate a variety of cell surface proteins through enhancement of their endocytosis and degradation. In this report we present data that while K5 modulation of major histocompatibility complex class I (MHC-I) closely mirrors the mechanisms used by K3, alternative molecular pathways are utilized by this E3 ligase in the down regulation of intercellular adhesion molecule 1 (ICAM-1) and B7.2. Internalization assays demonstrate that down regulation of each target can occur through increased endocytosis from the cell surface. However, mutation of a conserved tyrosine-based endocytosis motif in K5 resulted in a protein lacking the ability to direct an increased rate of MHC-I or ICAM-1 internalization but still able to down regulate B7.2 in a ubiquitin-dependent but endocytosis-independent manner. Further, mutation of two acidic clusters abolished K5-mediated MHC-I degradation while only slightly decreasing ICAM-1 or B7.2 protein destruction. This same mutant abolished detectable ubiquitylation of all targets. These data indicate that while K5 can act as an E3 ubiquitin ligase to directly mediate cell surface molecule destruction, regulation of its targets occurs through multiple pathways, including ubiquitin-independent mechanisms. PMID:17409151

  2. Molecular cloning and analysis of Ancylostoma ceylanicum glutamate-cysteine ligase.

    PubMed

    Wiśniewski, Marcin; Lapiński, Maciej; Zdziarska, Anna; Długosz, Ewa; Bąska, Piotr

    2014-08-01

    Glutamate-cysteine ligase (GCL) is a heterodimer enzyme composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM). This enzyme catalyses the synthesis of γ-glutamylcysteine, a precursor of glutathione. cDNAs of the putative glutamate-cysteine ligase catalytic (Ace-GCLC) and modifier subunits (Ace-GCLM) of Ancylostoma ceylanicum were cloned using the RACE-PCR amplification method. The Ace-gclc and Ace-gclm cDNAs encode proteins with 655 and 254 amino acids and calculated molecular masses of 74.76 and 28.51kDa, respectively. The Ace-GCLC amino acid sequence shares about 70% identity and 80% sequence similarity with orthologs in Loa loa, Onchocerca volvulus, Brugia malayi, and Ascaris suum, whereas the Ace-GCLM amino acid sequence has only about 30% sequence identity and 50% similarity to homologous proteins in those species. Real-time PCR analysis of mRNA expression in L3, serum stimulated L3 and adult stages of A. ceylanicum showed the highest level of Ace-GCLC and Ace-GCLM expression occurred in adult worms. No differences were detected among adult hookworms harvested 21 and 35dpi indicating expression of Ace-gclc and Ace-gclm in adult worms is constant during the course of infection. Positive interaction between two subunits of glutamate-cysteine ligase was detected using the yeast two-hybrid system, and by specific enzymatic reaction. Ace-GCL is an intracellular enzyme and is not exposed to the host immune system. Thus, as expected, we did not detect IgG antibodies against Ace-GCLC or Ace-GCLM on days 21, 60 and 120 of A. ceylanicum infection in hamsters. Furthermore, vaccination with one or both antigens did not reduce worm burdens, and resulted in no improvement of clinical parameters (hematocrit and hemoglobin) of infected hamsters. Therefore, due to the significant role of the enzyme in parasite metabolism, our analyses raises hope for the development of a successful new drug against ancylostomiasis based on the specific GCL inhibitor. PMID

  3. A portrayal of E3 ubiquitin ligases and deubiquitylases in cancer.

    PubMed

    Satija, Yatendra Kumar; Bhardwaj, Abhishek; Das, Sanjeev

    2013-12-15

    E3 ubiquitin ligases and deubiquitylating enzymes (DUBs) are the key components of ubiquitin proteasome system which plays a critical role in cellular protein homeostasis. Any shortcoming in their biological roles can lead to various diseases including cancer. The dynamic interplay between ubiquitylation and deubiquitylation determines the level and activity of several proteins including p53, which is crucial for cellular stress response and tumor suppression pathways. In this review, we describe the different types of E3 ubiquitin ligases including those targeting tumor suppressor p53, SCF ligases and RING type ligases and accentuate on biological functions of few important E3 ligases in the cellular regulatory networks. Tumor suppressor p53 level is tightly regulated by multiple E3 ligases including Mdm2, COP1, Pirh2, etc. SCF ubiquitin ligase complexes are key regulators of cell cycle and signal transduction. BRCA1 and VHL RING type ligases function as tumor suppressors and play an important role in DNA repair and hypoxia response respectively. Further, we discuss the biological consequences of deregulation of the E3 ligases and the implications for cancer development. We also describe deubiquitylases which reverse the process of ubiquitylation and regulate diverse cellular pathways including metabolism, cell cycle control and chromatin remodelling. As the E3 ubiquitin ligases and DUBs work in a substrate specific manner, an improved understanding of them can lead to better therapeutics for cancer.

  4. Parkin and relatives: the RBR family of ubiquitin ligases.

    PubMed

    Marín, Ignacio; Lucas, J Ignasi; Gradilla, Ana-Citlali; Ferrús, Alberto

    2004-05-19

    Mutations in the parkin gene cause autosomal-recessive juvenile parkinsonism. Parkin encodes a ubiquitin-protein ligase characterized by having the RBR domain, composed of two RING fingers plus an IBR/DRIL domain. The RBR family is defined as the group of genes whose products contain an RBR domain. RBR family members exist in all eukaryotic species for which significant sequence data is available, including animals, plants, fungi, and several protists. The integration of comparative genomics with structural and functional data allows us to conclude that RBR proteins have multiple roles, not only in protein quality control mechanisms, but also as indirect regulators of transcription. A recently formulated hypothesis, based on a case of gene fusion, suggested that RBR proteins may be often part of cullin-containing ubiquitin ligase complexes. Recent data on Parkin protein agrees with that hypothesis. We discuss the involvement of RBR proteins in several neurodegenerative diseases and cancer.

  5. Biotin deficiency in the cat and the effect on hepatic propionyl CoA carboxylase.

    PubMed

    Carey, C J; Morris, J G

    1977-02-01

    Biotin deficiency was produced in growing kittens by feeding a diet containing dried, raw egg white. After receiving either an 18.5% egg white diet for 25 weeks, or a 32% egg white diet for 12 weeks, they exhibited dermal lesions characterized by alopecia, scaly dermatitis and achromotrichia, which increased in severity with the deficiency. Females developed accumulations of dried salivary, nasal and lacrymal secretions in the facial region although a male did not. There was a loss of body weight in all cats as the deficiency progressed. Hepatic propionyl CoA carboxylase activities were measured on biopsy samples of liver during biotin deficiency and after biotin supplementation. In the deficient state, activities were 4% and 24% of that following biotin supplementation. Propionyl carboxylase activity in the liver of the cat was comparable to that reported in the rat and chick in the deficient and normal states. Subcutaneous injection of 0.25 mg biotin every other day while continuing to receive the egg white diet caused remission of clinical signs, a body weight gain and increased food intake.

  6. Clustering of mutations in methylmalonyl CoA mutase associated with mut- methylmalonic acidemia.

    PubMed Central

    Crane, A. M.; Ledley, F. D.

    1994-01-01

    Mutations have been described in human methylmalonyl CoA mutase (MCM) that exhibit partial defects in enzyme activity, including cobalamin-dependent (i.e., mut-) or interallelic complementation. This work describes mutations in cells from four patients, three of whom exhibit a cobalamin-dependent phenotype and all four of whom exhibit interallelic complementation. Four novel mutations (R694W, G648D, G630E, and G626C) are identified that cluster near the carboxyl terminus of the protein, a region close to another mut- mutation (G717V). Each of these mutations was shown to express a phenotype congruent with that of the parental cell line, after transfection into mut0 fibroblasts, and each exhibits interallelic complementation in cotransfection assays with clones bearing a R93H mutation. The activity of mutant enzymes expressed in Saccharomyces cerevisiae parallels the residual activity of the parental cell lines and exhibits novel sensitivities to pH and salt. The clustering of these mutations identifies a region of MCM that most likely represents the cobalamin-binding domain. The location of this domain, as well as the pattern of sequence preservation between the homologous human and Probiono-bacterium shermanii enzymes, suggests a mechanism for interallelic complementation in which the cobalamin-binding defect is complemented in trans from the heterologous subunits of the dimer. Images Figure 6 PMID:7912889

  7. Enhanced activity of acetyl CoA synthetase adsorbed on smart microgel: an implication for precursor biosynthesis.

    PubMed

    Dubey, Nidhi Chandrama; Tripathi, Bijay Prakash; Müller, Martin; Stamm, Manfred; Ionov, Leonid

    2015-01-28

    Acetyl coenzyme A (acetyl CoA) is an essential precursor molecule for synthesis of metabolites such as the polyketide-based drugs (tetracycline, mitharamycin, Zocor, etc.) fats, lipids, and cholesterol. Acetyl CoA synthetase (Acs) is one of the enzymes that catalyzes acetyl CoA synthesis, and this enzyme is essentially employed for continuous supply of the acetyl CoA for the production of these metabolites. To achieve reusable and a more robust entity of the enzyme, we carried out the immobilization of Acs on poly(N-isopropylacrylamide)-poly(ethylenimine) (PNIPAm-PEI) microgels via adsorption. Cationic PNIPAm-PEI microgel was synthesized by one-step graft copolymerization of NIPAm and N,N-methylene bis-acrylamide (MBA) from PEI. Adsorption studies of Acs on microgel indicated high binding of enzymes, with a maximum binding capacity of 286 μg/mg of microgel for Acs was achieved. The immobilized enzymes showed improved biocatalytic efficiency over free enzymes, beside this, the reaction parameters and circular dichroism (CD) spectroscopy studies indicated no significant changes in the enzyme structure after immobilization. This thoroughly characterized enzyme bioconjugate was further immobilized on an ultrathin membrane to assess the same reaction in flow through condition. Bioconjugate was covalently immobilized on a thin layer of preformed microgel support upon polyethylene terephthalate (PET) track etched membrane. The prepared membrane was used in a dead end filtration device to monitor the bioconversion efficiency and operational stability of cross-linked bioconjugate. The membrane reactor showed consistent operational stability and maintained >70% of initial activity after 7 consecutive operation cycles. PMID:25561344

  8. Genetic Diversity of Staphylocoagulase Genes (coa): Insight into the Evolution of Variable Chromosomal Virulence Factors in Staphylococcus aureus

    PubMed Central

    Watanabe, Shinya; Ito, Teruyo; Sasaki, Takashi; Li, Shanshuang; Uchiyama, Ikuo; Kishii, Kozue; Kikuchi, Ken; Skov, Robert Leo; Hiramatsu, Keiichi

    2009-01-01

    Background The production of staphylocoagulase (SC) causing the plasma coagulation is one of the important characteristics of Staphylococcus aureus. Although SCs have been classified into 10 serotypes based on the differences in the antigenicity, genetic bases for their diversities and relatedness to chromosome types are poorly understood. Methodology/Principal Findings We compared the nucleotide sequences of 105 SC genes (coa), 59 of which were determined in this study. D1 regions, which contain prothrombin-activating and -binding domains and are presumed to be the binding site of each type-specific antiserum, were classified into twelve clusters having more than 90% nucleotide identities, resulting to create two novel SC types, XI and XII, in addition to extant 10 types. Nine of the twelve SC types were further subdivided into subtypes based on the differences of the D2 or the central regions. The phylogenetical relations of the D1 regions did not correlate exactly with either one of agr types and multilocus sequence types (STs). In addition, genetic analysis showed that recombination events have occurred in and around coa. So far tested, STs of 126 S. aureus strains correspond to the combination of SC type and agr type except for the cases of CC1 and CC8, which contained two and three different SC types, respectively. Conclusion The data suggested that the evolution of coa was not monophyletic in the species. Chromosomal recombination had occurred at coa and agr loci, resulting in the carriage of the combinations of allotypically different important virulence determinants in staphylococcal chromosome. PMID:19492076

  9. Kinetic framework for ligation by an efficient RNA ligase ribozyme.

    PubMed

    Bergman, N H; Johnston, W K; Bartel, D P

    2000-03-21

    The class I RNA ligase ribozyme, isolated previously from random sequences, performs an efficient RNA ligation reaction. It ligates two substrate RNAs, promoting the attack of the 3'-hydroxyl of one substrate upon the 5'-triphosphate of the other substrate with release of pyrophosphate. This ligation reaction has similarities to the reaction catalyzed by RNA polymerases. Using data from steady-state kinetic measurements and pulse-chase/pH-jump experiments, we have constructed minimal kinetic frameworks for two versions of the class I ligase, named 207t and 210t. For both ligases, as well as for the self-ligating parent ribozyme, the rate constant for the chemical step (k(c)) is log-linear with pH in the range 5.7-8.0. At physiological pH, the k(c) is 100 min(-1), a value similar to those reported for the fastest naturally occurring ribozymes. At higher pH, product release is limiting for both 207t and 210t. The 210t ribozyme, with its faster product release, attains multiple-turnover rates (k(cat) = 360 min(-1), pH 9.0) exceeding those of 207t and other reported ribozyme reactions. The kinetic framework for the 210t ribozyme describes the limits of this catalysis and suggests how key steps can be targeted for improvement using design or combinatorial approaches. PMID:10715133

  10. SUBSURFACE WELL-LOG CORRELATION OF ARSENIC-BEARING LITHOFACIES IN THE PERMIAN GARBER SANDSTONE AND WELLINGTON FORMATION, CENTRAL OKLAHOMA AQUIFER (COA), CLEVELAND COUNTY, OKLAHOMA

    EPA Science Inventory

    The fluvial Garber Sandstone and the underlying Wellington Formation are important sources of drinking water in central Oklahoma. These formations, which make up much of the COA, consist of amalgamated sandstones with some interbedded mudstones, siltstones, and local mudstone- a...

  11. Biochemical characterization of recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala.

    PubMed

    Sonawane, Prashant; Vishwakarma, Rishi Kishore; Khan, Bashir M

    2013-07-01

    Recombinant cinnamoyl CoA reductase 1 (Ll-CCRH1) protein from Leucaena leucocephala was overexpressed in Escherichia coli BL21 (DE3) strain and purified to apparent homogeneity. Optimum pH for forward and reverse reaction was found to be 6.5 and 7.8 respectively. The enzyme was most stable around pH 6.5 at 25°C for 90 min. The enzyme showed Kcat/Km for feruloyl, caffeoyl, sinapoyl, coumaroyl CoA, coniferaldehyde and sinapaldehyde as 4.6, 2.4, 2.3, 1.7, 1.9 and 1.2 (×10(6) M(-1) s(-1)), respectively, indicating affinity of enzyme for feruloyl CoA over other substrates and preference of reduction reaction over oxidation. Activation energy, Ea for various substrates was found to be in the range of 20-50 kJ/mol. Involvement of probable carboxylate ion, histidine, lysine or tyrosine at the active site of enzyme was predicted by pH activity profile. SAXS studies of protein showed radius 3.04 nm and volume 49.25 nm(3) with oblate ellipsoid shape. Finally, metal ion inhibition studies revealed that Ll-CCRH1 is a metal independent enzyme. PMID:23541561

  12. C-terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin

    SciTech Connect

    Liu, Sicheng; Liu, Xunyue; Kamdar, Radhika Pankaj; Wanotayan, Rujira; Sharma, Mukesh Kumar; Adachi, Noritaka; Matsumoto, Yoshihisa

    2013-09-20

    Highlights: •Chromatin binding of XRCC4 is dependent on the presence of DNA ligase IV. •C-terminal region of DNA ligase IV alone can recruit itself and XRCC4 to chromatin. •Two BRCT domains of DNA ligase IV are essential for the chromatin binding of XRCC4. -- Abstract: DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ.

  13. Successful Conversion of the Bacillus subtilis BirA Group II Biotin Protein Ligase into a Group I Ligase

    PubMed Central

    Henke, Sarah K.; Cronan, John E.

    2014-01-01

    Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that allows transcriptional regulation of biotin biosynthetic and transport genes whereas Group I BPLs lack this N-terminal domain. The Bacillus subtilis BPL, BirA, is classified as a Group II BPL based on sequence predictions of an N-terminal helix-turn-helix motif and mutational alteration of its regulatory properties. We report evidence that B. subtilis BirA is a Group II BPL that regulates transcription at three genomic sites: bioWAFDBI, yuiG and yhfUTS. Moreover, unlike the paradigm Group II BPL, E. coli BirA, the N-terminal DNA binding domain can be deleted from Bacillus subtilis BirA without adverse effects on its ligase function. This is the first example of successful conversion of a Group II BPL to a Group I BPL with retention of full ligase activity. PMID:24816803

  14. Antitumor/Antifungal Celecoxib Derivative AR-12 is a Non-Nucleoside Inhibitor of the ANL-Family Adenylating Enzyme Acetyl CoA Synthetase

    PubMed Central

    2016-01-01

    AR-12/OSU-03012 is an antitumor celecoxib-derivative that has progressed to Phase I clinical trial as an anticancer agent and has activity against a number of infectious agents including fungi, bacteria and viruses. However, the mechanism of these activities has remained unclear. Based on a chemical-genetic profiling approach in yeast, we have found that AR-12 is an ATP-competitive, time-dependent inhibitor of yeast acetyl coenzyme A synthetase. AR-12-treated fungal cells show phenotypes consistent with the genetic reduction of acetyl CoA synthetase activity, including induction of autophagy, decreased histone acetylation, and loss of cellular integrity. In addition, AR-12 is a weak inhibitor of human acetyl CoA synthetase ACCS2. Acetyl CoA synthetase activity is essential in many fungi and parasites. In contrast, acetyl CoA is primarily synthesized by an alternate enzyme, ATP-citrate lyase, in mammalian cells. Taken together, our results indicate that AR-12 is a non-nucleoside acetyl CoA synthetase inhibitor and that acetyl CoA synthetase may be a feasible antifungal drug target. PMID:27088128

  15. Transport of long-chain fatty acids in Escherichia coli. Evidence for role of fadL gene product as long-chain fatty acid receptor.

    PubMed

    Nunn, W D; Colburn, R W; Black, P N

    1986-01-01

    Transport of long-chain fatty acids (LCFA) across the cytoplasmic membrane of Escherichia coli requires functional fadL and fadD genes. The fadD gene codes for an acyl-CoA synthetase (fatty acid: CoA ligase (AMP forming] which has broad chain length specificity and is loosely bound to the cytoplasmic membrane. The fadL gene codes for a 43,000-dalton cytoplasmic membrane protein which, acting by an unknown mechanism, is needed specifically for LCFA transport. As a first step to define the role of the fadL gene product, studies were performed to determine if it functions as a LCFA receptor. The LCFA-binding activity was quantitated in intact cells in the absence of LCFA transport by comparing the binding of LCFA in fadD fadL and fadD fadL+ strains. These studies revealed that (i) fadD fadL+ strains bind 6-fold more LCFA than fadD fadL strains; (ii) fadD fadL strains harboring a plasmid containing the fadL gene bind 16-fold more LCFA than fadD fadL strains harboring only the plasmid vector; and (iii) the fadL-specific LCFA-binding activity is regulated by the fadR gene and catabolite repression. Studies with fadL strains harboring fadL plasmids containing in vitro constructed deletions indicate that mutations which alter the physical properties of the 43,000-dalton fadL gene product also affect fadL gene product-specific LCFA-binding activity. Overall, these studies suggest that one role of the fadL gene product in the LCFA transport process is to sequester LCFA at sites in the cell membrane for transport.

  16. Identification of Amino Acids Conferring Chain Length Substrate Specificities on Fatty Alcohol-forming Reductases FAR5 and FAR8 from Arabidopsis thaliana*

    PubMed Central

    Chacón, Micaëla G.; Fournier, Ashley E.; Tran, Frances; Dittrich-Domergue, Franziska; Pulsifer, Ian P.; Domergue, Frédéric; Rowland, Owen

    2013-01-01

    Fatty alcohols play a variety of biological roles in all kingdoms of life. Fatty acyl reductase (FAR) enzymes catalyze the reduction of fatty acyl-coenzyme A (CoA) or fatty acyl-acyl carrier protein substrates to primary fatty alcohols. FAR enzymes have distinct substrate specificities with regard to chain length and degree of saturation. FAR5 (At3g44550) and FAR8 (At3g44560) from Arabidopsis thaliana are 85% identical at the amino acid level and are of equal length, but they possess distinct specificities for 18:0 or 16:0 acyl chain length, respectively. We used Saccharomyces cerevisiae as a heterologous expression system to assess FAR substrate specificity determinants. We identified individual amino acids that affect protein levels or 16:0-CoA versus 18:0-CoA specificity by expressing in yeast FAR5 and FAR8 domain-swap chimeras and site-specific mutants. We found that a threonine at position 347 and a serine at position 363 were important for high FAR5 and FAR8 protein accumulation in yeast and thus are likely important for protein folding and stability. Amino acids at positions 355 and 377 were important for dictating 16:0-CoA versus 18:0-CoA chain length specificity. Simultaneously converting alanine 355 and valine 377 of FAR5 to the corresponding FAR8 residues, leucine and methionine, respectively, almost fully converted FAR5 specificity from 18:0-CoA to 16:0-CoA. The reciprocal amino acid conversions, L355A and M377V, made in the active FAR8-S363P mutant background converted its specificity from 16:0-CoA to 18:0-CoA. This study is an important advancement in the engineering of highly active FAR proteins with desired specificities for the production of fatty alcohols with industrial value. PMID:24005667

  17. Defining interactions between DNA-PK and ligase IV/XRCC4

    SciTech Connect

    Hsu, Hsin-Ling; Yannone, Steven M.; Chen, David J.

    2001-04-10

    Non-homologous end joining (NHEJ) is a major pathway for the repair of DNA double-strand breaks in mammalian cells. DNA-dependent protein kinase (DNA-PK), ligase IV, and XRCC4 are all critical components of the NHEJ repair pathway. DNA-PK is composed of a heterodimeric DNA-binding component, Ku, and a large catalytic subunit, DNA-PKcs. Ligase IV and XRCC4 associate to form a multimeric complex that is also essential for NHEJ. DNA-PK and ligase IV/XRCC4 interact at DNA termini which results in stimulated ligase activity. Here we define interactions between the components of these two essential complexes, DNA-PK and ligase IV/XRCC4. We find that ligase IV/XRCC4 associates with DNA-PK in a DNA-independent manner. The specific protein-protein interactions that mediate the interaction between these two complexes are further identified. Direct physical interactions between ligase IV and Ku as well as between XRCC4 and DNA-PKcs are shown. No direct interactions are observed between ligase IV and DNA-PKcs or between XRCC4 and Ku. Our data defines the specific protein pairs involved in the association of DNA-PK and ligase IV/XRCC4, and suggests a molecular mechanism for coordinating the assembly of the DNA repair complex at DNA breaks.

  18. Up-regulation of an N-terminal truncated 3-hydroxy-3-methylglutaryl CoA reductase enhances production of essential oils and sterols in transgenic Lavandula latifolia.

    PubMed

    Muñoz-Bertomeu, Jesús; Sales, Ester; Ros, Roc; Arrillaga, Isabel; Segura, Juan

    2007-11-01

    Spike lavender (Lavandula latifolia) essential oil is widely used in the perfume, cosmetic, flavouring and pharmaceutical industries. Thus, modifications of yield and composition of this essential oil by genetic engineering should have important scientific and commercial applications. We generated transgenic spike lavender plants expressing the Arabidopsis thaliana HMG1 cDNA, encoding the catalytic domain of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR1S), a key enzyme of the mevalonic acid (MVA) pathway. Transgenic T0 plants accumulated significantly more essential oil constituents as compared to controls (up to 2.1- and 1.8-fold in leaves and flowers, respectively). Enhanced expression of HMGR1S also increased the amount of the end-product sterols, beta-sitosterol and stigmasterol (average differences of 1.8- and 1.9-fold, respectively), but did not affect the accumulation of carotenoids or chlorophylls. We also analysed T1 plants derived from self-pollinated seeds of T0 lines that flowered after growing for 2 years in the greenhouse. The increased levels of essential oil and sterols observed in the transgenic T0 plants were maintained in the progeny that inherited the HMG1 transgene. Our results demonstrate that genetic manipulation of the MVA pathway increases essential oil yield in spike lavender, suggesting a contribution for this cytosolic pathway to monoterpene and sesquiterpene biosynthesis in leaves and flowers of the species.

  19. Interactions of methylamine and ammonia with atmospheric nucleation precursor H2SO4 and common organic acids: Thermodynamics and atmospheric implications

    NASA Astrophysics Data System (ADS)

    Xu, Y.; Nadykto, A. B.; Jiang, L.; Bai, Z.

    2016-06-01

    Interactions of the two common atmospheric bases, ammonia (NH3) and methylamine MA (CH3NH2), which are considered to be important stabilizers of binary clusters in the Earth's atmosphere, with H2SO4, the key atmospheric precursor, and 14 common atmospheric organic acids (COA) (formic (CH2O2), acetic (C2H4O2), oxalic (C2H2O4), malonic (C3H4O4), succinic (C4H6O4), glutaric acid (C5H8O4), adipic (C6H10O4), benzoic (C6H5COOH), phenylacetic (C6H5CH2COOH), pyruvic (C3H4O3), maleic acid (C4H4O4), malic (C4H6O5), tartaric (C4H6O6) and pinonic acid (C10H16O3)) have been studied using the composite high-accuracy G3MP2 method. The thermodynamic stability of mixed (COA) (H2SO4), (COA)(B1) and (COA)(B2) dimers and (COA) (H2SO4) (B1) and (COA) (H2SO4) (B1) trimers, where B1 and B2 represent methylamine (CH3NH2) and ammonia (NH3), respectively, have been investigated and their impacts on the thermodynamic stability of clusters containing H2SO4 have been analyzed. It has been shown that in many cases the interactions of H2SO4 with COA, ammonia and methylamine lead to the formation of heteromolecular dimers and trimers, which are certainly more stable than (H2SO4)2 and (H2SO4)3. It has also been found that free energies of (COA) (H2SO4)+ CH3NH2⇔(COA) (H2SO4)(CH3NH2) reactions exceed 10-15 kcal mol-1. This is a clear indication that mixed trimers composed of COA, H2SO4 and methylamine are very stable and can thus serve as possible nucleation sites. The present study leads us to conclude that the interactions of COA coexisting with H2SO4 and common atmospheric bases in the Earth's atmosphere may be an important factor affecting the stability of nucleating sulfuric acid clusters and that the impacts of COA on atmospheric nucleation should be studied in further details.

  20. Rapid Assembly of DNA via Ligase Cycling Reaction (LCR).

    PubMed

    Chandran, Sunil

    2017-01-01

    The assembly of multiple DNA parts into a larger DNA construct is a requirement in most synthetic biology laboratories. Here we describe a method for the efficient, high-throughput, assembly of DNA utilizing the ligase chain reaction (LCR). The LCR method utilizes non-overlapping DNA parts that are ligated together with the guidance of bridging oligos. Using this method, we have successfully assembled up to 20 DNA parts in a single reaction or DNA constructs up to 26 kb in size. PMID:27671935

  1. E3 ubiquitin ligase gene CMPG1-V from Haynaldia villosa L. contributes to powdery mildew resistance in common wheat (Triticum aestivum L.).

    PubMed

    Zhu, Yanfei; Li, Yingbo; Fei, Fei; Wang, Zongkuan; Wang, Wei; Cao, Aizhong; Liu, Yuan; Han, Shuang; Xing, Liping; Wang, Haiyan; Chen, Wei; Tang, Sanyuan; Huang, Xiahe; Shen, Qianhua; Xie, Qi; Wang, Xiue

    2015-10-01

    Powdery mildew is one of the most devastating wheat fungal diseases. A diploid wheat relative, Haynaldia villosa L., is highly resistant to powdery mildew, and its genetic resource of resistances, such as the Pm21 locus, is now widely used in wheat breeding. Here we report the cloning of a resistance gene from H. villosa, designated CMPG1-V, that encodes a U-box E3 ubiquitin ligase. Expression of the CMPG1-V gene was induced in the leaf and stem of H. villosa upon inoculation with Blumeria graminis f. sp. tritici (Bgt) fungus, and the presence of Pm21 is essential for its rapid induction of expression. CMPG1-V has conserved key residues for E3 ligase, and possesses E3 ligase activity in vitro and in vivo. CMPG1-V is localized in the nucleus, endoplasmic reticulum, plasma membrane and partially in trans-Golgi network/early endosome vesicles. Transgenic wheat over-expressing CMPG1-V showed improved broad-spectrum powdery mildew resistance at seedling and adult stages, associated with an increase in expression of salicylic acid-responsive genes, H2 O2 accumulation, and cell-wall protein cross-linking at the Bgt infection sites, and the expression of CMPG1-V in H. villosa was increased when treated with salicylic acid, abscisic acid and H2 O2 . These results indicate the involvement of E3 ligase in defense responses to Bgt fungus in wheat, particularly in broad-spectrum disease resistance, and suggest association of reactive oxidative species and the phytohormone pathway with CMPG1-V-mediated powdery mildew resistance.

  2. Software interface for high-speed readout of particle detectors based on the CoaXPress communication standard

    NASA Astrophysics Data System (ADS)

    Hejtmánek, M.; Neue, G.; Voleš, P.

    2015-06-01

    This article is devoted to the software design and development of a high-speed readout application used for interfacing particle detectors via the CoaXPress communication standard. The CoaXPress provides an asymmetric high-speed serial connection over a single coaxial cable. It uses a widely available 75 Ω BNC standard and can operate in various modes with a data throughput ranging from 1.25 Gbps up to 25 Gbps. Moreover, it supports a low speed uplink with a fixed bit rate of 20.833 Mbps, which can be used to control and upload configuration data to the particle detector. The CoaXPress interface is an upcoming standard in medical imaging, therefore its usage promises long-term compatibility and versatility. This work presents an example of how to develop DAQ system for a pixel detector. For this purpose, a flexible DAQ card was developed using the XILINX Spartan 6 FPGA. The DAQ card is connected to the framegrabber FireBird CXP6 Quad, which is plugged in the PCI Express bus of the standard PC. The data transmission was performed between the FPGA and framegrabber card via the standard coaxial cable in communication mode with a bit rate of 3.125 Gbps. Using the Medipix2 Quad pixel detector, the framerate of 100 fps was achieved. The front-end application makes use of the FireBird framegrabber software development kit and is suitable for data acquisition as well as control of the detector through the registers implemented in the FPGA.

  3. Structure of the adenylation domain of NAD[superscript +]-dependent DNA ligase from Staphylococcus aureus

    SciTech Connect

    Han, Seungil; Chang, Jeanne S.; Griffor, Matt; Pfizer

    2010-09-17

    DNA ligase catalyzes phosphodiester-bond formation between immediately adjacent 5'-phosphate and 3''-hydroxyl groups in double-stranded DNA and plays a central role in many cellular and biochemical processes, including DNA replication, repair and recombination. Bacterial NAD{sup +}-dependent DNA ligases have been extensively characterized as potential antibacterial targets because of their essentiality and their structural distinction from human ATP-dependent DNA ligases. The high-resolution structure of the adenylation domain of Staphylococcus aureus NAD{sup +}-dependent DNA ligase establishes the conserved domain architecture with other bacterial adenylation domains. Two apo crystal structures revealed that the active site possesses the preformed NAD{sup +}-binding pocket and the 'C2 tunnel' lined with hydrophobic residues: Leu80, Phe224, Leu287, Phe295 and Trp302. The C2 tunnel is unique to bacterial DNA ligases and the Leu80 side chain at the mouth of the tunnel points inside the tunnel and forms a narrow funnel in the S. aureus DNA ligase structure. Taken together with other DNA ligase structures, the S. aureus DNA ligase structure provides a basis for a more integrated understanding of substrate recognition and catalysis and will be also be of help in the development of small-molecule inhibitors.

  4. Discovery of tumor-specific irreversible inhibitors of stearoyl CoA desaturase | Office of Cancer Genomics

    Cancer.gov

    A hallmark of targeted cancer therapies is selective toxicity among cancer cell lines. We evaluated results from a viability screen of over 200,000 small molecules to identify two chemical series, oxalamides and benzothiazoles, that were selectively toxic at low nanomolar concentrations to the same 4 of 12 human lung cancer cell lines. Sensitive cell lines expressed cytochrome P450 (CYP) 4F11, which metabolized the compounds into irreversible inhibitors of stearoyl CoA desaturase (SCD). SCD is recognized as a promising biological target in cancer and metabolic disease.

  5. Regulation of ubiquitin ligase dynamics by the nucleolus

    PubMed Central

    Mekhail, Karim; Khacho, Mireille; Carrigan, Amanda; Hache, Robert R.J.; Gunaratnam, Lakshman; Lee, Stephen

    2005-01-01

    Cellular pathways relay information through dynamic protein interactions. We have assessed the kinetic properties of the murine double minute protein (MDM2) and von Hippel-Lindau (VHL) ubiquitin ligases in living cells under physiological conditions that alter the stability of their respective p53 and hypoxia-inducible factor substrates. Photobleaching experiments reveal that MDM2 and VHL are highly mobile proteins in settings where their substrates are efficiently degraded. The nucleolar architecture converts MDM2 and VHL to a static state in response to regulatory cues that are associated with substrate stability. After signal termination, the nucleolus is able to rapidly release these proteins from static detention, thereby restoring their high mobility profiles. A protein surface region of VHL's β-sheet domain was identified as a discrete [H+]-responsive nucleolar detention signal that targets the VHL/Cullin-2 ubiquitin ligase complex to nucleoli in response to physiological fluctuations in environmental pH. Data shown here provide the first evidence that cells have evolved a mechanism to regulate molecular networks by reversibly switching proteins between a mobile and static state. PMID:16129783

  6. A design principle underlying the paradoxical roles of E3 ubiquitin ligases

    NASA Astrophysics Data System (ADS)

    Lee, Daewon; Kim, Minjin; Cho, Kwang-Hyun

    2014-07-01

    E3 ubiquitin ligases are important cellular components that determine the specificity of proteolysis in the ubiquitin-proteasome system. However, an increasing number of studies have indicated that E3 ubiquitin ligases also participate in transcription. Intrigued by the apparently paradoxical functions of E3 ubiquitin ligases in both proteolysis and transcriptional activation, we investigated the underlying design principles using mathematical modeling. We found that the antagonistic functions integrated in E3 ubiquitin ligases can prevent any undesirable sustained activation of downstream genes when E3 ubiquitin ligases are destabilized by unexpected perturbations. Interestingly, this design principle of the system is similar to the operational principle of a safety interlock device in engineering systems, which prevents a system from abnormal operation unless stability is guaranteed.

  7. Walnut oil increases cholesterol efflux through inhibition of stearoyl CoA desaturase 1 in THP-1 macrophage-derived foam cells

    PubMed Central

    2011-01-01

    Background Walnuts significantly decrease total and low-density lipoprotein cholesterol in normo- and hypercholesterolemic individuals. No study to date has evaluated the effects of walnuts on cholesterol efflux, the initial step in reverse cholesterol transport, in macrophage-derived foam cells (MDFC). The present study was conducted to investigate the mechanisms by which walnut oil affects cholesterol efflux. Methods The extract of English walnuts (walnut oil) was dissolved in DMSO and applied to cultured THP-1 MDFC cells (0.5 mg/mL). THP-1 MDFC also were treated with human sera (10%, v:v) taken from subjects in a walnut feeding study. Cholesterol efflux was examined by liquid scintillation counting. Changes in gene expression were quantified by real time PCR. Results Walnut oil treatment significantly increased cholesterol efflux through decreasing the expression of the lipogenic enzyme stearoyl CoA desaturase 1 (SCD1) in MDFC. Alpha-linolenic acid (ALA), the major n-3 polyunsaturated fatty acids found in walnuts, recaptured SCD1 reduction in MDFC, a mechanism mediated through activation of nuclear receptor farnesoid-X-receptor (FXR). Postprandial serum treatment also increased cholesterol efflux in MDFC. When categorized by baseline C-reactive protein (CRP; cut point of 2 mg/L), subjects in the lower CRP sub-group benefited more from dietary intervention, including a more increase in cholesterol efflux, a greater reduction in SCD1, and a blunted postprandial lipemia. Conclusion In conclusion, walnut oil contains bioactive molecules that significantly improve cholesterol efflux in MDFC. However, the beneficial effects of walnut intake may be reduced by the presence of a pro-inflammatory state. Trial Registration ClinicalTrials.gov: NCT00938340 PMID:21871057

  8. Molecular characterization of NAD+-dependent DNA ligase from Wolbachia endosymbiont of lymphatic filarial parasite Brugia malayi.

    PubMed

    Shrivastava, Nidhi; Nag, Jeetendra Kumar; Misra-Bhattacharya, Shailja

    2012-01-01

    The lymphatic filarial parasite, Brugia malayi contains Wolbachia endobacteria that are essential for development, viability and fertility of the parasite. Therefore, wolbachial proteins have been currently seen as the potential antifilarial drug targets. NAD(+)-dependent DNA ligase is characterized as a promising drug target in several organisms due to its crucial, indispensable role in DNA replication, recombination and DNA repair. We report here the cloning, expression and purification of NAD(+)-dependent DNA ligase of Wolbachia endosymbiont of B. malayi (wBm-LigA) for its molecular characterization. wBm-LigA has all the domains that are present in nearly all the eubacterial NAD(+)-dependent DNA ligases such as N-terminal adenylation domain, OB fold, helix-hairpin-helix (HhH) and BRCT domain except zinc-binding tetracysteine domain. The purified recombinant protein (683-amino acid) was found to be biochemically active and was present in its native form as revealed by the circular dichroism and fluorescence spectra. The purified recombinant enzyme was able to catalyze intramolecular strand joining on a nicked DNA as well as intermolecular joining of the cohesive ends of BstEII restricted lamda DNA in an in vitro assay. The enzyme was localized in the various life-stages of B. malayi parasites by immunoblotting and high enzyme expression was observed in Wolbachia within B. malayi microfilariae and female adult parasites along the hypodermal chords and in the gravid portion as evident by the confocal microscopy. Ours is the first report on this enzyme of Wolbachia and these findings would assist in validating the antifilarial drug target potential of wBm-LigA in future studies. PMID:22815933

  9. Ubiquitin E3 ligase FIEL1 regulates fibrotic lung injury through SUMO-E3 ligase PIAS4.

    PubMed

    Lear, Travis; McKelvey, Alison C; Rajbhandari, Shristi; Dunn, Sarah R; Coon, Tiffany A; Connelly, William; Zhao, Joe Y; Kass, Daniel J; Zhang, Yingze; Liu, Yuan; Chen, Bill B

    2016-05-30

    The E3 small ubiquitin-like modifier (SUMO) protein ligase protein inhibitor of activated STAT 4 (PIAS4) is a pivotal protein in regulating the TGFβ pathway. In this study, we discovered a new protein isoform encoded by KIAA0317, termed fibrosis-inducing E3 ligase 1 (FIEL1), which potently stimulates the TGFβ signaling pathway through the site-specific ubiquitination of PIAS4. FIEL1 targets PIAS4 using a double locking mechanism that is facilitated by the kinases PKCζ and GSK3β. Specifically, PKCζ phosphorylation of PIAS4 and GSK3β phosphorylation of FIEL1 are both essential for the degradation of PIAS4. FIEL1 protein is highly expressed in lung tissues from patients with idiopathic pulmonary fibrosis (IPF), whereas PIAS4 protein levels are significantly reduced. FIEL1 overexpression significantly increases fibrosis in a bleomycin murine model, whereas FIEL1 knockdown attenuates fibrotic conditions. Further, we developed a first-in-class small molecule inhibitor toward FIEL1 that is highly effective in ameliorating fibrosis in mice. This study provides a basis for IPF therapeutic intervention by modulating PIAS4 protein abundance.

  10. DNA ligase III and DNA ligase IV carry out genetically distinct forms of end joining in human somatic cells

    PubMed Central

    Oh, Sehyun; Harvey, Adam; Zimbric, Jacob; Wang, Yongbao; Nguyen, Thanh; Jackson, Pauline J.; Hendrickson, Eric A.

    2014-01-01

    Ku-dependent C-NHEJ (classic non-homologous end joining) is the primary DNA EJing (end joining) repair pathway in mammals. Recently, an additional EJing repair pathway (A-NHEJ; alternative-NHEJ) has been described. Currently, the mechanism of A-NHEJ is obscure although a dependency on LIGIII (DNA ligase III) is often implicated. To test the requirement for LIGIII in A-NHEJ we constructed a LIGIII conditionally-null human cell line using gene targeting. Nuclear EJing activity appeared unaffected by a deficiency in LIGIII as, surprisingly, so were random gene targeting integration events. In contrast, LIGIII was required for mitochondrial function and this defined the gene’s essential activity. Human Ku:LIGIII and Ku:LIGIV (DNA ligase IV) double knockout cell lines, however, demonstrated that LIGIII is required for the enhanced A-NHEJ activity that is observed in Ku-deficient cells. Most unexpectedly, however, the majority of EJing events remained LIGIV-dependent. In conclusion, although human LIGIII has an essential function in mitochondrial maintenance, it is dispensable for most types of nuclear DSB repair, except for the A-NHEJ events that are normally suppressed by Ku. Moreover, we describe that a robust Ku-independent, LIGIV-dependent repair pathway exists in human somatic cells. PMID:24837021

  11. Screening, identification, and characterization of mechanistically diverse inhibitors of the Mycobacterium tuberculosis enzyme, pantothenate kinase (CoaA).

    PubMed

    Venkatraman, Janani; Bhat, Jyothi; Solapure, Suresh M; Sandesh, Jatheendranath; Sarkar, Debasmita; Aishwarya, Sundaram; Mukherjee, Kakoli; Datta, Santanu; Malolanarasimhan, Krishnan; Bandodkar, Balachandra; Das, Kaveri S

    2012-03-01

    The authors describe the discovery of anti-mycobacterial compounds through identifying mechanistically diverse inhibitors of the essential Mycobacterium tuberculosis (Mtb) enzyme, pantothenate kinase (CoaA). Target-driven drug discovery technologies often work with purified enzymes, and inhibitors thus discovered may not optimally inhibit the form of the target enzyme predominant in the bacterial cell or may not be available at the desired concentration. Therefore, in addition to addressing entry or efflux issues, inhibitors with diverse mechanisms of inhibition (MoI) could be prioritized before hit-to-lead optimization. The authors describe a high-throughput assay based on protein thermal melting to screen large numbers of compounds for hits with diverse MoI. Following high-throughput screening for Mtb CoaA enzyme inhibitors, a concentration-dependent increase in protein thermal stability was used to identify true binders, and the degree of enhancement or reduction in thermal stability in the presence of substrate was used to classify inhibitors as competitive or non/uncompetitive. The thermal shift-based MoI assay could be adapted to screen hundreds of compounds in a single experiment as compared to traditional biochemical approaches for MoI determination. This MoI was confirmed through mechanistic studies that estimated K(ie) and K(ies) for representative compounds and through nuclear magnetic resonance-based ligand displacement assays.

  12. Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids

    SciTech Connect

    Melton, Elaina M.; Cerny, Ronald L.; DiRusso, Concetta C.; Black, Paul N.

    2013-11-01

    Highlights: •Roles of FATP2 in fatty acid transport/activation contribute to lipid homeostasis. •Use of 13C- and D-labeled fatty acids provide novel insights into FATP2 function. •FATP2-dependent trafficking of FA into phospholipids results in distinctive profiles. •FATP2 functions in the transport and activation pathways for exogenous fatty acids. -- Abstract: In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4 h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The

  13. CoaTx-II, a new dimeric Lys49 phospholipase A2 from Crotalus oreganus abyssus snake venom with bactericidal potential: Insights into its structure and biological roles.

    PubMed

    Almeida, J R; Lancellotti, M; Soares, A M; Calderon, L A; Ramírez, D; González, W; Marangoni, S; Da Silva, S L

    2016-09-15

    Snake venoms are rich and intriguing sources of biologically-active molecules that act on target cells, modulating a diversity of physiological functions and presenting promising pharmacological applications. Lys49 phospholipase A2 is one of the multifunctional proteins present in these complex secretions and, although catalytically inactive, has a variety of biological activities, including cytotoxic, antibacterial, inflammatory, antifungal activities. Herein, a Lys49 phospholipase A2, denominated CoaTx-II from Crotalus oreganus abyssus, was purified and structurally and pharmacologically characterized. CoaTx-II was isolated with a high degree of purity by a combination of two chromatographic steps; molecular exclusion and reversed-phase high performance liquid chromatography. This toxin is dimeric with a mass of 13868.2 Da (monomeric form), as determined by mass spectrometry. CoaTx-II is rich in Arg and Lys residues and displays high identity with other Lys49 PLA2 homologues, which have high isoelectric points. The structural model of dimeric CoaTx-II shows that the toxin is non-covalently stabilized. Despite its enzymatic inactivity, in vivo CoaTx-II caused local muscular damage, characterized by increased plasma creatine kinase and confirmed by histological alterations, in addition to an inflammatory activity, as demonstrated by mice paw edema induction and pro-inflammatory cytokine IL-6 elevation. CoaTx-II also presents antibacterial activity against gram negative (Pseudomonas aeruginosa 31NM, Escherichia coli ATCC 25922) and positive (Staphyloccocus aureus BEC9393 and Rib1) bacteria. Therefore, data show that this newly purified toxin plays a central role in mediating the degenerative events associated with envenomation, in addition to demonstrating antibacterial properties, with potential for use in the development of strategies for antivenom therapy and combating antibiotic-resistant bacteria. PMID:27530662

  14. CoaTx-II, a new dimeric Lys49 phospholipase A2 from Crotalus oreganus abyssus snake venom with bactericidal potential: Insights into its structure and biological roles.

    PubMed

    Almeida, J R; Lancellotti, M; Soares, A M; Calderon, L A; Ramírez, D; González, W; Marangoni, S; Da Silva, S L

    2016-09-15

    Snake venoms are rich and intriguing sources of biologically-active molecules that act on target cells, modulating a diversity of physiological functions and presenting promising pharmacological applications. Lys49 phospholipase A2 is one of the multifunctional proteins present in these complex secretions and, although catalytically inactive, has a variety of biological activities, including cytotoxic, antibacterial, inflammatory, antifungal activities. Herein, a Lys49 phospholipase A2, denominated CoaTx-II from Crotalus oreganus abyssus, was purified and structurally and pharmacologically characterized. CoaTx-II was isolated with a high degree of purity by a combination of two chromatographic steps; molecular exclusion and reversed-phase high performance liquid chromatography. This toxin is dimeric with a mass of 13868.2 Da (monomeric form), as determined by mass spectrometry. CoaTx-II is rich in Arg and Lys residues and displays high identity with other Lys49 PLA2 homologues, which have high isoelectric points. The structural model of dimeric CoaTx-II shows that the toxin is non-covalently stabilized. Despite its enzymatic inactivity, in vivo CoaTx-II caused local muscular damage, characterized by increased plasma creatine kinase and confirmed by histological alterations, in addition to an inflammatory activity, as demonstrated by mice paw edema induction and pro-inflammatory cytokine IL-6 elevation. CoaTx-II also presents antibacterial activity against gram negative (Pseudomonas aeruginosa 31NM, Escherichia coli ATCC 25922) and positive (Staphyloccocus aureus BEC9393 and Rib1) bacteria. Therefore, data show that this newly purified toxin plays a central role in mediating the degenerative events associated with envenomation, in addition to demonstrating antibacterial properties, with potential for use in the development of strategies for antivenom therapy and combating antibiotic-resistant bacteria.

  15. Stearoyl CoA desaturase is required to produce active, lipid-modified Wnt proteins.

    PubMed

    Rios-Esteves, Jessica; Resh, Marilyn D

    2013-09-26

    Wnt proteins contain palmitoleic acid, an unusual lipid modification. Production of an active Wnt signal requires the acyltransferase Porcupine and depends on the attachment of palmitoleic acid to Wnt. The source of this monounsaturated fatty acid has not been identified, and it is not known how Porcupine recognizes its substrate and whether desaturation occurs before or after fatty acid transfer to Wnt. Here, we show that stearoyl desaturase (SCD) generates a monounsaturated fatty acid substrate that is then transferred by Porcupine to Wnt. Treatment of cells with SCD inhibitors blocked incorporation of palmitate analogs into Wnt3a and Wnt5a and reduced Wnt secretion as well as autocrine and paracrine Wnt signaling. The SCD inhibitor effects were rescued by exogenous addition of monounsaturated fatty acids. We propose that SCD is a key molecular player responsible for Wnt biogenesis and processing and that SCD inhibition provides an alternative mechanism for blocking Wnt pathway activation.

  16. Stearoyl CoA desaturase is required to produce active, lipid-modified Wnt proteins

    PubMed Central

    Rios-Esteves, Jessica; Resh, Marilyn D.

    2013-01-01

    Summary Wnt proteins contain an unusual lipid modification, palmitoleic acid. Production of an active Wnt signal requires the acyltransferase Porcupine and depends on attachment of palmitoleic acid to Wnt. The source of this monounsaturated fatty acid has not been identified, and it is not known how Porcupine recognizes its substrate and whether desaturation occurs before or after fatty acid transfer to Wnt. Here we show that stearoyl desaturase (SCD) generates a monounsaturated fatty acid substrate which is then transferred by Porcupine to Wnt. Treatment of cells with SCD inhibitors blocked incorporation of palmitate analogs into Wnt3a and Wnt5a, and reduced Wnt secretion as well as autocrine and paracrine Wnt signaling. The SCD inhibitor effects were rescued by exogenous addition of monounsaturated fatty acids. We propose that SCD is a key molecular player responsible for Wnt biogenesis and processing and that SCD inhibition provides an alternative mechanism for blocking Wnt pathway activation. PMID:24055053

  17. Stearoyl CoA desaturase is required to produce active, lipid-modified Wnt proteins.

    PubMed

    Rios-Esteves, Jessica; Resh, Marilyn D

    2013-09-26

    Wnt proteins contain palmitoleic acid, an unusual lipid modification. Production of an active Wnt signal requires the acyltransferase Porcupine and depends on the attachment of palmitoleic acid to Wnt. The source of this monounsaturated fatty acid has not been identified, and it is not known how Porcupine recognizes its substrate and whether desaturation occurs before or after fatty acid transfer to Wnt. Here, we show that stearoyl desaturase (SCD) generates a monounsaturated fatty acid substrate that is then transferred by Porcupine to Wnt. Treatment of cells with SCD inhibitors blocked incorporation of palmitate analogs into Wnt3a and Wnt5a and reduced Wnt secretion as well as autocrine and paracrine Wnt signaling. The SCD inhibitor effects were rescued by exogenous addition of monounsaturated fatty acids. We propose that SCD is a key molecular player responsible for Wnt biogenesis and processing and that SCD inhibition provides an alternative mechanism for blocking Wnt pathway activation. PMID:24055053

  18. Unraveling Cholesterol Catabolism in Mycobacterium tuberculosis: ChsE4-ChsE5 α2β2 Acyl-CoA Dehydrogenase Initiates β-Oxidation of 3-Oxo-cholest-4-en-26-oyl CoA

    PubMed Central

    2016-01-01

    The metabolism of host cholesterol by Mycobacterium tuberculosis (Mtb) is an important factor for both its virulence and pathogenesis, although how and why cholesterol metabolism is required is not fully understood. Mtb uses a unique set of catabolic enzymes that are homologous to those required for classical β-oxidation of fatty acids but are specific for steroid-derived substrates. Here, we identify and assign the substrate specificities of two of these enzymes, ChsE4-ChsE5 (Rv3504-Rv3505) and ChsE3 (Rv3573c), that carry out cholesterol side chain oxidation in Mtb. Steady-state assays demonstrate that ChsE4-ChsE5 preferentially catalyzes the oxidation of 3-oxo-cholest-4-en-26-oyl CoA in the first cycle of cholesterol side chain β-oxidation that ultimately yields propionyl-CoA, whereas ChsE3 specifically catalyzes the oxidation of 3-oxo-chol-4-en-24-oyl CoA in the second cycle of β-oxidation that generates acetyl-CoA. However, ChsE4-ChsE5 can catalyze the oxidation of 3-oxo-chol-4-en-24-oyl CoA as well as 3-oxo-4-pregnene-20-carboxyl-CoA. The functional redundancy of ChsE4-ChsE5 explains the in vivo phenotype of the igr knockout strain of Mycobacterium tuberculosis; the loss of ChsE1-ChsE2 can be compensated for by ChsE4-ChsE5 during the chronic phase of infection. The X-ray crystallographic structure of ChsE4-ChsE5 was determined to a resolution of 2.0 Å and represents the first high-resolution structure of a heterotetrameric acyl-CoA dehydrogenase (ACAD). Unlike typical homotetrameric ACADs that bind four flavin adenine dinucleotide (FAD) cofactors, ChsE4-ChsE5 binds one FAD at each dimer interface, resulting in only two substrate-binding sites rather than the classical four active sites. A comparison of the ChsE4-ChsE5 substrate-binding site to those of known mammalian ACADs reveals an enlarged binding cavity that accommodates steroid substrates and highlights novel prospects for designing inhibitors against the committed β-oxidation step in the first

  19. Cross sections for production of the CO(A 1 Pi)-(X 1 Sigma) fourth positive band system and O(3 S) by photodissociation of CO2

    NASA Technical Reports Server (NTRS)

    Gentieu, E. P.; Mentall, J. E.

    1972-01-01

    The CO(A 1 Pi) cross sections reported here, along with previously determined electron impact results, establish the basis for calculating CO fourth positive system volume emission rates in the Martian dayglow. Calculated volume emission rates in turn determine relative distribution of photon vs. electron impact as mechanisms for producing CO(A 1 Pi) in the Mars atmosphere. The smallness of the O(1304) cross section confirms previous indirect evidence that photodissociative excitation of CO2 is not an important source of O(3 S) in the upper atmosphere of Mars.

  20. Human DNA Ligase III Recognizes DNA Ends by Dynamic Switching between Two DNA-Bound States

    SciTech Connect

    Cotner-Gohara, Elizabeth; Kim, In-Kwon; Hammel, Michal; Tainer, John A.; Tomkinson, Alan E.; Ellenberger, Tom

    2010-09-13

    Human DNA ligase III has essential functions in nuclear and mitochondrial DNA replication and repair and contains a PARP-like zinc finger (ZnF) that increases the extent of DNA nick joining and intermolecular DNA ligation, yet the bases for ligase III specificity and structural variation among human ligases are not understood. Here combined crystal structure and small-angle X-ray scattering results reveal dynamic switching between two nick-binding components of ligase III: the ZnF-DNA binding domain (DBD) forms a crescent-shaped surface used for DNA end recognition which switches to a ring formed by the nucleotidyl transferase (NTase) and OB-fold (OBD) domains for catalysis. Structural and mutational analyses indicate that high flexibility and distinct DNA binding domain features in ligase III assist both nick sensing and the transition from nick sensing by the ZnF to nick joining by the catalytic core. The collective results support a 'jackknife model' in which the ZnF loads ligase III onto nicked DNA and conformational changes deliver DNA into the active site. This work has implications for the biological specificity of DNA ligases and functions of PARP-like zinc fingers.

  1. The Gp78 ubiquitin ligase: probing endoplasmic reticulum complexity.

    PubMed

    St Pierre, Pascal; Nabi, Ivan R

    2012-02-01

    The endoplasmic reticulum (ER) has been classically divided, based on electron microscopy analysis, into parallel ribosome-studded rough ER sheets and a tubular smooth ER network. Recent studies have identified molecular constituents of the ER, the reticulons and DP1, that drive ER tubule formation and whose expression determines expression of ER sheets and tubules and thereby rough and smooth ER. However, segregation of the ER into only two domains remains simplistic and multiple functionally distinct ER domains necessarily exist. In this review, we will discuss the sub-organization of the ER in different domains focusing on the localization and role of the gp78 ubiquitin ligase in the mitochondria-associated smooth ER and on the evidence for a quality control ERAD domain.

