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Untreated HIV-1 infection leads to a slow decrease in CD4+ T cell lymphocytes over time resulting in increased susceptibility to opportunistic infections (acquired immunodeficiency syndrome, AIDS) and ultimately death of the infected individual. Initially, the host’s immune response controls the infection, but cannot eliminate the HIV-1 from the host. Cytotoxic lymphocytes are the key effector cells in this response and can mediate crucial antiviral responses through the release of a set of proteases called granzymes towards HIV-1-infected cells. However, little is known about the immunological molecular mechanisms by which granzymes could control HIV-1. Since we noted that HIV-1 subtype C (HIV-1C) Gag with the tetrapeptide insertion PYKE contains a putative granzyme M (GrM) cleavage site (KEPL) that overlaps with the PYKE insertion, we analyzed the proteolytic activity of GrM towards Gag. Immunoblot analysis showed that GrM could cleave Gag proteins from HIV-1B and variants from HIV-1C of which the Gag-PYKE variant was cleaved with extremely high efficiency. The main cleavage site was directly after the insertion after leucine residue 483. GrM-mediated cleavage of Gag was also observed in co-cultures using cytotoxic lymphocytes as effector cells and this cleavage could be inhibited by a GrM inhibitor peptide. Altogether, our data indicate towards a noncytotoxic immunological mechanism by which GrM-positive cytotoxic lymphocytes target the HIV-1 Gag protein within infected cells to potentially control HIV-1 infection. This mechanism could be exploited in new therapeutic strategies to treat HIV-1-infected patients to improve immunological control of the infection.

Auch nach dem Ausbruch der weltweiten Aquired Immune Deficiency Syndrome (AIDS) Pandemie in den 1980er Jahren, ist das Humane Immundefizienz-Virus Typ 1 (HIV-1) bis heute eine Bedrohung für die öffentliche Gesundheit. Eine weitreichende Erforschung, aber auch ein tieferes Verständnis der menschlichen Immunantwort bei Infektionen mit HIV-1 sind daher im Kampf gegen AIDS essenziell. Diese Bachelorarbeit hebt die Funktion von natürlichen Killerzellen (NK-Zellen) in der Immunantwort bei Infektionen mit HIV-1 hervor. NK-Zellen spielen eine Schlüsselrolle in der Immunantwort gegen Pathogene. Sie setzen Granzyme, unter anderem Granzym M (GrM), frei. Granzyme gehören zu den Serinproteasen. Die molekularen Prozesse von GrM in der Immunantwort gegen HIV-1 wurden bisher nur wenig erforscht. Dennoch gibt es Hinweise, dass GrM durchaus eine wichtige Rolle bei der Immunantwort spielen könnte: GrM wird bei HIV-1 positiven Patienten, welche die Viruslast ohne Einnahme von Medikamenten unter Kontrolle haben, unterschiedlich exprimiert. Um die Mechanismen von NK-Zellen und die Funktion von GrM besser zu verstehen, haben wir verschiedene Experimente durchgeführt, die zu beachtenswerten Ergebnissen führten: Durch das Freisetzen von GrM, können NK-Zellen die Reaktivierung von HIV-1 in J-Lat Zellen inhibieren. Des Weiteren wurde die virale Latenz von HIV-1 in Anwesenheit des pan-Caspase Inhibitors zVAD-FMK verstärkt rückgängig gemacht. Somit ist das ein potenzieller Drug Target. Diese Ergebnisse geben neue wichtige Einblicke in die menschliche Immunantwort gegen HIV-1 und könnten den Weg für zukünftige therapeutische Ansätze bereiten. After the onset of the worldwide acquired immune deficiency syndrome (AIDS) pandemic in the 1980s, human immunodeficiency virus type 1 (HIV-1) remains a threat to public health to this day. Further study and understanding of the molecular mechanisms of human immune responses towards HIV-1 are essential for the fight against AIDS. This thesis emphasises on the role of natural killer (NK) cells in the immune response towards HIV-1. NK cells play a key role in the human immune reaction towards pathogens. They release serine proteases called granzymes, including granzyme M (GrM). The molecular mechanisms of GrM in the immune response towards HIV-1 infection remain poorly studied. Nevertheless, differential GrM expression in HIV-1 infected individuals who are able to control their viral load without medication (Elite Controlers; EC), indicates that GrM indeed plays a role in immune response towards HIV-1. In order to study these mechanisms of NK cells, and subsequently GrM, we carried out several experiments that led to remarkable findings: NK cells are able to inhibit HIV-1 reactivation in J-Lat cells by releasing GrM. Furthermore, the presence of the pan-caspase inhibitor zVAD-FMK led to increased reversal of latency in HIV-1 infected cells, which is a potential drug target for the „shock and kill“ strategy. These findings provide important new insights into the immune response mechanisms, which may help pave the way towards future therapeutic interventions.

Granzyme M is a serine protease known to be often expressed by natural killer cells and induce target cells apoptosis in combination with perforin. However, we detected granzyme M expression in murine and human cancer cell lines and human tumor samples in our study. Granzyme M increased chemoresistance, colony-formation, cytokine secretion and invasiveness in vitro. Most importantly, granzyme M facilitated tumor growth and metastasis in vivo. Granzyme M induced the epithelial-mesenchymal transition (EMT) in cancer cells associated with STAT3 activation. Our study revealed the role of granzyme M expressed by tumor in chemoresistance, invasion, metastasis and EMT.

