RESUMEN
ADP-ribosylation has primarily been known as post-translational modification of proteins. As signalling strategy conserved in all domains of life, it modulates substrate activity, localisation, stability or interactions, thereby regulating a variety of cellular processes and microbial pathogenicity. Yet over the last years, there is increasing evidence of non-canonical forms of ADP-ribosylation that are catalysed by certain members of the ADP-ribosyltransferase family and go beyond traditional protein ADP-ribosylation signalling. New macromolecular targets such as nucleic acids and new ADP-ribose derivatives have been established, notably extending the repertoire of ADP-ribosylation signalling. Based on the physiological relevance known so far, non-canonical ADP-ribosylation deserves its recognition next to the traditional protein ADP-ribosylation modification and which we therefore review in the following.
Asunto(s)
ADP-Ribosilación/fisiología , ADP Ribosa Transferasas/química , ADP Ribosa Transferasas/clasificación , ADP Ribosa Transferasas/fisiología , Adenosina Difosfato/metabolismo , Guanosina/metabolismo , N-Glicosil Hidrolasas/fisiología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Secuencias Reguladoras de Ácido Ribonucleico , Transducción de Señal , Relación Estructura-Actividad , Timidina/metabolismo , Ubiquitina/metabolismoRESUMEN
Adprhl1, a member of the ADP-ribosylhydrolase protein family, is expressed exclusively in the developing heart of all vertebrates. In the amphibian Xenopus laevis, distribution of its mRNA is biased towards actively growing chamber myocardium. Morpholino oligonucleotide-mediated knockdown of all Adprhl1 variants inhibits striated myofibril assembly and prevents outgrowth of the ventricle. The resulting ventricles retain normal electrical conduction and express markers of chamber muscle differentiation but are functionally inert. Using a cardiac-specific Gal4 binary expression system, we show that the abundance of Adprhl1 protein in tadpole hearts is tightly controlled through a negative regulatory mechanism targeting the 5'-coding sequence of Xenopus adprhl1. Over-expression of full length (40kDa) Adprhl1 variants modified to escape such repression, also disrupts cardiac myofibrillogenesis. Disarrayed myofibrils persist that show extensive branching, with sarcomere division occurring at the actin-Z-disc boundary. Ultimately, Adprhl1-positive cells contain thin actin threads, connected to numerous circular branch points. Recombinant Adprhl1 can localize to stripes adjacent to the Z-disc, suggesting a direct role for Adprhl1 in modifying Z-disc and actin dynamics as heart chambers grow. Modelling the structure of Adprhl1 suggests this cardiac-specific protein is a pseudoenzyme, lacking key residues necessary for ADP-ribosylhydrolase catalytic activity.
Asunto(s)
Citoesqueleto de Actina/fisiología , Regulación del Desarrollo de la Expresión Génica , Miocardio/citología , N-Glicosil Hidrolasas/fisiología , Proteínas de Xenopus/fisiología , Animales , Animales Modificados Genéticamente , Técnicas de Silenciamiento del Gen , Corazón/embriología , Corazón/crecimiento & desarrollo , Ventrículos Cardíacos/embriología , Ventrículos Cardíacos/crecimiento & desarrollo , Humanos , Larva , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Ratones , Modelos Moleculares , Simulación de Dinámica Molecular , Morfolinos/farmacología , Mutación , Miocardio/metabolismo , N-Glicosil Hidrolasas/biosíntesis , N-Glicosil Hidrolasas/genética , Organogénesis , Conformación Proteica , ARN Mensajero/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Xenopus/biosíntesis , Proteínas de Xenopus/genética , Xenopus laevis/embriología , Xenopus laevis/crecimiento & desarrolloRESUMEN
Protein ADP-ribosylation is a reversible posttranslational modification of uncertain significance in cancer. In this study, we evaluated the consequences for cancer susceptibility in the mouse of a genetic deletion of the enzyme responsible for removing mono-ADP-ribose moieties from arginines in cellular proteins. Specifically, we analyzed cancer susceptibility in animals lacking the ADP-ribosylarginine hydrolase (ARH1) that cleaves the ADP ribose-protein bond. ARH1(-/-) cells or ARH1(-/-) cells overexpressing an inactive mutant ARH1 protein (ARH1(-/-)+dm) had higher proliferation rates than either wild-type ARH1(+/+) cells or ARH1(-/-) cells engineered to express the wild-type ARH1 enzyme. More significantly, ARH1(-/-) and ARH1(+/-) mice spontaneously developed lymphomas, adenocarcinomas, and metastases more frequently than wild-type ARH1(+/+) mice. In ARH1(+/-) mice, we documented in all arising tumors mutation of the remaining wild-type allele (or loss of heterozygosity), illustrating the strict correlation that existed between tumor formation and absence of ARH1 gene function. Our findings show that proper control of protein ADP-ribosylation levels affected by ARH1 is essential for cancer suppression.
