Bartonella Adhesin A Mediates a Proangiogenic Host Cell Response

Bartonella henselae causes vasculoproliferative disorders in humans. We identified a nonfimbrial adhesin of B. henselae designated as Bartonella adhesin A (BadA). BadA is a 340-kD outer membrane protein encoded by the 9.3-kb badA gene. It has a modular structure and contains domains homologous to the Yersinia enterocolitica nonfimbrial adhesin (Yersinia adhesin A). Expression of BadA was restored in a BadA-deficient transposon mutant by complementation in trans. BadA mediates the binding of B. henselae to extracellular matrix proteins and to endothelial cells, possibly via β1 integrins, but prevents phagocytosis. Expression of BadA is crucial for activation of hypoxia-inducible factor 1 in host cells by B. henselae and secretion of proangiogenic cytokines (e.g., vascular endothelial growth factor). BadA is immunodominant in B. henselae–infected patients and rodents, indicating that it is expressed during Bartonella infections. Our results suggest that BadA, the largest characterized bacterial protein thus far, is a major pathogenicity factor of B. henselae with a potential role in the induction of vasculoproliferative disorders

Drug-induced eRF1 degradation promotes readthrough and reveals a new branch of ribosome quality control.

Suppression of premature termination codons (PTCs) by translational readthrough is a promising strategy to treat a wide variety of severe genetic diseases caused by nonsense mutations. Here, we present two potent readthrough promoters-NVS1.1 and NVS2.1-that restore substantial levels of functional full-length CFTR and IDUA proteins in disease models for cystic fibrosis and Hurler syndrome, respectively. In contrast to other readthrough promoters that affect stop codon decoding, the NVS compounds stimulate PTC suppression by triggering rapid proteasomal degradation of the translation termination factor eRF1. Our results show that this occurs by trapping eRF1 in the terminating ribosome, causing ribosome stalls and subsequent ribosome collisions, and activating a branch of the ribosome-associated quality control network, which involves the translational stress sensor GCN1 and the catalytic activity of the E3 ubiquitin ligases RNF14 and RNF25

Identification of disease-associated MHC-ligands by mass spectrometry

In dieser Arbeit wurden neue T-zellunabhängige Methoden zur gezielten Identifizierung krankheitsassoziierter MHC-Liganden durch massenspektrometrische Verfahren entwickelt und getestet. Der 'Predict, Calibrate, Detect' (PCD)-Ansatz, eine Verbindung von Epitopvorhersage und HPLC-Massenspektrometrie, ermöglichte die Identifizierung neuer HLA-A2-Liganden aus den tumorassoziierten Proteinen CEA, p53 und MAGE-A1 mit einer Empfindlichkeit von bis zu <10 Kopien/Zelle. Für CEA694-702 konnte zudem auch die Präsentation auf einem soliden Gastrointestinalkarzinom nachgewiesen werden. MAGE-A1278-286 konnte inzwischen in transgenen Mäusen als CTL-Epitop eindeutig verifiziert werden, für die Liganden aus p53 und CEA sind Versuche dazu noch im Gange. Diese Peptide sind Kandidaten für eine peptidspezifische Immuntherapie bei Tumoren. Die Voraussetzung für den PCD-Ansatz und weitere Ansätze zur Identifizierung von CTL-Epitopen aus definierten Antigenen sind allelspezifische Peptidmotive, die eine Epitopvorhersage für diese Protein erlauben. Mit der Bestimmung des Peptidmotive für HLA-B*1510- und HLA-DQ(a1*0201/b1*0202) wurden hierfür die Grundlagen erweitert. Daneben gelang die Charakterisierung des ersten CTL-Epitops aus dem Borna Disease Virus, das zugleich den ersten virusspezifischen MHC-Liganden aus der Ratte darstellt. Weiterhin konnten durch Nanoelektrospray-MS/MS-Analyse MHC-Klasse II-Liganden aus dem IDDM-relevanten Autoantigen GAD65 identifiziert werden und damit eine neue Methode zur spezifischen Detektion von MHC-Liganden aus einem definierten, radioaktiv markierten Antigen erfolgreich getestet werden. Schließlich konnten mit der puromycin-sensitiven Aminopeptidase und der Bleomycinhydrolase zytosolische Proteasen durch Nanoelektrospray-MS/MS-Analyse identifiziert werden, die wahrscheinlich an der N-terminalen Verkürzung proteasomaler Verdauprodukte beteiligt sind und damit eine wichtige Rolle bei der MHC Klasse I-Antigenprozessierung spielen könnten.This thesis describes the development and evaluation of novel T cell-independent strategies for the identification of disease-associated MHC-ligands by mass spectrometry. The 'Predict, Calibrate and Detect' (PCD)-approach, a combination of epitope prediction and HPLC-mass spectrometry, allowed the identification of new HLA-A2-ligands from the tumor-associated proteins CEA, p53 and MAGE-A1 with a sensitivity of <10 copies/cell. Moreover, for CEA694-702 presentation on a solid gastrointestinal carcinoma could be shown. MAGE-A1278-286 could be verified as a CTL-epitope in transgenic mice, while the p53 and CEA-derived ligands are still under investigation. These peptides are candidates for the peptide-specific immunotherapy of tumors. Allel-specific peptide motifs are the main prerequisite for the PCD-approach as well as for other strategies that employ epitope prediction for the identification of CTL-epitopes from defined antigens. The determination of the peptide motifs of HLA-B*1510 and HLA-DQ(a1*0201/b1*0202) has paved the way for epitope prediction for these alleles. Moreover, the first Borna Disease Virus-derived CTL-epitope could be characterized. which also represents the first known virus-derived MHC-ligands from the rat. Furthermore, MHC class II-ligands from the diabetes-relevant autoantigen GAD65 could be identified by nanoelectrospray MSMS analysis, using a novel approach for the specific detection of MHC-ligands from a defined antigen by radioactive labeling of the protein. Finally, nanoelectrospray MSMS analysis allowed the identification of the cytosolic proteases puromycin-sensitive aminopeptidase and bleomycin hydrolase, that are probably involved in N-terminal trimming of proteasomal digestion products and might play an important role in the generation of MHC class I-ligands