Human cytomegalovirus (HCMV) reactivation can cause serious complications in allogeneic stem cell transplantation (SCT) patients. HCMV is controlled by cytotoxic lymphocytes that release antiviral granzymes. Recently, we have demonstrated that granzyme M (GrM) inhibits HCMV replication in vitro, however the physiological role of GrM and its cellular distribution during HCMV infection remains unknown. Here, we examined GrM expression in lymphocyte populations during HCMV infection. The percentage of GrM-expressing effector-memory CD4(+) T-cells was higher in HCMV latently-infected healthy individuals compared to that of uninfected individuals. SCT recipients had higher percentages of GrM-expressing CD4(+) T, CD8(+) T, γδT, and NKT cells. Despite lower total T-cell numbers, HCMV reactivation in SCT patients specifically associated with higher percentages of GrM-expressing CD4(+) (total and central-memory) T-cells. GrM was elevated in plasma during HCMV reactivation, pointing to extracellular perforin-independent functions of GrM. We conclude that GrM may be important in regulating HCMV latency and reactivation in SCT patients.

Immunotoxins are promising targeted therapeutic agents comprising an antibody-based ligand that specifically binds to diseased cells, and a pro-apoptotic protein. Toxic components from bacteria or plants can trigger a neutralizing immune response, so that human effector molecules are more suitable. In this context, the protease granzyme B has been successfully tested in cytotoxicity assays against different cancer cells in vitro and in vivo. Our aim here was to introduce granzyme M as an alternative and novel component of human cytolytic fusion proteins. We fused it to the humanized single-chain antibody fragment (scFv) H22 which specifically binds to CD64, an FcγRI receptor overexpressed on activated myeloid cells and leukemic cells. We show that the humanized cytolytic fusion protein Gm-H22(scFv) specifically targets the acute myeloid leukemia cell line HL60 in vitro and is cytotoxic with an IC50 between 1.2 and 6.4 nM. These findings were confirmed ex vivo using leukemic primary cells from patients, which were killed by granzyme M despite the presence of the granzyme B inhibitor serpin B9. In conclusion, granzyme M is a promising new cell-death inducing component for hCFPs because it specifically and efficiently kills target cells when fused to a targeting component.

Cytotoxic lymphocyte protease granzyme M (GrM) is a potent inducer of tumor cell death. The apoptotic phenotype and mechanism by which it induces cell death, however, remain poorly understood and controversial. Here, we show that GrM-induced cell death was largely caspase-dependent with various hallmarks of classical apoptosis, coinciding with caspase-independent G2/M cell cycle arrest. Using positional proteomics in human tumor cells, we identified the nuclear enzyme topoisomerase II alpha (topoIIα) as a physiological substrate of GrM. Cleavage of topoIIα by GrM at Leu(1280) separated topoIIα functional domains from the nuclear localization signals, leading to nuclear exit of topoIIα catalytic activity, thereby rendering it nonfunctional. Similar to the apoptotic phenotype of GrM, topoIIα depletion in tumor cells led to cell cycle arrest in G2/M, mitochondrial perturbations, caspase activation, and apoptosis. We conclude that cytotoxic lymphocyte protease GrM targets topoIIα to trigger cell cycle arrest and caspase-dependent apoptosis.

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 Leu439, 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 Leu439 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 granule exocytosis pathway of cytotoxic lymphocytes (Tc and NK cells) is critical for control of tumor development and viral infections. Granule-associated perforin and granzymes are key components in Tc cell-mediated function(s). On the basis of studies that showed granzymes A, B, C, K and M, to induce apoptosis in vitro, all granzymes were thought to also induce cell death in vivo. This review summarizes our present understanding of the biological processes elicited by purified granzyme A and granzyme as well as the processes induced by the more physiologically relevant cytotoxic cells secreting these proteases. The combined evidence supports the concept that the granule secretion pathway is not mono-specific but rather poly-functional including induction of pro-inflammatory cytokines, besides their widely appreciated apoptotic properties.

The granule-exocytosis pathway is the major mechanism for cytotoxic lymphocytes to kill tumor cells and virus-infected cells. Cytotoxic granules contain the pore-forming protein perforin and a set of structurally homologues serine proteases called granzymes. Perforin facilitates the entry of granzymes into a target cell, allowing these proteases to initiate distinct cell death routes by cleaving specific intracellular substrates. The family of granzymes consists of multiple members, of which granzyme A and granzyme B have been studied most extensively. Since the cloning of the granzyme M cDNA in the early 1990s, it has remained an "orphan" granzyme for many years and only during the past few years the interest in this protease has increased. Granzyme M appears to be a potent inducer of tumor cell death with morphological hallmarks that are unique among all granzymes. In this review, we summarize the characteristics of granzyme M that are currently known, including its cellular expression, substrate specificity, physiological functions, and inhibitors.

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 α-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, α-tubulin was more efficiently cleaved by GrM as compared with ezrin. Direct α-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.

The granzymes are cell death–inducing enzymes, stored in the cytotoxic granules of cytotoxic T lymphocytes and natural killer cells, that are released during granule exocytosis when a specific virus-infected or transformed target cell is marked for elimination. Recent work suggests that this homologous family of serine esterases can activate at least three distinct pathways of cell death. This redundancy likely evolved to provide protection against pathogens and tumors with diverse strategies for evading cell death. This review discusses what is known about granzyme-mediated pathways of cell death as well as recent studies that implicate granzymes in immune regulation and extracellular proteolytic functions in inflammation.

Natural killer (NK) cells play an important role in innate immunity against virally infected or transformed cells as the first defense line. Granzyme M (GzmM) is an orphan granzyme that is constitutively highly expressed in NK cells and is consistent with NK cell-mediated cytolysis. We recently demonstrated that GzmM induces caspase-dependent apoptosis with DNA fragmentation through direct cleavage of inhibitor of caspase-activated DNase (ICAD). However, the molecular mechanisms for GzmM-induced apoptosis are unclear. We found GzmM causes mitochondrial swelling and loss of mitochondrial transmembrane potential. Moreover, GzmM initiates reactive oxygen species (ROS) generation and cytochrome c release. Heat shock protein 75 (HSP75, also known as TRAP1) acts as an antagonist of ROS and protects cells from GzmM-mediated apoptosis. GzmM cleaves TRAP1 and abolishes its antagonistic function to ROS, resulting in ROS accumulation. Silencing TRAP1 through RNA interference increases ROS accumulation, whereas TRAP1 overexpression attenuates ROS production. ROS accumulation is in accordance with the release of cytochrome c from mitochondria and enhances GzmM-mediated apoptosis.

