Ralf Stohwasser

20S proteasome from LMP7 knock out mice reveals altered proteolytic activities and cleavage site preferences

20S proteasomes of tissues from LMP7 knock out mice which show reduced MHC class I restricted antigen presentation were analyzed with regard to their subunit composition, peptide hydrolyzing activity and their ability to cleave a synthetic 25‐mer polypeptide. LMP7 deficiency results in an enhanced incorporation of subunit MB1 and in a 2–3.8‐fold increase in V max for the Suc‐LLVY‐MCA hydrolyzing activity. Since LMP7 deficiency also affects the cleavage site preference of 20S proteasomes the reduced MHC class I antigen presentation of LMP7 knock out mice is most likely due to an impairment in peptide generation.

The role of the proteasome system and the proteasome activator PA28 complex in the cellular immune response.

Abstract The generation of antigenic peptides bound and presented to the immune system by MHC class I molecules predominantly depends on the function of the proteasome system. Stimulation of cells with interferon gamma induces the incorporation of three active site bearing β-subunits into the 20S proteasome and the formation of the PA28 proteasome modulator complex. PA28 alters the cleavage properties of the proteasome and enhances MHC class I antigen presentation. Thus, by cytokine induced change of the proteasome system cells may alter the proteolytic properties of the 20S proteasome and may render an organism more flexible in its peptide generation capacity.

Coding strategy of the S and M genomic segments of a hantavirus representing a new subtype of the Puumala serotype

SummaryThe hantavirus strain Vranica was previously reported to have been isolated from a bank vole in Bosnia-Hercegovina and associated with the occurrence of hemorrhagic fever with renal syndrome (HRFS) in humans. The complete cDNA nucleotide sequences of the small (S) and medium (M) genomic RNA segments of this virus were determined. Major open reading frames were found in the S and M segment between nucleotide positions 43 and 1341 coding for a polypeptide of 433 amino acid residues and between nucleotide positions 41 and 3 484 coding for 1 148 amino acid residues, respectively. The analysis and the alignment of the nucleotide and the derived amino acid sequences with known sequences of other hantavirus strains demonstrate that Vranica resembles Swedish strains and represents a new virus subtype of the Puumala serotype distinct from the subtypes represented by virus strains CG18–20 and Sotkamo.

Antigenicity of hantavirus nucleocapsid proteins expressed in E. coli

DNA clones representing the small genomic segment of Nephropathia epidemica virus strain Hällnäs B1 (NEV) and Hantaan virus strain 76-118 (HTV) encoding their nucleocapsid proteins were inserted into the E. coli vector pIN-III-ompA for secretion of proteins into the periplasmic space. The complete HTV and NEV nucleocapsid proteins and two truncated versions of the NEV nucleocapsid proteins were expressed as fusion proteins. Unexpectedly, all products accumulated as insoluble aggregates. Most of the ompA signal peptide remained uncleaved. However, nucleocapsid fusion proteins could be purified from the insoluble fraction by extraction with 8 M urea followed by separation on SDS-PAGE and electroelution. Rabbits were immunized with the eluted proteins and the resulting antibodies reacted specifically with authentic viral nucleocapsid proteins of HTV and NEV. The recombinant nucleocapsid proteins were found to react specifically with various hantavirus-immune sera, but not with human control sera, indicating their suitability as potential diagnostic antigens. This is the first report on the expression of a protein of a NEV serotype strain of hantaviruses by use of recombinant DNA techniques.

Identification of the gene encoding the major capsid protein of insect iridescent virus type 6 by polymerase chain reaction

The gene encoding the major capsid protein of Chilo iridescent virus (CIV) has been identified by PCR using oligonucleotide primers corresponding to different regions of the major capsid proteins of Tipula iridescent virus (TIV) and iridescent virus 22 (IV22). A DNA fragment of 0.5 kbp was amplified using two oligonucleotide primers corresponding to the amino acid positions 146 to 153 and 304 to 313 of the major capsid protein of TIV, respectively. The radioactively labelled DNA fragment derived from PCR was hybridized to a CIV gene library. This analysis revealed that only the EcoRI CIV DNA fragment X [2.85 kbp; 0.589 to 0.603 viral map units (m.u.)] hybridized to the amplified DNA fragment. An RNA transcript of about 1.5 kb was identified when the PCR product was used as a hybridization probe. The same RNA transcript was detected when the EcoRI fragments X and Q (5.9 kbp; 0.603 to 0.631 viral m.u.) were used as probes. This indicates that the expected gene is located within map coordinates 0.589 to 0.631 and harbours part of the DNA sequences of fragments Q and X. The analysis of the DNA sequences of this particular region of the CIV genome revealed the presence of one open reding frame of 1401 bp. The DNA sequences of this region encode a protein of 467 amino acid residues with an M(r) of 51.4K. A high degree (64.7%) of amino acid sequence identity was detected between the major capsid protein of TIV and/or IV22 and the amino acid composition of the identified CIV protein.

