Biomedical subjects

Markus Schirle

Publications and source records attributed to Markus Schirle.

13 recordsLinked to original sources

PubMed2006

Computational prediction of proteotypic peptides for quantitative proteomics.

Mass spectrometry-based quantitative proteomics has become an important component of biological and clinical research. Although such analyses typically assume that a protein's peptide fragments are observed with equal likelihood, only a few so-called 'proteotypic' peptides are repeatedly and consistently identified for any given protein present in a mixture. Using >600,000 peptide identifications generated by four proteomic platforms, we empirically identified >16,000 proteotypic peptides for 4,030 distinct yeast proteins. Characteristic physicochemical properties of these peptides were used to develop a computational tool that can predict proteotypic peptides for any protein from any organism, for a given platform, with >85% cumulative accuracy. Possible applications of proteotypic peptides include validation of protein identifications, absolute quantification of proteins, annotation of coding sequences in genomes, and characterization of the physical principles governing key elements of mass spectrometric workflows (e.g., digestion, chromatography, ionization and fragmentation).

Algorithms↗

PubMed2006

Proteome survey reveals modularity of the yeast cell machinery.

Protein complexes are key molecular entities that integrate multiple gene products to perform cellular functions. Here we report the first genome-wide screen for complexes in an organism, budding yeast, using affinity purification and mass spectrometry. Through systematic tagging of open reading frames (ORFs), the majority of complexes were purified several times, suggesting screen saturation. The richness of the data set enabled a de novo characterization of the composition and organization of the cellular machinery. The ensemble of cellular proteins partitions into 491 complexes, of which 257 are novel, that differentially combine with additional attachment proteins or protein modules to enable a diversification of potential functions. Support for this modular organization of the proteome comes from integration with available data on expression, localization, function, evolutionary conservation, protein structure and binary interactions. This study provides the largest collection of physically determined eukaryotic cellular machines so far and a platform for biological data integration and modelling.

Genome, Fungal↗

PubMed2005

Multiple synergizing factors contribute to the strength of the CD8+ T cell response against listeriolysin O.

Immunodominance in CD8+ T cell responses against Listeria monocytogenes is a well-recognized but still not fully understood phenomenon. From listeriolysin, the major virulence factor of L. monocytogenes, only a single epitope, pLLO91-99, is presented by MHC class I molecules in BALB/c mice which dominates the cytotoxic T cell response against this bacterial pathogen. To obtain more insights into the molecular and cellular mechanisms underlying immunodominance of this particular epitope, we compared the various steps involved in the presentation and recognition of pLLO91-99 derived from a wild-type toxin with an equivalent epitope from a mutated toxin. This fully functional variant contains within the pLLO91-99 epitope a conservative isoleucine to alanine replacement at the C-terminal anchor residue which results in loss of antigenicity. The binding properties of the variant peptide to soluble Kd remained unaffected and cytotoxic T cells capable of recognizing the pLLO99A/Kd complex were detectable in BALB/c mice. However, such T cells required higher concentrations of antigen in order to be optimally activated in vitro. A comparison between the TAP translocation efficiency of wild-type and mutant peptide demonstrated that the mutation at the C-terminus leads to a reduced transportation rate. Furthermore, the amino acid substitution changes the in vitro proteasomal cleavage pattern, resulting in a reduced liberation of the correct peptide from a polypeptide precursor. Thus, in all assays employed the immunodominant epitope performs optimally while the variant was found to be inferior. The synergy of all these steps most likely is the decisive factor in the immunodominance of pLLO91-99.

Amino Acid Substitution↗

PubMed2005

Scoring proteomes with proteotypic peptide probes.

Technologies for genome-wide analyses typically undergo a transition from a discovery phase to a scoring phase. In the discovery phase, the genomic universe is explored and all pertinent data are noted. In the scoring phase, relevant entities are screened to reveal groups of genes that are associated with specific biological processes or conditions. In this article, we propose that the transition from a discovery to a scoring phase is also essential, feasible and imminent for proteomics.

Amino Acid Sequence↗

PubMed2004

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 beta1 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.

Adhesins, Bacterial↗

PubMed2004

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.

ADP-Ribosylation Factor 1↗

PubMed2004

A 14-3-3 protein of Chlamydomonas reinhardtii associated with the endoplasmic reticulum: nucleotide sequence of the cDNA and the corresponding gene and derived amino acid sequence.

Two major 14-3-3 proteins of the unicellular green alga Chlamydomonas reinhardtii were purified and partially sequenced. The obtained data show that the 30-kDa isoform predominant in the cytosol is encoded by a previously cloned and sequenced 14-3-3 cDNA whereas the 27-kDa isoform represents a new 14-3-3 protein which is largely associated with the endoplasmic reticulum (ER). Therefore, the corresponding cDNA was cloned and sequenced. The nucleotide sequence of this new cDNA species and the derived amino acid sequence differ considerably from the previously cloned Chlamydomonas 14-3-3 cDNA. The conclusion that the divergent evolution of the corresponding genes must have started rather early as compared to the 14-3-3 genes of other organisms was corroborated by their different genomic organization. The amino acid sequences of both 14-3-3 isoforms were comparatively analysed to find differences which might be responsible for their differential binding to the ER.

14-3-3 Proteins↗

PubMed2004

A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway.

Signal transduction pathways are modular composites of functionally interdependent sets of proteins that act in a coordinated fashion to transform environmental information into a phenotypic response. The pro-inflammatory cytokine tumour necrosis factor (TNF)-alpha triggers a signalling cascade, converging on the activation of the transcription factor NF-kappa B, which forms the basis for numerous physiological and pathological processes. Here we report the mapping of a protein interaction network around 32 known and candidate TNF-alpha/NF-kappa B pathway components by using an integrated approach comprising tandem affinity purification, liquid-chromatography tandem mass spectrometry, network analysis and directed functional perturbation studies using RNA interference. We identified 221 molecular associations and 80 previously unknown interactors, including 10 new functional modulators of the pathway. This systems approach provides significant insight into the logic of the TNF-alpha/NF-kappa B pathway and is generally applicable to other pathways relevant to human disease.

