Search

Pathogen infection can activate multiple signaling cascades that ultimately alter the abundance of molecules in cells. This change can be measured both at the transcript and protein level. Studies analyzing the immune response at both levels are, however, rare. Here, we compare transcriptome and proteome data generated after infection of the nematode and model organism Caenorhabditis elegans with the Gram-positive pathogen Bacillus thuringiensis. Our analysis revealed a high overlap between abundance changes of corresponding transcripts and gene products, especially for genes encoding C-type lectin domain-containing proteins, indicating their particular role in worm immunity. We additionally identified a unique signature at the proteome level, suggesting that the C. elegans response to infection is shaped by changes beyond transcription. Such effects appear to be influenced by AMP-activated protein kinases (AMPKs), which may thus represent previously unknown regulators of C. elegans immune defense.

Many proteins are regulated by reversible O-glycosylation and O-phosphorylation. Whereas O-glycosylation of hydroxy-L-proline is common and well investigated, phosphorylation has not been proved so far in vivo, but this post-translational modification is entirely possible. As a first step to identify this phosphoamino acid, we describe both the syntheses of peptides phosphorylated at 4-hydroxy-L-proline and the 1H and 31P NMR parameters of these phosphopeptides. The model peptides were synthesized on solid-phase using Fmoc-strategy. Both natural isomers of 4-hydroxy-L-proline (containing the hydroxyl group in either the cis or trans position) were introduced without side-chain protection. All peptides were globally phosphorylated with O,O'-tert-butyl-N,N-diethylphosphoramidite on the solid phase and cleaved with trifluoroacetic acid. Additionally, we synthesized two classes of phosphonopeptides that mimic phosphopeptides, namely H- and methylphosphonopeptides. The NMR data were based on the model peptide Gly-Gly-Hyp-Ala, which is regarded as a typical random-coil sequence. The NMR parameters showed a significant influence of the phosphate group on the cis-trans isomerization of the Gly-Hyp bond, which may reflect a possible regulation of proteins by changing their local conformations. The 1H and 31P NMR parameters differed for each isomer, and were distinct from the parameters of phosphorylated serine, threonine and tyrosine. These known shifts can be used to identify both cis- and trans-O-phospho-4-hydroxy-L-proline in vivo.

We introduce solid-phase syntheses of H- and methylphosphonopeptides, giving access for the first time to a new class of mimics for o-phosphoamino acids. The model peptides H-GlyGlyXaaAla-OH (Xaa = Ser, Thr) were synthesized on a solid-phase using Fmoc/tBu strategy and HBTU/HOBt activation by incorporation of hydroxyl-protected serine and threonine. As selectively cleavable hydroxyl-protecting groups we used triphenylmethyl and tert-butyldimethylsilyl for both amino acids, as described in the literature. All peptides were phosphitilated with O, O-di-tert-butyl-N,N-diethylphosphoramidite and yielded H-phosphonopeptides after trifluoroacetic acid cleavage. Alternatively we phosphonylated the peptides with O-tert-butyl-N,N-diethyl-P-methylphosphonamidite, which was synthesized by a two-step one-pot procedure starting from commercially available chemicals. All H- and methylphosphonopeptides were obtained in high purities and yields, as shown by reversed-phase high-performance liquid chromatography and anion-exchange chromatography. The phosphonopeptides were characterized by 1H and 31P NMR. We confirmed their molecular masses by electrospray mass spectrometry and analyzed their fragmentation schemes, which seemed to be characteristic for each class of analogues. The H-phosphonopeptides lost phosphonic acid (H3PO3, 82 mass units) and the methylphosphonopeptides lost methylphosphonic acid (MeH2PO3, 96 mass units). Both H- and methylphosphonopeptides represent a new and simply accessible class of mimics for phosphopeptides. Compared with the corresponding phosphopeptides all phosphonopeptides were synthesized in higher yields and purities (> 80%).

Control of protein activity by phosphorylation appears to work principally by inducing conformational change, but the mechanisms so far reported are dependent on the structural context in which phosphorylation occurs. As the activity of many small peptides is also regulated by phosphorylation, we decided to investigate possible direct consequences of this on the preferred backbone conformation. We have performed 1H nuclear magnetic resonance (NMR) experiments with short model peptides of the pattern Gly-Ser-Xaa-Ser, where Xaa represents Ser, Thr, or Tyr in either phosphorylated or unphosphorylated form and with either free or blocked amino and carboxy termini. The chemical shifts of amide protons and the 3JNH-Hα coupling constants were estimated from one-dimensional and two-dimensional scalar correlated spectroscopy (COSY) spectra at different pH values. The results clearly indicate a direct structural effect of serine and threonine phosphorylation on the preferred backbone dihedrals independent of the presence of charged groups in the surrounding sequence. Tyrosine phosphorylation does not induce such a charge-independent effect. Additionally, experiments with p-fluoro- and p-nitro-phenylalanine-containing peptides showed that the mere presence of an electronegative group on the aromatic ring of tyrosine does not produce direct structural effects. In the case of serine and threonine phosphorylation a strong dependence of the conformational shift on the protonation level of the phosphoryl group could be observed, showing that phosphorylation induces the strongest effect in its dianionic, i.e., physiological, form. The data reveal a hitherto unknown mechanism that may be added to the repertoire of conformational control of peptides and proteins by phosphorylation.

