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Joseph A Trapani - One of the best experts on this subject based on the ideXlab platform.
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Perforin and Granzymes: function, dysfunction and human pathology
Nature reviews. Immunology, 2015Co-Authors: Ilia Voskoboinik, James C. Whisstock, Joseph A TrapaniAbstract:A defining property of cytotoxic lymphocytes is their expression and regulated secretion of potent toxins, including the pore-forming protein perforin and serine protease Granzymes. Until recently, mechanisms of pore formation and Granzyme transfer into the target cell were poorly understood, but advances in structural and cellular biology have now begun to unravel how synergy between perforin and Granzymes brings about target cell death. These and other advances are demonstrating the surprisingly broad pathophysiological roles of the perforin–Granzyme pathway, and this has important implications for understanding immune homeostasis and for developing immunotherapies for cancer and other diseases. In particular, we are beginning to define and understand a range of human diseases that are associated with a failure to deliver active perforin to target cells. In this Review, we discuss the current understanding of the structural, cellular and clinical aspects of perforin and Granzyme biology.
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Granzyme H is a novel protease expressed by human mast cells.
International archives of allergy and immunology, 2014Co-Authors: Elin Rönnberg, Joseph A Trapani, Vivien R Sutton, Gabriela Calounova, Ola Rollman, Eva Hagforsen, Gunnar PejlerAbstract:Background: Many of the functions attributed to mast cells depend on the various pro-inflammatory mediators that are secreted upon mast cell activation. These include a panel of mast cell-specific proteases. In addition, recent studies have indicated that murine mast cells also express Granzyme D, a protease previously thought to be confined to cytotoxic lymphocytes. Here, we address the human relevance of the latter findings by investigating whether human mast cells express Granzyme H, the Granzyme that may represent the functional counterpart to murine Granzyme D. Methods: Cord blood-derived mast cells, LAD2 cells and skin mast cells in situ were evaluated for their expression of Granzymes using quantitative PCR, Western blot analysis and immunostaining. Mast cells were activated by either calcium ionophore stimulation or IgE receptor cross-linking. Results: Cord blood-derived mast cells and LAD2 cells were shown to express Granzyme H and B mRNA, while Granzyme A, K and M expression was undetectable. Mast cell activation by either calcium ionophore or IgE receptor cross-linking caused down-regulated expression of Granzyme H. In contrast, Granzyme B expression was up-regulated by the same stimuli. Granzyme H expression was also confirmed at the protein level, as shown by both Western blot analysis and confocal microscopy. Further, we show that Granzyme H is expressed by human skin mast cells in situ. Conclusions: The present findings implicate Granzyme H as a novel protease expressed by human mast cells and support earlier findings obtained in natural killer cells suggesting that Granzymes B and H are reciprocally regulated.
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The perforin pore facilitates the delivery of cationic cargos.
The Journal of biological chemistry, 2014Co-Authors: Sarah E. Stewart, Joseph A Trapani, James C. Whisstock, Michelle A Dunstone, Michael E. D'angelo, Stephanie Kondos, Antony Yaron Matthews, Phillip I. BirdAbstract:Cytotoxic lymphocytes eliminate virally infected or neoplastic cells through the action of cytotoxic proteases (Granzymes). The pore-forming protein perforin is essential for delivery of Granzymes into the cytoplasm of target cells; however the mechanism of this delivery is incompletely understood. Perforin contains a membrane attack complex/perforin (MACPF) domain and oligomerizes to form an aqueous pore in the plasma membrane; therefore the simplest (and best supported) model suggests that Granzymes passively diffuse through the perforin pore into the cytoplasm of the target cell. Here we demonstrate that perforin preferentially delivers cationic molecules while anionic and neutral cargoes are delivered inefficiently. Furthermore, another distantly related pore-forming MACPF protein, pleurotolysin (from the oyster mushroom), also favors the delivery of cationic molecules, and efficiently delivers human Granzyme B. We propose that this facilitated diffusion is due to conserved features of oligomerized MACPF proteins, which may include an anionic lumen.