Asunto(s)
Transformación Celular Neoplásica/genética , N-Glicosil Hidrolasas/fisiología , Metástasis de la Neoplasia/genética , Proteínas de Neoplasias/fisiología , Neoplasias Experimentales/enzimología , Adenosina Difosfato Ribosa/análogos & derivados , Adenosina Difosfato Ribosa/metabolismo , Animales , División Celular , Transformación Celular Neoplásica/metabolismo , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Pérdida de Heterocigocidad , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , N-Glicosil Hidrolasas/deficiencia , N-Glicosil Hidrolasas/genética , Neoplasias Experimentales/genética , Procesamiento Proteico-Postraduccional , Ensayo de Tumor de Célula MadreRESUMEN
Maternal effects are defined by mutations that affect the next generation when they are maternally inherited. To date, most indepth studies of maternal effects in plants have attributed their origin to genomic imprinting that restricts expression to the maternal allele. The DNA glycosylase DEMETER (DME) removes methylated cytosine residues, causing transcriptional activation of the maternal allele of imprinted genes. In this study, we show that loss-of-function of the major DNA LIGASE I (AtLIG1) in Arabidopsis thaliana causes maternal effects in the endosperm, which is the seed tissue that nurtures embryo development. AtLIG1 expression is not imprinted and has a limited impact on imprinted gene expression. Genetic interaction analyses further indicate that AtLIG1 acts downstream of DME. The removal of methylated cytosine residues by DME involves the creation of DNA single-strand breaks and our results suggest that AtLIG1 repairs these breaks.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/embriología , Arabidopsis/enzimología , ADN Ligasas/fisiología , Semillas/enzimología , Semillas/crecimiento & desarrollo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , ADN Ligasa (ATP) , ADN Ligasas/genética , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Microscopía Confocal , Modelos Genéticos , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/fisiología , Fenotipo , Plantas Modificadas Genéticamente/embriología , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Polimorfismo Genético/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Semillas/genética , Semillas/metabolismo , Transactivadores/genética , Transactivadores/fisiologíaAsunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/genética , ADN (Citosina-5-)-Metiltransferasas/fisiología , Metilación de ADN , Proteínas de Unión al ADN/fisiología , N-Glicosil Hidrolasas/fisiología , Transactivadores/fisiología , Factores de Transcripción/fisiología , Adenosina Trifosfatasas/genética , Proteínas de Ciclo Celular/genética , Ensamble y Desensamble de Cromatina/genética , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/fisiología , Proteínas Nucleares/fisiología , Proteína Metiltransferasas , ARN de Planta/fisiología , Proteína que Contiene ValosinaRESUMEN
Pokeweed antiviral protein (PAP) isolated from Phytolacca americana is a ribosome-inactivating protein (RIP) that has RNA N-glycosidase (RNG) activity towards both eukaryotic and prokaryotic ribosomes. In contrast, karasurin-A (KRN), a RIP from Trichosanthes kirilowii var. japonica, is active only on eukaryotic ribosomes. Stepwise selection of chimera proteins between PAP and KRN indicated that the C-terminal region of PAP (residues 209-225) was critical for RNG activity toward prokaryotic ribosomes. When the region of PAP (residues 209-225) was replaced with the corresponding region of KRN the PAP chimera protein, like KRN, was active only on eukaryotic ribosomes. Furthermore, insertion of the region of PAP (residues 209-225) into the KRN chimera protein resulted not only in the detectable RNG activity toward prokaryotic ribosome, but also activity toward the eukaryotic ribosomes as well that was seven-fold higher than for the original KRN. In this study, the possibility of genetic manipulation of the activity and substrate specificity of RIPs is demonstrated.
