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Joseph A Trapani - One of the best experts on this subject based on the ideXlab platform.
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Lipid specificity of the immune effector Perforin
2020Co-Authors: Adrian Hodel, Ilia Voskoboinik, Joseph A Trapani, Jesse A. Rudd-schmidt, Bart W. HoogenboomAbstract:Perforin is a pore forming protein used by cytotoxic T lymphocytes to remove cancerous or virus-infected cells during immune response. During the response, the lymphocyte membrane becomes refractory to Perforin function by accumulating densely ordered lipid rafts and externalizing negatively charged lipid species. The dense membrane packing lowers the capacity of Perforin to bind, and negatively charged lipids scavenge any residual protein before pore formation. Using atomic force microscopy on model membrane systems, we here provide insight into the molecular basis of Perforin lipid specificity.
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Substituted arylsulphonamides as inhibitors of Perforin-mediated lysis.
European journal of medicinal chemistry, 2017Co-Authors: Julie Ann Spicer, Kylie A Browne, Christian K. Miller, Patrick D. O'connor, Jiney Jose, Kristiina M. Huttunen, Jagdish K. Jaiswal, William A. Denny, Hedieh Akhlaghi, Joseph A TrapaniAbstract:The structure-activity relationships for a series of arylsulphonamide-based inhibitors of the pore-forming protein Perforin have been explored. Perforin is a key component of the human immune response, however inappropriate activity has also been implicated in certain auto-immune and therapy-induced conditions such as allograft rejection and graft versus host disease. Since Perforin is expressed exclusively by cells of the immune system, inhibition of this protein would be a highly selective strategy for the immunosuppressive treatment of these disorders. Compounds from this series were demonstrated to be potent inhibitors of the lytic action of both isolated recombinant Perforin and Perforin secreted by natural killer cells in vitro. Several potent and soluble examples were assessed for in vivo pharmacokinetic properties and found to be suitable for progression to an in vivo model of transplant rejection.
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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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The Perforin pore facilitates the delivery of cationic cargos.
The Journal of biological chemistry, 2014Co-Authors: Sarah E. Stewart, James C. Whisstock, Michelle A Dunstone, Michael E. D'angelo, Joseph A Trapani, 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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protecting a serial killer pathways for Perforin trafficking and self defence ensure sequential target cell death
Trends in Immunology, 2012Co-Authors: Amelia J. Brennan, James C. Whisstock, Ilia Voskoboinik, Jamie A Lopez, Joseph A TrapaniAbstract:Considerable progress has been made in understanding how cytotoxic lymphocytes use the highly toxic pore-forming protein Perforin to eliminate dangerous cells, while remaining refractory to lysis. At least two mechanisms jointly preserve the killer cell: the C-terminal residues of Perforin dictate its rapid export from the endoplasmic reticulum (ER), whose milieu otherwise favours pore formation; Perforin is then stored in secretory granules whose acidity prevent its oligomerisation. Following exocytosis, Perforin delivers the proapoptotic protease, granzyme B, into the target cell by disrupting its plasma membrane. Although the precise mechanism of Perforin/granzyme synergy remains controversial, the recently defined crystal structure of the Perforin monomer and cryo-electron microscopy (EM) of the entire pore suggest that passive transmembrane granzyme diffusion is the dominant proapoptotic mechanism.
Ilia Voskoboinik - One of the best experts on this subject based on the ideXlab platform.
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Lipid specificity of the immune effector Perforin
2020Co-Authors: Adrian Hodel, Ilia Voskoboinik, Joseph A Trapani, Jesse A. Rudd-schmidt, Bart W. HoogenboomAbstract:Perforin is a pore forming protein used by cytotoxic T lymphocytes to remove cancerous or virus-infected cells during immune response. During the response, the lymphocyte membrane becomes refractory to Perforin function by accumulating densely ordered lipid rafts and externalizing negatively charged lipid species. The dense membrane packing lowers the capacity of Perforin to bind, and negatively charged lipids scavenge any residual protein before pore formation. Using atomic force microscopy on model membrane systems, we here provide insight into the molecular basis of Perforin lipid specificity.