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a role for Granzyme m in tlr4 driven inflammation and endotoxicosis
Journal of Immunology, 2010Co-Authors: Desiree A Anthony, Joseph A Trapani, Phillip I. Bird, Daniel M Andrews, Melvyn T Chow, Sally V Watt, Colin M House, Shizou Akira, Mark J. SmythAbstract: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.
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Functional dissection of the Granzyme family: cell death and inflammation.
Immunological reviews, 2010Co-Authors: Desiree A Anthony, Joseph A Trapani, Daniel M Andrews, Sally V Watt, Mark J. SmythAbstract:Cytotoxic lymphocytes rapidly respond and destroy both malignant cells and cells infected with intracellular pathogens. One mechanism, known as granule exocytosis, employs the secretory granules of these lymphocytes. These include the pore-forming protein perforin (pfp) and a family of serine proteases known as Granzymes that cleave and activate effector molecules within the target cell. Over the past two decades, the study of Granzymes has largely focused on the ability of these serine proteases to induce cell death. More recently, sophisticated mouse models of disease coupled with gene-targeted mice have allowed investigators to ask why Granzyme subfamilies are encoded on different chromosomal loci and what broader role these enzymes might play in inflammation and immune response. Here, we provide a brief overview of the Granzyme superfamily, their relationship to pfp, and their reported functions in apoptosis. This overview is followed by a comprehensive analysis of the less characterized and developing field regarding the non-apoptotic functions of Granzymes.
James C. Powers - One of the best experts on this subject based on the ideXlab platform.
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Dipeptidyl peptidase I: importance of proGranzyme activation sequences, other dipeptide sequences, and the N-terminal amino group of synthetic substrates for enzyme activity
Archives of Biochemistry and Biophysics, 2002Co-Authors: Tinh V. Tran, Karen A. Ellis, Dorothy Hudig, James C. PowersAbstract:Abstract The broadly reactive cysteine protease dipeptidyl peptidase I (DPPI, cathepsin C) is thought to activate all proGranzymes (zymogens of lymphocyte serine proteases) to form mature Granzymes. We synthesized dipeptide 7-amino-4-methylcoumarin (AMC) substrates containing proGranzyme activation sequences and showed that they were efficiently hydrolyzed by DPPI. However, DPPI will not hydrolyze Ile-Ile-AMC, the N-terminal dipeptide sequence found in mature Granzymes. Introduction of the nonphysiological homophenylalanine (Hph) residue at P1 resulted in the best substrate Ala-Hph-AMC for DPPI ( k cat / K m =9,000,000 M −1 s −1 ) . The charged N-terminal amino group of the substrate was essential and replacement of the NH 2 group with OH or NH(CH 3 ) in Gly-Phe-AMC reduced the k cat / K m value by two to three orders of magnitude. A hydrazide azaglycine analog, NH 2 NHCO-Phe-AMC, was not hydrolyzed at pH 5.5, but underwent slow hydrolysis at lower pHs where the amino group is partially protonated. DPPI also failed to hydrolyze NH 2 COCH 2 -Phe-AMC, where the NH 2 group is unprotonated. The results reported in this paper should be useful in the design of better DPPI inhibitors to block Granzyme maturation and Granzyme-dependent apoptosis.
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Granzymes (lymphocyte serine proteases): characterization with natural and synthetic substrates and inhibitors.
