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.

1999, Gröttrup, Marcus, Schmidtke, Gunter

Cytotoxic T cells are able to recognise whether a cell of our body is infected by a virus or whether it has acquired mutations leading to tumour formation. The cells show on their surface what kind of proteins are synthesised intracellularly and whether non-self proteins encoded by a virus or tumour antigens are among them. The proteins are presented not as functionally intact proteins but as peptide fragments which originate from their regular intracellular degradation. This fragmentation is accomplished by the proteasome, a large proteinase complex in the cytoplasm and nucleus of all cells. Upon stimulation with the antiviral cytokine interferon-gamma, subunits of the proteasome are exchanged, thus leading to optimised production of peptide antigens. In this review we introduce the system of antigen processing by the proteasome and sum up our latest results on the question how the interferon-gamma-mediated reorganisation of the proteasome occurs and what consequences and benefits this has for the cytotoxic immune response against viruses and tumours.

1997-04-25, Schmidtke, Gunter, Schmidt, Marion, Kloetzel, Peter M.

The maturation of the eukaryotic 20 S proteasome complex occurs via 13 S and 16 S precursor complexes in a multistep assembly pathway. These precursor complexes contain alpha-subunits as well as unprocessed beta-subunit proproteins. We have purified and characterized the different proteasome assembly intermediates and analysed their ability to support beta-subunit proprotein processing in vitro. Our data show that 13 S and 16 S proteasome precursor complexes differ not only in size but also in their protein content and behaviour during hydrophobic chromatography. By establishing conditions which allowed us to analyse beta-prosubunit maturation in vitro we demonstrate that the processing of the homologous proproteins of the beta-subunits LMP2 and delta essentially takes place in 16 S precursor complexes. No proprotein processing activity was observed in 13 S precursor complexes. Furthermore, proprotein processing in vitro can be inhibited with a proteasome specific inhibitor, but with different efficiency for LMP2 and delta. A peptide, which represents the sequence of the proprotein processing site HGTT, exhibited no inhibitory effect on the processing of either subunit. These data provide further evidence that proprotein processing occurs via an autocatalytic mechanism. Our experiments also demonstrate that the chaperone protein hsc73 is associated with 16 S but not with 13 S precursor complexes. In support of the specificity of this interaction incubation with ATP leads to the dissociation of hsc73 from 16 S complexes and to the formation of high molecular weight aggregates. Prosubunit processing in isolated 16 S complexes does not, however, result in the formation of proteolytically active 20 S proteasomes which may be due to the fact that not all beta-subunits can be efficiently processed in vitro. In contrast to previous assumptions subunit processing and formation of proteolytic activity do not coincide and final 20 S complex assembly seems to represent in part a separate event which requires additional factors or proteins which are not present or active in the purified 16 S precursor complexes.

1996-10-01, Feist, Eugen, Dörner, Thomas, Kuckelkorn, Ulrike, Schmidtke, Gunter, Micheel, B., Hiepe, Falk, Burmester, Gerd R., Kloetzel, Peter-Michael

Autoantibodies occur in low frequencies among patients with myositis characterizing only distinct subsets of this disease. Most of these known antibodies are directed to enzymatically active complexes. The 20S proteasome represents an essential cytoplasmatic protein complex for intracellular nonlysosomal protein degradation, and is involved in major histocompatibility complex class I restricted antigen processing. In this study we investigated whether the 20S proteasome complex is an antibody target in myositis and in other autoimmune diseases. 34 sera of poly/dermatomyositis patients were assayed for antiproteasomal antibodies using enzyme-linked immunosorbent assay, immunoblot, and two-dimensional non-equilibrium pH gradient electrophoresis (NEPHGE). Sera was from patients with systemic lupus erythematosus (SLE), mixed connective tissue disease, and rheumatoid arthritis; healthy volunteers served as controls. In 62% (21/34) of the cases sera from patients with myositis and in 58% (30/52) of the cases sera from patients with SLE reacted with the 20S proteasome. These frequencies exceeded those of sera from patients with mixed connective tissue disease, rheumatoid arthritis, and healthy controls. The alpha-type subunit C9 of the 20S proteasome was determined to be the predominant target of the autoimmune sera in myositis and SLE. Lacking other frequent autoantibodies in myositis, the antiproteasome antibodies are the most common humoral immune response so far detected in this disease entity.

1999, Gröttrup, Marcus, Schmidtke, Gunter

The proteasome is the main nonlysosomal endoprotease in the cytoplasm and nucleus of all eukaryotic cells. It is responsible for the generation of most antigenic peptides as ligands for major histocompatibility complex (MHC) class I proteins. The proteasome hence qualifies as a target for modifying or silencing antigen processing and presentation to cytotoxic T cells, which are important players in transplant rejection and autoimmune disease. The authors summarize recent progress in the understanding of antigen processing by the proteasome and discuss the potential of novel and selective proteasome inhibitors as drugs for suppressing or modifying the cytotoxic immune response.

1999, Raasi, Shahri, Schmidtke, Gunter, Giuli, Rita de, Gröttrup, Marcus

A means of regulating the fate of intracellular proteins is their covalent conjugation to ubiquitin-like proteins. A recently discovered ubiquitin-like protein is called "diubiquitin" because it consists of two ubiquitin-like domains in head-to-tail arrangement. Human diubiquitin is encoded at the telomeric end of the MHC class I locus and was previously found to be expressed in dendritic cells and mature B cells. We have extended the expression analysis of diubiquitin by reverse transcriptase-PCR and Northern blotting in primary endothelial cells and human cancer cell lines derived from nine different tissues. Diubiquitin expression was found to be generally and synergistically inducible with the cytokines IFN-gamma and TNF-alpha but not with IFN-alpha. Diubiquitin mRNA expression was induced within 2 h after cytokine stimulation and was independent of protein neosynthesis but dependent on proteasome activity. The mouse homologue of diubiquitin which is also encoded in the MHC class I locus was likewise induced with IFN-gamma and TNF-alpha. A general and synergistic induction with IFN-gamma and TNF-alpha suggests that diubiquitin may exert its functions in antigen presentation or other cellular processes controlled by these two cytokines.

