Proteasome-catalyzed peptide splicing (PCPS) represents an additional activity of mammalian 20S proteasomes recently identified in connection with antigen presentation. We show here that PCPS is not restricted to mammalians but that it is also a feature of yeast 20S proteasomes catalyzed by all three active site ␤ subunits. No major differences in splicing efficiency exist between human 20S standard-and immuno-proteasome or yeast 20S proteasome. Using H 2 18 O to monitor the splicing reaction we also demonstrate that PCPS occurs via direct transpeptidation that slightly favors the generation of peptides spliced in cis over peptides spliced in trans. The 20S proteasome with its proteolytically active site ␤-subunits (␤1, ␤2, and ␤5) is a N-terminal nucleophilic hydrolase, widely conserved during evolution from yeast to mammals. It is the central proteolytic machinery of the ubiquitin proteasome system and the catalytic core of the 26S proteasome that is built by the association of 19S regulator complexes with the 20S proteasome. As part of the 26S proteasome the 20S core degrades poly-ubiquitylated proteins to peptides of 3 to 20 residues in length (1). A small percentage of these peptides is transported to the endoplasmic reticulum, bound by major histocompatibility complex (MHC) 1 class I molecules, and presented at the cell surface to CD8ϩ cytotoxic T lymphocyte for immune recognition. This antigen presentation pathway is usually restricted to the proteasome-dependent processing of self-and viral-proteins (2). Antigen presentation is generally increased after IFN-␥ stimuli because it induces, among others, the synthesis of alternative catalytic subunits (␤1i, ␤2i, and ␤5i) and the concomitant formation of immunoproteasomes (i-proteasomes) (2).All active ␤ subunits carry an N-terminal threonine residue as reactive nucleophile. Therefore, their distinct cleavage preferences are determined by the structural features of the substrate binding pockets. In particular, the nonprimed substrate binding site of the active site ␤ subunits binds the residues of the peptide substrate that are located at the N-terminal side of the cleaved residue. The residues of the peptide located C-terminally of the cleavage site are bound by the primed substrate binding site. The binding to both substrate binding sites of the active site ␤ subunit provides the stability and the orientation of the substrate, which is mandatory to carry out the proteolytic cleavage (3).Peptides can be produced by proteasomes during the degradation of proteins or polypeptides by conventional peptide bond hydrolysis or by proteasome-catalyzed peptide splicing (PCPS). The latter has been demonstrated in vivo so far only for four MHC class I-restricted epitopes (4 -8), leading to the assumption that PCPS is most likely a rare event that lacks any wider functional importance (9). PCPS was suggested to occur in a direct transpeptidation reaction, in either cis or trans, by linking two proteasomal cleavage products (PCPs) derived either from the same or from two ...

Secreted proteins (the secretome) of the human pathogen Helicobacter pylori may mediate important pathogen-host interactions, but such proteins are technically difficult to analyze. Here, we report on a comprehensive secretome analysis that uses protein-free culture conditions to minimize autolysis, an efficient recovery method for extracellular proteins, and two-dimensional gel electrophoresis followed by peptide mass fingerprinting for protein resolution and identification. Twenty-six of the 33 separated secreted proteins were identified. Among them were six putative oxidoreductases that may be involved in the modification of protein-disulfide bonds, three flagellar proteins, three defined fragments of the vacuolating toxin VacA, the serine protease HtrA, and eight proteins of unknown function. A cleavage site for the amino-terminal passenger domain of VacA between amino acids 991 and 992 was determined by collision-induced dissociation mass spectrometry. Several of the secreted proteins are interesting targets for antimicrobial chemotherapy and vaccine development.The widespread human pathogen Helicobacter pylori is a major cause of gastric and duodenal ulcers and gastric cancer (48, 53). To identify factors of H. pylori that are potentially involved in pathogen-host interactions (11), proteome analysis has been successfully used by numerous groups (4,7,14,17,21,22,24,29,37,50). Secreted proteins (the secretome) may be of special importance, since these proteins come into direct contact with host compartments; however, technical difficulties have led to somewhat contradictory results and have prevented a comprehensive analysis (8,19,25,34,42,51). H. pylori is commonly cultivated in rich media complemented with various additions of serum containing numerous foreign proteins that are difficult to resolve from extracellular H. pylori proteins. Only a few studies have used protein-free media (1,19,26,38,49,51), and growth is usually much slower in such media. Moreover, H. pylori is particularly prone to spontaneous autolysis (34), resulting in the nonspecific release of numerous proteins; the latter makes the interpretation of protein patterns obtained from culture supernatants difficult.In this study, we optimized culture conditions for minimal autolysis, adapted a precipitation method for the optimal recovery of extracellular proteins, resolved the various secreted proteins by two-dimensional gel electrophoresis, and identified 26 protein species. Based on a comparison of the intensities of staining of specific protein species in supernatants and wholecell samples, we obtained a semiquantitative estimate for secretion selectivity. Among the secreted proteins were several redox-active enzymes, various components of the flagellar apparatus, three fragments of the vacuolating cytotoxin VacA, the serine protease and chaperone HtrA, and several previously uncharacterized proteins that are potential targets for therapy and vaccine development. To our knowledge, this is the first comprehensive analysis of the H. pylor...