Biochimica et Biophysica Acta, 2000Co-Authors: Dorothy Hudig, James C. PowersAbstract:Natural killer (NK) and cytotoxic T-lymphocytes (CTLs) kill cells within an organism to defend it against viral infections and the growth of tumors. One mechanism of killing involves exocytosis of lymphocyte granules which causes pores to form in the membranes of the attacked cells, fragments nuclear DNA and leads to cell death. The cytotoxic granules contain perforin, a pore-forming protein, and a family of at least 11 serine proteases termed Granzymes. Both perforin and Granzymes are involved in the lytic activity. Although the biological functions of most Granzymes remain to be resolved, Granzyme B clearly promotes DNA fragmentation and is directly involved in cell death. Potential natural substrates for Gr B include procaspases and other proteins involved in cell death. Activated caspases are involved in apoptosis. The search continues for natural substrates for the other Granzymes. The first Granzyme crystal structure remains to be resolved, but in the interim, molecular models of Granzymes have provided valuable structural information about their substrate binding sites. The information has been useful to predict the amino acid sequences that immediately flank each side of the scissile peptide bond of peptide and protein substrates. Synthetic substrates, such as peptide thioesters, nitroanilides and aminomethylcoumarins, have also been used to study the substrate specificity of Granzymes. The different Granzymes have one of four primary substrate specificities: tryptase (cleaving after Arg or Lys), Asp-ase (cleaving after Asp), Met-ase (cleaving after Met or Leu), and chymase (cleaving after Phe, Tyr, or Trp). Natural serpins and synthetic inhibitors (including isocoumarins, peptide chloromethyl ketones, and peptide phosphonates) inhibit Granzymes. Studies of substrate and inhibitor kinetics are providing valuable information to identify the most likely natural Granzyme substrates and provide tools for the study of key reactions in the cytolytic mechanism.
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the human cytotoxic t cell granule serine protease Granzyme h has chymotrypsin like chymase activity and is taken up into cytoplasmic vesicles reminiscent of Granzyme b containing endosomes
Journal of Biological Chemistry, 1999Co-Authors: Kirsten M Edwards, James C. Powers, Chihmin Kam, Joseph A TrapaniAbstract:Serine proteases (Granzymes) contained within the cytoplasmic granules of cytotoxic T cells and natural killer cells play a variety of roles including the induction of target cell apoptosis, breakdown of extracellular matrix proteins and induction of cytokine secretion by bystander leukocytes. Different Granzymes display proteolytic specificities that mimic the activities of trypsin or chymotrypsin, or may cleave substrates at acidic ("Asp-ase") or at long unbranched amino acids such as Met ("Met-ase"). Here, we report that recombinant Granzyme H has chymotrypsin-like (chymase) activity, the first report of a human Granzyme with this proteolytic specificity. Recombinant 32-kDa Granzyme H expressed in the baculovirus vector pBacPAK8 was secreted from Sf21 cells and recovered by Ni-affinity chromatography, using a poly-His tag encoded at the predicted carboxyl terminus of full-length Granzyme H cDNA. The Granzyme H efficiently cleaved Suc-Phe-Leu-Phe-SBzl (v = 185 nM/s at [S] = 0.217 mM) and also hydrolyzed Boc-Ala-Ala-X-SBzl (X = Phe, Tyr, Met, Nle, or Nva) with slower rates but had little tryptase or Asp-ase activity. Enzymatic activity was inhibited completely by 0.1 mM 3,4-dichloroisocoumarin and 84% by 1.0 mM phenylmethylsulfonyl fluoride. Fluoresceinated Granzyme H was internalized in a temperature-dependent manner by Jurkat cells into endosome-like vesicles, suggesting that it can bind to cell surface receptors similar to those that bind Granzyme B. This suggests a hitherto unsuspected intracellular function for Granzyme H.
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Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins.