Animals↗

PubMed2003

Profiling core proteomes of human cell lines by one-dimensional PAGE and liquid chromatography-tandem mass spectrometry.

Protein expression profiles vary considerably between human cell lines and tissues, which is in part a reflection of their specialized roles within an organism. It is of considerable practical use to establish which proteins constitute the primary components of the respective proteomes. When compiled into databases, such information can facilitate the assessment of selectivity and specificity of a wide range of proteomic experiments. Here we describe the major constituents of proteomes of six human immortalized cell lines. By employing a combination of one-dimensional SDS-PAGE and nanocapillary liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified up to 1785 non-redundant cytoplasmic and nuclear proteins from a single cell line using 50 and 30 microg of total protein from the corresponding fractions. Up to 38 proteins could be identified from a single band in one liquid chromatography-MS/MS experiment. When combined with systematic gridding of gel lanes into 48 slices, a dynamic range for protein identification of approximately 1:2000 can be envisaged for this approach. Identified proteins range from 4-553 kDa in size, cover the pI range between 3.4 and 12.8, and include 255 proteins with predicted transmembrane domains. Repeated analysis of peptides derived from the same gel band showed that the reproducibility of nanocapillary liquid chromatography-MS/MS of such complex mixtures is about 60-70% suggesting that a particular analytical experiment would need to be repeated about three times to arrive at a representative estimate of the set of highly abundant proteins in a given proteome. Given its technical simplicity, sensitivity, and wealth of generated information, we have adopted this experimental approach to characterize every cell line and tissue that is the subject of experimentation in our laboratory. The combined dataset for the six cell lines consists of 2341 non-redundant human proteins and thus constitutes one of the largest collections of human proteomic data published to date.

Cell Line↗

PubMed2002

Integrated functional genomics approach for the design of patient-individual antitumor vaccines.

Our aim is to identify as many candidates as possible for tumor-associated T-cell epitopes in individual patients. First, we performed expression profiling of tumor and normal tissue to identify genes exclusively expressed or overexpressed in the tumor sample. Then, using mass spectrometry, we characterized up to 77 different MHC ligands from the same tumor sample. Several of the MHC ligands were derived from overexpressed gene products, one was derived from a proto-oncogene, and another was derived from a frameshift mutation. At least one was identified as an actual T-cell epitope. Thus, we could show that by combining these two analytic tools, it is possible to propose several candidates for peptide-based immunotherapy. We envision the use of this novel integrated functional genomics approach for the design of antitumor vaccines tailored to suit the needs of each patient.

Antigens, Neoplasm↗

PubMed2002

Expression of the proteasome activator PA28 rescues the presentation of a cytotoxic T lymphocyte epitope on melanoma cells.

The proteasome system represents a major source of HLA class I- presented peptides exposed to CTLs. Stimulation of cells with IFN-gamma instantly induces the expression of the proteasome immunosubunits as well as the proteasome activator PA28. These proteins have been shown to optimize class I antigen presentation of several viral CTL epitopes; however, their contribution to tumor antigen processing remains poorly understood. Here, we analyzed the generation of an HLA-A*0201-presented epitope derived from the melanoma antigen tyrosinase-related protein 2 (TRP2). Melanoma cells that lacked the IFN-gamma-inducible proteasome activator PA28 and immunoproteasomes did not display the TRP2(360-368) epitope to specific CTLs. Our experiments demonstrate that epitope presentation correlated with the presence of PA28 and could be completely rescued by restoration of PA28 expression. In vitro digestion of TRP2 polypeptides with 20S proteasomes confirmed that PA28 is essential for epitope liberation. Thus, our experiments indicate that PA28 provides the threshold for CTL recognition of this epitope. Importantly, processing of a second TRP2-derived epitope, TRP2(288-296), was diminished in IFN-gamma-treated cells, even in the absence of immunoproteasome up-regulation. Therefore, the reported IFN-gamma-induced self-regulation of epitopes may not necessarily be a consequence of immunoproteasomes as suggested previously.

Amino Acid Sequence↗

PubMed2002

Identification of tumor-associated HLA-ligands in the post-genomic era.

Over 10 years ago, the identification of the first tumor-specific T cell epitope shed light on the molecular principles underlying the phenomenon of tumor eradication by the immune system. Since then, a considerable number of different approaches for this task have been introduced and employed successfully, reflecting the growing knowledge about the cellular processes preceding antigen presentation as well as significant technical developments. This review tries to give an overview over available conventional strategies as well as current developments that utilize the potent large-scale screening tools of the post-genomic era.

Animals↗

PubMed2001

The Biosynthesis of Vancomycin-Type Glycopeptide Antibiotics-The Order of the Cyclization Steps This work was supported by the Deutsche Forschungsgemeinschaft (SFB 323) and by a grant of the EU (MEGATOP, QLK3-1999-00650). R. D. S. gratefully acknowledges the support of a Feodor-Lynen Fellowship granted by the Alexander-von-Humboldt Stiftung. We thank Corina Bihlmaier and Volker Pfeifer for help with transformation and Southern hybridization, J. A. Moss (La Jolla (USA)) for critical comments on the manuscript and Prof. Dr. M. E. Maier and Prof. Dr. H.-P. Fiedler (Tübingen) for generous support.

Explore the source record for details and available documents.

Journal Article↗