  2. The Ubiquitin Ligase Hul5 Promotes Proteasomal Processivity▿

    PubMed Central

    Aviram, Sharon; Kornitzer, Daniel

    2010-01-01

    The 26S proteasome is a large cytoplasmic protease that degrades polyubiquitinated proteins to short peptides in a processive manner. The proteasome 19S regulatory subcomplex tethers the target protein via its polyubiquitin adduct and unfolds the target polypeptide, which is then threaded into the proteolytic site-containing 20S subcomplex. Hul5 is a 19S subcomplex-associated ubiquitin ligase that elongates ubiquitin chains on proteasome-bound substrates. We isolated hul5Δ as a mutation with which fusions of an unstable cyclin to stable reporter proteins accumulate as partially processed products. These products appear transiently in the wild type but are strongly stabilized in 19S ATPase mutants and in the hul5Δ mutant, supporting a role for the ATPase subunits in the unfolding of proteasome substrates before insertion into the catalytic cavity and suggesting a role for Hul5 in the processive degradation of proteins that are stalled on the proteasome. PMID:20008553

  3. The Gp78 ubiquitin ligase: probing endoplasmic reticulum complexity.

    PubMed

    St Pierre, Pascal; Nabi, Ivan R

    2012-02-01

    The endoplasmic reticulum (ER) has been classically divided, based on electron microscopy analysis, into parallel ribosome-studded rough ER sheets and a tubular smooth ER network. Recent studies have identified molecular constituents of the ER, the reticulons and DP1, that drive ER tubule formation and whose expression determines expression of ER sheets and tubules and thereby rough and smooth ER. However, segregation of the ER into only two domains remains simplistic and multiple functionally distinct ER domains necessarily exist. In this review, we will discuss the sub-organization of the ER in different domains focusing on the localization and role of the gp78 ubiquitin ligase in the mitochondria-associated smooth ER and on the evidence for a quality control ERAD domain. PMID:22045301

  4. Unusually divergent 4-coumarate:CoA-ligases from Ruta graveolens L.

    PubMed

    Endler, Alexander; Martens, Stefan; Wellmann, Frank; Matern, Ulrich

    2008-07-01

    Most angiosperms encode a small family of 4-coumarate:CoA-ligases (4CLs) activating hydroxycinnamic acids for lignin and flavonoid pathways. The common rue, Ruta graveolens L., additionally produces coumarins by cyclization of the 4-coumaroyl moiety, possibly involving the CoA-ester, as well as acridone and furoquinoline alkaloids relying on (N-methyl)anthraniloyl-CoA as the starter substrate for polyketide synthase condensation. The accumulation of alkaloids and coumarins, but not flavonoids, was enhanced in Ruta graveolens suspension cultures upon the addition of fungal elicitor. Total RNA of elicitor-treated Ruta cells was used as template for RT-PCR amplification with degenerate oligonucleotide primers inferred from conserved motifs in AMP-binding proteins, and two full-size cDNAs were generated through RACE and identified as 4-coumarate:CoA-ligases, Rg4CL1 and Rg4CL2, by functional expression in yeast cells. The recombinant enzymes differed considerably in their preferential affinities to cinnamate (Rg4CL1) or ferulate (RgCL2) besides 4-coumarate, but did not activate hydroxybenzoic or (N-methyl)anthranilic acid. Most notably, the Rg4CL1 polypeptide included an N-terminal extension suggesting a chloroplast transit peptide. The genes were cloned and revealed four exons, separated by 1056, 94 and 54 bp introns for RgCL1, while Rg4CL2 was composed of five exons interupted by four introns from 113 to 350 bp, and the divergent heritage of these genes was substantiated by phylogenetic analysis. Both genes were expressed in shoot, leaf and flower tissues of adult Ruta plants with preference in shoot and flower, whereas negligible expression occurred in the root. However, Rg4CL1 was expressed much stronger in the flower, while Rg4CL2 was expressed mostly in the shoot. Furthermore, considerable transient induction of only Rg4CL1 was observed upon elicitation of Ruta cells, which seems to support a role of Rg4CL1 in coumarin biosynthesis.

  5. The Arabidopsis MIEL1 E3 ligase negatively regulates ABA signalling by promoting protein turnover of MYB96.

    PubMed

    Lee, Hong Gil; Seo, Pil Joon

    2016-01-01

    The phytohormone abscisic acid (ABA) regulates plant responses to various environmental challenges. Controlled protein turnover is an important component of ABA signalling. Here we show that the RING-type E3 ligase MYB30-INTERACTING E3 LIGASE 1 (MIEL1) regulates ABA sensitivity by promoting MYB96 turnover in Arabidopsis. Germination of MIEL1-deficient mutant seeds is hypersensitive to ABA, whereas MIEL1-overexpressing transgenic seeds are less sensitive. MIEL1 can interact with MYB96, a regulator of ABA signalling, and stimulate its ubiquitination and degradation. Genetic analysis shows that MYB96 is epistatic to MIEL1 in the control of ABA sensitivity in seeds. While MIEL1 acts primarily via MYB96 in seed germination, MIEL1 regulates protein turnover of both MYB96 and MYB30 in vegetative tissues. We find that ABA regulates the expression of MYB30-responsive genes during pathogen infection and this regulation is partly dependent on MIEL1. These results suggest that MIEL1 may facilitate crosstalk between ABA and biotic stress signalling. PMID:27615387

  6. The Arabidopsis MIEL1 E3 ligase negatively regulates ABA signalling by promoting protein turnover of MYB96

    PubMed Central

    Lee, Hong Gil; Seo, Pil Joon

    2016-01-01

    The phytohormone abscisic acid (ABA) regulates plant responses to various environmental challenges. Controlled protein turnover is an important component of ABA signalling. Here we show that the RING-type E3 ligase MYB30-INTERACTING E3 LIGASE 1 (MIEL1) regulates ABA sensitivity by promoting MYB96 turnover in Arabidopsis. Germination of MIEL1-deficient mutant seeds is hypersensitive to ABA, whereas MIEL1-overexpressing transgenic seeds are less sensitive. MIEL1 can interact with MYB96, a regulator of ABA signalling, and stimulate its ubiquitination and degradation. Genetic analysis shows that MYB96 is epistatic to MIEL1 in the control of ABA sensitivity in seeds. While MIEL1 acts primarily via MYB96 in seed germination, MIEL1 regulates protein turnover of both MYB96 and MYB30 in vegetative tissues. We find that ABA regulates the expression of MYB30-responsive genes during pathogen infection and this regulation is partly dependent on MIEL1. These results suggest that MIEL1 may facilitate crosstalk between ABA and biotic stress signalling. PMID:27615387

  7. Protein Phosphatase 2A Stabilizes Human Securin, Whose Phosphorylated Forms Are Degraded via the SCF Ubiquitin Ligase

    PubMed Central

    Gil-Bernabé, Ana M.; Romero, Francisco; Limón-Mortés, M. Cristina; Tortolero, María

    2006-01-01

    Sister chromatid segregation is triggered at the metaphase-to-anaphase transition by the activation of the protease separase. For most of the cell cycle, separase activity is kept in check by its association with the inhibitory chaperone securin. Activation of separase occurs at anaphase onset, when securin is targeted for destruction by the anaphase-promoting complex or cyclosome E3 ubiquitin protein ligase. This results in the release of the cohesins from chromosomes, which in turn allows the segregation of sister chromatids to opposite spindle poles. Here we show that human securin (hSecurin) forms a complex with enzymatically active protein phosphatase 2A (PP2A) and that it is a substrate of the phosphatase, both in vitro and in vivo. Treatment of cells with okadaic acid, a potent inhibitor of PP2A, results in various hyperphosphorylated forms of hSecurin which are extremely unstable, due to the action of the Skp1/Cul1/F-box protein complex ubiquitin ligase. We propose that PP2A regulates hSecurin levels by counteracting its phosphorylation, which promotes its degradation. Misregulation of this process may lead to the formation of tumors, in which overproduction of hSecurin is often observed. PMID:16705156

  8. The U-Box/ARM E3 ligase PUB13 regulates cell death, defense, and flowering time in Arabidopsis.

    PubMed

    Li, Wei; Ahn, Il-Pyung; Ning, Yuese; Park, Chan-Ho; Zeng, Lirong; Whitehill, Justin G A; Lu, Haibin; Zhao, Qingzhen; Ding, Bo; Xie, Qi; Zhou, Jian-Min; Dai, Liangying; Wang, Guo-Liang

    2012-05-01

    The components in plant signal transduction pathways are intertwined and affect each other to coordinate plant growth, development, and defenses to stresses. The role of ubiquitination in connecting these pathways, particularly plant innate immunity and flowering, is largely unknown. Here, we report the dual roles for the Arabidopsis (Arabidopsis thaliana) Plant U-box protein13 (PUB13) in defense and flowering time control. In vitro ubiquitination assays indicated that PUB13 is an active E3 ubiquitin ligase and that the intact U-box domain is required for the E3 ligase activity. Disruption of the PUB13 gene by T-DNA insertion results in spontaneous cell death, the accumulation of hydrogen peroxide and salicylic acid (SA), and elevated resistance to biotrophic pathogens but increased susceptibility to necrotrophic pathogens. The cell death, hydrogen peroxide accumulation, and resistance to necrotrophic pathogens in pub13 are enhanced when plants are pretreated with high humidity. Importantly, pub13 also shows early flowering under middle- and long-day conditions, in which the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 and FLOWERING LOCUS T is induced while FLOWERING LOCUS C expression is suppressed. Finally, we found that two components involved in the SA-mediated signaling pathway, SID2 and PAD4, are required for the defense and flowering-time phenotypes caused by the loss of function of PUB13. Taken together, our data demonstrate that PUB13 acts as an important node connecting SA-dependent defense signaling and flowering time regulation in Arabidopsis.

  9. Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids.

    PubMed

    Melton, Elaina M; Cerny, Ronald L; DiRusso, Concetta C; Black, Paul N

    2013-11-01

    In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of

  10. Human ITCH E3 ubiquitin ligase deficiency causes syndromic multisystem autoimmune disease.

    PubMed

    Lohr, Naomi J; Molleston, Jean P; Strauss, Kevin A; Torres-Martinez, Wilfredo; Sherman, Eric A; Squires, Robert H; Rider, Nicholas L; Chikwava, Kudakwashe R; Cummings, Oscar W; Morton, D Holmes; Puffenberger, Erik G

    2010-03-12

    Ubiquitin ligases play an important role in the regulation of the immune system. Absence of Itch E3 ubiquitin ligase in mice has been shown to cause severe autoimmune disease. Using autozygosity mapping in a large Amish kindred, we identified a linkage region on chromosome 20 and selected candidate genes for screening. We describe, in ten patients, identification of a mutation resulting in truncation of ITCH. These patients represent the first reported human phenotype associated with ITCH deficiency. These patients not only have multisystem autoimmune disease but also display morphologic and developmental abnormalities. This disorder underscores the importance of ITCH ubiquitin ligase in many cellular processes. PMID:20170897

  11. Human ITCH E3 Ubiquitin Ligase Deficiency Causes Syndromic Multisystem Autoimmune Disease

    PubMed Central

    Lohr, Naomi J.; Molleston, Jean P.; Strauss, Kevin A.; Torres-Martinez, Wilfredo; Sherman, Eric A.; Squires, Robert H.; Rider, Nicholas L.; Chikwava, Kudakwashe R.; Cummings, Oscar W.; Morton, D. Holmes; Puffenberger, Erik G.

    2010-01-01

    Ubiquitin ligases play an important role in the regulation of the immune system. Absence of Itch E3 ubiquitin ligase in mice has been shown to cause severe autoimmune disease. Using autozygosity mapping in a large Amish kindred, we identified a linkage region on chromosome 20 and selected candidate genes for screening. We describe, in ten patients, identification of a mutation resulting in truncation of ITCH. These patients represent the first reported human phenotype associated with ITCH deficiency. These patients not only have multisystem autoimmune disease but also display morphologic and developmental abnormalities. This disorder underscores the importance of ITCH ubiquitin ligase in many cellular processes. PMID:20170897

  12. Characterization of Chlamydia MurC-Ddl, a fusion protein exhibiting D-alanyl-D-alanine ligase activity involved in peptidoglycan synthesis and D-cycloserine sensitivity.

    PubMed

    McCoy, Andrea J; Maurelli, Anthony T

    2005-07-01

    Recent characterization of chlamydial genes encoding functional peptidoglycan (PG)-synthesis proteins suggests that the Chlamydiaceae possess the ability to synthesize PG yet biochemical evidence for the synthesis of PG has yet to be demonstrated. The presence of D-amino acids in PG is a hallmark of bacteria. Chlamydiaceae do not appear to encode amino acid racemases however, a D-alanyl-D-alanine (D-Ala-D-Ala) ligase homologue (Ddl) is encoded in the genome. Thus, we undertook a genetics-based approach to demonstrate and characterize the D-Ala-D-Ala ligase activity of chlamydial Ddl, a protein encoded as a fusion with MurC. The full-length murC-ddl fusion gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible ara promoter and transformed into a D-Ala-D-Ala ligase auxotroph of Escherichia coli possessing deletions of both the ddlA and ddlB genes. Viability of the E. coliDeltaddlADeltaddlB mutant in the absence of exogenous D-Ala-D-Ala dipeptide became dependent on the expression of the chlamydial murC-ddl thus demonstrating functional ligase activity. Domain mapping of the full-length fusion protein and site-directed mutagenesis of the MurC domain revealed that the structure of the full fusion protein but not MurC enzymatic activity was required for ligase activity in vivo. Recombinant MurC-Ddl exhibited substrate specificity for D-Ala. Chlamydia growth is inhibited by D-cycloserine (DCS) and in vitro analysis provided evidence for the chlamydial MurC-Ddl as the target for DCS sensitivity. In vivo sensitivity to DCS could be reversed by addition of exogenous D-Ala and D-Ala-D-Ala. Together, these findings further support our hypothesis that PG is synthesized by members of the Chlamydiaceae family and suggest that D-amino acids, specifically D-Ala, are present in chlamydial PG.

  13. Human mitochondrial HMG CoA synthase: Liver cDNA and partial genomic cloning, chromosome mapping to 1p12-p13, and possible role in vertebrate evolution

    SciTech Connect

    Boukaftane, Y.; Robert, M.F.; Mitchell, G.A.

    1994-10-01

    Mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase (mHS) is the first enzyme of ketogenesis, whereas the cytoplasmic HS isozyme (cHS) mediates an early step in cholersterol synthesis. We here report the sequence of human and mouse liver mHS cDNAs, the sequence of an HS-like cDNA from Caenorhabditis elegans, the structure of a partial human mHS genomic clone, and the mapping of the human mHS gene to chromosome 1p12-p13. the nucleotide sequence of the human mHS cDNA encodes a mature mHS peptide of 471 residues, with a mean amino acid identity of 66.5% with cHS from mammals and chicken. Comparative analysis of all known mHS and cHS protein and DNA sequences shows a high degree of conservation near the N-terminus that decreases progressively toward the C-terminus and suggests that the two isozymes arose from a common ancestor gene 400-900 million years ago. Comparison of the gene structure of mHS and cHS is also consistant with a recent duplication event. We hypothesize that the physiologic result of the HS gene duplication was the appearance of HS within the mitochondria around the time of emergence of early vertebrates, which linked preexisting pathways of beta oxidation and leucine catabolism and created the HMG CoA pathway of ketogenesis, thus providing a lipid-derived energy source for the vertebrate brain. 56 refs., 4 figs., 2 tabs.

  14. Sequencing of the ddl gene and modeling of the mutated D-alanine:D-alanine ligase in glycopeptide-dependent strains of Enterococcus faecium.

    PubMed

    Gholizadeh, Y; Prevost, M; Van Bambeke, F; Casadewall, B; Tulkens, P M; Courvalin, P

    2001-04-01

    Glycopeptide dependence for growth in enterococci results from mutations in the ddl gene that inactivate the host D-Ala:D-Ala ligase. The strains require glycopeptides as inducers for synthesis of resistance proteins, which allows for the production of peptidoglycan precursors ending in D-Ala-D-Lac instead of D-Ala-D-Ala. The sequences of the ddl gene from nine glycopeptide-dependent Enterococcus faecium clinical isolates were determined. Each one had a mutation consisting either in a 5-bp insertion at position 41 leading to an early stop codon, an in-frame 6-bp deletion causing the loss of two residues (KDVA243-246 to KA), or single base-pair changes resulting in an amino acid substitution (E13 --> G, G99 --> R, V241 --> D, D295 --> G, P313 --> L). The potential consequences of the deletion and point mutations on the 3-D structure of the enzyme were evaluated by comparative molecular modeling of the E. faecium enzyme, using the X-ray structure of the homologous Escherichia coli D-Ala:D-Ala ligase DdlB as a template. All mutated residues were found either to interact directly with one of the substrates of the enzymatic reaction (E13 and D295) or to stabilize the position of critical residues in the active site. Maintenance of the 3-D structure in the vicinity of these mutations in the active site appears critical for D-Ala:D-Ala ligase activity.

  15. Arabidopsis RZFP34/CHYR1, a Ubiquitin E3 Ligase, Regulates Stomatal Movement and Drought Tolerance via SnRK2.6-Mediated Phosphorylation[OPEN

    PubMed Central

    2015-01-01

    Abscisic acid (ABA) is a phytohormone that plays a fundamental role in plant development and stress response, especially in the regulation of stomatal closure in response to water deficit stress. The signal transduction that occurs in response to ABA and drought stress is mediated by protein phosphorylation and ubiquitination. This research identified Arabidopsis thaliana RING ZINC-FINGER PROTEIN34 (RZP34; renamed here as CHY ZINC-FINGER AND RING PROTEIN1 [CHYR1]) as an ubiquitin E3 ligase. CHYR1 expression was significantly induced by ABA and drought, and along with its corresponding protein, was expressed mainly in vascular tissues and stomata. Analysis of CHYR1 gain-of-function and loss-of-function plants revealed that CHYR1 promotes ABA-induced stomatal closure, reactive oxygen species production, and plant drought tolerance. Furthermore, CHYR1 interacted with SNF1-RELATED PROTEIN KINASE2 (SnRK2) kinases and could be phosphorylated by SnRK2.6 on the Thr-178 residue. Overexpression of CHYR1T178A, a phosphorylation-deficient mutant, interfered with the proper function of CHYR1, whereas CHYR1T178D phenocopied the gain of function of CHYR1. Thus, this study identified a RING-type ubiquitin E3 ligase that functions positively in ABA and drought responses and detailed how its ubiquitin E3 ligase activity is regulated by SnRK2.6-mediated protein phosphorylation. PMID:26508764

  16. QSAR and Molecular Docking Studies of Oxadiazole-Ligated Pyrrole Derivatives as Enoyl-ACP (CoA) Reductase Inhibitors

    PubMed Central

    Asgaonkar, Kalyani D.; Mote, Ganesh D.; Chitre, Trupti S.

    2014-01-01

    A quantitative structure-activity relationship model was developed on a series of compounds containing oxadiazole-ligated pyrrole pharmacophore to identify key structural fragments required for anti-tubercular activity. Two-dimensional (2D) and three-dimensional (3D) QSAR studies were performed using multiple linear regression (MLR) analysis and k-nearest neighbour molecular field analysis (kNN-MFA), respectively. The developed QSAR models were found to be statistically significant with respect to training, cross-validation, and external validation. New chemical entities (NCEs) were designed based on the results of the 2D- and 3D-QSAR. NCEs were subjected to Lipinski’s screen to ensure the drug-like pharmacokinetic profile of the designed compounds in order to improve their bioavailability. Also, the binding ability of the NCEs with enoyl-ACP (CoA) reductase was assessed by docking. PMID:24634843

  17. Characterization of the JWST Pathfinder mirror dynamics using the center of curvature optical assembly (CoCOA)

    NASA Astrophysics Data System (ADS)

    Wells, Conrad; Hadaway, James B.; Olczak, Gene; Cosentino, Joseph; Johnston, John D.; Whitman, Tony; Connolly, Mark; Chaney, David; Knight, J. Scott; Telfer, Randal

    2016-07-01

    The James Webb Space Telescope (JWST) Optical Telescope Element (OTE) consists of a 6.6 m clear aperture, 18 segment primary mirror, all-reflective, three-mirror anastigmat operating at cryogenic temperatures. To verify performance of the primary mirror, a full aperture center of curvature optical null test is performed under cryogenic conditions in Chamber A at the National Aeronautics and Space Administration (NASA) Johnson Space Center (JSC) using an instantaneous phase measuring interferometer. After phasing the mirrors during the JWST Pathfinder testing, the interferometer is utilized to characterize the mirror relative piston and tilt dynamics under different facility configurations. The correlation between the motions seen on detectors at the focal plane and the interferometer validates the use of the interferometer for dynamic investigations. The success of planned test hardware improvements will be characterized by the multi-wavelength interferometer (MWIF) at the Center of Curvature Optical Assembly (CoCOA).

  18. Characterization of the JWST Pathfinder Mirror Dynamics Using the Center of Curvature Optical Assembly (CoCOA)

    NASA Technical Reports Server (NTRS)

    Wells, Conrad; Hadaway, James B.; Olczak, Gene; Cosentino, Joseph; Johnston, John D.; Whitman, Tony; Connolly, Mark; Chaney, David; Knight, J. Scott; Telfer, Randal

    2016-01-01

    The JWST (James Webb Space Telescope) Optical Telescope Element (OTE) consists of a 6.6 meter clear aperture, 18-segment primary mirror, all-reflective, three-mirror anastigmat operating at cryogenic temperatures. To verify performance of the primary mirror, a full aperture center of curvature optical null test is performed under cryogenic conditions in Chamber A at NASA Johnson Space Center using an instantaneous phase measuring interferometer. After phasing the mirrors during the JWST Pathfinder testing, the interferometer is utilized to characterize the mirror relative piston and tilt dynamics under different facility configurations. The correlation between the motions seen on detectors at the focal plane and the interferometer validates the use of the interferometer for dynamic investigations. The success of planned test hardware improvements will be characterized by the multi-wavelength interferometer (MWIF) at the Center of Curvature Optical Assembly (CoCOA).

  19. The RING E3 Ligase KEEP ON GOING Modulates JASMONATE ZIM-DOMAIN12 Stability1[OPEN

    PubMed Central

    Pauwels, Laurens; Ritter, Andrés; Goossens, Jonas; Durand, Astrid Nagels; Liu, Hongxia; Gu, Yangnan; Geerinck, Jan; Boter, Marta; Vanden Bossche, Robin; De Clercq, Rebecca; Van Leene, Jelle; Gevaert, Kris; De Jaeger, Geert; Solano, Roberto; Stone, Sophia; Innes, Roger W.; Callis, Judy; Goossens, Alain

    2015-01-01

    Jasmonate (JA) signaling in plants is mediated by the JASMONATE ZIM-DOMAIN (JAZ) proteins that repress the activity of several transcription factors regulating JA-inducible gene expression. The hormone JA-isoleucine triggers the interaction of JAZ repressor proteins with the F-box protein CORONATINE INSENSITIVE1 (COI1), part of an S-phase kinase-associated protein1/Cullin1/F-box protein COI1 (SCFCOI1) E3 ubiquitin ligase complex, and their degradation by the 26S proteasome. In Arabidopsis (Arabidopsis thaliana), the JAZ family consists of 13 members. The level of redundancy or specificity among these members is currently not well understood. Here, we characterized JAZ12, encoded by a highly expressed JAZ gene. JAZ12 interacted with the transcription factors MYC2, MYC3, and MYC4 in vivo and repressed MYC2 activity. Using tandem affinity purification, we found JAZ12 to interact with SCFCOI1 components, matching with observed in vivo ubiquitination and with rapid degradation after treatment with JA. In contrast to the other JAZ proteins, JAZ12 also interacted directly with the E3 RING ligase KEEP ON GOING (KEG), a known repressor of the ABSCISIC ACID INSENSITIVE5 transcription factor in abscisic acid signaling. To study the functional role of this interaction, we circumvented the lethality of keg loss-of-function mutants by silencing KEG using an artificial microRNA approach. Abscisic acid treatment promoted JAZ12 degradation, and KEG knockdown led to a decrease in JAZ12 protein levels. Correspondingly, KEG overexpression was capable of partially inhibiting COI1-mediated JAZ12 degradation. Our results provide additional evidence for KEG as an important factor in plant hormone signaling and a positive regulator of JAZ12 stability. PMID:26320228

  20. Structural Basis for Nick Recognition by a Minimal Pluripotent DNA Ligase

    SciTech Connect

    Nair,P.; Nandakumar, J.; Smith, P.; Odell, M.; Lima, C.; Shuman, S.

    2007-01-01

    Chlorella virus DNA ligase, the smallest eukaryotic ligase known, has pluripotent biological activity and an intrinsic nick-sensing function, despite having none of the accessory domains found in cellular ligases. A 2.3-{angstrom} crystal structure of the Chlorella virus ligase-AMP intermediate bound to duplex DNA containing a 3'-OH-5'-PO{sub 4} nick reveals a new mode of DNA envelopment, in which a short surface loop emanating from the OB domain forms a {beta}-hairpin 'latch' that inserts into the DNA major groove flanking the nick. A network of interactions with the 3'-OH and 5'-PO{sub 4} termini in the active site illuminates the DNA adenylylation mechanism and the crucial roles of AMP in nick sensing and catalysis. Addition of a divalent cation triggered nick sealing in crystallo, establishing that the nick complex is a bona fide intermediate in the DNA repair pathway.

  1. The CUL3-KLHL18 ligase regulates mitotic entry and ubiquitylates Aurora-A.

    PubMed

    Moghe, Saili; Jiang, Fei; Miura, Yoshie; Cerny, Ronald L; Tsai, Ming-Ying; Furukawa, Manabu

    2012-02-15

    The cullin-RING family of ubiquitin ligases regulates diverse cellular functions, such as cell cycle control, via ubiquitylation of specific substrates. CUL3 targets its substrates through BTB proteins. Here we show that depletion of CUL3 and the BTB protein KLHL18 causes a delay in mitotic entry. Centrosomal activation of Aurora-A, a kinase whose activity is required for entry into mitosis, is also delayed in depleted cells. Moreover, we identify Aurora-A as a KLHL18-interacting partner. Overexpression of KLHL18 and CUL3 promotes Aurora-A ubiquitylation in vivo, and the CUL3-KLHL18-ROC1 ligase ubiquitylates Aurora-A in vitro. Our study reveals that the CUL3-KLHL18 ligase is required for timely entry into mitosis, as well as for the activation of Aurora-A at centrosomes. We propose that the CUL3-KLHL18 ligase regulates mitotic entry through an Aurora-A-dependent pathway.

  2. Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells

    PubMed Central

    Arakawa, Hiroshi; Bednar, Theresa; Wang, Minli; Paul, Katja; Mladenov, Emil; Bencsik-Theilen, Alena A.; Iliakis, George

    2012-01-01

    In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-joining (NHEJ) pathway. The evolutionary younger DNA ligase III (LigIII) is restricted to higher eukaryotes and has been associated with base excision (BER) and single strand break repair (SSBR). Here, using conditional knockout strategies for LIG3 and concomitant inactivation of the LIG1 and LIG4 genes, we show that in DT40 cells LigIII efficiently supports semi-conservative DNA replication. Our observations demonstrate a high functional versatility for the evolutionary new LigIII in DNA replication and mitochondrial metabolism, and suggest the presence of an alternative pathway for Okazaki fragment ligation. PMID:22127868

  3. Structure of the DNA Ligase-Adenylate Intermediate: Lysine (ε-amino)-Linked Adenosine Monophosphoramidate*

    PubMed Central

    Gumport, Richard I.; Lehman, I. R.

    1971-01-01

    Proteolytic degradation of the Escherichia coli DNA ligase-adenylate intermediate releases adenosine 5′-monophosphate linked to the ε-amino group of lysine by a phosphoamide bond. Measurements of the rate of hydroxylaminolysis of the ligase-adenylate provide further support for a phosphoamide linkage in the native enzyme. Lysine (ε-amino)-linked adenosine monophosphoramidate has also been isolated from the T4 phage-induced ligase-adenylate intermediate. These results indicate that an initial step of the DNA ligase reaction consists of the nucleophilic attack of the ε-amino group of a lysine residue of the enzyme on the adenylyl phosphorus of DPN or ATP that leads to the formation of enzyme-bound lysine (εamino)-linked adenosine monophosphoramidate. PMID:4944632

  4. Biochemical characterization of an inhibitor of Escherichia coli UDP-N-acetylmuramyl-l-alanine ligase.

    PubMed

    Ehmann, David E; Demeritt, Julie E; Hull, Kenneth G; Fisher, Stewart L

    2004-05-01

    UDP-N-acetylmuramyl-l-alanine ligase (MurC) is an essential bacterial enzyme involved in peptidoglycan biosynthesis and a target for the discovery of novel antibacterial agents. As a result of a high-throughput screen (HTS) against a chemical library for inhibitors of MurC, a series of benzofuran acyl-sulfonamides was identified as potential leads. One of these compounds, Compound A, inhibited Escherichia coli MurC with an IC(50) of 2.3 microM. Compound A exhibited time-dependent, partially reversible inhibition of E. coli MurC. Kinetic studies revealed a mode of inhibition consistent with the compound acting competitively with the MurC substrates ATP and UDP-N-acetyl-muramic acid (UNAM) with a K(i) of 4.5 microM against ATP and 6.3 microM against UNAM. Fluorescence binding experiments yielded a K(d) of 3.1 microM for the compound binding to MurC. Compound A also exhibited high-affinity binding to bovine serum albumin (BSA) as evidenced by a severe reduction in MurC inhibition upon addition of BSA. This finding is consistent with the high lipophilicity of the compound. Advancement of this compound series for further drug development will require reduction of albumin binding. PMID:15134649

  5. Membrane-Associated Ubiquitin Ligase SAUL1 Suppresses Temperature- and Humidity-Dependent Autoimmunity in Arabidopsis.

    PubMed

    Disch, Eva-Maria; Tong, Meixuezi; Kotur, Tanja; Koch, Gerald; Wolf, Carl-Asmus; Li, Xin; Hoth, Stefan

    2016-01-01

    Plants have evolved elaborate mechanisms to regulate pathogen defense. Imbalances in this regulation may result in autoimmune responses that are affecting plant growth and development. In Arabidopsis, SAUL1 encodes a plant U-box ubiquitin ligase and regulates senescence and cell death. Here, we show that saul1-1 plants exhibit characteristics of an autoimmune mutant. A decrease in relative humidity or temperature resulted in reduced growth and systemic lesioning of saul1-1 rosettes. These physiological changes are associated with increased expression of salicylic acid-dependent and pathogenesis-related (PR) genes. Consistently, resistance of saul1-1 plants against Pseudomonas syringae pv. maculicola ES4326, P. syringae pv. tomato DC3000, or Hyaloperonospora arabidopsidis Noco2 was enhanced. Transmission electron microscopy revealed alterations in saul1-1 chloroplast ultrastructure and cell-wall depositions. Confocal analysis on aniline blue-stained leaf sections and cellular universal micro spectrophotometry further showed that these cell-wall depositions contain callose and lignin. To analyze signaling downstream of SAUL1, we performed epistasis analyses between saul1-1 and mutants in the EDS1/PAD4/SAG101 hub. All phenotypes observed in saul1-1 plants at low temperature were dependent on EDS1 and PAD4 but not SAG101. Taken together, SAUL1 negatively regulates immunity upstream of EDS1/PAD4, likely through the degradation of an unknown activator of the pathway.

  6. A Human Ubiquitin Conjugating Enzyme (E2)-HECT E3 Ligase Structure-function Screen*

    PubMed Central

    Sheng, Yi; Hong, Jenny H.; Doherty, Ryan; Srikumar, Tharan; Shloush, Jonathan; Avvakumov, George V.; Walker, John R.; Xue, Sheng; Neculai, Dante; Wan, Janet W.; Kim, Sung K.; Arrowsmith, Cheryl H.; Raught, Brian; Dhe-Paganon, Sirano

    2012-01-01

    Here we describe a systematic structure-function analysis of the human ubiquitin (Ub) E2 conjugating proteins, consisting of the determination of 15 new high-resolution three-dimensional structures of E2 catalytic domains, and autoubiquitylation assays for 26 Ub-loading E2s screened against a panel of nine different HECT (homologous to E6-AP carboxyl terminus) E3 ligase domains. Integration of our structural and biochemical data revealed several E2 surface properties associated with Ub chain building activity; (1) net positive or neutral E2 charge, (2) an “acidic trough” located near the catalytic Cys, surrounded by an extensive basic region, and (3) similarity to the previously described HECT binding signature in UBE2L3 (UbcH7). Mass spectrometry was used to characterize the autoubiquitylation products of a number of functional E2-HECT pairs, and demonstrated that HECT domains from different subfamilies catalyze the formation of very different types of Ub chains, largely independent of the E2 in the reaction. Our data set represents the first comprehensive analysis of E2-HECT E3 interactions, and thus provides a framework for better understanding the molecular mechanisms of ubiquitylation. PMID:22496338

  7. The -4 phenylalanine is required for substrate ubiquitination catalyzed by HECT ubiquitin ligases.

    PubMed

    Salvat, Catherine; Wang, Guangli; Dastur, Anahita; Lyon, Nancy; Huibregtse, Jon M

    2004-04-30

    The reaction cycle of HECT domain ubiquitin ligases consists of three steps: 1) binding of an E2 protein, 2) transfer of ubiquitin from E2 to the HECT domain, and 3) transfer of ubiquitin to the substrate. We report the identification of a determinant that is specifically required for the last step of this cycle, a phenylalanine residue located four amino acids from the C terminus of most HECT domains, referred to here as the -4F. Alteration of this residue in human E6AP and Saccharomyces cerevisae Rsp5p did not affect ubiquitin-thioester formation, but effectively blocked substrate ubiquitination. Alteration of the -4F to alanine with concomitant substitution of a nearby residue to phenylalanine only partially restored Rsp5p activity, indicating that precise spatial placement of this residue is important. C-terminally extended E6AP and Rsp5p proteins were also defective for substrate ubiquitination, providing a likely biochemical understanding of a previously isolated Angelman syndrome-associated mutation of E6AP that alters the stop codon of an otherwise wild-type gene. We propose that the -4F may play a role in orienting ubiquitin when it is tethered to the HECT active site cysteine. This may be necessary to allow for approach of the incoming lysine epsilon-amino group of the substrate.

  8. Proteomic analysis and identification of cellular interactors of the giant ubiquitin ligase HERC2.

    PubMed

    Galligan, Jeffrey T; Martinez-Noël, Gustavo; Arndt, Verena; Hayes, Sebastian; Chittenden, Thomas W; Harper, J Wade; Howley, Peter M

    2015-02-01

    HERC2 is a large E3 ubiquitin ligase with multiple structural domains that has been implicated in an array of cellular processes. Mutations in HERC2 are linked to developmental delays and impairment caused by nervous system dysfunction, such as Angelman Syndrome and autism-spectrum disorders. However, HERC2 cellular activity and regulation remain poorly understood. We used a broad proteomic approach to survey the landscape of cellular proteins that interact with HERC2. We identified nearly 300 potential interactors, a subset of which we validated binding to HERC2. The potential HERC2 interactors included the eukaryotic translation initiation factor 3 complex, the intracellular transport COPI coatomer complex, the glycogen regulator phosphorylase kinase, beta-catenin, PI3 kinase, and proteins involved in fatty acid transport and iron homeostasis. Through a complex bioinformatic analysis of potential interactors, we linked HERC2 to cellular processes including intracellular protein trafficking and transport, metabolism of cellular energy, and protein translation. Given its size, multidomain structure, and association with various cellular activities, HERC2 may function as a scaffold to integrate protein complexes and bridge critical cellular pathways. This work provides a significant resource with which to interrogate HERC2 function more deeply and evaluate its contributions to mechanisms governing cellular homeostasis and disease. PMID:25476789

  9. An allosteric inhibitor of substrate recognition by the SCF[superscript Cdc4] ubiquitin ligase

    SciTech Connect

    Orlicky, Stephen; Tang, Xiaojing; Neduva, Victor; Elowe, Nadine; Brown, Eric D.; Sicheri, Frank; Tyers, Mike

    2010-09-17

    The specificity of SCF ubiquitin ligase-mediated protein degradation is determined by F-box proteins. We identified a biplanar dicarboxylic acid compound, called SCF-I2, as an inhibitor of substrate recognition by the yeast F-box protein Cdc4 using a fluorescence polarization screen to monitor the displacement of a fluorescein-labeled phosphodegron peptide. SCF-I2 inhibits the binding and ubiquitination of full-length phosphorylated substrates by SCF{sup Cdc4}. A co-crystal structure reveals that SCF-I2 inserts itself between the {beta}-strands of blades 5 and 6 of the WD40 propeller domain of Cdc4 at a site that is 25 {angstrom} away from the substrate binding site. Long-range transmission of SCF-I2 interactions distorts the substrate binding pocket and impedes recognition of key determinants in the Cdc4 phosphodegron. Mutation of the SCF-I2 binding site abrogates its inhibitory effect and explains specificity in the allosteric inhibition mechanism. Mammalian WD40 domain proteins may exhibit similar allosteric responsiveness and hence represent an extensive class of druggable target.

  10. Transcriptional repressor NIR interacts with the p53-inhibiting ubiquitin ligase MDM2

    PubMed Central

    Heyne, Kristina; Förster, Juliane; Schüle, Roland; Roemer, Klaus

    2014-01-01

    NIR (novel INHAT repressor) can bind to p53 at promoters and inhibit p53-mediated gene transactivation by blocking histone acetylation carried out by p300/CBP. Like NIR, the E3 ubiquitin ligase MDM2 can also bind and inhibit p53 at promoters. Here, we present data indicating that NIR, which shuttles between the nucleolus and nucleoplasm, not only binds to p53 but also directly to MDM2, in part via the central acidic and zinc finger domain of MDM2 that is also contacted by several other nucleolus-based MDM2/p53-regulating proteins. Like some of these, NIR was able to inhibit the ubiquitination of MDM2 and stabilize MDM2; however, unlike these nucleolus-based MDM2 regulators, NIR did not inhibit MDM2 to activate p53. Rather, NIR cooperated with MDM2 to repress p53-induced transactivation. This cooperative repression may at least in part involve p300/CBP. We show that NIR can block the acetylation of p53 and MDM2. Non-acetylated p53 has been documented previously to more readily associate with inhibitory MDM2. NIR may thus help to sustain the inhibitory p53:MDM2 complex, and we present evidence suggesting that all three proteins can indeed form a ternary complex. In sum, our findings suggest that NIR can support MDM2 to suppress p53 as a transcriptional activator. PMID:24413661

  11. Fragment-based discovery of 6-azaindazoles as inhibitors of bacterial DNA ligase.

    PubMed

    Howard, Steven; Amin, Nader; Benowitz, Andrew B; Chiarparin, Elisabetta; Cui, Haifeng; Deng, Xiaodong; Heightman, Tom D; Holmes, David J; Hopkins, Anna; Huang, Jianzhong; Jin, Qi; Kreatsoulas, Constantine; Martin, Agnes C L; Massey, Frances; McCloskey, Lynn; Mortenson, Paul N; Pathuri, Puja; Tisi, Dominic; Williams, Pamela A

    2013-12-12

    Herein we describe the application of fragment-based drug design to bacterial DNA ligase. X-ray crystallography was used to guide structure-based optimization of a fragment-screening hit to give novel, nanomolar, AMP-competitive inhibitors. The lead compound 13 showed antibacterial activity across a range of pathogens. Data to demonstrate mode of action was provided using a strain of S. aureus, engineered to overexpress DNA ligase. PMID:24900632

  12. Fragment-Based Discovery of 6-Azaindazoles As Inhibitors of Bacterial DNA Ligase

    PubMed Central

    2013-01-01

    Herein we describe the application of fragment-based drug design to bacterial DNA ligase. X-ray crystallography was used to guide structure-based optimization of a fragment-screening hit to give novel, nanomolar, AMP-competitive inhibitors. The lead compound 13 showed antibacterial activity across a range of pathogens. Data to demonstrate mode of action was provided using a strain of S. aureus, engineered to overexpress DNA ligase. PMID:24900632

  13. Inhibitors of Fatty Acid Synthesis Induce PPAR α -Regulated Fatty Acid β -Oxidative Genes: Synergistic Roles of L-FABP and Glucose.

    PubMed

    Huang, Huan; McIntosh, Avery L; Martin, Gregory G; Petrescu, Anca D; Landrock, Kerstin K; Landrock, Danilo; Kier, Ann B; Schroeder, Friedhelm

    2013-01-01

    While TOFA (acetyl CoA carboxylase inhibitor) and C75 (fatty acid synthase inhibitor) prevent lipid accumulation by inhibiting fatty acid synthesis, the mechanism of action is not simply accounted for by inhibition of the enzymes alone. Liver fatty acid binding protein (L-FABP), a mediator of long chain fatty acid signaling to peroxisome proliferator-activated receptor- α (PPAR α ) in the nucleus, was found to bind TOFA and its activated CoA thioester, TOFyl-CoA, with high affinity while binding C75 and C75-CoA with lower affinity. Binding of TOFA and C75-CoA significantly altered L-FABP secondary structure. High (20 mM) but not physiological (6 mM) glucose conferred on both TOFA and C75 the ability to induce PPAR α transcription of the fatty acid β -oxidative enzymes CPT1A, CPT2, and ACOX1 in cultured primary hepatocytes from wild-type (WT) mice. However, L-FABP gene ablation abolished the effects of TOFA and C75 in the context of high glucose. These effects were not associated with an increased cellular level of unesterified fatty acids but rather by increased intracellular glucose. These findings suggested that L-FABP may function as an intracellular fatty acid synthesis inhibitor binding protein facilitating TOFA and C75-mediated induction of PPAR α in the context of high glucose at levels similar to those in uncontrolled diabetes.

  14. RNA nicking activity associated with DNA ligase of T4 infected E. coli: properties and influence on in vitro reactions of ligase.

    PubMed Central

    Sano, H; Feix, G

    1975-01-01

    Highly purified DNA ligase from T4 infected E. coli displays an RNA nicking activity which cleaves endonucleolytically the RNA of ribo-desoxy-and ribo-ribo type doublestranded structures to oligonucleotides with 5'phosphoryl-and 3'hydroxy termini. In the presence of ATP the generated nicks are repaired by the ligase except at the ends of the doublestranded regions where some short oligonucleotides are released before ligation can occur. As judged from its behaviour during the various purification steps and from some of its properties, the nicking activity seems to be different from known nicking enzymes. PMID:1101228

  15. Neuropsychological Outcomes in Fatty Acid Oxidation Disorders: 85 Cases Detected by Newborn Screening

    ERIC Educational Resources Information Center

    Waisbren, Susan E.; Landau, Yuval; Wilson, Jenna; Vockley, Jerry

    2013-01-01

    Mitochondrial fatty acid oxidation disorders include conditions in which the transport of activated acyl-Coenzyme A (CoA) into the mitochondria or utilization of these substrates is disrupted or blocked. This results in a deficit in the conversion of fat into energy. Most patients with fatty acid oxidation defects are now identified through…

  16. Discovery and design of DNA and RNA ligase inhibitors in infectious microorganisms

    PubMed Central

    Swift, Robert V.; Amaro, Rommie E.

    2009-01-01

    Background Members of the nucleotidyltransferase superfamily known as DNA and RNA ligases carry out the enzymatic process of polynucleotide ligation. These guardians of genomic integrity share a three-step ligation mechanism, as well as common core structural elements. Both DNA and RNA ligases have experienced a surge of recent interest as chemotherapeutic targets for the treatment of a range of diseases, including bacterial infection, cancer, and the diseases caused by the protozoan parasites known as trypanosomes. Objective In this review, we will focus on efforts targeting pathogenic microorganisms; specifically, bacterial NAD+-dependent DNA ligases, which are promising broad-spectrum antibiotic targets, and ATP-dependent RNA editing ligases from Trypanosoma brucei, the species responsible for the devastating neurodegenerative disease, African sleeping sickness. Conclusion High quality crystal structures of both NAD+-dependent DNA ligase and the Trypanosoma brucei RNA editing ligase have facilitated the development of a number of promising leads. For both targets, further progress will require surmounting permeability issues and improving selectivity and affinity. PMID:20354588

  17. Structure and two-metal mechanism of a eukaryal nick-sealing RNA ligase

    PubMed Central

    Unciuleac, Mihaela-Carmen; Goldgur, Yehuda; Shuman, Stewart

    2015-01-01

    ATP-dependent RNA ligases are agents of RNA repair that join 3′-OH and 5′-PO4 RNA ends. Naegleria gruberi RNA ligase (NgrRnl) exemplifies a family of RNA nick-sealing enzymes found in bacteria, viruses, and eukarya. Crystal structures of NgrRnl at three discrete steps along the reaction pathway—covalent ligase-(lysyl-Nζ)–AMP•Mn2+ intermediate; ligase•ATP•(Mn2+)2 Michaelis complex; and ligase•Mn2+ complex—highlight a two-metal mechanism of nucleotidyl transfer, whereby (i) an enzyme-bound “catalytic” metal coordination complex lowers the pKa of the lysine nucleophile and stabilizes the transition state of the ATP α phosphate; and (ii) a second metal coordination complex bridges the β- and γ-phosphates. The NgrRnl N domain is a distinctively embellished oligonucleotide-binding (OB) fold that engages the γ-phosphate and associated metal complex and orients the pyrophosphate leaving group for in-line catalysis with stereochemical inversion at the AMP phosphate. The unique domain architecture of NgrRnl fortifies the theme that RNA ligases have evolved many times, and independently, by fusions of a shared nucleotidyltransferase domain to structurally diverse flanking modules. The mechanistic insights to lysine adenylylation gained from the NgrRnl structures are likely to apply broadly to the covalent nucleotidyltransferase superfamily of RNA ligases, DNA ligases, and RNA capping enzymes. PMID:26512110

  18. Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi

    PubMed Central

    Unciuleac, Mihaela-Carmen; Shuman, Stewart

    2015-01-01

    The proteome of the amoebo-flagellate protozoan Naegleria gruberi is rich in candidate RNA repair enzymes, including 15 putative RNA ligases, one of which, NgrRnl, is a eukaryal homolog of Deinococcus radiodurans RNA ligase, DraRnl. Here we report that purified recombinant NgrRnl seals nicked 3′-OH/5′-PO4 duplexes in which the 3′-OH strand is RNA. It does so via the “classic” ligase pathway, entailing reaction with ATP to form a covalent NgrRnl–AMP intermediate, transfer of AMP to the nick 5′-PO4, and attack of the RNA 3′-OH on the adenylylated nick to form a 3′–5′ phosphodiester. Unlike members of the four known families of ATP-dependent RNA ligases, NgrRnl lacks a carboxy-terminal appendage to its nucleotidyltransferase domain. Instead, it contains a defining amino-terminal domain that we show is important for 3′-OH/5′-PO4 nick-sealing and ligase adenylylation, but dispensable for phosphodiester synthesis at a preadenylylated nick. We propose that NgrRnl, DraRnl, and their homologs from diverse bacteria, viruses, and unicellular eukarya comprise a new “Rnl5 family” of nick-sealing ligases with a signature domain organization. PMID:25740837

  19. Cullin 3 Ubiquitin Ligases in Cancer Biology: Functions and Therapeutic Implications

    PubMed Central

    Chen, Hsin-Yi; Chen, Ruey-Hwa

    2016-01-01

    Cullin-RING ubiquitin ligases are the largest E3 ligase family in eukaryotes and are multiprotein complexes. In these complexes, the Cullin protein serves as a scaffold to connect two functional modules of the ligases, the catalytic subunit and substrate-binding subunit. To date, eight members of the Cullin family proteins have been identified. In the Cul3 ubiquitin ligases, Bric-a-brac/Tramtrack/Broad complex (BTB) domain-containing proteins function as a bridge to connect Cul3 and substrates. While the BTB domain is responsible for Cul3 binding, these proteins usually contain an additional domain for substrate interaction, such as MATH, kelch, Zn finger, and PAM, Highwire, and RPM-1 (PHR domain). With the existence of a large number of BTB proteins in human, the Cul3 ubiquitin ligases ubiquitinate a wide range of substrates involving in diverse cellular functions. In this review, we will discuss recent advances on the functions of Cul3 ubiquitin ligases in cancer development, progression, and therapeutic response and the dysregulation of Cul3-mediated ubiquitination events in human malignancies. In particular, we will focus on three Cul3 substrate adaptors, kelch-like ECH-associated protein (Keap1), kelch-like family member 20 (KLHL20), and speckle type BTB/POZ protein (SPOP), with the intent to highlight novel targets in cancer therapy. PMID:27200299

  20. Activation of the E3 ubiquitin ligase Parkin.

    PubMed

    Caulfield, Thomas R; Fiesel, Fabienne C; Springer, Wolfdieter

    2015-04-01

    The PINK1 (phosphatase and tensin homologue-induced putative kinase 1)/Parkin-dependent mitochondrial quality control pathway mediates the clearance of damaged organelles, but appears to be disrupted in Parkinson's disease (PD) [Springer and Kahle (2011) Autophagy 7, 266-278]. Upon mitochondrial stress, PINK1 activates the E3 ubiquitin (Ub) ligase Parkin through phosphorylation of the Ub-like (UBL) domain of Parkin and of the small modifier Ub itself at a conserved residue [Sauvé and Gehring (2014) Cell Res. 24, 1025-1026]. Recently resolved partial crystal structures of Parkin showed a 'closed', auto-inhibited conformation, consistent with its notoriously weak enzymatic activity at steady state [Wauer and Komander (2013) EMBO J. 32, 2099-2112; Riley et al. (2013) Nat. Commun. 4, 1982; Trempe et al. (2013) Science 340, 1451-1455; Spratt et al. (2013) Nat. Commun. 4, 1983]. It has thus become clear that Parkin must undergo major structural rearrangements in order to unleash its catalytic functions. Recent published findings derived from X-ray structures and molecular modelling present a complete structural model of human Parkin at an all-atom resolution [Caulfield et al. (2014) PLoS Comput. Biol. 10, e1003935]. The results of the combined in silico simulations-based and experimental assay-based study indicates that PINK1-dependent Ser65 phosphorylation of Parkin is required for its activation and triggering of 'opening' conformations. Indeed, the obtained structures showed a sequential release of Parkin's intertwined domains and allowed docking of an Ub-charged E2 coenzyme, which could enable its enzymatic activity. In addition, using cell-based screening, select E2 enzymes that redundantly, cooperatively or antagonistically regulate Parkin's activation and/or enzymatic functions at different stages of the mitochondrial autophagy (mitophagy) process were identified [Fiesel et al. (2014) J. Cell Sci. 127, 3488-3504]. Other work that aims to pin-point the particular

  1. High fat fed heart failure animals have enhanced mitochondrial function and acyl-coa dehydrogenase activities

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have previously shown that administration of high fat in heart failure (HF) increased mitochondrial respiration and did not alter left ventricular (LV) function. PPARalpha is a nuclear transcription factor that activates expression of genes involved in fatty acid uptake and utilization. We hypoth...

  2. DNA ligase I is recruited to sites of DNA replication by an interaction with proliferating cell nuclear antigen: identification of a common targeting mechanism for the assembly of replication factories.

    PubMed

    Montecucco, A; Rossi, R; Levin, D S; Gary, R; Park, M S; Motycka, T A; Ciarrocchi, G; Villa, A; Biamonti, G; Tomkinson, A E

    1998-07-01

    In mammalian cells, DNA replication occurs at discrete nuclear sites termed replication factories. Here we demonstrate that DNA ligase I and the large subunit of replication factor C (RF-C p140) have a homologous sequence of approximately 20 amino acids at their N-termini that functions as a replication factory targeting sequence (RFTS). This motif consists of two boxes: box 1 contains the sequence IxxFF whereas box 2 is rich in positively charged residues. N-terminal fragments of DNA ligase I and the RF-C large subunit that contain the RFTS both interact with proliferating cell nuclear antigen (PCNA) in vitro. Moreover, the RFTS of DNA ligase I and of the RF-C large subunit is necessary and sufficient for the interaction with PCNA. Both subnuclear targeting and PCNA binding by the DNA ligase I RFTS are abolished by replacement of the adjacent phenylalanine residues within box 1. Since sequences similar to the RFTS/PCNA-binding motif have been identified in other DNA replication enzymes and in p21(CIP1/WAF1), we propose that, in addition to functioning as a DNA polymerase processivity factor, PCNA plays a central role in the recruitment and stable association of DNA replication proteins at replication factories.

  3. Identification of Erwinia stewartii by a ligase chain reaction assay.

    PubMed Central

    Wilson, W J; Wiedmann, M; Dillard, H R; Batt, C A

    1994-01-01

    A PCR-coupled ligase chain reaction (LCR) assay was developed to distinguish the plant pathogenic bacterium Erwinia stewartii from other erwiniae. This new technique allows discrimination to the species level on the basis of a single-base-pair difference in the 16S rRNA gene which is unique to E. stewartii. Portions of the 16S rRNA genes of E. stewartii and the closely related Erwinia herbicola were sequenced. From comparison of the two 16S rRNA gene regions, two primer pairs were constructed such that only E. stewartii DNA gave a product in the LCR assay. The ligated product was separated from the radioactively labelled primers by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. Twenty-four different Erwinia species and strains were tested by PCR-coupled LCR to verify the specificity of the assay, and only E. stewartii strains gave a positive reaction. In addition, infected and healthy plant material was also assayed. E. stewartii was detected in infected plant material, even when large populations of epiphytic bacteria were present. No enrichment was necessary for detection of the pathogen in corn leaves. This assay has potential as a diagnostic technique for the detection of E. stewartii in infected plant and vector material. Images PMID:7509585

  4. Structural basis of tubulin tyrosination by tubulin tyrosine ligase.

    PubMed

    Prota, Andrea E; Magiera, Maria M; Kuijpers, Marijn; Bargsten, Katja; Frey, Daniel; Wieser, Mara; Jaussi, Rolf; Hoogenraad, Casper C; Kammerer, Richard A; Janke, Carsten; Steinmetz, Michel O

    2013-02-01

    Tubulin tyrosine ligase (TTL) catalyzes the post-translational retyrosination of detyrosinated α-tubulin. Despite the indispensable role of TTL in cell and organism development, its molecular mechanism of action is poorly understood. By solving crystal structures of TTL in complex with tubulin, we here demonstrate that TTL binds to the α and β subunits of tubulin and recognizes the curved conformation of the dimer. Biochemical and cellular assays revealed that specific tubulin dimer recognition controls the activity of the enzyme, and as a consequence, neuronal development. The TTL-tubulin structure further illustrates how the enzyme binds the functionally crucial C-terminal tail sequence of α-tubulin and how this interaction catalyzes the tyrosination reaction. It also reveals how TTL discriminates between α- and β-tubulin, and between different post-translationally modified forms of α-tubulin. Together, our data suggest that TTL has specifically evolved to recognize and modify tubulin, thus highlighting a fundamental role of the evolutionary conserved tubulin tyrosination cycle in regulating the microtubule cytoskeleton. PMID:23358242

  5. The ubiquitin ligase Mindbomb 1 coordinates gastrointestinal secretory cell maturation

    PubMed Central

    Capoccia, Benjamin J.; Jin, Ramon U.; Kong, Young-Yun; Peek, Richard M.; Fassan, Matteo; Rugge, Massimo; Mills, Jason C.

    2013-01-01

    After cell fate specification, differentiating cells must amplify the specific subcellular features required for their specialized function. How cells regulate such subcellular scaling is a fundamental unanswered question. Here, we show that the E3 ubiquitin ligase Mindbomb 1 (MIB1) is required for the apical secretory apparatus established by gastric zymogenic cells as they differentiate from their progenitors. When Mib1 was deleted, death-associated protein kinase–1 (DAPK1) was rerouted to the cell base, microtubule-associated protein 1B (MAP1B) was dephosphorylated, and the apical vesicles that normally support mature secretory granules were dispersed. Consequently, secretory granules did not mature. The transcription factor MIST1 bound the first intron of Mib1 and regulated its expression. We further showed that loss of MIB1 and dismantling of the apical secretory apparatus was the earliest quantifiable aberration in zymogenic cells undergoing transition to a precancerous metaplastic state in mouse and human stomach. Our results reveal a mechanistic pathway by which cells can scale up a specific, specialized subcellular compartment to alter function during differentiation and scale it down during disease. PMID:23478405

  6. Biotin Protein Ligase Is a Target for New Antibacterials

    PubMed Central

    Feng, Jiage; Paparella, Ashleigh S.; Booker, Grant W.; Polyak, Steven W.; Abell, Andrew D.