Granzymes are serine proteases involved in killing of tumor cells and virally infected cells. However, granzymes are also upregulated in blood under inflammatory conditions and contribute to cytokine release and processing. Here, we show that granzyme M (GrM) and to a lesser extent GrK are transiently elevated in the circulation following LPS administration in humans. GrM is released upon stimulation of whole blood with LPS or the gram-negative bacteria Escherichia coli BL21, Pseudomonas aeruginosa, and Neisseria meningitidis. GrK is only released upon stimulation with P. aeruginosa. Thus, GrM and GrK are differentially released in response to LPS and gram-negative bacteria.

Expression of serine protease inhibitors (serpins) is one of the mechanisms used by tumour cells to escape immune surveillance. Previously, we have shown that expression of serpins SPI-6 and SPI-CI, respectively, renders tumour cells resistant to granzyme B (GrB)-mediated death and granzyme M (GrM)-mediated death. To obtain better insight into the interaction between serpins and their target proteases, we investigated the roles of protease inhibitor (PI)-9 and SPI-6 in the resistance to GrB-mediated and CD95-mediated death in further detail. Neither human PI-9 nor its murine orthologue SPI-6 was capable of preventing CD95-induced apoptosis in murine or human cells, indicating that these serpins do not inhibit the activation of apical caspases in this pathway. High expression of PI-9 or SPI-6 did prevent apoptosis induced by human GrB. Strikingly, only SPI-6, and not PI-9, was capable of inhibiting murine GrB, suggesting that a difference in enzymatic specificity exists between the mouse and the human granzymes. In agreement with this suggestion, murine GrB was clearly less effective in inducing apoptosis in human cells. Similar species specificity was also observed for SPI-CI and GrM when either their capacity to associate or the effectiveness of GrM-induced cytotoxicity was analysed. Our findings therefore indicate a species diversity that has a clear effect on mixed in vitro effector target settings.

Cytotoxic lymphocytes express a large family of granule serine proteases, including one member, granzyme (Grz)M, with a unique protease activity, restricted expression, and distinct gene locus. Although a number of Grzs, including GrzM, have been shown to mediate target cell apoptosis in the presence of perforin, the biological activity of Grz has been restricted to control of a number of viral pathogens, including two natural mouse pathogens, ectromelia, and murine CMV (MCMV). In this article, we describe the first reported gene targeting of GrzM in mice. GrzM-deficient mice display normal NK cell/T cell development and homeostasis and intact NK cell-mediated cytotoxicity of tumor targets as measured by membrane damage and DNA fragmentation. GrzM-deficient mice demonstrated increased susceptibility to MCMV infection typified by the presence of more viral inclusions and transiently higher viral burden in the visceral organs of GrzM-deficient mice compared with wild-type (WT) mice. The cytotoxicity of NK cells from MCMV-infected GrzM-deficient mice remained unchanged and, like WT control mice, GrzM-deficient mice eventually effectively cleared MCMV infection from the visceral organs. In contrast, GrzM-deficient mice were as resistant as WT control mice to mouse pox ectromelia infection, as well as challenge with a number of NK cell-sensitive tumors. These data confirm a role for GrzM in the host response to MCMV infection, but suggest that GrzM is not critical for NK cell-mediated cytotoxicity.

SummaryThe mechanism of target cell lysis in cytotoxic lymphocyte-mediated death is not well understood, and the role of granzymes in this process is unclear. Chemical functional probes were thus prepared for the major granzymes A and B to deconvolute their role in natural killer cell-mediated lysis of target cells. These biotinylated and substrate specificity-based diphenyl phosphonates allowed facile evaluation of selectivity through activity-based profiling in cell lysates and intact cells. Both inhibitors were found to be extremely selective in vitro and in cells. Use of these inhibitors in cell-based assays revealed granzyme A to be a minor effector and granzyme B to be a major effector of target cell lysis by natural killer cells. These studies indicate that the proapoptotic granzyme B functions also as a pronecrotic effector of target cell death.

Granzyme M is a trypsin-fold serine protease that is specifically found in the granules of natural killer cells. This enzyme has been implicated recently in the induction of target cell death by cytotoxic lymphocytes, but unlike granzymes A and B, the molecular mechanism of action of granzyme M is unknown. We have characterized the extended substrate specificity of human granzyme M by using purified recombinant enzyme, several positional scanning libraries of coumarin substrates, and a panel of individual p-nitroanilide and coumarin substrates. In contrast to previous studies conducted using thiobenzyl ester substrates (Smyth, M. J., O'Connor, M. D., Trapani, J. A., Kershaw, M. H., and Brinkworth, R. I. (1996) J. Immunol. 156, 4174–4181), a strong preference for leucine at P1 over methionine was demonstrated. The extended substrate specificity was determined to be lysine = norleucine at P4, broad at P3, proline > alanine at P2, and leucine > norleucine > methionine at P1. The enzyme activity was found to be highly dependent on the length and sequence of substrates, indicative of a regulatory function for human granzyme M. Finally, the interaction between granzyme M and the serpins α1-antichymotrypsin, α1 -proteinase inhibitor, and proteinase inhibitor 9 was characterized by using a candidate-based approach to identify potential endogenous inhibitors. Proteinase inhibitor 9 was effectively hydrolyzed and inactivated by human granzyme M, raising the possibility that this orphan granzyme bypasses proteinase inhibitor 9 inhibition of granzyme B.