Biochemistry, 1991Co-Authors: Shinjiro Odake, Lakshmi S. Narasimhan, Joseph T. Blake, Olivier Krahenbuhl, Juerg Tschopp, James C. PowersAbstract:: The active site structures of human Q31 Granzyme A, murine Granzymes (A, B, C, D, E, and F), and human Granzymes (A, B, and 3) isolated from cytotoxic T lymphocytes (CTL) were studied with peptide thioester substrates, peptide chloromethyl ketone, and isocoumarin inhibitors. Human Q31, murine, and human Granzyme A hydrolyzed Arg- or Lys-containing thioesters very efficiently with kcat/KM of 10(4)-10(5) M-1 s-1. Murine Granzyme B was found to have Asp-ase activity and hydrolyzed Boc-Ala-Ala-Asp-SBzl with a kcat/KM value of 2.3 X 10(5) M-1 s-1. The rate was accelerated 1.4-fold when the 0.05 M NaCl in the assay was replaced with CaCl2. The preparation of Granzyme B also had significant activity toward Boc-Ala-Ala-AA-SBzl substrates, where AA was Asn, Met, or Ser [kcat/KM = (4-5) X 10(4) M-1 s-1]. Murine Granzymes C, D, and E did not hydrolyze any thioester substrate but contained minor contaminating activity toward Arg- or Lys-containing thioesters. Murine Granzyme F had small activity toward Suc-Phe-Leu-Phe-SBzl, along with some contaminating trypsin-like activity. Human Q31 Granzyme A, murine, and human Granzyme A were inhibited quite efficiently by mechanism-based isocoumarin inhibitors substituted with basic groups (guanidino or isothiureidopropoxy). Although the general serine protease inhibitor 3,4-dichloroisocoumarin (DCI) inactivated these tryptases poorly, it was the best isocoumarin inhibitor for murine Granzyme B (kobs/[I] = 3700-4200 M-1 s-1). Murine and human Granzyme B were also inhibited by Boc-Ala-Ala-Asp-CH2Cl; however, the inhibition was less potent than that with DCI. DCI, 3-(3-amino-propoxy)-4-chloroisocoumarin, 4-chloro-3-(3-isothiureidopropoxy)isocoumarin, and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin inhibited Q31 cytotoxic T lymphocyte mediated lysis of human JY lymphoblasts (ED50 = 0.5-5.0 microM).
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Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins.
Biochemistry, 1991Co-Authors: Shinjiro Odake, Lakshmi S. Narasimhan, Joseph T. Blake, Olivier Krahenbuhl, Juerg Tschopp, James C. PowersAbstract:: The active site structures of human Q31 Granzyme A, murine Granzymes (A, B, C, D, E, and F), and human Granzymes (A, B, and 3) isolated from cytotoxic T lymphocytes (CTL) were studied with peptide thioester substrates, peptide chloromethyl ketone, and isocoumarin inhibitors. Human Q31, murine, and human Granzyme A hydrolyzed Arg- or Lys-containing thioesters very efficiently with kcat/KM of 10(4)-10(5) M-1 s-1. Murine Granzyme B was found to have Asp-ase activity and hydrolyzed Boc-Ala-Ala-Asp-SBzl with a kcat/KM value of 2.3 X 10(5) M-1 s-1. The rate was accelerated 1.4-fold when the 0.05 M NaCl in the assay was replaced with CaCl2. The preparation of Granzyme B also had significant activity toward Boc-Ala-Ala-AA-SBzl substrates, where AA was Asn, Met, or Ser [kcat/KM = (4-5) X 10(4) M-1 s-1]. Murine Granzymes C, D, and E did not hydrolyze any thioester substrate but contained minor contaminating activity toward Arg- or Lys-containing thioesters. Murine Granzyme F had small activity toward Suc-Phe-Leu-Phe-SBzl, along with some contaminating trypsin-like activity. Human Q31 Granzyme A, murine, and human Granzyme A were inhibited quite efficiently by mechanism-based isocoumarin inhibitors substituted with basic groups (guanidino or isothiureidopropoxy). Although the general serine protease inhibitor 3,4-dichloroisocoumarin (DCI) inactivated these tryptases poorly, it was the best isocoumarin inhibitor for murine Granzyme B (kobs/[I] = 3700-4200 M-1 s-1). Murine and human Granzyme B were also inhibited by Boc-Ala-Ala-Asp-CH2Cl; however, the inhibition was less potent than that with DCI. DCI, 3-(3-amino-propoxy)-4-chloroisocoumarin, 4-chloro-3-(3-isothiureidopropoxy)isocoumarin, and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin inhibited Q31 cytotoxic T lymphocyte mediated lysis of human JY lymphoblasts (ED50 = 0.5-5.0 microM).
Phillip I. Bird - One of the best experts on this subject based on the ideXlab platform.