Characterizing drug-target interactions in complex tissues

Recently, Perrin et al. reported that the coupling of the cellular shift assay with an unbiased proteomics read-out (Thermal Proteome Profiling, TPP) can be applied to complex tissue samples from model organisms and patients. This study demonstrates that TPP enables organ-specific target engagement and identification studies even in patient-derived material

Identifying Compound Efficacy Targets in Phenotypic Drug Discovery

The identification of the efficacy target(s) for hits from phenotypic compound screens remains a key step to progress compounds into drug development. In addition to efficacy targets, the characterization of epistatic nodes influencing compound activity often facilitates the elucidation of the underlying mechanism of action, and, further, early determination of off-targets that cause potentially unwanted secondary phenotypes helps in assessing potential liabilities of a scaffold. This short review discusses the most important technologies currently available for characterizing the direct and indirect target space of bioactive compounds following phenotypic screening. We present a comprehensive strategy leveraging complementary approaches to balance individual strengths and weaknesses

Disclosure of compounds to UCB for NB-CPACT

Previously blinded compounds that were screened: compounds to be disclosed and could be included in a future publication. Edited version of previous list: see OAK 35929 Compounds included here have been circulated to GDCLeaders.IP group and feedback included to modify disclosur

Arf1p Provides an Unexpected Link between COPI Vesicles and mRNA in Saccharomyces cerevisiae

The small GTPase Arf1p is involved in different cellular processes that require its accumulation at specific cellular locations. The recruitment of Arf1p to distinct points of action might be achieved by association of Arf1p with different proteins. To identify new interactors of Arf1p, we performed an affinity chromatography with GTP- or GDP-bound Arf1p proteins. A new interactor of Arf1p-GTP was identified as Pab1p, which binds to the polyA-tail of mRNAs. Pab1p was found to associate with purified COPI-coated vesicles generated from Golgi membranes in vitro. The stability of the Pab1p–Arf1p complex depends on the presence of mRNA. Both symmetrically distributed mRNAs as well as the asymmetrically localized ASH1 mRNA are found in association with Arf1p. Remarkably, Arf1p and Pab1p are both required to restrict ASH1 mRNA to the bud tip. Arf1p and coatomer play an unexpected role in localizing mRNA independent and downstream of the SHE machinery. Hereby acts the SHE machinery in long-range mRNA transport, whereas COPI vesicles could act as short-range and localization vehicles. The endoplasmic reticulum (ER)–Golgi shuttle might be involved in concentrating mRNA at the ER