Cell death is mediated by cytotoxic lymphocytes through various granule serine proteases released with perforin. The unique protease activity, restricted expression, and distinct gene locus of granzyme M suggested this enzyme might have a novel biological function or trigger a novel form of cell death. Herein, we demonstrate that in the presence of perforin, the protease activity of granzyme M rapidly and effectively induces target cell death. In contrast to granzyme B, cell death induced by granzyme M does not feature obvious DNA fragmentation, occurs independently of caspases, caspase activation, and perturbation of mitochondria and is not inhibited by overexpression of Bcl-2. These data raise the likelihood that granzyme M represents a third major and specialized perforin-dependent cell death pathway that plays a significant role in death mediated by NK cells.

Granzyme M is a member of a family of granule serine proteases that participate in target cell death initiated by cytotoxic lymphocytes. The enzyme is almost exclusively expressed in NK cell types. Granzyme M cleaves at the carboxy side of amino acids with long, hydrophobic side chains like Met, Leu, and Nle. To further study the substrate specificity of the enzyme, a series of peptide thiobenzyl esters was synthesized. The hydrolysis of the substrates with murine and human recombinant forms of granzyme M was observed. The results show that the enzyme has a strong preference for Pro at the P2 position and Ala, Ser, or Asp at the P3 position. These results suggest that the protein residues of the S2 and S3 subsites form important binding interactions that aid in the selection of specific natural substrates for granzyme M. A series of inhibitors was also tested with granzyme M. None of the inhibitors were effective inactivators of granzyme M, including the general serine protease inhibitor, 3,4-dichloroisocoumarin, which is usually a potent inactivator of serine proteases. This suggests that inhibition of granzyme M may be difficult. Also reported for the first time is the method utilized to isolate granzyme M used in this and previous publications. The observations in this paper will be valuable in development of new potent inhibitors for granzyme M as well as assist in determining the biological function of the enzyme.

Granzyme B is a caspase-like serine protease that is released by cytotoxic lymphocytes to kill virus-infected and tumor cells. Major recent advances in our understanding of granzyme B biochemistry, biology and function include an appreciation of its uptake into and trafficking within target cells, a thorough dissection of how cell death is triggered, and the identification of the serpin protease inhibitor PI-9, which regulates its function in lymphocytes and in other cells. The roles that granzyme B plays in human pathologies, such as transplant rejection, viral immunity and particularly tumor immune surveillance, remain a topic for vigorous debate and conjecture. The recent discovery of a triply mutated human granzyme B allele, whose product is predicted to possess a reduced capacity to induce cell death, opens the way for major progress in these areas in coming years.

Cytotoxic lymphocytes (CTLs), the key players of cell mediated immunity, induce apoptosis by engaging death receptors or through exocytosis of cytolytic granules containing granzyme (proteases) and pore-forming protein (perforin). The crystal structure of granzyme B from human (B(h)) and rat (B(r)), as well as that of pro-granzyme K (K(h)) has been reported recently. In the present communication, we describe the homology modeling of granzyme family (in particular Gzm A(h), M(h), B(m), and C(m) from human and mouse) based on the crystal structural coordinates of trypsin, granzyme K (K(h)), and granzyme B (B(h)). These models have been used for establishing phylogenetic relationship as well as identifying characteristic features for designing specific inhibitors. The paper also highlights key residues at the S1, S2, and S2(') binding subsites in all granzyme, which may be involved in the structure-function relationship of this enzyme family. The predicted 3D homology models show a conserved two similar domain structure, i.e., an N-terminal domain and a C-terminal domain comprising predominantly of beta-sheet structure with a little alpha-helical content. Micro-heterogeneities have been observed in the vicinity of the active site in all granzymes as compared to granzyme B(h). For example, in granzyme M(h), valine is present at the S1 subsite instead of arginine. Similarly differences at S2 (Leu-->Phe), S3 (Ser-->Gly), and S4 (Arg-->Asn) subsites are quite apparent and appear to hold the potential for selective designing of inhibitors for possible therapeutic applications. Furthermore, analysis of the electrostatic surface potential on the shape of granzyme-inhibitor binding groove reveals clear differences at the reactive site. Additionally the different posttranslational modification sites such as phosphorylation (e.g., in granzyme M Thr101, Ser109), myristoylation (Gly22, 117, and 131), and glycosylation (Ser160) have been identified, as very little is known about the functional significance of these modifications in the granzyme family. Thus, glycosylation at Ser160 in granzyme M may influence the net charge of the enzyme, resulting in altered substrate binding as compared to granzyme B. Also this modification may influence the rate of complexation and binding affinity with proteoglycans. These studies are expected to contribute towards the basic understanding of functional associations of the granzymes with other molecules and their possible role in apoptosis.

Granzyme A (GzmA) induces a caspase-independent cell death pathway characterized by single-stranded DNA nicks and other features of apoptosis. A GzmA-activated DNase (GAAD) is in an ER associated complex containing pp32 and the GzmA substrates SET, HMG-2, and Ape1. We show that GAAD is NM23-H1, a nucleoside diphosphate kinase implicated in suppression of tumor metastasis, and its specific inhibitor (IGAAD) is SET. NM23-H1 binds to SET and is released from inhibition by GzmA cleavage of SET. After GzmA loading or CTL attack, SET and NM23-H1 translocate to the nucleus and SET is degraded, allowing NM23-H1 to nick chromosomal DNA. GzmA-treated cells with silenced NM23-H1 expression are resistant to GzmA-mediated DNA damage and cytolysis, while cells overexpressing NM23-H1 are more sensitive.

