All human granzymes target hnRNP K that is essential for tumor cell viability.

Granule exocytosis by cytotoxic lymphocytes is the key mechanism to eliminate virus-infected cells and tumor cells. These lytic granules contain the pore-forming protein perforin and a set of five serine proteases called granzymes. All human granzymes display distinct substrate specificities and induce cell death by cleaving critical intracellular death substrates. In the present study, we show that all human granzymes directly cleaved the DNA/RNA-binding protein heterogeneous nuclear ribonucleoprotein K (hnRNP K), designating hnRNP K as the first known pan-granzyme substrate. Cleavage of hnRNP K was more efficient in the presence of RNA and occurred in two apparent proteolysis-sensitive amino acid regions, thereby dissecting the functional DNA/RNA-binding hnRNP K domains. HnRNP K was cleaved under physiological conditions when purified granzymes were delivered into living tumor cells and during lymphokine-activated killer cell-mediated attack. HnRNP K is essential for tumor cell viability, since knockdown of hnRNP K resulted in spontaneous tumor cell apoptosis with caspase activation and reactive oxygen species production. This apoptosis was more pronounced at low tumor cell density where hnRNP K knockdown also triggered a caspase-independent apoptotic pathway. This suggests that hnRNP K promotes tumor cell survival in the absence of cell-cell contact. Silencing of hnRNP K protein expression rendered tumor cells more susceptible to cellular cytotoxicity. We conclude that hnRNP K is indispensable for tumor cell viability and our data suggest that targeting of hnRNP K by granzymes contributes to or reinforces the cell death mechanisms by which cytotoxic lymphocytes eliminate tumor cells.

Intracellular serine protease inhibitor SERPINB4 inhibits granzyme M-induced cell death.

Granzyme-mediated cell death is the major pathway for cytotoxic lymphocytes to kill virus-infected and tumor cells. In humans, five different granzymes (i.e. GrA, GrB, GrH, GrK, and GrM) are known that all induce cell death. Expression of intracellular serine protease inhibitors (serpins) is one of the mechanisms by which tumor cells evade cytotoxic lymphocyte-mediated killing. Intracellular expression of SERPINB9 by tumor cells renders them resistant to GrB-induced apoptosis. In contrast to GrB, however, no physiological intracellular inhibitors are known for the other four human granzymes. In the present study, we show that SERPINB4 formed a typical serpin-protease SDS-stable complex with both recombinant and native human GrM. Mutation of the P2-P1-P1' triplet in the SERPINB4 reactive center loop completely abolished complex formation with GrM and N-terminal sequencing revealed that GrM cleaves SERPINB4 after P1-Leu. SERPINB4 inhibited GrM activity with a stoichiometry of inhibition of 1.6 and an apparent second order rate constant of 1.3×10(4) M(-1) s(-1). SERPINB4 abolished cleavage of the macromolecular GrM substrates α-tubulin and nucleophosmin. Overexpression of SERPINB4 in tumor cells inhibited recombinant GrM-induced as well as NK cell-mediated cell death and this inhibition depended on the reactive center loop of the serpin. As SERPINB4 is highly expressed by squamous cell carcinomas, our results may represent a novel mechanism by which these tumor cells evade cytotoxic lymphocyte-induced GrM-mediated cell death.

Proteomic profiling of the human failing heart after left ventricular assist device support.

BACKGROUND: Left ventricular assist device (LVAD) support is commonly used in patients with heart failure as a bridge to heart transplantation. Whereas myocardial gene expression profile changes have been well established after LVAD support, the consequences on the protein level largely remain unclear. METHODS: Pre-LVAD and post-LVAD myocardial tissue specimens from dilated cardiomyopathy (DCM) and ischemic heart disease (IHD) patients were analyzed by fluorescent 2-dimensional difference gel electrophoresis, and differentially expressed proteins were identified by mass spectrometry. RESULTS: In the DCM group, 16 proteins were detected that showed statistically significant downregulation from pre-LVAD to post-LVAD tissue. In IHD patients, 50 alterations were found, including upregulated (n = 12) and downregulated (n = 38) proteins. The identified proteins in both groups partially overlapped and included proteins from cytoskeleton and mitochondrial energy metabolism. The latter changes were paralleled by severe abnormalities in mitochondrial morphology, as shown by electron microscopy. Post-LVAD proteomes of both DCM and IHD patients largely mimicked the protein profiles of non-failing hearts. Downregulation of the serine protease inhibitor α-1-antichymotrypsin in both DCM and IHD patients after LVAD support was confirmed by immunosorbent assay. CONCLUSIONS: LVAD-induced cardiac remodeling in DCM and IHD patients is associated with downregulation of α-1-antichymotrypsin and specific atrophic changes in protein expression profiles predominantly involved in cytoskeleton integrity and mitochondrial energy metabolism.