Asunto(s)
Células Procariotas/fisiología , ARN , Proteínas Inactivadoras de Ribosomas Tipo 1/genética , Proteínas Inactivadoras de Ribosomas/fisiología , Ribosomas/fisiología , Secuencia de Aminoácidos , Cristalografía por Rayos X , Activación Enzimática , Modelos Moleculares , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/química , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/fisiología , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/fisiología , Conformación Proteica , ARN/química , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Inactivadoras de Ribosomas/química , Proteínas Inactivadoras de Ribosomas/genética , Proteínas Inactivadoras de Ribosomas Tipo 1/química , Proteínas Inactivadoras de Ribosomas Tipo 1/fisiología , Alineación de Secuencia , Relación Estructura-Actividad , Especificidad por SustratoAsunto(s)
Proteínas de Arabidopsis/antagonistas & inhibidores , Mutación , N-Glicosil Hidrolasas/antagonistas & inhibidores , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , ADN de Plantas , Genes de Plantas , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/fisiologíaRESUMEN
Alpha-sarcin and ricin represent two structurally and mechanistically distinct families of site-specific enzymes that block translation by irreversibly modifying the sarcin/ricin loop (SRL) of 23S-28S rRNA. alpha-Sarcin family enzymes are designated as ribotoxins and act as endonucleases. Ricin family enzymes are designated as ribosome inactivating proteins (RIP) and act as N-glycosidases. Recently, we demonstrated that basic surface residues of the ribotoxin restrictocin promote rapid and specific ribosome targeting by this endonuclease. Here, we report that three RIP: ricin A, saporin, and gypsophilin depurinate the ribosome with strong salt sensitivity and achieve unusually fast kcat/Km approximately 10(9)-10(10) M(-1) s(-1), implying that RIP share with ribotoxins a common mechanism of electrostatically facilitated ribosome targeting. Bioinformatics analysis of RIP revealed that surface charge properties correlate with the presence of the transport chain in the RIP molecule, suggesting a second role for the surface charge in RIP transport. These findings put forward surface electrostatics as an important determinant of RIP activity.
Asunto(s)
Endorribonucleasas/química , Proteínas Fúngicas/química , Familia de Multigenes/fisiología , N-Glicosil Hidrolasas/química , N-Glicosil Hidrolasas/fisiología , Proteínas de Plantas/química , Proteínas de Plantas/fisiología , Inhibidores de la Síntesis de la Proteína/química , Ribosomas/metabolismo , Ricina/química , Ésteres del Ácido Sulfúrico/química , Triterpenos/química , Endorribonucleasas/fisiología , Proteínas Fúngicas/fisiología , N-Glicosil Hidrolasas/clasificación , Proteínas de Plantas/clasificación , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1 , Ribosomas/química , Ricina/clasificación , Ricina/farmacología , Saporinas , Electricidad Estática , Ésteres del Ácido Sulfúrico/clasificación , Ésteres del Ácido Sulfúrico/farmacología , Propiedades de Superficie , Triterpenos/clasificación , Triterpenos/farmacologíaAsunto(s)
Replicación del ADN/genética , ADN Mitocondrial/genética , ADN Recombinante/genética , Dosificación de Gen , Adenosina Trifosfato/metabolismo , Animales , Ciclo del Ácido Cítrico/fisiología , Fragmentación del ADN , ADN Concatenado , ADN-(Sitio Apurínico o Apirimidínico) Liasa , ARN Polimerasas Dirigidas por ADN/fisiología , Humanos , Proteínas Mitocondriales , N-Glicosil Hidrolasas/fisiología , Proteínas Nucleares/fisiología , Estrés Oxidativo/fisiología , ARN/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Factores de Transcripción/fisiologíaAsunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Proteínas de Arabidopsis/fisiología , Arabidopsis/genética , Arabidopsis/inmunología , Inmunidad Innata/genética , N-Glicosil Hidrolasas/fisiología , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Proteínas Qa-SNARE/fisiología , Transportadoras de Casetes de Unión a ATP/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Mutación , N-Glicosil Hidrolasas/genética , Proteínas Qa-SNARE/genéticaRESUMEN