    2016-01-01

    There is a desperate need for novel antibiotic classes to combat the rise of drug resistant pathogenic bacteria, such as Staphylococcus aureus. Inhibitors of the essential metabolic enzyme biotin protein ligase (BPL) represent a promising drug target for new antibacterials. Structural and biochemical studies on the BPL from S. aureus have paved the way for the design and development of new antibacterial chemotherapeutics. BPL employs an ordered ligand binding mechanism for the synthesis of the reaction intermediate biotinyl-5′-AMP from substrates biotin and ATP. Here we review the structure and catalytic mechanism of the target enzyme, along with an overview of chemical analogues of biotin and biotinyl-5′-AMP as BPL inhibitors reported to date. Of particular promise are studies to replace the labile phosphoroanhydride linker present in biotinyl-5′-AMP with alternative bioisosteres. A novel in situ click approach using a mutant of S. aureus BPL as a template for the synthesis of triazole-based inhibitors is also presented. These approaches can be widely applied to BPLs from other bacteria, as well as other closely related metabolic enzymes and antibacterial drug targets. PMID:27463729

  7. Biotin Protein Ligase Is a Target for New Antibacterials.

    PubMed

    Feng, Jiage; Paparella, Ashleigh S; Booker, Grant W; Polyak, Steven W; Abell, Andrew D

    2016-01-01

    There is a desperate need for novel antibiotic classes to combat the rise of drug resistant pathogenic bacteria, such as Staphylococcus aureus. Inhibitors of the essential metabolic enzyme biotin protein ligase (BPL) represent a promising drug target for new antibacterials. Structural and biochemical studies on the BPL from S. aureus have paved the way for the design and development of new antibacterial chemotherapeutics. BPL employs an ordered ligand binding mechanism for the synthesis of the reaction intermediate biotinyl-5'-AMP from substrates biotin and ATP. Here we review the structure and catalytic mechanism of the target enzyme, along with an overview of chemical analogues of biotin and biotinyl-5'-AMP as BPL inhibitors reported to date. Of particular promise are studies to replace the labile phosphoroanhydride linker present in biotinyl-5'-AMP with alternative bioisosteres. A novel in situ click approach using a mutant of S. aureus BPL as a template for the synthesis of triazole-based inhibitors is also presented. These approaches can be widely applied to BPLs from other bacteria, as well as other closely related metabolic enzymes and antibacterial drug targets. PMID:27463729

  8. Ubiquitin-protein ligases in muscle wasting: multiple parallel pathways?

    NASA Technical Reports Server (NTRS)

    Lecker, Stewart H.; Goldberg, A. L. (Principal Investigator)

    2003-01-01

    PURPOSE OF REVIEW: Studies in a wide variety of animal models of muscle wasting have led to the concept that increased protein breakdown via the ubiquitin-proteasome pathway is responsible for the loss of muscle mass seen as muscle atrophy. The complexity of the ubiquitination apparatus has hampered our understanding of how this pathway is activated in atrophying muscles and which ubiquitin-conjugating enzymes in muscle are responsible. RECENT FINDINGS: Recent experiments have shown that two newly identified ubiquitin-protein ligases (E3s), atrogin-1/MAFbx and MURF-1, are critical in the development of muscle atrophy. Other in-vitro studies also implicated E2(14k) and E3alpha, of the N-end rule pathway, as playing an important role in the process. SUMMARY: It seems likely that multiple pathways of ubiquitin conjugation are activated in parallel in atrophying muscle, perhaps to target for degradation specific classes of muscle proteins. The emerging challenge will be to define the protein targets for, as well as inhibitors of, these E3s.

  9. Selective inhibition of Biotin Protein Ligase from Staphylococcus aureus*

    PubMed Central

    Soares da Costa, Tatiana P.; Tieu, William; Yap, Min Y.; Pendini, Nicole R.; Polyak, Steven W.; Sejer Pedersen, Daniel; Morona, Renato; Turnidge, John D.; Wallace, John C.; Wilce, Matthew C. J.; Booker, Grant W.; Abell, Andrew D.

    2012-01-01

    There is a well documented need to replenish the antibiotic pipeline with new agents to combat the rise of drug resistant bacteria. One strategy to combat resistance is to discover new chemical classes immune to current resistance mechanisms that inhibit essential metabolic enzymes. Many of the obvious drug targets that have no homologous isozyme in the human host have now been investigated. Bacterial drug targets that have a closely related human homologue represent a new frontier in antibiotic discovery. However, to avoid potential toxicity to the host, these inhibitors must have very high selectivity for the bacterial enzyme over the human homolog. We have demonstrated that the essential enzyme biotin protein ligase (BPL) from the clinically important pathogen Staphylococcus aureus could be selectively inhibited. Linking biotin to adenosine via a 1,2,3 triazole yielded the first BPL inhibitor selective for S. aureus BPL over the human equivalent. The synthesis of new biotin 1,2,3-triazole analogues using click chemistry yielded our most potent structure (Ki 90 nm) with a >1100-fold selectivity for the S. aureus BPL over the human homologue. X-ray crystallography confirmed the mechanism of inhibitor binding. Importantly, the inhibitor showed cytotoxicity against S. aureus but not cultured mammalian cells. The biotin 1,2,3-triazole provides a novel pharmacophore for future medicinal chemistry programs to develop this new antibiotic class. PMID:22437830

  10. UHRF2, another E3 ubiquitin ligase for p53

    SciTech Connect

    Bai, Lu; Wang, Xiaohui; Jin, Fangmin; Yang, Yan; Qian, Guanhua; Duan, Changzhu

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer UHRF2 associates with p53 in vivo and in vitro. Black-Right-Pointing-Pointer UHRF2 interacts with p53 through its SRA/YDG domain. Black-Right-Pointing-Pointer UHRF2 ubiquitinates p53 in vivo and in vitro. -- Abstract: UHRF2, ubiquitin-like with PHD and ring finger domains 2, is a nuclear E3 ubiquitin ligase, which is involved in cell cycle and epigenetic regulation. UHRF2 interacts with multiple cell cycle proteins, including cyclins (A2, B1, D1, and E1), CDK2, and pRb; moreover, UHRF2 could ubiquitinate cyclin D1 and cyclin E1. Also, UHRF2 has been shown to be implicated in epigenetic regulation by associating with DNMTs, G9a, HDAC1, H3K9me2/3 and hemi-methylated DNA. We found that UHRF2 associates with tumor suppressor protein p53, and p53 is ubiquitinated by UHRF2 in vivo and in vitro. Given that both UHRF2 and p53 are involved in cell cycle regulation, this study may suggest a novel signaling pathway on cell proliferation.

  11. Screening for E3-Ubiquitin ligase inhibitors: challenges and opportunities

    PubMed Central

    Landré, Vivien; Rotblat, Barak; Melino, Sonia; Bernassola, Francesca; Melino, Gerry

    2014-01-01

    The ubiquitin proteasome system (UPS) plays a role in the regulation of most cellular pathways, and its deregulation has been implicated in a wide range of human pathologies that include cancer, neurodegenerative and immunological disorders and viral infections. Targeting the UPS by small molecular regulators thus provides an opportunity for the development of therapeutics for the treatment of several diseases. The proteasome inhibitor Bortezomib was approved for treatment of hematologic malignancies by the FDA in 2003, becoming the first drug targeting the ubiquitin proteasome system in the clinic. Development of drugs targeting specific components of the ubiquitin proteasome system, however, has lagged behind, mainly due to the complexity of the ubiquitination reaction and its outcomes. However, significant advances have been made in recent years in understanding the molecular nature of the ubiquitination system and the vast variety of cellular signals that it produces. Additionally, improvement of screening methods, both in vitro and in silico, have led to the discovery of a number of compounds targeting components of the ubiquitin proteasome system, and some of these have now entered clinical trials. Here, we discuss the current state of drug discovery targeting E3 ligases and the opportunities and challenges that it provides. PMID:25237759

  12. Predicting the function of 4-coumarate:CoA ligase (LJ4CL1) in Lonicera japonica.

    PubMed

    Yuan, Yuan; Yu, Shulin; Yu, Jun; Zhan, Zhilai; Li, Minhui; Liu, Guiming; Wang, Xumin; Huang, Luqi

    2014-02-10

    4-Coumarate:CoA ligases (4CLs) are a group of essential enzymes involved in the pathway of phenylpropanoid-derived compound metabolisms; however it is still difficult to identify orthologs and paralogs of these important enzymes just based on sequence similarity of the conserved domains. Using sequence data of 20 plant species from the public databases and sequences from Lonicera japonica, we define 1252 adenosine monophosphate (AMP)-dependent synthetase/ligase sequences and classify them into three phylogenetic clades. 4CLs are in one of the four subgroups, according to their partitioning, with known proteins characterized in A. thaliana and Oryza sativa. We also defined 184 non-redundant sequences that encode proteins containing the GEICIRG motif and the taxonomic distribution of these GEICIRG-containing proteins suggests unique catalytic activities in plants. We further analyzed their transcription levels in L. japonica and L. japonica. var. chinensis flowers and chose the highest expressed genes representing the subgroups for structure and binding site predictions. Coupled with liquid chromatography-mass spectrometry (LC-MS) analysis of the L. japonica flowers, the structural study on putative substrate binding amino acid residues, ferulate, and 4-coumaric acid of the conserved binding-site of LJ4CL1 leads to a conclusion that this highly expressed protein group in the flowers may process 4-coumarate that represents 90% of the known phenylpropanoid-derived compounds. The activity of purified crude LJ4CL1 protein was analyzed using 4-coumarate as template and high activity indicating that 4-coumarate is one of the substrates of LJ4CL1.

  13. Predicting the Function of 4-Coumarate:CoA Ligase (LJ4CL1) in Lonicera japonica

    PubMed Central

    Yuan, Yuan; Yu, Shulin; Yu, Jun; Zhan, Zhilai; Li, Minhui; Liu, Guiming; Wang, Xumin; Huang, Luqi

    2014-01-01

    4-Coumarate:CoA ligases (4CLs) are a group of essential enzymes involved in the pathway of phenylpropanoid-derived compound metabolisms; however it is still difficult to identify orthologs and paralogs of these important enzymes just based on sequence similarity of the conserved domains. Using sequence data of 20 plant species from the public databases and sequences from Lonicera japonica, we define 1252 adenosine monophosphate (AMP)-dependent synthetase/ligase sequences and classify them into three phylogenetic clades. 4CLs are in one of the four subgroups, according to their partitioning, with known proteins characterized in A. thaliana and Oryza sativa. We also defined 184 non-redundant sequences that encode proteins containing the GEICIRG motif and the taxonomic distribution of these GEICIRG-containing proteins suggests unique catalytic activities in plants. We further analyzed their transcription levels in L. japonica and L. japonica. var. chinensis flowers and chose the highest expressed genes representing the subgroups for structure and binding site predictions. Coupled with liquid chromatography-mass spectrometry (LC-MS) analysis of the L. japonica flowers, the structural study on putative substrate binding amino acid residues, ferulate, and 4-coumaric acid of the conserved binding-site of LJ4CL1 leads to a conclusion that this highly expressed protein group in the flowers may process 4-coumarate that represents 90% of the known phenylpropanoid-derived compounds. The activity of purified crude LJ4CL1 protein was analyzed using 4-coumarate as template and high activity indicating that 4-coumarate is one of the substrates of LJ4CL1. PMID:24518682

  14. RBR E3 ubiquitin ligases: new structures, new insights, new questions

    PubMed Central

    Spratt, Donald E.; Walden, Helen; Shaw, Gary S.

    2014-01-01

    The RBR (RING-BetweenRING-RING) or TRIAD [two RING fingers and a DRIL (double RING finger linked)] E3 ubiquitin ligases comprise a group of 12 complex multidomain enzymes. This unique family of E3 ligases includes parkin, whose dysfunction is linked to the pathogenesis of early-onset Parkinson's disease, and HOIP (HOIL-1-interacting protein) and HOIL-1 (haem-oxidized IRP2 ubiquitin ligase 1), members of the LUBAC (linear ubiquitin chain assembly complex). The RBR E3 ligases share common features with both the larger RING and HECT (homologous with E6-associated protein C-terminus) E3 ligase families, directly catalysing ubiquitin transfer from an intrinsic catalytic cysteine housed in the C-terminal domain, as well as recruiting thioester-bound E2 enzymes via a RING domain. Recent three-dimensional structures and biochemical findings of the RBRs have revealed novel protein domain folds not previously envisioned and some surprising modes of regulation that have raised many questions. This has required renaming two of the domains in the RBR E3 ligases to more accurately reflect their structures and functions: the C-terminal Rcat (required-for-catalysis) domain, essential for catalytic activity, and a central BRcat (benign-catalytic) domain that adopts the same fold as the Rcat, but lacks a catalytic cysteine residue and ubiquitination activity. The present review discusses how three-dimensional structures of RBR (RING1-BRcat-Rcat) E3 ligases have provided new insights into our understanding of the biochemical mechanisms of these important enzymes in ubiquitin biology. PMID:24576094

  15. RBR E3 ubiquitin ligases: new structures, new insights, new questions.

    PubMed

    Spratt, Donald E; Walden, Helen; Shaw, Gary S

    2014-03-15

    The RBR (RING-BetweenRING-RING) or TRIAD [two RING fingers and a DRIL (double RING finger linked)] E3 ubiquitin ligases comprise a group of 12 complex multidomain enzymes. This unique family of E3 ligases includes parkin, whose dysfunction is linked to the pathogenesis of early-onset Parkinson's disease, and HOIP (HOIL-1-interacting protein) and HOIL-1 (haem-oxidized IRP2 ubiquitin ligase 1), members of the LUBAC (linear ubiquitin chain assembly complex). The RBR E3 ligases share common features with both the larger RING and HECT (homologous with E6-associated protein C-terminus) E3 ligase families, directly catalysing ubiquitin transfer from an intrinsic catalytic cysteine housed in the C-terminal domain, as well as recruiting thioester-bound E2 enzymes via a RING domain. Recent three-dimensional structures and biochemical findings of the RBRs have revealed novel protein domain folds not previously envisioned and some surprising modes of regulation that have raised many questions. This has required renaming two of the domains in the RBR E3 ligases to more accurately reflect their structures and functions: the C-terminal Rcat (required-for-catalysis) domain, essential for catalytic activity, and a central BRcat (benign-catalytic) domain that adopts the same fold as the Rcat, but lacks a catalytic cysteine residue and ubiquitination activity. The present review discusses how three-dimensional structures of RBR (RING1-BRcat-Rcat) E3 ligases have provided new insights into our understanding of the biochemical mechanisms of these important enzymes in ubiquitin biology.

  16. Endoplasmic Reticulum Exit of Golgi-resident Defective for SREBP Cleavage (Dsc) E3 Ligase Complex Requires Its Activity.

    PubMed

    Raychaudhuri, Sumana; Espenshade, Peter J

    2015-06-01

    Layers of quality control ensure proper protein folding and complex formation prior to exit from the endoplasmic reticulum. The fission yeast Dsc E3 ligase is a Golgi-localized complex required for sterol regulatory element-binding protein (SREBP) transcription factor activation that shows architectural similarity to endoplasmic reticulum-associated degradation E3 ligases. The Dsc E3 ligase consists of five integral membrane proteins (Dsc1-Dsc5) and functionally interacts with the conserved AAA-ATPase Cdc48. Utilizing an in vitro ubiquitination assay, we demonstrated that Dsc1 has ubiquitin E3 ligase activity that requires the E2 ubiquitin-conjugating enzyme Ubc4. Mutations that specifically block Dsc1-Ubc4 interaction prevent SREBP cleavage, indicating that SREBP activation requires Dsc E3 ligase activity. Surprisingly, Golgi localization of the Dsc E3 ligase complex also requires Dsc1 E3 ligase activity. Analysis of Dsc E3 ligase complex formation, glycosylation, and localization indicated that Dsc1 E3 ligase activity is specifically required for endoplasmic reticulum exit of the complex. These results define enzyme activity-dependent sorting as an autoregulatory mechanism for protein trafficking.

  17. Endoplasmic Reticulum Exit of Golgi-resident Defective for SREBP Cleavage (Dsc) E3 Ligase Complex Requires Its Activity*

    PubMed Central

    Raychaudhuri, Sumana; Espenshade, Peter J.

    2015-01-01

    Layers of quality control ensure proper protein folding and complex formation prior to exit from the endoplasmic reticulum. The fission yeast Dsc E3 ligase is a Golgi-localized complex required for sterol regulatory element-binding protein (SREBP) transcription factor activation that shows architectural similarity to endoplasmic reticulum-associated degradation E3 ligases. The Dsc E3 ligase consists of five integral membrane proteins (Dsc1–Dsc5) and functionally interacts with the conserved AAA-ATPase Cdc48. Utilizing an in vitro ubiquitination assay, we demonstrated that Dsc1 has ubiquitin E3 ligase activity that requires the E2 ubiquitin-conjugating enzyme Ubc4. Mutations that specifically block Dsc1-Ubc4 interaction prevent SREBP cleavage, indicating that SREBP activation requires Dsc E3 ligase activity. Surprisingly, Golgi localization of the Dsc E3 ligase complex also requires Dsc1 E3 ligase activity. Analysis of Dsc E3 ligase complex formation, glycosylation, and localization indicated that Dsc1 E3 ligase activity is specifically required for endoplasmic reticulum exit of the complex. These results define enzyme activity-dependent sorting as an autoregulatory mechanism for protein trafficking. PMID:25918164

  18. Acyl CoA synthetase 5 (ACSL5) ablation in mice increases energy expenditure and insulin sensitivity and delays fat absorption

    PubMed Central

    Bowman, Thomas A.; O'Keeffe, Kayleigh R.; D'Aquila, Theresa; Yan, Qing Wu; Griffin, John D.; Killion, Elizabeth A.; Salter, Deanna M.; Mashek, Douglas G.; Buhman, Kimberly K.; Greenberg, Andrew S.

    2016-01-01

    Objective The family of acyl-CoA synthetase enzymes (ACSL) activates fatty acids within cells to generate long chain fatty acyl CoA (FACoA). The differing metabolic fates of FACoAs such as incorporation into neutral lipids, phospholipids, and oxidation pathways are differentially regulated by the ACSL isoforms. In vitro studies have suggested a role for ACSL5 in triglyceride synthesis; however, we have limited understanding of the in vivo actions of this ACSL isoform. Methods To elucidate the in vivo actions of ACSL5 we generated a line of mice in which ACSL5 expression was ablated in all tissues (ACSL5−/−). Results Ablation of ACSL5 reduced ACSL activity by ∼80% in jejunal mucosa, ∼50% in liver, and ∼37% in brown adipose tissue lysates. Body composition studies revealed that ACSL5−/−, as compared to control ACSL5loxP/loxP, mice had significantly reduced fat mass and adipose fat pad weights. Indirect calorimetry studies demonstrated that ACSL5−/− had increased metabolic rates, and in the dark phase, increased respiratory quotient. In ACSL5−/− mice, fasting glucose and serum triglyceride were reduced; and insulin sensitivity was improved during an insulin tolerance test. Both hepatic mRNA (∼16-fold) and serum levels of fibroblast growth factor 21 (FGF21) (∼13-fold) were increased in ACSL5−/− as compared to ACSL5loxP/loxP. Consistent with increased FGF21 serum levels, uncoupling protein-1 gene (Ucp1) and PPAR-gamma coactivator 1-alpha gene (Pgc1α) transcript levels were increased in gonadal adipose tissue. To further evaluate ACSL5 function in intestine, mice were gavaged with an olive oil bolus; and the rate of triglyceride appearance in serum was found to be delayed in ACSL5−/− mice as compared to control mice. Conclusions In summary, ACSL5−/− mice have increased hepatic and serum FGF21 levels, reduced adiposity, improved insulin sensitivity, increased energy expenditure and delayed triglyceride absorption. These studies

  19. E3 ubiquitin-ligases and their target proteins during the regulation of plant innate immunity.

    PubMed

    Duplan, Vincent; Rivas, Susana

    2014-01-01

    Reversible protein ubiquitination plays a crucial role during the regulation of plant immune signaling. E3 ubiquitin (Ub)-ligase enzymes, which are classified into different families depending on their structural and functional features, confer the specificity of substrate and are the best characterized components of the ubiquitination cascade. E3 Ub-ligases of different families have been shown to be involved in all steps of plant immune responses. Indeed, they have been involved in the first steps of pathogen perception, as they appear to modulate perception of pathogen-associated molecular patterns by pattern-recognition receptors at the plasma membrane and to regulate the accumulation of nucleotide-binding leucine-rich repeat-type intracellular immune receptors. In addition, E3 Ub-ligase proteins are also involved in the regulation of the signaling responses downstream of pathogen perception through targeting vesicle trafficking components or nuclear transcription factors, for instance. Finally, we also discuss the case of microbial effector proteins that are able to target host E3 Ub-ligases, or to act themselves as E3 Ub-ligases, in their attempt to subvert the host proteasome to promote disease. PMID:24592270

  20. Structural and Functional Interaction Between the Human DNA Repair Proteins DNA ligase IV and XRCC4

    SciTech Connect

    Wu, P.; Meesala, S; Dauvillier, S; Modesti, M; Andres, S; Huang, Y; Sekiguchi, J; Calsou, P; Salles, B; Junop, M

    2009-01-01

    Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the high-resolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate that interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.

  1. A palmitoylated RING finger ubiquitin ligase and its homologue in the brain membranes.

    PubMed

    Araki, Kazuaki; Kawamura, Meiko; Suzuki, Toshiaki; Matsuda, Noriyuki; Kanbe, Daiji; Ishii, Kyoko; Ichikawa, Tomio; Kumanishi, Toshiro; Chiba, Tomoki; Tanaka, Keiji; Nawa, Hiroyuki

    2003-08-01

    Ubiquitin (Ub) ligation is implicated in active protein metabolism and subcellular trafficking and its impairment is involved in various neurologic diseases. In rat brain, we identified two novel Ub ligases, Momo and Sakura, carrying double zinc finger motif and RING finger domain. Momo expression is enriched in the brain gray matter and testis, and Sakura expression is more widely detected in the brain white matter as well as in many peripheral organs. Both proteins associate with the cell membranes of neuronal and/or glial cells. We examined their Ub ligase activity in vivo and in vitro using viral expression vectors carrying myc-tagged Momo and Sakura. Overexpression of either Momo or Sakura in mixed cortical cultures increased total polyubiquitination levels. In vitro ubiquitination assay revealed that the combination of Momo and UbcH4 and H5c, or of Sakura and UbcH4, H5c and H6 is required for the reaction. Deletion mutagenesis suggested that the E3 Ub ligase activity of Momo and Sakura depended on their C-terminal domains containing RING finger structure, while their N-terminal domains influenced their membrane association. In agreement, Sakura associating with the membrane was specifically palmitoylated. Although the molecular targets of their Ub ligation remain to be identified, these findings imply a novel function of the palmitoylated E3 Ub ligase(s).

  2. Regulation of neuronal survival and morphology by the E3 ubiquitin ligase RNF157

    PubMed Central

    Matz, A; Lee, S-J; Schwedhelm-Domeyer, N; Zanini, D; Holubowska, A; Kannan, M; Farnworth, M; Jahn, O; Göpfert, M C; Stegmüller, J

    2015-01-01

    Neuronal health is essential for the long-term integrity of the brain. In this study, we characterized the novel E3 ubiquitin ligase ring finger protein 157 (RNF157), which displays a brain-dominant expression in mouse. RNF157 is a homolog of the E3 ligase mahogunin ring finger-1, which has been previously implicated in spongiform neurodegeneration. We identified RNF157 as a regulator of survival in cultured neurons and established that the ligase activity of RNF157 is crucial for this process. We also uncovered that independently of its ligase activity, RNF157 regulates dendrite growth and maintenance. We further identified the adaptor protein APBB1 (amyloid beta precursor protein-binding, family B, member 1 or Fe65) as an interactor and proteolytic substrate of RNF157 in the control of neuronal survival. Here, the nuclear localization of Fe65 together with its interaction partner RNA-binding protein SART3 (squamous cell carcinoma antigen recognized by T cells 3 or Tip110) is crucial to trigger apoptosis. In summary, we described that the E3 ligase RNF157 regulates important aspects of neuronal development. PMID:25342469

  3. Regulation of neuronal survival and morphology by the E3 ubiquitin ligase RNF157.

    PubMed

    Matz, A; Lee, S-J; Schwedhelm-Domeyer, N; Zanini, D; Holubowska, A; Kannan, M; Farnworth, M; Jahn, O; Göpfert, M C; Stegmüller, J

    2015-04-01

    Neuronal health is essential for the long-term integrity of the brain. In this study, we characterized the novel E3 ubiquitin ligase ring finger protein 157 (RNF157), which displays a brain-dominant expression in mouse. RNF157 is a homolog of the E3 ligase mahogunin ring finger-1, which has been previously implicated in spongiform neurodegeneration. We identified RNF157 as a regulator of survival in cultured neurons and established that the ligase activity of RNF157 is crucial for this process. We also uncovered that independently of its ligase activity, RNF157 regulates dendrite growth and maintenance. We further identified the adaptor protein APBB1 (amyloid beta precursor protein-binding, family B, member 1 or Fe65) as an interactor and proteolytic substrate of RNF157 in the control of neuronal survival. Here, the nuclear localization of Fe65 together with its interaction partner RNA-binding protein SART3 (squamous cell carcinoma antigen recognized by T cells 3 or Tip110) is crucial to trigger apoptosis. In summary, we described that the E3 ligase RNF157 regulates important aspects of neuronal development. PMID:25342469

  4. Ulcerative dermatitis in C57BL/6 mice lacking stearoyl CoA desaturase 1.

    PubMed

    Krugner-Higby, Lisa; Brown, Richard; Rassette, Matthew; Behr, Melissa; Okwumabua, Ogi; Cook, Mark; Bell, Cynthia; Flowers, Matthew T; Ntambi, James; Gendron, Annette

    2012-08-01

    Ulcerative dermatitis (UD) is a common cause of morbidity and euthanasia in mice with a C57BL/6 (B6) background. The purposes of the current study were to determine whether UD lesions could be reliably produced in B6 mice lacking stearoyl-CoA desaturase 1 (SCD1(-/-) mice), to ascertain whether the UD lesions in SCD1(-/-) mice were similar to those found in other B6 mice, and to characterize the cell invasion phenotype of Staphlococcus xylosus cultured from the lesions. S. xylosus isolates from the environment and human skin were used as controls. SCD1(-/-) (n = 8 per group) and nontransgenic B6 control mice (n = 22 mice pooled from 3 groups that received different concentrations of conjugated linoleic acid) were fed standard rodent chow or a semipurified diet (NIH AIN76A) for 4 wk. Samples from other B6 mice with UD (field cases; n = 7) also were submitted for histology and culture. All of the SCD1(-/-) mice developed UD lesions by 4 wk on NIH AIN76A. None of SCD1(-/-) fed standard rodent chow and none of the wildtype B6 mice fed NIH AIN76A developed UD. Supplementation with conjugated linoleic acid did not affect ulcerogenesis. UD lesions in SCD1(-/-) mice and field cases were grossly and histologically similar. S. xylosus was isolated from SCD1(-/-) mice with UD (71%) and field cases of UD (43%). These isolates were the most cell-invasive, followed by the environmental isolate, and finally the human skin isolate. Our results provide a basis for further pathologic and clinical study of UD.

  5. Ulcerative Dermatitis in C57BL/6 Mice Lacking Stearoyl CoA Desaturase 1

    PubMed Central

    Krugner-Higby, Lisa; Brown, Richard; Rassette, Matthew; Behr, Melissa; Okwumabua, Ogi; Cook, Mark; Bell, Cynthia; Flowers, Matthew T; Ntambi, James; Gendron, Annette

    2012-01-01

    Ulcerative dermatitis (UD) is a common cause of morbidity and euthanasia in mice with a C57BL/6 (B6) background. The purposes of the current study were to determine whether UD lesions could be reliably produced in B6 mice lacking stearoyl-CoA desaturase 1 (SCD1–/– mice), to ascertain whether the UD lesions in SCD1–/– mice were similar to those found in other B6 mice, and to characterize the cell invasion phenotype of Staphlococcus xylosus cultured from the lesions. S. xylosus isolates from the environment and human skin were used as controls. SCD1–/– (n = 8 per group) and nontransgenic B6 control mice (n = 22 mice pooled from 3 groups that received different concentrations of conjugated linoleic acid) were fed standard rodent chow or a semipurified diet (NIH AIN76A) for 4 wk. Samples from other B6 mice with UD (field cases; n = 7) also were submitted for histology and culture. All of the SCD1–/– mice developed UD lesions by 4 wk on NIH AIN76A. None of SCD1–/– fed standard rodent chow and none of the wildtype B6 mice fed NIH AIN76A developed UD. Supplementation with conjugated linoleic acid did not affect ulcerogenesis. UD lesions in SCD1–/– mice and field cases were grossly and histologically similar. S. xylosus was isolated from SCD1–/– mice with UD (71%) and field cases of UD (43%). These isolates were the most cell-invasive, followed by the environmental isolate, and finally the human skin isolate. Our results provide a basis for further pathologic and clinical study of UD. PMID:23043777

  6. Nonthermal rotational distribution of CO/A 1Pi/ fragments produced by dissociative excitation of CO2 by electron impact. [in Mars atmosphere

    NASA Technical Reports Server (NTRS)

    Mumma, M. J.; Stone, E. J.; Zipf, E. C.

    1975-01-01

    Measurements were made of the rotational profiles of specific bands of the CO fourth-positive group (4PG). The CO 4PG bands were excited by electron impact dissociative excitation of CO2. The results are applicable to analysis of the Mariner observations of the CO 4PG in the dayglow of Mars. The results indicate that dissociative excitation of CO2 by electron impact leads to CO(A 1Pi) fragments with a rotational distribution that is highly nonthermal. The parent CO2 temperature was about 300 K in the experiment, while the fragment CO(A 1Pi) showed emission band profiles consistent with a rotational temperature greater than about 1500 K. Laboratory measurement of the reduced transmission of the hot bands by thermal CO appears to be the most direct way of determining the column density responsible for the CO(v',0) absorption of Mars.

  7. Substrate specificity and structure-function analysis of the 3'-phosphoesterase component of the bacterial NHEJ protein, DNA ligase D.

    PubMed

    Zhu, Hui; Shuman, Stewart

    2006-05-19

    DNA ligase D (LigD) performs end remodeling and end sealing reactions during nonhomologous end joining in bacteria. Pseudomonas aeruginosa LigD consists of a central ATP-dependent ligase domain fused to a C-terminal polymerase domain and an N-terminal phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at the 3' end of the primer strand of a primer-template. The phosphodiesterase cleaves a 3'-terminal diribonucleotide to yield a primer strand with a ribonucleoside 3'-PO4 terminus. The phosphomonoesterase converts a terminal ribonucleoside 3'-PO4 or deoxyribonucleoside 3'-PO4 of a primer-template to a 3'-OH. Here we report that the phosphodiesterase and phosphomonoesterase activities are both dependent on the presence and length of the 5' single-strand tail of the primer-template substrate. Although the phosphodiesterase activity is strictly dependent on the 2'-OH of the penultimate ribose, it is indifferent to a 2'-OH versus a2'-H on the terminal nucleoside. Incision at the ribonucleotide linkage is suppressed when the 2'-OH is moved by 1 nucleotide in the 5' direction, suggesting that LigD is an exoribonuclease that cleaves the 3'-terminal phosphodiester. We report the effects of conservative amino acid substitutions at residues: (i) His42, His48, Asp50, Arg52, His84, and Tyr88, which are essential for both the ribonuclease and 3'-phosphatase activities; (ii) Arg14, Asp15, Glu21, and Glu82, which are critical for 3'-phosphatase activity but not 3'-ribonucleoside removal; and (iii) at Lys66 and Arg76, which participate selectively in the 3'-ribonuclease reaction. The results suggest roles for individual functional groups in metal binding and/or phosphoesterase chemistry.

  8. Mitochondrial E3 Ubiquitin Protein Ligase 1 Mediates Cigarette Smoke-Induced Endothelial Cell Death and Dysfunction.

    PubMed

    Kim, Sun-Yong; Kim, Hyo Jeong; Park, Mi Kyeong; Huh, Jin Won; Park, Hye Yun; Ha, Sang Yun; Shin, Joo-Ho; Lee, Yun-Song

    2016-02-01

    By virtue of the critical roles of Akt in vascular endothelial cell (EC) survival and function, cigarette smoke-induced Akt reduction may contribute to EC death and dysfunction in smokers' lungs. One of the negative Akt regulatory mechanisms is K48-linked Akt ubiquitination and subsequent proteasomal degradation. Here, we assessed the involvement of mitochondrial E3 ubiquitin protein ligase 1 (MUL1), recently revealed as a novel Akt ubiquitin E3 ligase, in cigarette smoke-induced Akt ubiquitination and its contribution to pulmonary EC death and dysfunction. In human lung microvascular ECs (HLMVECs), cigarette smoke extract (CSE) noticeably elevated MUL1 expression and K48-linked Akt ubiquitination, whereas Akt, p-Akt, eNOS, and p-eNOS levels were decreased. MUL1 knockdown suppressed CSE-induced Akt ubiquitination/degradation and cytoplasmic reductions of Akt and p-Akt. Furthermore, MUL1 knockdown attenuated reductions of eNOS and p-eNOS and alleviated EC survival, migration, and tube formation in the presence of CSE exposure. In addition, overexpression of K284R Akt, a mutant for a MUL1-ubiquitination site, produced similar effects. In HLMVECs exposed to CSE, Akt-MUL1 interaction was increased in coimmunoprecipitation and in situ proximity ligation assays. Similarly, the proximity ligation assay signals were elevated in rat lungs exposed to cigarette smoke for 3 months, during which Mul1 levels were noticeably increased. Finally, we found that CSE-mediated MUL1 induction in HLMVECs is mediated by retinoic acid receptor-related orphan receptor α. Taken together, these data suggest that cigarette smoke-induced MUL1 elevation mediates Akt ubiquitination/degradation, potentially leading to pulmonary EC death and functional impairment.

  9. Enzymatic synthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) with CoA recycling using polyhydroxyalkanoate synthase and acyl-CoA synthetase.

    PubMed

    Satoh, Yasuharu; Murakami, Fumikazu; Tajima, Kenji; Munekata, Masanobu

    2005-05-01

    We succeeded in developing a novel method for in vitro poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3 HB-co-4 HB)] synthesis with CoA recycling using polyhydroxyalkanoate synthase and an acyl-CoA synthetase. Using this method, the monomer compositions in P(3 HB-co-4 HB)s could be controlled strictly by the ratios of the monomers in the reaction mixtures. PMID:16233824

  10. Multiple Src Homology 3 Binding to the Ubiquitin Ligase Itch Conserved Proline-Rich Region.

    PubMed

    Desrochers, Guillaume; Lussier-Price, Mathieu; Omichinski, James G; Angers, Annie

    2015-12-22

    Itch is a member of the C2-WW-HECT (CWH) family of ubiquitin ligases involved in the control of inflammatory signaling pathways, several transcription factors, and sorting of surface receptors to the degradative pathway. In addition to these common domains, Itch also contains a conserved proline-rich region (PRR) allowing its interaction with Src homology 3 (SH3) domain-containing proteins. This region is composed of 20 amino acids and contains one consensus class I and three class II SH3-binding motifs. Several SH3 domain-containing partners have been shown to recognize the Itch PRR, but their binding properties have been poorly defined. Here we compare a subset of endocytic SH3 domain-containing proteins using bioluminescence resonance energy transfer, isothermal titration calorimetry, and pull-down assays. Results indicate that Endophilin is a high-affinity binding partner of Itch both in vivo and in vitro, with a calculated KD placing this complex among the highest-affinity SH3 domain-mediated interactions reported to date. All of the SH3 domains tested here bind to Itch with a 1:1 stoichiometry, except for β-PIX that binds with a 2:1 stoichiometry. Together, these results indicate that Itch PRR is a versatile binding module that can accommodate several different SH3 domain-containing proteins but has a preference for Endophilin. Interestingly, the catalytic activity of Itch toward different SH3 domain-containing proteins was similar, except for β-PIX that was not readily ubiquitylated even though it could interact with an affinity comparable to those of other substrates tested. PMID:26613292

  11. Genetic dissection of methylcrotonyl CoA carboxylase indicates a complex role for mitochondrial leucine catabolism during seed development and germination.

    PubMed

    Ding, Geng; Che, Ping; Ilarslan, Hilal; Wurtele, Eve S; Nikolau, Basil J

    2012-05-01

    3-methylcrotonyl CoA carboxylase (MCCase) is a nuclear-encoded, mitochondrial-localized biotin-containing enzyme. The reaction catalyzed by this enzyme is required for leucine (Leu) catabolism, and it may also play a role in the catabolism of isoprenoids and the mevalonate shunt. In Arabidopsis, two MCCase subunits (the biotinylated MCCA subunit and the non-biotinylated MCCB subunit) are each encoded by single genes (At1g03090 and At4g34030, respectively). A reverse genetic approach was used to assess the physiological role of MCCase in plants. We recovered and characterized T-DNA and transposon-tagged knockout alleles of the MCCA and MCCB genes. Metabolite profiling studies indicate that mutations in either MCCA or MCCB block mitochondrial Leu catabolism, as inferred from the increased accumulation of Leu. Under light deprivation conditions, the hyper-accumulation of Leu, 3-methylcrotonyl CoA and isovaleryl CoA indicates that mitochondrial and peroxisomal Leu catabolism pathways are independently regulated. This biochemical block in mitochondrial Leu catabolism is associated with an impaired reproductive growth phenotype, which includes aberrant flower and silique development and decreased seed germination. The decreased seed germination phenotype is only observed for homozygous mutant seeds collected from a parent plant that is itself homozygous, but not from a parent plant that is heterozygous. These characterizations may shed light on the role of catabolic processes in growth and development, an area of plant biology that is poorly understood.

  12. Leucine-684: A conserved residue of an AMP-acetyl CoA synthetase (AceCS) from Leishmania donovani is involved in substrate recognition, catalysis and acetylation.

    PubMed

    Soumya, Neelagiri; Tandan, Hitendra; Damre, Mangesh V; Gangwal, Rahul P; Sangamwar, Abhay T; Singh, Sushma

    2016-04-15

    AMP-acetyl CoA synthetase (AMP-AceCS) is a key enzyme which catalyzes the activation of acetate to acetyl CoA, an important intermediate at the cross roads of various anabolic and catabolic pathways. Multiple sequence alignment of Leishmania donovani AceCS with other organisms revealed the presence of a highly conserved leucine residue at 684 position which is known to be crucial for acetylation by protein acetyl transferases in other organisms. In an attempt to understand the role of leucine residue at 684 position in L. donovani acetyl CoA synthetase (LdAceCS), it was mutated to proline (P) by site directed mutagenesis. Kinetic analysis of the L684P-LdAceCS mutant revealed approximately two fold increased binding affinity with acetate, whereas fivefold decreased affinity was observed with ATP. There was insignificant change in secondary structure as revealed by CD however, two fold decreased fluorescence intensity was observed at an emission maxima of 340 nm. Interestingly, L684P mutation abolished the acetylation of the mutant enzyme indicating the importance of L684 in acetylation of the enzyme. Changes in biochemical parameters of the mutant protein were validated by homology modeling of the wild type and mutant LdAceCS enzyme using Salmonella enterica AceCS crystal structure as template. Our data provides evidence for the role of leucine 684 residue in substrate recognition, catalysis and acetylation of the AceCS enzyme.

  13. CRP Is an Activator of Yersinia pestis Biofilm Formation that Operates via a Mechanism Involving gmhA and waaAE-coaD.

    PubMed

    Liu, Lei; Fang, Haihong; Yang, Huiying; Zhang, Yiquan; Han, Yanping; Zhou, Dongsheng; Yang, Ruifu

    2016-01-01

    gmhA encodes a phosphoheptose isomerase that catalyzes the biosynthesis of heptose, a conserved component of lipopolysaccharide (LPS). GmhA plays an important role in Yersinia pestis biofilm blockage in the flea gut. waaA, waaE, and coaD constitute a three-gene operon waaAE-coaD in Y. pestis. waaA encodes a transferase that is responsible for binding lipid-A to the core oligosaccharide of LPS. WaaA is a key determinant in Y. pestis biofilm formation, and the waaA expression is positively regulated by the two-component regulatory system PhoP/PhoQ. WaaE is involved in LPS modification and is necessary for Y. pestis biofilm production. In this study, the biofilm-related phenotypic assays indicate that the global regulator CRP stimulates Y. pestis biofilm formation in vitro and on nematodes, while it has no regulatory effect on the biosynthesis of the biofilm-signaling molecular 3',5'-cyclic diguanosine monophosphate. Further gene regulation experiments disclose that CRP does not regulate the hms genes at the transcriptional level but directly promotes the gmhA transcription and indirectly activates the waaAE-coaD transcription through directly acting on phoPQ-YPO1632. Thus, it is speculated that CRP-mediated carbon catabolite regulation of Y. pestis biofilm formation depends on the CRP-dependent carbon source metabolic pathways of the biosynthesis, modification, and transportation of biofilm exopolysaccharide. PMID:27014218

  14. Discrimination of Listeria monocytogenes from other Listeria species by ligase chain reaction.

    PubMed Central

    Wiedmann, M; Czajka, J; Barany, F; Batt, C A

    1992-01-01

    A ligase chain reaction assay based on a single-base-pair difference in the V9 region of the 16S rRNA gene (16S rDNA) was developed to distinguish between Listeria monocytogenes and other Listeria species. For this purpose, two pairs of primers were designed, with one primer of each pair being radioactively labeled. The ligated product was separated from the primers by denaturing polyacrylamide gel electrophoresis and then detected by autoradiography. To achieve a higher sensitivity, the 16S rDNA was initially amplified by polymerase chain reaction prior to the ligase chain reaction. The ligase chain reaction was tested on 19 different Listeria species and strains and proved to be a highly specific diagnostic method for the detection of L. monocytogenes. Images PMID:1482171

  15. Composition, Roles, and Regulation of Cullin-Based Ubiquitin E3 Ligases

    PubMed Central

    Choi, Christina M.; Gray, William M.; Mooney, Sutton; Hellmann, Hanjo

    2014-01-01

    Due to their sessile nature, plants depend on flexible regulatory systems that allow them to adequately regulate developmental and physiological processes in context with environmental cues. The ubiquitin proteasome pathway, which targets a great number of proteins for degradation, is cellular tool that provides the necessary flexibility to accomplish this task. Ubiquitin E3 ligases provide the needed specificity to the pathway by selectively binding to particular substrates and facilitating their ubiquitylation. The largest group of E3 ligases known in plants is represented by CULLIN-REALLY INTERESTING NEW GENE (RING) E3 ligases (CRLs). In recent years, a great amount of knowledge has been generated to reveal the critical roles of these enzymes across all aspects of plant life. This review provides an overview of the different classes of CRLs in plants, their specific complex compositions, the variety of biological processes they control, and the regulatory steps that can affect their activities. PMID:25505853

  16. Immunoprecipitation of Cullin-RING Ligases (CRLs) in Arabidopsis thaliana Seedlings.

    PubMed

    Franciosini, Anna; Serino, Giovanna

    2016-01-01

    CRL (Cullin-RING ubiquitin ligase) is the major class of plant E3 ubiquitin ligases. Immunoprecipitation-based methods are useful techniques for revealing interactions among Cullin-RING Ligase (CRL) subunits or between CRLs and other proteins, as well as for detecting poly-ubiquitin modifications of the CRLs themselves. Here, we describe two immunoprecipitation (IP) procedures suitable for CRLs in Arabidopsis: a procedure for IP analysis of CRL subunits and their interactors and a second procedure for in vivo ubiquitination analysis of the CRLs. Both protocols can be divided into two major steps: (1) preparation of cell extracts without disruption of protein interactions and (2) affinity purification of the protein complexes and subsequent detection. We provide a thorough description of all the steps, as well as advice on how to choose proper buffers for these analyses. We also suggest a series of negative controls that can be used to verify the specificity of the procedure. PMID:27424742

  17. Identification and Validation of Human DNA Ligase Inhibitors Using Computer-Aided Drug Design

    PubMed Central

    Zhong, Shijun; Chen, Xi; Zhu, Xiao; Dziegielewska, Barbara; Bachman, Kurtis E.; Ellenberger, Tom; Ballin, Jeff D.; Wilson, Gerald M.; Tomkinson, Alan E.; MacKerell, Alexander D.

    2009-01-01

    Linking together of DNA strands by DNA ligases is essential for DNA replication and repair. Since many therapies used to treat cancer act by causing DNA damage, there is growing interest in the development of DNA repair inhibitors. Accordingly, virtual database screening and experimental evaluation were applied to identify inhibitors of human DNA ligase I (hLigI). When a DNA binding site within the DNA binding domain (DBD) of hLigI was targeted, more than 1 million compounds were screened from which 192 were chosen for experimental evaluation. In DNA joining assays, 10 compounds specifically inhibited hLigI, 5 of which also inhibited the proliferation of cultured human cell lines. Analysis of the 10 active compounds revealed the utility of including multiple protein conformations and chemical clustering in the virtual screening procedure. The identified ligase inhibitors are structurally diverse and have druglike physical and molecular characteristics making them ideal for further drug development studies. PMID:18630893

  18. Structure of 5-formyltetrahydrofolate cyclo-ligase from Bacillus anthracis (BA4489)

    SciTech Connect

    Meier, Christoph; Carter, Lester G.; Winter, Graeme; Owens, Ray J.; Stuart, David I.; Esnouf, Robert M.

    2007-03-01

    The structure of 5-formyltetrahydrofolate cyclo-ligase from B. anthracis determined by X-ray crystallography at a resolution of 1.6 Å is described. Bacillus anthracis is a spore-forming bacterium and the causative agent of the disease anthrax. The Oxford Protein Production Facility has been targeting proteins from B. anthracis in order to develop high-throughput technologies within the Structural Proteomics in Europe project. As part of this work, the structure of 5-formyltetrahydrofolate cyclo-ligase (BA4489) has been determined by X-ray crystallography to 1.6 Å resolution. The structure, solved in complex with magnesium-ion-bound ADP and phosphate, gives a detailed picture of the proposed catalytic mechanism of the enzyme. Chemical differences from other cyclo-ligase structures close to the active site that could be exploited to design specific inhibitors are also highlighted.

  19. A Bacterial Inhibitor of Host Programmed Cell Death Defenses is an E3 Ubiquitin Ligase

    SciTech Connect

    Janjusevic,R.; Abramovitch, R.; Martin, G.; Stebbins, C.

    2005-01-01

    The Pseudomonas syringae protein AvrPtoB is translocated into plant cells, where it inhibits immunity-associated programmed cell death (PCD). The structure of a C-terminal domain of AvrPtoB that is essential for anti-PCD activity reveals an unexpected homology to the U-box and RING-finger components of eukaryotic E3 ubiquitin ligases, and we show that AvrPtoB has ubiquitin ligase activity. Mutation of conserved residues involved in the binding of E2 ubiquitin-conjugating enzymes abolishes this activity in vitro, as well as anti-PCD activity in tomato leaves, which dramatically decreases virulence. These results show that Pseudomonas syringae uses a mimic of host E3 ubiquitin ligases to inactivate plant defenses.

  20. Biochemical and structural characterization of DNA ligases from bacteria and archaea

    PubMed Central

    Pergolizzi, Giulia; Wagner, Gerd K.; Bowater, Richard P.

    2016-01-01

    DNA ligases are enzymes that seal breaks in the backbones of DNA, leading to them being essential for the survival of all organisms. DNA ligases have been studied from many different types of cells and organisms and shown to have diverse sizes and sequences, with well conserved specific sequences that are required for enzymatic activity. A significant number of DNA ligases have been isolated or prepared in recombinant forms and, here, we review their biochemical and structural characterization. All DNA ligases contain an essential lysine that transfers an adenylate group from a co-factor to the 5′-phosphate of the DNA end that will ultimately be joined to the 3′-hydroxyl of the neighbouring DNA strand. The essential DNA ligases in bacteria use β-nicotinamide adenine dinucleotide (β-NAD+) as their co-factor whereas those that are essential in other cells use adenosine-5′-triphosphate (ATP) as their co-factor. This observation suggests that the essential bacterial enzyme could be targeted by novel antibiotics and the complex molecular structure of β-NAD+ affords multiple opportunities for chemical modification. Several recent studies have synthesized novel derivatives and their biological activity against a range of DNA ligases has been evaluated as inhibitors for drug discovery and/or non-natural substrates for biochemical applications. Here, we review the recent advances that herald new opportunities to alter the biochemical activities of these important enzymes. The recent development of modified derivatives of nucleotides highlights that the continued combination of structural, biochemical and biophysical techniques will be useful in targeting these essential cellular enzymes. PMID:27582505

  1. Bioinformatics analysis identifies several intrinsically disordered human E3 ubiquitin-protein ligases

    PubMed Central

    Nielsen, Sofie V.; Lindorff-Larsen, Kresten; Hartmann-Petersen, Rasmus

    2016-01-01

    The ubiquitin-proteasome system targets misfolded proteins for degradation. Since the accumulation of such proteins is potentially harmful for the cell, their prompt removal is important. E3 ubiquitin-protein ligases mediate substrate ubiquitination by bringing together the substrate with an E2 ubiquitin-conjugating enzyme, which transfers ubiquitin to the substrate. For misfolded proteins, substrate recognition is generally delegated to molecular chaperones that subsequently interact with specific E3 ligases. An important exception is San1, a yeast E3 ligase. San1 harbors extensive regions of intrinsic disorder, which provide both conformational flexibility and sites for direct recognition of misfolded targets of vastly different conformations. So far, no mammalian ortholog of San1 is known, nor is it clear whether other E3 ligases utilize disordered regions for substrate recognition. Here, we conduct a bioinformatics analysis to examine >600 human and S. cerevisiae E3 ligases to identify enzymes that are similar to San1 in terms of function and/or mechanism of substrate recognition. An initial sequence-based database search was found to detect candidates primarily based on the homology of their ordered regions, and did not capture the unique disorder patterns that encode the functional mechanism of San1. However, by searching specifically for key features of the San1 sequence, such as long regions of intrinsic disorder embedded with short stretches predicted to be suitable for substrate interaction, we identified several E3 ligases with these characteristics. Our initial analysis revealed that another remarkable trait of San1 is shared with several candidate E3 ligases: long stretches of complete lysine suppression, which in San1 limits auto-ubiquitination. We encode these characteristic features into a San1 similarity-score, and present a set of proteins that are plausible candidates as San1 counterparts in humans. In conclusion, our work indicates that San1 is

  2. Identification and functional expression of the pepper RING type E3 ligase, CaDTR1, involved in drought stress tolerance via ABA-mediated signalling

    PubMed Central

    Joo, Hyunhee; Lim, Chae Woo; Lee, Sung Chul

    2016-01-01

    Drought negatively affects plant growth and development, thereby leading to loss of crop productivity. Several plant E3 ubiquitin ligases act as positive or negative regulators of abscisic acid (ABA) and thus play important roles in the drought stress response. Here, we show that the C3HC4-type RING finger E3 ligase, CaDTR1, regulates the drought stress response via ABA-mediated signalling. CaDTR1 contains an amino-terminal RING finger motif and two carboxyl-terminal hydrophobic regions; the RING finger motif functions during attachment of ubiquitins to the target proteins, and the carboxyl-terminal hydrophobic regions function during subcellular localisation. The expression of CaDTR1 was induced by ABA, drought, and NaCl treatments. CaDTR1 localised in the nucleus and displayed in vitro E3 ubiquitin ligase activity. CaDTR1-silenced pepper plants exhibited a drought-sensitive phenotype characterised by high levels of transpirational water loss. On the other hand, CaDTR1-overexpressing (OX) Arabidopsis plants exhibited an ABA-hypersensitive phenotype during the germinative and post-germinative growth stages. Moreover, in contrast to CaDTR1-silenced pepper plants, CaDTR1-OX plants exhibited a drought-tolerant phenotype characterised by low levels of transpirational water loss via increased stomatal closure and high leaf temperatures. Our data indicate that CaDTR1 functions as a positive regulator of the drought stress response via ABA-mediated signalling. PMID:27439598

  3. Identification and functional expression of the pepper RING type E3 ligase, CaDTR1, involved in drought stress tolerance via ABA-mediated signalling.

    PubMed

    Joo, Hyunhee; Lim, Chae Woo; Lee, Sung Chul

    2016-01-01

    Drought negatively affects plant growth and development, thereby leading to loss of crop productivity. Several plant E3 ubiquitin ligases act as positive or negative regulators of abscisic acid (ABA) and thus play important roles in the drought stress response. Here, we show that the C3HC4-type RING finger E3 ligase, CaDTR1, regulates the drought stress response via ABA-mediated signalling. CaDTR1 contains an amino-terminal RING finger motif and two carboxyl-terminal hydrophobic regions; the RING finger motif functions during attachment of ubiquitins to the target proteins, and the carboxyl-terminal hydrophobic regions function during subcellular localisation. The expression of CaDTR1 was induced by ABA, drought, and NaCl treatments. CaDTR1 localised in the nucleus and displayed in vitro E3 ubiquitin ligase activity. CaDTR1-silenced pepper plants exhibited a drought-sensitive phenotype characterised by high levels of transpirational water loss. On the other hand, CaDTR1-overexpressing (OX) Arabidopsis plants exhibited an ABA-hypersensitive phenotype during the germinative and post-germinative growth stages. Moreover, in contrast to CaDTR1-silenced pepper plants, CaDTR1-OX plants exhibited a drought-tolerant phenotype characterised by low levels of transpirational water loss via increased stomatal closure and high leaf temperatures. Our data indicate that CaDTR1 functions as a positive regulator of the drought stress response via ABA-mediated signalling. PMID:27439598

  4. The effects of simvastatin and pravastatin on objective and subjective measures of nocturnal sleep: a comparison of two structurally different HMG CoA reductase inhibitors in patients with primary moderate hypercholesterolaemia.

    PubMed Central

    Eckernäs, S A; Roos, B E; Kvidal, P; Eriksson, L O; Block, G A; Neafus, R P; Haigh, J R

    1993-01-01

    1. It has been suggested that HMG CoA reductase inhibitors which are administered as inactive, lipophilic lactones (e.g. simvastatin) have a greater propensity to evoke nocturnal sleep disturbances than pravastatin, an inhibitor given in the active, hydrophilic, open-acid form. 2. The effects of 4 weeks treatment with equipotent doses of simvastatin (20 mg day-1) and pravastatin (40 mg day-1) have been compared using polysomnography and subjective sleep assessments in a double-blind, placebo-controlled, two-period, incomplete block design study involving 24 male patients with primary moderate hypercholesterolaemia (mean LDL cholesterol 5.11 mmol l-1). 3. Analysis of sleep EEG measures relevant to insomnia provided no evidence of significant differences between pravastatin, simvastatin and placebo, except in terms of entries and latency to stage I sleep. The number of entries to stage I sleep was significantly greater after simvastatin treatment than after either pravastatin or placebo (P < 0.05), but by contrast the latency to stage I sleep was significantly prolonged only in the pravastatin group (P < 0.05 vs placebo). 4. Subjective ratings of sleep initiation, maintenance and quality made during and after therapy were not significantly different between the three treatment groups. 5. It appears that the inherent hydrophobicity of simvastatin does not increase the occurrence of sleep disturbances in this patient population at a dose shown to elicit a characteristic hypolipidaemic response. PMID:8471404

  5. Structural insight into β-Clamp and its interaction with DNA Ligase in Helicobacter pylori.

    PubMed

    Pandey, Preeti; Tarique, Khaja Faisal; Mazumder, Mohit; Rehman, Syed Arif Abdul; Kumari, Nilima; Gourinath, Samudrala

    2016-08-08

    Helicobacter pylori, a gram-negative and microaerophilic bacterium, is the major cause of chronic gastritis, gastric ulcers and gastric cancer. Owing to its central role, DNA replication machinery has emerged as a prime target for the development of antimicrobial drugs. Here, we report 2Å structure of β-clamp from H. pylori (Hpβ-clamp), which is one of the critical components of DNA polymerase III. Despite of similarity in the overall fold of eubacterial β-clamp structures, some distinct features in DNA interacting loops exists that have not been reported previously. The in silico prediction identified the potential binders of β-clamp such as alpha subunit of DNA pol III and DNA ligase with identification of β-clamp binding regions in them and validated by SPR studies. Hpβ-clamp interacts with DNA ligase in micromolar binding affinity. Moreover, we have successfully determined the co-crystal structure of β-clamp with peptide from DNA ligase (not reported earlier in prokaryotes) revealing the region from ligase that interacts with β-clamp.

