Journal articles: 'Granzymes'

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Joeckel, Lars T., and Phillip I. Bird. "Are all granzymes cytotoxic in vivo?" Biological Chemistry 395, no. 2 (February 1, 2014): 181–202. http://dx.doi.org/10.1515/hsz-2013-0238.

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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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Ebnet, K., C. N. Levelt, T. T. Tran, K. Eichmann, and M. M. Simon. "Transcription of granzyme A and B genes is differentially regulated during lymphoid ontogeny." Journal of Experimental Medicine 181, no. 2 (February 1, 1995): 755–63. http://dx.doi.org/10.1084/jem.181.2.755.

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During development, thymocytes express a number of genes typical for activated peripheral T lymphocytes, including granzymes. We have now analyzed by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and cytochemistry fetal liver cells and thymocytes at various developmental stages for the expression of granzyme A-G genes. At days 13-17 of gestation, only granzyme B but none of the other granzymes is expressed in fetal liver. In the most immature, Pgp-1+IL2R alpha-, thymocyte subpopulation mRNAs for granzymes A-C but not for granzymes D-G are detectable. Upon further differentiation via Pgp-1-IL-2R alpha + into more mature Pgp-1-IL-2R alpha- thymocytes the level of expression of granzymes A, B, and C gradually declines reaching its lowest level at the CD4+ 8+ double positive stage. In fetal thymic lobes depleted of lymphoid cells by treatment with deoxyguanosine, no transcripts for granzymes A, B, and C were found indicating that the PCR signals are derived exclusively from early precursor T/natural killer (NK) lineage cells rather than from residual stromal elements. In mature CD4+CD8- and CD4-CD8+ thymocytes, granzyme B mRNA is found at similar levels in both subsets whereas granzyme A mRNA is expressed selectively in the CD4-CD8+ subset. Enzymatic activity of granzyme A was only seen in a fraction of CD4-CD8+ thymocytes negative for heat stable antigen (HSA) but not in the more immature HSA+ fraction of CD4-CD8+ thymocytes. The data suggest that (a) granzyme B is a pro-thymocyte marker for all T/NK lineage cells; (b) granzyme A transcripts are associated with thymocytes with the potential to develop into the CD8+ lineage; and (c) granzyme A enzymatic activity is only expressed in the most mature CD4-CD8+ stage, suggesting that granzyme proteins are not involved in early stages of thymocyte development.

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Hay, Zachary L. Z., and Jill E. Slansky. "Granzymes: The Molecular Executors of Immune-Mediated Cytotoxicity." International Journal of Molecular Sciences 23, no. 3 (February 6, 2022): 1833. http://dx.doi.org/10.3390/ijms23031833.

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Cytotoxic T lymphocytes, differentiated CD8+ T cells, use multiple mechanisms to mediate their function, including release of granules containing perforin and granzymes at target cells. Granzymes are a family of cytotoxic proteases that each act on unique sets of biological substrates within target cells, usually to induce cell death. Granzymes are differentially expressed within T cells, depending on their environment and activation state, making the granzyme cytotoxic pathway dynamic and responsive to individual circumstances. In this review, we describe what is currently known about granzyme structure, processing, and granzyme-induced cell death in the context of cancer and in some other inflammatory diseases.

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Grossman, William J., James W. Verbsky, Benjamin L. Tollefsen, Claudia Kemper, John P. Atkinson, and Timothy J. Ley. "Differential expression of granzymes A and B in human cytotoxic lymphocyte subsets and T regulatory cells." Blood 104, no. 9 (November 1, 2004): 2840–48. http://dx.doi.org/10.1182/blood-2004-03-0859.

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Abstract 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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Zeberg, Lennart, and Tor Olofsson. "Differential Expression of Granzymes A and K in Subsets of Human T-Cells and NK-Cells." Blood 106, no. 11 (November 16, 2005): 3917. http://dx.doi.org/10.1182/blood.v106.11.3917.3917.

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Abstract Granzymes are highly specific proteases stored in secretory granule in T-cells and NK-cells. Five different granzymes are known in human (A, B, H, K and M). A and B are the best studied and both can induce apotosis when delivered to target cells. The role for granzyme H, K and M are principally unknown. We have studied the expression of granzymes in subsets of T-cells and NK-cells both in fresh circulating cells and cultured cells stimulated with IL-2, IL-15 and OKT3/antiCD28. Protein expression (biosynthesis/immunoprecipitation) and mRNA level (RT-PCR) was studied in parallel. This report focus on Granzyme A and K which are close relatives, both tryptases located 70 kb apart on chromosome 5. Granzyme A have the highest expression in NK-cells were granzyme K are barely detected. Granzyme K on the other hand have a particular high expression in CD8+ T cells. Stimulation with IL-2 or IL-15 give a strong upregulation of both enzymes in NK-cells. Stimulation of CD8+ T cells gives mainly upregulation of granzyme A. Fresh CD4+ T cells show very weak synthesis of granzymes but treatment with IL-2 or IL-15 give a clear upregulation of both granzyme A and K. T-cells (both CD4+ and CD8+) are highly responsive to stimulation with OKT3/anti CD28 producing granzyme B but not A and K. This rather discordant pattern of expression for granzyme A and K talks in favour of different modes of regulation and most likely different functions. A striking difference between granzyme A and K concern the propensity for granzyme A to be secreted into the culture medium after stimulation while this is not the case for granzyme K. The secreted form of granzyme A has a molecular weight slightly higher than the intracellular form indicating the presence of a proform. Secretion of granzymes to the extracellular medium is probably not only an in vitro phenomena since both granzyme A and B can be detected in serum from healthy individuals in picomolar concentrations. Moreover, significant elevation of granzyme levels in serum can bee seen in different diseases with activation of cellular immunity and in T- and NK-cells malignancies. We have previously reported that proforms of certain serine proteases including the five human granzymes have a downregulating activity on myeloid proliferation, a mechanism probably implicated in certain forms of neutropenia. We propose that secreted granzymes (especially A, B and H) constitute a part of T regulatory function working both locally at the site of inflammation and distantly via the circulation.

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Cao, Xuefang, Paula A. Revell, William J. Grossman, Dori A. Thomas, Zhi Hong Lu, and Timothy J. Ley. "Orphan Granzymes Downstream from Granzyme B Are Important for Tumor Clearance In Vivo and in Vitro." Blood 104, no. 11 (November 16, 2004): 2653. http://dx.doi.org/10.1182/blood.v104.11.2653.2653.

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Abstract 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<0.001). The reduction in granzyme B cluster-deficient killing is also seen with LAK cells against YAC-1 and RMA-S target cells (e.g. RMA-S killing at 4 hours, WT: 26%±1%, B only-deficient: 24%±1%, B cluster-deficient: 18%±1%, p<0.001). These results suggest that both allogeneic CTL and LAK cells require orphan granzymes (C and/or F) for optimal tumor cell killing. The defects in cytotoxicity detected by the FloKA assay have been confirmed to be biologically relevant (Revell et al, Blood2003, 102 (11): 1022) since granzyme B cluster-deficient mice cleared P815 cells less efficiently than either WT or granzyme B only-deficient mice (p<0.02). These studies suggest that the orphan granzymes are important for cytotoxic lymphocyte functions, and that they may provide a source of functional redundancy that would help protect from pathogens or tumor cells that express inhibitors of granzyme A or B.