Granzyme M (GM) is a novel serine protease whose expression is highly restricted to natural killer (NK) cells, CD3(+)CD56(+) T cells, and gamma delta T cells. Using a GM-specific monoclonal antibody, we analyzed the expression of GM in 214 mature T-cell and NK-cell lymphomas. GM was preferentially expressed in nasal NK/T-cell lymphomas (100%), gamma delta T-cell lymphomas (100%), and intestinal T-cell lymphomas (85%). In contrast, GM expression was present at low prevalence in mycosis fungoides/Sézary syndrome (3%), anaplastic large-cell lymphoma (6%), panniculitis-like T-cell lymphoma (11%), and angioimmunoblastic T-cell lymphoma (0%) cases. Peripheral T-cell lymphomas of unspecified subtype showed an intermediate frequency (37%) of GM expression, consistent with their heterogeneous origin. We conclude that GM expression is a distinctive feature of the nasal NK/T-cell, gamma delta T-cell, and intestinal T-cell lymphomas, and suggest that these tumors develop from lymphocytes involved in innate immunity.

Cytotoxic T lymphocytes (CTLs) provide potent defences against virus infection and intracellular pathogens. However, CTLs have a dark side--their lytic machinery can be directed against self-tissues in autoimmune disorders, transplanted cells during graft rejection and host tissues to cause graft-versus-host disease, which is one of the most serious diseases related to CTL function. Although this duplicitous behaviour might seem contradictory, both beneficial and detrimental effects are the result of the same effector proteins. So, an understanding of the mechanisms that are used by CTLs to destroy targets and a knowledge of pathogen immune-evasion strategies will provide vital information for the design of new therapies.

The cytotoxic T-lymphocyte protease granzyme A induces caspase-independent cell death in which DNA single-stranded nicking is observed instead of oligonucleosomal fragmentation. A 270- to 420-kDa endoplasmic reticulum-associated complex (SET complex) containing the nucleosome assembly protein SET, the tumor suppressor pp32, and the base excision repair enzyme APE can induce single-stranded DNA damage in isolated nuclei in a granzyme A-dependent manner. The normal functions of the SET complex are unknown, but the functions of its components suggest that it is involved in activating transcription and DNA repair. We now find that the SET complex contains DNA binding and bending activities mediated by the chromatin-associated protein HMG2. HMG2 facilitates assembly of nucleoprotein higher-order structures by bending and looping DNA or by stabilizing underwound DNA. HMG2 is in the SET complex and coprecipitates with SET. By confocal microscopy, it is observed that cytoplasmic HMG2 colocalizes with SET in association with the endoplasmic reticulum, but most nuclear HMG2 is unassociated with SET. This physical association suggests that HMG2 may facilitate the nucleosome assembly, transcriptional activation, and DNA repair functions of SET and/or APE. HMG2, like SET and APE, is a physiologically relevant granzyme A substrate in targeted cells. HMG1, however, is not a substrate. Granzyme A cleavage after Lys65 in the midst of HMG box A destroys HMG2-mediated DNA binding and bending functions. Granzyme A cleavage and functional disruption of key nuclear substrates, including HMG2, SET, APE, lamins, and histones, are likely to cripple the cellular repair response to promote cell death in this novel caspase-independent death pathway.

Cytotoxic T lymphocytes (CTL) induce apoptosis by engaging death receptors or by exocytosis of cytolytic granules containing granzyme (Gzm) proteases and perforin. The lamins, which maintain the structural integrity of the nuclear envelope, are cleaved by caspases during caspase-mediated apoptosis. Although death receptor engagement and GzmB activate caspases, CTL also induce apoptosis during caspase blockade. Both GzmA and GzmB directly and efficiently cleave laminB in vitro , in situ in isolated nuclei and in cells loaded with perforin and Gzms, even in the presence of caspase inhibitors. LaminB is cleaved by GzmA at concentrations of 3 nM, but GzmB is 50 times less active. GzmA cuts laminB at R392; GzmB cuts at the caspase VEVD231 site. Characteristic laminB fragments generated by Gzm proteolysis also are observed during CTL lysis, even in the presence of caspase inhibitors or in cells overexpressing bcl-2. Lamins A/C are direct substrates of GzmA, but not GzmB. GzmA and GzmB therefore directly target critical caspase substrates in caspase-resistant cells.

We have isolated cDNAs to two transcripts, granzyme M and alternative granzyme M mRNA from the mouse eye. Analysis of genomic DNA revealed these transcripts were derived from alternative transcription initiations. Northern blot analysis and reverse transcription-polymerase chain reactions revealed that both transcripts were expressed in the eye, though the alternative form was the major type. In situ hybridization studies demonstrated that alternative granzyme M mRNA localized exclusively in the photoreceptor cells in the retina and expressed only after the opening of the eye, suggesting that these transcripts are related to the maintenance of the retinal structure or functions of matured photoreceptor cells rather than the development or differentiation of retinal cells.

Granzyme (gzm) B–deficient cytotoxic lymphocytes (CTL) have a severe defect in the rapid induction of target cell apoptosis that is almost completely corrected by prolonged incubation of the CTL effectors and their targets. We show in this report that perforin-dependent, gzmB-independent cytotoxicity is caused by gzmA (or tightly linked genes). CTL deficient for gzmA and gzmB retain normal perforin function, but these CTL have a cytotoxic defect in vivo that is as severe as perforin-deficient CTL. Collectively, these results suggest that perforin provides target cell access and/or trafficking signals for the gzms, and that the gzms themselves deliver the lethal hits. The gzmA pathway appears to function independently from gzmB and may therefore provide a critical "back-up" system when gzmB is inhibited in the target cell.

