2019-09, Schmidtke, Gunter, Schregle, Richard, Alvarez Salinas, Gerardo Omar, Huber, Eva M., Gröttrup, Marcus

The 20S immunoproteasome (IP) is an interferon(IFN)-γ - and tumor necrosis factor (TNF) -inducible variant of the 20S constitutive proteasome (CP) in which all its peptidolytically active subunits β1, β2, and β5 are replaced by their cytokine inducible homologues β1i (LMP2), β2i (MECL-1), and β5i (LMP7). These subunit replacements alter the cleavage specificity of the proteasome and the spectrum of proteasome-generated peptide ligands of MHC class I molecules. In addition to antigen processing, the IP has recently been shown to serve unique functions in the generation of pro-inflammatory T helper cell subtypes and cytokines as well as in the pathogenesis of autoimmune diseases, but the mechanistic involvement of the IP in these processes has remained elusive. In this study we investigated whether the IP differs from the CP in the interaction with two IFN-γ/TNF inducible factors: the 11S proteasome regulator PA28αβ and the ubiquitin-like modifier FAT10 (ubiquitin D). Using thermophoresis, we determined the affinity of PA28αβ for the CP and IP to be 12.2nM +/- 2.8nM and 15.3nM +/- 2.7nM, respectively, which is virtually identical. Also the activation of the peptidolytic activities of the IP and CP by PA28αβ did not differ. For FAT10 we determined the degradation kinetics in cycloheximide chase experiments in cells expressing almost exclusively IP or CP as well as in IFN-γ stimulated and unstimulated cells and found no differences between the degradation rates. Taken together, we conclude that neither differences in the binding strength to, nor activation by PA28αβ, nor a difference in the rate of FAT10-mediated degradation can account for distinct functional capabilities of the IP as compared to the CP.

2008, Gröttrup, Marcus, Pelzer, Christiane, Schmidtke, Gunter, Hofmann, Kay

Since its discovery in 1981, ubiquitin-activating enzyme 1 was thought to be the only E1-type enzyme responsible for ubiquitin activation. Recently, a relatively uncharacterized E1 enzyme, designated ubiquitin-like modifier activating enzyme 6, was also shown to activate ubiquitin. Ubiquitin-activating enzyme 1 and ubiquitin-like modifier activating enzyme 6 are both essential proteins, and each uses a different spectrum of ubiquitin-conjugating (E2) enzymes. Ubiquitin-like modifier activating enzyme 6 activates not only ubiquitin, but also the ubiquitin-like modifier FAT10 (human leukocyte antigen F-associated transcript 10), which, similarly to ubiquitin, serves as a signal for proteasomal degradation. This new layer of regulation in ubiquitin activation markedly increases the versatility of the ubiquitin conjugation system.

2001, Khan, Selina, De Giuli, Rita, Schmidtke, Gunter, Bruns, Michael, Buchmeier, Michael, Van den Broek, Maries, Gröttrup, Marcus

CTLs recognize peptide epitopes which are proteolytically generated by the proteasome and presented on MHC class I molecules. According to the defective ribosomal product (DRiP) hypothesis, epitopes originate from newly synthesized polypeptides which are degraded shortly after their translation. The DRiP hypothesis would explain how epitopes can be generated from long-lived proteins. We examined whether neosynthesis is required for presentation of the immunodominant epitope NP118 of the lymphocytic choriomeningitis virus nucleoprotein, which has a half-life of >3 days. Two days after nucleoprotein biosynthesis was terminated in a tetracycline-regulated transfectant, the presentation of the NP118 epitope ceased. This indicates that NP118 epitopes are generated from newly synthesized nucleoproteins rather than from the long-lived pool of nucleoproteins in the cell. Therefore, the lymphocytic choriomeningitis virus nucleoprotein is the first substrate for which a major prediction of the DRiP hypothesis, namely the requirement for neosynthesis, is shown to hold true.