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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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protecting a serial killer pathways for Perforin trafficking and self defence ensure sequential target cell death
Trends in Immunology, 2012Co-Authors: Amelia J. Brennan, James C. Whisstock, Ilia Voskoboinik, Jamie A Lopez, Joseph A TrapaniAbstract:Considerable progress has been made in understanding how cytotoxic lymphocytes use the highly toxic pore-forming protein Perforin to eliminate dangerous cells, while remaining refractory to lysis. At least two mechanisms jointly preserve the killer cell: the C-terminal residues of Perforin dictate its rapid export from the endoplasmic reticulum (ER), whose milieu otherwise favours pore formation; Perforin is then stored in secretory granules whose acidity prevent its oligomerisation. Following exocytosis, Perforin delivers the proapoptotic protease, granzyme B, into the target cell by disrupting its plasma membrane. Although the precise mechanism of Perforin/granzyme synergy remains controversial, the recently defined crystal structure of the Perforin monomer and cryo-electron microscopy (EM) of the entire pore suggest that passive transmembrane granzyme diffusion is the dominant proapoptotic mechanism.
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The structural basis for membrane binding and pore formation by lymphocyte Perforin
Nature, 2010Co-Authors: Ruby H. P. Law, Natalya Lukoyanova, Ilia Voskoboinik, Tom T. Caradoc-davies, Katherine Baran, Michelle A Dunstone, Michael E. D'angelo, Elena V. Orlova, Fasseli Joseph Coulibaly, Sandra VerschoorAbstract:Natural killer cells and cytotoxic T lymphocytes accomplish the critically important function of killing virus-infected and neoplastic cells. They do this by releasing the pore-forming protein Perforin and granzyme proteases from cytoplasmic granules into the cleft formed between the abutting killer and target cell membranes. Perforin, a 67-kilodalton multidomain protein, oligomerizes to form pores that deliver the pro-apoptopic granzymes into the cytosol of the target cell. The importance of Perforin is highlighted by the fatal consequences of congenital Perforin deficiency, with more than 50 different Perforin mutations linked to familial haemophagocytic lymphohistiocytosis (type 2 FHL). Here we elucidate the mechanism of Perforin pore formation by determining the X-ray crystal structure of monomeric murine Perforin, together with a cryo-electron microscopy reconstruction of the entire Perforin pore. Perforin is a thin 'key-shaped' molecule, comprising an amino-terminal membrane attack complex Perforin-like (MACPF)/cholesterol dependent cytolysin (CDC) domain followed by an epidermal growth factor (EGF) domain that, together with the extreme carboxy-terminal sequence, forms a central shelf-like structure. A C-terminal C2 domain mediates initial, Ca(2+)-dependent membrane binding. Most unexpectedly, however, electron microscopy reveals that the orientation of the Perforin MACPF domain in the pore is inside-out relative to the subunit arrangement in CDCs. These data reveal remarkable flexibility in the mechanism of action of the conserved MACPF/CDC fold and provide new insights into how related immune defence molecules such as complement proteins assemble into pores.
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The Molecular Basis for Perforin Oligomerization and Transmembrane Pore Assembly
Immunity, 2009Co-Authors: Katherine Baran, Natalya Lukoyanova, Helen R. Saibil, Annette Ciccone, James C. Whisstock, Michelle A Dunstone, Kylie A Browne, Jenny Chia, Christopher J.p. Clarke, Ilia VoskoboinikAbstract:Perforin, a pore-forming protein secreted by cytotoxic lymphocytes, is indispensable for destroying virus-infected cells and for maintaining immune homeostasis. Perforin polymerizes into transmembrane channels that inflict osmotic stress and facilitate target cell uptake of proapoptotic granzymes. Despite this, the mechanism through which Perforin monomers self-associate remains unknown. Our current study establishes the molecular basis for Perforin oligomerization and pore assembly. We show that after calcium-dependent membrane binding, direct ionic attraction between the opposite faces of adjacent Perforin monomers was necessary for pore formation. By using mutagenesis, we identified the opposing charges on residues Arg213 (positive) and Glu343 (negative) to be critical for intermolecular interaction. Specifically, disrupting this interaction had no effect on Perforin synthesis, folding, or trafficking in the killer cell, but caused a marked kinetic defect of oligomerization at the target cell membrane, severely disrupting lysis and granzyme B-induced apoptosis. Our study provides important insights into Perforin's mechanism of action.
Mark J Smyth - One of the best experts on this subject based on the ideXlab platform.