Caspase-mediated proteolysis of downstream substrates is a critical element of the execution pathway common to all forms of apoptosis studied to date. While this caspase-dependent pathway is activated during cytotoxic lymphocyte granule–induced cell death, recent studies have also provided evidence for caspase-independent pathways. However, the mechanisms mediating these additional pathways have not been defined. The current study demonstrates that DNA-PK cs and NuMA are directly and efficiently cleaved by granzyme B in vitro and in vivo, generating unique substrate fragments not observed during other forms of apoptosis. This direct, caspase-independent ability of granzyme B to cleave downstream death substrates constitutes an apoptotic effector mechanism that is insensitive to inhibitors of the signaling or execution components of the endogenous apoptotic cascade.

The release of cytotoxic granule contents by cytotoxic T lymphocytes triggers apoptotic target cell death. Cytotoxic granules contain a pore-forming protein, perforin, and a group of serine proteases called granzymes. We expressed human granzyme A in bacteria as a proenzyme capable ofin vitroactivation by enterokinase. The recombinant activated enzyme has catalytic activity against substrates with Arg, preferably, or Lys at the P1 position, comparable to trypsin. An enzymatically inactive recombinant granzyme A, with the active site Ser mutated to Ala, was produced and used with affinity chromatography to identify potential substrates. Two granzyme A-binding cytoplasmic proteins of molecular mass 33 and 44 kDa were isolated and identified by tryptic fragment sequencing as PHAP I and II, ubiquitous putative HLA-associated proteins, previously coisolated by binding to an HLA class II peptide. PHAP II forms an SDS-stable complex with recombinant mutant granzyme A and coprecipitates with it from cytoplasmic extracts. PHAP II, either purified or in cell lysates, is cleaved by the recombinant enzyme at nanomolar concentrations to a 25-kDa fragment. PHAP II begins to be degraded within minutes of initiation of cytotoxic T lymphocyte attack. PHAP I and II are candidate participants in the granzyme A pathway of cell-mediated cytotoxicity.

The granule-exocytosis pathway is the major mechanism via which cytotoxic lymphocytes eliminate virus-infected and tumor cells. In this pathway, cytotoxic lymphocytes release granules containing the pore-forming protein perforin and a family of serine proteases known as granzymes into the immunological synapse. Pore-formation by perforin facilitates entry of granzymes into the target cell, where they can activate various (death) pathways. Humans express five different granzymes, of which granzymes A and B have been most extensively characterized. However, much less is known about granzyme M (GrM). Recently, structural analysis and advanced proteomics approaches have determined the primary and extended specificity of GrM. GrM functions have expanded over the past few years: not only can GrM efficiently induce cell death in tumor cells, it can also inhibit cytomegalovirus replication in a noncytotoxic manner. Finally, a role for GrM in lipopolysaccharide-induced inflammatory responses has been proposed. In this review, we recapitulate the current status of GrM expression, substrate specificity, functions, and inhibitors.

Granzymes are serine proteases mainly found in cytotoxic lymphocytes. The most-studied member of this group is granzyme B, which is a potent cytotoxin that has set the paradigm that all granzymes are cyototoxic. In the last 5 years, this paradigm has become controversial. On one hand, there is a plethora of sometimes contradictory publications showing mainly caspase-independent cytotoxic effects of granzyme A and the so-called orphan granzymes in vitro. On the other hand, there are increasing numbers of reports of granzymes failing to induce cell death in vitro unless very high (potentially supra-physiological) concentrations are used. Furthermore, experiments with granzyme A or granzyme M knock-out mice reveal little or no deficit in their cytotoxic lymphocytes’ killing ability ex vivo, but indicate impairment in the inflammatory response. These findings of non-cytotoxic effects of granzymes challenge dogma, and thus require alternative or additional explanations to be developed of the role of granzymes in defeating pathogens. Here we review evidence for granzyme cytotoxicity, give an overview of their non-cytotoxic functions, and suggest technical improvements for future investigations.

Cytolytic lymphocytes are of cardinal importance in the recovery from primary viral infections. Both natural killer cells and cytolytic T cells mediate at least part of their effector function by target cell lysis and DNA fragmentation. Two proteins, perforin and granzyme B, contained within the cytoplasmic granules of these cytolytic effector cells have been shown to be directly involved in these processes. A third protein contained within these granules, granzyme A, has so far not been attributed with any biological relevance. Using mice deficient for granzyme A, we show here that granzyme A plays a crucial role in recovery from the natural mouse pathogen, ectromelia, by mechanisms other than cytolytic activity.

Inflammatory bowel disease (IBD) is an immunoregulatory disorder, associated with a chronic and inappropriate mucosal immune response to commensal bacteria, underlying disease states such as ulcerative colitis (UC) and Crohn’s disease (CD) in humans. Granzyme M (GrzM) is a serine protease expressed by cytotoxic lymphocytes, in particular natural killer (NK) cells. Granzymes are thought to be involved in triggering cell death in eukaryotic target cells; however, some evidence supports their role in inflammation. The role of GrzM in the innate immune response to mucosal inflammation has never been examined. Here, we discover that patients with UC, unlike patients with CD, display high levels of GrzM mRNA expression in the inflamed colon. By taking advantage of well-established models of experimental UC, we revealed that GrzM-deficient mice have greater levels of inflammatory indicators during dextran sulfate sodium (DSS)-induced IBD, including increased weight loss, greater colon length reduction and more severe intestinal histopathology. The absence of GrzM expression also had effects on gut permeability, tissue cytokine/chemokine dynamics, and neutrophil infiltration during disease. These findings demonstrate, for the first time, that GrzM has a critical role during early stages of inflammation in UC, and that in its absence colonic inflammation is enhanced.