Noncytotoxic inhibition of cytomegalovirus replication through NK cell protease granzyme M-mediated cleavage of viral phosphoprotein 71.

Granzyme M (GrM) is highly expressed in cytotoxic granules of NK cells, which provide the first line of defense against viral pathogens. GrM knockout mice show increased susceptibility toward murine CMV infection. Although GrM is a potent inducer of cell death, the mechanism by which GrM eliminates viruses remains elusive. In this paper, we show that purified human GrM in combination with the perforin-analog streptolysin O (SLO) strongly inhibited human CMV (HCMV) replication in fibroblasts in the absence of host cell death. In a proteomic approach, GrM was highly specific toward the HCMV proteome and most efficiently cleaved phosphoprotein 71 (pp71), an HCMV tegument protein that is critical for viral replication. Cleavage of pp71 occurred when viral lysates were incubated with purified GrM, when intact cells expressing recombinant pp71 were challenged with living cytotoxic effector cells, and when HCMV-infected fibroblasts were incubated with SLO and purified GrM. GrM directly cleaved pp71 after Leu(439), which coincided with aberrant cellular localization of both pp71 cleavage fragments as determined by confocal immunofluorescence. In a luciferase reporter assay, cleavage of pp71 after Leu(439) by GrM completely abolished the ability of pp71 to transactivate the HCMV major immediate-early promoter, which is indispensable for effective HCMV replication. Finally, GrM decreased immediate-early 1 protein expression in HCMV-infected fibroblasts. These results indicate that the NK cell protease GrM mediates cell death-independent antiviral activity by direct cleavage of a viral substrate.

The cytotoxic protease granzyme M is expressed by lymphocytes of both the innate and adaptive immune system.

The cytotoxic serine protease granzyme M (GrM) is one of the five human granzymes, which are mainly expressed by cytotoxic T lymphocytes and/or NK cells. Upon perforin-dependent entry into a target cell, GrM cleaves specific substrates resulting in the onset of a unique cell death mechanism. However, the role of GrM in pathophysiological conditions is not clear yet. Knowledge of the expression and regulation of GrM by lymphocyte populations is instrumental for a better understanding of the contribution of this unique granzyme in health and disease. Two previous studies demonstrated GrM protein expression by lymphocytes of the innate immune system, i.e., NK cells, NKT cells, and gammadelta T cells, whereas its expression by CD8(+) T cells remained controversial. In the present study, we have investigated the expression and regulation of GrM in lymphocyte subsets in more detail. Flow cytometry analysis with a novel specific antibody against human GrM confirmed high expression of this protease by NK cells, NKT cells, and gammadelta T cells. CD8(+) T cells also expressed GrM and comparing the naive to early effector-memory, to late effector-memory, to effector subset, this expression gradually increased during differentiation. In contrast, CD4(+) T cells hardly expressed GrM. Quantitative PCR analysis for GrM mRNA levels in the diverse lymphocyte sub-populations confirmed the FACS results. GrM protein expression by lymphocyte populations was not significantly affected by a panel of GrB-inducing cytokines, indicating that GrM expression is differentially regulated as compared to GrB. In conclusion, the human cytotoxic protease GrM is, besides by innate immune cells, also expressed by CD8(+) effector T cells, in particular by the differentiated effector CD27(-) CD45RO(-) subset. Our current findings support not only a role for GrM in the innate but also in the adaptive immune response.

Downregulation of SERPINB13 expression in head and neck squamous cell carcinomas associates with poor clinical outcome.

Tumorigenesis of head and neck squamous cell carcinomas (HNSCC) is associated with various genetic changes such as loss of heterozygosity (LOH) on human chromosome 18q21. This chromosomal region maps a gene cluster coding for a family of intracellular serine protease inhibitors (serpins), including SERPINB13. As SERPINB13 expression in HNSCC has recently been shown to be downregulated both at the mRNA and protein levels, here we investigated if such a low SERPINB13 expression is associated with histopathological and clinical parameters of HNSCC tumors and patient survival. By generating specific antibodies followed by immunohistochemistry on a well-defined cohort of 99 HNSCC of the oral cavity and oropharynx, SERPINB13 expression was found to be partially or totally downregulated in 75% of the HNSCC as compared with endogenous expression in non-neoplastic epithelial cells. Downregulation of SERPINB13 protein expression in HNSCC was significantly associated with the presence of LOH at the SERPINB13 gene in the tumors (p = 0.006), a poor differentiation grade of the tumors (p = 0.001), the presence of a lymph node metastasis (p = 0.012), and a decreased disease-free (p = 0.033) as well as overall (p = 0.018) survival of the patients. This is the first report demonstrating that downregulation of SERPINB13 protein expression in HNSCC is positively associated with poor clinical outcome. Therefore, SERPINB13 seems to act as an important protease inhibitor involved in the progression of HNSCC.

