Cytoxic T lymphocytes (CTLs) recognize peptides presented by HLA class I molecules on the cell surface. The C-terminus of these CTL epitopes is predominantly generated by the proteasome.Here we demonstrate that the cytosolic endopeptidases nardilysin and thimet oligopeptidase (TOP) complemented proteasome activity. Nardilysin and TOP were required, either together or alone, for the generation of a tumor-specific CTL epitope from PRAME, an immunodominant CTL epitope from Epstein-Barr virus, and a clinically important epitope from melanoma-protein MART-1. TOP functioned as C-terminal trimming peptidase in antigen processing and nardilysin contributed to both C-terminal and N-terminal CTL epitope generation. By broadening the antigenic peptide repertoire, nardilysin and TOP strengthen the immune defense against intracellular pathogens and cancer.

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The oligopeptidase tripeptidyl-peptidase II (TPP II) is upregulated Burkitt's lymphoma (BL) cells that overexpress the c-myc proto-oncogene and is required for their growth and survival. Here we show that overexpression of TPP II induces accelerated growth and resistance to apoptosis in human embryonic kidney 293 cells. This correlates with the appearance of multiple chromosomal aberrations, numerical and structural centrosome abnormalities, and multipolar cell divisions. Similar mitotic aberrations were also observed in a panel of BL lines and were suppressed, in parallel with TPP II down-regulation, upon reversion of BL-like characteristics in EBV-immortalized B lymphocytes carrying a tetracyclineregulated c-myc. Functional TPP II knockdown by small interfering RNA expression in BL cells caused the appearance of giant polynucleated cells that failed to complete cell division. Collectively, these data point to a role of TPP II in the regulation of centrosome homeostasis and mitotic fidelity suggesting that this enzyme may be a critical player in the induction and/or maintenance of genetic instability in malignant cells. (Cancer Res 2005; 65(4): 1361-8)

Tripeptidyl peptidase II is an extralysosomal serine peptidase of an unusually large size, i.e. Mr greater than 10(6) for the native enzyme and Mr 135000 for the subunit. The enzyme from human erythrocytes was studied by electron microscopy on samples negatively stained by ammonium molybdate. Two different structural representations of the purified enzyme were obtained, both with a length of about 50 nm, and consisting of repetitive substructures. Upon dialysis of the enzyme against a Tris/HCl buffer, the activity was gradually decreased. This decrease was shown to parallel the dissociation of the large enzyme structures into smaller ones, the smallest measuring 3 nm by 10 nm and apparently corresponding to the repetitive substructures. The results indicate that a large polymeric form of the enzyme is a prerequisite for full activity.

Tripeptidyl peptidase II is a high molecular weight serine exopeptidase, which has been purified from rat liver and human erythrocytes. Four clones, representing 4453 bp, or 90% of the mRNA of the human enzyme, have been isolated from two different cDNA libraries. One clone, designated A2, was obtained after screening a human B-lymphocyte cDNA library with a degenerated oligonucleotide mixture. The B-lymphocyte cDNA library and a cDNA library, obtained from human fibroblasts, were rescreened with a 147 bp fragment from the 5' part of the A2 clone, whereby three different overlapping cDNA clones could be isolated. The deduced amino acid sequence, 1196 amino acid residues, corresponding to the longest open reading frame of the assembled nucleotide sequence, was compared to sequences of current databases. This revealed a 56% similarity between the bacterial enzyme subtilisin and the N-terminal part of tripeptidyl peptidase II. The enzyme was found to be represented by two different mRNAs of 4.2 and 5.0 kilobases, respectively, which probably result from the utilization of two different polyadenylation sites. Furthermore, cDNA corresponding to both the N-terminal and C-terminal part of tripeptidyl peptidase II hybridized with genomic DNA from mouse, horse, calf, and hen, even under fairly high stringency conditions, indicating that tripeptidyl peptidase II is highly conserved.