Identifizierung von Substratproteinen von Mitogen-aktivierten Proteinkinasen durch Massenspektrometrie

Mitogen-aktivierte Proteinkinasen (MAPKs) sind in eukaryotischen Organismen vorkommende Proteine, die der Weiterleitung exogener Signale an entsprechende Effektoren dienen. Die von pflanzlichen Genomen kodierten MAPK-Familien sind besonders umfangreich; in Arabidopsis thaliana, beispielsweise, existieren 20 MAPK-Gene. Bis auf wenige Ausnahmen sind die von pflanzlichen MAPKs regulierten Zielproteine unbekannt. In der vorliegenden Arbeit wurden potentielle MAPK-Substratproteine analysiert. Diese waren in einer vorangegangenen Studie anhand des Auftretens von Phosphopeptiden, deren Sequenz dem von MAPKs erkannten Konsensusmotif entspricht, ausgewählt worden. Für acht potentielle Substratproteine wurden rekombinante Proteine aufgereinigt und auf Phosphorylierbarkeit durch die MAPKs MPK3, MPK4 und MPK6 in in vitro-Kinase- Experimenten getestet. Dabei zeigten die drei MAPKs deutliche Unterschiede hinsichtlich ihrer Spezifität für diese acht Substrate. Bei fünf der Kandidaten konnten die phosphorylierten Gruppen durch Flüssigchromatographie-Tandem- Massenspektrometrie (LC-MS/MS/MS) den entsprechenden Aminosäureresten zugeordnet werden. Dies sind die Proteine: Phosphoglucose-Isomerase 1 (PGI1), das putative Golgi SNARE protein GOS12, der mechanosensitive Ionenkanal MSL9, eine Histon-Deacetylase (HDT2/HD2b) sowie Phosphatidylinositol-4-phosphat-Kinase 4ß1 (PI-4Kß1). Die LC-MS/MS/MS-Analysie zeigte außerdem, dass jedes der untersuchten MAPK-Substratproteine mehrere phosphorylierte Reste enthielt. In in vitro Kinase-Experimenten wurde für fast alle der in vivo-identifizierten Phosphopeptide eine Modifizierung durch MAPKs beobachtet. Dies deutet darauf hin, dass die untersuchten Proteine potentielle in vivo-Substrate für MAPKs darstellen. Mit Hilfe von fluoreszierenden Fusionsproteinen an HDT2 und seiner an der phosphorylierbaren Aminosäure mutierten Derivate konnte gezeigt werden, dass Verlust der Phosphorylierung zur partiellen Exclusion des Proteins vom Nukleolus führt. Weiters wurde für GOS12 eine phosphorylierungsabhängige Lokalisation im Golgi-Apparat beobachtet und eine potentielle Rolle von GOS12 im Erhalt der Golgi- Struktur abgeleitet. Die Behandlung von Protoplasten mit dem bakteriellen Elicitor flg22 führte zur Relokalisierung von GOS12 aus Nukleus und Cytoplasma zum Golgi-Apparat. Zusammengefasst, konnten durch die auf MS-Daten basierende Herangehensweise MAPK Substrate ihren spezifischen MAPKs zugeordnet werden. Eine potentielle Rolle von MAPKs in der Regulation des Vesikeltransports und der Histon-Deacetylierung in Pflanzen wurde aufgedecktMitogen-activated protein kinases (MAPKs) transmit numerous incoming signals to downstream targets in animals and yeasts. Plant genomes encode the largest families of MAPKs in all eukaryotes, with 20 members in Arabidopsis thaliana. With a few exceptions, however, downstream targets have remained undiscovered. Here, we present a systematic screen for Arabidopsis MAPK substrates. Potential MAPK targets were selected on the basis of previously identified in vivo phosphorylation sites that match a MAPK motif. Eight candidates were purified and tested in in vitro kinase assays for phosphorylation by MPK3, -4, -6. These MAPKs clearly differed in their specificity towards these substrates. Phosphorylation sites were mapped by liquid chromatography-tandem mass spectrometry (LC-MS/MS/MS) in phosphoglucose isomerase 1 (PGI1), the putative Golgi SNARE protein GOS12, the mechanosensitive ion channel MSL9, the histone deacetylase HDT2/HD2B and phosphatidylinositol 4-phosphate kinase 4ß1 (PI-4Kß1). This analysis showed that MAPKs phosphorylate multiple residues in each substrate. Moreover, nearly all in vivo sites were phosphorylated in vitro by MAPKs, suggesting that these candidates could be in vivo targets. The phosphosite mapping and subsequent mutagenesis showed that several candidates were phosphorylated on multiple residues. The use of a fluorescent protein reporter showed that a phosphorylation site mutant of HDT2 excludes a subpool of the protein from the nucleolus. Moreover, the use of phosphosite mutants of GOS12 showed that phosphorylation of GOS12 likely mediates Golgi localization and that GOS12 is involved in maintaining Golgi morphology. Treatment of protoplasts with flg22 caused relocalization of GOS12 from the nucleus and cytosol to the Golgi, similar to the phosphomimicking mutant. This selective MS-based approach to connect MAPK isoforms with these MAPK substrates revealed a potential MAPK network regulating vesicular trafficking and histone deacetilation in plants

The Dark Side of the Salad: Salmonella typhimurium Overcomes the Innate Immune Response of Arabidopsis thaliana and Shows an Endopathogenic Lifestyle