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Substrate specificities of the Granzyme tryptases A and K.
Journal of proteome research, 2014Co-Authors: Kim Plasman, Phillip I. Bird, Kris Gevaert, Hans Demol, Petra Van DammeAbstract:The physiological roles of the Granzymes A and K have been debated, especially concerning their involvement in cytotoxic and inflammatory processes. By performing N-terminal COFRADIC assisted N-terminomics on the homologous human Granzymes A and K, we here provide detailed data on their substrate repertoires, their specificities, and differences in efficiency by which they cleave their substrates, all of which may aid in elucidating their key substrates. In addition, the so far uncharacterized mouse Granzyme K was profiled alongside its human orthologue. While the global primary specificity profiles of these Granzymes appear quite similar as they revealed only subtle differences and pointed to substrate occupancies in the P1, P1', and P2' position as the main determinants for substrate recognition, differential analyses unveiled distinguishing substrate subsite features, some of which were confirmed by the more selective cleavage of specifically designed probes.
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The perforin pore facilitates the delivery of cationic cargos.
The Journal of biological chemistry, 2014Co-Authors: Sarah E. Stewart, Joseph A Trapani, James C. Whisstock, Michelle A Dunstone, Michael E. D'angelo, Stephanie Kondos, Antony Yaron Matthews, Phillip I. BirdAbstract:Cytotoxic lymphocytes eliminate virally infected or neoplastic cells through the action of cytotoxic proteases (Granzymes). The pore-forming protein perforin is essential for delivery of Granzymes into the cytoplasm of target cells; however the mechanism of this delivery is incompletely understood. Perforin contains a membrane attack complex/perforin (MACPF) domain and oligomerizes to form an aqueous pore in the plasma membrane; therefore the simplest (and best supported) model suggests that Granzymes passively diffuse through the perforin pore into the cytoplasm of the target cell. Here we demonstrate that perforin preferentially delivers cationic molecules while anionic and neutral cargoes are delivered inefficiently. Furthermore, another distantly related pore-forming MACPF protein, pleurotolysin (from the oyster mushroom), also favors the delivery of cationic molecules, and efficiently delivers human Granzyme B. We propose that this facilitated diffusion is due to conserved features of oligomerized MACPF proteins, which may include an anionic lumen.
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Are all Granzymes cytotoxic in vivo
Biological chemistry, 2014Co-Authors: Lars T. Joeckel, Phillip I. BirdAbstract:Abstract 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.
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Conservation of the Extended Substrate Specificity Profiles Among Homologous Granzymes Across Species
Molecular & cellular proteomics : MCP, 2013Co-Authors: Kim Plasman, Phillip I. Bird, Sebastian Maurer-stroh, Jamshaid Ahmad, Han Hao, Dion Kaiserman, Fernanda L. Sirota, Veronique Jonckheere, Kris Gevaert, Petra Van DammeAbstract:Granzymes are structurally related serine proteases involved in cell death and immunity. To date four out of five human Granzymes have assigned orthologs in mice; however for Granzyme H, no murine ortholog has been suggested and its role in cytotoxicity remains controversial. Here, we demonstrate that, as is the case for Granzyme C, human Granzyme H is an inefficient cytotoxin that together with their similar pattern of GrB divergence and functional similarity strongly hint to their orthologous relationship. Besides analyzing the substrate specificity profile of Granzyme H by substrate phage display, substrate cleavage susceptibility of human Granzyme H and mouse Granzyme C was assessed on a proteome-wide level. The extended specificity profiles of Granzymes C and H (i.e. beyond cleavage positions P4-P4′) match those previously observed for Granzyme B. We demonstrate conservation of these extended specificity profiles among various Granzymes as Granzyme B cleavage susceptibility of an otherwise Granzyme H/C specific cleavage site can simply be conferred by altering the P1-residue to aspartate, the preferred P1-residue of Granzyme B. Our results thus indicate a conserved, but hitherto underappreciated specificity-determining role of extended protease-substrate contacts in steering cleavage susceptibility.