  6. Structural insight into β-Clamp and its interaction with DNA Ligase in Helicobacter pylori

    PubMed Central

    Pandey, Preeti; Tarique, Khaja Faisal; Mazumder, Mohit; Rehman, Syed Arif Abdul; kumari, Nilima; Gourinath, Samudrala

    2016-01-01

    Helicobacter pylori, a gram-negative and microaerophilic bacterium, is the major cause of chronic gastritis, gastric ulcers and gastric cancer. Owing to its central role, DNA replication machinery has emerged as a prime target for the development of antimicrobial drugs. Here, we report 2Å structure of β-clamp from H. pylori (Hpβ-clamp), which is one of the critical components of DNA polymerase III. Despite of similarity in the overall fold of eubacterial β-clamp structures, some distinct features in DNA interacting loops exists that have not been reported previously. The in silico prediction identified the potential binders of β-clamp such as alpha subunit of DNA pol III and DNA ligase with identification of β-clamp binding regions in them and validated by SPR studies. Hpβ-clamp interacts with DNA ligase in micromolar binding affinity. Moreover, we have successfully determined the co-crystal structure of β-clamp with peptide from DNA ligase (not reported earlier in prokaryotes) revealing the region from ligase that interacts with β-clamp. PMID:27499105

  7. Crystallization and preliminary crystallographic analysis of d-alanine-d-alanine ligase from Streptococcus mutans

    SciTech Connect

    Lu, Yong-Zhi; Sheng, Yu; Li, Lan-Fen; Tang, De-Wei; Liu, Xiang-Yu; Zhao, Xiaojun; Liang, Yu-He Su, Xiao-Dong

    2007-09-01

    A potential target for antibiotic drug design, d-alanine-d-alanine ligase from S. mutans, was expressed in E. coli, purified and crystallized. Diffraction data were collected to 2.4 Å resolution. d-Alanine-d-alanine ligase is encoded by the gene ddl (SMU-599) in Streptococcus mutans. This ligase plays a very important role in cell-wall biosynthesis and may be a potential target for drug design. To study the structure and function of this ligase, the gene ddl was amplified from S. mutans genomic DNA and cloned into the expression vector pET28a. The protein was expressed in soluble form in Escherichia coli strain BL21 (DE3). Homogeneous protein was obtained using a two-step procedure consisting of Ni{sup 2+}-chelating and size-exclusion chromatography. Purified protein was crystallized and the cube-shaped crystal diffracted to 2.4 Å. The crystal belongs to space group P3{sub 1}21 or P3{sub 2}21, with unit-cell parameters a = b = 79.50, c = 108.97 Å. There is one molecule per asymmetric unit.

  8. THE ROLE OF E3 LIGASES IN THE UBIQUITIN-DEPENDENT REGULATION OF SPERMATOGENESIS*

    PubMed Central

    Richburg, John H.; Myers, Jessica L.; Bratton, Shawn B.

    2014-01-01

    The ubiquitination of proteins is a post-translational modification that was first described as a means to target misfolded or unwanted proteins for degradation by the proteasome. It is now appreciated that the ubiquitination of proteins also serves as a mechanism to modify protein function and cellular functions such as protein trafficking, cell signaling, DNA repair, chromatin modifications, cell-cycle progression and cell death. The ubiquitination of proteins occurs through the hierarchal transfer of ubiquitin from an E1 ubiquitin-activating enzyme to an E2 ubiquitin-conjugating enzyme and finally to an E3 ubiquitin ligase that transfers the ubiquitin to its target protein. It is the final E3 ubiquitin ligase that confers the substrate specificity for ubiquitination and is the focus of this review. Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells undergo mitotic proliferation and expansion of the diploid spermatogonial population, differentiate into spermatocytes and progress through two meiotic divisions to produce haploid spermatids that proceed through a final morphogenesis to generate mature spermatozoa. The ubiquitination of proteins in the cells of the testis occurs in many of the processes required for the progression of mature spermatozoa. Since it is the E3 ubiquitin ligase that recognizes the target protein and provides the specificity and selectivity for ubiquitination, this review highlights known examples of E3 ligases in the testis and the differing roles that they play in maintaining functional spermatogenesis. PMID:24632385

  9. The prolific ATL family of RING-H2 ubiquitin ligases

    PubMed Central

    Guzmán, Plinio

    2012-01-01

    An abundant class of E3 ubiquitin ligases encodes the RING-finger domain. The RING finger binds to the E2 ubiquitin-conjugating enzyme and brings together both the E2 and substrate. It is predicted that 477 RING finger E3 ligases exist in Arabidopsis thaliana. A particular family among them, named Arabidopsis Tóxicos en Levadura (ATL), consists of 91 members that contain the RING-H2 variation and a hydrophobic domain located at the N-terminal end. Transmembrane E3 ligases are important in several biological processes. For instance, some transmembrane RING finger E3 ligases are main participants in the endoplasmic reticulum-associated degradation pathway that targets misfolded proteins. Functional analysis of a number of ATLs has shown that some of them regulate distinct pathways in plants. Several ATLs have been shown to participate in defense responses, while others play a role in the regulation of the carbon/nitrogen response during post-germinative seedling growth transition, in the regulation of cell death during root development, in endosperm development, or in the transition to flowering under short day conditions. The ATL family has also been instrumental in evolution studies for showing how gene families are expanded in plant genomes. PMID:22827943

  10. SILENCING OF 4-COUMARATE-CoA LIGASE IN PINUS RADIATA, A CONIFEROUS GYMNOSPERM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The enzyme 4-coumarate-CoA ligase (4CL) is involved in the general phenylpropanoid pathway and provides monolignol precursors such as p-coumaroyl-CoA, ultimately for the biosynthesis of lignin. Recombinant studies designed to assess the role of 4CL in the lignification process have focused on angios...

  11. Structural insight into β-Clamp and its interaction with DNA Ligase in Helicobacter pylori.

    PubMed

    Pandey, Preeti; Tarique, Khaja Faisal; Mazumder, Mohit; Rehman, Syed Arif Abdul; Kumari, Nilima; Gourinath, Samudrala

    2016-01-01

    Helicobacter pylori, a gram-negative and microaerophilic bacterium, is the major cause of chronic gastritis, gastric ulcers and gastric cancer. Owing to its central role, DNA replication machinery has emerged as a prime target for the development of antimicrobial drugs. Here, we report 2Å structure of β-clamp from H. pylori (Hpβ-clamp), which is one of the critical components of DNA polymerase III. Despite of similarity in the overall fold of eubacterial β-clamp structures, some distinct features in DNA interacting loops exists that have not been reported previously. The in silico prediction identified the potential binders of β-clamp such as alpha subunit of DNA pol III and DNA ligase with identification of β-clamp binding regions in them and validated by SPR studies. Hpβ-clamp interacts with DNA ligase in micromolar binding affinity. Moreover, we have successfully determined the co-crystal structure of β-clamp with peptide from DNA ligase (not reported earlier in prokaryotes) revealing the region from ligase that interacts with β-clamp. PMID:27499105

  12. Another tier for caspase regulation: IAPs as NEDD8 E3 ligases.

    PubMed

    Benjamin, Sigi; Steller, Hermann

    2010-12-14

    Many inhibitor of apoptosis proteins (IAPs) function as E3 ligases to ubiquitinate important cell death proteins, including caspases. Broemer et al. (2010) report recently in Molecular Cell that IAPs can also inhibit caspases by promoting conjugation of the UBL NEDD8.

  13. In vitro selection of optimal DNA substrates for T4 RNA ligase

    NASA Technical Reports Server (NTRS)

    Harada, Kazuo; Orgel, Leslie E.

    1993-01-01

    We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 RNA ligase. We find that the ensemble of selected sequences ligated about 10 times as efficiently as the random mixture of sequences used as the input for selection. Surprisingly, the majority of the selected sequences approximated a well-defined consensus sequence.

  14. A novel Fbxo25 acts as an E3 ligase for destructing cardiac specific transcription factors.

    PubMed

    Jang, Jae-Woo; Lee, Won-Young; Lee, Jae-Ho; Moon, Sung-Hwan; Kim, Chang-Hoon; Chung, Hyung-Min

    2011-07-01

    Alterations in ubiquitin-proteasome system (UPS) have been implicated in the etiology of human cardiovascular diseases. Skp1/Cul1/F-box (SCF) ubiquitin E3 ligase complex plays a pivotal role in ubiquitination of cardiac proteins. However, a specific ubiquitin E3 ligase responsible for the destruction of cardiac transcription factors such as Nkx2-5, Isl1, Mef2C, and Tbx5 remains elusive to date. Here, we show that a novel F-box containing Fbxo25 is cardiac-specific and acts as an ubiquitin E3 ligase for cardiac transcription factors. Fbxo25 expression was nuclei-specific in vitro and cardiomyocytes. Expression level of Fbxo25 was higher in a fetal heart than an adult. Moreover, Fbxo25 expression was increased along with those of cardiac-specific genes during cardiomyocyte development from ESCs. Fbxo25 expression facilitated protein degradation of Nkx2-5, Isl1, Hand1, and Mef2C. Especially, Fbxo25 ubiquitinated Nkx2-5, Isl1, and Hand1. Altogether, Fbxo25 acts as an ubiquitin E3 ligase to target cardiac transcription factors including Nkx2-5, Isl1, and Hand1, indicating that cardiac protein homeostasis through Fbxo25 has a pivotal impact on cardiac development.

  15. Yeast SREBP cleavage activation requires the Golgi Dsc E3 ligase complex.

    PubMed

    Stewart, Emerson V; Nwosu, Christine C; Tong, Zongtian; Roguev, Assen; Cummins, Timothy D; Kim, Dong-Uk; Hayles, Jacqueline; Park, Han-Oh; Hoe, Kwang-Lae; Powell, David W; Krogan, Nevan J; Espenshade, Peter J

    2011-04-22

    Mammalian lipid homeostasis requires proteolytic activation of membrane-bound sterol regulatory element binding protein (SREBP) transcription factors through sequential action of the Golgi Site-1 and Site-2 proteases. Here we report that while SREBP function is conserved in fungi, fission yeast employs a different mechanism for SREBP cleavage. Using genetics and biochemistry, we identified four genes defective for SREBP cleavage, dsc1-4, encoding components of a transmembrane Golgi E3 ligase complex with structural homology to the Hrd1 E3 ligase complex involved in endoplasmic reticulum-associated degradation. The Dsc complex binds SREBP and cleavage requires components of the ubiquitin-proteasome pathway: the E2-conjugating enzyme Ubc4, the Dsc1 RING E3 ligase, and the proteasome. dsc mutants display conserved aggravating genetic interactions with components of the multivesicular body pathway in fission yeast and budding yeast, which lacks SREBP. Together, these data suggest that the Golgi Dsc E3 ligase complex functions in a post-ER pathway for protein degradation.

  16. ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair

    PubMed Central

    Gracheva, Ekaterina; Chitale, Shalaka; Wilhelm, Thomas; Rapp, Alexander; Byrne, Jonathan; Stadler, Jens; Medina, Rebeca; Cardoso, M. Cristina

    2016-01-01

    Faithful DNA repair is essential to maintain genome integrity. Ultraviolet (UV) irradiation elicits both the recruitment of DNA repair factors and the deposition of histone marks such as monoubiquitylation of histone H2A at lesion sites. Here, we report how a ubiquitin E3 ligase complex specific to DNA repair is remodeled at lesion sites in the global genome nucleotide excision repair (GG-NER) pathway. Monoubiquitylation of histone H2A (H2A-ubiquitin) is catalyzed predominantly by a novel E3 ligase complex consisting of DDB2, DDB1, CUL4B, and RING1B (UV–RING1B complex) that acts early during lesion recognition. The H2A-ubiquitin binding protein ZRF1 mediates remodeling of this E3 ligase complex directly at the DNA lesion site, causing the assembly of the UV–DDB–CUL4A E3 ligase complex (DDB1–DDB2–CUL4A-RBX1). ZRF1 is an essential factor in GG-NER, and its function at damaged chromatin sites is linked to damage recognition factor XPC. Overall, the results shed light on the interplay between epigenetic and DNA repair recognition factors at DNA lesion sites. PMID:27091446

  17. Identification of dynamical hinge points of the L1 ligase molecular switch.

    PubMed

    Giambasu, George M; Lee, Tai-Sung; Sosa, Carlos P; Robertson, Michael P; Scott, William G; York, Darrin M

    2010-04-01

    The L1 ligase is an in vitro selected ribozyme that uses a noncanonically base-paired ligation site to catalyze regioselectively and regiospecifically the 5' to 3' phosphodiester bond ligation, a reaction relevant to origin of life hypotheses that invoke an RNA world scenario. The L1 ligase crystal structure revealed two different conformational states that were proposed to represent the active and inactive forms. It remains an open question as to what degree these two conformers persist as stable conformational intermediates in solution, and along what pathway are they able to interconvert. To explore these questions, we have performed a series of molecular dynamics simulations in explicit solvent of the inactive-active conformational switch in L1 ligase. Four simulations were performed departing from both conformers in both the reactant and product states, in addition to a simulation where local unfolding in the active state was induced. From these simulations, along with crystallographic data, a set of four virtual torsion angles that span two evolutionarily conserved and restricted regions were identified as dynamical hinge points in the conformational switch transition. The ligation site visits three distinct states characterized by hydrogen bond patterns that are correlated with the formation of specific contacts that may promote catalysis. The insights gained from these simulations contribute to a more detailed understanding of the coupled catalytic/conformational switch mechanism of L1 ligase that may facilitate the design and engineering of new catalytic riboswitches.

  18. Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expands the Versatility of a Quality Control Ubiquitin Ligase.

    PubMed

    Weber, Annika; Cohen, Itamar; Popp, Oliver; Dittmar, Gunnar; Reiss, Yuval; Sommer, Thomas; Ravid, Tommer; Jarosch, Ernst

    2016-09-01

    The Doa10 quality control ubiquitin (Ub) ligase labels proteins with uniform lysine 48-linked poly-Ub (K48-pUB) chains for proteasomal degradation. Processing of Doa10 substrates requires the activity of two Ub conjugating enzymes. Here we show that the non-canonical conjugating enzyme Ubc6 attaches single Ub molecules not only to lysines but also to hydroxylated amino acids. These Ub moieties serve as primers for subsequent poly-ubiquitylation by Ubc7. We propose that the evolutionary conserved propensity of Ubc6 to mount Ub on diverse amino acids augments the number of ubiquitylation sites within a substrate and thereby increases the target range of Doa10. Our work provides new insights on how the consecutive activity of two specialized conjugating enzymes facilitates the attachment of poly-Ub to very heterogeneous client molecules. Such stepwise ubiquitylation reactions most likely represent a more general cellular phenomenon that extends the versatility yet sustains the specificity of the Ub conjugation system.

  19. Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expands the Versatility of a Quality Control Ubiquitin Ligase.

    PubMed

    Weber, Annika; Cohen, Itamar; Popp, Oliver; Dittmar, Gunnar; Reiss, Yuval; Sommer, Thomas; Ravid, Tommer; Jarosch, Ernst

    2016-09-01

    The Doa10 quality control ubiquitin (Ub) ligase labels proteins with uniform lysine 48-linked poly-Ub (K48-pUB) chains for proteasomal degradation. Processing of Doa10 substrates requires the activity of two Ub conjugating enzymes. Here we show that the non-canonical conjugating enzyme Ubc6 attaches single Ub molecules not only to lysines but also to hydroxylated amino acids. These Ub moieties serve as primers for subsequent poly-ubiquitylation by Ubc7. We propose that the evolutionary conserved propensity of Ubc6 to mount Ub on diverse amino acids augments the number of ubiquitylation sites within a substrate and thereby increases the target range of Doa10. Our work provides new insights on how the consecutive activity of two specialized conjugating enzymes facilitates the attachment of poly-Ub to very heterogeneous client molecules. Such stepwise ubiquitylation reactions most likely represent a more general cellular phenomenon that extends the versatility yet sustains the specificity of the Ub conjugation system. PMID:27570077

  20. Characterization of the tRNA ligases of pathogenic fungi Aspergillus fumigatus and Coccidioides immitis.

    PubMed

    Remus, Barbara S; Schwer, Beate; Shuman, Stewart

    2016-10-01

    Yeast tRNA ligase (Trl1) is an essential trifunctional enzyme that repairs RNA breaks with 2',3'-cyclic-PO4 and 5'-OH ends. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase domains that heal the broken ends to generate the 3'-OH, 2'-PO4, and 5'-PO4 termini required for sealing by an N-terminal ligase domain. Trl1 enzymes are found in all human fungal pathogens and they are promising targets for antifungal drug discovery because: (i) their domain structures and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme; and (ii) there are no obvious homologs of the Trl1 ligase domain in mammalian proteomes. Here we characterize the tRNA ligases of two human fungal pathogens: Coccidioides immitis and Aspergillus fumigatus The biological activity of CimTrl1 and AfuTrl1 was verified by showing that their expression complements a Saccharomyces cerevisiae trl1Δ mutant. Purified recombinant AfuTrl1 and CimTrl1 proteins were catalytically active in joining 2',3'-cyclic-PO4 and 5'-OH ends in vitro, either as full-length proteins or as a mixture of separately produced healing and sealing domains. The biochemical properties of CimTrl1 and AfuTrl1 are similar to those of budding yeast Trl1, particularly with respect to their preferential use of GTP as the phosphate donor for the polynucleotide kinase reaction. Our findings provide genetic and biochemical tools to screen for inhibitors of tRNA ligases from pathogenic fungi.

  1. Very long chain fatty acid synthesis in sunflower kernels.

    PubMed

    Salas, Joaquín J; Martínez-Force, Enrique; Garcés, Rafael

    2005-04-01

    Most common seed oils contain small amounts of very long chain fatty acids (VLCFAs), the main components of oils from species such as Brassica napus or Lunnaria annua. These fatty acids are synthesized from acyl-CoA precursors in the endoplasmic reticulum through the activity of a dissociated enzyme complex known as fatty acid elongase. We studied the synthesis of the arachidic, behenic, and lignoceric VLCFAs in sunflower kernels, in which they account for 1-3% of the saturated fatty acids. These VLCFAs are synthesized from 18:0-CoA by membrane-bound fatty acid elongases, and their biosynthesis is mainly dependent on NADPH equivalents. Two condensing enzymes appear to be responsible for the synthesis of VLCFAs in sunflower kernels, beta-ketoacyl-CoA synthase-I (KCS-I) and beta-ketoacyl-CoA synthase-II (KCS-II). Both of these enzymes were resolved by ion exchange chromatography and display different substrate specificities. While KCS-I displays a preference for 20:0-CoA, 18:0-CoA was more efficiently elongated by KCS-II. Both enzymes have different sensitivities to pH and Triton X-100, and their kinetic properties indicate that both are strongly inhibited by the presence of their substrates. In light of these results, the VLCFA composition of sunflower oil is considered in relation to that in other commercially exploited oils.

  2. Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation.

    PubMed

    Lechtenberg, Bernhard C; Rajput, Akhil; Sanishvili, Ruslan; Dobaczewska, Małgorzata K; Ware, Carl F; Mace, Peter D; Riedl, Stefan J

    2016-01-28

    Ubiquitination is a central process affecting all facets of cellular signalling and function. A critical step in ubiquitination is the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to a substrate or a growing ubiquitin chain, which is mediated by E3 ubiquitin ligases. RING-type E3 ligases typically facilitate the transfer of ubiquitin from the E2 directly to the substrate. The RING-between-RING (RBR) family of RING-type E3 ligases, however, breaks this paradigm by forming a covalent intermediate with ubiquitin similarly to HECT-type E3 ligases. The RBR family includes Parkin and HOIP, the central catalytic factor of the LUBAC (linear ubiquitin chain assembly complex). While structural insights into the RBR E3 ligases Parkin and HHARI in their overall auto-inhibited forms are available, no structures exist of intact fully active RBR E3 ligases or any of their complexes. Thus, the RBR mechanism of action has remained largely unknown. Here we present the first structure, to our knowledge, of the fully active human HOIP RBR in its transfer complex with an E2~ubiquitin conjugate, which elucidates the intricate nature of RBR E3 ligases. The active HOIP RBR adopts a conformation markedly different from that of auto-inhibited RBRs. HOIP RBR binds the E2~ubiquitin conjugate in an elongated fashion, with the E2 and E3 catalytic centres ideally aligned for ubiquitin transfer, which structurally both requires and enables a HECT-like mechanism. In addition, three distinct helix-IBR-fold motifs inherent to RBRs form ubiquitin-binding regions that engage the activated ubiquitin of the E2~ubiquitin conjugate and, surprisingly, an additional regulatory ubiquitin molecule. The features uncovered reveal critical states of the HOIP RBR E3 ligase cycle, and comparison with Parkin and HHARI suggests a general mechanism for RBR E3 ligases. PMID:26789245

  3. Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation

    PubMed Central

    Lechtenberg, Bernhard C.; Rajput, Akhil; Sanishvili, Ruslan; Dobaczewska, Małgorzata K.; Ware, Carl F.; Mace, Peter D.; Riedl, Stefan J.

    2015-01-01

    Ubiquitination is a central process affecting all facets of cellular signaling and function1. A critical step in ubiquitination is the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to a substrate or a growing ubiquitin chain, which is mediated by E3 ubiquitin ligases. RING-type E3 ligases typically facilitate the transfer of ubiquitin from the E2 directly to the substrate2,3. The RBR family of RING-type E3 ligases, however, breaks this paradigm by forming a covalent intermediate with ubiquitin similarly to HECT-type E3 ligases4–6. The RBR family includes Parkin4 and HOIP, the central catalytic factor of the linear ubiquitin chain assembly complex (LUBAC)7. While structural insights into the RBR E3 ligases Parkin and HHARI in their overall autoinhibited forms are available8–13, no structures exist of intact fully active RBR E3 ligases or any of their complexes. Thus, the RBR mechanism of action has remained largely enigmatic. Here we present the first structure of the fully active HOIP-RBR in its transfer complex with an E2~ubiquitin conjugate, which elucidates the intricate nature of RBR E3 ligases. The active HOIP-RBR adopts a conformation markedly different from that of autoinhibited RBRs. HOIP-RBR binds the E2~ubiquitin conjugate in an elongated fashion, with the E2 and E3 catalytic centers ideally aligned for ubiquitin transfer, which structurally both requires and enables a HECT-like mechanism. In addition, surprisingly, three distinct helix–IBR-fold motifs inherent to RBRs form ubiquitin-binding regions that engage the activated ubiquitin of the E2~Ub conjugate as well as an additional regulatory ubiquitin molecule. The features uncovered reveal critical states of the HOIP-RBR E3 ligase cycle, and comparison with Parkin and HHARI suggests a general mechanism for RBR E3 ligases. PMID:26789245

  4. The cowpea RING ubiquitin ligase VuDRIP interacts with transcription factor VuDREB2A for regulating abiotic stress responses.

    PubMed

    Sadhukhan, Ayan; Panda, Sanjib Kumar; Sahoo, Lingaraj

    2014-10-01

    Cowpea (Vigna unguiculata L. Walp) is an important grain legume cultivated in drought-prone parts of the world, having higher tolerance to heat and drought than many other crops. The transcription factor, Dehydration-Responsive Element-Binding protein 2A (DREB2A), controls expression of many genes involved in osmotic and heat stress responses of plants. In Arabidopsis, DREB2A-interacting proteins (DRIPs), which function as E3 ubiquitin ligases (EC 6.3.2.19), regulate the stability of DREB2A by targeting it for proteasome-mediated degradation. In this study, we cloned the cowpea ortholog of DRIP (VuDRIP) using PCR based methods. The 1614 bp long VuDRIP mRNA encoded a protein of 433 amino acids having a C3HC4-type Really Interesting New Gene (RING) domain in the N-terminus and a C-terminal conserved region, similar to Arabidopsis DRIP1 and DRIP2. We found VuDRIP up-regulation in response to various abiotic stresses and phytohormones. Using yeast (Saccharomyces cerevisae) two-hybrid analysis, VuDRIP was identified as a VuDREB2A-interacting protein. The results indicate negative regulation of VuDREB2A by ubiquitin ligases in cowpea similar to Arabidopsis along with their other unknown roles in stress and hormone signaling pathways. PMID:25090086

  5. Detection of T4 DNA ligase using a solid-state electrochemiluminescence biosensing switch based on ferrocene-labeled molecular beacon.

    PubMed

    Wang, Xiaoying; Dong, Ping; Yun, Wen; Xu, Ying; He, Pingang; Fang, Yuzhi

    2010-03-15

    A solid-state electrochemiluminescence (ECL) biosensing switch based on special ferrocene-labeled molecular beacon (Fc-MB) has been successfully developed for T4 DNA ligase detection. Such special switch system consisted of two main parts, an ECL substrate and an ECL intensity switch. The ECL substrate was made by modifying the complex of Au nanoparticle and Ruthenium (II) tris-(bipyridine) (Ru(bpy)(3)(2+)-AuNPs) onto Au electrode. A molecular beacon labeled by ferrocene as the ECL intensity switch. The molecular beacon is designed with special base sequence, which could combine with its target biomolecule via the reaction of the repair and recombination of nucleic acids by DNA ligase. During the reaction, the molecular beacon opened its stem-loop, and the labeled Fc was consequently kept away from the ECL substrate. Such structural change resulted in an obvious increment in ECL intensity due to the decreased Fc quenching effect to the ECL substrate. The analysis results are sensitive and specific.

  6. The cowpea RING ubiquitin ligase VuDRIP interacts with transcription factor VuDREB2A for regulating abiotic stress responses.

    PubMed

    Sadhukhan, Ayan; Panda, Sanjib Kumar; Sahoo, Lingaraj

    2014-10-01

    Cowpea (Vigna unguiculata L. Walp) is an important grain legume cultivated in drought-prone parts of the world, having higher tolerance to heat and drought than many other crops. The transcription factor, Dehydration-Responsive Element-Binding protein 2A (DREB2A), controls expression of many genes involved in osmotic and heat stress responses of plants. In Arabidopsis, DREB2A-interacting proteins (DRIPs), which function as E3 ubiquitin ligases (EC 6.3.2.19), regulate the stability of DREB2A by targeting it for proteasome-mediated degradation. In this study, we cloned the cowpea ortholog of DRIP (VuDRIP) using PCR based methods. The 1614 bp long VuDRIP mRNA encoded a protein of 433 amino acids having a C3HC4-type Really Interesting New Gene (RING) domain in the N-terminus and a C-terminal conserved region, similar to Arabidopsis DRIP1 and DRIP2. We found VuDRIP up-regulation in response to various abiotic stresses and phytohormones. Using yeast (Saccharomyces cerevisae) two-hybrid analysis, VuDRIP was identified as a VuDREB2A-interacting protein. The results indicate negative regulation of VuDREB2A by ubiquitin ligases in cowpea similar to Arabidopsis along with their other unknown roles in stress and hormone signaling pathways.

  7. COA User's Guide

    SciTech Connect

    Fox, B.; Pautz, J.; Sellers, C.

    1999-01-28

    The Department of Energy (DOE) has one of the largest and most complete collections of information on crude oil composition that is available to the public. The computer program that manages this database of crude oil analyses has recently been rewritten to allow easier access to this information. This report describes how the new system can be accessed and how the information contained in the Crude Oil Analysis Data Bank can be obtained.

  8. Steady state fluorescence studies of wild type recombinant cinnamoyl CoA reductase (Ll-CCRH1) and its active site mutants.

    PubMed

    Sonawane, Prashant; Vishwakarma, Rishi Kishore; Singh, Somesh; Gaikwad, Sushama; Khan, Bashir M

    2014-05-01

    Fluorescence quenching and time resolved fluorescence studies of wild type recombinant cinnamoyl CoA reductase (Ll-CCRH1), a multitryptophan protein from Leucaena leucocephala and 10 different active site mutants were carried out to investigate tryptophan environment. The enzyme showed highest affinity for feruloyl CoA (K(a)  = 3.72 × 10(5) M(-1)) over other CoA esters and cinnamaldehydes, as determined by fluorescence spectroscopy. Quenching of the fluorescence by acrylamide for wild type and active site mutants was collisional with almost 100% of the tryptophan fluorescence accessible under native condition and remained same after denaturation of protein with 6 M GdnHCl. In wild type Ll-CCRH1, the extent of quenching achieved with iodide (f(a) = 1.0) was significantly higher than cesium ions (f(a) = 0.33) suggesting more density of positive charge around surface of trp conformers under native conditions. Denaturation of wild type protein with 6 M GdnHCl led to significant increase in the quenching with cesium (f(a) = 0.54), whereas quenching with iodide ion was decreased (f(a) = 0.78), indicating reorientation of charge density around trp from positive to negative and heterogeneity in trp environment. The Stern-Volmer plots for wild type and mutants Ll-CCRH1 under native and denatured conditions, with cesium ion yielded biphasic quenching profiles. The extent of quenching for cesium and iodide ions under native and denatured conditions observed in active site mutants was significantly different from wild type Ll-CCRH1 under the same conditions. Thus, single substitution type mutations of active site residues showed heterogeneity in tryptophan microenvironment and differential degree of conformation of protein under native or denatured conditions. PMID:24322526

  9. Effect of Genistein and L-Carnitine and Their Combination on Gene Expression of Hepatocyte HMG-COA Reductase and LDL Receptor in Experimental Nephrotic Syndrome

    PubMed Central

    YOUSEFINEJAD, Abbas; SIASSI, Fereydoon; MIRSHAFIEY, Abbas; ESHRAGHIAN, Mohammad-Reza; KOOHDANI, Fariba; JAVANBAKHT, Mohammad Hassan; SEDAGHAT, Reza; RAMEZANI, Atena; ZAREI, Mahnaz; DJALALI, Mahmoud

    2015-01-01

    Background: Nephrotic syndrome is a disorder that leads to hyperlipidemia. L-carnitine and genistein can effect on lipid metabolism and the syndrome. In the present study, we have delved into the separate and the twin-effects of L-carnitine and genistein on the gene expressions of HMG-COA reductase and LDL receptor in experimental nephrotic syndrome. Methods: In this controlled experimental study, 50 male Sprague–Dawley rats were randomly divided into five groups: NC (normal-control), PC (patient-control), LC (L-carnitine), G (genistein), LCG (L-carnitine-genistein). Adriamycin was used for inducing nephrotic syndrome and the spot urine samples and urine protein-to-creatinine ratio were measured. Hepatocytic RNA was extracted and real-time PCR was used for HMG-COA Reductase and LDL receptor gene Expression measurement. Results: The final weight of the patients groups were lower than the NC group (P=0.001), and weight gain of the NC group was higher than the other groups (P<0.001). The proteinuria and urine protein-to-creatinine ratio showed significant differences between PC group and LC, G and LCG groups at week 7 (P<0.001). The expression of HMGCOA Reductase mRNA down regulated in LC, G and LCG groups in comparison with PC group (P<0.001). ΔCT of LDLr mRNA showed significant differences between the PC group and the other patient groups (P<0.001). Conclusion: This study shows a significant decreasing (P<0.001) and non-significant increasing trend in HMG-COA Reductase and LDLr gene expression, respectively, and synergistic effect of L-carnitine and genistein on these genes in experimental nephrotic syndrome. PMID:26576346

  10. CRP Is an Activator of Yersinia pestis Biofilm Formation that Operates via a Mechanism Involving gmhA and waaAE-coaD

    PubMed Central

    Liu, Lei; Fang, Haihong; Yang, Huiying; Zhang, Yiquan; Han, Yanping; Zhou, Dongsheng; Yang, Ruifu

    2016-01-01

    gmhA encodes a phosphoheptose isomerase that catalyzes the biosynthesis of heptose, a conserved component of lipopolysaccharide (LPS). GmhA plays an important role in Yersinia pestis biofilm blockage in the flea gut. waaA, waaE, and coaD constitute a three-gene operon waaAE-coaD in Y. pestis. waaA encodes a transferase that is responsible for binding lipid-A to the core oligosaccharide of LPS. WaaA is a key determinant in Y. pestis biofilm formation, and the waaA expression is positively regulated by the two-component regulatory system PhoP/PhoQ. WaaE is involved in LPS modification and is necessary for Y. pestis biofilm production. In this study, the biofilm-related phenotypic assays indicate that the global regulator CRP stimulates Y. pestis biofilm formation in vitro and on nematodes, while it has no regulatory effect on the biosynthesis of the biofilm-signaling molecular 3′,5′-cyclic diguanosine monophosphate. Further gene regulation experiments disclose that CRP does not regulate the hms genes at the transcriptional level but directly promotes the gmhA transcription and indirectly activates the waaAE-coaD transcription through directly acting on phoPQ-YPO1632. Thus, it is speculated that CRP-mediated carbon catabolite regulation of Y. pestis biofilm formation depends on the CRP-dependent carbon source metabolic pathways of the biosynthesis, modification, and transportation of biofilm exopolysaccharide. PMID:27014218

  11. The apparent coupling between synthesis and posttranslational modification of Escherichia coli acyl carrier protein is due to inhibition of amino acid biosynthesis.

    PubMed

    Keating, D H; Zhang, Y; Cronan, J E

    1996-05-01

    Acyl carrier protein (ACP) is modified on serine 36 by the covalent posttranslational attachment of 4'-phosphopantetheine from coenzyme A (CoA), and this modification is required for lipid biosynthesis. Jackowski and Rock (J. Biol. Chem 258:15186-15191, 1983) reported that upon depletion of the CoA pool by starvation for a CoA precursor, no accumulation of the unmodified form of ACP (apo-ACP) was detected. We report that this lack of apo-ACP accumulation results from decreased translation of the acpP mRNAs because of the limitation of the synthesis of glutamate and other amino acids made directly from tricarboxylic acid cycle intermediates.

  12. Structural and Functional Impact of Parkinson Disease-Associated Mutations in the E3 Ubiquitin Ligase Parkin.

    PubMed

    Fiesel, Fabienne C; Caulfield, Thomas R; Moussaud-Lamodière, Elisabeth L; Ogaki, Kotaro; Dourado, Daniel F A R; Flores, Samuel C; Ross, Owen A; Springer, Wolfdieter

    2015-08-01

    Mutations in the PARKIN/PARK2 gene that result in loss-of-function of the encoded, neuroprotective E3 ubiquitin ligase Parkin cause recessive, familial early-onset Parkinson disease. As an increasing number of rare Parkin sequence variants with unclear pathogenicity are identified, structure-function analyses will be critical to determine their disease relevance. Depending on the specific amino acids affected, several distinct pathomechanisms can result in loss of Parkin function. These include disruption of overall Parkin folding, decreased solubility, and protein aggregation. However pathogenic effects can also result from misregulation of Parkin autoinhibition and of its enzymatic functions. In addition, interference of binding to coenzymes, substrates, and adaptor proteins can affect its catalytic activity too. Herein, we have performed a comprehensive structural and functional analysis of 21 PARK2 missense mutations distributed across the individual protein domains. Using this combined approach, we were able to pinpoint some of the pathogenic mechanisms of individual sequence variants. Similar analyses will be critical in gaining a complete understanding of the complex regulations and enzymatic functions of Parkin. These studies will not only highlight the important residues, but will also help to develop novel therapeutics aimed at activating and preserving an active, neuroprotective form of Parkin.

  13. Degradation of the ABA co-receptor ABI1 by PUB12/13 U-box E3 ligases

    PubMed Central

    Kong, Lingyao; Cheng, Jinkui; Zhu, Yujuan; Ding, Yanglin; Meng, Jingjing; Chen, Zhizhong; Xie, Qi; Guo, Yan; Li, Jigang; Yang, Shuhua; Gong, Zhizhong

    2015-01-01

    Clade A protein phosphatase 2Cs (PP2Cs) are abscisic acid (ABA) co-receptors that block ABA signalling by inhibiting the downstream protein kinases. ABA signalling is activated after PP2Cs are inhibited by ABA-bound PYR/PYL/RCAR ABA receptors (PYLs) in Arabidopsis. However, whether these PP2Cs are regulated by other factors remains unknown. Here, we report that ABI1 (ABA-INSENSITIVE 1) can interact with the U-box E3 ligases PUB12 and PUB13, but is ubiquitinated only when it interacts with ABA receptors in an in vitro assay. A mutant form of ABI1-1 that is unable to interact with PYLs is more stable than the wild-type protein. Both ABI1 degradation and all tested ABA responses are reduced in pub12 pub13 mutants compared with the wild type. Introducing the abi1-3 loss-of-function mutation into pub12 pub13 mutant recovers the ABA-insensitive phenotypes of the pub12 pub13 mutant. We thus uncover an important regulatory mechanism for regulating ABI1 levels by PUB12 and PUB13. PMID:26482222

  14. Regulating ehrlich and demethiolation pathways for alcohols production by the expression of ubiquitin-protein ligase gene HUWE1

    PubMed Central

    Zhang, Quan; Jia, Kai-Zhi; Xia, Shi-Tao; Xu, Yang-Hua; Liu, Rui-Sang; Li, Hong-Mei; Tang, Ya-Jie

    2016-01-01

    Ehrlich and demethiolation pathways as two competing branches converted amino acid into alcohols. Controlling both pathways offers considerable potential for industrial applications including alcohols overproduction, flavor-quality control and developing new flavors. While how to regulate ehrlich and demethiolation pathways is still not applicable. Taking the conversion of methionine into methionol and methanethiol for example, we constructed two suppression subtractive cDNA libraries of Clonostachys rosea by using suppression subtractive hybridization (SSH) technology for screening regulators controlling the conversion. E3 ubiquitin-protein ligase gene HUWE1 screened from forward SSH library was validated to be related with the biosynthesis of end products. Overexpressing HUWE1 in C. rosea and S. cerevisiae significantly increased the biosynthesis of methanethiol and its derivatives in demethiolation pathway, while suppressed the biosynthesis of methional and methionol in ehrlich pathway. These results attained the directional regulation of both pathways by overexpressing HUWE1. Thus, HUWE1 has potential to be a key target for controlling and enhancing alcohols production by metabolic engineering. PMID:26860895

  15. ATDC (Ataxia Telangiectasia Group D Complementing) Promotes Radioresistance through an Interaction with the RNF8 Ubiquitin Ligase.

    PubMed

    Yang, Huibin; Palmbos, Phillip L; Wang, Lidong; Kim, Evelyn H; Ney, Gina M; Liu, Chao; Prasad, Jayendra; Misek, David E; Yu, Xiaochun; Ljungman, Mats; Simeone, Diane M

    2015-11-01

    Induction of DNA damage by ionizing radiation (IR) and/or cytotoxic chemotherapy is an essential component of cancer therapy. The ataxia telangiectasia group D complementing gene (ATDC, also called TRIM29) is highly expressed in many malignancies. It participates in the DNA damage response downstream of ataxia telangiectasia-mutated (ATM) and p38/MK2 and promotes cell survival after IR. To elucidate the downstream mechanisms of ATDC-induced IR protection, we performed a mass spectrometry screen to identify ATDC binding partners. We identified a direct physical interaction between ATDC and the E3 ubiquitin ligase and DNA damage response protein, RNF8, which is required for ATDC-induced radioresistance. This interaction was refined to the C-terminal portion (amino acids 348-588) of ATDC and the RING domain of RNF8 and was disrupted by mutation of ATDC Ser-550 to alanine. Mutations disrupting this interaction abrogated ATDC-induced radioresistance. The interaction between RNF8 and ATDC, which was increased by IR, also promoted downstream DNA damage responses such as IR-induced γ-H2AX ubiquitination, 53BP1 phosphorylation, and subsequent resolution of the DNA damage foci. These studies define a novel function for ATDC in the RNF8-mediated DNA damage response and implicate RNF8 binding as a key determinant of the radioprotective function of ATDC.

  16. “Ubiquitylation: mechanism and functions“ Review series: RBR E3-ligases at work

    PubMed Central

    Smit, Judith J; Sixma, Titia K

    2014-01-01

    The RING-in-between-RING (RBR) E3s are a curious family of ubiquitin E3-ligases, whose mechanism of action is unusual in several ways. Their activities are auto-inhibited, causing a requirement for activation by protein-protein interactions or posttranslational modifications. They catalyse ubiquitin conjugation by a concerted RING/HECT-like mechanism in which the RING1 domain facilitates E2-discharge to directly form a thioester intermediate with a cysteine in RING2. This short-lived, HECT-like intermediate then modifies the target. Uniquely, the RBR ligase HOIP makes use of this mechanism to target the ubiquitin amino-terminus, by presenting the target ubiquitin for modification using its distinctive LDD region. PMID:24469331

  17. AMP-dependent DNA relaxation catalyzed by DNA ligase occurs by a nicking-closing mechanism.

    PubMed Central

    Montecucco, A; Ciarrocchi, G

    1988-01-01

    In the presence of AMP and Mg2+, a covalently closed duplex DNA containing negative superhelical turns was treated with DNA ligase isolated from bacteriophage T4-infected E. coli. This resulted in the gradual and not sudden loss of superhelical turns as for example in the case of type I DNA topoisomerase. All DNA products remain covalently closed. Since T4 enzyme-mediated DNA relaxation is inhibited by both pyrophosphate and by ATP this suggests that DNA relaxing and DNA joining activities probably coincide. EDTA addition in the presence of a large excess of enzyme, induces the formation of nicked DNA products while protein denaturing treatments are not very effective. Our observations might suggest an involvement of the relaxing activity of DNA ligase during the ligation process. Images PMID:3137526

  18. The interplay between DSL proteins and ubiquitin ligases in Notch signaling.

    PubMed

    Pitsouli, Chrysoula; Delidakis, Christos

    2005-09-01

    Lateral inhibition is a pattern refining process that generates single neural precursors from a field of equipotent cells and is mediated via Notch signaling. Of the two Notch ligands Delta and Serrate, only the former was thought to participate in this process. We now show that macrochaete lateral inhibition involves both Delta and Serrate. In this context, Serrate interacts with Neuralized, a ubiquitin ligase that was heretofore thought to act only on Delta. Neuralized physically associates with Serrate and stimulates its endocytosis and signaling activity. We also characterize a mutation in mib1, a Drosophila homolog of mind bomb, another Delta-targeting ubiquitin ligase from zebrafish. Mib1 affects the signaling activity of Delta and Serrate in both lateral inhibition and wing dorsoventral boundary formation. Simultaneous absence of neuralized and mib1 completely abolishes Notch signaling in both aforementioned contexts, making it likely that ubiquitination is a prerequisite for Delta/Serrate signaling.

  19. Inhibitor of apoptosis proteins as E3 ligases for ubiquitin and NEDD8.

    PubMed

    Kamada, Shinji

    2013-04-01

    The inhibitors of apoptosis proteins (IAPs) are endogenous inhibitors for apoptosis. Apoptosis is carried out by caspases, which are the family of cystein proteases. IAPs regulate caspases through two conserved regions, the baculovirus IAP repeats (BIRs) and the really interesting new gene (RING) domains. Although the BIRs are responsible for binding to caspases, the RING domain can act as a ubiquitin-E3 ligase, leading to ubiquitylation of IAPs themselves and their pro-apoptotic IAP counterparts such as caspases. Recently, it is reported that another ubiquitin-like protein, neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8), is also involved in the regulation of apoptosis through neddylation of caspases mediated by IAPs. On the contrary, the results against the function of IAPs as a NEDD8-E3 ligase are also suggested. This review presents the summary of IAPs, caspases, and the ubiquitin-proteasome system and how their interactions influence the regulation of apoptosis.

  20. IAPs as E3 ligases of Rac1: shaping the move.

    PubMed

    Oberoi-Khanuja, Tripat Kaur; Rajalingam, Krishnaraj

    2012-01-01

    Inhibitors of Apoptosis Proteins (IAPs) are well-studied E3 ubiquitin ligases predominantly known for regulation of apoptosis. We uncovered that IAPs can function as a direct E3 ubiquitin ligase of RhoGTPase Rac1. cIAP1 and XIAP directly conjugate polyubiquitin chains to Lysine 147 of activated Rac1 and target it for proteasomal degradation. Consistently, loss of these IAPs by various strategies led to stabilization of Rac1 and mesenchymal mode of migration in tumor cells. IAPs also regulate Rac1 degradation upon RhoGDI1 depletion and CNF1 toxin treatment. Our observations revealed an evolutionarily conserved role of IAPs in regulating Rac1 stability shedding light on to the mechanisms behind ubiquitination-dependent inactivation of Rac1 signaling.

  1. Structurally complex and highly active RNA ligases derived from random RNA sequences

    NASA Technical Reports Server (NTRS)

    Ekland, E. H.; Szostak, J. W.; Bartel, D. P.

    1995-01-01

    Seven families of RNA ligases, previously isolated from random RNA sequences, fall into three classes on the basis of secondary structure and regiospecificity of ligation. Two of the three classes of ribozymes have been engineered to act as true enzymes, catalyzing the multiple-turnover transformation of substrates into products. The most complex of these ribozymes has a minimal catalytic domain of 93 nucleotides. An optimized version of this ribozyme has a kcat exceeding one per second, a value far greater than that of most natural RNA catalysts and approaching that of comparable protein enzymes. The fact that such a large and complex ligase emerged from a very limited sampling of sequence space implies the existence of a large number of distinct RNA structures of equivalent complexity and activity.

  2. An improved smaller biotin ligase for BioID proximity labeling

    PubMed Central

    Kim, Dae In; Jensen, Samuel C.; Noble, Kyle A.; KC, Birendra; Roux, Kenneth H.; Motamedchaboki, Khatereh; Roux, Kyle J.

    2016-01-01

    The BioID method uses a promiscuous biotin ligase to detect protein–protein associations as well as proximate proteins in living cells. Here we report improvements to the BioID method centered on BioID2, a substantially smaller promiscuous biotin ligase. BioID2 enables more-selective targeting of fusion proteins, requires less biotin supplementation, and exhibits enhanced labeling of proximate proteins. Thus BioID2 improves the efficiency of screening for protein–protein associations. We also demonstrate that the biotinylation range of BioID2 can be considerably modulated using flexible linkers, thus enabling application-specific adjustment of the biotin-labeling radius. PMID:26912792

  3. Biochemical Competition Makes Fatty-Acid β-Oxidation Vulnerable to Substrate Overload

    PubMed Central

    van Eunen, Karen; Simons, Sereh M. J.; Gerding, Albert; Bleeker, Aycha; den Besten, Gijs; Touw, Catharina M. L.; Houten, Sander M.; Groen, Bert K.; Krab, Klaas; Reijngoud, Dirk-Jan; Bakker, Barbara M.

    2013-01-01

    Fatty-acid metabolism plays a key role in acquired and inborn metabolic diseases. To obtain insight into the network dynamics of fatty-acid β-oxidation, we constructed a detailed computational model of the pathway and subjected it to a fat overload condition. The model contains reversible and saturable enzyme-kinetic equations and experimentally determined parameters for rat-liver enzymes. It was validated by adding palmitoyl CoA or palmitoyl carnitine to isolated rat-liver mitochondria: without refitting of measured parameters, the model correctly predicted the β-oxidation flux as well as the time profiles of most acyl-carnitine concentrations. Subsequently, we simulated the condition of obesity by increasing the palmitoyl-CoA concentration. At a high concentration of palmitoyl CoA the β-oxidation became overloaded: the flux dropped and metabolites accumulated. This behavior originated from the competition between acyl CoAs of different chain lengths for a set of acyl-CoA dehydrogenases with overlapping substrate specificity. This effectively induced competitive feedforward inhibition and thereby led to accumulation of CoA-ester intermediates and depletion of free CoA (CoASH). The mitochondrial [NAD+]/[NADH] ratio modulated the sensitivity to substrate overload, revealing a tight interplay between regulation of β-oxidation and mitochondrial respiration. PMID:23966849

  4. Biochemical competition makes fatty-acid β-oxidation vulnerable to substrate overload.

    PubMed

    van Eunen, Karen; Simons, Sereh M J; Gerding, Albert; Bleeker, Aycha; den Besten, Gijs; Touw, Catharina M L; Houten, Sander M; Groen, Bert K; Krab, Klaas; Reijngoud, Dirk-Jan; Bakker, Barbara M

    2013-01-01

    Fatty-acid metabolism plays a key role in acquired and inborn metabolic diseases. To obtain insight into the network dynamics of fatty-acid β-oxidation, we constructed a detailed computational model of the pathway and subjected it to a fat overload condition. The model contains reversible and saturable enzyme-kinetic equations and experimentally determined parameters for rat-liver enzymes. It was validated by adding palmitoyl CoA or palmitoyl carnitine to isolated rat-liver mitochondria: without refitting of measured parameters, the model correctly predicted the β-oxidation flux as well as the time profiles of most acyl-carnitine concentrations. Subsequently, we simulated the condition of obesity by increasing the palmitoyl-CoA concentration. At a high concentration of palmitoyl CoA the β-oxidation became overloaded: the flux dropped and metabolites accumulated. This behavior originated from the competition between acyl CoAs of different chain lengths for a set of acyl-CoA dehydrogenases with overlapping substrate specificity. This effectively induced competitive feedforward inhibition and thereby led to accumulation of CoA-ester intermediates and depletion of free CoA (CoASH). The mitochondrial [NAD⁺]/[NADH] ratio modulated the sensitivity to substrate overload, revealing a tight interplay between regulation of β-oxidation and mitochondrial respiration.

  5. Identification of HECT E3 ubiquitin ligase family genes involved in stem cell regulation and regeneration in planarians.

    PubMed

    Henderson, Jordana M; Nisperos, Sean V; Weeks, Joi; Ghulam, Mahjoobah; Marín, Ignacio; Zayas, Ricardo M

    2015-08-15

    E3 ubiquitin ligases constitute a large family of enzymes that modify specific proteins by covalently attaching ubiquitin polypeptides. This post-translational modification can serve to regulate protein function or longevity. In spite of their importance in cell physiology, the biological roles of most ubiquitin ligases remain poorly understood. Here, we analyzed the function of the HECT domain family of E3 ubiquitin ligases in stem cell biology and tissue regeneration in planarians. Using bioinformatic searches, we identified 17 HECT E3 genes that are expressed in the Schmidtea mediterranea genome. Whole-mount in situ hybridization experiments showed that HECT genes were expressed in diverse tissues and most were expressed in the stem cell population (neoblasts) or in their progeny. To investigate the function of all HECT E3 ligases, we inhibited their expression using RNA interference (RNAi) and determined that orthologs of huwe1, wwp1, and trip12 had roles in tissue regeneration. We show that huwe1 RNAi knockdown led to a significant expansion of the neoblast population and death by lysis. Further, our experiments showed that wwp1 was necessary for both neoblast and intestinal tissue homeostasis as well as uncovered an unexpected role of trip12 in posterior tissue specification. Taken together, our data provide insights into the roles of HECT E3 ligases in tissue regeneration and demonstrate that planarians will be a useful model to evaluate the functions of E3 ubiquitin ligases in stem cell regulation.

  6. Enzyme–adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding surface

    PubMed Central

    Williamson, Adele; Rothweiler, Ulli; Schrøder Leiros, Hanna-Kirsti

    2014-01-01

    DNA ligases are a structurally diverse class of enzymes which share a common catalytic core and seal breaks in the phosphodiester backbone of double-stranded DNA via an adenylated intermediate. Here, the structure and activity of a recombinantly produced ATP-dependent DNA ligase from the bacterium Psychromonas sp. strain SP041 is described. This minimal-type ligase, like its close homologues, is able to ligate singly nicked double-stranded DNA with high efficiency and to join cohesive-ended and blunt-ended substrates to a more limited extent. The 1.65 Å resolution crystal structure of the enzyme–adenylate complex reveals no unstructured loops or segments, and suggests that this enzyme binds the DNA without requiring full encirclement of the DNA duplex. This is in contrast to previously characterized minimal DNA ligases from viruses, which use flexible loop regions for DNA interaction. The Psychromonas sp. enzyme is the first structure available for the minimal type of bacterial DNA ligases and is the smallest DNA ligase to be crystallized to date. PMID:25372693

  7. Orally administered rosmarinic acid is present as the conjugated and/or methylated forms in plasma, and is degraded and metabolized to conjugated forms of caffeic acid, ferulic acid and m-coumaric acid.

    PubMed

    Baba, Seigo; Osakabe, Naomi; Natsume, Midori; Terao, Junji

    2004-05-28

    Rosmarinic acid (RA) is contained in various Lamiaceae herbs used commonly as culinary herbs. Although RA has various potent physiological actions, little is known on its bioavailability. We therefore investigated the absorption and metabolism of orally administered RA in rats. After being deprived of food for 12 h, RA (50 mg/kg body weight) or deionized water was administered orally to rats. Blood samples were collected from a cannula inserted in the femoral artery before and at designated time intervals after administration of RA. Urine excreted within 0 to 8 h and 8 to 18 h post-administration was also collected. RA and its related metabolites in plasma and urine were measured by LC-MS after treatment with sulfatase and/or beta-glucuronidase. RA, mono-methylated RA (methyl-RA) and m-coumaric acid (COA) were detected in plasma, with peak concentrations being reached at 0.5, 1 and 8 h after RA administration, respectively. RA, methyl-RA, caffeic acid (CAA), ferulic acid (FA) and COA were detected in urine after RA administration. These components in plasma and urine were present predominantly as conjugated forms such as glucuronide or sulfate. The percentage of the original oral dose of RA excreted in the urine within 18 h of administration as free and conjugated forms was 0.44 +/- 0.21% for RA, 1.60 +/- 0.74% for methyl-RA, 1.06 +/- 0.35% for CAA, 1.70 +/- 0.45% for FA and 0.67 +/- 0.29% for COA. Approximately 83% of the total amount of these metabolites was excreted in the period 8 to 18 h after RA administration. These results suggest that RA was absorbed and metabolized as conjugated and/or methylated forms, and that the majority of RA absorbed was degraded into conjugated and/or methylated forms of CAA, FA and COA before being excreted gradually in the urine. PMID:15120569

  8. Proteolytic regulation of metabolic enzymes by E3 ubiquitin ligase complexes: lessons from yeast.

    PubMed

    Nakatsukasa, Kunio; Okumura, Fumihiko; Kamura, Takumi

    2015-01-01

    Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes. Coarse metabolic control can be achieved through the degradation of metabolic enzymes by the ubiquitin-proteasome system (UPS), in which substrates are specifically ubiquitinated by an E3 ubiquitin ligase and targeted for proteasomal degradation. Here, we review select multi-protein E3 ligase complexes that directly regulate metabolic enzymes in Saccharomyces cerevisiae. The first part of the review focuses on the endoplasmic reticulum (ER) membrane-associated Hrd1 and Doa10 E3 ligase complexes. In addition to their primary roles in the ER-associated degradation pathway that eliminates misfolded proteins, recent quantitative proteomic analyses identified native substrates of Hrd1 and Doa10 in the sterol synthesis pathway. The second part focuses on the SCF (Skp1-Cul1-F-box protein) complex, an abundant prototypical multi-protein E3 ligase complex. While the best-known roles of the SCF complex are in the regulation of the cell cycle and transcription, accumulating evidence indicates that the SCF complex also modulates carbon metabolism pathways. The increasing number of metabolic enzymes whose stability is directly regulated by the UPS underscores the importance of the proteolytic regulation of metabolic processes for the acclimation of cells to environmental changes. PMID:26362128

  9. Substrate Trapping Proteomics Reveals Targets of the βTrCP2/FBXW11 Ubiquitin Ligase

    PubMed Central

    Kim, Tai Young; Siesser, Priscila F.; Rossman, Kent L.; Goldfarb, Dennis; Mackinnon, Kathryn; Yan, Feng; Yi, XianHua; MacCoss, Michael J.; Moon, Randall T.; Der, Channing J.

