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The tripeptidyl-peptidase II complex consists of repeated 138 kDa subunits, assembled into two twisted strands that form a high molecular weight complex (>5 MDa). TPPII, like many other cytosolic peptidases, plays a role in the ubiquitin-proteasome pathway downstream of the proteasome as well as in the production and destruction of MHC class I antigens and degradation of neuropeptides. Tripeptidyl-peptidase II activity is increased in cells with an increased demand for protein degradation, but whether degradation of cytosolic peptides is the only cell biological role for TPPII has remained unclear. Recent data indicated that TPPII translocates into the nucleus to control DNA damage responses in malignant cells, supporting that cytosolic "housekeeping peptidases" may have additional roles in cell biology, besides their contribution to protein turnover. Overall, TPPII has an emerging importance in several cancer-related fields, such as metabolism, cell death control, and control of genome integrity; roles that are not understood in detail. The present paper reviews the cell biology of TPPII and discusses distinct roles for TPPII in the nucleus and cytosol.

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Reactive oxygen species (ROS) are a continuous hazard in eukaroytic cells by their ability to cause damage to biomolecules, in particular to DNA. Previous data indicated that the cytosolic serine peptidase tripeptidyl-peptidase II (TPPII) translocates into the nucleus of most tumor cell lines in response to gamma-irradiation and ROS production; an event that promoted p53 expression as well as caspase-activation. We here observed that nuclear translocation of TPPII was dependent on signaling by MAP kinases, including p38MAPK. Further, this was caused by several types of DNA-damaging drugs, a DNA cross-linker (cisplatinum), an inhibitor of topoisomerase II (etoposide), and to some extent also by nucleoside-analogues (5-fluorouracil, hydroxyurea). In the minority of tumor cell lines where TPPII was not translocated into the nucleus in response to DNA damage we observed reduced intracellular ROS levels, and the expression levels of redox defense systems were increased. Further, treatment with the ROS-inducer gamma-hexa-chloro-cyclohexane (gamma-HCH, lindane), an inhibitor of GAP junctions, restored nuclear translocation of TPPII in these cell lines upon gamma-irradiation. Moreover, blocking nuclear translocation of TPPII in etoposide-treated cells, by using a peptide-derived inhibitor (Z-Gly-Leu-Ala-OH), attenuated expression of gamma-H2AX in gamma-irradiated melanoma cells. Our results indicated a role for TPPII in MAPK-dependent DNA damage signaling.

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Responses to DNA damage are influenced by cellular metabolism through the continuous production of reactive oxygen species (ROS), of which most are by-products of mitochondrial respiration. ROS have a strong influence on signaling pathways during responses to DNA damage, by relatively unclear mechanisms. Previous reports have shown conflicting data on a possible role for tripeptidyl-peptidase II (TPPII), a large cytosolic peptidase, within the DNA damage response. Here we show that TPPII translocated into the nucleus in a p160-ROCK-dependent fashion in response to gamma-irradiation, and that nuclear expression of TPPII was present in most gamma-irradiated transformed cell lines. We used a panel of nine cell lines of diverse tissue origin, including four lymphoma cell lines (T, B and Hodgkins lymphoma), a melanoma, a sarcoma, a colon and two breast carcinomas, where seven out of nine cell lines showed nuclear TPPII expression after gamma-irradiation. Further, this required cellular production of ROS; treatment with either N-acetyl-Cysteine (anti-oxidant) or Rotenone (inhibitor of mitochondrial respiration) inhibited nuclear accumulation of TPPII. The local density of cells was important for nuclear accumulation of TPPII at early time-points following gamma-irradiation (at 1-4h), indicating a bystander effect. Further, we showed that the peptide-based inhibitor Z-Gly-Leu-Ala-OH, but not its analogue Z-Gly-(D)-Leu-Ala-OH, excluded TPPII from the nucleus. This correlated with reduced nuclear expression of p53 as well as caspase-3 and -9 activation in gamma-irradiated lymphoma cells. Our data suggest a role for TPPII in ROS-dependent DNA damage responses, through alteration of its localization from the cytosol into the nucleus.

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Cellular responses to gamma-irradiation exposure are controlled by phosphatidylinositol 3-kinase-related kinases (PIKK) in the nucleus, and in addition, cytosolic PIKKs may have a role in such responses. Here, we show that the expression of tripeptidyl-peptidase II (TPPII), a high molecular weight cytosolic peptidase, required PIKK signaling and that TPPII was rapidly translocated into the nucleus of gamma-irradiated cells. These events were dependent on mammalian target of rapamycin, a cytosolic/mitochondrial PIKK that is activated by gamma-irradiation. Lymphoma cells with inhibited expression of TPPII failed to efficiently stabilize p53 and had reduced ability to arrest proliferation in response to gamma-irradiation. We observed that TPPII contains a BRCA COOH-terminal-like motif, contained within sequences of several proteins involved in DNA damage signaling pathways, and this motif was important for nuclear translocation of TPPII and stabilization of p53. Novel tripeptide-based inhibitors of TPPII caused complete in vivo tumor regression in mice in response to relatively low doses of gamma-irradiation (3-4 Gy/wk). This was observed with established mouse and human tumors of diverse tissue backgrounds, with no tumor regrowth after cancellation of treatment. These TPPII inhibitors had minor effects on tumor growth as single agent and had low cellular toxicity. Our data indicated that TPPII connects signaling by cytosolic/mitochondrial and nuclear PIKK-dependent pathways and that TPPII can be targeted for inhibition of tumor therapy resistance.

