In vitro reconstitution of hnRNP particles

AbstractThe assembly of hnRNP-like particles was studied by in vitro reconstitution, UV-crosslinking and CsCl-equilibrium centrifugation. Using total nuclear protein and RNA extracts from HeLa cells for RNP reconstitution, RNP particles sedimenting with the same buoyant density of ϱ=1.4 g/cm3 as ‘native’ 40 S core hnRNPs were obtained. Under the stringent reconstitution conditions used, hnRNP complexes containing only the C1-core hnRNP protein could be identified

Interferon y Stimulation Modulates the Proteolytic Activity and Cleavage Site Preference of 20S Mouse Proteasomes

The proteasome is a 700-kD multisubunit enzyme complex with several proteolytically active sites. The enzyme complex is involved in both ubiquitin-dependent and -independent protein degradation and may contribute to the processing of antigens presented by major histocompatibility complex (MHC) class I molecules. Here we demonstrate that treatment of mouse fibroblast cells with 20 U interferon qr (IFN-y) for 3 d induces a change in the proteasome subunit composition and that the B-type subunit LMP2, which is encoded in the MHC class II region, is incorporated into the enzyme complex. This is paralleled by reduction of the homologous 6-subunit. IFN-3' stimulation results in a downregulation of the chymotrypsin-like Suc-LLVY-MCA peptide hydrolyzing activity of 20S proteasomes whereas the trypsin-like activity remains unaffected. When tested as a substrate a synthetic 25-mer polypeptide whose sequence covers the antigenic nonapeptide YPHFMPTNL of the MCMV pp89, 20S proteasomes of IFN-3'-induced cells exhibit altered chymotrypsin-like cleavage site preferences. In the absence of IFN-qr induction, the naturally processed nonamer peptide that is presented by MHC class.I molecules appears as a minor cleavage product. IFN-'y activation does not result in an increase of the final peptide but results in a different set of peptides. We hypothesize that these peptides represent precursor peptides that can be trimmed to final peptide size

The proteasome inhibitor PI31 competes with PA28 for binding to 20S proteasomes

AbstractPI31 is a previously described inhibitor of 20S proteasomes. Using recombinant PI31 we have analyzed its effect on proteasomal hydrolyzing activity of short fluorogenic substrates and of a synthetic 40-mer polypeptide. In addition, we investigated its influence on the activation of 20S proteasome by the proteasome activator PA28. PI31 inhibits polypeptide degradation already at concentrations which only partially inhibit fluorogenic substrate turnover and immunosubunits do not influence the PI31 binding affinity. Furthermore our data demonstrate that PI31 is a potent competitor of PA28-mediated activation

Exposure to Melan-A/MART-126-35 tumor epitope specific CD8+T cells reveals immune escape by affecting the ubiquitin-proteasome system (UPS)

Efficient processing of target antigens by the ubiquitin-proteasome-system (UPS) is essential for treatment of cancers by T cell therapies. However, immune escape due to altered expression of IFN-γ-inducible components of the antigen presentation machinery and consequent inefficient processing of HLA- dependent tumor epitopes can be one important reason for failure of such therapies. Here, we show that short-term co-culture of Melan-A/MART-1 tumor antigen-expressing melanoma cells with Melan-A/MART-126-35-specific cytotoxic T lymphocytes (CTL) led to resistance against CTL-induced lysis because of impaired Melan-A/MART-126-35 epitope processing. Interestingly, deregulation of p97/VCP expression, which is an IFN-γ-independent component of the UPS and part of the ER-dependent protein degradation pathway (ERAD), was found to be essentially involved in the observed immune escape. In support, our data demonstrate that re-expression of p97/VCP in Melan-A/MART-126-35 CTL-resistant melanoma cells completely restored immune recognition by Melan-A/MART-126-35 CTL. In conclusion, our experiments show that impaired expression of IFN-γ-independent components of the UPS can exert rapid immune evasion of tumor cells and suggest that tumor antigens processed by distinct UPS degradation pathways should be simultaneously targeted in T cell therapies to restrict the likelihood of immune evasion due to impaired antigen processing

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 α and β 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

Multi-level Strategy for Identifying Proteasome-Catalyzed Spliced Epitopes Targeted by CD8+ T Cells during Bacterial Infection

Proteasome-catalyzed peptide splicing (PCPS) generates peptides that are presented by MHC class I molecules, but because their identification is challenging, the immunological relevance of spliced peptides remains unclear. Here, we developed a reverse immunology-based multi-level approach to identify proteasome-generated spliced epitopes. Applying this strategy to a murine Listeria monocytogenes infection model, we identified two spliced epitopes within the secreted bacterial phospholipase PlcB that primed antigen-specific CD8+ T cells in L. monocytogenes-infected mice. While reacting to the spliced epitopes, these CD8+ T cells failed to recognize the non-spliced peptide parts in the context of their natural flanking sequences. Thus, we here show that PCPS expands the CD8+ T cell response against L. monocytogenes by exposing spliced epitopes on the cell surface. Moreover, our multi-level strategy opens up opportunities to systematically investigate proteins for spliced epitope candidates and thus strategies for immunotherapies or vaccine design

The 20S Proteasome Splicing Activity Discovered by SpliceMet

The identification of proteasome-generated spliced peptides (PSP) revealed a new unpredicted activity of the major cellular protease. However, so far characterization of PSP was entirely dependent on the availability of patient-derived cytotoxic CD8+ T lymphocytes (CTL) thus preventing a systematic investigation of proteasome-catalyzed peptide splicing (PCPS). For an unrestricted PSP identification we here developed SpliceMet, combining the computer-based algorithm ProteaJ with in vitro proteasomal degradation assays and mass spectrometry. By applying SpliceMet for the analysis of proteasomal processing products of four different substrate polypeptides, derived from human tumor as well as viral antigens, we identified fifteen new spliced peptides generated by PCPS either by cis or from two separate substrate molecules, i.e., by trans splicing. Our data suggest that 20S proteasomes represent a molecular machine that, due to its catalytic and structural properties, facilitates the generation of spliced peptides, thereby providing a pool of qualitatively new peptides from which functionally relevant products may be selected