    2014-01-01

    Defining the full complement of substrates for each ubiquitin ligase remains an important challenge. Improvements in mass spectrometry instrumentation and computation and in protein biochemistry methods have resulted in several new methods for ubiquitin ligase substrate identification. Here we used the parallel adapter capture (PAC) proteomics approach to study βTrCP2/FBXW11, a substrate adaptor for the SKP1–CUL1–F-box (SCF) E3 ubiquitin ligase complex. The processivity of the ubiquitylation reaction necessitates transient physical interactions between FBXW11 and its substrates, thus making biochemical purification of FBXW11-bound substrates difficult. Using the PAC-based approach, we inhibited the proteasome to “trap” ubiquitylated substrates on the SCFFBXW11 E3 complex. Comparative mass spectrometry analysis of immunopurified FBXW11 protein complexes before and after proteasome inhibition revealed 21 known and 23 putatively novel substrates. In focused studies, we found that SCFFBXW11 bound, polyubiquitylated, and destabilized RAPGEF2, a guanine nucleotide exchange factor that activates the small GTPase RAP1. High RAPGEF2 protein levels promoted cell-cell fusion and, consequently, multinucleation. Surprisingly, this occurred independently of the guanine nucleotide exchange factor (GEF) catalytic activity and of the presence of RAP1. Our data establish new functions for RAPGEF2 that may contribute to aneuploidy in cancer. More broadly, this report supports the continued use of substrate trapping proteomics to comprehensively define targets for E3 ubiquitin ligases. All proteomic data are available via ProteomeXchange with identifier PXD001062. PMID:25332235

  10. Proteolytic regulation of metabolic enzymes by E3 ubiquitin ligase complexes: lessons from yeast.

    PubMed

    Nakatsukasa, Kunio; Okumura, Fumihiko; Kamura, Takumi

    2015-01-01

    Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes. Coarse metabolic control can be achieved through the degradation of metabolic enzymes by the ubiquitin-proteasome system (UPS), in which substrates are specifically ubiquitinated by an E3 ubiquitin ligase and targeted for proteasomal degradation. Here, we review select multi-protein E3 ligase complexes that directly regulate metabolic enzymes in Saccharomyces cerevisiae. The first part of the review focuses on the endoplasmic reticulum (ER) membrane-associated Hrd1 and Doa10 E3 ligase complexes. In addition to their primary roles in the ER-associated degradation pathway that eliminates misfolded proteins, recent quantitative proteomic analyses identified native substrates of Hrd1 and Doa10 in the sterol synthesis pathway. The second part focuses on the SCF (Skp1-Cul1-F-box protein) complex, an abundant prototypical multi-protein E3 ligase complex. While the best-known roles of the SCF complex are in the regulation of the cell cycle and transcription, accumulating evidence indicates that the SCF complex also modulates carbon metabolism pathways. The increasing number of metabolic enzymes whose stability is directly regulated by the UPS underscores the importance of the proteolytic regulation of metabolic processes for the acclimation of cells to environmental changes.

  11. Unexpected substrate specificity of T4 DNA ligase revealed by in vitro selection

    NASA Technical Reports Server (NTRS)

    Harada, Kazuo; Orgel, Leslie E.

    1993-01-01

    We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 DNA ligase. We find that the ensemble of selected sequences ligates about 50 times as efficiently as the random mixture of sequences used as the input for selection. Surprisingly many of the selected sequences failed to produce a match at or close to the ligation junction. None of the 20 selected oligomers that we sequenced produced a match two bases upstream from the ligation junction.

  12. Structure And Function of the Yeast U-Box-Containing Ubiquitin Ligase Ufd2p

    SciTech Connect

    Tu, D.; Li, W.; Ye, Y.; Brunger, A.T.

    2009-06-04

    Proteins conjugated by Lys-48-linked polyubiquitin chains are preferred substrates of the eukaryotic proteasome. Polyubiquitination requires an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3). Occasionally, these enzymes only assemble short ubiquitin oligomers, and their extension to full length involves a ubiquitin elongating factor termed E4. Ufd2p, as the first E4 identified to date, is involved in the degradation of misfolded proteins of the endoplasmic reticulum and of a ubiquitin-{beta}-GAL fusion substrate in Saccharomyces cerevisiae. The mechanism of action of Ufd2p is unknown. Here we describe the crystal structure of the full-length yeast Ufd2p protein. Ufd2p has an elongated shape consisting of several irregular Armadillo-like repeats with two helical hairpins protruding from it and a U-box domain flexibly attached to its C terminus. The U-box of Ufd2p has a fold similar to that of the RING (Really Interesting New Gene) domain that is present in certain ubiquitin ligases. Accordingly, Ufd2p has all of the hallmarks of a RING finger-containing ubiquitin ligase: it associates with its cognate E2 Ubc4p via its U-box domain and catalyzes the transfer of ubiquitin from the E2 active site to Ufd2p itself or to an acceptor ubiquitin molecule to form unanchored diubiquitin oligomers. Thus, Ufd2p can function as a bona fide E3 ubiquitin ligase to promote ubiquitin chain elongation on a substrate.

  13. Cell cycle-dependent localization and properties of a second mitochondrial DNA ligase in Crithidia fasciculata.

    PubMed

    Sinha, Krishna Murari; Hines, Jane C; Ray, Dan S

    2006-01-01

    The mitochondrial DNA in kinetoplastid protozoa is contained in a single highly condensed structure consisting of thousands of minicircles and approximately 25 maxicircles. The disk-shaped structure is termed kinetoplast DNA (kDNA) and is located in the mitochondrial matrix near the basal body. We have previously identified a mitochondrial DNA ligase (LIG kbeta) in the trypanosomatid Crithidia fasciculata that localizes to antipodal sites flanking the kDNA disk where several other replication proteins are localized. We describe here a second mitochondrial DNA ligase (LIG kalpha). LIG kalpha localizes to the kinetoplast primarily in cells that have completed mitosis and contain either a dividing kinetoplast or two newly divided kinetoplasts. Essentially all dividing or newly divided kinetoplasts show localization of LIG kalpha. The ligase is present on both faces of the kDNA disk and at a high level in the kinetoflagellar zone of the mitochondrial matrix. Cells containing a single nucleus show localization of the LIG kalpha to the kDNA but at a much lower frequency. The mRNA level of LIG kalpha varies during the cell cycle out of phase with that of LIG kbeta. LIG kalpha transcript levels are maximal during the phase when cells contain two nuclei, whereas LIG kbeta transcript levels are maximal during S phase. The LIG kalpha protein decays with a half-life of 100 min in the absence of protein synthesis. The periodic expression of the LIG kalpha transcript and the instability of the LIG kalpha protein suggest a possible role of the ligase in regulating minicircle replication.

  14. Structural basis for catalytic activation by the human ZNF451 SUMO E3 ligase

    PubMed Central

    Cappadocia, Laurent; Pichler, Andrea; Lima, Christopher D.

    2015-01-01

    E3 protein ligases enhance transfer of ubiquitin-like (Ubl) proteins from E2 conjugating enzymes to substrates by stabilizing the thioester-charged E2~Ubl in a closed configuration optimally aligned for nucleophilic attack. Here, we report biochemical and structural data that define the N-terminal domain of the Homo sapiens ZNF451 as the catalytic module for SUMO E3 ligase activity. ZNF451 catalytic module contains tandem SUMO interaction motifs (SIMs) bridged by a Proline-Leucine-Arginine-Proline (PLRP) motif. The first SIM and PLRP motif engage thioester charged E2~SUMO while the next SIM binds a second molecule of SUMO bound to the backside of E2. We show that ZNF451 is SUMO2 specific and that SUMO-modification of ZNF451 may contribute to activity by providing a second molecule of SUMO that interacts with E2. Our results are consistent with ZNF451 functioning as a bona fide SUMO E3 ligase. PMID:26524494

  15. E3 Ubiquitin Ligases Pellinos as Regulators of Pattern Recognition Receptor Signaling and Immune responses

    PubMed Central

    Medvedev, Andrei E.; Murphy, Michael; Zhou, Hao; Li, Xiaoxia

    2015-01-01

    SUMMARY Pellinos are a family of E3 ubiquitin ligases discovered for their role in catalyzing K63-linked polyubiquitination of Pelle, an IL-1 receptor-associated kinase homologue in the Drosophila Toll pathway. Subsequent studies have revealed the central and non-redundant roles of mammalian Pellino-1, Pellino-2 and Pelino-3 in signaling pathways emanating from IL-1 receptors, Toll-like receptors, NOD-like receptors, T- and B-cell receptors. While Pellinos ability to interact with many signaling intermediates suggested their scaffolding roles, recent findings in mice expressing ligase-inactive Pellinos demonstrated the importance of Pellino ubiquitin ligase activity. Cell-specific functions of Pellinos have emerged, e.g., Pellino-1 being a negative regulator in T-lymphocytes and a positive regulator in myeloid cells, and details of molecular regulation of receptor signaling by various members of the Pellino family have been revealed. In this review, we have summarized current information about Pellino-mediated regulation of signaling by pattern recognition receptors, T-cell and B-cell receptors and TNF receptors, and discuss Pellino’s role in sepsis and infectious diseases, as well as in autoimmune, inflammatory and allergic disorders. We also provide our perspective on the potential of targeting Pellinos with peptide- or small molecule-based drug compounds as a new therapeutic approach for septic shock and autoimmune pathologies. PMID:26085210

  16. Disinhibition of the HECT E3 ubiquitin ligase WWP2 by polymerized Dishevelled

    PubMed Central

    Mund, Thomas; Graeb, Michael; Mieszczanek, Juliusz; Gammons, Melissa; Pelham, Hugh R. B.; Bienz, Mariann

    2015-01-01

    Dishevelled is a pivot in Wnt signal transduction, controlling both β-catenin-dependent transcription to specify proliferative cell fates, and cell polarity and other non-nuclear events in post-mitotic cells. In response to Wnt signals, or when present at high levels, Dishevelled forms signalosomes by dynamic polymerization. Its levels are controlled by ubiquitylation, mediated by various ubiquitin ligases, including NEDD4 family members that bind to a conserved PPxY motif in Dishevelled (mammalian Dvl1–3). Here, we show that Dvl2 binds to the ubiquitin ligase WWP2 and unlocks its ligase activity from autoinhibition. This disinhibition of WWP2 depends on several features of Dvl2 including its PPxY motif and to a lesser extent its DEP domain, but crucially on the ability of Dvl2 to polymerize, indicating that WWP2 is activated in Wnt signalosomes. We show that Notch intracellular domains are substrates for Dvl-activated WWP2 and their transcriptional activity is consequently reduced, providing a molecular mechanism for cross-talk between Wnt and Notch signalling. These regulatory interactions are conserved in Drosophila whose WWP2 orthologue, Suppressor-of-deltex, downregulates Notch signalling upon activation by Dishevelled in developing wing tissue. Attentuation of Notch signalling by Dishevelled signalosomes could be important during the transition of cells from the proliferative to the post-mitotic state. PMID:26701932

  17. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide.

    PubMed

    Fischer, Eric S; Böhm, Kerstin; Lydeard, John R; Yang, Haidi; Stadler, Michael B; Cavadini, Simone; Nagel, Jane; Serluca, Fabrizio; Acker, Vincent; Lingaraju, Gondichatnahalli M; Tichkule, Ritesh B; Schebesta, Michael; Forrester, William C; Schirle, Markus; Hassiepen, Ulrich; Ottl, Johannes; Hild, Marc; Beckwith, Rohan E J; Harper, J Wade; Jenkins, Jeremy L; Thomä, Nicolas H

    2014-08-01

    In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide, these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-deletion-associated dysplasia. IMiDs target the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4(CRBN)) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4(CRBN). Here we present crystal structures of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes that CRBN is a substrate receptor within CRL4(CRBN) and enantioselectively binds IMiDs. Using an unbiased screen, we identified the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4(CRBN). Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4(CRBN) while the ligase complex is recruiting IKZF1 or IKZF3 for degradation. This dual activity implies that small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins.

  18. TRAIP is a PCNA-binding ubiquitin ligase that protects genome stability after replication stress

    PubMed Central

    Hoffmann, Saskia; Smedegaard, Stine; Nakamura, Kyosuke; Mortuza, Gulnahar B.; Räschle, Markus; Ibañez de Opakua, Alain; Oka, Yasuyoshi; Feng, Yunpeng; Blanco, Francisco J.; Mann, Matthias; Montoya, Guillermo; Groth, Anja; Bekker-Jensen, Simon

    2016-01-01

    Cellular genomes are highly vulnerable to perturbations to chromosomal DNA replication. Proliferating cell nuclear antigen (PCNA), the processivity factor for DNA replication, plays a central role as a platform for recruitment of genome surveillance and DNA repair factors to replication forks, allowing cells to mitigate the threats to genome stability posed by replication stress. We identify the E3 ubiquitin ligase TRAIP as a new factor at active and stressed replication forks that directly interacts with PCNA via a conserved PCNA-interacting peptide (PIP) box motif. We show that TRAIP promotes ATR-dependent checkpoint signaling in human cells by facilitating the generation of RPA-bound single-stranded DNA regions upon replication stress in a manner that critically requires its E3 ligase activity and is potentiated by the PIP box. Consequently, loss of TRAIP function leads to enhanced chromosomal instability and decreased cell survival after replication stress. These findings establish TRAIP as a PCNA-binding ubiquitin ligase with an important role in protecting genome integrity after obstacles to DNA replication. PMID:26711499

  19. SIVA1 directs the E3 ubiquitin ligase RAD18 for PCNA monoubiquitination

    PubMed Central

    Han, Jinhua; Liu, Ting; Huen, Michael S.Y.; Hu, Lin; Chen, Zhiqiu

    2014-01-01

    Translesion DNA synthesis (TLS) is a universal DNA damage tolerance mechanism conserved from yeast to mammals. A key event in the regulation of TLS is the monoubiquitination of proliferating cell nuclear antigen (PCNA). Extensive evidence indicates that the RAD6–RAD18 ubiquitin-conjugating/ligase complex specifically monoubiquitinates PCNA and regulates TLS repair. However, the mechanism by which the RAD6–RAD18 complex is targeted to PCNA has remained elusive. In this study, we used an affinity purification approach to isolate the PCNA-containing complex and have identified SIVA1 as a critical regulator of PCNA monoubiquitination. We show that SIVA1 constitutively interacts with PCNA via a highly conserved PCNA-interacting peptide motif. Knockdown of SIVA1 compromised RAD18-dependent PCNA monoubiquitination and Polη focus formation, leading to elevated ultraviolet sensitivity and mutation. Furthermore, we demonstrate that SIVA1 interacts with RAD18 and serves as a molecular bridge between RAD18 and PCNA, thus targeting the E3 ligase activity of RAD18 onto PCNA. Collectively, our results provide evidence that the RAD18 E3 ligase requires an accessory protein for binding to its substrate PCNA. PMID:24958773

  20. Solving the SAT problem using a DNA computing algorithm based on ligase chain reaction.

    PubMed

    Wang, Xiaolong; Bao, Zhenmin; Hu, Jingjie; Wang, Shi; Zhan, Aibin

    2008-01-01

    A new DNA computing algorithm based on a ligase chain reaction is demonstrated to solve an SAT problem. The proposed DNA algorithm can solve an n-variable m-clause SAT problem in m steps and the computation time required is O (3m+n). Instead of generating the full-solution DNA library, we start with an empty test tube and then generate solutions that partially satisfy the SAT formula. These partial solutions are then extended step by step by the ligation of new variables using Taq DNA ligase. Correct strands are amplified and false strands are pruned by a ligase chain reaction (LCR) as soon as they fail to satisfy the conditions. If we score and sort the clauses, we can use this algorithm to markedly reduce the number of DNA strands required throughout the computing process. In a computer simulation, the maximum number of DNA strands required was 2(0.48n) when n=50, and the exponent ratio varied inversely with the number of variables n and the clause/variable ratio m/n. This algorithm is highly space-efficient and error-tolerant compared to conventional brute-force searching, and thus can be scaled-up to solve large and hard SAT problems. PMID:17904730

  1. Self-clearance mechanism of mitochondrial E3 ligase MARCH5 contributes to mitochondria quality control.

    PubMed

    Kim, Song-Hee; Park, Yong-Yea; Yoo, Young-Suk; Cho, Hyeseong

    2016-01-01

    MARCH5, a mitochondrial E3 ubiquitin ligase, controls mitochondrial dynamics proteins and misfolded proteins, and has been proposed to play a role in mitochondria quality control. However, it remains unclear how mutant MARCH5 found in cancer tissues is removed from cells. Here, we show that mutation in the MARCH5 ligase domain increased its half-life fourfold, resulting in a drastic increase in its protein level. Abnormal accumulation of the E3 ligase-defective MARCH5 mutants MARCH5(H43W) and MARCH5(C65/68S) was diminished by overexpression of active MARCH5(WT) ; the mutant proteins were degraded through the ubiquitin-proteasome pathway. Coimmunoprecipitation revealed that MARCH5 forms homodimers, and that substitution of Gly to Leu at the first putative GxxxG dimerization motif, but not the second, resulted in a loss of dimeric interaction. Moreover, overexpression of the dimerization-defective mutant MARCH5(4GL) could not decrease the level of accumulated MARCH5(H43W) , suggesting that dimerization of MARCH5 is necessary for self-clearance. Abnormal accumulation of MARCH5(H43W) and mitochondrial hyperfusion led to NF-ĸB activation, which was suppressed by overexpression of MARCH5(WT) . Together, the data reveal a self-protective mechanism involving MARCH5, which can target its own dysfunctional mutant for degradation in order to maintain mitochondrial homeostasis.

  2. RNF38 encodes a nuclear ubiquitin protein ligase that modifies p53

    SciTech Connect

    Sheren, Jamie E.; Kassenbrock, C. Kenneth

    2013-11-01

    Highlights: •RNF38 is shown to be a nuclear protein with a bipartite nuclear localization signal. •RNF38 protein is purified and shown to have ubiquitin protein ligase (E3) activity. •We show that RNF38 binds p53 and can ubiquitinate p53 in vitro. •Overexpression of RNF38 increases p53 ubiquitination in HEK293T cells. •Overexpression of RNF38 in HEK293T cells alters p53 localization. -- Abstract: The RNF38 gene encodes a RING finger protein of unknown function. Here we demonstrate that RNF38 is a functional ubiquitin protein ligase (E3). We show that RNF38 isoform 1 is localized to the nucleus by a bipartite nuclear localization sequence (NLS). We confirm that RNF38 is a binding partner of p53 and demonstrate that RNF38 can ubiquitinate p53 in vitro and in vivo. Finally, we show that overexpression of RNF38 in HEK293T cells results in relocalization of p53 to discrete foci associated with PML nuclear bodies. These results suggest RNF38 is an E3 ubiquitin ligase that may play a role in regulating p53.

  3. E3 ubiquitin ligases Pellinos as regulators of pattern recognition receptor signaling and immune responses.

    PubMed

    Medvedev, Andrei E; Murphy, Michael; Zhou, Hao; Li, Xiaoxia

    2015-07-01

    Pellinos are a family of E3 ubiquitin ligases discovered for their role in catalyzing K63-linked polyubiquitination of Pelle, an interleukin-1 (IL-1) receptor-associated kinase homolog in the Drosophila Toll pathway. Subsequent studies have revealed the central and non-redundant roles of mammalian Pellino-1, Pellino-2, and Pelino-3 in signaling pathways emanating from IL-1 receptors, Toll-like receptors, NOD-like receptors, T- and B-cell receptors. While Pellinos ability to interact with many signaling intermediates suggested their scaffolding roles, recent findings in mice expressing ligase-inactive Pellinos demonstrated the importance of Pellino ubiquitin ligase activity. Cell-specific functions of Pellinos have emerged, e.g. Pellino-1 being a negative regulator in T lymphocytes and a positive regulator in myeloid cells, and details of molecular regulation of receptor signaling by various members of the Pellino family have been revealed. In this review, we summarize current information about Pellino-mediated regulation of signaling by pattern recognition receptors, T-cell and B-cell receptors and tumor necrosis factor receptors, and discuss Pellinos roles in sepsis and infectious diseases, as well as in autoimmune, inflammatory, and allergic disorders. We also provide our perspective on the potential of targeting Pellinos with peptide- or small molecule-based drug compounds as a new therapeutic approach for septic shock and autoimmune pathologies.

  4. The Hypoxia-controlled FBXL14 Ubiquitin Ligase Targets SNAIL1 for Proteasome Degradation*

    PubMed Central

    Viñas-Castells, Rosa; Beltran, Manuel; Valls, Gabriela; Gómez, Irene; García, José Miguel; Montserrat-Sentís, Bàrbara; Baulida, Josep; Bonilla, Félix; de Herreros, Antonio García; Díaz, Víctor M.

    2010-01-01

    The transcription factor SNAIL1 is a master regulator of epithelial to mesenchymal transition. SNAIL1 is a very unstable protein, and its levels are regulated by the E3 ubiquitin ligase β-TrCP1 that interacts with SNAIL1 upon its phosphorylation by GSK-3β. Here we show that SNAIL1 polyubiquitylation and degradation may occur in conditions precluding SNAIL1 phosphorylation by GSK-3β, suggesting that additional E3 ligases participate in the control of SNAIL1 protein stability. In particular, we demonstrate that the F-box E3 ubiquitin ligase FBXl14 interacts with SNAIL1 and promotes its ubiquitylation and proteasome degradation independently of phosphorylation by GSK-3β. In vivo, inhibition of FBXl14 using short hairpin RNA stabilizes both ectopically expressed and endogenous SNAIL1. Moreover, the expression of FBXl14 is potently down-regulated during hypoxia, a condition that increases the levels of SNAIL1 protein but not SNAIL1 mRNA. FBXL14 mRNA is decreased in tumors with a high expression of two proteins up-regulated in hypoxia, carbonic anhydrase 9 and TWIST1. In addition, Twist1 small interfering RNA prevents hypoxia-induced Fbxl14 down-regulation and SNAIL1 stabilization in NMuMG cells. Altogether, these results demonstrate the existence of an alternative mechanism controlling SNAIL1 protein levels relevant for the induction of SNAIL1 during hypoxia. PMID:19955572

  5. Mechanistic Details of Glutathione Biosynthesis Revealed by Crystal Structures of Saccharomyces cerevisiae Glutamate Cysteine Ligase

    SciTech Connect

    Biterova, Ekaterina I.; Barycki, Joseph J.

    2009-12-01

    Glutathione is a thiol-disulfide exchange peptide critical for buffering oxidative or chemical stress, and an essential cofactor in several biosynthesis and detoxification pathways. The rate-limiting step in its de novo biosynthesis is catalyzed by glutamate cysteine ligase, a broadly expressed enzyme for which limited structural information is available in higher eukaryotic species. Structural data are critical to the understanding of clinical glutathione deficiency, as well as rational design of enzyme modulators that could impact human disease progression. Here, we have determined the structures of Saccharomyces cerevisiae glutamate cysteine ligase (ScGCL) in the presence of glutamate and MgCl{sub 2} (2.1 {angstrom}; R = 18.2%, R{sub free} = 21.9%), and in complex with glutamate, MgCl{sub 2}, and ADP (2.7 {angstrom}; R = 19.0%, R{sub free} = 24.2%). Inspection of these structures reveals an unusual binding pocket for the {alpha}-carboxylate of the glutamate substrate and an ATP-independent Mg{sup 2+} coordination site, clarifying the Mg{sup 2+} dependence of the enzymatic reaction. The ScGCL structures were further used to generate a credible homology model of the catalytic subunit of human glutamate cysteine ligase (hGCLC). Examination of the hGCLC model suggests that post-translational modifications of cysteine residues may be involved in the regulation of enzymatic activity, and elucidates the molecular basis of glutathione deficiency associated with patient hGCLC mutations.

  6. Nedd4, a human ubiquitin ligase, affects actin cytoskeleton in yeast cells.

    PubMed

    Stawiecka-Mirota, Marta; Kamińska, Joanna; Urban-Grimal, Daniele; Haines, Dale S; Zoładek, Teresa

    2008-11-01

    Human Nedd4 ubiquitin ligase is involved in protein trafficking, signal transduction and oncogenesis. Nedd4 with an inactive WW4 domain is toxic to yeast cells. We report here that actin cytoskeleton is abnormal in yeast cells expressing the NEDD4 or NEDD4w4 gene and these cells are more sensitive to Latrunculin A, an actin-depolymerizing drug. These phenotypes are less pronounced when a mutation inactivating the catalytic domain of the ligase has been introduced. In contrast, overexpression of the LAS17 gene, encoding an activator of the Arp2/3 actin nucleating complex, is detrimental to NEDD4w4-expressing cells. The level of Las17p is increased in cells overproducing Nedd4w4 and this depends partially on its catalytic domain. Expression of genes encoding Nedd4 variants, like overexpression of LAS17, suppresses the growth defect of the arp2-1 strain. Our results suggest that human Nedd4 ligase inhibits yeast cell growth by disturbing the actin cytoskeleton, in part by increasing Las17p level, and that Nedd4 ubiquitination targets may include actin cytoskeleton-associated proteins conserved in evolution. PMID:18804462

  7. Mutational analyses of the thermostable NAD+-dependent DNA ligase from Thermus filiformis.

    PubMed

    Jeon, Hyo Jeong; Shin, Hea-Jin; Choi, Jeong Jin; Hoe, Hyang-Sook; Kim, Hyun-Kyu; Suh, Se Won; Kwon, Suk-Tae

    2004-08-01

    The crystal structure of NAD+-dependent DNA ligase from Thermus filiformis (Tfi) revealed that the protein comprised four structural domains. In order to investigate the biochemical activities of these domains, seven deletion mutants were constructed from the Tfi DNA ligase. The mutants Tfi-M1 (residues 1-581), Tfi-M2 (residues 1-448), Tfi-M3 (residues 1-403) and Tfi-M4 (residues 1-314) showed the same adenylation activity as that of wild-type. This result indicates that only the adenylation domain (domain 1) is essential for the formation of enzyme-AMP complex. It was found that the zinc finger and helix-hairpin-helix (HhH) motif domain (domain 3) and the oligomer binding (OB)-fold domain (domain 2) are important for the formation of enzyme-DNA complex. The mutant Tfi-M1 alone showed the activities for in vitro nick-closing and in vivo complementation in Escherichia coli as those of wild-type. These results indicate that the BRCT domain (domain 4) of Tfi DNA ligase is not essential for the enzyme activity. The enzymatic properties of Tfi-M1 mutant (deleted the BRCT domain) were slightly different from those of wild-type and the nick-closing activity of Tfi-M1 mutant was approximately 50% compared with that of wild-type. PMID:15268945

  8. Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining

    PubMed Central

    Kukshal, Vandna; Kim, In-Kwon; Hura, Gregory L.; Tomkinson, Alan E.; Tainer, John A.; Ellenberger, Tom

    2015-01-01

    Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation. PMID:26130724

  9. E3 Ubiquitin Ligase RLIM Negatively Regulates c-Myc Transcriptional Activity and Restrains Cell Proliferation

    PubMed Central

    Wang, Lan; Cai, Hao; Zhu, Jingjing; Yu, Long

    2016-01-01

    RNF12/RLIM is a RING domain-containing E3 ubiquitin ligase whose function has only begun to be elucidated recently. Although RLIM was reported to play important roles in some biological processes such as imprinted X-chromosome inactivation and regulation of TGF-β pathway etc., other functions of RLIM are largely unknown. Here, we identified RLIM as a novel E3 ubiquitin ligase for c-Myc, one of the most frequently deregulated oncoproteins in human cancers. RLIM associates with c-Myc in vivo and in vitro independently of the E3 ligase activity of RLIM. Moreover, RLIM promotes the polyubiquitination of c-Myc protein independently of Ser62 and Thr58 phosphorylation of c-Myc. However, RLIM-mediated ubiquitination does not affect c-Myc stability. Instead, RLIM inhibits the transcriptional activity of c-Myc through which RLIM restrains cell proliferation. Our results suggest that RLIM may function as a tumor suppressor by controlling the activity of c-Myc oncoprotein. PMID:27684546

  10. Covalent ISG15 conjugation positively regulates the ubiquitin E3 ligase activity of parkin

    PubMed Central

    Im, Eunju; Yoo, Lang; Hyun, Minju; Shin, Woo Hyun

    2016-01-01

    Parkinson's disease (PD) is characterized by selective loss of dopaminergic neurons in the pars compacta of the substantia nigra and accumulation of ubiquitinated proteins in aggregates called Lewy bodies. Several mutated genes have been found in familial PD patients, including SNCA (α-synuclein), PARK2 (parkin), PINK1, PARK7 (DJ-1), LRRK2 and ATP13A2. Many pathogenic mutations of PARK2, which encodes the ubiquitin E3 ligase parkin, result in loss of function, leading to accumulation of parkin substrates and consequently contributing to dopaminergic cell death. ISG15 is a member of the ubiquitin-like modifier family and is induced by stimulation with type I interferons. Similar to ubiquitin and ubiquitination, covalent conjugation of ISG15 to target proteins (ISGylation) regulates their biochemical properties. In this study, we identified parkin as a novel target of ISGylation specifically mediated by the ISG15-E3 ligase HERC5. In addition, we identified two ISGylation sites, Lys-349 and Lys-369, in the in-between-ring domain of parkin. ISGylation of these sites promotes parkin's ubiquitin E3 ligase activity by suppressing the intramolecular interaction that maintains its autoinhibited conformation and increases its cytoprotective effect. In conclusion, covalent ISG15 conjugation is a novel mode of modulating parkin activity, and alteration in this pathway may be associated with PD pathogenesis. PMID:27534820

  11. Coevolution of RtcB and Archease created a multiple-turnover RNA ligase.

    PubMed

    Desai, Kevin K; Beltrame, Amanda L; Raines, Ronald T

    2015-11-01

    RtcB is a noncanonical RNA ligase that joins either 2',3'-cyclic phosphate or 3'-phosphate termini to 5'-hydroxyl termini. The genes encoding RtcB and Archease constitute a tRNA splicing operon in many organisms. Archease is a cofactor of RtcB that accelerates RNA ligation and alters the NTP specificity of the ligase from Pyrococcus horikoshii. Yet, not all organisms that encode RtcB also encode Archease. Here we sought to understand the differences between Archease-dependent and Archease-independent RtcBs so as to illuminate the evolution of Archease and its function. We report on the Archease-dependent RtcB from Thermus thermophilus and the Archease-independent RtcB from Thermobifida fusca. We find that RtcB from T. thermophilus can catalyze multiple turnovers only in the presence of Archease. Remarkably, Archease from P. horikoshii can activate T. thermophilus RtcB, despite low sequence identity between the Archeases from these two organisms. In contrast, RtcB from T. fusca is a single-turnover enzyme that is unable to be converted into a multiple-turnover ligase by Archease from either P. horikoshii or T. thermophilus. Thus, our data indicate that Archease likely evolved to support multiple-turnover activity of RtcB and that coevolution of the two proteins is necessary for a functional interaction.

  12. A common fold for peptide synthetases cleaving ATP to ADP: glutathione synthetase and D-alanine:d-alanine ligase of Escherichia coli.

    PubMed Central

    Fan, C; Moews, P C; Shi, Y; Walsh, C T; Knox, J R

    1995-01-01

    Examination of x-ray crystallographic structures shows the tertiary structure of D-alanine:D-alanine ligase (EC 6.3.2.4). a bacterial cell wall synthesizing enzyme, is similar to that of glutathione synthetase (EC 6.32.3) despite low sequence homology. Both Escherichia coli enzymes, which convert ATP to ADP during ligation to produce peptide products, are made of three domains, each folded around a 4-to 6-stranded beta-sheet core. Sandwiched between the beta-sheets of the C-terminal and central domains of each enzyme is a nonclassical ATP-binding site that contains a common set of spatially equivalent amino acids. In each enzyme, two loops are proposed to exhibit a required flexibility that allows entry of ATP and substrates, provides protection of the acylphosphate intermediate and tetrahedral adduct from hydrolysis during catalysis, and then permits release of products. PMID:7862655

  13. Chlorsulfuron modifies biosynthesis of acyl Acid substituents of sucrose esters secreted by tobacco trichomes.

    PubMed

    Kandra, L; Wagner, G J

    1990-11-01

    Sucrose esters and duvatrienediol diterpenes are principal constituents formed in and secreted outside head cells of trichomes occurring on surfaces of Nicotiana tabacum. Using trichome-bearing epidermal peels prepared from midveins of N. tabacum cv T.I. 1068 leaves, we found that chlorsulfuron reduced and modified radiolabeling of sucrose ester acyl acids derived from branched-chain amino acid metabolism. The herbicide did not effect formation and exudation of diterpenes which are products of isoprenoid metabolism. Treatment with 1.0 micromolar chlorsulfuron affected 8.5- and 6.3-fold reductions in radiolabeling of methylvaleryl and methylbutyryl groups of sucrose esters, respectively, and concomitant increases of 9- and 9.8-fold in radiolabeling of straight chain valeryl and butyryl groups, respectively. These results and others indicate that inhibition of acetolactate synthase causes an accumulation of 2-oxo-butyric acid that is utilized by enzymes common to Leu biosynthesis to form 2-oxo-valeric acid. Coenzyme A (CoA) activation of this keto acid gives rise to butyryl CoA, which is utilized to form butyryl containing sucrose esters. Alternatively, reutilization of 2-oxo-valeric acid by the same enzymes followed by CoA activation leads to valeryl containing sucrose esters. We propose that in trichome secretory cells synthase, isomerase and dehydrogenase enzymes which catalyze Leu synthesis/degredation in most tissues, convert iso-branched, anteiso-branched and straight-chain keto acids in the formation of sucrose ester acyl groups. PMID:16667871

  14. Chlorsulfuron Modifies Biosynthesis of Acyl Acid Substituents of Sucrose Esters Secreted by Tobacco Trichomes

    PubMed Central

    Kandra, Lili; Wagner, George J.

    1990-01-01

    Sucrose esters and duvatrienediol diterpenes are principal constituents formed in and secreted outside head cells of trichomes occurring on surfaces of Nicotiana tabacum. Using trichome-bearing epidermal peels prepared from midveins of N. tabacum cv T.I. 1068 leaves, we found that chlorsulfuron reduced and modified radiolabeling of sucrose ester acyl acids derived from branched-chain amino acid metabolism. The herbicide did not effect formation and exudation of diterpenes which are products of isoprenoid metabolism. Treatment with 1.0 micromolar chlorsulfuron affected 8.5- and 6.3-fold reductions in radiolabeling of methylvaleryl and methylbutyryl groups of sucrose esters, respectively, and concomitant increases of 9- and 9.8-fold in radiolabeling of straight chain valeryl and butyryl groups, respectively. These results and others indicate that inhibition of acetolactate synthase causes an accumulation of 2-oxo-butyric acid that is utilized by enzymes common to Leu biosynthesis to form 2-oxo-valeric acid. Coenzyme A (CoA) activation of this keto acid gives rise to butyryl CoA, which is utilized to form butyryl containing sucrose esters. Alternatively, reutilization of 2-oxo-valeric acid by the same enzymes followed by CoA activation leads to valeryl containing sucrose esters. We propose that in trichome secretory cells synthase, isomerase and dehydrogenase enzymes which catalyze Leu synthesis/degredation in most tissues, convert iso-branched, anteiso-branched and straight-chain keto acids in the formation of sucrose ester acyl groups. PMID:16667871

  15. Post-Transcriptional Coordination of the Arabidopsis Iron Deficiency Response is Partially Dependent on the E3 Ligases RING DOMAIN LIGASE1 (RGLG1) and RING DOMAIN LIGASE2 (RGLG2).

    PubMed

    Pan, I-Chun; Tsai, Huei-Hsuan; Cheng, Ya-Tan; Wen, Tuan-Nan; Buckhout, Thomas J; Schmidt, Wolfgang

    2015-10-01

    Acclimation to changing environmental conditions is mediated by proteins, the abundance of which is carefully tuned by an elaborate interplay of DNA-templated and post-transcriptional processes. To dissect the mechanisms that control and mediate cellular iron homeostasis, we conducted quantitative high-resolution iTRAQ proteomics and microarray-based transcriptomic profiling of iron-deficient Arabidopsis thaliana plants. A total of 13,706 and 12,124 proteins was identified with a quadrupole-Orbitrap hybrid mass spectrometer in roots and leaves, respectively. This deep proteomic coverage allowed accurate estimates of post-transcriptional regulation in response to iron deficiency. Similarly regulated transcripts were detected in only 13% (roots) and 11% (leaves) of the 886 proteins that differentially accumulated between iron-sufficient and iron-deficient plants, indicating that the majority of the iron-responsive proteins was post-transcriptionally regulated. Mutants harboring defects in the RING DOMAIN LIGASE1 (RGLG1)(1) and RING DOMAIN LIGASE2 (RGLG2) showed a pleiotropic phenotype that resembled iron-deficient plants with reduced trichome density and the formation of branched root hairs. Proteomic and transcriptomic profiling of rglg1 rglg2 double mutants revealed that the functional RGLG protein is required for the regulation of a large set of iron-responsive proteins including the coordinated expression of ribosomal proteins. This integrative analysis provides a detailed catalog of post-transcriptionally regulated proteins and allows the concept of a chiefly transcriptionally regulated iron deficiency response to be revisited. Protein data are available via ProteomeXchange with identifier PXD002126.

  16. Post-Transcriptional Coordination of the Arabidopsis Iron Deficiency Response is Partially Dependent on the E3 Ligases RING DOMAIN LIGASE1 (RGLG1) and RING DOMAIN LIGASE2 (RGLG2)*

    PubMed Central

    Pan, I-Chun; Tsai, Huei-Hsuan; Cheng, Ya-Tan; Wen, Tuan-Nan; Buckhout, Thomas J.; Schmidt, Wolfgang

    2015-01-01

    Acclimation to changing environmental conditions is mediated by proteins, the abundance of which is carefully tuned by an elaborate interplay of DNA-templated and post-transcriptional processes. To dissect the mechanisms that control and mediate cellular iron homeostasis, we conducted quantitative high-resolution iTRAQ proteomics and microarray-based transcriptomic profiling of iron-deficient Arabidopsis thaliana plants. A total of 13,706 and 12,124 proteins was identified with a quadrupole-Orbitrap hybrid mass spectrometer in roots and leaves, respectively. This deep proteomic coverage allowed accurate estimates of post-transcriptional regulation in response to iron deficiency. Similarly regulated transcripts were detected in only 13% (roots) and 11% (leaves) of the 886 proteins that differentially accumulated between iron-sufficient and iron-deficient plants, indicating that the majority of the iron-responsive proteins was post-transcriptionally regulated. Mutants harboring defects in the RING DOMAIN LIGASE1 (RGLG1)1 and RING DOMAIN LIGASE2 (RGLG2) showed a pleiotropic phenotype that resembled iron-deficient plants with reduced trichome density and the formation of branched root hairs. Proteomic and transcriptomic profiling of rglg1 rglg2 double mutants revealed that the functional RGLG protein is required for the regulation of a large set of iron-responsive proteins including the coordinated expression of ribosomal proteins. This integrative analysis provides a detailed catalog of post-transcriptionally regulated proteins and allows the concept of a chiefly transcriptionally regulated iron deficiency response to be revisited. Protein data are available via ProteomeXchange with identifier PXD002126. PMID:26253232

  17. p38 MAP kinase-dependent phosphorylation of the Gp78 E3 ubiquitin ligase controls ER-mitochondria association and mitochondria motility.

    PubMed

    Li, Lei; Gao, Guang; Shankar, Jay; Joshi, Bharat; Foster, Leonard J; Nabi, Ivan R

    2015-11-01

    Gp78 is an ERAD-associated E3 ubiquitin ligase that induces degradation of the mitofusin mitochondrial fusion proteins and mitochondrial fission. Gp78 is localized throughout the ER; however, the anti-Gp78 3F3A monoclonal antibody (mAb) recognizes Gp78 selectively in mitochondria-associated ER domains. Epitope mapping localized the epitope of 3F3A and a commercial anti-Gp78 mAb to an 8-amino acid motif (533-541) in mouse Gp78 isoform 2 that forms part of a highly conserved 41-amino acid region containing 14-3-3- and WW-binding domains and a p38 MAP kinase (p38 MAPK) consensus site on Ser-538 (S538). 3F3A binds selectively to nonphosphorylated S538 Gp78. Using 3F3A as a reporter, we induced Gp78 S538 phosphorylation by serum starvation and showed it to be mediated by p38 MAPK. Mass spectroscopy analysis of Gp78 phosphopeptides confirmed S538 as a major p38 MAPK phosphorylation site on Gp78. Gp78 S538 phosphorylation limited its ability to induce mitochondrial fission and degrade MFN1 and MFN2 but did not affect in vitro Gp78 ubiquitin E3 ligase activity. Phosphomimetic Gp78 S538D mutation prevented Gp78 promotion of ER-mitochondria interaction, and SB203580 inhibition of p38 MAPK increased ER-mitochondria association. p38 MAPK phosphorylation of Gp78 S538 therefore regulates Gp78-dependent ER-mitochondria association and mitochondria motility.

  18. Ligase-independent cloning of amylase gene from a local Bacillus subtilis isolate and biochemical characterization of the purified enzyme.

    PubMed

    Tuzlakoglu Ozturk, Merve; Akbulut, Nagihan; Issever Ozturk, Saliha; Gumusel, Fusun

    2013-09-01

    Five hundred ninety-seven bacterial isolates from Turkish hot spring water sources were screened for their ability to produce extracellular α-amylase. Among them, a high enzyme-producing Bacillus subtilis isolate, A28, was selected, and its α-amylase gene was cloned and expressed in Escherichia coli by a ligase-independent method. α-Amylase from the recombinant strain was purified to homogeneity by Q-Sepharose anion exchange and Sephacryl S-100 gel filtration chromatographies. The final yield of the enzyme was about 22.5 % of the initial activity, with a 16.4-fold increase in specific activity compared with the culture lysate. The optimum temperature and pH of the enzyme were 70 °C and 6.0, respectively. The enzyme was highly active at acidic-neutral pH range of 4.5-7.0. The amy28 α-amylase retained 100 % of its activity after incubation at 50 °C for 90 min. Co(+2), Cu(2+), Fe(2+), Fe(3+), Ni(+2), and Zn(+2) caused significant inhibition in enzyme activity, which was not affected by Na(+), Mg(2+), Li(+), and Ba(2+). The activity was inhibited about 70 % upon treatment of the enzyme with 10 mM ethylenediaminetetraacetic acid. However, Ca(2+) ions known as high temperature stabilizer for other amylases did not stimulate the activity of the enzyme. Due to pH stability and thermostability of the recombinant amylase, this enzyme may be suitable in starch processing, brewing, and food industries.

  19. Peptide Arrays for Binding Studies of E3 Ubiquitin Ligases.

    PubMed

    Klecker, Maria; Dissmeyer, Nico

    2016-01-01

    The automated SPOT (synthetic peptide arrays on membrane support technique) synthesis technology has entrenched as a rapid and robust method to generate peptide libraries on cellulose membrane supports. The synthesis method is based on conventional Fmoc chemistry building up peptides with free N-terminal amino acids starting at their cellulose-coupled C-termini. Several hundreds of peptide sequences can be assembled with this technique on one membrane comprising a strong binding potential due to high local peptide concentrations. Peptide orientation on SPOT membranes qualifies this array type for assaying substrate specificities of N-recognins, the recognition elements of the N-end rule pathway of targeted protein degradation (NERD). Pioneer studies described binding capability of mammalian and yeast enzymes depending on a peptide's N-terminus. SPOT arrays have been successfully used to describe substrate specificity of N-recognins which are the recognition elements of the N-end rule pathway of targeted protein degradation (NERD). Here, we describe the implementation of SPOT binding assays with focus on the identification of N-recognin substrates, applicable also for plant NERD enzymes. PMID:27424747

  20. A Family of Salmonella Virulence Factors Functions as a Distinct Class of Autoregulated E3 Ubiquitin Ligases

    SciTech Connect

    Quezada, C.; Hicks, S; Galan, J; Stebbins, C

    2009-01-01

    Processes as diverse as receptor binding and signaling, cytoskeletal dynamics, and programmed cell death are manipulated by mimics of host proteins encoded by pathogenic bacteria. We show here that the Salmonella virulence factor SspH2 belongs to a growing class of bacterial effector proteins that harness and subvert the eukaryotic ubiquitination pathway. This virulence protein possesses ubiquitination activity that depends on a conserved cysteine residue. A crystal structure of SspH2 reveals a canonical leucine-rich repeat (LRR) domain that interacts with a unique E{sub 3} ligase [which we have termed NEL for Novel E{sub 3} Ligase] C-terminal fold unrelated to previously observed HECT or RING-finger E{sub 3} ligases. Moreover, the LRR domain sequesters the catalytic cysteine residue contained in the NEL domain, and we suggest a mechanism for activation of the ligase requiring a substantial conformational change to release the catalytic domain for function. We also show that the N-terminal domain targets SspH2 to the apical plasma membrane of polarized epithelial cells and propose a model whereby binding of the LRR to proteins at the target site releases the ligase domain for site-specific function.

  1. Structural Insight into the Human Immunodeficiency Virus Vif SOCS Box and Its Role in Human E3 Ubiquitin Ligase Assembly

    SciTech Connect

    Stanley,B.; Ehrlich, E.; Short, L.; Yu, Y.; Xiao, Z.; Yu, X.; Xiong, Y.

    2008-01-01

    Human immunodeficiency virus (HIV) virion infectivity factor (Vif) causes the proteasome-mediated destruction of human antiviral protein APOBEC3G by tethering it to a cellular E3 ubiquitin ligase composed of ElonginB, ElonginC, Cullin5, and Rbx2. It has been proposed that HIV Vif hijacks the E3 ligase through two regions within its C-terminal domain: a BC box region that interacts with ElonginC and a novel zinc finger motif that interacts with Cullin5. We have determined the crystal structure of the HIV Vif BC box in complex with human ElonginB and ElonginC. This complex presents direct structural evidence of the recruitment of a human ubiquitin ligase by a viral BC box protein that mimics the conserved interactions of cellular ubiquitin ligases. We further mutated conserved hydrophobic residues in a region downstream of the Vif BC box. These mutations demonstrate that this region, the Vif Cullin box, composes a third E3-ligase recruiting site critical for interaction between Vif and Cullin5. Furthermore, our homology modeling reveals that the Vif Cullin box and zinc finger motif may be positioned adjacent to the N terminus of Cullin5 for interaction with loop regions in the first cullin repeat of Cullin5.

  2. CREB SUMOylation by the E3 ligase PIAS1 enhances spatial memory.

    PubMed

    Chen, Yan-Chu; Hsu, Wei-Lun; Ma, Yun-Li; Tai, Derek J C; Lee, Eminy H Y

    2014-07-16

    cAMP-responsive element binding protein (CREB) phosphorylation and signaling plays an important role in long-term memory formation, but other posttranslational modifications of CREB are less known. Here, we found that CREB1Δ, the short isoform of CREB, could be sumoylated by the small ubiquitin-like modifier (SUMO) E3 ligase protein inhibitor of activated STAT1 (PIAS1) at Lys271 and Lys290 and PIAS1 SUMOylation of CREB1Δ increased the expression level of CREB1Δ. CREB1Δ could also be sumoylated by other PIAS family proteins, but not by the E3 ligases RanBP2 and Pc2 or by the E2 ligase Ubc9. Furthermore, water maze training increased the level of endogenous CREB SUMOylation in rat CA1 neurons determined by in vitro SUMOylation assay, but this effect was not observed in other brain areas. Moreover, transduction of Lenti-CREBWT to rat CA1 area facilitated, whereas transduction of Lenti-CREB double sumo-mutant (CREBK271RK290R) impaired, spatial learning and memory performance. Transduction of Lenti-CREBWT-SUMO1 fusion vector to rat CA1 area showed a more significant effect in enhancing spatial learning and memory and CREB SUMOylation. Lenti-CREBWT transduction increased, whereas Lenti-CREBK271RK290R transduction decreased, CREB DNA binding to the brain-derived neurotrophic factor (bdnf) promoter and decreased bdnf mRNA expression. Knock-down of PIAS1 expression in CA1 area by PIAS1 siRNA transfection impaired spatial learning and memory and decreased endogenous CREB SUMOylation. In addition, CREB SUMOylation was CREB phosphorylation dependent and lasted longer. Therefore, CREB phosphorylation may be responsible for signal transduction during the early phase of long-term memory formation, whereas CREB SUMOylation sustains long-term memory.

  3. UV-B induction of the E3 ligase ARIADNE12 depends on CONSTITUTIVELY PHOTOMORPHOGENIC 1

    PubMed Central

    Xie, Lisi; Lang-Mladek, Christina; Richter, Julia; Nigam, Neha; Hauser, Marie-Theres

    2015-01-01

    The UV-B inducible ARIADNE12 (ARI12) gene of Arabidopsis thaliana is a member of the RING-between-RING (RBR) family of E3 ubiquitin ligases for which a novel ubiquitination mechanism was identified in mammalian homologs. This RING-HECT hybrid mechanism needs a conserved cysteine which is replaced by serine in ARI12 and might affect the E3 ubiquitin ligase activity. We have shown that under photomorphogenic UV-B, ARI12 is a downstream target of the classical ultraviolet B (UV-B) UV RESISTANCE LOCUS 8 (UVR8) pathway. However, under high fluence rate of UV-B ARI12 was induced independently of UVR8 and the UV-A/blue light and red/far-red photoreceptors. A key component of several light signaling pathways is CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1). Upon UV-B COP1 is trapped in the nucleus through interaction with UVR8 permitting the activation of genes that regulate the biosynthesis of UV-B protective metabolites and growth adaptations. To clarify the role of COP1 in the regulation of ARI12 mRNA expression and ARI12 protein stability, localization and interaction with COP1 was assessed with and without UV-B. We found that COP1 controls ARI12 in white light, low and high fluence rate of UV-B. Furthermore we show that ARI12 is indeed an E3 ubiquitin ligase which is mono-ubiquitinated, a prerequisite for the RING-HECT hybrid mechanism. Finally, genetic analyses with transgenes expressing a genomic pmARI12:ARI12-GFP construct confirm the epistatic interaction between COP1 and ARI12 in growth responses to high fluence rate UV-B. PMID:25817546

  4. UV-B induction of the E3 ligase ARIADNE12 depends on CONSTITUTIVELY PHOTOMORPHOGENIC 1.

    PubMed

    Xie, Lisi; Lang-Mladek, Christina; Richter, Julia; Nigam, Neha; Hauser, Marie-Theres

    2015-08-01

    The UV-B inducible ARIADNE12 (ARI12) gene of Arabidopsis thaliana is a member of the RING-between-RING (RBR) family of E3 ubiquitin ligases for which a novel ubiquitination mechanism was identified in mammalian homologs. This RING-HECT hybrid mechanism needs a conserved cysteine which is replaced by serine in ARI12 and might affect the E3 ubiquitin ligase activity. We have shown that under photomorphogenic UV-B, ARI12 is a downstream target of the classical ultraviolet B (UV-B) UV Resistance Locus 8 (UVR8) pathway. However, under high fluence rate of UV-B ARI12 was induced independently of UVR8 and the UV-A/blue light and red/far-red photoreceptors. A key component of several light signaling pathways is Constitutively Photomorphogenic 1 (COP1). Upon UV-B COP1 is trapped in the nucleus through interaction with UVR8 permitting the activation of genes that regulate the biosynthesis of UV-B protective metabolites and growth adaptations. To clarify the role of COP1 in the regulation of ARI12 mRNA expression and ARI12 protein stability, localization and interaction with COP1 was assessed with and without UV-B. We found that COP1 controls ARI12 in white light, low and high fluence rate of UV-B. Furthermore we show that ARI12 is indeed an E3 ubiquitin ligase which is mono-ubiquitinated, a prerequisite for the RING-HECT hybrid mechanism. Finally, genetic analyses with transgenes expressing a genomic pmARI12:ARI12-GFP construct confirm the epistatic interaction between COP1 and ARI12 in growth responses to high fluence rate UV-B.

  5. DD-ligases as a potential target for antibiotics: past, present and future.

    PubMed

    Tytgat, I; Colacino, E; Tulkens, P M; Poupaert, J H; Prévost, M; Van Bambeke, F

    2009-01-01

    DD-ligases catalyze the synthesis of the D-Ala-D-Ala and D-Ala-D-Ser dipeptides or the D Ala-D-Lac depsipeptide in an early step of peptidoglycan synthesis. Their function is essential for bacterial growth and specific to bacteria, making them attractive targets for the development of novel antibiotics. This review examines the biochemical and structural features of these enzymes and presents the main families of inhibitors described so far. Over the last 20 years, 7 structures of DD-ligases have been solved by X-ray crystallography, giving a detailed view of the general topology of the active site and of the residues in the catalytic pocket that play a central role in substrate recognition. This has paved the way to the rational design of inhibitors, which can be classified as (i) analogues of substrates, (ii) analogues of the product of the reaction, (iii) analogues of the transition state, and (iv) original scaffolds discovered by screening or by rational computer-aided design. The three first strategies have led to molecules that are polar by nature and have therefore poor access to their cytosolic target. The fourth one is potentially most promising as it yields more diverse structures. The most active molecules show affinity constants in the microM range, but microbiological evaluation remains scarce (typical MIC 1-8 mg/L for the tested compounds). These data strongly suggest targeting DD-ligases is a promising approach for discovery of new antibiotics. Future research should, however, aim at finding more potent inhibitors endowed with the appropriate pharmacokinetic properties that ensure access to their intracellular target.

  6. Targeting Neddylation Pathways to Inactivate Cullin-RING Ligases for Anticancer Therapy

    PubMed Central

    Zhao, Yongchao; Morgan, Meredith A.

    2014-01-01

    Abstract Significance: Protein neddylation is catalyzed by an E1 NEDD8-activating enzyme (NAE), an E2 NEDD8-conjugating enzyme, and an E3 NEDD8 ligase. Known physiological substrates of neddylation are cullin family members. Cullin neddylation leads to activation of cullin-RING ligases (CRLs), the largest family of E3 ubiquitin ligases responsible for ubiquitylation and degradation of many key signaling/regulatory proteins. Thus, through modulating CRLs, neddylation regulates many biological processes, including cell cycle progression, signal transduction, and tumorigenesis. Given that NEDD8 is overexpressed and CRLs are abnormally activated in many human cancers, targeting protein neddylation, in general, and cullin neddylation, in particular, appears to be an attractive anticancer approach. Recent Advances: MLN4924, a small molecule inhibitor of NAE, was discovered that inactivates CRLs and causes accumulation of CRL substrates to suppress tumor cell growth both in vitro and in vivo. Promising preclinical results advanced MLN4924 to several clinical trials for anticancer therapy. Critical Issues: In preclinical settings, MLN4924 effectively suppresses tumor cell growth by inducing apoptosis, senescence, and autophagy, and causes sensitization to chemoradiation therapies in a cellular context-dependent manner. Signal molecules that determine the cell fate upon MLN4924 treatment, however, remain elusive. Cancer cells develop MLN4924 resistance by selecting target mutations. Future Directions: In the clinical side, several Phase 1b trials are under way to determine the safety and efficacy of MLN4924, acting alone or in combination with conventional chemotherapy, against human solid tumors. In the preclinical side, the efforts are being made to develop additional neddylation inhibitors by targeting NEDD8 E2s and E3s. Antioxid. Redox Signal. 21, 2383–2400. PMID:24410571

  7. Aβ-Induced Synaptic Alterations Require the E3 Ubiquitin Ligase Nedd4-1

    PubMed Central

    Rodrigues, Elizabeth M.; Scudder, Samantha L.; Goo, Marisa S.

    2016-01-01

    Alzheimer's disease (AD) is a neurodegenerative disease in which patients experience progressive cognitive decline. A wealth of evidence suggests that this cognitive impairment results from synaptic dysfunction in affected brain regions caused by cleavage of amyloid precursor protein into the pathogenic peptide amyloid-β (Aβ). Specifically, it has been shown that Aβ decreases surface AMPARs, dendritic spine density, and synaptic strength, and also alters synaptic plasticity. The precise molecular mechanisms by which this occurs remain unclear. Here we demonstrate a role for ubiquitination in Aβ-induced synaptic dysfunction in cultured rat neurons. We find that Aβ promotes the ubiquitination of AMPARs, as well as the redistribution and recruitment of Nedd4-1, a HECT E3 ubiquitin ligase we previously demonstrated to target AMPARs for ubiquitination and degradation. Strikingly, we show that Nedd4-1 is required for Aβ-induced reductions in surface AMPARs, synaptic strength, and dendritic spine density. Our findings, therefore, indicate an important role for Nedd4-1 and ubiquitin in the synaptic alterations induced by Aβ. SIGNIFICANCE STATEMENT Synaptic changes in Alzheimer's disease (AD) include surface AMPAR loss, which can weaken synapses. In a cell culture model of AD, we found that AMPAR loss correlates with increased AMPAR ubiquitination. In addition, the ubiquitin ligase Nedd4-1, known to ubiquitinate AMPARs, is recruited to synapses in response to Aβ. Strikingly, reducing Nedd4-1 levels in this model prevented surface AMPAR loss and synaptic weakening. These findings suggest that, in AD, Nedd4-1 may ubiquitinate AMPARs to promote their internalization and weaken synaptic strength, similar to what occurs in Nedd4-1's established role in homeostatic synaptic scaling. This is the first demonstration of Aβ-mediated control of a ubiquitin ligase to regulate surface AMPAR expression. PMID:26843640

  8. Genome-wide identification and characterization of the apple (Malus domestica) HECT ubiquitin-protein ligase family and expression analysis of their responsiveness to abiotic stresses.