1999-07, Küttner, G., Kramer, Achim, Schmidtke, Gunter, Giessmann, Elke, Dong, L., Roggenbuck, D., Scholz, C., Seifert, Martina, Stigler, Rolf-Dietrich, Schneider-Mergener, Jens

Single-chain Fv fragments (scFv) were generated from two murine monoclonal antibodies directed to the neutralizing epitopes of the pre-S1 and pre-S2 region of hepatitis B virus, respectively, using different assembly cloning strategies. The scFv fragments were solubly expressed in E. coli. Dissociation constants were in the nanomolar range for all forms (whole IgG antibodies, Fab fragment and scFv fragments). The epitopes of both antibodies were mapped using solid phase peptide synthesis on continuous cellulose membranes and turned out to be linear determinants. The minimal epitope for the anti-pre-S2 antibody 1F6 was identified to be DPRVRGLYF (amino acid 133-141 of the pre-S region). For the anti-pre-S1 antibody MA 18/7 the minimal epitope proved to be the hexamer LDPAFR (amino acid 30-35 of the pre-S region). Complete substitutional analyses as well as truncation experiments revealed key residues for these antibody-antigen interactions. On the basis of those results we used computer-assisted modeling techniques to suggest models for both antibody-peptide interactions providing insight into the structural basis of these molecular recognitions.

2019, Aichem, Annette, Boehm, Annika N., Catone, Nicola, Schmidtke, Gunter, Gröttrup, Marcus

The ubiquitin-like modifier FAT10 (also called ubiquitin D (UBD)) interacts noncovalently with a substantial number of proteins and also gets covalently conjugated to many substrate proteins, leading to their degradation by the 26S proteasome. FAT10 comprises two loosely folded ubiquitin-like domains that are connected by a flexible linker, and this unusual structure makes it highly prone to aggregation. Here, we report methods to purify high amounts of soluble recombinant FAT10 for various uses, such as in vitro FAT10ylation assays. In addition, we describe how to generate and handle overexpressed as well as endogenous FAT10 in cellulo for use in immunoprecipitations, Western blot analyses, and FAT10 degradation studies.

2008, Lukasiak, Sebastian, Breuhahn, Kai, Schiller, Claudia, Schmidtke, Gunter, Gröttrup, Marcus

Due to the fact that mutations and up- or downregulation of genes can lead to the development of cancer, quantitative comparison of relative gene expression in healthy and cancerous tissue can gain valuable insights into tumorigenesis. While the semi-quantitative DNA microarrays are being used to identify differentially expressed genes on a genomic scale, real-time RT-PCR provides a powerful tool for quantitative measurement of gene expression. Presently, it is the most sensitive method available. Here we describe in detail a SYBR GreenI-based assay using the LightCycler® instrument to measure the levels of mRNA for the ubiquitin-like protein FAT10 relative to 18S rRNA in human hepatocellular carcinoma tissue. This method can be easily adapted to any tissue (human or mouse, rat, etc.) and any gene.

2000, Schwarz, Katrin, Giuli, Rita de, Schmidtke, Gunter, Kostka, Susanne, Broek, Maries van den, Kim, Kyung Bo, Crews, Craig M., Kraft, Regine, Gröttrup, Marcus

The complete inhibition of proteasome activities interferes with the production of most MHC class I peptide ligands as well as with cellular proliferation and survival. In this study we have investigated how partial and selective inhibition of the chymotrypsin-like activity of the proteasome by the proteasome inhibitors lactacystin or epoxomicin would affect Ag presentation. At 0.5-1 microM lactacystin, the presentation of the lymphocytic choriomeningitis virus-derived epitopes NP118 and GP33 and the mouse CMV epitope pp89-168 were reduced and were further diminished in a dose-dependent manner with increasing concentrations. Presentation of the lymphocytic choriomeningitis virus-derived epitope GP276, in contrast, was markedly enhanced at low, but abrogated at higher, concentrations of either lactacystin or epoxomicin. The inhibitor-mediated effects were thus epitope specific and did not correlate with the degradation rates of the involved viral proteins. Although neither apoptosis induction nor interference with cellular proliferation was observed at 0.5-1 microM lactacystin in vivo, this concentration was sufficient to alter the fragmentation of polypeptides by the 20S proteasome in vitro. Our results indicate that partial and selective inhibition of proteasome activity in vivo is a valid approach to modulate Ag presentation, with potential applications for the treatment of autoimmune diseases and the prevention of transplant rejection.