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Kaiserman, Dion, Catherina H. Bird, Jiuru Sun, Antony Matthews, Kheng Ung, James C. Whisstock, Philip E. Thompson, Joseph A. Trapani, and Phillip I. Bird. "The major human and mouse granzymes are structurally and functionally divergent." Journal of Cell Biology 175, no. 4 (November 20, 2006): 619–30. http://dx.doi.org/10.1083/jcb.200606073.

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Approximately 2% of mammalian genes encode proteases. Comparative genomics reveals that those involved in immunity and reproduction show the most interspecies diversity and evidence of positive selection during evolution. This is particularly true of granzymes, the cytotoxic proteases of natural killer cells and CD8+ T cells. There are 5 granzyme genes in humans and 10 in mice, and it is suggested that granzymes evolve to meet species-specific immune challenge through gene duplication and more subtle alterations to substrate specificity. We show that mouse and human granzyme B have distinct structural and functional characteristics. Specifically, mouse granzyme B is 30 times less cytotoxic than human granzyme B and does not require Bid for killing but regains cytotoxicity on engineering of its active site cleft. We also show that mouse granzyme A is considerably more cytotoxic than human granzyme A. These results demonstrate that even “orthologous” granzymes have species-specific functions, having evolved in distinct environments that pose different challenges.

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Thiery, Jerome, Dennis Keefe, Saviz Saffarian, Denis Martinvalet, Michael Walch, Emmanuel Boucrot, Tomas Kirchhausen, and Judy Lieberman. "Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis." Blood 115, no. 8 (February 25, 2010): 1582–93. http://dx.doi.org/10.1182/blood-2009-10-246116.

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Abstract Cytotoxic T lymphocytes and natural killer cells destroy target cells via the polarized exocytosis of lytic effector proteins, perforin and granzymes, into the immunologic synapse. How these molecules enter target cells is not fully understood. It is debated whether granzymes enter via perforin pores formed at the plasma membrane or whether perforin and granzymes are first endocytosed and granzymes are then released from endosomes into the cytoplasm. We previously showed that perforin disruption of the plasma membrane induces a transient Ca2+ flux into the target cell that triggers a wounded membrane repair response in which lysosomes and endosomes donate their membranes to reseal the damaged membrane. Here we show that perforin activates clathrin- and dynamin-dependent endocytosis, which removes perforin and granzymes from the plasma membrane to early endosomes, preserving outer membrane integrity. Inhibiting clathrin- or dynamin-dependent endocytosis shifts death by perforin and granzyme B from apoptosis to necrosis. Thus by activating endocytosis to preserve membrane integrity, perforin facilitates granzyme uptake and avoids the proinflammatory necrotic death of a membrane-damaged cell.

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Nakajima, H., H. L. Park, and P. A. Henkart. "Synergistic roles of granzymes A and B in mediating target cell death by rat basophilic leukemia mast cell tumors also expressing cytolysin/perforin." Journal of Experimental Medicine 181, no. 3 (March 1, 1995): 1037–46. http://dx.doi.org/10.1084/jem.181.3.1037.

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We have studied the cytotoxic activity of rat basophilic leukemia (RBL) cells transfected with cDNAs for the cytotoxic T lymphocyte (CTL) granule components, cytolysin (perforin), granzyme A, and granzyme B. With red cell targets, cytolysin expression conferred potent hemolytic activity, which was not influenced by coexpression of granzymes. With tumor targets, RBL cells expressing cytolysin alone were weakly cytotoxic, but both cytolytic and nucleolytic activity were enhanced by coexpression of granzyme B. RBL cells expressing all three CTL granule components showed still higher cytotoxic activities, with apoptotic target death. Analysis of the cytotoxic activity of individual transfectant clones showed that cytolytic and nucleolytic activity correlated with granzyme expression but was independent of cytolysin expression within the range examined. A synergism between granzymes A and B was apparent when the triple transfectant was compared with RBL cells expressing cytolysin and one granzyme. These data implicate granzymes as the major mediators of tumor target damage by cytotoxic lymphocytes.

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Griffiths, G. M., and S. Isaaz. "Granzymes A and B are targeted to the lytic granules of lymphocytes by the mannose-6-phosphate receptor." Journal of Cell Biology 120, no. 4 (February 15, 1993): 885–96. http://dx.doi.org/10.1083/jcb.120.4.885.

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To investigate the question of whether lytic granules share a common biogenesis with lysosomes, cloned cytolytic T cell lines were derived from a patient with I-cell disease. The targeting of two soluble lytic granule components, granzymes A and B, was studied in these cells which lack a functional mannose-6-phosphate (Man-6-P) receptor-mediated pathway to lysosomes. Using antibodies and enzymatic substrates to detect the lytic proteins, I-cells were found to constitutively secrete granzymes A and B in contrast to normal cells in which these proteins were stored for regulated secretion. These results suggest that granzymes A and B are normally targeted to the lytic granules of activated lymphocytes by the Man-6-P receptor. In normal cells, the granzymes bear Man-6-P residues, since the oligosaccharide side chains of granzymes A and B, as well as radioactive phosphate on granzyme A from labeled cells, were removed by endoglycosidase H (Endo H). However, in I-cells, granzymes cannot bear Man-6-P and granzyme B acquires complex glycans, becoming Endo H resistant. Although the levels of granzymes A and B in cytolytic I-cell lymphocytes are < 30% of the normal levels, immunolocalization and cell fractionation of granzyme A demonstrated that this reduced amount is correctly localized in the lytic granules. Therefore, a Man-6-P receptor-independent pathway to the lytic granules must also exist. Cathepsin B colocalizes with granzyme A in both normal and I-cells indicating that lysosomal proteins can also use the Man-6-P receptor-independent pathway in these cells. The complete overlap of these lysosomal and lytic markers implies that the lytic granules perform both lysosomal and secretory roles in cytolytic lymphocytes. The secretory role of lytic granules formed by the Man-6-P receptor-independent pathway is intact as assessed by the ability of I-cell lymphocytes to lyse target cells by regulated secretion.

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Loh, Joy, Dori A. Thomas, Paula A. Revell, Timothy J. Ley, and Herbert W. Virgin. "Granzymes and Caspase 3 Play Important Roles in Control of Gammaherpesvirus Latency." Journal of Virology 78, no. 22 (November 15, 2004): 12519–28. http://dx.doi.org/10.1128/jvi.78.22.12519-12528.2004.