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Perforin-mediated target-cell death and immune homeostasis
Nature reviews. Immunology, 2006Co-Authors: Ilia Voskoboinik, Mark J Smyth, Joseph A TrapaniAbstract:The granule exocytosis pathway of cytotoxic lymphocytes is crucial for immune surveillance and homeostasis. The trafficking of granule components, including the membrane-disruptive protein Perforin, to the immunological synapse leads to the delivery of granule proteases (granzymes) into the target cell and its destruction through apoptosis. Several independent molecular abnormalities associated with defects of either granule trafficking or Perforin function can cause cytotoxic lymphocyte dysfunction. In humans, inherited Perforin mutations result in severe immune dysregulation that manifests as familial haemophagocytic lymphohistiocytosis. This Review describes recent progress in defining the structure, function, biochemistry and cell biology of Perforin.
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calcium dependent plasma membrane binding and cell lysis by Perforin are mediated through its c2 domain a critical role for aspartate residues 429 435 483 and 485 but not 491
Journal of Biological Chemistry, 2005Co-Authors: Ilia Voskoboinik, Annette Ciccone, Marieclaude Thia, Kylie A Browne, Mark J Smyth, Jamie I Fletcher, Joseph A TrapaniAbstract:The lymphocyte pore-forming protein Perforin is essential for maintaining immune homeostasis and for effective defense against intracellular pathogens. To date, there have been no reported structure-function studies to substantiate the function of any putative domains of Perforin, which have been postulated totally on primary sequence similarities with domains in other proteins. In this report, we have used recently developed modalities for expressing full-length Perforin and robust functional assays to investigate one of the hallmarks of Perforin function: its absolute dependence on calcium for lipid binding and cell lysis. We provide, for the first time, experimental evidence that the predicted C-terminal C2 motif constitutes a functional domain that is responsible for membrane binding of Perforin. Whereas conserved aspartate residues at positions 429, 435, 483, and 485 were essential for calcium-dependent plasma membrane binding and cell lysis, the contribution of Asp-491 was limited. Finally, after experimentally verifying an optimized three-dimensional model, we have made predictions on the impact of two inherited Perforin mutations of the C2 domain on calcium-dependent lipid binding and cell lysis.
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functional significance of the Perforin granzyme cell death pathway
Nature Reviews Immunology, 2002Co-Authors: Joseph A Trapani, Mark J SmythAbstract:Perforin/granzyme-induced apoptosis is the main pathway used by cytotoxic lymphocytes to eliminate virus-infected or transformed cells. Studies in gene-disrupted mice indicate that Perforin is vital for cytotoxic effector function; it has an indispensable, but undefined, role in granzyme-mediated apoptosis. Despite its vital importance, the molecular and cellular functions of Perforin and the basis of Perforin and granzyme synergy remain poorly understood. The purpose of this review is to evaluate critically recent findings on cytotoxic granule-mediated cell death and to assess the functional significance of postulated cell-death pathways in appropriate pathophysiological contexts, including virus infection and susceptibility to experimental or spontaneous tumorigenesis.
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Functional significance of the Perforin/granzyme cell death pathway
Nature reviews. Immunology, 2002Co-Authors: Joseph A Trapani, Mark J SmythAbstract:Perforin/granzyme-induced apoptosis is the main pathway used by cytotoxic lymphocytes to eliminate virus-infected or transformed cells. Studies in gene-disrupted mice indicate that Perforin is vital for cytotoxic effector function; it has an indispensable, but undefined, role in granzyme-mediated apoptosis. Despite its vital importance, the molecular and cellular functions of Perforin and the basis of Perforin and granzyme synergy remain poorly understood. The purpose of this review is to evaluate critically recent findings on cytotoxic granule-mediated cell death and to assess the functional significance of postulated cell-death pathways in appropriate pathophysiological contexts, including virus infection and susceptibility to experimental or spontaneous tumorigenesis.
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Perforin is a major contributor to nk cell control of tumor metastasis
Journal of Immunology, 1999Co-Authors: Mark J Smyth, Kevin Y T Thia, Erika Cretney, Janice M Kelly, Marie B Snook, Catherine A Forbes, Anthony A ScalzoAbstract:We provide the first demonstration, using experimental and spontaneous models of metastasis in C57BL/6 (B6) (RM-1 prostate carcinoma) and BALB/c (DA3 mammary carcinoma) mice, that tumor metastasis is primarily controlled by Perforin-dependent cytotoxicity mediated by NK1.1+ cells. MHC class Ilow RM-1 and DA3 tumor cells were sensitive in vitro to Fas-mediated lysis or spleen NK cells in a Perforin-dependent fashion. Perforin-deficient NK cells did not lyse these tumors, and Perforin-deficient mice were 10-100-fold less proficient than wild-type mice in rejecting the metastasis of tumor cells to the lung. Fas ligand mutant gld mice displayed uncompromised protection against tumor metastasis. Depletion of NK subsets resulted in greater numbers of metastases than observed in Perforin-deficient mice, suggesting that Perforin-independent effector functions of NK cells may also contribute to protection from tumor metastasis.
