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Biomedical subjects

Thomas Ruppert

Publications and source records attributed to Thomas Ruppert.

16 recordsLinked to original sources
PubMed2006

Deletion of the gliP gene of Aspergillus fumigatus results in loss of gliotoxin production but has no effect on virulence of the fungus in a low-dose mouse infection model.

Gliotoxin is a secondary metabolite produced by several fungi including the opportunistic human pathogen Aspergillus fumigatus. As gliotoxin exerts immunosuppressive effects in vitro and in vivo, a role as a virulence determinant in invasive aspergillosis has been discussed for a long time but evidence has not been provided until now. Here, by the use of different selection marker genes A. fumigatus knock-out strains were generated that are deficient for the non-ribosomal peptide synthetase GliP, the putative key enzyme of the gliotoxin biosynthesis. Deletion of the gliP gene resulted in loss of gliotoxin production, as analysed by high performance liquid chromatography and tandem mass spectrometry. No differences in morphology or growth kinetics between wild-type and gliP-deletion strains were observed. In vitro, the culture supernatant of the gliP-deficient strains showed a reduced cytotoxic effect on both macrophage-like cells and T cell lines. In a low-dose murine infection model of invasive aspergillosis, gliotoxin was detected in the lung and absent when mice were infected with the gliP deletion strain. However, gliP deletion strains showed no difference in virulence compared with the corresponding wild-type strains. Taken together, the non-ribosomal peptide synthetase GliP is essential for gliotoxin production in A. fumigatus. Gliotoxin is not required for pathogenicity of the fungus in immunocompromised mice, despite the fact that a reduced cytotoxicity of the culture supernatant of gliP deletion strains was demonstrated.

Animals↗
PubMed2006

Proteomics fingerprinting of phagosome maturation and evidence for the role of a Galpha during uptake.

Phagocytosis, whether of food particles in protozoa or bacteria and cell remnants in the metazoan immune system, is a conserved process. The particles are taken up into phagosomes, which then undergo complex remodeling of their components, called maturation. By using two-dimensional gel electrophoresis and mass spectrometry combined with genomic data, we identified 179 phagosomal proteins in the amoeba Dictyostelium, including components of signal transduction, membrane traffic, and the cytoskeleton. By carrying out this proteomics analysis over the course of maturation, we obtained time profiles for 1,388 spots and thus generated a dynamic record of phagosomal protein composition. Clustering of the time profiles revealed five clusters and 24 functional groups that were mapped onto a flow chart of maturation. Two heterotrimeric G protein subunits, Galpha4 and Gbeta, appeared at the earliest times. We showed that mutations in the genes encoding these two proteins produce a phagocytic uptake defect in Dictyostelium. This analysis of phagosome protein dynamics provides a reference point for future genetic and functional investigations.

Animals↗
PubMed2006

Comparative proteomics of glycosomes from bloodstream form and procyclic culture form Trypanosoma brucei brucei.

Peroxisomes are present in nearly every eukaryotic cell and compartmentalize a wide range of important metabolic processes. Glycosomes of Kinetoplastid parasites are peroxisome-like organelles, characterized by the presence of the glycolytic pathway. The two replicating stages of Trypanosoma brucei brucei, the mammalian bloodstream form (BSF) and the insect (procyclic) form (PCF), undergo considerable adaptations in metabolism when switching between the two different hosts. These adaptations involve also substantial changes in the proteome of the glycosome. Comparative (non-quantitative) analysis of BSF and PCF glycosomes by nano LC-ESI-Q-TOF-MS resulted in the validation of known functional aspects of glycosomes and the identification of novel glycosomal constituents.

Animals↗
PubMed2006

Proteomic analysis of mitochondrial protein turnover: identification of novel substrate proteins of the matrix protease pim1.

ATP-dependent oligomeric proteases are major components of cellular protein quality control systems. To investigate the role of proteolytic processes in the maintenance of mitochondrial functions, we analyzed the dynamic behavior of the mitochondrial proteome of Saccharomyces cerevisiae by two-dimensional (2D) polyacrylamide gel electrophoresis. By a characterization of the influence of temperature on protein turnover in isolated mitochondria, we were able to define four groups of proteins showing a differential susceptibility to proteolysis. The protein Pim1/LON has been shown to be the main protease in the mitochondrial matrix responsible for the removal of damaged or nonnative proteins. To assess the substrate range of Pim1 under in vivo conditions, we performed a quantitative comparison of the 2D protein spot patterns between wild-type and pim1Delta mitochondria. We were able to identify a novel subset of mitochondrial proteins that are putative endogenous substrates of Pim1. Using an in organello degradation assay, we confirmed the Pim1-specific, ATP-dependent proteolysis of the newly identified substrate proteins. We could demonstrate that the functional integrity of the Pim1 substrate proteins, in particular, the presence of intact prosthetic groups, had a major influence on the susceptibility to proteolysis.