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a role for Granzyme m in tlr4 driven inflammation and endotoxicosis
Journal of Immunology, 2010Co-Authors: Desiree A Anthony, Joseph A Trapani, Phillip I. Bird, Daniel M Andrews, Melvyn T Chow, Sally V Watt, Colin M House, Shizou Akira, Mark J. SmythAbstract: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.
Graham P Cook - One of the best experts on this subject based on the ideXlab platform.
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identification of the bcl2 adenovirus e1b 19k protein interacting protein 2 bnip 2 as a Granzyme b target during human natural killer cell mediated killing
Biochemical Journal, 2010Co-Authors: Gina B Scott, Paul A Bowles, Erica B Wilson, Josephine L Meade, Boon Chuan Low, Adam Davison, Eric G Blair, Graham P CookAbstract:Cytotoxic lymphocytes eliminate infected cells and tumours via the perforin-mediated delivery of pro-apoptotic serine proteases known as Granzymes. Granzyme B triggers apoptosis via the cleavage of a repertoire of cellular proteins, leading to caspase activation and mitochondrial depolarization. A simple bioinformatics strategy identified a candidate Granzyme B cleavage site in the widely expressed BNIP-2 (BCL2/adenovirus E1B-19K protein-interacting protein 2). Granzyme B cleaved recombinant BNIP-2 in vitro and endogenous BNIP-2 was cleaved during the NK (natural killer) cell-mediated killing of tumour cells. Cleavage required the site identified in the bioinformatics screen and was caspase-independent. Expression of either full-length BNIP-2 or a truncated molecule mimicking the Granzyme B cleaved form was pro-apoptotic and led to the caspase-dependent cleavage of BNIP-2 at a site distinct from Granzyme B cleavage. Inhibition of BNIP-2 expression did not affect the susceptibility to NK cell-mediated killing. Furthermore, target cells in which BID (BH3-interacting domain death agonist) expression was inhibited also remained highly susceptible to NK cell-mediated killing, revealing redundancy in the pro-apoptotic response to human cytotoxic lymphocytes. Such redundancy reduces the opportunity for escape from apoptosis induction and maximizes the chances of immune-mediated clearance of infected cells or tumour cells.
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Identification of the BCL2/Adenovirus E1B-19K protein-interacting protein 2 (BNIP-2) as a Granzyme B target during human natural killer cell-mediated killing.
Biochemical Journal, 2010Co-Authors: Gina B Scott, Paul A Bowles, Erica B Wilson, Josephine L Meade, Boon Chuan Low, Adam Davison, G. Eric Blair, Graham P CookAbstract:Cytotoxic lymphocytes eliminate infected cells and tumours via the perforin mediated delivery of pro-apoptotic serine proteases known as Granzymes. Granzyme B triggers apoptosis via the cleavage of a repertoire of cellular proteins, leading to caspase activation and mitochondrial depolarisation. A simple bioinformatics strategy identified a candidate Granzyme B cleavage site in the widely expressed BCL2/Adenovirus E1B 19K protein-interacting protein-2 (BNIP-2). Granzyme B cleaved recombinant BNIP-2 in vitro and endogenous BNIP-2 was cleaved during the NK cell mediated killing of tumour cells. Cleavage required the site identified in the bioinformatics screen and was caspase-independent. Expression of either full length BNIP-2 or a truncated molecule mimicking the Granzyme B cleaved form was pro-apoptotic and led to the caspase-dependent cleavage of BNIP-2 at a site distinct from Granzyme B cleavage. Inhibition of BNIP-2 expression did not affect the susceptibility to NK cell-mediated killing. Furthermore, target cells in which BID expression was inhibited also remained highly susceptible to NK cell-mediated killing, revealing redundancy in the pro-apoptotic response to human cytotoxic lymphocytes. Such redundancy reduces the opportunity for escape from apoptosis induction and maximises the chances of immune-mediated clearance of infected cells or tumour cells.
Timothy J. Ley - One of the best experts on this subject based on the ideXlab platform.