    PubMed

    Xu, Jianing; Xing, Shanshan; Cui, Haoran; Chen, Xuesen; Wang, Xiaoyun

    2016-04-01

    The ubiquitin-protein ligases (E3s) directly participate in ubiquitin (Ub) transferring to the target proteins in the ubiquitination pathway. The HECT ubiquitin-protein ligase (UPL), one type of E3s, is characterized as containing a conserved HECT domain of approximately 350 amino acids in the C terminus. Some UPLs were found to be involved in trichome development and leaf senescence in Arabidopsis. However, studies on plant UPLs, such as characteristics of the protein structure, predicted functional motifs of the HECT domain, and the regulatory expression of UPLs have all been limited. Here, we present genome-wide identification of the genes encoding UPLs (HECT gene) in apple. The 13 genes (named as MdUPL1-MdUPL13) from ten different chromosomes were divided into four groups by phylogenetic analysis. Among these groups, the encoding genes in the intron-exon structure and the included additional functional domains were quite different. Notably, the F-box domain was first found in MdUPL7 in plant UPLs. The HECT domain in different MdUPL groups also presented different spatial features and three types of conservative motifs were identified. The promoters of each MdUPL member carried multiple stress-response related elements by cis-acting element analysis. Experimental results demonstrated that the expressions of several MdUPLs were quite sensitive to cold-, drought-, and salt-stresses by qRT-PCR assay. The results of this study helped to elucidate the functions of HECT proteins, especially in Rosaceae plants.

  9. Crystallization and preliminary X-ray diffraction studies of the WW4 domain of the Nedd4-2 ubiquitin–protein ligase

    SciTech Connect

    Umadevi, N.; Kumar, S.; Narayana, N.

    2005-12-01

    The first crystallographic study of an isolated WW domain is reported. Single crystals of the WW4 domain of the Nedd4-2 ubiquitin–protein ligase contain a high solvent content of 74% and diffract X-rays to 2.5 Å resolution. Ubiquitin-mediated protein modification via covalent attachment of ubiquitin has emerged as one of the most common regulatory processes in all eukaryotes. Nedd4-2, closely related to neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4), is a multimodular ubiquitin–protein ligase comprised of four WW domains and a Hect domain. The WW domains recognize the proline-rich motifs on the multi-subunit amiloride-sensitive epithelial sodium channel (ENaC). To gain insights into the binding of the WW domain to proline-rich peptides, a protein fragment (78 amino acids) containing the fourth WW domain (WW4) of the Nedd4-2 protein was purified and crystallized and X-ray diffraction data were collected. A data set was obtained to 2.5 Å resolution from a cryocooled single crystal at a synchrotron source. The crystals belong to the tetragonal space group P4{sub 1}2{sub 1}2 (or P4{sub 3}2{sub 1}2), with unit-cell parameters a = b = 113.43, c = 103.21 Å. Analysis of the self-rotation function suggests the presence of four WW4 molecules in the asymmetric unit, with a high unit-cell solvent content of 74%.

  10. Versatile and Efficient Site-Specific Protein Functionalization by Tubulin Tyrosine Ligase.

    PubMed

    Schumacher, Dominik; Helma, Jonas; Mann, Florian A; Pichler, Garwin; Natale, Francesco; Krause, Eberhard; Cardoso, M Cristina; Hackenberger, Christian P R; Leonhardt, Heinrich

    2015-11-01

    A novel chemoenzymatic approach for simple and fast site-specific protein labeling is reported. Recombinant tubulin tyrosine ligase (TTL) was repurposed to attach various unnatural tyrosine derivatives as small bioorthogonal handles to proteins containing a short tubulin-derived recognition sequence (Tub-tag). This novel strategy enables a broad range of high-yielding and fast chemoselective C-terminal protein modifications on isolated proteins or in cell lysates for applications in biochemistry, cell biology, and beyond, as demonstrated by the site-specific labeling of nanobodies, GFP, and ubiquitin. PMID:26404067

  11. The ubiquitin ligase APC/CCdh1 puts the brakes on DNA-end resection

    PubMed Central

    Lafranchi, Lorenzo; Sartori, Alessandro A

    2015-01-01

    DNA double-strand breaks (DSBs) are highly deleterious lesions and their misrepair can promote genomic instability, a hallmark of cancer. DNA-end resection is a cell cycle-regulated mechanism that is required for the faithful repair of DSBs. We recently discovered that the anaphase-promoting complex/cyclosome-Cdh1 (APC/CCdh1) ubiquitin ligase is responsible for the timely degradation of CtBP-interacting protein (CtIP), a key DNA-end resection factor, providing a new layer of regulation of DSB repair in human cells. PMID:27308488

  12. DATEL: A Scarless and Sequence-Independent DNA Assembly Method Using Thermostable Exonucleases and Ligase.

    PubMed

    Jin, Peng; Ding, Wenwen; Du, Guocheng; Chen, Jian; Kang, Zhen

    2016-09-16

    DNA assembly is a pivotal technique in synthetic biology. Here, we report a scarless and sequence-independent DNA assembly method using thermal exonucleases (Taq and Pfu DNA polymerases) and Taq DNA ligase (DATEL). Under the optimized conditions, DATEL allows rapid assembly of 2-10 DNA fragments (1-2 h) with high accuracy (between 74 and 100%). Owing to the simple operation system with denaturation-annealing-cleavage-ligation temperature cycles in one tube, DATEL is expected to be a desirable choice for both manual and automated high-throughput assembly of DNA fragments, which will greatly facilitate the rapid progress of synthetic biology and metabolic engineering. PMID:27230689

  13. Fatty acid biosynthesis in pea root plastids

    SciTech Connect

    Stahl, R.J.; Sparace, S.A. )

    1989-04-01

    Fatty acid biosynthesis from (1-{sup 14}C)acetate was optimized in plastids isolated from primary root tips of 7-day-old germinating pea seeds. Fatty acid synthesis was maximum at approximately 80 nmoles/hr/mg protein in the presence of 200 {mu}M acetate, 0.5 mM each of NADH, NADPH and CoA, 6 mM each of ATP and MgCl{sub 2}, 1 mM each of the MnCl{sub 2} and glycerol-3-phosphate, 15 mM KHCO{sub 3}, and 0.1M Bis-tris-propane, pH 8.0 incubated at 35C. At the standard incubation temperature of 25C, fatty acid synthesis was linear from up to 6 hours with 80 to 100 {mu}g/mL plastid protein. ATP and CoA were absolute requirements, whereas KHCO{sub 3}, divalent cations and reduced nucleotides all improved activity by 80 to 85%. Mg{sup 2+} and NADH were the preferred cation and nucleotide, respectively. Dithiothreitol and detergents were generally inhibitory. The radioactive products of fatty acid biosynthesis were approximately 33% 16:0, 10% 18:0 and 56% 18:1 and generally did not vary with increasing concentrations of each cofactor.

  14. Regulation of Synthesis of the Branched-Chain Amino Acids and Cognate Aminoacyl-Transfer Ribonucleic Acid Synthetases of Escherichia coli: a Common Regulatory Element

    PubMed Central

    Jackson, Julius; Williams, L. S.; Umbarger, H. E.

    1974-01-01

    Regulation of isoleucine, valine, and leucine biosynthesis and isoleucyl-, valyl-, and leucyl-transfer ribonucleic acid (tRNA) synthetase formation was examined in two mutant strains of Escherichia coli. One mutant was selected for growth resistance to the isoleucine analogue, ketomycin, and the other was selected for growth resistance to both trifluoroleucine and valine. Control of the synthesis of the branched-chain amino acids by repression was altered in both of these mutants. They also exhibited altered control of formation of isoleucyl-tRNA synthetase (EC 6.1.15, isoleucine:sRNA ligase, AMP), valyl-tRNA synthetase (EC 6.1.1.9, valine:sRNA ligase, AMP), and leucyl-tRNA synthetase (EC 6.1.1.4, leucine:sRNA ligase, AMP). These results suggest the existence of a common element for the control of these two classes of enzymes in Escherichia coli. PMID:4612020

  15. The E3 Ubiquitin Protein Ligase HERC2 Modulates the Activity of Tumor Protein p53 by Regulating Its Oligomerization*

    PubMed Central

    Cubillos-Rojas, Monica; Amair-Pinedo, Fabiola; Peiró-Jordán, Roser; Bartrons, Ramon; Ventura, Francesc; Rosa, Jose Luis

    2014-01-01

    The tumor suppressor p53 is a transcription factor that coordinates the cellular response to several kinds of stress. p53 inactivation is an important step in tumor progression. Oligomerization of p53 is critical for its posttranslational modification and its ability to regulate the transcription of target genes necessary to inhibit tumor growth. Here we report that the HECT E3 ubiquitin ligase HERC2 interacts with p53. This interaction involves the CPH domain of HERC2 (a conserved domain within Cul7, PARC, and HERC2 proteins) and the last 43 amino acid residues of p53. Through this interaction, HERC2 regulates p53 activity. RNA interference experiments showed how HERC2 depletion reduces the transcriptional activity of p53 without affecting its stability. This regulation of p53 activity by HERC2 is independent of proteasome or MDM2 activity. Under these conditions, up-regulation of cell growth and increased focus formation were observed, showing the functional relevance of the HERC2-p53 interaction. This interaction was maintained after DNA damage caused by the chemotherapeutic drug bleomycin. In these stressed cells, p53 phosphorylation was not impaired by HERC2 knockdown. Interestingly, p53 mutations that affect its tetramerization domain disrupted the HERC2-p53 interaction, suggesting a role for HERC2 in p53 oligomerization. This regulatory role was shown using cross-linking assays. Thus, the inhibition of p53 activity after HERC2 depletion can be attributed to a reduction in p53 oligomerization. Ectopic expression of HERC2 (residues 2292–2923) confirmed these observations. Together, these results identify HERC2 as a novel regulator of p53 signaling. PMID:24722987

  16. The E3 ubiquitin ligase HOS1 regulates low ambient temperature-responsive flowering in Arabidopsis thaliana.

    PubMed

    Lee, Jeong Hwan; Kim, Jae Joon; Kim, Soo Hyun; Cho, Hyun Jung; Kim, Joonki; Ahn, Ji Hoon

    2012-10-01

    Ubiquitin-dependent proteolysis regulates multiple aspects of plant growth and development, but little is known about its role in ambient temperature-responsive flowering. In addition to being regulated by daylength, the onset of flowering in many plants can also be delayed by low ambient temperatures. Here, we show that HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1), which encodes an E3 ubiquitin ligase, controls flowering time in response to ambient temperatures (16 and 23°C) and intermittent cold. hos1 mutants flowered early, and were insensitive to ambient temperature, but responded normally to vernalization and gibberellic acid. Genetic analyses suggested that this ambient temperature-insensitive flowering was independent of FLOWERING LOCUS C (FLC). Also, FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) expression was up-regulated in hos1 mutants at both temperatures. The ft tsf mutation almost completely suppressed the early flowering of hos1 mutants at different temperatures, suggesting that FT and TSF are downstream of HOS1 in the ambient temperature response. A lesion in CONSTANS (CO) did not affect the ambient temperature-insensitive flowering phenotype of hos1-3 mutants. In silico analysis showed that FVE was spatiotemporally co-expressed with HOS1. A HOS1-green fluorescent protein (GFP) fusion co-localized with FVE-GFP in the nucleus at both 16 and 23°C. HOS1 physically interacted with FVE and FLK in yeast two-hybrid and co-immunoprecipitation assays. Moreover, hos1 mutants were insensitive to intermittent cold. Collectively, our results suggest that HOS1 acts as a common regulator in the signaling pathways that control flowering time in response to low ambient temperature.

  17. Structure-guided mutational analysis of the OB, HhH, and BRCT domains of Escherichia coli DNA ligase.

    PubMed

    Wang, Li Kai; Nair, Pravin A; Shuman, Stewart

    2008-08-22

    NAD(+)-dependent DNA ligases (LigAs) are ubiquitous in bacteria and essential for growth. LigA enzymes have a modular structure in which a central catalytic core composed of nucleotidyltransferase and oligonucleotide-binding (OB) domains is linked via a tetracysteine zinc finger to distal helix-hairpin-helix (HhH) and BRCT (BRCA1-like C-terminal) domains. The OB and HhH domains contribute prominently to the protein clamp formed by LigA around nicked duplex DNA. Here we conducted a structure-function analysis of the OB and HhH domains of Escherichia coli LigA by alanine scanning and conservative substitutions, entailing 43 mutations at 22 amino acids. We thereby identified essential functional groups in the OB domain that engage the DNA phosphodiester backbone flanking the nick (Arg(333)); penetrate the minor grove and distort the nick (Val(383) and Ile(384)); or stabilize the OB fold (Arg(379)). The essential constituents of the HhH domain include: four glycines (Gly(455), Gly(489), Gly(521), Gly(553)), which bind the phosphate backbone across the minor groove at the outer margins of the LigA-DNA interface; Arg(487), which penetrates the minor groove at the outer margin on the 3 (R)-OH side of the nick; and Arg(446), which promotes protein clamp formation via contacts to the nucleotidyltransferase domain. We find that the BRCT domain is required in its entirety for effective nick sealing and AMP-dependent supercoil relaxation. PMID:18515356

  18. RMND5 from Xenopus laevis is an E3 ubiquitin-ligase and functions in early embryonic forebrain development.

    PubMed

    Pfirrmann, Thorsten; Villavicencio-Lorini, Pablo; Subudhi, Abinash K; Menssen, Ruth; Wolf, Dieter H; Hollemann, Thomas

    2015-01-01

    In Saccharomyces cerevisiae the Gid-complex functions as an ubiquitin-ligase complex that regulates the metabolic switch between glycolysis and gluconeogenesis. In higher organisms six conserved Gid proteins form the CTLH protein-complex with unknown function. Here we show that Rmnd5, the Gid2 orthologue from Xenopus laevis, is an ubiquitin-ligase embedded in a high molecular weight complex. Expression of rmnd5 is strongest in neuronal ectoderm, prospective brain, eyes and ciliated cells of the skin and its suppression results in malformations of the fore- and midbrain. We therefore suggest that Xenopus laevis Rmnd5, as a subunit of the CTLH complex, is a ubiquitin-ligase targeting an unknown factor for polyubiquitination and subsequent proteasomal degradation for proper fore- and midbrain development. PMID:25793641

  19. RMND5 from Xenopus laevis Is an E3 Ubiquitin-Ligase and Functions in Early Embryonic Forebrain Development

    PubMed Central

    Pfirrmann, Thorsten; Villavicencio-Lorini, Pablo; Subudhi, Abinash K.; Menssen, Ruth; Wolf, Dieter H.; Hollemann, Thomas

    2015-01-01

    In Saccharomyces cerevisiae the Gid-complex functions as an ubiquitin-ligase complex that regulates the metabolic switch between glycolysis and gluconeogenesis. In higher organisms six conserved Gid proteins form the CTLH protein-complex with unknown function. Here we show that Rmnd5, the Gid2 orthologue from Xenopus laevis, is an ubiquitin-ligase embedded in a high molecular weight complex. Expression of rmnd5 is strongest in neuronal ectoderm, prospective brain, eyes and ciliated cells of the skin and its suppression results in malformations of the fore- and midbrain. We therefore suggest that Xenopus laevis Rmnd5, as a subunit of the CTLH complex, is a ubiquitin-ligase targeting an unknown factor for polyubiquitination and subsequent proteasomal degradation for proper fore- and midbrain development. PMID:25793641

  20. Structure of a Putative Lipoate Protein Ligase from Thermoplasma acidophilum and the Mechanism of Target Selection for Post-Translational Modification

    SciTech Connect

    McManus,E.; Luisi, B.; Perham, R.

    2006-01-01

    Lipoyl-lysine swinging arms are crucial to the reactions catalysed by the 2-oxo acid dehydrogenase multienzyme complexes. A gene encoding a putative lipoate protein ligase (LplA) of Thermoplasma acidophilum was cloned and expressed in Escherichia coli. The recombinant protein, a monomer of molecular mass 29 kDa, was catalytically inactive. Crystal structures in the absence and presence of bound lipoic acid were solved at 2.1 Angstroms resolution. The protein was found to fall into the a/{beta} class and to be structurally homologous to the catalytic domains of class II aminoacyl-tRNA synthases and biotin protein ligase, BirA. Lipoic acid in LplA was bound in the same position as biotin in BirA. The structure of the T. acidophilum LplA and limited proteolysis of E. coli LplA together highlighted some key features of the post-translational modification. A loop comprising residues 71-79 in the T. acidophilumligase is proposed as interacting with the dithiolane ring of lipoic acid and discriminating against the entry of biotin. A second loop comprising residues 179-193 was disordered in the T. acidophilum structure; tryptic cleavage of the corresponding loop in the E. coli LplA under non-denaturing conditions rendered the enzyme catalytically inactive, emphasizing its importance. The putative LplA of T. acidophilum lacks a C-terminal domain found in its counterparts in E. coli (Gram-negative) or Streptococcus pneumoniae (Gram-positive). A gene encoding a protein that appears to have structural homology to the additional domain in the E. coli and S. pneumoniae enzymes was detected alongside the structural gene encoding the putative LplA in the T. acidophilum genome. It is likely that this protein is required to confer activity on the LplA as currently purified, one protein perhaps catalysing the formation of the obligatory lipoyl-AMP intermediate, and the other transferring the lipoyl group from it to the specific lysine residue in the target protein.

  1. In vitro synthesis of arachidonoyl amino acids by cytochrome c.

    PubMed

    McCue, Jeffrey M; Driscoll, William J; Mueller, Gregory P

    2009-11-01

    Arachidonoyl amino acids are a class of endogenous lipid messengers that are expressed in the mammalian central nervous system and peripherally. While several of their prominent pharmacologic effects have been documented, the mechanism by which arachidonoyl amino acids are biosynthesized has not been defined. We have previously observed that the mitochondrial protein, cytochrome c, is capable of catalyzing the formation of the prototypic arachidonoyl amino acid, arachidonoyl glycine, utilizing arachidonoyl CoA and glycine as substrates, in the presence of hydrogen peroxide. Here we report that cytochrome c is similarly able to catalyze the formation of N-arachidonoyl serine, N-arachidonoyl alanine, and N-arachidonoyl gamma aminobutyric acid from arachidonoyl CoA and the respective amino acids. The identities of the arachidonoyl amino acid products were verified by mass spectral fragmentation pattern analysis. The synthetic reactions exhibited Michaelis-Menten kinetics and continued favorably at physiologic temperature and pH. Spectral data indicate that both cytochrome c protein structure and a +3 heme iron oxidation state are required for the reaction mechanism to proceed optimally. Reactions designed to catalyze the formation of N-arachidonoyl dopamine were not efficient due to the rapid oxidation of dopamine substrate by hydrogen peroxide, consuming both reactants. Finally, under standard assay conditions, arachidonoyl CoA and ethanolamine were found to react spontaneously to form anandamide, independent of cytochrome c and hydrogen peroxide. Accordingly, it was not possible to demonstrate a potential role for cytochrome c in the biosynthetic mechanism for either arachidonoyl dopamine or anandamide. However, the ability of cytochrome c to effectively catalyze the formation of N-arachidonoyl serine, N-arachidonoyl alanine, and N-arachidonoyl gamma aminobutyric acid in vitro highlights its potential role for the generation of these lipid messengers in vivo.

  2. Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I.

    PubMed

    Lama, Lodoe; Ryan, Kevin

    2016-01-01

    Many high-throughput small RNA next-generation sequencing protocols use 5' preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter's 5' end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5' adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3' blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3' ends to be 5' adenylylated at >90% efficiency. PMID:26567315

  3. Structural basis for ligase-specific conjugation of linear ubiquitin chains by HOIP

    PubMed Central

    Koliopoulos, Marios G.; Morris-Davies, Aylin C.; Schaeffer, Veronique; Christodoulou, Evangelos; Howell, Steven; Brown, Nicholas R.; Dikic, Ivan; Rittinger, Katrin

    2013-01-01

    Linear ubiquitin chains are important regulators of cellular signaling pathways that control innate immunity and inflammation through NF-κB activation and protection against TNFα-induced apoptosis1-5. They are synthesized by HOIP, which belongs to the RBR (RING-between-RING) family of E3 ligases and is the catalytic component of LUBAC (linear ubiquitin chain assembly complex), a multi-subunit E3 ligase6. RBR family members act as RING/HECT hybrids, employing RING1 to recognize ubiquitin-loaded E2 while a conserved cysteine in RING2 subsequently forms a thioester intermediate with the transferred or “donor” ubiquitin7. Here we report the crystal structure of the catalytic core of HOIP in its apo form and in complex with ubiquitin. The C-terminal portion of HOIP adopts a novel fold that, together with a zinc finger, forms an ubiquitin-binding platform which orients the acceptor ubiquitin and positions its α-amino group for nucleophilic attack on the E3~ubiquitin thioester. The carboxy-terminal tail of a second ubiquitin molecule is located in close proximity to the catalytic cysteine providing a unique snapshot of the ubiquitin transfer complex containing both donor and acceptor ubiquitin. These interactions are required for activation of the NF-kB pathway in vivo and explain the determinants of linear ubiquitin chain specificity by LUBAC. PMID:24141947

  4. The relationship between polymorphisms in the glutamate cysteine ligase gene and asthma susceptibility.

    PubMed

    Polonikov, A V; Ivanov, V P; Solodilova, M A; Khoroshaya, I V; Kozhuhov, M A; Panfilov, V I

    2007-11-01

    The present study was designed to investigate an association of common -588C/T and -23G/T polymorphisms within glutamate cysteine ligase modifier subunit gene with susceptibility to bronchial asthma. A total of 435 ethnically Russian subjects were recruited in this study, including 221 patients with asthma and 214 sex and age matched healthy subjects. As previously reported, the -588C/T and -23G/T polymorphisms were completely linked. The -588TT/-23TT genotype was found to be associated with decreased risk of allergic asthma after adjustment for age, gender and smoking status using multivariate logistic regression analysis (OR=0.33 95% CI 0.15-0.70, p=0.036). However, the -588CT/-23GT genotype was associated with increased risk of non-allergic asthma (OR=2.03 95% CI 1.05-3.90, p=0.06). This is a first study reporting the association between genetic variations in the glutamate cysteine ligase gene and susceptibility to bronchial asthma. PMID:17643973

  5. Cullin-RING ubiquitin E3 ligase regulation by the COP9 signalosome.

    PubMed

    Cavadini, Simone; Fischer, Eric S; Bunker, Richard D; Potenza, Alessandro; Lingaraju, Gondichatnahalli M; Goldie, Kenneth N; Mohamed, Weaam I; Faty, Mahamadou; Petzold, Georg; Beckwith, Rohan E J; Tichkule, Ritesh B; Hassiepen, Ulrich; Abdulrahman, Wassim; Pantelic, Radosav S; Matsumoto, Syota; Sugasawa, Kaoru; Stahlberg, Henning; Thomä, Nicolas H

    2016-03-31

    The cullin-RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The CUL4A-RBX1-DDB1-DDB2 complex (CRL4A(DDB2)) monitors the genome for ultraviolet-light-induced DNA damage. CRL4A(DBB2) is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4A(DDB2) and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 Å resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN.

  6. A complex ligase ribozyme evolved in vitro from a group I ribozyme domain

    NASA Technical Reports Server (NTRS)

    Jaeger, L.; Wright, M. C.; Joyce, G. F.; Bada, J. L. (Principal Investigator)

    1999-01-01

    Like most proteins, complex RNA molecules often are modular objects made up of distinct structural and functional domains. The component domains of a protein can associate in alternative combinations to form molecules with different functions. These observations raise the possibility that complex RNAs also can be assembled from preexisting structural and functional domains. To test this hypothesis, an in vitro evolution procedure was used to isolate a previously undescribed class of complex ligase ribozymes, starting from a pool of 10(16) different RNA molecules that contained a constant region derived from a large structural domain that occurs within self-splicing group I ribozymes. Attached to this constant region were three hypervariable regions, totaling 85 nucleotides, that gave rise to the catalytic motif within the evolved catalysts. The ligase ribozymes catalyze formation of a 3',5'-phosphodiester linkage between adjacent template-bound oligonucleotides, one bearing a 3' hydroxyl and the other a 5' triphosphate. Ligation occurs in the context of a Watson-Crick duplex, with a catalytic rate of 0.26 min(-1) under optimal conditions. The constant region is essential for catalytic activity and appears to retain the tertiary structure of the group I ribozyme. This work demonstrates that complex RNA molecules, like their protein counterparts, can share common structural domains while exhibiting distinct catalytic functions.

  7. Polyubiquitylation of AMF requires cooperation between the gp78 and TRIM25 ubiquitin ligases.

    PubMed

    Wang, Ying; Ha, Seung-Wook; Zhang, Tianpeng; Kho, Dhong-Hyo; Raz, Avraham; Xie, Youming

    2014-04-30

    gp78 is a ubiquitin ligase that plays a vital role in endoplasmic reticulum (ER)-associated degradation (ERAD). Here we report that autocrine motility factor (AMF), also known as phosphoglucose isomerase (PGI), is a novel substrate of gp78. We show that polyubiquitylation of AMF requires cooperative interaction between gp78 and the ubiquitin ligase TRIM25 (tripartite motif-containing protein 25). While TRIM25 mediates the initial round of ubiquitylation, gp78 catalyzes polyubiquitylation of AMF. The E4-like activity of gp78 was illustrated by an in vitro polyubiquitylation assay using Ub-DHFR as a model substrate. We further demonstrate that TRIM25 ubiquitylates gp78 and that overexpression of TRIM25 accelerates the degradation of gp78. Our data suggest that TRIM25 not only cooperates with gp78 in polyubiquitylation of AMF but also gauges the steady-state level of gp78. This study uncovers a previously unknown functional link between gp78 and TRIM25 and provides mechanistic insight into gp78-mediated protein ubiquitylation.

  8. E3 ubiquitin ligase SP1 regulates peroxisome biogenesis in Arabidopsis

    DOE PAGES

    Pan, Ronghui; Satkovich, John; Hu, Jianping

    2016-10-31

    Peroxisomes are ubiquitous eukaryotic organelles that play pivotal roles in a suite of metabolic processes and often act coordinately with other organelles, such as chloroplasts and mitochondria. Peroxisomes import proteins to the peroxisome matrix by peroxins (PEX proteins), but how the function of the PEX proteins is regulated is poorly understood. In this study, we identified the Arabidopsis RING (really interesting new gene) type E3 ubiquitin ligase SP1 [suppressor of plastid protein import locus 1 (ppi1) 1] as a peroxisome membrane protein with a regulatory role in peroxisome protein import. SP1 interacts physically with the two components of the peroxisomemore » protein docking complex PEX13–PEX14 and the (RING)-finger peroxin PEX2. Loss of SP1 function suppresses defects of the pex14-2 and pex13-1 mutants, and SP1 is involved in the degradation of PEX13 and possibly PEX14 and all three RING peroxins. An in vivo ubiquitination assay showed that SP1 has the ability to promote PEX13 ubiquitination. Our study has revealed that, in addition to its previously reported function in chloroplast biogenesis, SP1 plays a role in peroxisome biogenesis. The same E3 ubiquitin ligase promotes the destabilization of components of two distinct protein-import machineries, indicating that degradation of organelle biogenesis factors by the ubiquitin–proteasome system may constitute an important regulatory mechanism in coordinating the biogenesis of metabolically linked organelles in eukaryotes.« less

  9. Smurf E3 ubiquitin ligases at the cross roads of oncogenesis and tumor suppression.

    PubMed

    David, Diana; Nair, S Asha; Pillai, M Radhakrishna

    2013-01-01

    Smad ubiquitin regulatory factors (Smurfs) belong to the HECT- family of E3 ubiquitin ligases and comprise mainly of two members, Smurf1 and Smurf2. Initially, Smurfs have been implicated in determining the competence of cells to respond to TGF-β/BMP signaling pathway. Nevertheless, the intrinsic catalytic activity has extended the repertoire of Smurf substrates beyond the TGF-β/BMP super family expanding its realm further to epigenetic modifications of histones governing the chromatin landscape. Through regulation of a large number of proteins in multiple cellular compartments, Smurfs regulate diverse cellular processes, including cell-cycle progression, cell proliferation, differentiation, DNA damage response, maintenance of genomic stability, and metastasis. As the genomic ablation of Smurfs leads to global changes in histone modifications and predisposition to a wide spectrum of tumors, Smurfs are also considered to have a novel tumor suppressor function. This review focuses on regulation network and biological functions of Smurfs in connection with its role in cancer progression. By providing a portrait of their protein targets, we intend to link the substrate specificity of Smurfs with their contribution to tumorigenesis. Since the regulation and biological functions of Smurfs are quite complex, understanding the oncogenic potential of these E3 ubiquitin ligases may facilitate the development of mechanism-based drugs in cancer treatment.

  10. [The applications of thermostable ligase chain reaction in facilitating DNA recombination].

    PubMed

    Xiangda, Zhou; Xiao, Song; Cong, Huai; Haiyan, Sun; Hongyan, Chen; Daru, Lu

    2016-02-01

    The traditional Type Ⅱ restriction enzyme-based method is restricted by the purification steps, and therefore, cannot be applied to specific DNA assembly in chaotic system. To solve this problem, Thermostable Ligase Chain Reaction (TLCR) was introduced in the process of DNA assembly and capture. This technique combines the feature of thermostable DNA ligase and sequence specific oligo ligation template, "Helper", to achieve specific assembly of target fragments and exponential increase of products in multiple thermocyclings. Two plasmid construction experiments were carried out in order to test the feasibility and practical performance of TLCR. One was that, TLCR was used to specifically capture a 1.5 kb fragment into vector from an unpurified chaotic system which contained 7 different sizes of fragments. The results showed that the capturing accuracy was around 80%, which proved the feasibility and accuracy of using TLCR to specific assembly of DNA fragments in a complicated mixed system. In the other experiment, TLCR was used to capture two fragments (total length was 27 kb) from Hind Ⅲ digestion of Lambda genome into vector by order. The results also showed an accuracy of around 80%. As demonstrated in the results, TLCR can simplify the process of DNA recombination experiments and is suitable for the assembly of multiple and large DNA fragments. This technique can provide convenience to biological experiments.

  11. Characterization of the mammalian family of DCN-type NEDD8 E3 ligases

    PubMed Central

    Keuss, Matthew J.; Thomas, Yann; Mcarthur, Robin; Wood, Nicola T.; Knebel, Axel; Kurz, Thimo

    2016-01-01

    ABSTRACT Cullin-RING ligases (CRL) are ubiquitin E3 enzymes that bind substrates through variable substrate receptor proteins and are activated by attachment of the ubiquitin-like protein NEDD8 to the cullin subunit. DCNs are NEDD8 E3 ligases that promote neddylation. Mammalian cells express five DCN-like (DCNL) proteins but little is known about their specific functions or interaction partners. We found that DCNLs form stable stoichiometric complexes with CAND1 and cullins that can only be neddylated in the presence of a substrate adaptor. These CAND–cullin–DCNL complexes might represent ‘reserve’ CRLs that can be rapidly activated when needed. We further found that all DCNLs interact with most cullin subtypes, but that they are probably responsible for the neddylation of different subpopulations of any given cullin. This is consistent with the fact that the subcellular localization of DCNLs in tissue culture cells differs and that they show unique tissue-specific expression patterns in mice. Thus, the specificity between DCNL-type NEDD8 E3 enzymes and their cullin substrates is only apparent in well-defined physiological contexts and related to their subcellular distribution and restricted expression. PMID:26906416

  12. A novel effect of thalidomide and its analogs: suppression of cereblon ubiquitination enhances ubiquitin ligase function.

    PubMed

    Liu, Yaobin; Huang, Xiangao; He, Xian; Zhou, Yanqing; Jiang, Xiaogang; Chen-Kiang, Selina; Jaffrey, Samie R; Xu, Guoqiang

    2015-12-01

    The immunomodulatory drug (IMiD) thalidomide and its structural analogs lenalidomide and pomalidomide are highly effective in treating clinical indications. Thalidomide binds to cereblon (CRBN), a substrate receptor of the cullin-4 really interesting new gene (RING) E3 ligase complex. Here, we examine the effect of thalidomide and its analogs on CRBN ubiquitination and its functions in human cell lines. We find that the ubiquitin modification of CRBN includes K48-linked polyubiquitin chains and that thalidomide blocks the formation of CRBN-ubiquitin conjugates. Furthermore, we show that ubiquitinated CRBN is targeted for proteasomal degradation. Treatment of human myeloma cell lines such as MM1.S, OPM2, and U266 with thalidomide (100 μM) and its structural analog lenalidomide (10 μM) results in stabilization of CRBN and elevation of CRBN protein levels. This in turn leads to the reduced level of CRBN target proteins and enhances the sensitivity of human multiple myeloma cells to IMiDs. Our results reveal a novel mechanism by which thalidomide and its analogs modulate the CRBN function in cells. Through inhibition of CRBN ubiquitination, thalidomide and its analogs allow CRBN to accumulate, leading to the increased cullin-4 RING E3 ligase-mediated degradation of target proteins.

  13. Ubiquitin ligase gp78 targets unglycosylated prion protein PrP for ubiquitylation and degradation.

    PubMed

    Shao, Jia; Choe, Vitnary; Cheng, Haili; Tsai, Yien Che; Weissman, Allan M; Luo, Shiwen; Rao, Hai

    2014-01-01

    Prion protein PrP is a central player in several devastating neurodegenerative disorders, including mad cow disease and Creutzfeltd-Jacob disease. Conformational alteration of PrP into an aggregation-prone infectious form PrPSc can trigger pathogenic events. How levels of PrP are regulated is poorly understood. Human PrP is known to be degraded by the proteasome, but the specific proteolytic pathway responsible for PrP destruction remains elusive. Here, we demonstrate that the ubiquitin ligase gp78, known for its role in protein quality control, is critical for unglycosylated PrP ubiquitylation and degradation. Furthermore, C-terminal sequences of PrP protein are crucial for its ubiquitylation and degradation. Our study reveals the first ubiquitin ligase specifically involved in prion protein PrP degradation and PrP sequences crucial for its turnover. Our data may lead to a new avenue to control PrP level and pathogenesis. PMID:24714645

  14. Ubiquitin ligase parkin promotes Mdm2-arrestin interaction but inhibits arrestin ubiquitination

    PubMed Central

    Ahmed, M. Rafiuddin; Zhan, Xuanzhi; Song, Xiufeng; Kook, Seunghyi; Gurevich, Vsevolod V.; Gurevich, Eugenia V.

    2011-01-01

    Numerous mutations in E3 ubiquitin ligase parkin were shown to associate with familial Parkinson's disease. Here we show that parkin binds arrestins, versatile regulators of cell signaling. Arrestin-parkin interaction was demonstrated by coimmuno-precipitation of endogenous proteins from brain tissue, and shown to be direct using purified proteins. Parkin binding enhances arrestin interactions with another E3 ubiquitin ligase, Mdm2, apparently by shifting arrestin conformational equilibrium to the basal state preferred by Mdm2. Although Mdm2 was reported to ubiquitinate arrestins, parkin-dependent increase in Mdm2 binding dramatically reduces the ubiquitination of both non-visual arrestins, basal and stimulated by receptor activation, without affecting receptor internalization. Several disease-associated parkin mutations differentially affect the stimulation of Mdm2 binding. All parkin mutants tested effectively suppress arrestin ubiquitination, suggesting that bound parkin shields arrestin lysines targeted by Mdm2. Parkin binding to arrestins along with its effects on arrestin interaction with Mdm2 and ubiquitination is a novel function of this protein with implications for Parkinson's disease pathology. PMID:21466165

  15. The Ubiquitin Ligase Siah2 Regulates Obesity-induced Adipose Tissue Inflammation

    PubMed Central

    Kilroy, Gail; Carter, Lauren E.; Newman, Susan; Burk, David H.; Manuel, Justin; Möller, Andreas; Bowtell, David D.; Mynatt, Randall L.; Ghosh, Sujoy; Floyd, Z. Elizabeth

    2015-01-01

    Objective Chronic, low-grade adipose tissue inflammation associated with adipocyte hypertrophy is an important link in the relationship between obesity and insulin resistance. Although ubiquitin ligases regulate inflammatory processes, the role of these enzymes in metabolically driven adipose tissue inflammation is relatively unexplored. Herein, we examined the effect of the ubiquitin ligase Siah2 on obesity-related adipose tissue inflammation. Methods Wild-type and Siah2KO mice were fed a low or high fat diet for 16 weeks. Indirect calorimetry, body composition, glucose and insulin tolerance were assayed along with glucose and insulin levels. Gene and protein expression, immunohistochemistry, adipocyte size distribution and lipolysis were also analyzed. Results Enlarged adipocytes in obese Siah2KO mice are not associated with obesity-induced insulin resistance. Proinflammatory gene expression, stress kinase signaling, fibrosis and crown-like structures are reduced in the Siah2KO adipose tissue and Siah2KO adipocytes are more responsive to insulin-dependent inhibition of lipolysis. Loss of Siah2 increases expression of PPARγ target genes involved in lipid metabolism and decreases expression of proinflammatory adipokines regulated by PPARγ. Conclusions Siah2 links adipocyte hypertrophy with adipocyte dysfunction and recruitment of proinflammatory immune cells to adipose tissue. Selective regulation of PPARγ activity is a Siah2-mediated mechanism contributing to obesity-induced adipose tissue inflammation. PMID:26380945

  16. A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation.

    PubMed

    Li, Li; Martinez, Silvia Sanchez; Hu, Wenxin; Liu, Zhe; Tjian, Robert

    2015-01-01

    TFIID-a complex of TATA-binding protein (TBP) and TBP-associated factors (TAFs)-is a central component of the Pol II promoter recognition apparatus. Recent studies have revealed significant downregulation of TFIID subunits in terminally differentiated myocytes, hepatocytes and adipocytes. Here, we report that TBP protein levels are tightly regulated by the ubiquitin-proteasome system. Using an in vitro ubiquitination assay coupled with biochemical fractionation, we identified Huwe1 as an E3 ligase targeting TBP for K48-linked ubiquitination and proteasome-mediated degradation. Upregulation of Huwe1 expression during myogenesis induces TBP degradation and myotube differentiation. We found that Huwe1 activity on TBP is antagonized by the deubiquitinase USP10, which protects TBP from degradation. Thus, modulating the levels of both Huwe1 and USP10 appears to fine-tune the requisite degradation of TBP during myogenesis. Together, our study unmasks a previously unknown interplay between an E3 ligase and a deubiquitinating enzyme regulating TBP levels during cellular differentiation.

  17. A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation

    PubMed Central

    Li, Li; Martinez, Silvia Sanchez; Hu, Wenxin; Liu, Zhe; Tjian, Robert

    2015-01-01

    TFIID—a complex of TATA-binding protein (TBP) and TBP-associated factors (TAFs)—is a central component of the Pol II promoter recognition apparatus. Recent studies have revealed significant downregulation of TFIID subunits in terminally differentiated myocytes, hepatocytes and adipocytes. Here, we report that TBP protein levels are tightly regulated by the ubiquitin-proteasome system. Using an in vitro ubiquitination assay coupled with biochemical fractionation, we identified Huwe1 as an E3 ligase targeting TBP for K48-linked ubiquitination and proteasome-mediated degradation. Upregulation of Huwe1 expression during myogenesis induces TBP degradation and myotube differentiation. We found that Huwe1 activity on TBP is antagonized by the deubiquitinase USP10, which protects TBP from degradation. Thus, modulating the levels of both Huwe1 and USP10 appears to fine-tune the requisite degradation of TBP during myogenesis. Together, our study unmasks a previously unknown interplay between an E3 ligase and a deubiquitinating enzyme regulating TBP levels during cellular differentiation. DOI: http://dx.doi.org/10.7554/eLife.08536.001 PMID:26393420

  18. KBTBD13 interacts with Cullin 3 to form a functional ubiquitin ligase.

    PubMed

    Sambuughin, Nyamkhishig; Swietnicki, Wieslaw; Techtmann, Stephen; Matrosova, Vera; Wallace, Tarina; Goldfarb, Lev; Maynard, Ernest

    2012-05-18

    Autosomal dominant mutations in BTB and Kelch domain containing 13 protein (KBTBD13) are associated with a new type of Nemaline Myopathy (NEM). NEM is a genetically heterogeneous group of muscle disorders. Mutations causing phenotypically distinct NEM variants have previously been identified in components of muscle thin filament. KBTBD13 is a muscle specific protein composed of an N terminal BTB domain and a C terminal Kelch-repeat domain. The function of this newly identified protein in muscle remained unknown. In this study, we show that KBTBD13 interacts with Cullin 3 (Cul3) and the BTB domain mediates this interaction. Using ubiquitination assays, we determined that KBTBD13 participates in the formation of a Cul3 based RING ubiquitin ligase (Cul3-RL) capable of ubiquitin conjugation. Confocal microscopy of transiently expressed KBTBD13 revealed its co-localization with ubiquitin. Taken together, our results demonstrate that KBTBD13 is a putative substrate adaptor for Cul3-RL that functions as a muscle specific ubiquitin ligase, and thereby implicate the ubiquitin proteasome pathway in the pathogenesis of KBTBD13-associated NEM. PMID:22542517

  19. Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I.

    PubMed

    Lama, Lodoe; Ryan, Kevin

    2016-01-01

    Many high-throughput small RNA next-generation sequencing protocols use 5' preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter's 5' end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5' adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3' blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3' ends to be 5' adenylylated at >90% efficiency.

  20. RNF185, a Novel Mitochondrial Ubiquitin E3 Ligase, Regulates Autophagy through Interaction with BNIP1

    PubMed Central

    Tang, Fei; Wang, Bin; Li, Na; Wu, Yanfang; Jia, Junying; Suo, Talin; Chen, Quan; Liu, Yong-Jun; Tang, Jie

    2011-01-01

    Autophagy is an evolutionarily conserved catabolic process that allows recycling of cytoplasmic organelles, such as mitochondria, to offer a bioenergetically efficient pathway for cell survival. Considerable progress has been made in characterizing mitochondrial autophagy. However, the dedicated ubiquitin E3 ligases targeting mitochondria for autophagy have not been revealed. Here we show that human RNF185 is a mitochondrial ubiquitin E3 ligase that regulates selective mitochondrial autophagy in cultured cells. The two C-terminal transmembrane domains of human RNF185 mediate its localization to mitochondrial outer membrane. RNF185 stimulates LC3II accumulation and the formation of autophagolysosomes in human cell lines. We further identified the Bcl-2 family protein BNIP1 as one of the substrates for RNF185. Human BNIP1 colocalizes with RNF185 at mitochondria and is polyubiquitinated by RNF185 through K63-based ubiquitin linkage in vivo. The polyubiquitinated BNIP1 is capable of recruiting autophagy receptor p62, which simultaneously binds both ubiquitin and LC3 to link ubiquitination and autophagy. Our study might reveal a novel RNF185-mediated mechanism for modulating mitochondrial homeostasis through autophagy. PMID:21931693

  1. SpyLigase peptide–peptide ligation polymerizes affibodies to enhance magnetic cancer cell capture

    PubMed Central

    Fierer, Jacob O.; Veggiani, Gianluca; Howarth, Mark

    2014-01-01

    Individual proteins can now often be modified with atomic precision, but there are still major obstacles to connecting proteins into larger assemblies. To direct protein assembly, ideally, peptide tags would be used, providing the minimal perturbation to protein function. However, binding to peptides is generally weak, so assemblies are unstable over time and disassemble with force or harsh conditions. We have recently developed an irreversible protein–peptide interaction (SpyTag/SpyCatcher), based on a protein domain from Streptococcus pyogenes, that locks itself together via spontaneous isopeptide bond formation. Here we develop irreversible peptide–peptide interaction, through redesign of this domain and genetic dissection into three parts: a protein domain termed SpyLigase, which now ligates two peptide tags to each other. All components expressed efficiently in Escherichia coli and peptide tags were reactive at the N terminus, at the C terminus, or at internal sites. Peptide–peptide ligation enabled covalent and site-specific polymerization of affibodies or antibodies against the tumor markers epidermal growth factor receptor (EGFR) and HER2. Magnetic capture of circulating tumor cells (CTCs) is one of the most promising approaches to improve cancer prognosis and management, but CTC capture is limited by inefficient recovery of cells expressing low levels of tumor antigen. SpyLigase-assembled protein polymers made possible the isolation of cancerous cells expressing lower levels of tumor antigen and should have general application in enhancing molecular capture. PMID:24639550

  2. Cullin-RING ubiquitin E3 ligase regulation by the COP9 signalosome.

    PubMed

    Cavadini, Simone; Fischer, Eric S; Bunker, Richard D; Potenza, Alessandro; Lingaraju, Gondichatnahalli M; Goldie, Kenneth N; Mohamed, Weaam I; Faty, Mahamadou; Petzold, Georg; Beckwith, Rohan E J; Tichkule, Ritesh B; Hassiepen, Ulrich; Abdulrahman, Wassim; Pantelic, Radosav S; Matsumoto, Syota; Sugasawa, Kaoru; Stahlberg, Henning; Thomä, Nicolas H

    2016-03-31

    The cullin-RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The CUL4A-RBX1-DDB1-DDB2 complex (CRL4A(DDB2)) monitors the genome for ultraviolet-light-induced DNA damage. CRL4A(DBB2) is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4A(DDB2) and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 Å resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN. PMID:27029275

  3. The SUMO (Small Ubiquitin-like Modifier) Ligase PIAS3 Primes ATR for Checkpoint Activation.

    PubMed

    Wu, Ching-Shyi; Zou, Lee

    2016-01-01

    The maintenance of genomic stability relies on the concerted action of DNA repair and DNA damage signaling pathways. The PIAS (protein inhibitor of activated STAT) family of SUMO (small ubiquitin-like modifier) ligases has been implicated in DNA repair, but whether it plays a role in DNA damage signaling is still unclear. Here, we show that the PIAS3 SUMO ligase is important for activation of the ATR (ataxia telangiectasia and Rad3 related)-regulated DNA damage signaling pathway. PIAS3 is the only member of the PIAS family that is indispensable for ATR activation. In response to different types of DNA damage and replication stress, PIAS3 plays multiple roles in ATR activation. In cells treated with camptothecin (CPT), PIAS3 contributes to formation of DNA double-stranded breaks. In UV (ultraviolet light)- or HU (hydroxyurea)-treated cells, PIAS3 is required for efficient ATR autophosphorylation, one of the earliest events during ATR activation. Although PIAS3 is dispensable for ATRIP (ATR-interacting protein) SUMOylation and the ATR-ATRIP interaction, it is required for maintaining the basal kinase activity of ATR prior to DNA damage. In the absence of PIAS3, ATR fails to display normal kinase activity after DNA damage, which accompanies with reduced phosphorylation of ATR substrates. Together, these results suggest that PIAS3 primes ATR for checkpoint activation by sustaining its basal kinase activity, revealing a new function of the PIAS family in DNA damage signaling. PMID:26565033

  4. Ubiquitin-protein ligase WWP2 binds to and downregulates the epithelial Na(+) channel.

    PubMed

    McDonald, Fiona J; Western, Andrea H; McNeil, John D; Thomas, Brittany C; Olson, Diane R; Snyder, Peter M

    2002-09-01

    The epithelial Na(+) channel (ENaC) is a critical component of the pathway maintaining salt and water balance. The channel is regulated by members of the Nedd4 family of ubiquitin-protein ligases, which bind to channel subunits and catalyze channel internalization and degradation. ENaC mutations that abolish this interaction cause Liddle's syndrome, a genetic form of hypertension. Here, we test the hypothesis that WW domain-containing protein 2 (WWP2), a member of the Nedd4 family of ubiquitin-protein ligases, is a candidate to regulate ENaC. Consistent with this hypothesis, we found that WWP2 is expressed in epithelial tissues that express ENaC, as well as in a wide variety of other tissues. WWP2 contains four WW domains, three of which bound differentially to ENaC subunits. In contrast, all four human Nedd4-2 WW domains bound to ENaC. WWP2 inhibited ENaC when coexpressed in epithelia, requiring a direct interaction between the proteins; mutation of the ENaC PY motifs abolished inhibition. Thus expression, binding, and functional data all suggest that WWP2 is a candidate to regulate ENaC-mediated Na(+) transport in epithelia. PMID:12167593

  5. The E3 ubiquitin ligase Mib1 regulates Plk4 and centriole biogenesis.

    PubMed

    Čajánek, Lukas; Glatter, Timo; Nigg, Erich A

    2015-05-01

    Centrioles function as core components of centrosomes and as basal bodies for the formation of cilia and flagella. Thus, effective control of centriole numbers is essential for embryogenesis, tissue homeostasis and genome stability. In mammalian cells, the centriole duplication cycle is governed by Polo-like kinase 4 (Plk4). Here, we identify the E3 ubiquitin ligase Mind bomb (Mib1) as a new interaction partner of Plk4. We show that Mib1 localizes to centriolar satellites but redistributes to centrioles in response to conditions that induce centriole amplification. The E3 ligase activity of Mib1 triggers ubiquitylation of Plk4 on multiple sites, causing the formation of Lys11-, Lys29- and Lys48-ubiquitin linkages. These modifications control the abundance of Plk4 and its ability to interact with centrosomal proteins, thus counteracting centriole amplification induced by excess Plk4. Collectively, these results identify the interaction between Mib1 and Plk4 as a new and important element in the control of centriole homeostasis.

  6. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide

    PubMed Central

    Fischer, Eric S.; Böhm, Kerstin; Lydeard, John R.; Yang, Haidi; Stadler, Michael B.; Cavadini, Simone; Nagel, Jane; Serluca, Fabrizio; Acker, Vincent; Lingaraju, Gondichatnahalli M.; Tichkule, Ritesh B.; Schebesta, Michael; Forrester, William C.; Schirle, Markus; Hassiepen, Ulrich; Ottl, Johannes; Hild, Marc; Beckwith, Rohan E. J.; Harper, J. Wade; Jenkins, Jeremy L.; Thomä, Nicolas H.

    2015-01-01

    In the 1950s the drug thalidomide administered as a sedative to pregnant women led to the birth of thousands of children with multiple defects. Despite its teratogenicity, thalidomide and its derivatives lenalidomide and pomalidomide (together known as Immunomodulatory Drugs: IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-dysplasia. IMiDs target the CUL4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase and promote the ubiquitination of Ikaros/Aiolos transcription factors by CRL4CRBN. Here we present the crystal structure of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes CRBN as a CRL4CRBN substrate receptor, which enantioselectively binds IMiDs. Through an unbiased screen we identify the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4CRBN. Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4CRBN when recruiting Ikaros/Aiolos for degradation. This dual activity implies that small molecules can principally modulate a ligase to up- or down-regulate the ubiquitination of proteins. PMID:25043012

  7. An Arabidopsis SUMO E3 Ligase, SIZ1, Negatively Regulates Photomorphogenesis by Promoting COP1 Activity

    PubMed Central

    Lin, Xiao-Li; Niu, De; Hu, Zi-Liang; Kim, Dae Heon; Jin, Yin Hua; Cai, Bin; Liu, Peng; Miura, Kenji; Yun, Dae-Jin; Kim, Woe-Yeon; Lin, Rongcheng

    2016-01-01

    COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1), a ubiquitin E3 ligase, is a central negative regulator of photomorphogenesis. However, how COP1 activity is regulated by post-translational modifications remains largely unknown. Here we show that SUMO (small ubiquitin-like modifier) modification enhances COP1 activity. Loss-of-function siz1 mutant seedlings exhibit a weak constitutive photomorphogenic phenotype. SIZ1 physically interacts with COP1 and mediates the sumoylation of COP1. A K193R substitution in COP1 blocks its SUMO modification and reduces COP1 activity in vitro and in planta. Consistently, COP1 activity is reduced in siz1 and the level of HY5, a COP1 target protein, is increased in siz1. Sumoylated COP1 may exhibits higher transubiquitination activity than does non-sumoylated COP1, but SIZ1-mediated SUMO modification does not affect COP1 dimerization, COP1-HY5 interaction, and nuclear accumulation of COP1. Interestingly, prolonged light exposure reduces the sumoylation level of COP1, and COP1 mediates the ubiquitination and degradation of SIZ1. These regulatory mechanisms may maintain the homeostasis of COP1 activity, ensuing proper photomorphogenic development in changing light environment. Our genetic and biochemical studies identify a function for SIZ1 in photomorphogenesis and reveal a novel SUMO-regulated ubiquitin ligase, COP1, in plants. PMID:27128446

  8. A sensitive ligase-based ATP electrochemical assay using molecular beacon-like DNA.

    PubMed

    Wang, Yonghong; He, Xiaoxiao; Wang, Kemin; Ni, Xiaoqi

    2010-05-15

    A sensitive and selective ligase-based signal-on electrochemical sensing method for adenosine-5'-triphosphate (ATP) detection had been developed using molecular beacon (MB)-like DNA. In this method, the biotin-tagged MB-like DNA was self-assembled onto a gold electrode to form a stem-loop structure by means of facile gold-thiol chemistry, which resulted in blockage of electronic transmission. It was eT OFF state. In the presence of ATP, two nucleotide fragments which were complementary to the loop of the MB-like DNA could be ligated by the ATP-dependent T4 DNA ligase. Hybridization of the ligated DNA with the MB-like DNA induced a significant conformational change in this surface-confined DNA structure, which in turn released the biotin from the surface allowing free exchange of electrons with the electrode generating a measurable electrochemical signal (eT ON). The resulting change in electron transfer efficiency was readily measured by differential pulse voltammetry at target ATP concentrations as low as 0.05 nM and with linear response range from 0.1 to 1000 nM. Moreover, it was also able to discriminate ATP from its analogues. The proposed method had been successfully applied to the determination of ATP in the Escherichia coli O157:H7 extracts of water samples, and the linear response was found between the concentrations of 10(3) and 10(7) cfu/mL.