Most of the peptides presented by major histocompatibility complex (MHC) class I molecules require processing by proteasomes. Tripeptidyl peptidase II (TPPII), an aminopeptidase with endoproteolytic activity, may also have a role in antigen processing. Here, we analyzed the processing and presentation of the immunodominant human immunodeficiency virus epitope HIV-Nef(73-82) in human dendritic cells. We found that inhibition of proteasome activity did not impair Nef(73-82) epitope presentation. In contrast, specific inhibition of TPPII led to a reduction of Nef(73-82) epitope presentation. We propose that TPPII can act in combination with or independent of the proteasome system and can generate epitopes that evade generation by the proteasome-system.

Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100mel47–52/40–42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100mel47–52/40–42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8+ T cell response. Importantly, we demonstrate that different gp100mel-derived spliced epitopes are generated and presented to CD8+ T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100mel-derived spliced epitopes trigger activation of CD8+ T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes.

Self-assembly of beta-sheet domains resulting in the formation of pathogenic, fibrillar protein aggregates (amyloids) is a characteristic feature of various medical disorders. These include neurodegenerative diseases, such as Alzheimer's, Huntington's, and Creutzfeldt-Jacob's. A significant problem in studying such aggregation processes is the poor solubility of these beta-sheet complexes. The present work describes water-soluble de novo beta-sheet peptides which self-assemble into fibrillar structures. The model peptides enable studies of the relationship between beta-sheet stability and association behavior. The peptides [DPKGDPKG-(VT)n-GKGDPKPD-NH2, n = 3-8] are composed of a central beta-sheet-forming domain (VT-sequence), and N- and C-terminal nonstructured octapeptide sequences which promote water solubility. Conformational analyses by circular dichroism and Fourier transform infrared spectroscopy indicate the influence of peptide length, D-amino acid substitution, and concentration on the ability of the peptides to form stable beta-sheet structures. The association behavior investigated by analytical ultracentrifugation and dynamic light scattering was found to correlate strongly with the stability of a beta-sheet conformation. Model peptides with n >/= 6 form stable, water-soluble beta-sheet complexes with molecular masses of more than 2000 kDa, which are organized in fibrillar structures. The fibrils examined by Congo Red staining and electron microscopy show some similarities with naturally occurring amyloid fibrils.

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This article describes a simple procedure for the detection of phosphorylated peptides by comparable positive and negative ion mode matrix-assisted laser desorption/ionization mass spectrometry measurements. Based on studies with phosphorylated peptides (EAIXAAPFAK, X = pS, pT, pY) and their corresponding non-phosphorylated analogs, it was found that phosphopeptides, which are characterized by a low ionization efficiency in the positive ion mode, exhibit drastically increased signal intensities in the negative ion mode compared to their non-phosphorylated analogs. The effect was successfully used to identify phosphorylated sequences of the commonly used phosphoprotein standards, protein kinase A and beta-casein, by peptide mass fingerprint analyses of the corresponding Lys C and trypsin digests using both (positive and negative) ion modes. The comparison of positive and negative ion spectra of a given protein digest (relative intensity([M - H]-)/relative intensity([M + H]+)) can be used to identify any phosphopeptides present which may then be separated and analyzed further.

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.