2008, Schmidtke, Gunter, Gröttrup, Marcus

The 26S proteasome is the executing protease of the ubiquitin-dependent degradation system. It consists of one or two 19S regulatory sub-complexes and one 20S proteolytic sub-complex (1). The 20S proteasome is a barrel-shaped cylinder which consists or four stacked rings (2). Each of the two outer rings consists of seven different alpha-subunits, whereas each of the two inner rings is formed by seven different beta-subunits (3). Only three of these beta-subunits bear a catalytically active N-terminal threonine (4,5). Under normal conditions, these are beta1 (delta), beta2 (Z), and beta5 (mb1). However, by induction of some cytokines, e.g., interferon-gamma, these subunits are exchanged against beta1i(LMP2), beta2i (Mecl1), and beta5i (LMP7) and the so-called immunoproteasome is formed (6,7). To investigate the role of LMP7 in MHC class I-restricted immunology, we decided to generate a transgenic mouse which constitutively expresses LMP7 in all tissues. To get the highest possible expression, we bread the mice to be homozygous for the transgene LMP7. These mice cannot be identified by conventional polymerase chain reaction (PCR). So far, Southern blotting was the only possible method to quantify the DNA content. Here, we describe the analysis of these mice by quantitative PCR using sequence specific fluorescence resonance energy transfer-primers to reliably detect a difference in DNA content as small as a factor of 2 or only one PCR cycle.

2001, Gröttrup, Marcus, Broek, Marlies van den, Schwarz, Katrin, Macagno, Annalisa, Khan, Selina, Giuli, Rita de, Schmidtke, Gunter

The proteasome is the main provider of peptide ligands for major histocompatibility complex class I molecules. During an immune response to pathogens, the proinflammatory cytokine interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha are released, which induce the proteasome subunits LMP2, LMP7, and MECL-1. These replace the constitutively expressed active site subunits of the proteasome (delta, MB1, and Z) leading to a marked change in the cleavage preference of the proteasome and the production of T-cell epitopes. Proteasome activity is further changed by the IFN-gamma-mediated induction of the proteasome regulator PA28alpha/beta and the downregulation of PA28gamma. Why such an extensive exchange of proteasome active site subunits and regulators occurs is still poorly understood. In this article we discuss recent insights in the structural consequences of proteasome reorganization and their effects on epitope generation and shaping of the cytotoxic immune response. Moreover, we review the latest data on how the ubiquitin pathway targets protein antigens for peptide processing and discuss the potential of proteasome inhibitors for the modulation of antigen presentation.

2000, Schwarz, Katrin, Van den Broek, Maries, Kostka, Susanne, Kraft, Regine, Soza, Andrea, Schmidtke, Gunter, Klötzel, Peter, Gröttrup, Marcus

The proteasome is a large protease complex that generates most of the peptide ligands of MHC class I molecules either in their final form or in the form of N-terminally extended precursors. Upon the stimulation of cells with IFN-gamma, three constitutively expressed subunits of the 20S proteasome are replaced by the inducible subunits LMP2 (low-molecular mass polypeptide 2), LMP7, and MECL-1 (multicatalytic endopeptidase complex-like-1) to form so-called immunoproteasomes. We show in this study that overexpression of these three subunits in triple transfectants led to a marked enhancement in the H-2Ld-restricted presentation of the immunodominant nonameric epitope NP118, which is derived from the nucleoprotein (NP) of lymphocytic choriomeningitis virus. Overexpression of the alpha and beta subunits of the IFN-gamma-inducible proteasome regulator PA28, in contrast, did not have a comparable effect. In vitro, immunoproteasomes as compared with constitutive proteasomes generated higher amounts of 11- and 12-mer fragments containing the NP118 epitope. These are likely to be cytosolic precursors of NP118, as a proline anchor residue in the second position of NP118 may interfere with TAP-mediated transport of the nonameric epitope itself. In conclusion, we provide evidence that up-regulation of the three inducible subunits, LMP2, LMP7, and MECL-1, can result in a marked improvement of Ag presentation and that, depending on the epitope, PA28 and immunoproteasomes may differentially affect Ag processing.