We have generated H-2b mice with a homozygous null mutation in the granzyme (gzm) B gene. Gzm B is a neutral serine protease with Aspase activity that is found only in the granules of activated cytolytic T cells, natural killer cells, and lymphokine-activated killer cells. Gzm B-/- mice develop normally and have normal hematopoiesis and lymphopoiesis. In vitro, cytotoxic T lymphocytes (CTL) derived from gzm B-/- animals are able to induce 51Cr release from allotarget cells, but with reduced efficiency. However, gzm B-/- CTL have a profound defect in their ability to induce rapid DNA fragmentation and apoptosis in allogeneic target cells. This defect is kinetic since DNA fragmentation is partially compensated and 51Cr release is completely rescued with long incubation times. We conclude that gzm B serves a critical and nonredundant role for the rapid induction of target cell DNA fragmentation and apoptosis by alloreactive cytotoxic T lymphocytes.

Granzyme M (GzmM), an orphan Gzm, is constitutively and abundantly expressed in innate effector natural killer cells. We previously demonstrated that GzmM induces caspase (casp)-dependent apoptosis and cytochrome c release from mitochondria. We also resolved the crystal structure for GzmM and generated its specific inhibitor. However, how GzmM causes casp activation has not been defined. Here we found that casp-8 is an initiator caspase in GzmM-induced casp cascade, which causes other casp activation and Bid cleavage. GzmM does not directly cleave procaspase-3 and Bid, whose processing is casp dependent. Casp-8 knockdown or deficient cells attenuate or abolish GzmM-induced proteolysis of procaspase-3 and Bid. Extrinsic death receptor pathway adaptor Fas-associated protein with death domain (FADD) contributes to GzmM-induced casp-8 activation. GzmM specifically cleaves FADD after Met 196 to generate truncated FADD (tFADD) that enhances its self-association for oligomerization. The oligomerized tFADD facilitates procaspase-8 recruitment to promote its auto-processing leading to casp activation cascade. FADD-deficient cells abrogate GzmM-induced activation of casp-8 and apoptosis as well as significantly inhibit lymphokine-activated killer cell-mediated cytotoxicity. FADD processing by GzmM can potentiate killing efficacy against tumor cells and intracellular pathogens.

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 1.3x10(4) M(-1) s(-1). SERPINB4 abolished cleavage of the macromolecular GrM substrates alpha-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.

Cytotoxic lymphocyte protease GrM (granzyme M) is a potent inducer of tumour cell death and a key regulator of inflammation. Although hGrM (human GrM) and mGrM (mouse GrM) display extensive sequence homology, the substrate specificity of mGrM remains unknown. In the present study, we show that hGrM and mGrM have diverged during evolution. Positional scanning libraries of tetrapeptide substrates revealed that mGrM is preferred to cleave after a methionine residue, whereas hGrM clearly favours a leucine residue at the P1 position. The kinetic optimal non-prime subsites of both granzymes were also distinct. Gel-based and complementary positional proteomics showed that hGrM and mGrM have a partially overlapping set of natural substrates and a diverged prime and non-prime consensus cleavage motif with leucine and methionine residues being major P1 determinants. Consistent with positional scanning libraries of tetrapeptide substrates, P1 methionine was more frequently used by mGrM as compared with hGrM. Both hGrM and mGrM cleaved α-tubulin with similar kinetics. Strikingly, neither hGrM nor mGrM hydrolysed mouse NPM (nucleophosmin), whereas human NPM was hydrolysed efficiently by GrM from both species. Replacement of the putative P1′–P2′ residues in mouse NPM with the corresponding residues of human NPM restored cleavage of mouse NPM by both granzymes. This further demonstrates the importance of prime sites as structural determinants for GrM substrate specificity. GrM from both species efficiently triggered apoptosis in human but not in mouse tumour cells. These results indicate that hGrM and mGrM not only exhibit divergent specificities but also trigger species-specific functions.

Lymphocyte perforin and serine protease granzymes are well-recognized extrinsic mediators of apoptosis. We now demonstrate that cytotoxic lymphocyte granule components profoundly augment the myeloid cell inflammatory cytokine cascade in response to TLR4 ligation. Whereas caspase-1–deficient mice were completely resistant to LPS, reduced serum cytokine production and resistance to lethal endotoxicosis were also obtained with perforin-deficient mice, indicating a role for granzymes. Consistently, a lack of granzyme M (GrzM) resulted in reduced serum IL-1α, IL-1β, TNF, and IFN-γ levels and significantly reduced susceptibility to lethal endotoxicosis. These altered responses were also observed in granzyme A-deficient but not granzyme B-deficient mice. A role for APC–NK cell cross-talk in the inflammatory cascade was highlighted, as GrzM was exclusively expressed by NK cells and resistance to LPS was also observed on a RAG-1/GrzM-double deficient background. Collectively, the data suggest that NK cell GrzM augments the inflammatory cascade downstream of LPS-TLR4 signaling, which ultimately results in lethal endotoxicosis. Most importantly, these data demonstrate that granzymes should no longer be considered solely as mediators of apoptosis, but additionally as potential key regulators of inflammation.

Granzyme M (GzmM) is a chymotrypsin-like serine protease that preferentially cuts its substrates after Met or Leu. GzmM is constitutively expressed in activated innate effector natural killer (NK) cells. GzmM-induced cell death is consistent with the kinetics of cytotoxicity of NK cells. These suggest that GzmM may play an important role in innate immunity. Our previous work demonstrated that GzmM induces caspase-dependent apoptosis. However, it is unknown about how GzmM causes caspase activation. Here, we showed that the inhibitor of the apoptosis gene family member Survivin is a physiological substrate for GzmM. GzmM hydrolyzes Survivin at Leu-138 to remove the last four C-terminal residues. The truncated form (sur-TF) is more rapidly hydrolyzed through proteasome-mediated degradation. In addition, Survivin is in complex with X-linked inhibitor of apoptosis protein (XIAP) to inhibit caspase activation as an endogenous inhibitor. Survivin cleavage by GzmM abolishes the stability of the Survivin-XIAP complex and enhances XIAP hydrolysis, which amplifies caspase-9 and 3 activation of target tumor cells. The noncleavable L138A Survivin overexpression can significantly inhibit GzmM-mediated XIAP degradation, caspase activation, and GzmM- and NK cell-induced cytotoxicity. Moreover, Survivin silencing promotes XIAP degradation and enhances GzmM-induced caspase activation as well as GzmM- and NK cell-induced cytolysis of target tumor cells.