The conserved signal recognition particle targets ribosomes synthesizing presecretory proteins to the endoplasmic reticulum membrane. Key to the activity of SRP is its ability to bind the ribosome at distant locations, the signal sequence exit and elongation factor-binding sites. These contacts are made by the S and Alu domains of SRP, respectively. We tested earlier secondary structure predictions of the Saccharomyces cerevisiae SRP RNA, scR1, and provide and test a consensus structure. The structure contains four non-conserved insertions, helices 9-12, into the core SRP RNA fold, and an extended helix 7. Using a series of scR1 mutants lacking part or all of these structural elements, we find that they are important for the RNA in both function and assembly of the RNP. About 20% of the RNA, corresponding to the outer regions of these helices, is dispensable for function. Further, we examined the role of several features within the S-domain section of the core, helix 5, and find that its length and flexibility are important for proper SRP function and become essential in the absence of helix 10, 11 and/or 7 regions. Overall, the genetic data indicate that regions of scR1 distant in both primary sequence and secondary structure have interrelated roles in the function of the complex, and possibly mediate communication between Alu and S domains during targeting.
Asunto(s)
N-Glicosil Hidrolasas/química , Conformación de Ácido Nucleico , ARN de Hongos/química , Proteínas de Saccharomyces cerevisiae/química , Partícula de Reconocimiento de Señal/química , Secuencia de Bases , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Datos de Secuencia Molecular , Mutación , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/fisiología , ARN de Hongos/genética , ARN de Hongos/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Partícula de Reconocimiento de Señal/genética , Partícula de Reconocimiento de Señal/fisiologíaRESUMEN
RCL is a c-Myc target with tumorigenic potential. Genome annotation predicted that RCL belonged to the N-deoxyribosyltransferase family. However, its putative relationship to this class of enzymes did not lead to its precise biochemical function. The purified native or N-terminal His-tagged recombinant rat RCL protein expressed in Escherichia coli exhibits the same enzyme activity, deoxynucleoside 5'-monophosphate N-glycosidase, never before described. dGMP appears to be the best substrate. RCL opens a new route in the nucleotide catabolic pathways by cleaving the N-glycosidic bond of deoxynucleoside 5'-monophosphates to yield two reaction products, deoxyribose 5-phosphate and purine or pyrimidine base. Biochemical studies show marked differences in the terms of the structure and catalytic mechanism between RCL and of its closest enzyme family neighbor, N-deoxyribosyltransferase. The reaction products of this novel enzyme activity have been implicated in purine or pyrimidine salvage, glycolysis, and angiogenesis, and hence are all highly relevant for tumorigenesis.
Asunto(s)
Regulación de la Expresión Génica , N-Glicosil Hidrolasas/fisiología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Secuencia de Aminoácidos , Animales , Escherichia coli/metabolismo , Genoma , Enlace de Hidrógeno , Cinética , Lactobacillus/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis , N-Glicosil Hidrolasas/genética , Pentosiltransferasa/química , Proteínas Proto-Oncogénicas/química , Ratas , Proteínas Recombinantes/químicaRESUMEN
Exogenous thymine must be converted to thymidine to enable a thyA (thymidylate synthase) mutant to grow. The deoxyribose in the thymidine comes from dUMP, which must first be dephosphorylated. The nucleotidase YjjG is critical for this step. A yjjG thyA mutant cannot use thymine for growth on a glucose minimal medium.