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ABSTRACT Gammaherpesviruses can establish lifelong latent infections in lymphoid cells of their hosts despite active antiviral immunity. Identification of the immune mechanisms which regulate gammaherpesvirus latent infection is therefore essential for understanding how gammaherpesviruses persist for the lifetime of their host. Recently, an individual with chronic active Epstein-Barr virus infection was found to have mutations in perforin, and studies using murine gammaherpesvirus 68 (γHV68) as a small-animal model for gammaherpesvirus infection have similarly revealed a critical role for perforin in regulating latent infection. These results suggest involvement of the perforin/granzyme granule exocytosis pathway in immune regulation of gammaherpesvirus latent infection. In this study, we examined γHV68 infection of knockout mice to identify specific molecules within the perforin/granzyme pathway which are essential for regulating gammaherpesvirus latent infection. We show that granzymes A and B and the granzyme B substrate, caspase 3, are important for regulating γHV68 latent infection. Interestingly, we show for the first time that orphan granzymes encoded in the granzyme B gene cluster are also critical for regulating viral infection. The requirement for specific granzymes differs for early versus late forms of latent infection. These data indicate that different granzymes play important and distinct roles in regulating latent gammaherpesvirus infection.

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Weston, G. Scott, and Robert D. Sindelar. "Granzymes as Potential Targets for Rational Drug Design." Current Medicinal Chemistry 3, no. 1 (February 1996): 37–46. http://dx.doi.org/10.2174/092986730301220224163207.

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Abstract: A family of serine proteases, the granzymes, found in the dense cytoplasmic granules of lymphocytes/granulocytes have become targets of interest for rational drug design because of their role in cell-mediated cytotoxicity. Granzymes represent potential late-stage, specific targets for immunomodulation with the promise of fewer side-effects than agents acting at earlier stages of the immune response, such as cyclosporine A (Neoral®, Sandimmune®) and tacrolimus/FK-506 (Prograf®). Recent reports suggesting other possible biological roles for granzymes, including antigen processing and lymphocyte recruitment, and the increasing evidence for the involvement of aspartic acid-specific proteases, such as granzyme B and the interleukin converting enzyme (ICE), in apoptosis or programmed cell death pathways have increased the interest in this growing family of enzymes. This review summarizes current progress in granzyme research and outlines some of the prospects for future work in this area.

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Yarosh, I. V., V. A. Misyurin, and I. I. Krasnyuk. "NOVEL IMMUNOTHERAPEUTIC TARGETED GRANZYME DELIVERY SYSTEMS IN TREATMENT OF MALIGNANT TUMORS." Russian Journal of Biotherapy 20, no. 2 (July 14, 2021): 31–41. http://dx.doi.org/10.17650/1726-9784-2021-20-2-31-41.

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Cytotoxicity is the main human killer cell property. The cytotoxicity reaction of human killer cells is achieved through a complex of molecules, including perforins, granzyme, cathepsin and others. However, only one molecule is enough for target cell death: granzyme. Other molecules are intended for granzyme activation and its delivery to the target cell cytoplasm. Granzymes are a whole family of serine proteases that perform their function in the human body as integral cytolytic effectors during programmed cell death of cancer and pathogen-infected cells. Secreted mainly by cytotoxic T-lymphocytes and NK-cells, granzymes initiate apoptosis via caspase-dependent and caspase-independent pathways. These natural properties make granzymes one of the most promising human enzymes for use in the development of targeted therapeutic strategies in the treatment of various types of cancer.The most promising is granzyme B, because it has the most powerful effector properties. Due to the initiation of cascade reactions that activate apoptosis, granzyme is attractive as a basis for the development of medicines applicable in clinical oncology. At this time, several approaches have been developed for delivering granzyme molecules to tumor cells and facilitating its penetration through the cell membrane. Moreover, some solutions are proposed to overcome the resistance of target cells to granzyme-mediated apoptosis. These approaches are discussed in this review.The purpose of this review was to systematize information on the use of granzyme B as a nanostructured drug delivery system in the treatment of solid and hematological malignancies. In addition, this review discusses ways to overcome the resistance of granzyme penetration into target cells.

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Nakajima, H., P. Golstein, and P. A. Henkart. "The target cell nucleus is not required for cell-mediated granzyme- or Fas-based cytotoxicity." Journal of Experimental Medicine 181, no. 5 (May 1, 1995): 1905–9. http://dx.doi.org/10.1084/jem.181.5.1905.

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The requirement for target cell nuclei in the two apoptotic death pathways used by cytotoxic lymphocytes was tested using model effector systems in which the granzyme and Fas pathways of target damage are isolated. Mast cell tumors expressing granzymes A and B in addition to cytolysin/perforin lysed tumor target cells about 10-fold more efficiently than comparable effector cells without granzymes. Enucleated cytoplast targets derived from these cells were also lysed with a similar 10-fold effect of granzymes. In contrast to cytoplasts, effector granzyme expression did not influence lysis of red cell targets. The Fas pathway was assessed using the selected cytotoxic T lymphocyte hybridoma subline d11S, which lysed target cells expressing Fas but not those lacking Fas. Similarly, cytoplasts derived from Fas+ but not Fas- cells were also readily lysed by these effector cells. Thus, neither the nucleus itself nor the characteristic apoptotic nuclear damage associated with the two major cell death pathways used by cytotoxic lymphocytes are required for cell death per se.

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Prunk, Mateja, Milica Perisic Nanut, Jerica Sabotic, Urban Svajger, and Janko Kos. "Increased cystatin F levels correlate with decreased cytotoxicity of cytotoxic T cells." Radiology and Oncology 53, no. 1 (March 3, 2019): 57–68. http://dx.doi.org/10.2478/raon-2019-0007.

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AbstractBackgroundCystatin F is a protein inhibitor of cysteine peptidases, expressed predominantly in immune cells and localised in endosomal/lysosomal compartments. In cytotoxic immune cells cystatin F inhibits both the major pro-granzyme convertases, cathepsins C and H that activate granzymes, and cathepsin L, that acts as perforin activator. Since perforin and granzymes are crucial molecules for target cell killing by cytotoxic lymphocytes, defects in the activation of either granzymes or perforin can affect their cytotoxic potential.Materials and methodsLevels of cystatin F were assessed by western blot and interactions of cystatin F with cathepsins C, H and L were analysed by immunoprecipitation and confocal microscopy. In TALL-104 cells specific activities of the cathepsins and granzyme B were determined using peptide substrates.ResultsTwo models of reduced T cell cytotoxicity of TALL-104 cell line were established, either by treatment by ionomycin or by immunosuppressive transforming growth factor beta. Reduced cytotoxicity correlated with increased levels of cystatin F and with attenuated activities of cathepsins C, H and L and of granzyme B. Co-localisation of cystatin F and cathepsins C, H and L and interactions between cystatin F and cathepsins C and H were demonstrated.ConclusionsCystatin F is designated as a possible regulator of T cell cytotoxicity, similar to its role in natural killer cells.

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MacIvor, Debra M., Christine T. N. Pham, and Timothy J. Ley. "The 5′ Flanking Region of the Human Granzyme H Gene Directs Expression to T/Natural Killer Cell Progenitors and Lymphokine-Activated Killer Cells in Transgenic Mice." Blood 93, no. 3 (February 1, 1999): 963–73. http://dx.doi.org/10.1182/blood.v93.3.963.