We report the purification from a rat natural killer (RNK) large granular lymphocyte leukemia of a 32-kD granule protein that induces rapid DNA fragmentation and apoptosis. The protein, which we have called "fragmentin," was capable of causing DNA from intact YAC-1 cells to be cleaved into oligonucleosomal-sized fragments and producing severe chromatin condensation within 1 h. Amino acid sequence of tryptic peptides indicated that fragmentin was highly homologous to the NK and T cell granule serine proteases RNK protease 1 and mouse cytotoxic T cell protease I (CCPI)/granzyme B. Preincubation with the serine esterase inhibitor 3,4-dichloroisocoumarin blocked fragmentin-induced DNA damage, but had no effect on cytolysin. Fragmentin activity against four lymphoma target cells was completely dependent on the presence of cytolysin. Fragmentin produced rapid membrane damage as well as DNA fragmentation at nonlytic cytolysin doses, suggesting that fragmentin activity was not limited to its effects on the nucleus. Fragmentin and cytolysin activity were completely inhibited by EGTA, indicating the process was Ca2+ dependent. A role for cytolysin in endocytosis of fragmentin was suggested by the observation that treatment of YAC-1 with cytochalasin B or sodium azide and 2-deoxyglucose blocked DNA fragmentation but not cytolysin activity. A 30-kD N alpha-CBZ-L-lysine thiobenzyl esterase, which copurified with fragmentin, was inactive on its own but was able to synergistically amplify the DNA damage induced by fragmentin in the presence of cytolysin. Fragmentin activity was not dependent on protein synthesis, as cycloheximide treatment of YAC-1 cells did not prevent DNA damage. We postulate that fragmentin is the molecular mediator of NK cell-mediated DNA fragmentation and apoptosis.

Granzymes kill cells in a variety of ways. They induce mitochondrial dysfunction through caspase dependent and caspase-independent pathways and destroy DNA and the integrity of the nucleus. For gaining a better understanding of the molecular function of granzyme M and its NK cell specificity, structural characterization of this enzyme by molecular modeling as well as its detailed comparison with other granzymes is presented in this study. The study includes mode of action of granzyme M using cationic binding sites, substrate specificity, post-translational structural modification and its functional relationship and interaction of the enzyme with inhibitor in an attempt to explore how the activity of human granzyme M is controlled under physiological conditions. It is concluded from the present study that the post-translational modification, including Oglycosylation of serine, phosphorylation of serine and threonine and myristoylation of glycine, play an important role in the interaction of enzyme with the cell surface membrane and regulate protein trafficking and stability. Phosphorylated serine and threonine also plays a role in tumor elimination, viral clearance and tissue repair. In Gzm M there are cationic sites, cs1 and cs2 that may participate in binding of Gzm M to the cell surface, thereby promoting its uptake and eventual release into the cytoplasm. Gzm M shows apoptotic activity both by caspase dependent and independent pathways. Modeling of inhibitors bound to the granzyme active site shows that the dimer also contributes to substrate specificity in a unique manner by extending the active-site cleft.

Granzyme M (GzmM), a unique serine protease constitutively expressed in NK cells, is important for granule-mediated cytolysis and can induce rapid caspase-dependent apoptosis of tumor cells. However, few substrates of GzmM have been reported to date, and the mechanism by which this enzyme recognizes and hydrolyzes substrates is unknown. To provide structural insights into the proteolytic specificity of human GzmM (hGzmM), crystal structures of wild-type hGzmM, the inactive D86N-GzmM mutant with bound peptide substrate, and the complexes with a catalytic product and with a tetrapeptide chloromethylketone inhibitor were solved to 1.96 Å, 2.30 Å, 2.17 Å and 2.70 Å, respectively. Structure-based mutagenesis revealed that the N terminus and catalytic triad of hGzmM are most essential for proteolytic function. In particular, D86N-GzmM was found to be an ideal inactive enzyme for functional studies. Structural comparisons indicated a large conformational change of the L3 loop upon substrate binding, and suggest this loop mediates the substrate specificity of hGzmM. Based on the complex structure of GzmM with its catalytic product, a tetrapeptide chloromethylketone inhibitor was designed and found to specifically block the catalytic activity of hGzmM.

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.

Natural killer (NK) cells kill virus-infected or transformed target cells by delivering cytotoxic proteases called granzymes to the target cell cytosol. One of these proteases, granzyme M, is specifically expressed in NK cells and is thought to instigate a form of cell death distinct from that mediated by granzyme A or granzyme B. However, the mechanism of granzyme M-induced cell death is unclear at present, and few substrates for this granzyme have been reported to date. Here we show that the abundant nucleolar phosphoprotein, nucleophosmin (NPM), is cleaved and inactivated by granzyme M. NPM is essential for cell viability as RNA interference-mediated ablation of NPM expression in human cells resulted in spontaneous apoptosis. Significantly, overexpression of wild-type NPM rescued cells treated with NPM small interference RNA, whereas overexpression of the granzyme M-cleaved form of NPM did not. Because NPM is essential for cell viability, these data suggest that targeting of NPM by granzyme M may contribute to tumor cell eradication by abolishing NPM function.