The role of TPPII in apoptosis control and treatment of malignant disease

Abstract

Degradation of cytosolic proteins depends largely on the proteasome. This complex contains a catalytic core (20S) and an accessory complex (19S) that recognizes ubiquitinated substrates and translocates these to the catalytic sites in the 20S particle. The proteasome is responsible for most of cytosolic protein degradation in eukaryotic cells and some of the degradation fragments are presented bound to MHC class I molecules. In the case of an immunological challenge, mammalian cells express IFN-inducible proteasomal subunits.

Impaired proteasomal activity can be functionally compensated, at least in part, by another large cytosolic peptidase, tripeptidyl-peptidase II (TPPII). This peptidase degrades cytosolic polypeptides and generates certain MHC class I ligands. Further, TPPII participates in regulation of apoptosis and its overexpression in lymphoma cells increases tumor growth in vivo. Increased expression of TPPII is induced by proteotoxic and oncogenic stress as well as starvation.

EL-4 lymphoma cells adapted to growth in the presence of inhibitor maintain cytosolic proteolysis and cell viability by a mechanism that includes compensatory upregulation of TPPII. This phenotype contributed to escape from tumor rejection in tumor graft experiments in syngeneic C57B1/6 mice. Using a GFP reporter to measure proteolysis, we show in live cells that non-proteasomal serine peptidase activity participated in protein degradation, but inhibition of these enzymes failed to have a significant effect on the assembly of H-2Kb molecules. Continued proteolysis in proteasome-impaired cells afforded the cell proliferation while preventing the full display of the usual set of MHC class I-restricted epitopes.

Many tumors overexpress members of the inhibitor of apoptosis protein (IAP) family. IAPs contribute to tumor cell apoptosis resistance by the inhibition of caspases, and are degraded by the proteasome to allow further progression of apoptosis. Impaired proteasomal activity is associated with induction of alternative cytosolic peptidases, such as TPPII and iso-peptidases. In second study we have analysed apoptosis control in tumor cells with high TPP II activity, which can grow despite proteasomal inhibition. We find that EL-4 lymphoma cells that can grow in the presence of low proteasomal activity acquire apoptosis resistance due to a failure in degradation of IAPs. The rate of in vivo tumor growth of such cells was strongly increased. Rapid tumor growth, as well as a delayed degradation of IAPs, could be induced by transfection of TPP H. In addition, in cells derived from large in vivo tumors we observed an upregulated TPPII activity and a slower degradation of IAPs. Our data suggest a novel mechanism for apoptosis resistance in tumors.

Cancer therapy frequently depends on the induction of DNA damage, e.g. treatment with γ-irradiation. Responses to DNA damage are controlled by Phospho-Inositide-3-0H-Kinase-related-kinases (PIKKs). These kinases are crucial for maintenance of DNA stability and are constitutively activated in many cancer cells. We have explored the involvement of TPPII in response to DNA damage. We found that the expression of TPPII is controlled by PIKKs. TPPII is required for stabilization of p53 and p2 1, and for lymphoma cell cycle arrest in response to stress. We also studied that TPPII contains a putative BRCT domain; a targeting domain for ATM-kinase phosphorylated proteins. This domain was important for p53 stabilization. We found a TPPII-dependent interaction between p53 and ATM, as well as DNA repair foci components Mre11 and 53BP1 Novel peptide-based inhibitors of TPPII caused complete in vivo tumor regression in mice, in response to relatively low dose of γ-irradiation. This was observed with established mouse and human tumors of diverse tissue backgrounds. Our data suggest that TPPII has an essential role in DNA damage responses, and that this peptidase can be targeted for treatment of tumors.

At last we have studied the activity of the ubiquitin-proteasome pathway in stressed cells. We observed that TPPII was essential for down regulation of proteasomal substrate degradation, and targeting of proteasomal complexes to the nucleus during stress. Our data indicate that TPPII-mediated suppression of substrate degradation may have a substantial impact on cellular processes and that this may contribute to transduction of cellular stress signals.

In conclusion, our results indicate a crucial role for TPPII in signal transduction during cellular stress, and that this peptidase complex is suitable for drug targeting.