MHC-Klasse-I präsentierte Epitope werden überwiegend durch den proteasomalen Abbau von Poly-Ubiquitin markierten Proteinen und defekten ribosomalen Produkten (DRiPs) generiert. Die post-proteasomale Prozessierung durch zytosolische Exo- und Endopeptidasen führt jedoch hauptsächlich zur Epitop-Zerstörung und nur ein sehr geringer Anteil der Peptide entkommt der Degradation. Bisher ist noch unklar, wie die enzymatischen Aktivitäten des heterogenen Peptidase-Pools im Zytosol die finale Epitop-Prozessierung beeinflussen. In der vorliegenden Arbeit wurden heteromere Interaktionen von zytosolischen Peptidasen analysiert und ihre Wirkung auf die Prozessierung und Präsentation von proteasomal generier-ten Vorläuferpeptiden in Bezug auf die HCMVpp65495-503 Epitop-Generierung untersucht. Glycerolgradientenzentrifugationen und Immunpräzipitationsexperimente zeigten, dass die zytosolischen Peptidasen Nardilysin (NRDc) und Aminopeptidase-B (AP-B) in den gleichen Fraktionen sedimentieren und zu heteromeren Komplexen interagieren. Die siRNA- abhängige Reduktion der Proteinexpression beider Peptidasen hatte einen positiven Effekt auf die HCMVpp65 spezifische CTL-Antwort. Demnach vermindert der Peptidase-Komplex die HCMVpp65-spezifische Epitop-Präsentation auf der Zelloberfläche. Im Gegensatz dazu bewirkte ein in vitro rekonstituierter trimerer Peptidase-Komplex jedoch die verstärkte HCMVpp65 Epitop-Generierung aus einem proteasomal generierten Vorläuferpeptid. Auf der anderen Seite führte gereinigte AP-B zu der anhaltenden Zerstörung des Epitops. Die Ergeb-nisse deuten somit darauf hin, dass sowohl einzelne als auch verschiedene Interaktionen von zytosolischen Peptidasen die Prozessierung und Präsentation des HCMVpp65-Epitops unterschiedlich modulieren und somit die HCMVpp65-spezifische antivirale Immunantwort beeinflussen. / MHC class I presented antigens are generated by the degradation of poly- ubiquitinated pro-teins and defective ribosomal products (DRiPs) by a major protease, the 26S proteasome. However, the post- proteasomal processing by cytosolic exo-and endopeptidases mainly leads to epitope destruction and only a very small proportion of the peptides escape degradation. So far, it is still unclear how the enzymatic activity of the heterogeneous pool of peptidases in the cytosol affects final epitope processing and therewith immune response. In the present work heteromeric interactions of cytosolic peptidases and their effect on pro-cessing and presentation of proteasomal generated peptides were analysed with regard to HCMVpp65495-503 epitope generation. Glycerol gradient centrifugation and immunoprecipitation experiments indicate that the cyto-solic peptidases Nardilysine (NRDc) and Aminopeptidase B (AP-B) sediment in the same fractions and interact to heteromeric complexes. The siRNA dependent reduction of protein expression of these two peptidases had a positive effect on the HCMVpp65 specific CTL re-sponse. Thus the peptidase complex reduces HCMVpp65 epitope presentation on the cell sur-face possibly due to epitope destruction. In contrast to the findings of the CTL assays, an in vitro reconstituted trimeric peptidase complex resulted in the increased generation of HCMVpp65 epitopes from a proteasomal generated peptide precursor. On the other hand pu-rified AP-B led to the ongoing destruction of the epitope. The findings obtained show that single cytosolic peptidases and various interactions of cytosolic peptidases regulate the pro-cessing and presentation of the HCMVpp65 epitope differently, thereby influencing the HCMV-specific antiviral immune response.