1997-03, Schmidt, Marion, Schmidtke, Gunter, Kloetzel, Peter-M.

Eukaryotic 20S proteasomes are complex oligomeric proteins. The maturation process of the 14 different alpha- and beta-subunits has to occur in a highly coordinate manner. In addition beta-subunits are synthesized as proproteins and correct processing has to be guaranteed during complex maturation. The structure formation can be subdivided in different phases. The knowledge of the individual phases is summarized in this publication. As a first step the newly synthesized monomers have to adopt the correct tertiary structure, a process that might be supported in the case of the beta-subunits by the intramolecular chaperone activity postulated for the prosequences. Subsequently the alpha-subunits form ring-like structures thereby providing docking sites for the different beta-subunits. The result most likely is a double ring structure (13S precursor) representing half-proteasomes, which contain immature proproteins. Two 13S precursors associate to form the proteolytically inactive 16S assembly intermediate which still contains unprocessed beta-monomers. In addition the chaperone Hsc73 is present within these particles suggesting an essential role during the structure formation process. The processing of monomers with an N-terminal threonine occurs within the 16S particles and is achieved autocatalytically by two subsequent processing events finally leading to the mature, active 20S proteasome.

1999, Schmidtke, Gunter, Holzhütter, Hermann-Georg, Bogyo, Matthew, Kairies, Norman, Groll, Michael, Giuli, Rita de, Emch, Sabine, Gröttrup, Marcus

The human immunodeficiency virus, type I protease inhibitor Ritonavir has been used successfully in AIDS therapy for 4 years. Clinical observations suggested that Ritonavir may exert a direct effect on the immune system unrelated to inhibition of the human immunodeficiency virus, type I protease. In fact, Ritonavir inhibited the major histocompatibility complex class I restricted presentation of several viral antigens at therapeutically relevant concentrations (5 microM). In search of a molecular target we found that Ritonavir inhibited the chymotrypsin-like activity of the proteasome whereas the tryptic activity was enhanced. In this study we kinetically analyzed how Ritonavir modulates proteasome activity and what consequences this has on cellular functions of the proteasome. Ritonavir is a reversible effector of proteasome activity that protected the subunits MB-1 (X) and/or LMP7 from covalent active site modification with the vinyl sulfone inhibitor(125)I-NLVS, suggesting that they are the prime targets for competitive inhibition by Ritonavir. At low concentrations of Ritonavir (5 microM) cells were more sensitive to canavanine but proliferated normally whereas at higher concentrations (50 microM) protein degradation was affected, and the cell cycle was arrested in the G(1)/S phase. Ritonavir thus modulates antigen processing at concentrations at which vital cellular functions of the proteasome are not yet severely impeded. Proteasome modulators may hence qualify as therapeutics for the control of the cytotoxic immune response.

1998, Schmidtke, Gunter, Eggers, Maren, Ruppert, Thomas, Gröttrup, Marcus, Koszinowski, Ulrich, Kloetzel, Peter-M.

Proteasomes generate peptides bound by major histocompatibility complex (MHC) class I molecules. Avoiding proteasome inhibitors, which in most cases do not distinguish between individual active sites within the cell, we used a molecular genetic approach that allowed for the first time the in vivo analysis of defined proteasomal active sites with regard to their significance for antigen processing. Functional elimination of the delta/low molecular weight protein (LMP) 2 sites by substitution with a mutated inactive LMP2 T1A subunit results in reduced cell surface expression of the MHC class I H-2Ld and H-2Dd molecules. Surface levels of H-2Ld and H-2Dd molecules were restored by external loading with peptides. However, as a result of the active site mutation, MHC class I presentation of a 9-mer peptide derived from a protein of murine cytomegalovirus was enhanced about three- to fivefold. Our experiments provide evidence that the delta/LMP2 active site elimination limits the processing and presentation of several peptides, but may be, nonetheless, beneficial for the generation and presentation of others.

1996-12-16, Schmidtke, Gunter, Kraft, R., Kostka, S., Henklein, Petra, Frömmel, Cornelius, Löwe, J., Huber, R., Kloetzel, Peter-Michael, Schmidt, M.

Maturation of eukaryotic 20S proteasomes involves the processing of beta-subunits by limited proteolysis. To study the processing mechanism we analysed different point mutations of the beta-subunit LMP2 in transfected human T2 cells. Here we show that the presence of the intact Gly-1Thr1 consensus motif and Lys33 are essential for correct processing. Mutation of Thr1, the active site residue in mature subunits, or of Lys33, results in complete inhibition of processing at the consensus site. In addition, proprotein processing in vitro of wild-type LMP2, incorporated in immature 16S precursor complexes, can be blocked by a proteasome-specific inhibitor. While the processing of inhibitor-treated wild-type proprotein was completely prevented, the site-directed mutagenesis of LMP2 results in processing intermediates carrying an extension of 8-10 residues preceding Thr1, suggesting an additional cleavage event within the prosequence. Furthermore, exchange of mammalian prosequences interferes with processing efficiency and suggests subunit specificity. Based on our data we propose a model for self-activation of proteasomal beta-subunits in which residue Thr1 serves as nucleophile and Lys33 as proton donor/acceptor. We provide evidence that subunit processing of mammalian beta-subunits proceeds via a novel ordered two-step mechanism involving autocatalysis.