ATP-Dependent Proteases↗
PubMed2005

GOME level 1-to-2 data processor version 3.0: a major upgrade of the GOME/ERS-2 total ozone retrieval algorithm.

The global ozone monitoring experiment (GOME) was launched in April 1995, and the GOME data processor (GDP) retrieval algorithm has processed operational total ozone amounts since July 1995. GDP level 1-to-2 is based on the two-step differential optical absorption spectroscopy (DOAS) approach, involving slant column fitting followed by air mass factor (AMF) conversions to vertical column amounts. We present a major upgrade of this algorithm to version 3.0. GDP 3.0 was implemented in July 2002, and the 9-year GOME data record from July 1995 to December 2004 has been processed using this algorithm. The key component in GDP 3.0 is an iterative approach to AMF calculation, in which AMFs and corresponding vertical column densities are adjusted to reflect the true ozone distribution as represented by the fitted DOAS effective slant column. A neural network ensemble is used to optimize the fast and accurate parametrization of AMFs. We describe results of a recent validation exercise for the operational version of the total ozone algorithm; in particular, seasonal and meridian errors are reduced by a factor of 2. On a global basis, GDP 3.0 ozone total column results lie between -2% and +4% of ground-based values for moderate solar zenith angles lower than 70 degrees. A larger variability of about +5% and -8% is observed for higher solar zenith angles up to 90 degrees.

Air Pollutants↗
PubMed2005

Irreversible inactivation of trypanothione reductase by unsaturated Mannich bases: a divinyl ketone as key intermediate.

Trypanothione reductase is a flavoenzyme unique to trypanosomatid parasites. Here we show that unsaturated Mannich bases irreversibly inactivate trypanothione reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. The inhibitory potency of the compounds strongly increased upon storage of the DMSO stock solutions. HPLC, NMR, and mass spectrometry data of potential intermediates revealed a divinyl ketone as the active compound inactivating the enzyme. ESI- and MALDI-TOF mass spectrometry of trypanothione reductase modified by the Mannich base or the divinyl ketone showed specific alkylation of the active site Cys52 by a 5-(2'chlorophenyl)-3-oxo-4-pentenyl substituent. The reaction mechanism and the site of alkylation differ from those in Plasmodium falciparum thioredoxin reductase where the C-terminal redox active dithiol is modified. After deamination, unsaturated Mannich bases are highly reactive in polycondensation with trypanothione. Interaction of these compounds with both trypanothione and trypanothione reductase could account for their potent trypanocidal effect against Trypanosoma brucei.

Animals↗
PubMed2005

Experimental proof for a signal peptidase I like activity in Mycoplasma pneumoniae, but absence of a gene encoding a conserved bacterial type I SPase.

Although the annotation of the complete genome sequence of Mycoplasma pneumoniae did not reveal a bacterial type I signal peptidase (SPase I) we showed experimentally that such an activity must exist in this bacterium, by determining the N-terminus of the N-terminal gene product P40 of MPN142, formerly called ORF6 gene. Combining mass spectrometry with a method for sulfonating specifically the free amino terminal group of proteins, the cleavage site for a typical signal peptide was located between amino acids 25 and 26 of the P40 precursor protein. The experimental results were in agreement with the cleavage site predicted by computational methods providing experimental confirmation for these theoretical analyses.

Amino Acid Sequence↗
PubMed2004

Hepatitis C virus mutation affects proteasomal epitope processing.

The high incidence of hepatitis C virus (HCV) persistence raises the question of how HCV interferes with host immune responses. Studying a single-source HCV outbreak, we identified an HCV mutation that impaired correct carboxyterminal cleavage of an immunodominant HLA-A2-restricted CD8 cell epitope that is frequently recognized by recovered patients. The mutation, a conservative HCV nonstructural protein 3 (NS3) tyrosine to phenylalanine substitution, was absent in 54 clones of the infectious source, but present in 15/21 (71%) HLA-A2-positive and in 11/24 (46%) HLA-A2-negative patients with chronic hepatitis C. In order to analyze whether the mutation affected the processing of the HLA-A2-restricted CD8 cell epitope, mutant and wild-type NS3 polypeptides were digested in vitro with 20S constitutive proteasomes and with immunoproteasomes. The presence of the mutation resulted in impaired carboxyterminal cleavage of the epitope. In order to analyze whether impaired epitope processing affected T cell priming in vivo, HLA-A2-transgenic mice were infected with vaccinia viruses encoding either wild-type or mutant HCV NS3. The mutant induced fewer epitope-specific, IFN-gamma;-producing and fewer tetramer(+) cells than the wild type. These data demonstrate how a conservative mutation in the flanking region of an HCV epitope impairs the induction of epitope-specific CD8(+) T cells and reveal a mechanism that may contribute to viral sequence evolution in infected patients.