Background The human neuroblastoma cell line, SH-SY5Y, is a commonly used cell line in studies related to neurotoxicity, oxidative stress, and neurodegenerative diseases. Although this cell line is often used as a cellular model for Parkinson’s disease, the relevance of this cellular model in the context of Parkinson’s disease (PD) and other neurodegenerative diseases has not yet been systematically evaluated. Results We have used a systems genomics approach to characterize the SH-SY5Y cell line using whole-genome sequencing to determine the genetic content of the cell line and used transcriptomics and proteomics data to determine molecular correlations. Further, we integrated genomic variants using a network analysis approach to evaluate the suitability of the SH-SY5Y cell line for perturbation experiments in the context of neurodegenerative diseases, including PD. Conclusions The systems genomics approach showed consistency across different biological levels (DNA, RNA and protein concentrations). Most of the genes belonging to the major Parkinson’s disease pathways and modules were intact in the SH-SY5Y genome. Specifically, each analysed gene related to PD has at least one intact copy in SH-SY5Y. The disease-specific network analysis approach ranked the genetic integrity of SH-SY5Y as higher for PD than for Alzheimer’s disease but lower than for Huntington’s disease and Amyotrophic Lateral Sclerosis for loss of function perturbation experiments. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-1154) contains supplementary material, which is available to authorized users.

In dieser Studie sollten Erkenntnisse über Schutzmechanismen gegenüber oxidativem Stress gewonnen werden. Als Induktor oxidativen Stresses wurde die hyperbare Hyperoxie verwendet. Zunächst wurde anhand von Probanden bestätigt, dass hyperbare Oxygenation in vivo in hoch signifikantem Maß oxidative Schädigung in PBMCs induzieren kann. Weiterhin wurde die in der Literatur beschriebene Möglichkeit der körpereigenen Adaptation in prospektivem Ansatz überprüft, indem die basale oxidative Schädigung in PBMCs von Probanden nach repetitiver Sauerstoffexposition sowie die Vulnerabilität dieser PBMCs gegenüber hyperbarer Oxygenation untersucht wurde. Die Ergebnisse legten nahe, dass bis etwa zur dritten Woche ein wirkungsvoller adaptiver Schutz besteht. Ursächlich für diesen adaptiven Schutz sind entsprechend der in der Literatur vorherrschenden Meinung die Aktivierung von antioxidativ wirksamen Enzymen sowie die Induktion der Translation spezialisierter antioxidativ wirksamer Proteine. Diesbezüglich kommt auch eine Akkumulation von Antioxidantien, z. B. den antioxidativen Vitaminen C und E in Frage. Anknüpfend an die in der Literatur beschriebene mögliche Schutzwirkung von Vitamin C (VC) wurde im Modell der hyperbaren Oxigenierung anhand von PBMCs gefunden, dass VC in moderaten Konzentrationen DNA-Schädigung reduzieren kann, dass jedoch sehr hohe VC-Konzentrationen zu einer Vermehrung der DNA-Schädigung führen. Die Ergebnisse dieser Studie legen nahe, dass eine Adaptation an oxidativen Stress durch Sauerstofftraining möglich ist, sowie, dass Vitamin C über eine antioxidative Kapazität verfügt.

Proteasen übernehmen als hydrolytische Enzyme zentrale Funktionen in den verschiedensten physiologischen sowie pathologischen Prozessen und sind deshalb von sehr großem Interesse für die Entwicklung neuer Krankheitstherapien. Zur Analyse proteasekatalysierter Reaktionen gewann die Massenspektrometrie in den letzten Jahren zunehmend an Bedeutung. In der vorliegenden Arbeit wurden auf dieser Technologie basierende Methoden entwickelt und auf drei verschiedene Fragestellungen angewendet: (i) Bestimmung der Spaltspezifität von ADAM10 und ADAM17, (ii) Zeitaufgelöste Analyse proteasekatalysierter Reaktionen mittels LC-MS und (iii) Identifizierung von Meprin-alpha/-beta Spaltstellen in Interleukin-6. Proteases are key mediators in a variety of physiological and pathological processes. For this reason the hydrolytic enzymes present as an interesting target for the development of novel drugs. Mass spectrometry-based methods became increasingly important for the analysis of protease-catalyzed reactions within the last years. The present work focuses on the development and application of mass spectrometry-based strategies with regard to three different projects: (i) deciphering the cleavage site specificities of ADAM10 and ADAM17, (ii) time-dependent monitoring of protease-catalyzed reactions by LC-MS and (iii) identification of meprin-alpha/-beta cleavage sites in interleukin-6.