Asunto(s)
Nucleótidos de Desoxiuracil/metabolismo , Proteínas de Escherichia coli/fisiología , Escherichia coli/metabolismo , N-Glicosil Hidrolasas/fisiología , Timina/metabolismo , Fosforilación , Timidilato Sintasa/fisiologíaRESUMEN
Pfs expression is required for several metabolic pathways and limits the production of autoinducer-2, a molecule proposed to play a central role in interspecies quorum sensing. The present study reveals physiological conditions and promoter DNA elements that regulate Escherichia coli pfs transcription. Pfs transcription is shown to rely on both sigma 70 and sigma 38 (rpoS), and the latter is subject to induction that increases pfs expression. Transcription is maximal as the cells approach stationary phase, and this level can be increased by salt stress through induction of sigma 38-dependent expression. The pfs promoter is shown to contain both positive and negative elements, which can be used by both forms of RNA polymerase. The negative element is contained within the overlapping dgt promoter, which is involved in purine metabolism. Consideration of the physiological roles of sigma 38 and dgt leads to a model for how autoinducer production is controlled under changing physiological conditions.
Asunto(s)
Escherichia coli/fisiología , Regulación Bacteriana de la Expresión Génica , Homoserina/análogos & derivados , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/fisiología , Purinas/metabolismo , Transcripción Genética , Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Homoserina/biosíntesis , Lactonas , N-Glicosil Hidrolasas/biosíntesis , Regiones Promotoras Genéticas , ARN Bacteriano/análisis , ARN Bacteriano/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Factor sigma/metabolismoRESUMEN
Bgp, one of the surface-localized glycosaminoglycan-binding proteins of the Lyme disease spirochete, Borrelia burgdorferi, exhibited nucleosidase activity. Infection of SCID mice with B. burgdorferi strain N40 mutants harboring a targeted insertion in bgp and apparently retaining all endogenous plasmids revealed that Bgp is not essential for colonization of immunocompromised mice.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas Portadoras/fisiología , Enfermedad de Lyme/etiología , N-Glicosil Hidrolasas/fisiología , Animales , Borrelia burgdorferi/genética , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos C3H , Ratones SCID , PlásmidosRESUMEN
ADP-ribosylation is a post-translational modification resulting from transfer of the ADP-ribose moiety of NAD to protein. Mammalian cells contain mono-ADP-ribosyltransferases that catalyze the formation of ADP-ribose-(arginine) protein, which can be cleaved by a 39-kDa ADP-ribose-(arginine) protein hydrolase (ARH1), resulting in release of free ADP-ribose and regeneration of unmodified protein. Enzymes involved in poly(ADP-ribosylation) participate in several critical physiological processes, including DNA repair, cellular differentiation, and carcinogenesis. Multiple poly(ADP-ribose) polymerases have been identified in the human genome, but there is only one known poly(ADP-ribose) glycohydrolase (PARG), a 111-kDa protein that degrades the (ADP-ribose) polymer to ADP-ribose. We report here the identification of an ARH1-like protein, termed poly(ADP-ribose) hydrolase or ARH3, which exhibited PARG activity, generating ADP-ribose from poly-(ADP-ribose), but did not hydrolyze ADP-ribose-arginine, -cysteine, -diphthamide, or -asparagine bonds. The 39-kDa ARH3 shares amino acid sequence identity with both ARH1 and the catalytic domain of PARG. ARH3 activity, like that of ARH1, was enhanced by Mg(2+). Critical vicinal acidic amino acids in ARH3, identified by mutagenesis (Asp(77) and Asp(78)), are located in a region similar to that required for activity in ARH1 but different from the location of the critical vicinal glutamates in the PARG catalytic site. All findings are consistent with the conclusion that ARH3 has PARG activity but is structurally unrelated to PARG.