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Abstract Human granzyme H is a neutral serine protease that is expressed predominantly in the lymphokine-activated killer (LAK)/natural killer (NK) compartment of the immune system. The gene that encodes this granzyme is located between the granzyme B and cathepsin G genes on human chromosome 14q11.2. Although the murine orthologue of human granzyme H has not yet been identified, murine granzymes C, D, E, F, and G also lie between the murine granzyme B and cathepsin G genes on murine chromosome 14; murine granzymes C, D, and F are also highly expressed in LAK cells, but minimally in cytotoxic T lymphocytes (CTL). We therefore tested whether the 5′ flanking region of human granzyme H contains the cis-acting DNA sequences necessary to target a reporter gene to the LAK/NK compartment of transgenic mice. A 1.2-kb fragment of 5′ flanking human granzyme H sequence was linked to an SV40 large T-antigen (TAg) reporter gene and used to create six transgenic founder lines. SV40 TAg was specifically expressed in the LAK cells of these mice, but not in resting T or NK cells, in CTL, or in any other tissues. Most mice eventually developed a fatal illness characterized by massive hepatosplenomegaly and disseminated organ infiltration by large malignant lymphocytes. Cell lines derived from splenic tumors were TAg+ and NK1.1+ large granular lymphocytes and displayed variable expression of CD3, CD8, and CD16. Although these cell lines contained perforin and expressed granzymes A, B, C, D, and F, they did not exhibit direct cytotoxicity. Collectively, these results suggest that the 5′ flanking sequences of the human granzyme H gene target expression to an NK/T progenitor compartment and to activated NK (LAK) cells. Mice and humans may therefore share a regulatory “program” for the transcription of NK/LAK specific granzyme genes.

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MacIvor, Debra M., Christine T. N. Pham, and Timothy J. Ley. "The 5′ Flanking Region of the Human Granzyme H Gene Directs Expression to T/Natural Killer Cell Progenitors and Lymphokine-Activated Killer Cells in Transgenic Mice." Blood 93, no. 3 (February 1, 1999): 963–73. http://dx.doi.org/10.1182/blood.v93.3.963.403k18_963_973.

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Human granzyme H is a neutral serine protease that is expressed predominantly in the lymphokine-activated killer (LAK)/natural killer (NK) compartment of the immune system. The gene that encodes this granzyme is located between the granzyme B and cathepsin G genes on human chromosome 14q11.2. Although the murine orthologue of human granzyme H has not yet been identified, murine granzymes C, D, E, F, and G also lie between the murine granzyme B and cathepsin G genes on murine chromosome 14; murine granzymes C, D, and F are also highly expressed in LAK cells, but minimally in cytotoxic T lymphocytes (CTL). We therefore tested whether the 5′ flanking region of human granzyme H contains the cis-acting DNA sequences necessary to target a reporter gene to the LAK/NK compartment of transgenic mice. A 1.2-kb fragment of 5′ flanking human granzyme H sequence was linked to an SV40 large T-antigen (TAg) reporter gene and used to create six transgenic founder lines. SV40 TAg was specifically expressed in the LAK cells of these mice, but not in resting T or NK cells, in CTL, or in any other tissues. Most mice eventually developed a fatal illness characterized by massive hepatosplenomegaly and disseminated organ infiltration by large malignant lymphocytes. Cell lines derived from splenic tumors were TAg+ and NK1.1+ large granular lymphocytes and displayed variable expression of CD3, CD8, and CD16. Although these cell lines contained perforin and expressed granzymes A, B, C, D, and F, they did not exhibit direct cytotoxicity. Collectively, these results suggest that the 5′ flanking sequences of the human granzyme H gene target expression to an NK/T progenitor compartment and to activated NK (LAK) cells. Mice and humans may therefore share a regulatory “program” for the transcription of NK/LAK specific granzyme genes.

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Rönnberg, Elin, Gabriela Calounova, Bengt Guss, Anders Lundequist, and Gunnar Pejler. "Granzyme D Is a Novel Murine Mast Cell Protease That Is Highly Induced by Multiple Pathways of Mast Cell Activation." Infection and Immunity 81, no. 6 (March 25, 2013): 2085–94. http://dx.doi.org/10.1128/iai.00290-13.

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ABSTRACTGranzymes are serine proteases known mostly for their role in the induction of apoptosis. Granzymes A and B have been extensively studied, but relatively little is known about granzymes C to G and K to M. T cells, lymphohematopoietic stromal cells, and granulated metrial gland cells express granzyme D, but the function of granzyme D is unknown. Here we show that granzyme D is expressed by murine mast cells and that its level of expression correlates positively with the extent of mast cell maturation. Coculture of mast cells with live, Gram-positive bacteria caused a profound, Toll-like receptor 2 (TLR2)-dependent induction of granzyme D expression. Granzyme D expression was also induced by isolated bacterial cell wall components, including lipopolysaccharide (LPS) and peptidoglycan, and by stem cell factor, IgE receptor cross-linking, and calcium ionophore stimulation. Granzyme D was released into the medium in response to mast cell activation. Granzyme D induction was dependent on protein kinase C and nuclear factor of activated T cells (NFAT). Together, these findings identify granzyme D as a novel murine mast cell protease and implicate granzyme D in settings where mast cells are activated, such as bacterial infection and allergy.

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Waterhouse, Nigel J., Vivien R. Sutton, Karin A. Sedelies, Annette Ciccone, Misty Jenkins, Stephen J. Turner, Phillip I. Bird, and Joseph A. Trapani. "Cytotoxic T lymphocyte–induced killing in the absence of granzymes A and B is unique and distinct from both apoptosis and perforin-dependent lysis." Journal of Cell Biology 173, no. 1 (April 10, 2006): 133–44. http://dx.doi.org/10.1083/jcb.200510072.

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Cytotoxic T lymphocyte (CTL)–induced death triggered by the granule exocytosis pathway involves the perforin-dependent delivery of granzymes to the target cell. Gene targeting has shown that perforin is essential for this process; however, CTL deficient in the key granzymes A and B maintain the ability to kill their targets by granule exocytosis. It is not clear how granzyme AB−/− CTLs kill their targets, although it has been proposed that this occurs through perforin-induced lysis. We found that purified granzyme B or CTLs from wild-type mice induced classic apoptotic cell death. Perforin-induced lysis was far more rapid and involved the formation of large plasma membrane protrusions. Cell death induced by granzyme AB−/− CTLs shared similar kinetics and morphological characteristics to apoptosis but followed a distinct series of molecular events. Therefore, CTLs from granzyme AB−/− mice induce target cell death by a unique mechanism that is distinct from both perforin lysis and apoptosis.

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Veugelers, Kirstin, Bruce Motyka, Christine Frantz, Irene Shostak, Tracy Sawchuk, and R. Chris Bleackley. "The granzyme B–serglycin complex from cytotoxic granules requires dynamin for endocytosis." Blood 103, no. 10 (May 15, 2004): 3845–53. http://dx.doi.org/10.1182/blood-2003-06-2156.