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Differential Expression of Granzyme B and C in Murine Cytotoxic Lymphocytes
Journal of immunology (Baltimore Md. : 1950), 2009Co-Authors: Sheng F Cai, Xuefang Cao, Todd A Fehniger, Joshua C. Mayer, Joel D. Brune, Anthony R. French, Timothy J. LeyAbstract:Cytotoxic lymphocytes use the granule exocytosis pathway to kill pathogen-infected cells and tumor cells. Although many genes in this pathway have been extensively characterized (e.g., perforin, Granzymes A and B), the role of Granzyme C is less clear. We therefore developed a Granzyme C-specific mAb and used flow cytometry to examine the expression of Granzyme B and C in the lymphocyte compartments of wild-type and mutant GzmB−/− cre mice, which have a small deletion in the Granzyme B gene. We detected Granzyme B and C expression in CD4+ and CD8+ T cells activated with CD3/CD28 beads or MLRs. Stimulation of NK cells in vitro with IL-15 also induced expression of both Granzymes. Granzyme C up-regulation was delayed relative to Granzyme B in wild-type lymphocytes, whereas GzmB−/− cre cells expressed Granzyme C earlier and more abundantly on a per-cell basis, suggesting that the deleted 350-bp region in the Granzyme B gene is important for the regulation of both Granzymes B and C. Quantitative RT-PCR revealed that Granzyme C protein levels were regulated by mRNA abundance. In vivo, a population of wild-type CD8αα+ intraepithelial lymphocytes constitutively expressed Granzyme B and GzmB−/− cre intraepithelial lymphocytes likewise expressed Granzyme C. Using a model of a persistent murine CMV infection, we detected delayed expression of Granzyme C in NK cells from infected hosts. Taken together, these findings suggest that Granzyme C is activated with persistent antigenic stimulation, providing nonredundant backup protection for the host when Granzyme B fails.
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Orphan Granzymes Downstream from Granzyme B Are Important for Tumor Clearance In Vivo and in Vitro.
Blood, 2004Co-Authors: Xuefang Cao, William J. Grossman, Paula A. Revell, Dori A. Thomas, Timothy J. LeyAbstract:Cytotoxic lymphocytes (Natural Killer cells and Cytotoxic T lymphocytes) can utilize the perforin/Granzyme pathway as a major mechanism to kill pathogen-infected cells and tumor cells. Perforin is responsible for delivering and/or trafficking the Granzymes (a family of neutral serine proteases) to the target cells. In the target cell cytoplasm and nucleus, the Granzymes deliver the lethal hits. Granzymes A and B are the best characterized Granzymes, and they can cleave a variety of important protein substrates to execute the target cells. However, some tumors and viruses have developed potent Granzyme inhibitors that may allow them to evade cytotoxic lymphocyte-induced death. Interestingly, additional Granzyme genes downstream from Granzyme B (C, F, G, and D) on murine chromosome 14 are also expressed in cytotoxic lymphocytes, and are referred to as “orphans” since their functions have not been defined. We have developed two kinds of Granzyme B knockout mice in the 129/SvJ background (H-2b) and examined their expression of Granzyme B and orphan Granzymes using quantitative RT-PCR and Western Blotting. In the first mouse (Gzm B−/−/+PGK-neo) a PGK-neo cassette was retained in the Granzyme B gene, which caused a neighborhood effect, with significantly reduced expression of orphan Granzymes C and F in cytotoxic lymphocytes (this mouse is referred to as “B cluster” deficient); In the second mouse (Gzm B−/−/ΔPGK-neo) the PGK-neo cassette was removed by Cre/loxP technology, which restored expression of Granzymes C and F in cytotoxic lymphocytes (referred to as “B only” deficient). Both mutations completely abolish Granzyme B expression. Using a Flow-based Killing Assay (FloKA), we have examined the cytotoxic functions of lymphocytes derived from mixed lymphocyte reactions (MLR) and 10-day lymphokine activated killer (LAK) cultures. We have found that Granzyme B cluster-deficient cytotoxic lymphocytes (H-2b) generated by MLR kill allogeneic P815 or TA-3 tumor cells (H-2d) less efficiently than those deficient for Granzyme B only (e.g. P815 killing at 3 hours, WT: 35%±1%, B only-deficient: 24%±5%, B cluster-deficient: 14%±3%, p
