Biomedical subjects

Thomas Ruppert

Publications and source records attributed to Thomas Ruppert.

8 recordsLinked to original sources

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↗