Salmonella enterica serovar typhimurium contaminated vegetables and fruits are considerable sources of human infections. Bacteria present in raw plant-derived nutrients cause salmonellosis, the world wide most spread food poisoning. This facultative endopathogen enters and replicates in host cells and actively suppresses host immune responses. Although Salmonella survives on plants, the underlying bacterial infection mechanisms are only poorly understood. In this report we investigated the possibility to use Arabidopsis thaliana as a genetically tractable host system to study Salmonella-plant interactions. Using green fluorescent protein (GFP) marked bacteria, we show here that Salmonella can infect various Arabidopsis tissues and proliferate in intracelullar cellular compartments. Salmonella infection of Arabidopsis cells can occur via intact shoot or root tissues resulting in wilting, chlorosis and eventually death of the infected organs. Arabidopsis reacts to Salmonella by inducing the activation of mitogen-activated protein kinase (MAPK) cascades and enhanced expression of pathogenesis related (PR) genes. The induction of defense responses fails in plants that are compromised in ethylene or jasmonic acid signaling or in the MKK3-MPK6 MAPK pathway. These findings demonstrate that Arabidopsis represents a true host system for Salmonella, offering unique possibilities to study the interaction of this human pathogen with plants at the molecular level for developing novel drug targets and addressing current safety issues in human nutrition

Tractatus || ALEXANDRI || CARERII I. C.|| PATAVINI, DIVI || ANDREAE PRAE-||POSITI || De Sponsalibus et Matrimoniis,|| Libri Quinque.|| IN QVIBVS ... || OMNIA IN || sponsalitiis & matrimonialibus caussis || scitu necessaria explicantur & || docentur.|| Iamprimum in vsum Iurisconsultorum Germaniae || recusi, duplici#́[que] indice donati.||

Variante zu ZV 3089. Erkennungslesart: Leclerum.Vorlageform des Erscheinungsvermerks: 1599.|| FRANCOFVRTI,|| Apud Ioann. Leclerum, Sumptibus Petri Kopffij.|

Impact on respiratory tract infections of heptavalent pneumococcal conjugate vaccine administered at 3, 5 and 11 months of age

Abstract Background Medical and public health importance of pneumococcal infections justifies the implementation of measures capable of reducing their incidence and severity, and explains why the recently marketed heptavalent pneumococcal conjugate vaccine (PCV-7) has been widely studied by pediatricians. This study was designed to evaluate the impact of PCV-7 administered at 3, 5 and 11 months of age on respiratory tract infections in very young children. Methods A total of 1,571 healthy infants (910 males) aged 75–105 days (median 82 days) were enrolled in this prospective cohort trial to receive a hexavalent vaccine (DTaP/IPV/HBV/Hib) and PCV-7 (n = 819) or the hexavalent vaccine alone (n = 752) at 3, 5 and 11 months of age. Morbidity was recorded for the 24 months following the second dose by monthly telephone interviews conducted by investigators blinded to the study treatment assignment using standardised questionnaires. During these interviews, the caregivers and the children's pediatricians were questioned about illnesses and the use of antibiotics since the previous telephone call. All of the data were analysed using SAS Windows v.12. Results Among the 1,555 subjects (98.9%) who completed the study, analysis of the data by the periods of follow-up demonstrated that radiologically confirmed community-acquired pneumonia (CAP) was significantly less frequent in the PCV-7 group during the follow-up as a whole and during the last period of follow-up. Moreover, there were statistically significant between-group differences in the incidence of acute otitis media (AOM) in each half-year period of follow-up except the first, with significantly lower number of episodes in children receiving PCV-7 than in controls. Furthermore, the antibiotic prescription data showed that the probability of receiving an antibiotic course was significantly lower in the PCV-7 group than in the control group. Conclusion Our findings show the effectiveness of the simplified PCV-7 schedule (three doses administered at 3, 5 and 11–12 months of age) in the prevention of CAP and AOM, diseases in which Streptococcus pneumoniae plays a major etiological role. A further benefit is that the use of PCV-7 reduces the number of antibiotic prescriptions. All of these advantages may also be important from an economic point of view.</p

Ciencias sociais: Ciencias políticas e da administración

International audienc

MAP KINASE PHOSPHATASE1 and PROTEIN TYROSINE PHOSPHATASE1 Are Repressors of Salicylic Acid Synthesis and SNC1-Mediated Responses in Arabidopsis[C][W]

Mitogen-activated protein (MAP) kinase phosphatases are important negative regulators of the levels and kinetics of MAP kinase activation that modulate cellular responses. The dual-specificity phosphatase MAP KINASE PHOSPHATASE1 (MKP1) was previously shown to regulate MAP KINASE6 (MPK6) activation levels and abiotic stress responses in Arabidopsis thaliana. Here, we report that the mkp1 null mutation in the Columbia (Col) accession results in growth defects and constitutive biotic defense responses, including elevated levels of salicylic acid, camalexin, PR gene expression, and resistance to the bacterial pathogen Pseudomonas syringae. PROTEIN TYROSINE PHOSPHATASE1 (PTP1) also interacts with MPK6, but the ptp1 null mutant shows no aberrant growth phenotype. However, the pronounced constitutive defense response of the mkp1 ptp1 double mutant reveals that MKP1 and PTP1 repress defense responses in a coordinated fashion. Moreover, mutations in MPK3 and MPK6 distinctly suppress mkp1 and mkp1 ptp1 phenotypes, indicating that MKP1 and PTP1 act as repressors of inappropriate MPK3/MPK6-dependent stress signaling. Finally, we provide evidence that the natural modifier of mkp1 in Col is largely the disease resistance gene homolog SUPPRESSOR OF npr1-1, CONSTITUTIVE 1 (SNC1) that is absent in the Wassilewskija accession. Our data thus indicate a major role of MKP1 and PTP1 in repressing salicylic acid biosynthesis in the autoimmune-like response caused by SNC1