Amino Acid Sequence↗
PubMed2003

Analysis of proteins in the spent culture medium of Lupinus albus by electrospray ionisation tandem mass spectrometry.

Lupinus albus cell cultures secrete a large set of hydrolases into their medium with a small number of highly abundant proteins. We have investigated the protein composition of the medium with two different methods, two-dimensional gel electrophoresis-electrospray ionisation tandem mass spectrometry (ESI-MS-MS) and enzymatic analysis. The proteomic approach revealed the presence of several abundant proteins that had been overlooked using standard enzyme assays, e.g. subtilisin-like protease, glucan 1,3-beta-glucosidase, alpha-amylase, chitinase, thaumatin-like protein, and a secretory pathogenesis-related protein. Several low-abundant proteins were readily detectable by enzymatic assays (peroxidases, phosphatase), but could not be found by ESI-MS-MS. Both data sets support the assumed lytic function of the medium, which appears to be similar to that of the plant vacuole.

Culture Media↗
PubMed2003

The roles of intersubunit interactions in exosome stability.

In eukaryotes, at least 10 proteins associate in a 3'-5' exonuclease complex, the exosome, which is involved in the processing of many RNA species. A recent model for the exosome placed six RNase PH-related components in a hexameric ring core structure, with three S1 domain proteins associated with the ring surface. So far, however, this model lacks experimental support. Using a combination of RNA interference, complex affinity purification, and yeast two-hybrid approaches, we show here that the RNase PH homologues are important for maintenance of complex integrity. In contrast, the S1 domain proteins are not required for complex stability, although they are required for exosome function. Our results are partially consistent with the proposed model of the exosome, but indicate a different arrangement of the RNase PH proteins.

Amino Acid Sequence↗
PubMed2002

Evidence for a copper-binding superfamily of the amyloid precursor protein.

The amyloid precursor protein (APP) copper-binding domain (CuBD) has been shown to reduce Cu(II) to Cu(I) and to mediate copper-induced oxidation in vitro. However, little is known about copper binding to the homologous domains of APP and APP family paralogs and orthologs (including amyloid precursor-like proteins from Drosophila melanogaster, Xenopus laevis, and Caenorhabditis elegans) and their effects on Cu-induced oxidation and Cu(I) formation. Here, we show that APP homologues with and without conserved histidine residues at positions 147, 149, and 151 all bind Cu(II). Oxidized peptides were the kinetically favored products of the redox reaction of CuBDs promoting the reduction of Cu(II) to Cu(I). These results reveal a molecular phylogeny-based divergence that has taken place between the ancestral Drosophila APPL and C. elegans APL-1 and the recently evolved APP lineage of CuBDs. Whereas higher species CuBDs have a decreased affinity for Cu(II) and high Cu(II) reducing activities, ancestral CuBDs form very tight binding sites for Cu(II) ions and have low Cu(II) reducing activities. Thus, the APP lineage displays a gain in activity toward promoting Cu(II) reduction and Cu(I) release. The findings suggests that the Cu(II)-binding equilibrium at the phylogenetic stage of Drosophila APPL and C. elegans APL-1 is shifted from the exchangeable Cu(II) pool to the tightly bound, nonexchangeable pool and that ancestral CuBDs may exert antioxidation activities in vivo. The more recently evolved homologues of human APP appear to take advantage of unique redox properties for yet unknown biological functions.

Amino Acid Sequence↗
PubMed2002

Link between organ-specific antigen processing by 20S proteasomes and CD8(+) T cell-mediated autoimmunity.