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Abstract Cytotoxic T lymphocytes and natural killer cells destroy target cells via the directed exocytosis of lytic effector molecules such as perforin and granzymes. The mechanism by which these proteins enter targets is uncertain. There is ongoing debate over whether the most important endocytic mechanism is nonspecific or is dependent on the cation-independent mannose 6-phosphate receptor. This study tested whether granzyme B endocytosis is facilitated by dynamin, a key factor in many endocytic pathways. Uptake of and killing by the purified granzyme B molecule occurred by both dynamin-dependent and -independent mechanisms. However most importantly, serglycin-bound granzyme B in high-molecular-weight degranulate material from cytotoxic T lymphocytes predominantly followed a dynamin-dependent pathway to kill target cells. Similarly, killing by live cytotoxic T lymphocytes was attenuated by a defect in the dynamin endocytic pathway, and in particular, the pathways characteristically activated by granzyme B were affected. We therefore propose a model where degranulated serglycin-bound granzymes require dynamin for uptake.

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Johnson, Hillary, Luca Scorrano, Stanley J. Korsmeyer, and Timothy J. Ley. "Cell death induced by granzyme C." Blood 101, no. 8 (April 15, 2003): 3093–101. http://dx.doi.org/10.1182/blood-2002-08-2485.

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Abstract 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.

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Yamanaka, Gaku, Shinichiro Morichi, Tomoko Takamatsu, Ryou Takahashi, Yusuke Watanabe, Yu Ishida, Mika Takeshita, et al. "Granzyme A Participates in the Pathogenesis of Infection-Associated Acute Encephalopathy." Journal of Child Neurology 35, no. 3 (November 11, 2019): 208–14. http://dx.doi.org/10.1177/0883073819886217.

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Objective: The present study aimed to determine whether granzymes are implicated in the pathogenesis of infection-associated acute encephalopathy (AE). Methods: We investigated granzyme and cytokine levels in the cerebrospinal fluid of patients with acute encephalopathy or complex febrile seizures (cFS). A total of 24 acute encephalopathy patients and 22 complex febrile seizures patients were included in the present study. Levels of granzymes A and B were measured using enzyme-linked immunosorbent assay, and levels of tumor necrosis factor α (TNF-α), interferon-γ (IFN-γ), interleukin 1β (IL-1β), IL-1 receptor antagonist (IL-1RA), IL-4, IL-6, IL-8, and IL-10 were assessed using the Bio-Plex suspension array system. Results: Cerebrospinal fluid levels of granzyme A were significantly higher, and those of TNF-α and IL-1RA were significantly lower in the AE group than in the cFS group; however, no significant differences in the levels of granzyme B, IFN-γ, IL-1β, IL-4, IL-6, IL-8, and IL-10 were observed between the 2 groups. In addition, no significant differences in granzyme A, granzyme B, or cytokine levels were observed between acute encephalopathy patients with and those without neurologic sequelae. Conclusions: Our findings indicate the involvement of granzyme A in the pathogenesis of acute encephalopathy.

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Casciola-Rosen, Livia, Margarita Garcia-Calvo, Herbert G. Bull, Joseph W. Becker, Tonie Hines, Nancy A. Thornberry, and Antony Rosen. "Mouse and Human Granzyme B Have Distinct Tetrapeptide Specificities and Abilities to Recruit the Bid Pathway." Journal of Biological Chemistry 282, no. 7 (December 19, 2006): 4545–52. http://dx.doi.org/10.1074/jbc.m606564200.

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Granzyme B is an important mediator of cytotoxic lymphocyte granule-induced death of target cells, accomplishing this through cleavage of Bid and cleavage and activation of caspases as well as direct cleavage of downstream substrates. Significant controversy exists regarding the primary pathways used by granzyme B to induce cell death, perhaps arising from the use of different protease/substrate combinations in different studies. The primary sequence of human, rat, and mouse granzymes B is well conserved, and the substrate specificity and crystal structure of the human and rat proteases are extremely similar. Although little is known about the substrate specificity of mouse granzyme B, recent studies suggest that it may differ significantly from the human protease. In these studies we show that the specificities of human and mouse granzymes B differ significantly. Human and mouse granzyme B cleave species-specific procaspase-3 more efficiently than the unmatched substrates. The distinct specificities of human and mouse granzyme B highlight a previously unappreciated requirement for Asp192 in the acquisition of catalytic activity upon cleavage of procaspase-3 at Asp175. Although human granzyme B efficiently cleaves human or mouse Bid, these substrates are highly resistant to cleavage by the mouse protease, strongly indicating that the Bid pathway is not a major primary mediator of the effects of mouse granzyme B. These studies provide important insights into the substrate specificity and function of the granzyme B pathway in different species and highlight that caution is essential when designing and interpreting experiments with different forms of granzyme B.

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Zhou, Zhiwei, Huabin He, Kun Wang, Xuyan Shi, Yupeng Wang, Ya Su, Yao Wang, et al. "Granzyme A from cytotoxic lymphocytes cleaves GSDMB to trigger pyroptosis in target cells." Science 368, no. 6494 (April 16, 2020): eaaz7548. http://dx.doi.org/10.1126/science.aaz7548.

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Cytotoxic lymphocyte–mediated immunity relies on granzymes. Granzymes are thought to kill target cells by inducing apoptosis, although the underlying mechanisms are not fully understood. Here, we report that natural killer cells and cytotoxic T lymphocytes kill gasdermin B (GSDMB)–positive cells through pyroptosis, a form of proinflammatory cell death executed by the gasdermin family of pore-forming proteins. Killing results from the cleavage of GSDMB by lymphocyte-derived granzyme A (GZMA), which unleashes its pore-forming activity. Interferon-γ (IFN-γ) up-regulates GSDMB expression and promotes pyroptosis. GSDMB is highly expressed in certain tissues, particularly digestive tract epithelia, including derived tumors. Introducing GZMA-cleavable GSDMB into mouse cancer cells promotes tumor clearance in mice. This study establishes gasdermin-mediated pyroptosis as a cytotoxic lymphocyte–killing mechanism, which may enhance antitumor immunity.

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Kiselevsky, D. B. "Granzymes and Mitochondria." Biochemistry (Moscow) 85, no. 2 (February 2020): 131–39. http://dx.doi.org/10.1134/s0006297920020017.

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Dressel, Ralf, Srikumar M. Raja, Stefan Höning, Tim Seidler, Christopher J. Froelich, Kurt von Figura, and Eberhard Günther. "Granzyme-mediated Cytotoxicity Does Not Involve the Mannose 6-Phosphate Receptors on Target Cells." Journal of Biological Chemistry 279, no. 19 (February 25, 2004): 20200–20210. http://dx.doi.org/10.1074/jbc.m313108200.

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Cytotoxic T lymphocytes (CTL) and natural killer cells secrete granzymes to kill infected or transformed cells. The mannose 6-phosphate receptor (Mpr) 300 on target cells has been reported to function as receptor for secreted granzyme B. Using lymphoblasts and mouse embryonal fibroblast lines from Mpr300 and Mpr46 knockout mice, we show here that both receptors are not essential for CTL-induced apoptosis. Similarly, cells exposed to either monomeric granzyme B or granzyme B-serglycin complexes readily internalize the granzyme and undergo apoptosis in the absence of Mpr300 and Mpr46. Further, no colocalization of granzyme B and Mpr300 could be observed in target cells after internalization. In conclusion, these results strongly argue against an Mpr300- or Mpr46-dependent pathway of granzyme-mediated killing and provide new insight in the internalization of monomeric and complexed granzyme B.