Granzyme K displays highly restricted substrate specificity that only partially overlaps with granzyme A.

Granzymes are serine proteases stored in cytolytic granules of cytotoxic lymphocytes that eliminate virus-infected and tumor cells. Little is known about the molecular mechanism and function of granzyme (Gr)K. GrK is similar to GrA in that they are the only granzymes that display tryptase-like activity. Both granzymes induce cell death by single-stranded nicking of the chromosomal DNA by cleaving the same components of the endoplasmic reticulum-associated SET complex. Therefore, GrK may provide a backup and failsafe mechanism for GrA with redundant specificity. In the present study, we addressed the question of whether GrK displays identical substrate specificity as GrA. In peptide- and protease-proteomic screens, GrK and GrA displayed highly restricted substrate specificities that overlapped only partially. Whereas GrK and GrA cleave SET with similar efficiencies likely at the same sites, both granzymes cleaved the pre-mRNA-binding protein heterogeneous ribonuclear protein K with different kinetics at distinct sites. GrK was markedly more efficient in cleaving heterogeneous ribonuclear protein K than GrA. GrK, but not GrA, cleaved the microtubule network protein beta-tubulin after two distinct Arg residues. Neither GrK cleavage sites in beta-tubulin nor a peptide-based proteomic screen revealed a clear GrK consensus sequence around the P1 residue, suggesting that GrK specificity depends on electrostatic interactions between exosites of the substrate and the enzyme. We hypothesize that GrK not only constitutes a redundant functional backup mechanism that assists GrA-induced cell death but that it also displays a unique function by cleaving its own specific substrates.

NK cell protease granzyme M targets alpha-tubulin and disorganizes the microtubule network.

Serine protease granzyme M (GrM) is highly expressed in the cytolytic granules of NK cells, which eliminate virus-infected cells and tumor cells. The molecular mechanisms by which GrM induces cell death, however, remain poorly understood. In this study we used a proteomic approach to scan the native proteome of human tumor cells for intracellular substrates of GrM. Among other findings, this approach revealed several components of the cytoskeleton. GrM directly and efficiently cleaved the actin-plasma membrane linker ezrin and the microtubule component alpha-tubulin by using purified proteins, tumor cell lysates, and tumor cells undergoing cell death induced by perforin and GrM. These cleavage events occurred independently of caspases or other cysteine proteases. Kinetically, alpha-tubulin was more efficiently cleaved by GrM as compared with ezrin. Direct alpha-tubulin proteolysis by GrM is complex and occurs at multiple cleavage sites, one of them being Leu at position 269. GrM disturbed tubulin polymerization dynamics in vitro and induced microtubule network disorganization in tumor cells in vivo. We conclude that GrM targets major components of the cytoskeleton that likely contribute to NK cell-induced cell death.

Cytosolic beta-glycosidases for activation of glycoside prodrugs of daunorubicin.

Human cytosolic beta-glycosidase is a small monomeric enzyme that is active under physiological conditions, which might be ideal for enzyme-prodrug therapy. We have previously reported the synthesis of a galactoside (DNR-GlA3) and a glucoside (DNR-GsA3) prodrug of daunorubicin. In the present study, we established that cellular uptake of DNR-GlA3 and DNR-GsA3 was low in contrast to that of daunorubicin. Recombinant human beta-glycosidase converted both prodrugs to daunorubicin as shown by liquid chromatography. The kinetics of the conversion of DNR-GlA3 and DNR-GsA3 by human beta-glycosidase, however, was unfavorable as the K(m) values were, respectively, 3- and 6-fold higher than those of another mammalian beta-glycosidase of bovine origin. The V(max) values were, respectively, 3.3 and 8.5nmol/hr/mg as compared to 158.3 and 147.8nmol/hr/mg of the bovine enzyme. Treatment of OVCAR-3 cells with human beta-glycosidase (0.5U/mL) and 0.5 microM DNR-GlA3 or DNR-GsA3 resulted in, respectively, 86 and 81% cell growth inhibition, while the prodrugs alone inhibited growth to only 19 and 1%. Treatment of cells with the bovine enzyme and the prodrugs inhibited cell growth more efficiently. We conclude that the endogenous intracellular beta-glycosidase is not available for extracellular prodrug activation. Thus, the incorporation of the enzyme in enzyme-prodrug therapy might be an elegant approach to achieve tumor-specific prodrug conversion. The efficiency of glycoside prodrug conversion might be improved by design of a prodrug that is more readily activated by human beta-glycosidase or by evolution of the enzyme into a mutant form that displays high activity towards these prodrugs.