Katharina Janek

Proteome Analysis of Secreted Proteins of the Gastric Pathogen Helicobacter pylori

ABSTRACT 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.

Water-soluble beta-sheet models which self-assemble into fibrillar structures.

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 Alzheimers, Huntingtons, and Creutzfeldt-Jacobs. 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.

The Bipartite Nuclear Localization Sequence of Rpn2 Is Required for Nuclear Import of Proteasomal Base Complexes via Karyopherin αβ and Proteasome Functions

26 S proteasomes fulfill final steps in the ubiquitin-dependent degradation pathway by recognizing and hydrolyzing ubiquitylated proteins. As the 26 S proteasome mainly localizes to the nucleus in yeast, we addressed the question how this 2-MDa multisubunit complex is imported into the nucleus. 26 S proteasomes consist of a 20 S proteolytically active core and 19 S regulatory particles, the latter composed of two subcomplexes, namely the base and lid complexes. We have shown that 20 S core particles are translocated into the nucleus as inactive precursor complexes via the classic karyopherin αβ import pathway. Here, we provide evidence that nuclear import of base and lid complexes also depends on karyopherin αβ. Potential classic nuclear localization sequences (NLSs) of base subunits were analyzed. Rpn2 and Rpt2, a non-ATPase subunit and an ATPase subunit of the base complex, harbor functional NLSs. The Rpt2 NLS deletion yielded wild type localization. However, the deletion of the Rpn2 NLS resulted in improper nuclear proteasome localization and impaired proteasome function. Our data support the model by which nuclear 26 S proteasomes are assembled from subcomplexes imported by karyopherin αβ

Mapping of O-GlcNAc Sites of 20 S Proteasome Subunits and Hsp90 by a Novel Biotin-Cystamine Tag

The post-translational modification of proteins with O-GlcNAc is involved in various cellular processes including signal transduction, transcription, translation, and nuclear transport. This transient protein modification enables cells or tissues to adapt to nutrient conditions or stress. O-Glycosylation of the 26 S proteasome ATPase subunit Rpt2 is known to influence the stability of proteins by reducing their proteasome-dependent degradation. In contrast, knowledge of the sites of O-GlcNAcylation on the subunits of the catalytic core of the 26 S proteasome, the 20 S proteasome, and the impact on proteasome activity is very limited. This is predominantly because O-GlcNAc modifications are often substoichiometric and because 20 S proteasomes represent a complex protein mixture of different subtypes. Therefore, identification of O-GlcNAcylation sites on proteasome subunits essentially requires effective enrichment strategies. Here we describe an adapted β-elimination-based derivatization method of O-GlcNAc peptides using a novel biotin-cystamine tag. The specificity of the reaction was increased by differential isotopic labeling with either “light” biotin-cystamine or deuterated “heavy” biotin-cystamine. The enriched peptides were analyzed by LC-MALDI-TOF/TOF-MS and relatively quantified. The method was optimized using bovine α-crystallin and then applied to murine 20 S proteasomes isolated from spleen and brain and murine Hsp90 isolated from liver. Using this approach, we identified five novel and one known O-GlcNAc sites within the murine 20 S proteasome core complex that are located on five different subunits and in addition two novel O-GlcNAc sites on murine Hsp90β, of which one corresponds to a previously described phosphorylation site.

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.

Ecm29 Fulfils Quality Control Functions in Proteasome Assembly

The proteasome, the central protease of eukaryotic cells, is composed of one core particle (CP) and one or two adjacent regulatory particles (RP), which contain multiple subunits. Several proteasome-dedicated chaperones govern the assembly of CP and RP, respectively. We sought for proteins that regulate final steps of RP-CP assembly in yeast and found Ecm29, a conserved HEAT-like repeat protein. Here, we show that Ecm29 controls the integrity of RP-CP assemblies. Ecm29 recognizes RP-CP species in which CP maturation is stalled due to the lack of distinct beta subunits. Reconstitution assays revealed that Ecm29 functions as scaffold protein during the remodeling of incompletely matured RP-CP assemblies into regular enzymes. Upon the completion of CP maturation, Ecm29 is degraded and RP-CP is dissociated.