The cytosolic protein degradation pathway, performed by ATP-dependent proteases and ATP-independent peptidases, plays important roles in several cellular activities, e.g. cell division, cell cycle progression, intracellular signaling, MHC class I antigen presentation, host-pathogen interactions, etc. The roles of ATP-dependent proteases during stress and infection have been studied in great detail but the functional roles of ATP-independent peptidases are not clearly understood. In this study, the functional roles of E. coli or S. typhimurium encoded Peptidase N (PepN), an ATP-independent enzyme belonging to theM1 family of metallopeptidases, were investigated. The thesis will address four different aspects. (i) In the first part, the utility of using E coli ∆pepN to identify and characterize novel peptidases will be shown. It is known that deletion of pepN leads to inability to cleave the majority of in vitro peptidase substrates in E. coli and S. typhimurium. To study the differences between two closely related paralogs of the M17 family, E. coli encoded pepA and pepB were cloned in pBAD24 vector and introduced in E. coli ∆pepN. Peptidase A (PepA) and Peptidase B (PepB) expression increases the cleavage of several aminopeptidase substrates and partially rescues growth of ∆pepN during nutritional downshift and high temperature stress (NDHT), a dual stress involving growth in minimal media at 42°C. Purified PepA and PepB enzymes display broad substrate specificity; however, distinct differences are observed between these two paralogs: PepA is more stable at high temperature whereas PepB displays broader substrate specificity as it cleaves Asp and Insulin B chain peptide. The strategy utilized in this study, i.e. overexpression of peptidases in ∆pepN followed by screening for substrate specificities in total cell extracts, may be used to rapidly identify the substrate preferences of novel peptidases encoded in genomes of different organisms. (ii) The second aspect investigates the functional roles of PepN during stress and infection in S. typhimurium. PepN has two conserved signature motifs of the M1 family, GAMEN and HEXXH, which play roles in substrate recognition and catalysis. To address the roles of catalytic activity of PepN, the residue E-298, which is present in the HEXXH motif and acts as a general base during catalysis, was mutated to A-298 by site-specific mutagenesis and introduced into ∆pepN (pBR322/pepNE298A). Biochemical and biophysical analysis of purified PepN (WT and E298A) revealed loss of catalytic activity of E298A but no major structural changes were observed in comparison to the WT protein. The functional roles of this mutation using ∆pepN expressing pBR322/pepN or pBR322/pepNE298A were investigated using two conditions: (i) Nutritional downshift high temperature (NDHT)stress and (ii) systemic infection in mice. Monitoring growth profiles of different strains demonstrated the requirement of the enzymatic activity of PepN for adaptation and growth to NDHT stress. Earlier studies have shown that S. typhimurium ∆pepN hyper proliferates in peripheral organs during systemic infection in mice. However, expression of wild type (WT)or E298A PepN led to lower colony forming units (CFU), demonstrating that the decrease in CFU is independent of catalytic activity. These observations are consistent with lower serum amounts of inflammatory cytokines, lower tissue damage and increase in survival of mice infected with S. typhimurium expressing WT or E298A PepN. (iii) Although pathogen encoded peptidases are known to be important during infection, their roles in modulating host responses in immunocompromised individuals are not well studied. In the third part of this thesis, the roles of S. typhimurium encoded PepN were studied in mice lacking Interferon-γ (Ifnγ), a cytokine important for immunity. S. typhimurium lacking pepN displays enhanced CFU compared to WT in peripheral organs during systemic infection in C57BL/6 mice. However, Ifnγ-/-mice show higher CFU compared to C57BL/6 mice, resulting in lower fold differences between WT and ∆pepN. Concomitantly, reintroduction of pepN in ∆pepN reduces CFU, demonstrating pepN dependence. In addition, three distinct differences were observed between infection ofC57BL/6 and Ifnγ-/-mice upon infection with different S. typhimurium strains: (i) cytokine profiles, (ii) histological analysis and (iii) mice survival. Overall, the roles of the host encoded Ifnγ during infection with S. typhimurium strains with varying degrees of virulence will be highlighted. (iv) The final aspect of this study reveals differences in gene expression between S. typhimurium grown in rich medium (Luria-Bertani) versus NDHT stress. This adaptation affects several pathways and the gene expression of secretory proteins that are important for virulence in S. typhimurium are greatly reduced during NDHT stress. Also, analysis of secretory protein amounts in different media conditions shows reduction during growth in minimal media plus high temperature stress. The functional consequences of this reduction in secretory protein amounts lead to lower bacterial replication after infection of RAW cells or mice infected via the oral route. In addition, the differences in gene expression between WT and ∆pepN during these conditions were studied. Interestingly, there is reduction in expression of flagellar genes whereas the genes involved in nitrogen metabolism are upregulated in ∆pepN upon exposure to NDHT stress. Further studies were performed by quantifying the motility of different S. typhimurium strains grown in a variety of culture conditions. Overall, this part of the study attempts to compare and contrast the possible adaptive responses of WT and ∆pepN to NDHT stress. Together, this thesis addresses multiple aspects of the biochemistry and roles of the enigmatic PepN during stress and infection.

Expression of the kallikrein-related peptidase 7 (KLK7) is dysregulated in ovarian cancer. We assessed KLK7 expression by ELISA and quantitative immunohistochemistry and analyzed its association with clinicopathological parameters and patients’ outcome. KLK7 antigen concentrations were determined in tumor tissue extracts of 98 ovarian cancer patients by ELISA. For analysis of KLK7 immunoexpression in ovarian cancer tissue microarrays, a manual quantitative scoring system as well as a software tool for quantitative high-throughput automated image analysis was used. In immunohistochemical analyses, expression levels of KLK7 were not associated with patients’ outcome. However, in multivariate analyses, KLK7 antigen levels in tumor tissue extracts were significantly associated with both overall and progression-free survival: ovarian cancer patients with high KLK7 levels had a significantly, 2-fold lower risk of death [hazard ratio (HR) = 0.51, 95% confidence interval (CI) = 0.29–0.90, ρ = 0.019] or relapse [HR = 0.47, 95% CI = 0.25–0.91, ρ = 0.024), as compared with patients who displayed low KLK7 levels. Our results indicate that – in contrast to earlier findings – high KLK7 antigen levels in tumor tissue extracts may be associated with a better prognosis of ovarian cancer patients.