  9. Addition of oligonucleotides to the 5'-terminus of DNA by T4 RNA ligase.

    PubMed Central

    Higgins, N P; Geballe, A P; Cozzarelli, N R

    1979-01-01

    Bacteriophage T4-induced RNA ligase catalyzes the controlled template-independent addition of RNA to the 5'-phosphoryl end of large DNA molecules. Restriction enzyme-generated fragments of Co1E1 DNA with completely basepaired or cohesive ends and linear single-stranded øX174 viral DNA were all good substrates. DNA molecules from 10 to 6000 nucleotides long were quantitatively joined in an hour to a number of different RNA homopolymers. The most efficient of these was A(pA)5; I(pI)5 and C(pC)5 were also utilized while U(pU)5 was not. The optimum ribohomopolymer length was six nucleotides. Joining of ribohomopolymers between 10 and 20 nucleotides long occurred at approximately 1/2 the maximal rate and a trimer was the shortest substrate. Thus RNA ligase provides a method for generating extensions of predetermined length and base composition at the 5'-end of large DNA molecules that complements the available procedures for extending the 3'-hydroxyl terminus of DNA. Images PMID:375192

  10. CUL4A ubiquitin ligase: a promising drug target for cancer and other human diseases

    PubMed Central

    Sharma, Puneet; Nag, Alo

    2014-01-01

    The ability of cullin 4A (CUL4A), a scaffold protein, to recruit a repertoire of substrate adaptors allows it to assemble into distinct E3 ligase complexes to mediate turnover of key regulatory proteins. In the past decade, a considerable wealth of information has been generated regarding its biology, regulation, assembly, molecular architecture and novel functions. Importantly, unravelling of its association with multiple tumours and modulation by viral proteins establishes it as one of the key proteins that may play an important role in cellular transformation. Considering the role of its substrate in regulating the cell cycle and maintenance of genomic stability, understanding the detailed aspects of these processes will have significant consequences for the treatment of cancer and related diseases. This review is an effort to provide a broad overview of this multifaceted ubiquitin ligase and addresses its critical role in regulation of important biological processes. More importantly, its tremendous potential to be exploited for therapeutic purposes has been discussed. PMID:24522884

  11. Datasets from an interaction proteomics screen for substrates of the SCFβTrCP ubiquitin ligase

    PubMed Central

    Magliozzi, Roberto; Peng, Mao; Mohammed, Shabaz; Guardavaccaro, Daniele; Heck, Albert J.R.; Low, Teck Yew

    2015-01-01

    An affinity purification-mass spectrometry (AP-MS) method was employed to identify novel substrates of the SCFβTrCP ubiquitin ligase. A FLAG-HA tagged version of the F-box protein βTrCP2, the substrate recognition subunit of SCFβTrCP, was used as bait. βTrCP2 wild type and the two mutants βTrCP2-R447A and βTrCP2-ΔF were expressed and purified from HEK293T cells to be able to discriminate between potential substrates of SCFβTrCP and unspecific binders. Affinity-purified samples were analyzed by mass spectrometry-based proteomics, applying ultra-high performance liquid chromatography (UHPLC) coupled to high-resolution tandem mass spectrometry. The raw mass spectrometry data have been deposited to the PRIDE partner repository with the identifiers PXD001088 and PXD001224. The present dataset is associated with a research resource published in T.Y. Low, M. Peng, R. Magliozzi, S. Mohammed, D. Guardavaccaro, A.J.R. Heck, A systems-wide screen identifies substrates of the SCFβTrCP ubiquitin ligase. Sci. Signal. 7 (2014) rs8–rs8, 10.1126/scisignal.2005882. PMID:26217795

  12. Ubiquitin-Activated Interaction Traps (UBAITs) identify E3 ligase binding partners.

    PubMed

    O'Connor, Hazel F; Lyon, Nancy; Leung, Justin W; Agarwal, Poonam; Swaim, Caleb D; Miller, Kyle M; Huibregtse, Jon M

    2015-12-01

    We describe a new class of reagents for identifying substrates, adaptors, and regulators of HECT and RING E3s. UBAITs (Ubiquitin-Activated Interaction Traps) are E3-ubiquitin fusion proteins and, in an E1- and E2-dependent manner, the C-terminal ubiquitin moiety forms an amide linkage to proteins that interact with the E3, enabling covalent co-purification of the E3 with partner proteins. We designed UBAITs for both HECT (Rsp5, Itch) and RING (Psh1, RNF126, RNF168) E3s. For HECT E3s, trapping of interacting proteins occurred in vitro either through an E3 thioester-linked lariat intermediate or through an E2 thioester intermediate, and both WT and active-site mutant UBAITs trapped known interacting proteins in yeast and human cells. Yeast Psh1 and human RNF126 and RNF168 UBAITs also trapped known interacting proteins when expressed in cells. Human RNF168 is a key mediator of ubiquitin signaling that promotes DNA double-strand break repair. Using the RNF168 UBAIT, we identify H2AZ--a histone protein involved in DNA repair--as a new target of this E3 ligase. These results demonstrate that UBAITs represent powerful tools for profiling a wide range of ubiquitin ligases.

  13. An E4 Ligase Facilitates Polyubiquitination of Plant Immune Receptor Resistance Proteins in Arabidopsis[W

    PubMed Central

    Huang, Yan; Minaker, Sean; Roth, Charlotte; Huang, Shuai; Hieter, Philip; Lipka, Volker; Wiermer, Marcel; Li, Xin

    2014-01-01

    Proteins with nucleotide binding and leucine-rich repeat domains (NLRs) serve as immune receptors in animals and plants that recognize pathogens and activate downstream defense responses. As high accumulation of NLRs can result in unwarranted autoimmune responses, their cellular concentrations must be tightly regulated. However, the molecular mechanisms of this process are poorly detailed. The F-box protein Constitutive expressor of PR genes 1 (CPR1) was previously identified as a component of a Skp1, Cullin1, F-box protein E3 complex that targets NLRs, including Suppressor of NPR1, Constitutive 1 (SNC1) and Resistance to Pseudomonas syringae 2 (RPS2), for ubiquitination and further protein degradation. From a forward genetic screen, we identified Mutant, snc1-enhancing 3 (MUSE3), an E4 ubiquitin ligase involved in polyubiquitination of its protein targets. Knocking out MUSE3 in Arabidopsis thaliana results in increased levels of NLRs, including SNC1 and RPS2, whereas overexpressing MUSE3 together with CPR1 enhances polyubiquitination and protein degradation of these immune receptors. This report on the functional role of an E4 ligase in plants provides insight into the scarcely understood NLR degradation pathway. PMID:24449689

  14. A conserved role for the ARC1 E3 ligase in Brassicaceae self-incompatibility.

    PubMed

    Indriolo, Emily; Goring, Daphne R

    2014-01-01

    Ubiquitination plays essential roles in the regulation of many processes in plants including pollen rejection in self-incompatible species. In the Brassicaceae (mustard family), self-incompatibility drives the rejection of self-pollen by preventing pollen hydration following pollen contact with the stigmatic surface. Self-pollen is recognized by a ligand-receptor pair: the pollen S-locus cysteine rich/S-locus protein 11 (SCR/SP11) ligand and the pistil S receptor kinase (SRK). Following self-pollen contact, the SCR/SP11 ligand on the pollen surface binds to SRK on the pistil surface, and the SRK-activated signaling pathway is initiated. This pathway includes the armadillo repeat containing 1 (ARC1) protein, a member of the plant U-box (PUB) family of E3 ubiquitin ligases. ARC1 is a functional E3 ligase and is required downstream of SRK for the self-incompatibility response. This mini review highlights our recent progress in establishing ARC1's conserved role in self-pollen rejection in Brassica and Arabidopsis species and discusses future research directions in this field. PMID:24847339

  15. Genomic and functional analysis of the E3 ligase PARK2 in glioma

    PubMed Central

    Lin, De-Chen; Xu, Liang; Chen, Ye; Yan, Haiyan; Hazawa, Masaharu; Doan, Ngan; Said, Jonathan W.; Ding, Ling-Wen; Liu, Li-Zhen; Yang, Henry; Yu, Shizhu; Kahn, Michael; Yin, Dong; Koeffler, H. Phillip

    2015-01-01

    PARK2 (PARKIN) is an E3 ubiquitin ligase whose dysfunction has been associated with the progression of Parkinsonism and human malignancies, and its role in cancer remains to be explored. In this study, we report that PARK2 is frequently deleted and underexpressed in human glioma, and low PARK2 expression is associated with poor survival. Restoration of PARK2 significantly inhibited glioma cell growth both in vitro and in vivo, while depletion of PARK2 promoted cell proliferation. PARK2 attenuated both Wnt- and EGF-stimulated pathways through down-regulating the intracellular level of β-catenin and EGFR. Notably, PARK2 physically interacted with both β-catenin and EGFR. We further found that PARK2 promoted the ubiquitination of these two proteins in an E3 ligase activity dependent manner. Finally, inspired by these newly identified tumor suppressive functions of PARK2, we tested and proved that combination of small-molecule inhibitors targeting both Wnt-β-catenin and EGFR-AKT pathways synergistically impaired glioma cell viability. Together, our findings uncover novel cancer-associated functions of PARK2 and provide a potential therapeutic approach to treat glioma. PMID:25877876

  16. Probes of Ubiquitin E3 ligases distinguish different stages of Parkin activation

    PubMed Central

    Pao, Kuan-Chuan; Stanley, Mathew; Han, Cong; Lai, Yu-Chiang; Murphy, Paul; Balk, Kristin; Wood, Nicola T.; Corti, Olga; Corvol, Jean-Christophe; Muqit, Miratul M.K.; Virdee, Satpal

    2016-01-01

    E3 ligases represent an important class of enzymes, yet there are currently no chemical probes to profile their activity. We develop a new class of activity-based probe by reengineering of a ubiquitin-charged E2 conjugating enzyme and demonstrate their utility by profiling the transthiolation activity of the RING-in-between-RING (RBR) E3 ligase Parkin in vitro and in cellular extracts. Our study provides valuable insight into the roles, and cellular hierarchy, of distinct phosphorylation events in Parkin activation. We also profile Parkin patient disease-associated mutations and strikingly demonstrate that they largely mediate their effect by altering transthiolation activity. Furthermore, our probes enable direct and quantitative measurement of endogenous Parkin activity revealing that endogenous Parkin is activated in neuronal cell lines (≥75 %) in response to mitochondrial depolarization. This new technology also holds promise as a novel biomarker of PINK1-Parkin signalling as demonstrated by compatibility with Parkinson’s disease patient-derived samples. PMID:26928937

  17. A Korean patient with glutaric aciduria type 1 with a novel mutation in the glutaryl CoA dehydrogenase gene.

    PubMed

    Kim, Hee Su; Yu, Hee Joon; Lee, Jeehun; Park, Hyung-Doo; Kim, Ji Hye; Shin, Hyung-Jin; Jin, Dong Kyu; Lee, Munhyang

    2014-01-01

    Mutations in the glutaryl-CoA dehydrogenase gene can result in Glutaric aciduria type 1(GA 1) by accumulation of glutaric acid, 3-hydroxyglutaric acid (3-OH-GA), and glutarylcarnitine (C5DC). GA 1 is characterized by macrocephaly, subdural hemorrhage (SDH), and dystonic movement disorder after acute encephalopathic crisis. We report a Korean patient with GA1 and a novel mutation. A 16-month-old boy presented with SDH, macrocephaly, and developmental delay. In the neurologic examination, the patient had mild axial hypotonia, but otherwise normal neurologic functions. The brain MRI showed large amounts of bilateral SDH and high signal intensity in both basal ganglia and thalamus. Metabolic screening tests detected highly elevated urinary GA levels but 3-OH-glutaric acid was normal. C5DC was 0.94 μM/L (reference range < 0.3 μM/L). The patient had compound heterozygous mutations of the GCDH gene: p.Arg257Gln (c.770G>A) and p.Cys308Arg (c.922T>C). p.Cys308Arg is a novel mutation; reports of p.Arg257Gln were also rare both in Caucasians and Asian populations. In summary, we hereby report one Korean patient with GA1 with clinical, biochemical, and radiologic characteristics confirmed by genetic analysis. PMID:24795062

  18. Possible mechanism for species difference on the toxicity of pivalic acid between dogs and rats

    SciTech Connect

    Yamaguchi, Toshiro . E-mail: toshiro.yamaguchi@shionogi.co.jp; Nakajima, Yoshitsugu; Nakamura, Yutaka

    2006-07-01

    In a high dose toxicity study of pivalic acid (PA), PA caused skeletal muscle disorder in dog, and a significant increase of pivaloyl carnitine (PC) was observed in canine muscle, but not in rat muscle. In order to understand species difference of the toxicity of PA, we compared the in vitro metabolism of PA among dog, rat and rabbit, especially focussing on the carnitine conjugate. Canine muscle showed low, but significant carnitine conjugating activity, while that of rat was negligible. Canine kidney mitochondria had significant activity in the pivaloyl CoA synthesis (7 nmol/mg protein/h), but muscle mitochondria showed only trace activity. Both kidney and muscle mitochondria displayed similar carnitine acyltransferase activity (2-3 nmol/mg protein/h) towards pivaloyl CoA. On the other hand, with respect to the activity of carnitine acyltransferase in the reverse direction using PC as substrate, canine muscle mitochondria showed higher activity than that of kidney mitochondria. This means that PC is not the final stable metabolite, but is converted easily to pivaloyl CoA in canine muscle. These results suggest one of the possible mechanisms for canine selective muscle disorder to be as follows. Only canine muscle can metabolize PA to its carnitine conjugate slowly, but significantly. In canine muscle, PC is not the final stable metabolite; it is easily converted to pivaloyl CoA. As carnitine conjugation is thought to be the only detoxification metabolic route in canine muscle, under certain circumstances such as carnitine deficiency, the risk of exposure with toxic pivaloyl CoA might increase and the CoASH pool in canine muscle might be exhausted, resulting in toxicity in canine muscle.

  19. Effect of pravastatin, an HMG CoA reductase inhibitor, and cholestyramine, a bile acid sequestrant, on lipoprotein particles defined by their apolipoprotein composition.

    PubMed

    Bard, J M; Parra, H J; Douste-Blazy, P; Fruchart, J C

    1990-03-01

    This study compares the effects of cholestyramine (16 g/d) and pravastatin (40 mg/d) on lipoprotein particles defined by their apolipoprotein composition (Lp A-I, Lp A-II:A-I, Lp E:B, and Lp C-III:B). Analysis was performed after 4, 8, and 12 weeks of therapy. Low-density lipoprotein (LDL) cholesterol decreased by 25.1% to 35.0% with cholestyramine and 26.2% to 30.7% with pravastatin, while triglycerides decreased slightly with pravastatin therapy and increased slightly during cholestyramine administration. The fall in cholesterol was mainly due to a decrease in very-low-density lipoprotein (VLDL) and LDL cholesterol; high-density lipoprotein (HDL) cholesterol increased. Apolipoprotein B was reduced dramatically (by 21.7% to 30.5% with cholestyramine and 27.7% to 37.4% with pravastatin). No significant effect on apolipoproteins C-III and E was observed with cholestyramine, while pravastatin reduced these parameters slightly. Apolipoprotein A-I increased during therapy with both drugs, while apolipoprotein A-II was slightly decreased. Although the drugs had nearly the same effects on plasma lipids, their influence on lipoprotein particles defined by their apolipoprotein composition was substantially different. Lp A-II:A-I was increased by both drugs (+8.1% to +41.2% for cholestyramine and +7.2% to +32.6% for pravastatin). Lp A-I was also increased with both drugs, but cholestyramine had a more constant and pronounced effect than pravastatin (+15.1% to +21.7% for cholestyramine and +1.7% to +13.0% for pravastatin). Lp E:B and Lp C-III:B were consistently decreased by pravastatin (-10.2% to -36.5% for LP E:B and -7.2% to -20.9% for Lp C-III:B), while cholestyramine had variable effects on these particles.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Sequence analysis and genetic variability of stearoyl CoA desaturase (SCD) gene in the Italian Mediterranean river buffalo.

    PubMed

    Pauciullo, A; Cosenza, G; D'Avino, A; Colimoro, L; Nicodemo, D; Coletta, A; Feligini, M; Marchitelli, C; Di Berardino, D; Ramunno, L

    2010-12-01

    Stearoyl-CoA desaturase (SCD) plays a key metabolic role by changing the saturated FA content of ruminant milk and meat. In this study we characterized for the first time the stearoyl-CoA desaturase (SCD) gene in river buffalo (Bubalus bubalis) and investigated its genetic variability. On a total of 78 buffaloes, 15 SNPs were detected and 6 of them were preliminarily genotyped. In particular, the g.133A>C SNP was found to create a new consensus site for the SP1 binding site, thus generating a new tandem repeat in the promoter region. A preliminary association study with the milk fatty acid content showed that the C allele significantly affects the total desaturation index (P<0.01). Linkage disequilibrium analysis allowed identification of 7 haplotypes and 4 tag SNPs. Such polymorphisms could represent useful genetic markers for association studies with fatty acid composition, but further studies are needed to evaluate their potential use to improve the nutritional quality of the dairy products. PMID:20691778

  1. Comparison of a Ligase Chain Reaction-Based Assay and Cell Culture for Detection of Pharyngeal Carriage of Chlamydia trachomatis

    PubMed Central

    Winter, Andrew J.; Gilleran, Gerry; Eastick, Kirstine; Ross, Jonathan D. C.

    2000-01-01

    In 264 genitourinary medicine clinic attenders reporting recent fellatio, the prevalence of pharyngeal Chlamydia trachomatis determined by an expanded standard including cell culture and two in-house PCR tests was 1.5% in 194 women and zero in 70 men. The ligase chain reaction (Abbott LCx) had a specificity of 99.2% and a positive predictive value of 60%. PMID:10970416

  2. Universal Labeling of 5′-Triphosphate RNAs by Artificial RNA Ligase Enzyme with Broad Substrate Specificity

    PubMed Central

    Haugner, John C.; Seelig, Burckhard

    2013-01-01

    An artificial RNA ligase specific to RNA with a 5′-triphosphate (PPP-RNA) exhibits broad sequence specificity on model substrates and secondary siRNAs with direct applications in the identification of PPP-RNAs through sequencing. PMID:23851643

  3. A simple template-dependent ligase ribozyme as the RNA replicase emerging first in the RNA world.

    PubMed

    Ma, Wentao; Yu, Chunwu; Zhang, Wentao; Hu, Jiming

    2010-05-01

    The "RNA world" hypothesis has offered a framework for both experimental and theoretical work in the field of the origin of life. An important concern about the hypothesis is how the RNA world could originate. It has long been speculated that a template-dependent RNA synthetase ribozyme, which catalyzed its own replication (thus, an "RNA replicase"), should have emerged first. However, experimental searches for such a replicase have so far been unsuccessful. This is primarily because of the large sequence length of candidate ribozymes, which mainly work in a polymerase-like way. Here, we propose that the replicase that emerged first would be a simple template-dependent ligase ribozyme, which loosely binds to template RNA and has a relatively low efficiency of catalyzing the formation of phosphodiester bonds between adjacently aligned nucleotides or oligonucleotides. We conducted a computer simulation to support this proposal and considered the factors that might affect the emergence of the ribozyme based on the parameter analysis in the simulation. We conclude that (1) a template-dependent ligase may be more likely than a template-dependent polymerase as an early replicase in the emergence of RNA-based replication; (2) such a ligase ribozyme could emerge and be stable against parasites under a broad range of parameters in our model; (3) the conditions shown to favor the initial appearance of a template-dependent ligase ribozyme do not favor its spread.

  4. Regulation of 4CL, encoding 4-coumarate: coenzyme A ligase, expression in kenaf under diverse stress conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We cloned the full length 4CL ortholog encoding 4-coumarate: coenzymeA ligase from kenaf (Hibiscus cannabiuns) using degenerate primers and RACE (rapid amplification of cDNA ends) systems. The 4CL is a key regulatory enzyme of the phenylpropanoid pathway that regulates the activation of cinnamic ac...

  5. Sorghum Brown midrib 2 (Bmr2) gene encodes the major 4-coumarate Coenzyme A ligase involved in lignin synthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Successful modification of plant cell wall composition without compromising plant integrity is dependent on being able to modify the expression of specific genes, but can be very challenging when the target genes are members of multigene families. 4-Coumarate:CoA ligase (4CL) catalyzes the formatio...

  6. Heterologous expression of rice SUMO E3 ligase (OsSIZ1) enhances drought and heat tolerance in transgenic cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Arabidopsis gene AtSIZ1 encodes a SUMO E3 ligase that plays important roles in plant response to abiotic stresses such as drought, heat, cold, salt, and nutrient starvation. Loss of function in AtSIZ1 leads to increased sensitivity to drought, heat, and salt stresses, whereas overexpression of t...

  7. Characterization of a novel RING-type ubiquitin E3 ligase GhRING2 differentially expressed in cotton fiber

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin-proteasome proteolysis pathway is responsible for the degradation of abnormal and short-lived proteins to regulate many important biochemical activities in eukaryotes. By employing affymetrix microarray analysis, we have identified a novel ubiquitin ligase E3 gene GhRING2 that is diffe...

  8. Identifying the trigger of c-IAPs: structural and functional characterization of CARD-mediated modulation of ubiquitin ligase activity.

    PubMed

    Oetjen, Karolyn A; Duckett, Colin S

    2011-06-10

    In this issue of Molecular Cell, Lopez et al. (2011) examine the caspase-recruitment domain (CARD) of c-IAP1 to reveal an intriguing mechanism in which conformational changes of the CARD determine c-IAP1's ubiquitin ligase activity, with implications for regulation of cell proliferation and survival by the IAPs.

  9. A Simple Template-Dependent Ligase Ribozyme as the RNA Replicase Emerging First in the RNA World

    NASA Astrophysics Data System (ADS)

    Ma, Wentao; Yu, Chunwu; Zhang, Wentao; Hu, Jiming

    2010-05-01

    The "RNA world" hypothesis has offered a framework for both experimental and theoretical work in the field of the origin of life. An important concern about the hypothesis is how the RNA world could originate. It has long been speculated that a template-dependent RNA synthetase ribozyme, which catalyzed its own replication (thus, an "RNA replicase"), should have emerged first. However, experimental searches for such a replicase have so far been unsuccessful. This is primarily because of the large sequence length of candidate ribozymes, which mainly work in a polymerase-like way. Here, we propose that the replicase that emerged first would be a simple template-dependent ligase ribozyme, which loosely binds to template RNA and has a relatively low efficiency of catalyzing the formation of phosphodiester bonds between adjacently aligned nucleotides or oligonucleotides. We conducted a computer simulation to support this proposal and considered the factors that might affect the emergence of the ribozyme based on the parameter analysis in the simulation. We conclude that (1) a template-dependent ligase may be more likely than a template-dependent polymerase as an early replicase in the emergence of RNA-based replication; (2) such a ligase ribozyme could emerge and be stable against parasites under a broad range of parameters in our model; (3) the conditions shown to favor the initial appearance of a template-dependent ligase ribozyme do not favor its spread.

  10. The MLLE Domain of the Ubiquitin Ligase UBR5 Binds to Its Catalytic Domain to Regulate Substrate Binding*

    PubMed Central

    Muñoz-Escobar, Juliana; Matta-Camacho, Edna; Kozlov, Guennadi; Gehring, Kalle

    2015-01-01

    E3 ubiquitin ligases catalyze the transfer of ubiquitin from an E2-conjugating enzyme to a substrate. UBR5, homologous to the E6AP C terminus (HECT)-type E3 ligase, mediates the ubiquitination of proteins involved in translation regulation, DNA damage response, and gluconeogenesis. In addition, UBR5 functions in a ligase-independent manner by prompting protein/protein interactions without ubiquitination of the binding partner. Despite recent functional studies, the mechanisms involved in substrate recognition and selective ubiquitination of its binding partners remain elusive. The C terminus of UBR5 harbors the HECT catalytic domain and an adjacent MLLE domain. MLLE domains mediate protein/protein interactions through the binding of a conserved peptide motif, termed PAM2. Here, we characterize the binding properties of the UBR5 MLLE domain to PAM2 peptides from Paip1 and GW182. The crystal structure with a Paip1 PAM2 peptide reveals the network of hydrophobic and ionic interactions that drive binding. In addition, we identify a novel interaction of the MLLE domain with the adjacent HECT domain mediated by a PAM2-like sequence. Our results confirm the role of the MLLE domain of UBR5 in substrate recruitment and suggest a potential role in regulating UBR5 ligase activity. PMID:26224628

  11. Competing E3 Ubiquitin Ligases Determine Circadian Period by Regulated Degradation of CRY in Nucleus and Cytoplasm

    PubMed Central

    Yoo, Seung-Hee; Mohawk, Jennifer A.; Siepka, Sandra M.; Shan, Yongli; Huh, Seong Kwon; Hong, Hee-Kyung; Kornblum, Izabela; Kumar, Vivek; Koike, Nobuya; Xu, Ming; Nussbaum, Justin; Liu, Xinran; Chen, Zheng; Chen, Zhijian J.; Green, Carla B.; Takahashi, Joseph S.

    2013-01-01

    SUMMARY Period determination in the mammalian circadian clock involves the turnover rate of the repressors, CRY and PER. Here we show that CRY ubiquitination engages two competing E3 ligase complexes that either lengthen or shorten circadian period in mice. Cloning of a short-period circadian mutant, Past-time, revealed a glycine to glutamate (G149E) missense mutation in Fbxl21, an F-box protein gene that is a paralog of Fbxl3 that targets the CRY proteins for degradation. While loss-of-function of FBXL3 leads to period lengthening, mutation of Fbxl21 causes period shortening. FBXL21 forms an SCF E3 ligase complex that slowly degrades CRY in the cytoplasm, but antagonizes the stronger E3 ligase activity of FBXL3 in the nucleus. FBXL21 plays a dual role: protecting CRY from FBXL3 degradation in the nucleus and promoting CRY degradation within the cytoplasm. Thus, the balance and cellular compartmentalization of competing E3 ligases for CRY determine circadian period of the clock in mammals. PMID:23452855

  12. The MLLE domain of the ubiquitin ligase UBR5 binds to its catalytic domain to regulate substrate binding.

    PubMed

    Muñoz-Escobar, Juliana; Matta-Camacho, Edna; Kozlov, Guennadi; Gehring, Kalle

    2015-09-11

    E3 ubiquitin ligases catalyze the transfer of ubiquitin from an E2-conjugating enzyme to a substrate. UBR5, homologous to the E6AP C terminus (HECT)-type E3 ligase, mediates the ubiquitination of proteins involved in translation regulation, DNA damage response, and gluconeogenesis. In addition, UBR5 functions in a ligase-independent manner by prompting protein/protein interactions without ubiquitination of the binding partner. Despite recent functional studies, the mechanisms involved in substrate recognition and selective ubiquitination of its binding partners remain elusive. The C terminus of UBR5 harbors the HECT catalytic domain and an adjacent MLLE domain. MLLE domains mediate protein/protein interactions through the binding of a conserved peptide motif, termed PAM2. Here, we characterize the binding properties of the UBR5 MLLE domain to PAM2 peptides from Paip1 and GW182. The crystal structure with a Paip1 PAM2 peptide reveals the network of hydrophobic and ionic interactions that drive binding. In addition, we identify a novel interaction of the MLLE domain with the adjacent HECT domain mediated by a PAM2-like sequence. Our results confirm the role of the MLLE domain of UBR5 in substrate recruitment and suggest a potential role in regulating UBR5 ligase activity.

  13. Aberrant Lipid Metabolism in Anaplastic Thyroid Carcinoma Reveals Stearoyl CoA Desaturase 1 as a Novel Therapeutic Target

    PubMed Central

    von Roemeling, Christina A.; Marlow, Laura A.; Pinkerton, Anthony B.; Crist, Angela; Miller, James; Tun, Han W.; Smallridge, Robert C.

    2015-01-01

    Context: Currently there are no efficacious therapies for patients with anaplastic thyroid carcinoma (ATC) that result in long-term disease stabilization or regression. Objective: We sought to identify pathways critical for ATC cell progression and viability in an effort to develop new therapeutic strategies. We investigated the effects of targeted inhibition of stearoyl-CoA desaturase 1 (SCD1), a constituent of fatty acid metabolism overexpressed in ATC. Design: A gene array of ATC and normal thyroid tissue was performed to identify gene transcripts demonstrating altered expression in tumor samples. Effects of pharmacological and the genetic inhibition of SCD1 on tumor cell viability as well as cell signaling responses to therapy were evaluated in in vitro and in vivo models of this rare, lethal malignancy. Results: The gene array analysis revealed consistent distortion of fatty acid metabolism and overexpression of SCD1 in ATC and well-differentiated thyroid carcinomas. SCD1 is critical for ATC cell survival and proliferation, the inhibition of which induced endoplasmic reticulum stress, activation of the unfolded protein response, and apoptosis. Combined suppression of endoplasmic reticulum-associated degradation, a prosurvival component of the unfolded protein response, using proteasome inhibitors resulted in a synergistic decrease in tumor cell proliferation and increased cell death. Conclusions: SCD1 is a novel oncogenic factor specifically required for tumor cell viability in ATC. Furthermore, the expression of SCD1 appears to be correlated with thyroid tumor aggressiveness and may serve as a prognostic biomarker. These findings substantiate SCD1 as a novel tumor-specific target for therapy in patients with ATC and should be further investigated in a clinical setting. PMID:25675381

  14. Nonhost resistance of tomato to the bean pathogen Pseudomonas syringae pv. syringae B728a is due to a defective E3 ubiquitin ligase domain in avrptobb728a.

    PubMed

    Chien, Ching-Fang; Mathieu, Johannes; Hsu, Chun-Hua; Boyle, Patrick; Martin, Gregory B; Lin, Nai-Chun

    2013-04-01

    The bean pathogen Pseudomonas syringae pv. syringae B728a expresses homologs of the type III effectors AvrPto and AvrPtoB, either of which can trigger resistance in tomato cultivars expressing Pto and Prf genes. We found that strain B728a also elicits nonhost resistance in tomato cultivars VFNT Cherry and Moneymaker that lack Pto but express other members of the Pto family (e.g., SlFen and SlPtoC). Here, we show that the AvrPtoB homolog from B728a, termed AvrPtoBB728a (also known as HopAB1), is recognized by 'VFNT Cherry' and 'Moneymaker' when the effector is expressed in P. syringae pv. syringae 61, a strain lacking the avrPto or avrPtoB homolog. Using a gene-silencing approach, this recognition was shown to involve one or more Pto family members and Prf. AvrPtoBB728a interacted with SlFen, SlPtoC, and SlPtoD, in addition to Pto, in a yeast two-hybrid assay. In P. syringae pv. tomato DC3000, the C-terminal domain of AvrPtoB is an E3 ubiquitin ligase that ubiquitinates Fen, causing its degradation and leading to disease susceptibility. Although the C-terminal domain of AvrPtoBB728a shares 69% amino acid identity with that of AvrPtoB, we found that it has greatly reduced E3 ligase activity and is unable to ubiquitinate Fen in an in vitro ubiquitination assay. Thus, the nonhost resistance of 'VFNT Cherry' and 'Moneymaker' to B728a appears to be due to recognition of AvrPtoBB728 as a result of the effector's reduced E3 ligase activity, which prevents it from facilitating degradation of a Pto family member. We speculate that the primary plant host of B728a lacks a Fen-like protein and that, therefore, the E3 ligase of AvrPtoBB728 was unnecessary for pathogenicity and has diverged and become ineffective. PMID:23252461

  15. Abiotic stress induces change in Cinnamoyl CoA Reductase (CCR) protein abundance and lignin deposition in developing seedlings of Leucaena leucocephala.

    PubMed

    Srivastava, Sameer; Vishwakarma, Rishi K; Arafat, Yasir Ali; Gupta, Sushim K; Khan, Bashir M

    2015-04-01

    Aboitic stress such as drought and salinity are class of major threats, which plants undergo through their lifetime. Lignin deposition is one of the responses to such abiotic stresses. The gene encoding Cinnamoyl CoA Reductase (CCR) is a key gene for lignin biosynthesis, which has been shown to be over-expressed under stress conditions. In the present study, developing seedlings of Leucaena leucocephala (Vernacular name: Subabul, White popinac) were treated with 1 % mannitol and 200 mM NaCl to mimic drought and salinity stress conditions, respectively. Enzyme linked immunosorbant assay (ELISA) based expression pattern of CCR protein was monitored coupled with Phlorogucinol/HCl activity staining of lignin in transverse sections of developing L. leucocephala seedlings under stress. Our result suggests a differential lignification pattern in developing root and stem under stress conditions. Increase in lignification was observed in mannitol treated stems and corresponding CCR protein accumulation was also higher than control and salt stress treated samples. On the contrary CCR protein was lower in NaCl treated stems and corresponding lignin deposition was also low. Developing root tissue showed a high level of CCR content and lignin deposition than stem samples under all conditions tested. Overall result suggested that lignin accumulation was not affected much in case of developing root however developing stems were significantly affected under drought and salinity stress condition. PMID:25931776

  16. Moonlighting proteins Hal3 and Vhs3 form a heteromeric PPCDC with Ykl088w in yeast CoA biosynthesis.

    PubMed

    Ruiz, Amparo; González, Asier; Muñoz, Ivan; Serrano, Raquel; Abrie, J Albert; Strauss, Erick; Ariño, Joaquín

    2009-12-01

    Unlike most other organisms, the essential five-step coenzyme A biosynthetic pathway has not been fully resolved in yeast. Specifically, the genes encoding the phosphopantothenoylcysteine decarboxylase (PPCDC) activity still remain unidentified. Sequence homology analyses suggest three candidates-Ykl088w, Hal3 and Vhs3-as putative PPCDC enzymes in Saccharomyces cerevisiae. Notably, Hal3 and Vhs3 have been characterized as negative regulatory subunits of the Ppz1 protein phosphatase. Here we show that YKL088w does not encode a third Ppz1 regulatory subunit, and that the essential roles of Ykl088w and the Hal3 and Vhs3 pair are complementary, cannot be interchanged and can be attributed to PPCDC-related functions. We demonstrate that while known eukaryotic PPCDCs are homotrimers, the active yeast enzyme is a heterotrimer that consists of Ykl088w and Hal3/Vhs3 monomers that separately provides two essential catalytic residues. Our results unveil Hal3 and Vhs3 as moonlighting proteins involved in both CoA biosynthesis and protein phosphatase regulation.

  17. HMG CoA lyase deficiency: identification of five causal point mutations in codons 41 and 42, including a frequent Saudi Arabian mutation, R41Q.

    PubMed

    Mitchell, G A; Ozand, P T; Robert, M F; Ashmarina, L; Roberts, J; Gibson, K M; Wanders, R J; Wang, S; Chevalier, I; Plöchl, E; Miziorko, H

    1998-02-01

    The hereditary deficiency of 3-hydroxy-3-methylglutaryl (HMG) CoA lyase (HL; OMIM 246450 [http://www3.ncbi.nlm.nih. gov:80/htbin-post/Omim/dispmim?246450]) results in episodes of hypoketotic hypoglycemia and coma and is reported to be frequent and clinically severe in Saudi Arabia. We found genetic diversity among nine Saudi HL-deficient probands: six were homozygous for the missense mutation R41Q, and two were homozygous for the frameshift mutation F305fs(-2). In 32 non-Saudi HL-deficient probands, we found three R41Q alleles and also discovered four other deleterious point mutations in codons 41 and 42: R41X, D42E, D42G, and D42H. In purified mutant recombinant HL, all four missense mutations in codons 41 and 42 cause a marked decrease in HL activity. We developed a screening procedure for HL missense mutations that yields residual activity at levels comparable to those obtained using purified HL peptides. Codons 41 and 42 are important for normal HL catalysis and account for a disproportionate 21 (26%) of 82 of mutant alleles in our group of HL-deficient probands.

  18. Detection of Listeria monocytogenes with a nonisotopic polymerase chain reaction-coupled ligase chain reaction assay.

    PubMed Central

    Wiedmann, M; Barany, F; Batt, C A

    1993-01-01

    A polymerase chain reaction (PCR)-coupled ligase chain reaction (LCR) assay for the specific detection of Listeria monocytogenes (M. Wiedmann, J. Czajka, F. Barany, and C. A. Batt, Appl. Environ. Microbiol. 58:3443-3447, 1992) has been modified for detection of the LCR products with a nonisotopic readout. When a chemiluminescent or a colorimetric substrate for the nonisotopic detection of the LCR products was used, the PCR-coupled LCR gave a sensitivity of 10 CFU of L. monocytogenes. The detection method with the chemiluminescent substrate Lumi-Phos 530 permitted detection of the LCR products in less than 3 h, so that the whole assay can be completed within 10 h. Images PMID:8368859

  19. The ubiquitin ligase Phr1 regulates axon outgrowth through modulation of microtubule dynamics.

    PubMed

    Lewcock, Joseph W; Genoud, Nicolas; Lettieri, Karen; Pfaff, Samuel L

    2007-11-21

    To discover new genes involved in axon navigation, we conducted a forward genetic screen for recessive alleles affecting motor neuron pathfinding in GFP reporter mice mutagenized with ENU. In Magellan mutant embryos, motor axons were error prone and wandered inefficiently at choice points within embryos, but paradoxically responded to guidance cues with normal sensitivity in vitro. We mapped the Magellan mutation to the Phr1 gene encoding a large multidomain E3 ubiquitin ligase. Phr1 is associated with the microtubule cytoskeleton within neurons and selectively localizes to axons but is excluded from growth cones. Motor and sensory neurons from Magellan mutants display abnormal morphologies due to a breakdown in the polarized distribution of components that segregate between axons and growth cones. The Magellan phenotype can be reversed by stabilizing microtubules with taxol or inhibiting p38MAPK activity. Thus, efficacious pathfinding requires Phr1 activity for coordinating the cytoskeletal organization that distinguishes axons from growth cones.

  20. Bisubstrate Inhibitors of Biotin Protein Ligase in Mycobacterium tuberculosis Resistant to Cyclonucleoside Formation

    PubMed Central

    2013-01-01

    Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, is the leading cause of bacterial infectious disease mortality. Biotin protein ligase (BirA) globally regulates lipid metabolism in Mtb through the posttranslational biotinylation of acyl coenzyme A carboxylases (ACCs) involved in lipid biosynthesis and is essential for Mtb survival. We previously developed a rationally designed bisubstrate inhibitor of BirA that displays potent enzyme inhibition and whole-cell activity against multidrug resistant and extensively drug resistant Mtb strains. Here we present the design, synthesis, and evaluation of a focused series of inhibitors, which are resistant to cyclonucleoside formation, a key decomposition pathway of our initial analogue. Improved chemical stability is realized through replacement of the adenosyl N-3 nitrogen and C-5′ oxygen atom with carbon as well as incorporation of a bulky group on the nucleobase to prevent the required syn-conformation necessary for proper alignment of N-3 with C-5′. PMID:24363833

  1. Cortical dynamics during cell motility are regulated by CRL3(KLHL21) E3 ubiquitin ligase.

    PubMed

    Courtheoux, Thibault; Enchev, Radoslav I; Lampert, Fabienne; Gerez, Juan; Beck, Jochen; Picotti, Paola; Sumara, Izabela; Peter, Matthias

    2016-01-01

    Directed cell movement involves spatial and temporal regulation of the cortical microtubule (Mt) and actin networks to allow focal adhesions (FAs) to assemble at the cell front and disassemble at the rear. Mts are known to associate with FAs, but the mechanisms coordinating their dynamic interactions remain unknown. Here we show that the CRL3(KLHL21) E3 ubiquitin ligase promotes cell migration by controlling Mt and FA dynamics at the cell cortex. Indeed, KLHL21 localizes to FA structures preferentially at the leading edge, and in complex with Cul3, ubiquitylates EB1 within its microtubule-interacting CH-domain. Cells lacking CRL3(KLHL21) activity or expressing a non-ubiquitylatable EB1 mutant protein are unable to migrate and exhibit strong defects in FA dynamics, lamellipodia formation and cortical plasticity. Our study thus reveals an important mechanism to regulate cortical dynamics during cell migration that involves ubiquitylation of EB1 at focal adhesions. PMID:27641145

  2. Structural and functional insights into the E3 ligase, RNF126

    PubMed Central

    Krysztofinska, Ewelina M.; Martínez-Lumbreras, Santiago; Thapaliya, Arjun; Evans, Nicola J.; High, Stephen; Isaacson, Rivka L.

    2016-01-01

    RNF126 is an E3 ubiquitin ligase that collaborates with the BAG6 sortase complex to ubiquitinate hydrophobic substrates in the cytoplasm that are destined for proteasomal recycling. Composed of a trimeric complex of BAG6, TRC35 and UBL4A the BAG6 sortase is also associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates. Here we solve the solution structure of the RNF126 zinc finger domain in complex with the BAG6 UBL domain. We also characterise an interaction between RNF126 and UBL4A and analyse the competition between SGTA and RNF126 for the N-terminal BAG6 binding site. This work sheds light on the sorting mechanism of the BAG6 complex and its accessory proteins which, together, decide the fate of stray hydrophobic proteins in the aqueous cytoplasm. PMID:27193484

  3. Cortical dynamics during cell motility are regulated by CRL3KLHL21 E3 ubiquitin ligase

    PubMed Central

    Courtheoux, Thibault; Enchev, Radoslav I.; Lampert, Fabienne; Gerez, Juan; Beck, Jochen; Picotti, Paola; Sumara, Izabela; Peter, Matthias

    2016-01-01

    Directed cell movement involves spatial and temporal regulation of the cortical microtubule (Mt) and actin networks to allow focal adhesions (FAs) to assemble at the cell front and disassemble at the rear. Mts are known to associate with FAs, but the mechanisms coordinating their dynamic interactions remain unknown. Here we show that the CRL3KLHL21 E3 ubiquitin ligase promotes cell migration by controlling Mt and FA dynamics at the cell cortex. Indeed, KLHL21 localizes to FA structures preferentially at the leading edge, and in complex with Cul3, ubiquitylates EB1 within its microtubule-interacting CH-domain. Cells lacking CRL3KLHL21 activity or expressing a non-ubiquitylatable EB1 mutant protein are unable to migrate and exhibit strong defects in FA dynamics, lamellipodia formation and cortical plasticity. Our study thus reveals an important mechanism to regulate cortical dynamics during cell migration that involves ubiquitylation of EB1 at focal adhesions. PMID:27641145

  4. Cortical dynamics during cell motility are regulated by CRL3(KLHL21) E3 ubiquitin ligase.

    PubMed

    Courtheoux, Thibault; Enchev, Radoslav I; Lampert, Fabienne; Gerez, Juan; Beck, Jochen; Picotti, Paola; Sumara, Izabela; Peter, Matthias

    2016-09-19

    Directed cell movement involves spatial and temporal regulation of the cortical microtubule (Mt) and actin networks to allow focal adhesions (FAs) to assemble at the cell front and disassemble at the rear. Mts are known to associate with FAs, but the mechanisms coordinating their dynamic interactions remain unknown. Here we show that the CRL3(KLHL21) E3 ubiquitin ligase promotes cell migration by controlling Mt and FA dynamics at the cell cortex. Indeed, KLHL21 localizes to FA structures preferentially at the leading edge, and in complex with Cul3, ubiquitylates EB1 within its microtubule-interacting CH-domain. Cells lacking CRL3(KLHL21) activity or expressing a non-ubiquitylatable EB1 mutant protein are unable to migrate and exhibit strong defects in FA dynamics, lamellipodia formation and cortical plasticity. Our study thus reveals an important mechanism to regulate cortical dynamics during cell migration that involves ubiquitylation of EB1 at focal adhesions.

  5. Rational optimization of the DSL ligase ribozyme with GNRA/receptor interacting modules.

    PubMed

    Ishikawa, Junya; Matsumura, Shigeyoshi; Jaeger, Luc; Inoue, Tan; Furuta, Hiroyuki; Ikawa, Yoshiya

    2009-10-15

    The DSL ribozyme is a class of artificial ligase ribozymes with a highly modular architecture, which catalyzes template-directed RNA ligation on a helical substrate module that can be either covalently connected (cis-DSL) or physically separated (trans-DSL) from the catalytic module. Substrate recognition by the catalytic module is promoted by one or two sets of GNRA/receptor interactions acting as clamps in the cis or trans configurations, respectively. In this study, we have rationally designed and analyzed the catalytic and self-assembly properties of several trans-DSL ribozymes with different sets of natural and artificial GNRA-receptor clamps. Two variants newly designed in this study showed significantly enhanced catalytic properties with respect of the original trans-DSL construct. While this work allows dissection of the turnover and catalytic properties of the trans-DSL ribozyme, it also emphasizes the remarkable modularity of RNA tertiary structure for nano-construction of complex functions.

  6. Detection of bovine leukocyte adhesion deficiency by nonisotopic ligase chain reaction.

    PubMed

    Batt, C A; Wagner, P; Wiedmann, M; Luo, J; Gilbert, R

    1994-04-01

    A nonisotopic ligase chain reaction (LCR) assay was developed to detect the mutation (D128G; Shuster et al. (1992) PNAS 89, 9225-9) for bovine leukocyte adhesion deficiency (BLAD). Two sets of diagonally opposed discriminating LCR primers that differentiate the normal and BLAD allele were designed so that the 3' end of each primer overlapped the D128G mutation. These discriminating primers were synthesized with a 5' biotin and could be captured using streptavidin-coated microtitre wells. A common set of primers that abut these discriminating primers were also synthesized and 3'-tailed with digoxigenin-ddUTP. Captured LCR products were then detected using antidigoxigenin antibodies coupled to alkaline phosphatase. The assay readout was a chemiluminescent signal generated by the hydrolysis of Lumi-Phos 530 and the entire assay including DNA isolation can be completed within 8 h. PMID:7912052

  7. Novel roles of Skp2 E3 ligase in cellular senescence, cancer progression, and metastasis

    PubMed Central

    Wang, Guocan; Chan, Chia-Hsin; Gao, Yuan; Lin, Hui-Kuan

    2012-01-01

    S-phase kinase-associated protein 2 (Skp2) belongs to the F-box protein family. It is a component of the SCF E3 ubiquitin ligase complex. Skp2 has been shown to regulate cellular proliferation by targeting several cell cycle-regulated proteins for ubiquitination and degradation, including cyclin-dependent kinase inhibitor p27. Skp2 has also been demonstrated to display an oncogenic function since its overexpression has been observed in many human cancers. This review discusses the recent discoveries on the novel roles of Skp2 in regulating cellular senescence, cancer progression, and metastasis, as well as the therapeutic potential of targeting Skp2 for human cancer treatment. PMID:22200179

  8. The antiobesity factor WDTC1 suppresses adipogenesis via the CRL4WDTC1 E3 ligase.

    PubMed

    Groh, Beezly S; Yan, Feng; Smith, Matthew D; Yu, Yanbao; Chen, Xian; Xiong, Yue

    2016-05-01

    WDTC1/Adp encodes an evolutionarily conserved suppressor of lipid accumulation. While reduced WDTC1 expression is associated with obesity in mice and humans, its cellular function is unknown. Here, we demonstrate that WDTC1 is a component of a DDB1-CUL4-ROC1 (CRL4) E3 ligase. Using 3T3-L1 cell culture model of adipogenesis, we show that disrupting the interaction between WDTC1 and DDB1 leads to a loss of adipogenic suppression by WDTC1, increased triglyceride accumulation and adipogenic gene expression. We show that the CRL4(WDTC) (1) complex promotes histone H2AK119 monoubiquitylation, thus suggesting a role for this complex in transcriptional repression during adipogenesis. Our results identify a biochemical role for WDTC1 and extend the functional range of the CRL4 complex to the suppression of fat accumulation. PMID:27113764

  9. Release from myosin V via regulated recruitment of an E3 ubiquitin ligase controls organelle localization.

    PubMed

    Yau, Richard G; Peng, Yutian; Valiathan, Rajeshwari R; Birkeland, Shanda R; Wilson, Thomas E; Weisman, Lois S

    2014-03-10

    Molecular motors transport organelles to specific subcellular locations. Upon arrival at their correct locations, motors release organelles via unknown mechanisms. The yeast myosin V, Myo2, binds the vacuole-specific adaptor Vac17 to transport the vacuole from the mother cell to the bud. Here, we show that vacuole detachment from Myo2 occurs in multiple regulated steps along the entire pathway of vacuole transport. Detachment initiates in the mother cell with the phosphorylation of Vac17 that recruits the E3 ligase Dma1 to the vacuole. However, Dma1 recruitment also requires the assembly of the vacuole transport complex and is first observed after the vacuole enters the bud. Dma1 remains on the vacuole until the bud and mother vacuoles separate. Subsequently, Dma1 targets Vac17 for proteasomal degradation. Notably, we find that the termination of peroxisome transport also requires Dma1. We predict that this is a general mechanism that detaches myosin V from select cargoes.

  10. Merlin's tumor suppression linked to inhibition of the E3 ubiquitin ligase CRL4DCAF1

    PubMed Central

    Li, Wei

    2010-01-01

    The mechanism by which the FERM domain protein Merlin, encoded by the tumor suppressor NF2, restrains cell proliferation is poorly understood. Prior studies have suggested that Merlin exerts its antimitogenic effect by interacting with multiple signaling proteins located at or near the plasma membrane. We have recently observed that Merlin translocates into the nucleus and binds to and inhibits the E3 ubiquitin ligase CRL4DCAF1. Genetic evidence indicates that inactivation of Merlin induces oncogenic gene expression, hyperproliferation, and tumorigenicity by unleashing the activity of CRL4DCAF1. In addition to providing a potential explanation for the diverse effects that loss of Merlin exerts in multiple cell types, these findings suggest that compounds inhibiting CRL4DCAF1 may display therapeutic efficacy in Neurofibromatosis type 2 and other cancers driven by Merlin inactivation. PMID:21084862

  11. Binding of Nickel to Testicular Glutamate–Ammonia Ligase Inhibits Its Enzymatic Activity

    PubMed Central

    SUN, YINGBIAO; OU, YOUNG; CHENG, MIN; RUAN, YIBING; VAN DER HOORN, FRANS A.

    2016-01-01

    SUMMARY Exposure to nickel has been shown to cause damage to the testis in several animal models. It is not known if the testis expresses protein(s) that can bind nickel. To test this, we used a nickel-binding assay to isolate testicular nickel-binding proteins. We identified glutamate–ammonia ligase (GLUL) as a prominent nickel-binding protein by mass spectrometry. Protein analysis and reverse transcriptase polymerase chain reaction showed that GLUL is expressed in the testis, predominantly in interstitial cells. We determined that GLUL has a higher affinity for nickel than for its regular co-factor manganese. We produced an enzymatically active, recombinant GLUL protein. Upon binding, nickel interferes with the manganese-catalyzed enzymatic activity of recombinant GLUL protein. We also determined that GLUL activity in testes of animals exposed to nickel sulfate is reduced. Our results identify testicular GLUL as the first testicular protein shown to be affected by nickel exposure. PMID:21254280

  12. Further analysis of XBAT32, an Arabidopsis RING E3 ligase, involved in ethylene biosynthesis.

    PubMed

    Prasad, Madhulika E; Stone, Sophia L

    2010-11-01

    The Arabidopsis RING E3 ligase, XBAT32, was previously characterized as a regulator of lateral root initiation. However, how XBAT32 function to modulate lateral root initiation was unknown. In our recent paper, we demonstrated that XBAT32 is involved in ethylene biosynthesis and it is through this function that XBAT32 is able to regulate lateral root production. Here we discuss a few other findings, observed in the ethylene overproducing mutant, xbat32, that reflect the effect of elevated ethylene levels on plant growth and development. Ethylene signaling also regulates plant responses to adverse environmental conditions such as high salinity. Consistent with ethylene's role as a stress hormone, xbat32 exhibited increased sensitivity to salt stress during seed germination and postgerminative growth. Thus, XBAT32 may also play a role in ethylene mediated response to abiotic stresses.

  13. The E3 ligase ube3a is required for learning in Drosophila melanogaster.

    PubMed

    Chakraborty, Moumita; Paul, Blesson K; Nayak, Tanmoyita; Das, Aniruddha; Jana, Nihar R; Bhutani, Supriya

    2015-06-19

    Angelman syndrome and autism are neurodevelopmental disorders linked to mutations and duplications of an E3 ligase called ube3a respectively. Since cognitive deficits and learning disabilities are hallmark symptoms of both these disorders, we investigated a role for dube3a in the learning ability of flies using the aversive phototaxis suppression assay. We show that down and up-regulation of dube3a are both detrimental to learning in larvae and adults. Using conditional gene expression we found that dube3a is required for normal brain development and during adulthood. Furthermore, we suggest that dube3a could be interacting with other learning and memory genes such as derailed. Along with firmly establishing dube3a as a gene that is required for learning, our work also opens avenues for further understanding the role played by this gene in brain development and behavior. PMID:25935478

  14. Single Quantum Dot Analysis Enables Multiplexed Point Mutation Detection by Gap Ligase Chain Reaction

    PubMed Central

    Song, Yunke; Zhang, Yi; Wang, Tza-Huei

    2014-01-01

    Gene point mutations present important biomarkers for genetic diseases. However, existing point mutation detection methods suffer from low sensitivity, specificity, and tedious assay processes. In this report, we propose an assay technology which combines the outstanding specificity of gap ligase chain reaction (Gap-LCR), the high sensitivity of single molecule coincidence detection and superior optical properties of quantum dots (QDs) for multiplexed detection of point mutations in genomic DNA. Mutant-specific ligation products are generated by Gap-LCR and subsequently captured by QDs to form DNA-QD nanocomplexes that are detected by single molecule spectroscopy (SMS) through multi-color fluorescence burst coincidence analysis, allowing for multiplexed mutation detection in a separation-free format. The proposed assay is capable of detecting zeptomoles of KRAS codon 12 mutation variants with near 100% specificity. Its high sensitivity allows direct detection of KRAS mutation in crude genomic DNA without PCR pre-amplification. PMID:23239594

  15. An immobilized biotin ligase: surface display of Escherichia coli BirA on Saccharomyces cerevisiae.

    PubMed

    Parthasarathy, Ranganath; Bajaj, Jitin; Boder, Eric T

    2005-01-01

    The Escherichia coli biotin ligase enzyme BirA has been extensively used in recent years to generate site-specifically biotinylated proteins via a biotin acceptor peptide tag. In the present study, BirA was displayed for the first time on the yeast Saccharomyces cerevisiae using the Aga1p-Aga2p platform and assayed using a peptide-tagged protein as the substrate. The enzyme is fully functional and resembles the soluble form in many of its properties, but the yeast-displayed enzyme demonstrates stability and reusability on the time scale of weeks. Thus, the yeast-displayed BirA system represents a facile and highly economical alternative for producing site-specifically biotinylated proteins.

  16. β-Cell Insulin Secretion Requires the Ubiquitin Ligase COP1.

    PubMed

    Suriben, Rowena; Kaihara, Kelly A; Paolino, Magdalena; Reichelt, Mike; Kummerfeld, Sarah K; Modrusan, Zora; Dugger, Debra L; Newton, Kim; Sagolla, Meredith; Webster, Joshua D; Liu, Jinfeng; Hebrok, Matthias; Dixit, Vishva M

    2015-12-01

    A variety of signals finely tune insulin secretion by pancreatic β cells to prevent both hyper-and hypoglycemic states. Here, we show that post-translational regulation of the transcription factors ETV1, ETV4, and ETV5 by the ubiquitin ligase COP1 (also called RFWD2) in β cells is critical for insulin secretion. Mice lacking COP1 in β cells developed diabetes due to insulin granule docking defects that were fully rescued by genetic deletion of Etv1, Etv4, and Etv5. Genes regulated by ETV1, ETV4, or ETV5 in the absence of mouse COP1 were enriched in human diabetes-associated genes, suggesting that they also influence human β-cell pathophysiology. In normal β cells, ETV4 was stabilized upon membrane depolarization and limited insulin secretion under hyperglycemic conditions. Collectively, our data reveal that ETVs negatively regulate insulin secretion for the maintenance of normoglycemia.