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Differential expression of Granzymes A and B in human cytotoxic lymphocyte subsets and T regulatory cells
Blood, 2004Co-Authors: William J. Grossman, Benjamin L. Tollefsen, James W Verbsky, Claudia Kemper, John P. Atkinson, Timothy J. LeyAbstract:Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use the perforin/Granzyme pathway as a major mechanism to kill pathogen-containing cells and tumor cells.(1,2) Dysregulation of this pathway results in several human diseases, such as hemophagocytic lymphohistiocytosis. Here we characterize the single-cell expression pattern of Granzymes A and B in human lymphocytes using a flow cytometry-based assay. We demonstrate that most circulating CD56(+)8(-) NK cells, and approximately half of circulating CD8(+) T lymphocytes, coexpressed both Granzymes A and B. In contrast, few circulating CD4(+) T lymphocytes expressed Granzymes A or B. Activation of CD8(+) T lymphocytes with concanavalin A (ConA)/interleukin-2 (IL-2), and activation of CD4(+) T lymphocytes with antibodies to CD3/CD28 or CD3/CD46 (to generate T regulatory [Tr1] cells), induced substantial expression of Granzyme B, but not Granzyme A. Naive CD4(+)CD45RA(+) cells stimulated with antibodies to CD3/CD46 strongly expressed Granzyme B, while CD3/CD28 stimulation was ineffective. Finally, we show that Granzyme B-expressing CD4(+) Tr1 cells are capable of killing target cells in a perforin-dependent, but major histocompatibility complex (MHC)/T-cell receptor (TCR)-independent, manner. Our results demonstrate discordant expression of Granzymes A and B in human lymphocyte subsets and T regulatory cells, which suggests that different Granzymes may play unique roles in immune system responses and regulation.
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The orphan Granzymes of humans and mice.
Current opinion in immunology, 2003Co-Authors: William Grossman, Paula A. Revell, Hillary Johnson, Andrew J. Bredemeyer, Timothy J. LeyAbstract:The Granzyme/perforin pathway is a central pathway for lymphocyte-mediated killing in both the innate and adaptive immune systems. This pathway is important in a variety of host defenses, including viral clearance and tumor cell killing, and its dysregulation results in several human and rodent diseases. To date, the majority of reports in this field have concentrated on the functions of Granzymes A and B. Recent reports, however, suggest that the non-A/non-B 'orphan' Granzymes found in both humans and mice are potentially significant. Although the functions of these orphan Granzymes have yet to be fully established, initial data suggests their importance in both immune and nonimmune cells.
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Cell death induced by Granzyme C.
Blood, 2002Co-Authors: Hillary Johnson, Luca Scorrano, Stanley J. Korsmeyer, Timothy J. LeyAbstract:Although the functions of Granzymes A and B have been defined, the functions of the other highly expressed Granzymes (Gzms) of murine cytotoxic lymphocytes (C, D, and F) have not yet been evaluated. In this report, we describe the ability of murine GzmC (which is most closely related to human Granzyme H) to cause cell death. The induction of death requires its protease activity and is characterized by the rapid externalization of phosphatidylserine, nuclear condensation and collapse, and single-stranded DNA nicking. The kinetics of these events are similar to those caused by Granzyme B, and its potency (defined on a molar basis) is also equivalent. The induction of death did not involve the activation of caspases, the cleavage of BID, or the activation of the CAD nuclease. However, Granzyme C did cause rapid mitochondrial swelling and depolarization in intact cells or in isolated mitochondria, and this mitochondrial damage was not prevented by cyclosporin A pretreatment. These results suggest that Granzyme C rapidly induces target cell death by attacking nuclear and mitochondrial targets and that these targets are distinct from those used by Granzyme B to cause classical apoptosis.