James C. Whisstock - One of the best experts on this subject based on the ideXlab platform.
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Perforin proteostasis is regulated through its C2 domain: supra-physiological cell death mediated by T431D-Perforin.
Cell death and differentiation, 2018Co-Authors: Amelia J. Brennan, Hideo Yagita, Ruby H. P. Law, Paul J. Conroy, Tahereh Noori, Natalya Lukoyanova, Helen R. Saibil, Annette Ciccone, Sandra Verschoor, James C. WhisstockAbstract:The pore forming, Ca2+-dependent protein, Perforin, is essential for the function of cytotoxic lymphocytes, which are at the frontline of immune defence against pathogens and cancer. Perforin is a glycoprotein stored in the secretory granules prior to release into the immune synapse. Congenital Perforin deficiency causes fatal immune dysregulation, and is associated with various haematological malignancies. At least 50% of pathological missense mutations in Perforin result in protein misfolding and retention in the endoplasmic reticulum. However, the regulation of Perforin proteostasis remains unexplored. Using a variety of biochemical assays that assess protein stability and acquisition of complex glycosylation, we demonstrated that the binding of Ca2+ to the C2 domain stabilises Perforin and regulates its export from the endoplasmic reticulum to the secretory granules. As Perforin is a thermo-labile protein, we hypothesised that by altering its C2 domain it may be possible to improve protein stability. On the basis of the X-ray crystal structure of the Perforin C2 domain, we designed a mutation (T431D) in the Ca2+ binding loop. Mutant Perforin displayed markedly enhanced thermal stability and lytic function, despite its trafficking from the endoplasmic reticulum remaining unchanged. Furthermore, by introducing the T431D mutation into A90V Perforin, a pathogenic mutation, which results in protein misfolding, we corrected the A90V folding defect and completely restored Perforin's cytotoxic function. These results revealed an unexpected role for the Ca2+-dependent C2 domain in maintaining Perforin proteostasis and demonstrated the possibility of designing Perforin with supra-physiological cytotoxic function through stabilisation of the C2 domain.
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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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The Perforin pore facilitates the delivery of cationic cargos.
The Journal of biological chemistry, 2014Co-Authors: Sarah E. Stewart, James C. Whisstock, Michelle A Dunstone, Michael E. D'angelo, Joseph A Trapani, 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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protecting a serial killer pathways for Perforin trafficking and self defence ensure sequential target cell death
Trends in Immunology, 2012Co-Authors: Amelia J. Brennan, James C. Whisstock, Ilia Voskoboinik, Jamie A Lopez, Joseph A TrapaniAbstract:Considerable progress has been made in understanding how cytotoxic lymphocytes use the highly toxic pore-forming protein Perforin to eliminate dangerous cells, while remaining refractory to lysis. At least two mechanisms jointly preserve the killer cell: the C-terminal residues of Perforin dictate its rapid export from the endoplasmic reticulum (ER), whose milieu otherwise favours pore formation; Perforin is then stored in secretory granules whose acidity prevent its oligomerisation. Following exocytosis, Perforin delivers the proapoptotic protease, granzyme B, into the target cell by disrupting its plasma membrane. Although the precise mechanism of Perforin/granzyme synergy remains controversial, the recently defined crystal structure of the Perforin monomer and cryo-electron microscopy (EM) of the entire pore suggest that passive transmembrane granzyme diffusion is the dominant proapoptotic mechanism.