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Balkow, Sandra, Astrid Kersten, Thi Thanh Thao Tran, Thomas Stehle, Philipp Grosse, Crisan Museteanu, Olaf Utermöhlen, et al. "Concerted Action of the FasL/Fas and Perforin/Granzyme A and B Pathways Is Mandatory for the Development of Early Viral Hepatitis but Not for Recovery from Viral Infection." Journal of Virology 75, no. 18 (September 15, 2001): 8781–91. http://dx.doi.org/10.1128/jvi.75.18.8781-8791.2001.

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ABSTRACT Cytotoxic T lymphocytes (CTL) play a major role in the recovery from primary viral infections and the accompanying tissue injuries. However, it is unclear to what extent the two main cytolytic pathways, perforin-granzyme A and B exocytosis and Fas ligand (FasL)-Fas interaction, contribute to these processes. Here we have employed mouse strains with either spontaneous mutations or targeted gene defects in one or more components of either of the two cytolytic pathways to analyze the molecular basis of viral clearance and induction of hepatitis during lymphocytic choriomeningitis virus infection. Our results reveal that viral clearance is solely dependent on perforin but that virus-induced liver damage only occurs when both the FasL/Fas and the perforin pathways, including granzymes A and B, are simultaneously activated. The finding that development of hepatitis but not viral clearance is dependent on the concomitant activation of FasL-Fas and perforin-granzymes may be helpful in designing novel strategies to prevent hepatic failures during viral infections.

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Browne, Kylie A., Elizabeth Blink, Vivien R. Sutton, Christopher J. Froelich, David A. Jans, and Joseph A. Trapani. "Cytosolic Delivery of Granzyme B by Bacterial Toxins: Evidence that Endosomal Disruption, in Addition to Transmembrane Pore Formation, Is an Important Function of Perforin." Molecular and Cellular Biology 19, no. 12 (December 1, 1999): 8604–15. http://dx.doi.org/10.1128/mcb.19.12.8604.

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ABSTRACT Granule-mediated cell killing by cytotoxic lymphocytes requires the combined actions of a membranolytic protein, perforin, and granule-associated granzymes, but the mechanism by which they jointly kill cells is poorly understood. We have tested a series of membrane-disruptive agents including bacterial pore-forming toxins and hemolytic complement for their ability to replace perforin in facilitating granzyme B-mediated cell death. As with perforin, low concentrations of streptolysin O and pneumolysin (causing <10%51Cr release) permitted granzyme B-dependent apoptosis of Jurkat and Yac-1 cells, but staphylococcal alpha-toxin and complement were ineffective, regardless of concentration. The ensuing nuclear apoptotic damage was caspase dependent and included cleavage of poly(ADP-ribose) polymerase, suggesting a mode of action similar to that of perforin. The plasma membrane lesions formed at low dose by perforin, pneumolysin, and streptolysin did not permit diffusion of fluorescein-labeled proteins as small as 8 kDa into the cell, indicating that large membrane defects are not necessary for granzymes (32 to 65 kDa) to enter the cytosol and induce apoptosis. The endosomolytic toxin, listeriolysin O, also effected granzyme B-mediated cell death at concentrations which produced no appreciable cell membrane damage. Cells pretreated with inhibitors of endosomal trafficking such as brefeldin A took up granzyme B normally but demonstrated seriously impaired nuclear targeting of granzyme B when perforin was also added, indicating that an important role of perforin is to disrupt vesicular protein trafficking. Surprisingly, cells exposed to granzyme B with perforin concentrations that produced nearly maximal 51Cr release (1,600 U/ml) also underwent apoptosis despite excluding a 8-kDa fluorescein-labeled protein marker. Only at concentrations of >4,000 U/ml were perforin pores demonstrably large enough to account for transmembrane diffusion of granzyme B. We conclude that pore formation may allow granzyme B direct cytosolic access only when perforin is delivered at very high concentrations, while perforin’s ability to disrupt endosomal trafficking may be crucial when it is present at lower concentrations or in killing cells that efficiently repair perforin pores.

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Bots, M., and J. P. Medema. "Granzymes at a glance." Journal of Cell Science 119, no. 24 (December 15, 2006): 5011–14. http://dx.doi.org/10.1242/jcs.03239.

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Hwang, Joo-Hee, Jeong-Hwan Hwang, and Chang-Seop Lee. "Elevated Extracellular Levels of Granzymes in Patients with Scrub Typhus." American Journal of Tropical Medicine and Hygiene 105, no. 6 (December 1, 2021): 1680–83. http://dx.doi.org/10.4269/ajtmh.20-1369.

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ABSTRACT. Scrub typhus is an acute febrile disease caused by Orientia tsutsugamushi, which is transmitted through chigger mites. Delayed treatment results in various complications and, in severe cases, death. Granzymes are secreted by cytotoxic T lymphocytes or natural killer cells and are known to play an important role in controlling intracellular pathogens. To date, few studies have been done on granzymes in patients with scrub typhus. In this study, granzymes A and B showed a significant increase during the acute stage of scrub typhus compared with healthy control subjects, and decreased sharply after treatment. In addition, granzymes A and B were significantly high in the moderately elevated liver enzyme group. In conclusion, it appears that the host during the acute phase of scrub typhus increases cytotoxic T-cell activity to control infection.

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Wensink, Annette C., C. Erik Hack, and Niels Bovenschen. "Granzymes Regulate Proinflammatory Cytokine Responses." Journal of Immunology 194, no. 2 (January 2, 2015): 491–97. http://dx.doi.org/10.4049/jimmunol.1401214.

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Bovenschen, Niels, and J. Alain Kummer. "Orphan granzymes find a home." Immunological Reviews 235, no. 1 (April 28, 2010): 117–27. http://dx.doi.org/10.1111/j.0105-2896.2010.00889.x.

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Romero, V., and F. Andrade. "Non-apoptotic functions of granzymes." Tissue Antigens 71, no. 5 (May 2008): 409–16. http://dx.doi.org/10.1111/j.1399-0039.2008.01013.x.

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Sower, Laurie E., Gary R. Klimpel, William Hanna, and Christopher J. Froelich. "Extracellular Activities of Human Granzymes." Cellular Immunology 171, no. 1 (July 1996): 159–63. http://dx.doi.org/10.1006/cimm.1996.0187.

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Ewen, C. L., K. P. Kane, and R. C. Bleackley. "A quarter century of granzymes." Cell Death & Differentiation 19, no. 1 (November 4, 2011): 28–35. http://dx.doi.org/10.1038/cdd.2011.153.

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Kaiserman, D., and P. I. Bird. "Control of granzymes by serpins." Cell Death & Differentiation 17, no. 4 (November 6, 2009): 586–95. http://dx.doi.org/10.1038/cdd.2009.169.