Adoptive transfer of cross-reactive HSP60-specific CD8(+) T cells into immunodeficient mice causes autoimmune intestinal pathology restricted to the small intestine. We wondered whether local immunopathology induced by CD8(+) T cells can be explained by tissue-specific differences in proteasome-mediated processing of major histocompatibility complex class I T cell epitopes. Our experiments demonstrate that 20S proteasomes of different organs display a characteristic composition of alpha and beta chain subunits and produce distinct peptide fragments with respect to both quality and quantity. Digests of HSP60 polypeptides by 20S proteasomes show most efficient generation of the pathology related CD8(+) T cell epitope in the small intestine. Further, we demonstrate that the organ-specific potential to produce defined T cell epitopes reflects quantities that are relevant for cytotoxic T lymphocyte recognition. We propose tissue-specific antigen processing by 20S proteasomes as a potential mechanism to control organ-specific immune responses.

Amino Acid Sequence↗
PubMed2002

Intramembrane cleavage of microneme proteins at the surface of the apicomplexan parasite Toxoplasma gondii.

Apicomplexan parasites actively secrete proteins at their apical pole as part of the host cell invasion process. The adhesive micronemal proteins are involved in the recognition of host cell receptors. Redistribution of these receptor-ligand complexes toward the posterior pole of the parasites is powered by the actomyosin system of the parasite and is presumed to drive parasite gliding motility and host cell penetration. The microneme protein protease termed MPP1 is responsible for the removal of the C-terminal domain of TgMIC2 and for shedding of the protein during invasion. In this study, we used site-specific mutagenesis to determine the amino acids essential for this cleavage to occur. Mapping of the cleavage site on TgMIC6 established that this processing occurs within the membrane-spanning domain, at a site that is conserved throughout all apicomplexan microneme proteins. The fusion of the surface antigen SAG1 with these transmembrane domains excluded any significant role for the ectodomain in the cleavage site recognition and provided evidence that MPP1 is constitutively active at the surface of the parasites, ready to sustain invasion at any time.

Amino Acid Sequence↗
PubMed2002

Protein-protein cross-linking in the use of the eukaryotic eGST-fusion system.

We describe here an unusual phenomenon in the isolation of protein complexes from eukaryotic cells using expressed GST-fusion proteins. Protein complexes are involved in a large number of regulatory mechanisms. Therefore, the use of tagged fusion proteins is an important tool for isolation of such protein complexes. For this purpose, we used the nuclear factor Alien, described as a corepressor for the thyroid hormone receptor, fused to the eukaryotic eGST and expressed this fusion in human cells. After affinity purification over glutathione-Sepharose using stringent washing steps, we observed several co-purifying bands migrating at molecular weights higher than the GST-Alien fusion protein. These bands appeared specifically in the GST-Alien transfected cell preparations. Surprisingly, using both Western blotting and MALDI-analyses, we revealed that these bands are composed of the GST-Alien protein itself. We hypothesize that overexpressed factors may generate unexpected cross-linking products which can confound the analyses of such affinity-purified complexes. The cross-linking products could not be eliminated by using beta-mercaptoethanol in the gel system and by boiling in SDS-sample buffer. Also, we demonstrate that Western blotting analyses using antibodies directed against both the tag-epitope and the expressed protein of interest can rapidly, reliably, and in a cost-saving manner identify such artifacts, eliminating them from the analyses of potentially interesting interaction partners. Our findings clearly show that the overexpression and purification of proteins from eukaryotic cells may generate unusual structural features that strongly influence complex formation and the migration in SDS-PAGE.

Artifacts↗
PubMed2001

The glycoprotein gp48 of murine cytomegalovirusL proteasome-dependent cytosolic dislocation and degradation.

Degradation of misfolded or unassembled proteins that are co-translationally inserted into the endoplasmic reticulum involves the cytosolic proteasome system. Different principles may exist for the export of proteins into the cytosol for proteasomal degradation. Here we studied the degradation pathway of the viral glycoprotein gp48, a type I transmembrane protein, encoded by the m06 gene of murine cytomegalovirus. In cells stably transfected with the cytomegalovirus m06 gene or infected with the virus itself, two populations of gp48 can be distinguished that have different fates. Complexes of gp48 and the major histocompatibility complex (MHC) class I molecule, are transported to the lysosome for degradation. Unassembled gp48 is degraded by the cytosolic proteasome. Proteasomal inhibitors stabilize the unassembled gp48 in its core-glycosylated and membrane-associated form in the endoplasmic reticulum (ER)-Golgi intermediate compartment. This implicates that both endoplasmic reticulum and ER-Golgi intermediate compartment export gp48 and that degradation is coupled to a functional proteasome. Analysis of gp48 mutants revealed that the cytosolic part of gp48 was not responsible for the proteasome-dependent substrate transport out of the ER-Golgi intermediate compartment. Thus an indirect interaction between the proteasome and its substrate has to be discussed.

3T3 Cells↗