Driving Forces of Proteasome-catalyzed Peptide Splicing in Yeast and Humans

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 β subunits. No major differences in splicing efficiency exist between human 20S standard- and immuno-proteasome or yeast 20S proteasome. Using H218O 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. Splicing efficiency itself is shown to be controlled by proteasomal cleavage site preference as well as by the sequence characteristics of the spliced peptides. By use of kinetic data and quantitative analyses of PCPS obtained by mass spectrometry we developed a structural model with two PCPS binding sites in the neighborhood of the active Thr1.

Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes.

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.

Characterization of intermolecular ?-sheet peptides by mass spectrometry and hydrogen isotope exchange

The self-assembly of beta-sheet peptide domains resulting in the formation of fibrillar aggregates (amyloids) is a feature of various neurodegenerative disorders. In order to evaluate mass spectrometric methods for the characterization of intermolecular beta-sheet structures the hydrogen/deuterium exchange behaviour of model peptides DPKGDPKG-(VT)(n)-GKGDPKPD-amide (n = 3,4,5,6,7,8), (VT)(n)-peptides, composed of a central beta-sheet-forming domain and N- and C-terminal nonstructured octapeptide sequences, was measured by electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The kinetic analysis of the hydrogen/deuterium exchange (HX) shows that intermolecular beta-sheet structures contain slowly exchanging protons (k </=0.001 1/min). Localization of beta-sheet domains was achieved by monitoring the hydrogen exchange of peptide fragments generated via collision-induced dissociation (CID) or post source decay (PSD). The hydrogen exchange kinetics and the beta-sheet domains determined by ESI- and MALDI-MS were found to correlate with the length and stability of the beta-structure domain of the (VT)(n)-peptides.

The Efficiency of Human Cytomegalovirus pp65 495-503 CD8 + T Cell Epitope Generation Is Determined by the Balanced Activities of Cytosolic and Endoplasmic Reticulum-Resident Peptidases

Control of human CMV (HCMV) infection depends on the cytotoxic activity of CD8+ CTLs. The HCMV phosphoprotein (pp)65 is a major CTL target Ag and pp65495–503 is an immunodominant CTL epitope in infected HLA-A*0201 individuals. As immunodominance is strongly determined by the surface abundance of the specific epitope, we asked for the components of the cellular Ag processing machinery determining the efficacy of pp65495–503 generation, in particular, for the proteasome, cytosolic peptidases, and endoplasmic reticulum (ER)-resident peptidases. In vitro Ag processing experiments revealed that standard proteasomes and immunoproteasomes generate the minimal 9-mer peptide epitope as well as N-terminal elongated epitope precursors of different lengths. These peptides are largely degraded by the cytosolic peptidases leucine aminopeptidase and tripeptidyl peptidase II, as evidenced by increased pp65495–503 epitope presentation after leucine aminopeptidase and tripeptidyl peptidase II knockdown. Additionally, with prolyl oligopeptidase and aminopeptidase B we identified two new Ag processing machinery components, which by destroying the pp65495–503 epitope limit the availability of the specific peptide pool. In contrast to cytosolic peptidases, silencing of ER aminopeptidases 1 and 2 strongly impaired pp65495–503-specific T cell activation, indicating the importance of ER aminopeptidases in pp65495–503 generation. Thus, cytosolic peptidases primarily interfere with the generation of the pp65495–503 epitope, whereas ER-resident aminopeptidases enhance such generation. As a consequence, our experiments reveal that the combination of cytosolic and ER-resident peptidase activities strongly shape the pool of specific antigenic peptides and thus modulate MHC class I epitope presentation efficiency.