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Cullen, S. P., M. Brunet, and S. J. Martin. "Granzymes in cancer and immunity." Cell Death & Differentiation 17, no. 4 (January 15, 2010): 616–23. http://dx.doi.org/10.1038/cdd.2009.206.

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Chen, G., L. Shi, D. W. Litchfield, and A. H. Greenberg. "Rescue from granzyme B-induced apoptosis by Wee1 kinase." Journal of Experimental Medicine 181, no. 6 (June 1, 1995): 2295–300. http://dx.doi.org/10.1084/jem.181.6.2295.

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Granzymes are a family of granule-associated serine esterases that mediate apoptosis by cytotoxic T lymphocytes and natural killer cells. We have previously shown that cdc2, the mitosis-regulating cyclin-dependent kinase, is required for granzyme B-induced apoptosis in target cells. In addition, granzyme B induces premature activation and tyrosine dephosphorylation of cdc2 during apoptosis. Throughout most of the cell cycle and until the cell is prepared to enter mitosis, cdc2 kinase activity is negatively regulated by phosphorylation of a residue within its adenosine triphosphate-binding domain by Wee1, a nuclear kinase that maintains mitotic timing in eukaryotic cells. We have transiently expressed c-myc epitope-tagged Wee1 cDNA in BHK cells. Cells that expressed Wee1 in the nucleus became resistant to apoptosis induced by granzyme B and perforin. Wee1-transfected cells also exhibited markedly increased cdc2 tyrosine phosphorylation. Thus, Wee1 can rescue cells from granzyme-induced apoptosis by preventing cdc2 dephosphorylation.

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Feehan, David D., Khusraw Jamil, Maria J. Polyak, Henry Ogbomo, Mark Hasell, Shu Shun LI, Richard F. Xiang, et al. "Natural killer cells kill extracellular Pseudomonas aeruginosa using contact-dependent release of granzymes B and H." PLOS Pathogens 18, no. 2 (February 24, 2022): e1010325. http://dx.doi.org/10.1371/journal.ppat.1010325.

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Pseudomonas aeruginosa is an opportunistic pathogen that often infects individuals with the genetic disease cystic fibrosis, and contributes to airway blockage and loss of lung function. Natural killer (NK) cells are cytotoxic, granular lymphocytes that are part of the innate immune system. NK cell secretory granules contain the cytolytic proteins granulysin, perforin and granzymes. In addition to their cytotoxic effects on cancer and virally infected cells, NK cells have been shown to play a role in an innate defense against microbes, including bacteria. However, it is not known if NK cells kill extracellular P. aeruginosa or how bacterial killing might occur at the molecular level. Here we show that NK cells directly kill extracellular P. aeruginosa using NK effector molecules. Live cell imaging of a co-culture of YT cells, a human NK cell line, and GFP-expressing P. aeruginosa in the presence of the viability dye propidium iodide demonstrated that YT cell killing of P. aeruginosa is contact-dependent. CRISPR knockout of granulysin or perforin in YT cells had no significant effect on YT cell killing of P. aeruginosa. Pre-treatment of YT and NK cells with the serine protease inhibitor 3,4-dichloroisocoumarin (DCI) to inhibit all granzymes, resulted in an inhibition of killing. Although singular CRISPR knockout of granzyme B or H had no effect, knockout of both in YT cells completely abrogated killing of P. aeruginosa in comparison to wild type YT cell controls. Nitrocefin assays suggest that the bacterial membrane is damaged. Inhibition of killing by antioxidants suggest that ROS are required for the bactericidal mode-of-action. Taken together, these results identify that NK cells kill P. aeruginosa through a membrane damaging, contact-dependent process that requires granzyme induced ROS production, and moreover, that granzyme B and H are redundant in this killing process.

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Sattar, Rabia, S. Abid Ali, and Atiya Abbasi. "Bioinformatics of granzymes: sequence comparison and structural studies on granzyme family by homology modeling." Biochemical and Biophysical Research Communications 308, no. 4 (September 2003): 726–35. http://dx.doi.org/10.1016/s0006-291x(03)01458-x.

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Dutta, Dibyendu, In Park, Hiwot Guililat, Samuel Sang, Arpita Talapatra, Barkha Singhal, and Nathaniel C. Mills. "Testosterone regulates granzyme K expression in rat testes." Endocrine Regulations 51, no. 4 (October 26, 2017): 193–204. http://dx.doi.org/10.1515/enr-2017-0020.

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Abstract Objective. Testosterone depletion induces increased germ cell apoptosis in testes. However, limited studies exist on genes that regulate the germ cell apoptosis. Granzymes (GZM) are serine proteases that induce apoptosis in various tissues. Multiple granzymes, including GZMA, GZMB and GZMN, are present in testes. Th us, we investigated which granzyme may be testosterone responsive and possibly may have a role in germ cell apoptosis aft er testosterone depletion. Methods. Ethylene dimethane sulfonate (EDS), a toxicant that selectively ablates the Leydig cells, was injected into rats to withdraw the testosterone. The testosterone depletion effects after 7 days post-EDS were verified by replacing the testosterone exogenously into EDS-treated rats. Serum or testicular testosterone was measured by radioimmunoassay. Using qPCR, mRNAs of granzyme variants in testes were quantified. The germ cell apoptosis was identified by TUNEL assay and the localization of GZMK was by immunohistochemistry. Results. EDS treatment eliminated the Leydig cells and depleted serum and testicular testosterone. At 7 days post-EDS, testis weights were reduced 18% with increased germ cell apoptosis plus elevation GZMK expression. GZMK was not associated with TUNEL-positive cells, but was localized to stripped cytoplasm of spermatids. In addition, apoptotic round spermatids were observed in the caput epididymis. Conclusions. GZMK expression in testes is testosterone dependent. GZMK is located adjacent to germ cells in seminiferous tubules and the presence of apoptotic round spermatids in the epididymis suggest its role in the degradation of microtubules in ectoplasmic specializations. Thus, overexpression of GZMK may indirectly regulate germ cell apoptosis by premature release of round spermatids from seminiferous tubule lumen.

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Pączek, Sara, Marta Łukaszewicz-Zając, and Barbara Mroczko. "Granzymes—Their Role in Colorectal Cancer." International Journal of Molecular Sciences 23, no. 9 (May 9, 2022): 5277. http://dx.doi.org/10.3390/ijms23095277.

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Colorectal cancer (CRC) is among the most common malignancies worldwide. CRC is considered a heterogeneous disease due to various clinical symptoms, biological behaviours, and a variety of mutations. A number of studies demonstrate that as many as 50% of CRC patients have distant metastases at the time of diagnosis. However, despite the fact that social and medical awareness of CRC has increased in recent years and screening programmes have expanded, there is still an urgent need to find new diagnostic tools for early detection of CRC. The effectiveness of the currently used classical tumour markers in CRC diagnostics is very limited. Therefore, new proteins that play an important role in the formation and progression of CRC are being sought. A number of recent studies show the potential significance of granzymes (GZMs) in carcinogenesis. These proteins are released by cytotoxic lymphocytes, which protect the body against viral infection as well specific signalling pathways that ultimately lead to cell death. Some studies suggest a link between GZMs, particularly the expression of Granzyme A, and inflammation. This paper summarises the role of GZMs in CRC pathogenesis through their involvement in the inflammatory process. Therefore, it seems that GZMs could become the focus of research into new CRC biomarkers.