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Chronic malaria infection is characterized by polyclonal B cell activation, hyperglobulinemia, and elevated titers of autoantibodies. We have recently identified the cysteine-rich interdomain region 1alpha (CIDR1alpha) of the Plasmodium falciparum erythrocyte membrane protein 1 as a T cell-independent polyclonal B cell activator and Ig binding protein. Here, we show that, although the binding affinity of CIDR1alpha to human IgM and IgG is relatively low, B cell activation still proceeds. CIDR1alpha rescues tonsillar B cells from apoptosis, and increases the proportion of cycling cells. Comparison of the impact on naive and memory B cell compartment indicated that CIDR1alpha preferentially activates memory B lymphocytes. Analysis of the gene expression profiles induced by CIDR1alpha and anti-Ig activation using a cDNA microarray demonstrated a low degree of homology in the signatures imposed by both stimuli. The microarray data correlate with the functional analysis demonstrating that CIDR1alpha activates various immunological pathways and protects B cells from apoptosis. Together, the results provide evidence for a role of malaria in preferentially activating the memory B cell compartment. The polyclonal B cell activation and augmented survival induced by CIDR1alpha is of relevance for understanding the mechanisms behind the increased risk of Burkitt's lymphoma in malaria endemic areas.

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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. Here we show that tumor cells can alter the specificity of cytosolic proteolysis in order to acquire apoptosis resistance, which promotes formation of rapidly growing tumors. Survival of tumor cells with low proteasomal activity can occur in the presence of high expression of Tri-peptidyl-peptidase II (TPP II), a large subtilisin-like peptidase that complements proteasomal activity. We find that this state leaves tumor cells unable of effectively degrading IAPs, and that cells in this state form rapidly growing tumors in vivo. We also find, in studies of apoptosis resistant cells derived from large in vivo tumors, that these have acquired an altered peptidase activity, with up-regulation of TPP II activity and decreased proteasomal activity. Importantly, we find that growth of subcutaneous tumors is limited by maintenance of the apoptosis resistant phenotype. The apoptosis resistant phenotype was reversed by increased expression of Smac/DIABLO, an antagonist of IAP molecules. Our data suggest a reversible mechanism in regulation of apoptosis resistance that drives tumor progression in vivo. These data are relevant in relation to the multitude of therapy-resistant clinical tumors that have increased levels of IAP molecules.

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Degradation of cytosolic proteins depends largely on the proteasome, and a fraction of the cleavage products are presented as major histocompatibility complex (MHC) class I-bound ligands at the cell surface of antigen presenting cells. Proteolytic pathways accessory to the proteasome contribute to protein turnover, and their up-regulation may complement the proteasome when proteasomal proteolysis is impaired. Here we show that reduced reliance on proteasomal proteolysis allowed a reduced efficiency of MHC class I ligand production, whereas protein turnover and cellular proliferation were maintained. Using the proteasomal inhibitor adamantane-acetyl-(6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulphone, we show that covalent inhibition of all three types of proteasomal beta-subunits (beta(1), beta(2), and beta(5)) was compatible with continued growth in cells that up-regulate accessory proteolytic pathways, which include cytosolic proteases as well as deubiquitinating enzymes. However, under these conditions, we observed poor assembly of H-2D(b) molecules and inhibited presentation of endogenous tumor antigens. Thus, the tight link between protein turnover and production of MHC class I ligands can be broken by enforcing the substitution of the proteasome with alternative proteolytic pathways.

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Cytotoxic T lymphocyte (CTL)-mediated immune responses rely on the efficiency of MHC class I ligand generation and presentation by antigen presenting cells (APCs). Whereas the abnormal expression of MHC molecules and transporters associated with antigen processing (TAPs) are commonly discussed as factors that modulate antigen presentation, much less is known about possible regulatory mechanisms at the level of proteolysis responsible for the generation of antigenic peptides. The ubiquitin-proteasome system is recognized as the major component responsible for this process in the cytosol and its activity can be regulated by cytokines, such as IFN-gamma. However, new evidence suggests the involvement of other proteases that can contribute to cytosolic proteolysis and therefore, to the quality and quantity of antigen production. Here, we review recent findings on an increasing number of proteolytic enzymes linked to antigen presentation, and we discuss how regulation of cytosolic protease activities might have implications for immune escape mechanisms that could be used by tumor cells and pathogens.

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