Hepatitis B virus HBx peptide 116-138 and proteasome activator PA28 compete for binding to the proteasome alpha4/MC6 subunit.

Abstract PA28 is a modulator of the 20S proteasome. The PA28 binding sites on the 20S proteasome are still not well defined. Using yeast two-hybrid interaction assays and proteasome inactivation kinetics we provide evidence that the proteasome α4 subunit is one of the PA28 binding sites. This finding is supported by the observation that a hepatitis Bvirus X protein-derived polypeptide habouring the α4 proteasome subunit binding motif impairs the activation of 20S proteasomes by PA28.

Polymerase Chain Reaction for Detection of Hantaviruses

The diagnosis of hantaviruses as the etiologic agent of hemorrhagic fever with renal syndrome (HFRS) so far has relied on immunofluorescence assays which require cells infected with pathogenic viruses. In this chapter we describe the use of gene amplification by the polymerase chain reaction (PCR) to diagnose hantavirus infections rapidly. In combination with direct nucleotide sequence analyses or differential oligonucleotide hybridization, PCR can also be used to identify different hantavirus strains and even to detect previously unknown hantaviruses.

Evidence for anti-apoptotic roles of proteasome activator 28γ via inhibiting caspase activity

Proteasome activator PA28γ (REGγ, Ki antigen) has recently been demonstrated to display anti-apoptotic properties via enhancing Mdm2-p53 interaction, thereby facilitating ubiquitination and down-regulation of the tumor suppressor p53. In this study we demonstrate a correlation between cellular PA28γ levels and the sensitivity of cells towards apoptosis in different cellular contexts thereby confirming a role of proteasome activator PA28γ as an anti-apoptotic regulator. We investigated the anti-apoptotic role of PA28γ upon UV-C stimulation in B8 mouse fibroblasts stably overexpressing the PA28γ-encoding PSME3 gene and upon butyrate-induced apoptosis in human HT29 adenocarcinoma cells with silenced PSME3 gene. Interestingly, our results demonstrate that PA28γ has a strong influence on different apoptotic hallmarks, especially p53 phosphorylation and caspase activation. In detail, PA28γ and effector caspases mutually restrict each other. PA28γ is a caspase substrate, if PA28γ levels are low. In contrast, PA28γ overexpression reduces caspase activities, including the caspase-dependent processing of PA28γ. Furthermore, overexpression of PA28γ resulted in a nuclear accumulation of transcriptional active p53. In summary, our findings indicate that even in a p53-dominated cellular context, pro-apoptotic signaling might be overcome by PA28γ-mediated caspase inhibition.

Multiplex localization of sequential peptide epitopes by use of a planar microbead chip

Epitope mapping is crucial for the characterization of protein-specific antibodies. Commonly, small overlapping peptides are chemically synthesized and immobilized to determine the specific peptide sequence. In this study, we report the use of a fast and inexpensive planar microbead chip for epitope mapping. We developed a generic strategy for expressing recombinant peptide libraries instead of using expensive synthetic peptide libraries. A biotin moiety was introduced in vivo at a defined peptide position using biotin ligase. Peptides in crude Escherichia coli lysate were coupled onto streptavidin-coated microbeads by incubation, thereby avoiding tedious purification procedures. For read-out we used a multiplex planar microbead chip with size- and fluorescence-encoded microbead populations. For epitope mapping, up to 18 populations of peptide-loaded microbeads (at least 20 microbeads per peptide) displaying the primary sequence of a protein were analyzed simultaneously. If an epitope was recognized by an antibody, a secondary fluorescence-labeled antibody generated a signal that was quantified, and the mean value of all microbeads in the population was calculated. We mapped the epitopes for rabbit anti-PA28γ (proteasome activator 28γ) polyclonal serum, for a murine monoclonal antibody against PA28γ, and for a murine monoclonal antibody against the hamster polyoma virus major capsid protein VP1 as models. In each case, the identification of one distinct peptide sequence out of up to 18 sequences was possible. Using this approach, an epitope can be mapped multiparametrically within three weeks.