Asunto(s)
Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/fisiología , N-Glicosil Hidrolasas/química , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/fisiología , Secuencia de Aminoácidos , Animales , Autorradiografía , Western Blotting , Dominio Catalítico , Bovinos , Línea Celular , Cromatografía Líquida de Alta Presión , Cartilla de ADN/química , Ditiotreitol/farmacología , Glicósido Hidrolasas/metabolismo , Humanos , Hidrólisis , Magnesio/química , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutagénesis , Mutación , N-Glicosil Hidrolasas/metabolismo , Hibridación de Ácido Nucleico , Poli(ADP-Ribosa) Polimerasas/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Ribosa/química , Homología de Secuencia de Aminoácido , Timo/metabolismo , Factores de TiempoRESUMEN
mRNA degradation occurs through distinct pathways, one primarily from the 5' end of the mRNA and the second from the 3' end. Decay from the 3' end generates the m7GpppN cap dinucleotide, which is subsequently hydrolyzed to m7Gp and ppN in Saccharomyces cerevisiae by a scavenger decapping activity termed Dcs1p. Although Dcs1p functions in the last step of mRNA turnover, we demonstrate that its activity modulates earlier steps of mRNA decay. Disruption of the DCS1 gene manifests a threefold increase of the TIF51A mRNA half-life. Interestingly, the hydrolytic activity of Dcs1p was essential for the altered mRNA turnover, as Dcs1p, but not a catalytically inactive Dcs1p mutant, complemented the increased mRNA stability. Mechanistic analysis revealed that 5' to 3' exoribonucleolytic activity was impeded in the dcs1Delta strain, resulting in the accumulation of uncapped mRNA. These data define a new role for the Dcs1p scavenger decapping enzyme and demonstrate a novel mechanism whereby the final step in the 3' mRNA decay pathway can influence 5' to 3' exoribonucleolytic activity.
Asunto(s)
N-Glicosil Hidrolasas/fisiología , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Northern Blotting , Western Blotting , Catálisis , ADN Complementario/metabolismo , Genotipo , Hidrólisis , Inmunoprecipitación , Metionina/química , Modelos Biológicos , N-Glicosil Hidrolasas/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Plásmidos/metabolismo , Poli A/química , Biosíntesis de Proteínas , ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Tiempo , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
Nonhost resistance describes the immunity of an entire plant species against nonadapted pathogen species. We report that Arabidopsis PEN2 restricts pathogen entry of two ascomycete powdery mildew fungi that in nature colonize grass and pea species. The PEN2 glycosyl hydrolase localizes to peroxisomes and acts as a component of an inducible preinvasion resistance mechanism. Postinvasion fungal growth is blocked by a separate resistance layer requiring the EDS1-PAD4-SAG101 signaling complex, which is known to function in basal and resistance (R) gene-triggered immunity. Concurrent impairment of pre- and postinvasion resistance renders Arabidopsis a host for both nonadapted fungi.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/inmunología , N-Glicosil Hidrolasas/fisiología , Enfermedades de las Plantas/microbiología , Proteínas Qa-SNARE/fisiología , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Ascomicetos/fisiología , Hidrolasas de Éster Carboxílico/fisiología , Proteínas de Unión al ADN/fisiología , Mutación , Peroxisomas/fisiología , Phytophthora/fisiología , Proteínas Qa-SNARE/genética , Transducción de SeñalRESUMEN
Nitrogen fixation in Azospirillum brasilense is regulated at transcriptional and post-translational levels. Post-translational control occurs through the reversible ADP-ribosylation of dinitrogenase reductase (Fe Protein), mediated by the dinitrogenase reductase ADP-ribosyltransferase (DraT) and dinitrogenase reductase glycohydrolase (DraG). Although the DraT and DraG activities are regulated in vivo, the molecules responsible for such regulation remain unknown. We have constructed broad-host-range plasmids capable of over-expressing, upon IPTG induction, the regulatory enzymes DraT and DraG as six-histidine-N-terminal fused proteins (His). Both DraT-His and DraG-His are functional in vivo. We have analyzed the effects of DraT-His and DraG-His over-expression on the post-translational modification of Fe Protein. The DraT-His over-expression led to Fe Protein modification in the absence of ammonium addition, while cells over-expressing DraG-His showed only partial ADP-ribosylation of Fe Protein by adding ammonium. These results suggest that both DraT-His and DraG-His lose their regulation upon over-expression, possible by titrating out negative regulators.