  17. Development of ligase-assisted spacer addition for the measurement of microsatellites.

    PubMed

    Brockhurst, V; Barnard, R; Wolter, L; Giffard, P; Timms, P

    2001-07-01

    Conventional methods for detecting differences in microsatellite repeat lengths rely on electrophoretic fractionation on long denaturing polyacrylamide gels, a time-consuming and labor-intensive method. Therefore, there is a need for the development of new and rapid approaches to routinely detect such length polymorphisms. The advent of techniques allowing the coupling of DNA molecules to solid surfaces has provided new prospects in the area of mutation detection. We describe here the development and optimization of the ligase-assisted spacer addition (LASA) method, a novel and rapid procedure based on an ELISA format to measure microsatellite repeat lengths. The LASA assay was successfully applied to a set of 11 bird samples to assess its capabilities as a genotyping method. PMID:11464526

  18. The E3 ligase Itch and deubiquitinase Cyld co-operatively regulate Tak1 and inflammation

    PubMed Central

    Ahmed, Neesar; Zeng, Minghui; Sinha, Indrajit; Polin, Lisa; Wei, Wei-Zen; Rathinam, Chozhavendan; Flavell, Richard; Massoumi, Ramin; Venuprasad, K

    2011-01-01

    Chronic inflammation has been strongly associated with tumor progression, but the underlying mechanisms remain elusive. Here we demonstrate that E3 ligase Itch and deubiquitinase Cyld form a complex via the interaction through ‘WW-PPXY’ motifs. The Itch-Cyld complex sequentially cleaved K63-linked ubiquitin chains and catalyzed K48-linked ubiquitination on the kinase Tak1 to terminate inflammatory tumor necrosis factor signaling. Reconstitution of wild-type Cyld but not mutant Cyld(Y485A), which cannot associate with Itch, blocked the sustained Tak1 activation and proinflammatory cytokine production by Cyld−/− bone marrow-derived macrophages. Itch or Cyld deficiency resulted in chronic production of tumor-promoting cytokines by the tumor-associated macrophages and aggressive growth of lung carcinoma. Thus, we have uncovered an Itch-Cyld mediated regulatory mechanism in innate inflammatory cells. PMID:22057290

  19. Identification of 3-sulfinopropionyl coenzyme A (CoA) desulfinases within the Acyl-CoA dehydrogenase superfamily.

    PubMed

    Schürmann, Marc; Demming, Rebecca Michaela; Krewing, Marco; Rose, Judith; Wübbeler, Jan Hendrik; Steinbüchel, Alexander

    2014-02-01

    In a previous study, the essential role of 3-sulfinopropionyl coenzyme A (3SP-CoA) desulfinase acyl-CoA dehydrogenase (Acd) in Advenella mimigardefordensis strain DPN7(T) (AcdDPN7) during degradation of 3,3'-dithiodipropionic acid (DTDP) was elucidated. DTDP is a sulfur-containing precursor substrate for biosynthesis of polythioesters (PTEs). AcdDPN7 showed high amino acid sequence similarity to acyl-CoA dehydrogenases but was unable to catalyze a dehydrogenation reaction. Hence, it was investigated in the present study whether 3SP-CoA desulfinase activity is an uncommon or a widespread property within the acyl-CoA dehydrogenase superfamily. Therefore, proteins of the acyl-CoA dehydrogenase superfamily from Advenella kashmirensis WT001, Bacillus cereus DSM31, Cupriavidus necator N-1, Escherichia coli BL21, Pseudomonas putida KT2440, Burkholderia xenovorans LB400, Ralstonia eutropha H16, Variovorax paradoxus B4, Variovorax paradoxus S110, and Variovorax paradoxus TBEA6 were expressed in E. coli strains. All purified acyl-CoA dehydrogenases appeared as homotetramers, as revealed by size exclusion chromatography. AcdS110, AcdB4, AcdH16, and AcdKT2440 were able to dehydrogenate isobutyryl-CoA. AcdKT2440 additionally dehydrogenated butyryl-CoA and valeryl-CoA, whereas AcdDSM31 dehydrogenated only butyryl-CoA and valeryl-CoA. No dehydrogenation reactions were observed with propionyl-CoA, isovaleryl-CoA, succinyl-CoA, and glutaryl-CoA for any of the investigated acyl-CoA dehydrogenases. Only AcdTBEA6, AcdN-1, and AcdLB400 desulfinated 3SP-CoA and were thus identified as 3SP-CoA desulfinases within the acyl-CoA dehydrogenase family, although none of these three Acds dehydrogenated any of the tested acyl-CoA thioesters. No appropriate substrates were identified for AcdBL21 and AcdWT001. Spectrophotometric assays provided apparent Km and Vmax values for active substrates and indicated the applicability of phylogenetic analyses to predict the substrate range of

  20. The Anaphase-Promoting Complex (APC) ubiquitin ligase affects chemosensory behavior in C. elegans.

    PubMed

    Wang, Julia; Jennings, Alexandra K; Kowalski, Jennifer R

    2016-01-01

    The regulation of fundamental aspects of neurobiological function has been linked to the ubiquitin signaling system (USS), which regulates the degradation and activity of proteins and is catalyzed by E1, E2, and E3 enzymes. The Anaphase-Promoting Complex (APC) is a multi-subunit E3 ubiquitin ligase that controls diverse developmental and signaling processes in post-mitotic neurons; however, potential roles for the APC in sensory function have yet to be explored. In this study, we examined the effect of the APC ubiquitin ligase on chemosensation in Caenorhabditis elegans by testing chemotaxis to the volatile odorants, diacetyl, pyrazine, and isoamyl alcohol, to which wild-type worms are attracted. Animals with loss of function mutations in either of two alleles (g48 and ye143) of the gene encoding the APC subunit EMB-27 APC6 showed increased chemotaxis towards diacetyl and pyrazine, odorants sensed by AWA neurons, but exhibited normal chemotaxis to isoamyl alcohol, which is sensed by AWC neurons. The statistically significant increase in chemotaxis in the emb-27 APC6 mutants suggests that the APC inhibits AWA-mediated chemosensation in C. elegans. Increased chemotaxis to pyrazine was also seen with mutants lacking another essential APC subunit, MAT-2 APC1; however, mat-2 APC1 mutants exhibited wild type responses to diacetyl. The difference in responsiveness of these two APC subunit mutants may be due to differential strength of these hypomorphic alleles or may indicate the presence of functional sub-complexes of the APC at work in this process. These findings are the first evidence for APC-mediated regulation of chemosensation and lay the groundwork for further studies aimed at identifying the expression levels, function, and targets of the APC in specific sensory neurons. Because of the similarity between human and C. elegans nervous systems, the role of the APC in sensory neurons may also advance our understanding of human sensory function and disease. PMID

  1. Enzymatic Analysis of PTEN Ubiquitylation by WWP2 and NEDD4-1 E3 Ligases.

    PubMed

    Chen, Zan; Thomas, Stefani N; Bolduc, David M; Jiang, Xuejun; Zhang, Xiangbin; Wolberger, Cynthia; Cole, Philip A

    2016-07-01

    PTEN is a lipid phosphatase that converts phosphatidylinositol 3,4,5-phosphate (PIP3) to phosphatidylinositol 4,5-phosphate (PIP2) and plays a critical role in the regulation of tumor growth. PTEN is subject to regulation by a variety of post-translational modifications, including phosphorylation on a C-terminal cluster of four Ser/Thr residues (380, 382, 383, and 385) and ubiquitylation by various E3 ligases, including NEDD4-1 and WWP2. It has previously been shown that C-terminal phosphorylation of PTEN can increase its cellular half-life. Using in vitro ubiquitin transfer assays, we show that WWP2 is more active than NEDD4-1 in ubiquitylating unphosphorylated PTEN. The mapping of ubiquitylation sites in PTEN by mass spectrometry showed that both NEDD4-1 and WWP2 can target a broad range of Lys residues in PTEN, although NEDD4-1 versus WWP2 showed a stronger preference for ubiquitylating PTEN's C2 domain. Whereas tetraphosphorylation of PTEN did not significantly affect its ubiquitylation by NEDD4-1, it inhibited PTEN ubiquitylation by WWP2. Single-turnover and pull-down experiments suggested that tetraphosphorylation of PTEN appears to weaken its interaction with WWP2. These studies reveal how the PTEN E3 ligases WWP2 and NEDD4-1 exhibit distinctive properties in Lys selectivity and sensitivity to PTEN phosphorylation. Our findings also provide a molecular mechanism for the connection between PTEN Ser/Thr phosphorylation and PTEN's cellular stability.

  2. An E3 ligase complex regulates SET-domain polycomb group protein activity in Arabidopsis thaliana

    PubMed Central

    Jeong, Cheol Woong; Roh, Hyungmin; Dang, Tuong Vi; Choi, Yang Do; Fischer, Robert L.; Lee, Jong Seob; Choi, Yeonhee

    2011-01-01

    Transcriptional repression via methylation of histone H3 lysine 27 (H3K27) by the polycomb repressive complex 2 (PRC2) is conserved in higher eukaryotes. The Arabidopsis PRC2 controls homeotic gene expression, flowering time, and gene imprinting. Although downstream target genes and the regulatory mechanism of PRC2 are well understood, much less is known about the significance of posttranslational regulation of PRC2 protein activity. Here, we show the posttranslational regulation of CURLY LEAF (CLF) SET-domain polycomb group (PcG) protein by the F-box protein, UPWARD CURLY LEAF1 (UCL1). Overexpression of UCL1 generates mutant phenotypes similar to those observed in plants with a loss-of-function mutation in the CLF gene. Leaf curling and early flowering phenotypes of UCL1 overexpression mutants, like clf mutants, are rescued by mutations in the AGAMOUS and FLOWERING LOCUS T genes, which is consistent with UCL1 and CLF functioning in the same genetic pathway. Overexpression of UCL1 reduces the level of CLF protein and alters expression and H3K27 methylation of CLF-target genes in transgenic plants, suggesting that UCL1 negatively regulates CLF. Interaction of UCL1 with CLF was detected in plant nuclei and in the yeast two-hybrid system. The UCL1 F-box binds in vivo to components of the E3 ligase complex, which ubiquitylate proteins that are subsequently degraded via the ubiquitin-26S proteasome pathway. Taken together, these results demonstrate the posttranslational regulation of the CLF SET-domain PcG activity by the UCL1 F-box protein in the E3 ligase complex. PMID:21518870

  3. Highly precise and developmentally programmed genome assembly in Paramecium requires ligase IV-dependent end joining.

    PubMed

    Kapusta, Aurélie; Matsuda, Atsushi; Marmignon, Antoine; Ku, Michael; Silve, Aude; Meyer, Eric; Forney, James D; Malinsky, Sophie; Bétermier, Mireille

    2011-04-01

    During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5' overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi-mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5'-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3' ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a "cut-and-close" mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms

  4. The Anaphase-Promoting Complex (APC) ubiquitin ligase affects chemosensory behavior in C. elegans

    PubMed Central

    Wang, Julia; Jennings, Alexandra K.

    2016-01-01

    The regulation of fundamental aspects of neurobiological function has been linked to the ubiquitin signaling system (USS), which regulates the degradation and activity of proteins and is catalyzed by E1, E2, and E3 enzymes. The Anaphase-Promoting Complex (APC) is a multi-subunit E3 ubiquitin ligase that controls diverse developmental and signaling processes in post-mitotic neurons; however, potential roles for the APC in sensory function have yet to be explored. In this study, we examined the effect of the APC ubiquitin ligase on chemosensation in Caenorhabditis elegans by testing chemotaxis to the volatile odorants, diacetyl, pyrazine, and isoamyl alcohol, to which wild-type worms are attracted. Animals with loss of function mutations in either of two alleles (g48 and ye143) of the gene encoding the APC subunit EMB-27 APC6 showed increased chemotaxis towards diacetyl and pyrazine, odorants sensed by AWA neurons, but exhibited normal chemotaxis to isoamyl alcohol, which is sensed by AWC neurons. The statistically significant increase in chemotaxis in the emb-27 APC6 mutants suggests that the APC inhibits AWA-mediated chemosensation in C. elegans. Increased chemotaxis to pyrazine was also seen with mutants lacking another essential APC subunit, MAT-2 APC1; however, mat-2 APC1 mutants exhibited wild type responses to diacetyl. The difference in responsiveness of these two APC subunit mutants may be due to differential strength of these hypomorphic alleles or may indicate the presence of functional sub-complexes of the APC at work in this process. These findings are the first evidence for APC-mediated regulation of chemosensation and lay the groundwork for further studies aimed at identifying the expression levels, function, and targets of the APC in specific sensory neurons. Because of the similarity between human and C. elegans nervous systems, the role of the APC in sensory neurons may also advance our understanding of human sensory function and disease. PMID

  5. SGR9, a RING type E3 ligase, modulates amyloplast dynamics important for gravity sensing.

    NASA Astrophysics Data System (ADS)

    Morita, Miyo T.; Nakamura, Moritaka; Tasaka, Masao

    Gravitropism is triggered when the directional change of gravity is sensed in the specific cells, called statocytes. In higher plants, statocytes contain sinking heavier amyloplasts which are particular plastids accumulating starch granules. The displacement of amyloplasts within the statocytes is thought to be the initial event of gravity perception. We have demonstrated that endodermal cells are most likely to be the statocytes in Arabidop-sis shoots. Live cell imaging of the endodermal cell of stem has shown that most amyloplasts are sediment to the direction of gravity but they are not static. Several amyloplasts move dynamically in an actin filament (F-actin) dependent manner. In the presence of actin poly-merization inhibitor, all amyloplasts become static and sediment to the direction of gravity. In addition, stems treated with the inhibitor can exhibit gravitropism. These results suggest that F-actin-dependent dynamic movement of amyloplasts is not essential for gravity sensing. sgr (shoot gravitropism) 9 mutant exhibits greatly reduced shoot gravitropism. In endodermal cells of sgr9, dynamic amyloplast movement was predominantly observed and amyloplasts did not sediment to the direction of gravity. Interestingly, inhibition of actin polymerization re-stored both gravitropism and amyloplast sedimentation in sgr9. The SGR9 encodes a novel RING finger protein, which is localized to amyloplasts in endodermal cells. SGR9 showed ubiq-uitin E3 ligase activity in vitro. Together with live cell imaging of amyloplasts and F-actin, our data suggest that SGR9 modulate interaction between amyloplasts and F-actin on amylo-plasts. SGR9 positively act on amyloplasts sedimentation, probably by releasing amyloplasts from F-actin. SGR9 that is localized to amyloplast, possibly degrades unknown substrates by its E3 ligase activity, and this might promote release of amyloplasts from F-actin.

  6. Mutations in CUL4B, which encodes a ubiquitin E3 ligase subunit, cause an X-linked mental retardation syndrome associated with aggressive outbursts, seizures, relative macrocephaly, central obesity, hypogonadism, pes cavus, and tremor.

    PubMed

    Tarpey, Patrick S; Raymond, F Lucy; O'Meara, Sarah; Edkins, Sarah; Teague, Jon; Butler, Adam; Dicks, Ed; Stevens, Claire; Tofts, Calli; Avis, Tim; Barthorpe, Syd; Buck, Gemma; Cole, Jennifer; Gray, Kristian; Halliday, Kelly; Harrison, Rachel; Hills, Katy; Jenkinson, Andrew; Jones, David; Menzies, Andrew; Mironenko, Tatiana; Perry, Janet; Raine, Keiran; Richardson, David; Shepherd, Rebecca; Small, Alexandra; Varian, Jennifer; West, Sofie; Widaa, Sara; Mallya, Uma; Moon, Jenny; Luo, Ying; Holder, Susan; Smithson, Sarah F; Hurst, Jane A; Clayton-Smith, Jill; Kerr, Bronwyn; Boyle, Jackie; Shaw, Marie; Vandeleur, Lucianne; Rodriguez, Jayson; Slaugh, Rachel; Easton, Douglas F; Wooster, Richard; Bobrow, Martin; Srivastava, Anand K; Stevenson, Roger E; Schwartz, Charles E; Turner, Gillian; Gecz, Jozef; Futreal, P Andrew; Stratton, Michael R; Partington, Michael

    2007-02-01

    We have identified three truncating, two splice-site, and three missense variants at conserved amino acids in the CUL4B gene on Xq24 in 8 of 250 families with X-linked mental retardation (XLMR). During affected subjects' adolescence, a syndrome emerged with delayed puberty, hypogonadism, relative macrocephaly, moderate short stature, central obesity, unprovoked aggressive outbursts, fine intention tremor, pes cavus, and abnormalities of the toes. This syndrome was first described by Cazebas et al., in a family that was included in our study and that carried a CUL4B missense variant. CUL4B is a ubiquitin E3 ligase subunit implicated in the regulation of several biological processes, and CUL4B is the first XLMR gene that encodes an E3 ubiquitin ligase. The relatively high frequency of CUL4B mutations in this series indicates that it is one of the most commonly mutated genes underlying XLMR and suggests that its introduction into clinical diagnostics should be a high priority. PMID:17236139

  7. Real-time monitoring of nucleic acid ligation in homogenous solutions using molecular beacons.

    PubMed

    Tang, Zhiwen; Wang, Kemin; Tan, Weihong; Li, Jun; Liu, Lingfeng; Guo, Qiuping; Meng, Xiangxian; Ma, Changbei; Huang, Shasheng

    2003-12-01

    Nucleic acids ligation is a vital process in the repair, replication and recombination of nucleic acids. Traditionally, it is assayed by denatured gel electrophoresis and autoradiography, which are not sensitive, and are complex and discontinuous. Here we report a new approach for ligation monitoring using molecular beacon DNA probes. The molecular beacon, designed in such a way that its sequence is complementary with the product of the ligation process, is used to monitor the nucleic acid ligation in a homogeneous solution and in real-time. Our method is fast and simple. We are able to study nucleic acids ligation kinetics conveniently and to determine the activity of DNA ligase accurately. We have studied different factors that influence DNA ligation catalyzed by T4 DNA ligase. The major advantages of our method are its ultrasensitivity, excellent specificity, convenience and real-time monitoring in homogeneous solution. This method will be widely useful for studying nucleic acids ligation process and other nucleic acid interactions.

  8. The Functions of the HIV1 protein Vpr and its action through the DCAF1•DDB1•Cullin4 ubiquitin ligase

    PubMed Central

    Casey, Laurieann; Wen, Xiaoyun; de Noronha, Carlos M.C.

    2010-01-01

    Among the proteins encoded by human and simian immunodeficiency viruses (HIV and SIV) at least three, Vif, Vpu and Vpr, subvert cellular ubiquitin ligases to block the action of anti-viral defenses. This review focuses on Vpr and its HIV2/SIV counterparts, Vpx and Vpr, which all engage the DDB1•Cullin4 ubiquitin ligase complex through the DCAF1 adaptor protein. Here, we discuss the multiple functions that have been linked to Vpr expression and summarize the current knowledge on the role of the ubiquitin ligase complex in carrying out a subset of these activities. PMID:20347598

  9. Type 1 diacylglycerol acyltransferases of Brassica napus preferentially incorporate oleic acid into triacylglycerol

    PubMed Central

    Aznar-Moreno, Jose; Denolf, Peter; Van Audenhove, Katrien; De Bodt, Stefanie; Engelen, Steven; Fahy, Deirdre; Wallis, James G.; Browse, John

    2015-01-01

    DGAT1 enzymes (acyl-CoA:diacylglycerol acyltransferase 1, EC 2.3.1.20) catalyse the formation of triacylglycerols (TAGs), the most abundant lipids in vegetable oils. Thorough understanding of the enzymology of oil accumulation is critical to the goal of modifying oilseeds for improved vegetable oil production. Four isoforms of BnDGAT1, the final and rate-limiting step in triacylglycerol synthesis, were characterized from Brassica napus, one of the world’s most important oilseed crops. Transcriptional profiling of developing B. napus seeds indicated two genes, BnDGAT1-1 and BnDGAT1-2, with high expression and two, BnDGAT1-3 and BnDGAT1-4, with low expression. The activities of each BnDGAT1 isozyme were characterized following expression in a strain of yeast deficient in TAG synthesis. TAG from B. napus seeds contain only 10% palmitic acid (16:0) at the sn-3 position, so it was surprising that all four BnDGAT1 isozymes exhibited strong (4- to 7-fold) specificity for 16:0 over oleic acid (18:1) as the acyl-CoA substrate. However, the ratio of 18:1-CoA to 16:0-CoA in B. napus seeds during the peak period of TAG synthesis is 3:1. When substrate selectivity assays were conducted with 18:1-CoA and 16:0-CoA in a 3:1 ratio, the four isozymes incorporated 18:1 in amounts 2- to 5-fold higher than 16:0. This strong sensitivity of the BnDGAT1 isozymes to the relative concentrations of acyl-CoA substrates substantially explains the observed fatty acid composition of B. napus seed oil. Understanding these enzymes that are critical for triacylglycerol synthesis will facilitate genetic and biotechnological manipulations to improve this oilseed crop. PMID:26195728

  10. Type 1 diacylglycerol acyltransferases of Brassica napus preferentially incorporate oleic acid into triacylglycerol.

    PubMed

    Aznar-Moreno, Jose; Denolf, Peter; Van Audenhove, Katrien; De Bodt, Stefanie; Engelen, Steven; Fahy, Deirdre; Wallis, James G; Browse, John

    2015-10-01

    DGAT1 enzymes (acyl-CoA:diacylglycerol acyltransferase 1, EC 2.3.1.20) catalyse the formation of triacylglycerols (TAGs), the most abundant lipids in vegetable oils. Thorough understanding of the enzymology of oil accumulation is critical to the goal of modifying oilseeds for improved vegetable oil production. Four isoforms of BnDGAT1, the final and rate-limiting step in triacylglycerol synthesis, were characterized from Brassica napus, one of the world's most important oilseed crops. Transcriptional profiling of developing B. napus seeds indicated two genes, BnDGAT1-1 and BnDGAT1-2, with high expression and two, BnDGAT1-3 and BnDGAT1-4, with low expression. The activities of each BnDGAT1 isozyme were characterized following expression in a strain of yeast deficient in TAG synthesis. TAG from B. napus seeds contain only 10% palmitic acid (16:0) at the sn-3 position, so it was surprising that all four BnDGAT1 isozymes exhibited strong (4- to 7-fold) specificity for 16:0 over oleic acid (18:1) as the acyl-CoA substrate. However, the ratio of 18:1-CoA to 16:0-CoA in B. napus seeds during the peak period of TAG synthesis is 3:1. When substrate selectivity assays were conducted with 18:1-CoA and 16:0-CoA in a 3:1 ratio, the four isozymes incorporated 18:1 in amounts 2- to 5-fold higher than 16:0. This strong sensitivity of the BnDGAT1 isozymes to the relative concentrations of acyl-CoA substrates substantially explains the observed fatty acid composition of B. napus seed oil. Understanding these enzymes that are critical for triacylglycerol synthesis will facilitate genetic and biotechnological manipulations to improve this oilseed crop.

  11. Underlying Resistance Mechanisms in the Cynosurus echinatus Biotype to Acetyl CoA Carboxylase-Inhibiting Herbicides.

    PubMed

    Fernández, Pablo; Alcántara-de la Cruz, Ricardo; Cruz-Hipólito, Hugo; Osuna, María D; De Prado, Rafael

    2016-01-01

    Hedgehog dogtail (Cynosurus echinatus) is an annual grass, native to Europe, but also widely distributed in North and South America, South Africa, and Australia. Two hedgehog dogtail biotypes, one diclofop-methyl (DM)-resistant and one DM-susceptible were studied in detail for experimental dose-response resistance mechanisms. Herbicide rates that inhibited shoot growth by 50% (GR50) were determined for DM, being the resistance factor (GR50R/GR50S) of 43.81. When amitrole (Cyt. P450 inhibitor) was applied before treatment with DM, the R biotype growth was significantly inhibited (GR50 of 1019.9 g ai ha(-1)) compared with the GR50 (1484.6 g ai ha(-1)) found for the R biotype without pretreatment with amitrole. However, GR50 values for S biotype do not vary with or without amitrole pretreatment. Dose-response experiments carried out to evaluate cross-resistance, showed resistance to aryloxyphenoxypropionate (APP), cyclohexanedione (CHD) and phenylpyrazoline (PPZ) inhibiting herbicides. Both R and S biotypes had a similar (14)C-DM uptake and translocation. The herbicide was poorly distributed among leaves, the rest of the shoot and roots with unappreciable acropetal and/or basipetal DM translocation at 96 h after treatment (HAT). The metabolism of (14)C-DM, D-acid and D-conjugate metabolites were identified by thin-layer chromatography. The results showed that DM resistance in C. echinatus is likely due to enhanced herbicide metabolism, involving Cyt. P450 as was demonstrated by indirect assays (amitrole pretreatment). The ACCase in vitro assays showed that the target site was very sensitive to APP, CHD and PPZ herbicides in the C. echinatus S biotype, while the R biotype was insensitive to the previously mentioned herbicides. DNA sequencing studies confirmed that C. echinatus cross-resistance to ACCase inhibitors has been conferred by specific ACCase double point mutations Ile-2041-Asn and Cys-2088-Arg. PMID:27148285

  12. Underlying Resistance Mechanisms in the Cynosurus echinatus Biotype to Acetyl CoA Carboxylase-Inhibiting Herbicides

    PubMed Central

    Fernández, Pablo; Alcántara-de la Cruz, Ricardo; Cruz-Hipólito, Hugo; Osuna, María D.; De Prado, Rafael

    2016-01-01

    Hedgehog dogtail (Cynosurus echinatus) is an annual grass, native to Europe, but also widely distributed in North and South America, South Africa, and Australia. Two hedgehog dogtail biotypes, one diclofop-methyl (DM)-resistant and one DM-susceptible were studied in detail for experimental dose-response resistance mechanisms. Herbicide rates that inhibited shoot growth by 50% (GR50) were determined for DM, being the resistance factor (GR50R/GR50S) of 43.81. When amitrole (Cyt. P450 inhibitor) was applied before treatment with DM, the R biotype growth was significantly inhibited (GR50 of 1019.9 g ai ha-1) compared with the GR50 (1484.6 g ai ha-1) found for the R biotype without pretreatment with amitrole. However, GR50 values for S biotype do not vary with or without amitrole pretreatment. Dose-response experiments carried out to evaluate cross-resistance, showed resistance to aryloxyphenoxypropionate (APP), cyclohexanedione (CHD) and phenylpyrazoline (PPZ) inhibiting herbicides. Both R and S biotypes had a similar 14C-DM uptake and translocation. The herbicide was poorly distributed among leaves, the rest of the shoot and roots with unappreciable acropetal and/or basipetal DM translocation at 96 h after treatment (HAT). The metabolism of 14C-DM, D-acid and D-conjugate metabolites were identified by thin-layer chromatography. The results showed that DM resistance in C. echinatus is likely due to enhanced herbicide metabolism, involving Cyt. P450 as was demonstrated by indirect assays (amitrole pretreatment). The ACCase in vitro assays showed that the target site was very sensitive to APP, CHD and PPZ herbicides in the C. echinatus S biotype, while the R biotype was insensitive to the previously mentioned herbicides. DNA sequencing studies confirmed that C. echinatus cross-resistance to ACCase inhibitors has been conferred by specific ACCase double point mutations Ile-2041-Asn and Cys-2088-Arg. PMID:27148285

  13. E3 SUMO ligase AtSIZ1 positively regulates SLY1-mediated GA signalling and plant development.

    PubMed

    Kim, Sung-Il; Park, Bong Soo; Kim, Do Youn; Yeu, Song Yion; Song, Sang Ik; Song, Jong Tae; Seo, Hak Soo

    2015-07-15

    Gibberellins affect various plant development processes including germination, cell division and elongation, and flowering. A large number of studies have been carried out to address the molecular mechanisms that mediate gibberellin signalling effects on plant growth. However, such studies have been limited to DELLA protein degradation; the regulatory mechanisms controlling how the stability and function of SLEEPY1 (SLY1), a protein that interacts with target DELLA proteins as components of the Skp, Cullin, F-box (SCF)(SLY1) complex, are modulated at the post-translational level have not been addressed. In the present study, we show that the E3 SUMO (small ubiquitin-related modifier) ligase AtSIZ1 regulates gibberellic acid signalling in Arabidopsis species by sumoylating SLY1. SLY1 was less abundant in siz1-2 mutants than in wild-type plants, but the DELLA protein repressor of ga1-3 (RGA) was more abundant in siz1-2 mutants than in wild-type plants. SLY1 also accumulated to a high level in the SUMO protease mutant esd4. Transgenic sly1-13 mutants over-expressing SLY1 were phenotypically similar to wild-type plants; however, sly1-13 plants over-expressing a mutated mSLY1 protein (K122R, a mutation at the sumoylation site) retained the mutant dwarfing phenotype. Over-expression of SLY1 in sly1-13 mutants resulted in a return of RGA levels to wild-type levels, but RGA accumulated to high levels in mutants over-expressing mSLY1. RGA was clearly detected in Arabidopsis co-expressing AtSIZ1 and mSLY1, but not in plants co-expressing AtSIZ1 and SLY1. In addition, sumoylated SLY1 interacted with RGA and SLY1 sumoylation was significantly increased by GA. Taken together, our results indicate that, in Arabidopsis, AtSIZ1 positively controls GA signalling through SLY1 sumoylation.

  14. Reverse genetic characterization of two paralogous acetoacetyl CoA thiolase genes in Arabidopsis reveals their importance in plant growth and development

    SciTech Connect

    Jin, Huanan; Song, Zhihong; Nikolau, Basil J.

    2012-03-31

    Acetoacetyl CoA thiolase (AACT, EC 2.3.1.9) catalyzes the condensation of two acetyl CoA molecules to form acetoacetyl CoA. Two AACT‐encoding genes, At5g47720 (AACT1) and At5g48230 (AACT2), were functionally identified in the Arabidopsis genome by direct enzymological assays and functional expression in yeast. Promoter::GUS fusion experiments indicated that AACT1 is primarily expressed in the vascular system and AACT2 is highly expressed in root tips, young leaves, top stems and anthers. Characterization of T‐DNA insertion mutant alleles at each AACT locus established that AACT2 function is required for embryogenesis and for normal male gamete transmission. In contrast, plants lacking AACT1 function are completely viable and show no apparent growth phenotypes, indicating that AACT1 is functionally redundant with respect to AACT2 function. RNAi lines that express reduced levels of AACT2 show pleiotropic phenotypes, including reduced apical dominance, elongated life span and flowering duration, sterility, dwarfing, reduced seed yield and shorter root length. Microscopic analysis reveals that the reduced stature is caused by a reduction in cell size and fewer cells, and male sterility is caused by loss of the pollen coat and premature degeneration of the tapetal cells. Biochemical analyses established that the roots of AACT2 RNAi plants show quantitative and qualitative alterations in phytosterol profiles. These phenotypes and biochemical alterations are reversed when AACT2 RNAi plants are grown in the presence of mevalonate, which is consistent with the role of AACT2 in generating the bulk of the acetoacetyl CoA precursor required for the cytosol‐localized, mevalonate‐derived isoprenoid biosynthetic pathway.

  15. Lysine 63-Linked TANK-Binding Kinase 1 Ubiquitination by Mindbomb E3 Ubiquitin Protein Ligase 2 Is Mediated by the Mitochondrial Antiviral Signaling Protein

    PubMed Central

    Ye, Jung Sook; Kim, Nari; Lee, Kyoung Jin; Nam, Young Ran; Lee, Uk

    2014-01-01

    ABSTRACT Beta interferon (IFN-β) is involved in a wide range of cellular functions, and its secretion must be tightly controlled to inhibit viral spreading while minimizing cellular damage. Intracellular viral replication triggers cellular signaling cascades leading to the activation of the transcription factors NF-κB and interferon regulatory factor 3 (IRF3) and IRF7 (IRF3/7), which synergistically bind to the IFN-β gene promoter to induce its expression. The mitochondrial antiviral signaling protein (MAVS) is a governing adaptor protein that mediates signaling communications between virus-sensing proteins and transcription factors. The activity of MAVS in the regulation of IFN-β secretion is affected by many cellular factors. However, the mechanism of MAVS-mediated IRF3/7 activation is not completely understood. Here, we identified a highly conserved DLAIS motif at amino acid positions 438 to 442 of MAVS that is indispensable for IRF3/7 activation. Specifically, the L439S and A440R mutations suppress IRF3/7 activation. Pulldown experiments using wild-type and mutant MAVS showed that mindbomb E3 ubiquitin protein ligase 2 (MIB2) binds to the DLAIS motif. Furthermore, the DLAIS motif was found to be critical for MIB2 binding, the ligation of K63-linked ubiquitin to TANK-binding kinase 1, and phosphorylation-mediated IRF3/7 activation. Our results suggest that MIB2 plays a putative role in MAVS-mediated interferon signaling. IMPORTANCE Mitochondrial antiviral signaling protein (MAVS) mediates signaling from virus-sensing proteins to transcription factors for the induction of beta interferon. However, the mechanism underlying activation of MAVS-mediated interferon regulatory factors 3 and 7 (IRF3/7) is not completely understood. We found a highly conserved DLAIS motif in MAVS that is indispensable for IRF3/7 activation through TANK-binding kinase 1 (TBK1) and identified it as the binding site for mindbomb E3 ubiquitin protein ligase 2 (MIB2). The mutations that

  16. Proteomic discovery of MNT as a novel interacting partner of E3 ubiquitin ligase E6AP and a key mediator of myeloid differentiation

    PubMed Central

    Kapoor, Isha; Kanaujiya, Jitendra; Kumar, Yogesh; Thota, Jagadeshwar Reddy; Bhatt, Madan L.B.; Chattopadhyay, Naibedya; Sanyal, Sabyasachi; Trivedi, Arun Kumar

    2016-01-01

    Perturbed stability of regulatory proteins is a major cause of transformations leading to cancer, including several leukemia subtypes. Here, for the first time we demonstrate that E6-associated protein (E6AP), an E3 ubiquitin ligase negatively targets MAX binding protein MNT for ubiquitin-mediated proteasome degradation and impedes ATRA mediated myeloid cell differentiation. MNT is a member of the Myc/Max/Mad network of transcription factor that regulates cell proliferation, differentiation, cellular transformation and tumorigenesis. Wild-type E6AP promoted proteasome dependent degradation of MNT, while catalytically inactive E6AP having cysteine replaced with alanine at amino-acid 843 position (E6APC843A) rather stabilized it. Further, these proteins physically associated with each other both in non-myeloid (HEK293T) and myeloid cells. MNT overexpression induced G0-G1 growth arrest and promoted myeloid differentiation while its knockdown mitigated even ATRA induced differentiation suggesting MNT to be crucial for myeloid differentiation. We further showed that ATRA inhibited E6AP and stabilized MNT expression by protecting it from E6AP mediated ubiquitin-proteasome degradation. Notably, E6AP knockdown in HL60 cells restored MNT expression and promoted myeloid differentiation. Taken together, our data demonstrated that E6AP negatively regulates granulocytic differentiation by targeting MNT for degradation which is required for growth arrest and subsequent myeloid differentiation by various differentiation inducing agents. PMID:26506232

  17. The autoantigen Ro52 is an E3 ligase resident in the cytoplasm but enters the nucleus upon cellular exposure to nitric oxide

    SciTech Connect

    Espinosa, Alexander; Oke, Vilija; Elfving, Ase; Nyberg, Filippa; Covacu, Ruxandra; Wahren-Herlenius, Marie

    2008-12-10

    Patients with the systemic autoimmune diseases Sjoegrens's syndrome and systemic lupus erythematosus often have autoantibodies against the intracellular protein Ro52. Ro52 is an E3 ligase dependent on the ubiquitin conjugation enzymes UBE2D1 and UBE2E1. While Ro52 and UBE2D1 are cytoplasmic proteins, UBE2E1 is localized to the nucleus. Here, we investigate how domains of human Ro52 regulate its intracellular localization. By expressing fluorescently labeled Ro52 and Ro52 mutants in HeLa cells, an intact coiled-coil domain was found to be necessary for the cytoplasmic localization of Ro52. The amino acids 381-470 of the B30.2 region were essential for translocation into the nucleus. Furthermore, after exposure of HeLa cells to the inflammatory mediator nitric oxide (NO), Ro52 translocated to the nucleus. A nuclear localization of Ro52 in inflamed tissue expressing inducible NO synthetase (iNOS) from cutaneous lupus patients was observed by immunohistochemistry and verified in NO-treated cultures of patient-derived primary keratinocytes. Our results show that the localization of Ro52 is regulated by endogenous sequences, and that nuclear translocation is induced by an inflammatory mediator. This suggests that Ro52 has both cytoplasmic and nuclear substrates, and that Ro52 mediates ubiquitination through UBE2D1 in the cytoplasm and through UBE2E1 in the nucleus.

  18. Rice root curling, a response to mechanosensing, is modulated by the rice E3-ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 (OsHOS1).

    PubMed

    Lourenço, T F; Serra, T S; Cordeiro, A M; Swanson, S J; Gilroy, S; Saibo, N J M; Oliveira, M M

    2016-08-01

    Plant development depends on the perception of external cues, such as light, gravity, touch, wind or nutrients, among others. Nevertheless, little is known regarding signal transduction pathways integrating these stimuli. Recently, we have reported the involvement of a rice E3-ubiquitin ligase (OsHOS1, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1), previously associated with abiotic stress response, in root responses to mechanical stimuli. We showed that OsHOS1 is involved in the regulation of root curling after mechanosensing and that RNAi::OsHOS1 plants failed to exhibit the root curling phenotype observed in WT. Interestingly, the straight root phenotype of these transgenics correlated with the up-regulation of rice ROOT MEANDER CURLING (OsRMC, a negative regulator of rice root curling) and was reverted by the exogenous application of jasmonic acid. Altogether, our results highlight the role of the proteasome modulating plant responses to mechanical stimuli and suggest that OsHOS1 is a hub integrating environmental and hormonal signaling into plant growth and development. PMID:27467198

  19. The RING finger ubiquitin E3 ligase SDIR1 targets SDIR1-INTERACTING PROTEIN1 for degradation to modulate the salt stress response and ABA signaling in Arabidopsis.

    PubMed

    Zhang, Huawei; Cui, Feng; Wu, Yaorong; Lou, Lijuan; Liu, Lijing; Tian, Miaomiao; Ning, Yuese; Shu, Kai; Tang, Sanyuan; Xie, Qi

    2015-01-01

    The plant hormone abscisic acid (ABA) regulates many aspects of plant development and the stress response. The intracellular E3 ligase SDIR1 (SALT- AND DROUGHT-INDUCED REALLY INTERESTING NEW GENE FINGER1) plays a key role in ABA signaling, regulating ABA-related seed germination and the stress response. In this study, we found that SDIR1 is localized on the endoplasmic reticulum membrane in Arabidopsis thaliana. Using cell biology, molecular biology, and biochemistry approaches, we demonstrated that SDIR1 interacts with and ubiquitinates its substrate, SDIRIP1 (SDIR1-INTERACTING PROTEIN1), to modulate SDIRIP1 stability through the 26S proteasome pathway. SDIRIP1 acts genetically downstream of SDIR1 in ABA and salt stress signaling. In detail, SDIRIP1 selectively regulates the expression of the downstream basic region/leucine zipper motif transcription factor gene ABA-INSENSITIVE5, rather than ABA-RESPONSIVE ELEMENTS BINDING FACTOR3 (ABF3) or ABF4, to regulate ABA-mediated seed germination and the plant salt response. Overall, the SDIR1/SDIRIP1 complex plays a vital role in ABA signaling through the ubiquitination pathway. PMID:25616872

  20. The RING Finger Ubiquitin E3 Ligase SDIR1 Targets SDIR1-INTERACTING PROTEIN1 for Degradation to Modulate the Salt Stress Response and ABA Signaling in Arabidopsis

    PubMed Central

    Zhang, Huawei; Cui, Feng; Wu, Yaorong; Lou, Lijuan; Liu, Lijing; Tian, Miaomiao; Ning, Yuese; Shu, Kai; Tang, Sanyuan; Xie, Qi

    2015-01-01

    The plant hormone abscisic acid (ABA) regulates many aspects of plant development and the stress response. The intracellular E3 ligase SDIR1 (SALT- AND DROUGHT-INDUCED REALLY INTERESTING NEW GENE FINGER1) plays a key role in ABA signaling, regulating ABA-related seed germination and the stress response. In this study, we found that SDIR1 is localized on the endoplasmic reticulum membrane in Arabidopsis thaliana. Using cell biology, molecular biology, and biochemistry approaches, we demonstrated that SDIR1 interacts with and ubiquitinates its substrate, SDIRIP1 (SDIR1-INTERACTING PROTEIN1), to modulate SDIRIP1 stability through the 26S proteasome pathway. SDIRIP1 acts genetically downstream of SDIR1 in ABA and salt stress signaling. In detail, SDIRIP1 selectively regulates the expression of the downstream basic region/leucine zipper motif transcription factor gene ABA-INSENSITIVE5, rather than ABA-RESPONSIVE ELEMENTS BINDING FACTOR3 (ABF3) or ABF4, to regulate ABA-mediated seed germination and the plant salt response. Overall, the SDIR1/SDIRIP1 complex plays a vital role in ABA signaling through the ubiquitination pathway. PMID:25616872

  1. In vitro construction of bacteriophage lambda carrying segments of the Escherichia coli chromosome: selection of hybrids containing the gene for DNA ligase.

    PubMed

    Cameron, J R; Panasenko, S M; Lehman, I R; Davis, R W

    1975-09-01

    DNA from lambdagt-lambdaB bacteriophage was cleaved with EcoRI endonuclease and fragments from EcoRI-digested E. coli DNA were inserted. This DNA was used to infect E. coli, and phages containing the gene for DNA ligase were isolated by genetic selection. Two different hybrids were found with the same E. coli segment inserted in opposite orientations. Both hybrids produced similar levels of ligase as measured in crude extracts of infected cells.

  2. In vitro construction of bacteriophage lambda carrying segments of the Escherichia coli chromosome: selection of hybrids containing the gene for DNA ligase.

    PubMed Central

    Cameron, J R; Panasenko, S M; Lehman, I R; Davis, R W

    1975-01-01

    DNA from lambdagt-lambdaB bacteriophage was cleaved with EcoRI endonuclease and fragments from EcoRI-digested E. coli DNA were inserted. This DNA was used to infect E. coli, and phages containing the gene for DNA ligase were isolated by genetic selection. Two different hybrids were found with the same E. coli segment inserted in opposite orientations. Both hybrids produced similar levels of ligase as measured in crude extracts of infected cells. Images PMID:1103146

  3. Negishi cross-coupling enabled synthesis of novel NAD(+)-dependent DNA ligase inhibitors and SAR development.

    PubMed

    Murphy-Benenato, Kerry E; Gingipalli, Lakshmaiah; Boriack-Sjodin, P Ann; Martinez-Botella, Gabriel; Carcanague, Dan; Eyermann, Charles J; Gowravaram, Madhu; Harang, Jenna; Hale, Michael R; Ioannidis, Georgine; Jahic, Harris; Johnstone, Michele; Kutschke, Amy; Laganas, Valerie A; Loch, James T; Miller, Matthew D; Oguto, Herbert; Patel, Sahil Joe

    2015-11-15

    Two novel compounds, pyridopyrimidines (1) and naphthyridines (2) were identified as potent inhibitors of bacterial NAD(+)-dependent DNA ligase (Lig) A in a fragment screening. SAR was guided by molecular modeling and X-ray crystallography. It was observed that the diaminonitrile pharmacophore made a key interaction with the ligase enzyme, specifically residues Glu114, Lys291, and Leu117. Synthetic challenges limited opportunities for diversification of the naphthyridine core, therefore most of the SAR was focused on a pyridopyrimidine scaffold. The initial diversification at R(1) improved both enzyme and cell potency. Further SAR developed at the R(2) position using the Negishi cross-coupling reaction provided several compounds, among these compounds 22g showed good enzyme potency and cellular potency.

  4. A tail of two sites: a bipartite mechanism for recognition of notch ligands by mind bomb E3 ligases.

    PubMed

    McMillan, Brian J; Schnute, Björn; Ohlenhard, Nadja; Zimmerman, Brandon; Miles, Laura; Beglova, Natalia; Klein, Thomas; Blacklow, Stephen C

    2015-03-01

    Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. We present here crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular, and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membrane proximal region and the other near the C terminus. Together, these studies provide insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential target for therapeutic modulation of Notch signal transduction in disease.

  5. The highly conserved orthopoxvirus 68k ankyrin-like protein is part of a cellular SCF ubiquitin ligase complex.

    PubMed

    Sperling, Karin M; Schwantes, Astrid; Schnierle, Barbara S; Sutter, Gerd

    2008-05-10

    The 68k ankyrin-like protein (68k-ank) of unknown function is highly conserved among orthopoxviruses and contains ankyrin repeats and an F-box-like domain. We performed a yeast-two-hybrid screen with 68k-ank to find interacting proteins. From a human and a murine cDNA library, 99% of the interaction partners were S-phase kinase-associated protein 1a (Skp1a), a part of the SCF ubiquitin ligase complex. 68k-ank co-immunoprecipitated with components of the endogenous, mammalian SCF ubiquitin ligase. This interaction was F-box domain dependent and could also be observed in infected cells, indicating that SCF complex formation might be important for the viral life cycle.

  6. The tumour antigen PRAME is a subunit of a Cul2 ubiquitin ligase and associates with active NFY promoters

    PubMed Central

    Costessi, Adalberto; Mahrour, Nawel; Tijchon, Esther; Stunnenberg, Rieka; Stoel, Marieke A; Jansen, Pascal W; Sela, Dotan; Martin-Brown, Skylar; Washburn, Michael P; Florens, Laurence; Conaway, Joan W; Conaway, Ronald C; Stunnenberg, Hendrik G

    2011-01-01

    The human tumour antigen PRAME (preferentially expressed antigen of melanoma) is frequently overexpressed in tumours. High PRAME levels correlate with poor clinical outcome of several cancers, but the mechanisms by which PRAME could be involved in tumourigenesis remain largely elusive. We applied protein-complex purification strategies and identified PRAME as a substrate recognition subunit of a Cullin2-based E3 ubiquitin ligase. PRAME can be recruited to DNA in vitro, and genome-wide chromatin immunoprecipitation experiments revealed that PRAME is specifically enriched at transcriptionally active promoters that are also bound by NFY and at enhancers. Our results are consistent with a role for the PRAME ubiquitin ligase complex in NFY-mediated transcriptional regulation. PMID:21822215

  7. HIV-1 Vpr Protein Inhibits Telomerase Activity via the EDD-DDB1-VPRBP E3 Ligase Complex*

    PubMed Central

    Wang, Xin; Singh, Shailbala; Jung, Hae-Yun; Yang, Guojun; Jun, Sohee; Sastry, K. Jagannadha; Park, Jae-Il

    2013-01-01

    Viral pathogens utilize host cell machinery for their benefits. Herein, we identify that HIV-1 Vpr (viral protein R) negatively modulates telomerase activity. Telomerase enables stem and cancer cells to evade cell senescence by adding telomeric sequences to the ends of chromosomes. We found that Vpr inhibited telomerase activity by down-regulating TERT protein, a catalytic subunit of telomerase. As a molecular adaptor, Vpr enhanced the interaction between TERT and the VPRBP substrate receptor of the DYRK2-associated EDD-DDB1-VPRBP E3 ligase complex, resulting in increased ubiquitination of TERT. In contrast, the Vpr mutant identified in HIV-1-infected long-term nonprogressors failed to promote TERT destabilization. Our results suggest that Vpr inhibits telomerase activity by hijacking the host E3 ligase complex, and we propose the novel molecular mechanism of telomerase deregulation in possibly HIV-1 pathogenesis. PMID:23612978

  8. Structure of the Siz/PIAS SUMO E3 Ligase Siz1 and Determinants Required for SUMO Modification of PCNA

    SciTech Connect

    Yunus, Ali A.; Lima, Christopher D.

    2010-01-12

    Siz1 is a founding member of the Siz/PIAS RING family of SUMO E3 ligases. The X-ray structure of an active Siz1 ligase revealed an elongated tripartite architecture comprised of an N-terminal PINIT domain, a central zinc-containing RING-like SP-RING domain, and a C-terminal domain we term the SP-CTD. Structure-based mutational analysis and biochemical studies show that the SP-RING and SP-CTD are required for activation of the E2SUMO thioester, while the PINIT domain is essential for redirecting SUMO conjugation to the proliferating cell nuclear antigen (PCNA) at lysine 164, a nonconsensus lysine residue that is not modified by the SUMO E2 in the absence of Siz1. Mutational analysis of Siz1 and PCNA revealed surfaces on both proteins that are required for efficient SUMO modification of PCNA in vitro and in vivo.

  9. Effects of chenodeoxycholic acid and deoxycholic acid on cholesterol absorption and metabolism in humans.

    PubMed

    Wang, Yanwen; Jones, Peter J H; Woollett, Laura A; Buckley, Donna D; Yao, Lihang; Granholm, Norman A; Tolley, Elizabeth A; Heubi, James E

    2006-07-01

    Quantitative and qualitative differences in intralumenal bile acids may affect cholesterol absorption and metabolism. To test this hypothesis, 2 cross-over outpatient studies were conducted in adults with apo-A IV 1/1 or apo-E 3/3 genotypes. Study 1 included 11 subjects 24 to 37 years of age, taking 15 mg/kg/day chenodeoxycholic acid (CDCA) or no bile acid for 20 days while being fed a controlled diet. Study 2 included 9 adults 25 to 38 years of age, taking 15 mg/kg/day deoxycholic acid (DCA) or no bile acid, following the same experimental design and procedures as study 1. CDCA had no effect on plasma lipid concentrations, whereas DCA decreased (P < 0.05) plasma high-density lipoprotein (HDL)-cholesterol and tended to decrease (P = 0.15) low-density lipoprotein (LDL)-cholesterol. CDCA treatment enriched (P < 0.0001) bile with CDCA and increased cholesterol concentration in micelles, whereas meal-stimulated bile acid concentrations were decreased. DCA treatment enriched (P < 0.0001) bile with DCA and tended to increase intralumenal cholesterol solubilized in micelles (P = 0.06). No changes were found in cholesterol absorption, free cholesterol fractional synthetic rate (FSR), or 3-hydroxy-3 methylglutaryl (HMG) CoA reductase and LDL receptor messenger ribonucleic acid (mRNA) levels after CDCA treatment. DCA supplementation tended to decrease cholesterol absorption and reciprocally increase FSR and HMG CoA reductase and LDL receptor mRNA levels. Results of these 2 studies suggest that the solubilization of cholesterol in the intestinal micelles is not a rate-limiting step for its absorption.

  10. CUL4-DDB1-CDT2 E3 Ligase Regulates the Molecular Clock Activity by Promoting Ubiquitination-Dependent Degradation of the Mammalian CRY1.

    PubMed

    Tong, Xin; Zhang, Deqiang; Guha, Anirvan; Arthurs, Blake; Cazares, Victor; Gupta, Neil; Yin, Lei

    2015-01-01

    The CUL4-DDB1 E3 ligase complex serves as a critical regulator in various cellular processes, including cell proliferation, DNA damage repair, and cell cycle progression. However, whether this E3 ligase complex regulates clock protein turnover and the molecular clock activity in mammalian cells is unknown. Here we show that CUL4-DDB1-CDT2 E3 ligase ubiquitinates CRY1 and promotes its degradation both in vitro and in vivo. Depletion of the major components of this E3 ligase complex, including Ddb1, Cdt2, and Cdt2-cofactor Pcna, leads to CRY1 stabilization in cultured cells or in the mouse liver. CUL4A-DDB1-CDT2 E3 ligase targets lysine 585 within the C-terminal region of CRY1 protein, shown by the CRY1 585KA mutant's resistance to ubiquitination and degradation mediated by the CUL4A-DDB1 complex. Surprisingly, both depletion of Ddb1 and over-expression of Cry1-585KA mutant enhance the oscillatory amplitude of the Bmal1 promoter activity without altering its period length, suggesting that CUL4A-DDB1-CDT2 E3 targets CRY1 for degradation and reduces the circadian amplitude. All together, we uncovered a novel biological role for CUL4A-DDB1-CDT2 E3 ligase that regulates molecular circadian behaviors via promoting ubiquitination-dependent degradation of CRY1.

  11. HOIL-1L Functions as the PKCζ Ubiquitin Ligase to Promote Lung Tumor Growth

    PubMed Central

    Queisser, Markus A.; Dada, Laura A.; Deiss-Yehiely, Nimrod; Angulo, Martin; Zhou, Guofei; Kouri, Fotini M.; Knab, Lawrence M.; Liu, Jing; Stegh, Alexander H.; DeCamp, Malcolm M.; Budinger, G. R. Scott; Chandel, Navdeep S.; Ciechanover, Aaron; Iwai, Kazuhiro

    2014-01-01

    Rationale: Protein kinase C zeta (PKCζ) has been reported to act as a tumor suppressor. Deletion of PKCζ in experimental cancer models has been shown to increase tumor growth. However, the mechanisms of PKCζ down-regulation in cancerous cells have not been previously described. Objectives: To determine the molecular mechanisms that lead to decreased PKCζ expression and thus increased survival in cancer cells and tumor growth. Methods: The levels of expression of heme-oxidized IRP2 ubiquitin ligase 1L (HOIL-1L), HOIL-1–interacting protein (HOIP), Shank-associated RH domain-interacting protein (SHARPIN), and PKCζ were analyzed by Western blot and/or quantitative real-time polymerase chain reaction in different cell lines. Coimmunoprecipitation experiments were used to demonstrate the interaction between HOIL-1L and PKCζ. Ubiquitination was measured in an in vitro ubiquitination assay and by Western blot with specific antibodies. The role of hypoxia-inducible factor (HIF) was determined by gain/loss-of-function experiments. The effect of HOIL-1L expression on cell death was investigated using RNA interference approaches in vitro and on tumor growth in mice models. Increased HOIL-1L and decreased PKCζ expression was assessed in lung adenocarcinoma and glioblastoma multiforme and documented in several other cancer types by oncogenomic analysis. Measurements and Main Results: Hypoxia is a hallmark of rapidly growing solid tumors. We found that during hypoxia, PKCζ is ubiquitinated and degraded via the ubiquitin ligase HOIL-1L, a component of the linear ubiquitin chain assembly complex (LUBAC). In vitro ubiquitination assays indicate that HOIL-1L ubiquitinates PKCζ at Lys-48, targeting it for proteasomal degradation. In a xenograft tumor model and lung cancer model, we found that silencing of HOIL-1L increased the abundance of PKCζ and decreased the size of tumors, suggesting that lower levels of HOIL-1L promote survival. Indeed, mRNA transcript levels of HOIL

  12. Chlorovirus Skp1-Binding Ankyrin Repeat Protein Interplay and Mimicry of Cellular Ubiquitin Ligase Machinery

    PubMed Central

    Noel, Eric A.; Kang, Ming; Adamec, Jiri; Oyler, George A.

    2014-01-01

    ABSTRACT The ubiquitin-proteasome system is targeted by many viruses that have evolved strategies to redirect host ubiquitination machinery. Members of the genus Chlorovirus are proposed to share an ancestral lineage with a broader group of related viruses, nucleo-cytoplasmic large DNA viruses (NCLDV). Chloroviruses encode an Skp1 homolog and ankyrin repeat (ANK) proteins. Several chlorovirus-encoded ANK repeats contain C-terminal domains characteristic of cellular F-boxes or related NCLDV chordopox PRANC (pox protein repeats of ankyrin at C-terminal) domains. These observations suggested that this unique combination of Skp1 and ANK repeat proteins might form complexes analogous to the cellular Skp1-Cul1-F-box (SCF) ubiquitin ligase complex. We identified two ANK proteins from the prototypic chlorovirus Paramecium bursaria chlorella virus-1 (PBCV-1) that functioned as binding partners for the virus-encoded Skp1, proteins A682L and A607R. These ANK proteins had a C-terminal Skp1 interactional motif that functioned similarly to cellular F-box domains. A C-terminal motif of ANK protein A682L binds Skp1 proteins from widely divergent species. Yeast two-hybrid analyses using serial domain deletion constructs confirmed the C-terminal localization of the Skp1 interactional motif in PBCV-1 A682L. ANK protein A607R represents an ANK family with one member present in all 41 sequenced chloroviruses. A comprehensive phylogenetic analysis of these related ANK and viral Skp1 proteins suggested partnered function tailored to the host alga or common ancestral heritage. Here, we show protein-protein interaction between corresponding family clusters of virus-encoded ANK and Skp1 proteins from three chlorovirus types. Collectively, our results indicate that chloroviruses have evolved complementing Skp1 and ANK proteins that mimic cellular SCF-associated proteins. IMPORTANCE Viruses have evolved ways to direct ubiquitination events in order to create environments conducive to their

  13. Highly Precise and Developmentally Programmed Genome Assembly in Paramecium Requires Ligase IV–Dependent End Joining

    PubMed Central

    Marmignon, Antoine; Ku, Michael; Silve, Aude; Meyer, Eric; Forney, James D.; Malinsky, Sophie; Bétermier, Mireille

    2011-01-01

    During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5′ overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi–mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5′-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3′ ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a “cut-and-close” mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new

  14. Characterization and identification of ubiquitin conjugation sites with E3 ligase recognition specificities

    PubMed Central

    2015-01-01

    Background In eukaryotes, ubiquitin-conjugation is an important mechanism underlying proteasome-mediated degradation of proteins, and as such, plays an essential role in the regulation of many cellular processes. In the ubiquitin-proteasome pathway, E3 ligases play important roles by recognizing a specific protein substrate and catalyzing the attachment of ubiquitin to a lysine (K) residue. As more and more experimental data on ubiquitin conjugation sites become available, it becomes possible to develop prediction models that can be scaled to big data. However, no development that focuses on the investigation of ubiquitinated substrate specificities has existed. Herein, we present an approach that exploits an iteratively statistical method to identify ubiquitin conjugation sites with substrate site specificities. Results In this investigation, totally 6259 experimentally validated ubiquitinated proteins were obtained from dbPTM. After having filtered out homologous fragments with 40% sequence identity, the training data set contained 2658 ubiquitination sites (positive data) and 5532 non-ubiquitinated sites (negative data). Due to the difficulty in characterizing the substrate site specificities of E3 ligases by conventional sequence logo analysis, a recursively statistical method has been applied to obtain significant conserved motifs. The profile hidden Markov model (profile HMM) was adopted to construct the predictive models learned from the identified substrate motifs. A five-fold cross validation was then used to evaluate the predictive model, achieving sensitivity, specificity, and accuracy of 73.07%, 65.46%, and 67.93%, respectively. Additionally, an independent testing set, completely blind to the training data of the predict