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Andzelm, Milena M., Xi Chen, Konrad Krzewski, Jordan S. Orange, and Jack L. Strominger. "Myosin IIA is required for cytolytic granule exocytosis in human NK cells." Journal of Experimental Medicine 204, no. 10 (September 17, 2007): 2285–91. http://dx.doi.org/10.1084/jem.20071143.

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Natural killer (NK) cell cytotoxicity involves the formation of an activating immunological synapse (IS) between the effector and target cell through which granzymes and perforin contained in lytic granules are delivered to the target cell via exocytosis. Inhibition of nonmuscle myosin II in human NK cells with blebbistatin or ML-9 impaired neither effector–target cell conjugation nor formation of a mature activating NK cell IS (NKIS; formation of an actin ring and polarization of the microtubule-organizing center and cytolytic granules to the center of the ring). However, membrane fusion of lytic granules, granzyme secretion, and NK cell cytotoxicity were all effectively blocked. Specific knockdown of the myosin IIA heavy chain by RNA interference impaired cytotoxicity, membrane fusion of lytic granules, and granzyme secretion. Thus, myosin IIA is required for a critical step between NKIS formation and granule exocytosis.

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Futas, Ján, Jan Oppelt, Pamela Anna Burger, and Petr Horin. "A Deadly Cargo: Gene Repertoire of Cytotoxic Effector Proteins in the Camelidae." Genes 12, no. 2 (February 21, 2021): 304. http://dx.doi.org/10.3390/genes12020304.

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Cytotoxic T cells and natural killer cells can kill target cells based on their expression and release of perforin, granulysin, and granzymes. Genes encoding these molecules have been only poorly annotated in camelids. Based on bioinformatic analyses of genomic resources, sequences corresponding to perforin, granulysin, and granzymes were identified in genomes of camelids and related ungulate species, and annotation of the corresponding genes was performed. A phylogenetic tree was constructed to study evolutionary relationships between the species analyzed. Re-sequencing of all genes in a panel of 10 dromedaries and 10 domestic Bactrian camels allowed analyzing their individual genetic polymorphisms. The data showed that all extant Old World camelids possess functional genes for two pore-forming proteins (PRF1, GNLY) and six granzymes (GZMA, GZMB, GZMH, GZMK, GZMM, and GZMO). All these genes were represented as single copies in the genome except the GZMH gene exhibiting interspecific differences in the number of loci. High protein sequence similarities with other camelid and ungulate species were observed for GZMK and GZMM. The protein variability in dromedaries and Bactrian camels was rather low, except for GNLY and chymotrypsin-like granzymes (GZMB, GZMH).

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Fehniger, Todd A., Sheng F. Cai, Xuefang Cao, Andrew J. Bredemeyer, Rachel M. Presti, Anthony R. French, and Timothy James Ley. "Murine NK Cells Require Activation-Dependent Expression of Granzyme B and Perforin To Become Potent Cytotoxic Effectors." Blood 108, no. 11 (November 16, 2006): 920. http://dx.doi.org/10.1182/blood.v108.11.920.920.

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Abstract NK cells predominantly utilize the granule exocytosis pathway to kill virus-infected and malignant target cells. Current paradigms suggest that resting NK cells have pre-formed granules containing granzymes A, B, and perforin and are ready to kill targets immediately upon proper recognition by NK receptors. Here, we report that resting murine NK cells in the spleen exhibit poor cytotoxicity (5.4±1.6% target cell death, 20:1 E:T ratio and 4 hour incubation), compared with cytokine-activated (IL-15, 48 hours) splenic NK cells (59.7±10.6% target cell death), against the RMAS tumor cell line in vitro as measured by a flow-based killing assay. In addition, using intracellular flow cytometric analysis with monoclonal antibodies specific for granzymes A, B, and perforin, we find that resting murine NK cells express abundant granzyme A (86.2±1.9% positive), but little or no granzyme B (4.4±5.4% positive) or perforin (2.6±1.8% positive). Activation of murine NK cells with IL-15 induces robust expression of both perforin (59.1±2.0% positive) and granzyme B (91.5±7.9% positive), which correlates with increased cytotoxicity. Further, granzyme B cluster −/− (26±6.7% target cell death) and perforin −/− (5.7±1.3% target cell death) NK cells have poor cytotoxicity in vitro despite IL-15 activation. Poly I:C simulates RNA virus infection and activates NK cell cytotoxicity in vivo through TLR3 and cytokine cascades. NK cell granzyme B and perforin expression is induced in vivo 24 hours after poly I:C injection, correlating with increased in vitro NK killing of tumor targets. In wild type mice infected with murine cytomegalovirus (MCMV), NK cell expression of both perforin (83.5±4.9% positive) and granzyme B (89.3±2.1% positive) is upregulated in the spleen, peaking 2–4 days post-infection and returning to baseline by 8 days post-infection. In addition, MCMV titers are significantly elevated at day 3 post-infection in both granzyme B cluster −/− (P<0.01) and perforin −/− (P<0.01) mice, compared to wild type mice. Moreover, survival following MCMV infection was significantly lower in granzyme B cluster −/− and perforin −/− mice, compared with wild type mice (P<0.001, see survival curve). Thus, our findings show that murine NK cells require the activation of granzyme B and perforin to become potent cytotoxic effectors. We also demonstrate for the first time that granzyme B is critical for early host defense against MCMV. These findings explain the long-standing observation that murine NK cells require prior activation for potent natural killing of tumor targets in vitro. Further, this requirement for activation-dependent granzyme B and perforin expression in NK cells may influence outcomes in murine models of innate immune anti-tumor and anti-viral responses. Figure Figure

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Vahedi, Fatemeh, Nya Fraleigh, Caitlyn Vlasschaert, Janet McElhaney, and Pejman Hanifi-Moghaddam. "Human granzymes: Related but far apart." Medical Hypotheses 83, no. 6 (December 2014): 688–93. http://dx.doi.org/10.1016/j.mehy.2014.09.019.

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47

Salvesen, G. "Cytosolic protease cascades: activation by granzymes." Biochemistry and Cell Biology 75, no. 4 (August 1, 1997): 475. http://dx.doi.org/10.1139/abstract26.

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Martinvalet, Denis, and Jérôme Thiery. "Comment les granzymes tuent leurs cibles." médecine/sciences 24, no. 11 (November 2008): 901–3. http://dx.doi.org/10.1051/medsci/20082411901.

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Granville, D. J. "Granzymes in disease: bench to bedside." Cell Death & Differentiation 17, no. 4 (March 15, 2010): 565–66. http://dx.doi.org/10.1038/cdd.2009.218.

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Zeglinski, Matthew R., and David J. Granville. "Granzymes in cardiovascular injury and disease." Cellular Signalling 76 (December 2020): 109804. http://dx.doi.org/10.1016/j.cellsig.2020.109804.

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Journal articles on the topic 'Granzymes'

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