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The Molecular Basis for Perforin Oligomerization and Transmembrane Pore Assembly
Immunity, 2009Co-Authors: Katherine Baran, Natalya Lukoyanova, Helen R. Saibil, Annette Ciccone, James C. Whisstock, Michelle A Dunstone, Kylie A Browne, Jenny Chia, Christopher J.p. Clarke, Ilia VoskoboinikAbstract:Perforin, a pore-forming protein secreted by cytotoxic lymphocytes, is indispensable for destroying virus-infected cells and for maintaining immune homeostasis. Perforin polymerizes into transmembrane channels that inflict osmotic stress and facilitate target cell uptake of proapoptotic granzymes. Despite this, the mechanism through which Perforin monomers self-associate remains unknown. Our current study establishes the molecular basis for Perforin oligomerization and pore assembly. We show that after calcium-dependent membrane binding, direct ionic attraction between the opposite faces of adjacent Perforin monomers was necessary for pore formation. By using mutagenesis, we identified the opposing charges on residues Arg213 (positive) and Glu343 (negative) to be critical for intermolecular interaction. Specifically, disrupting this interaction had no effect on Perforin synthesis, folding, or trafficking in the killer cell, but caused a marked kinetic defect of oligomerization at the target cell membrane, severely disrupting lysis and granzyme B-induced apoptosis. Our study provides important insights into Perforin's mechanism of action.
Eckhard R. Podack - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of intracellular bacterial replication in fibroblasts is dependent on the Perforin-like protein (Perforin-2) encoded by macrophage expressed gene 1
Journal of innate immunity, 2012Co-Authors: Ryan M. Mccormack, Motoaki Shiratsuchi, Lesley R. De Armas, Jahir Ramos, Eckhard R. PodackAbstract:Fibroblasts are known to eliminate intracellular bacteria, but the lethal hit of the bactericidal mechanism has not been defined. We show that primary embryonic and established fibroblasts can be induced by interferons or by intracellular bacterial infection to express a Perforin-like mRNA previously described as macrophage-expressed gene 1 (Mpeg1). The presence and level of the Perforin-like mRNA correlate with the ability of primary mouse embryonic fibroblasts (MEF) to eliminate intracellular bacteria. In addition, siRNA knockdown of the Perforin-like molecule abolishes bactericidal activity and allows intracellular bacterial replication. Complementation of MEF in which the endogenous Perforin-like molecule has been knocked down with a red fluorescent protein-tagged version restores bactericidal activity. The Perforin-like molecule has broad bactericidal specificity for pathogenic and non-pathogenic bacteria, including Gram-positive and -negative, and acid fast bacteria. The Perforin-like molecule renders previously lysozyme-resistant bacteria sensitive to lysis by lysozyme suggesting physical damage of the outer cell wall by the Perforin-like protein. MEF damage cell walls of intracellular bacteria by insertion, polymerization, and pore formation of the Perforin-like protein, analogous to pore formers of complement and Perforin-1 of cytolytic lymphocytes. We propose the name Perforin-2.
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Progesterone directly and indirectly affects Perforin expression in cytolytic cells.
American journal of reproductive immunology (New York N.Y. : 1989), 1999Co-Authors: Gordana Laškarin, Eckhard R. Podack, Natasa Strbo, Zs. Faust, Vlatka Sotošek, Julia Szekeres-bartho, Daniel RukavinaAbstract:Laskarin G, Faust Zs, Strbo N, SotoSek V, Szekeres-Bartho J, Podack ER, Rukavina D. Progesterone directly and indirectly affects Perforin expression in cytolytic cells. AJRI 1999; 42:312–320 © Munksgaard, Copenhagen PROBLEM: Decidual lymphocytes (DL) expressing the cytolytic molecule Perforin represent approximately 55% of DL in the first trimester of human pregnancy. Progesterone dominates this phase of pregnancy and controls the production of uterine cytokines and growth factors. The aim of this study was to investigate the role of progesterone and progesterone-induced blocking factor (PIBF) on Perforin expression in DL and peripheral blood lymphocytes (PBL). METHOD OF STUDY: Perforin expression was analyzed in PBL and DL incubated either in culture medium or with decidual adherent cells (DAC) and peripheral blood adherent cells (PBAC) and their supernatants with or without progesterone or PIBF. Perforin was detected by flow cytometry in PB and in decidual first trimester pregnancy lymphocytes. RESULTS: Progesterone in high concentrations directly affects Perforin expression in DL but not in PBL. Progesterone in a concentration dependent manner indirectly blocks Perforin expression in DL and PBL cultured with adherent cells or their supernatants. PIBF blocked upregulation of Perforin expression of DL cultured with DAC, but none of those cultured with PBAC. Similarly, PIBF was inefficient when PBL or DL were cultured with PBAC. CONCLUSION: Progesterone present in a high concentration locally at the maternal-fetal interface modulates Perforin expression in the first trimester pregnancy DL.
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Transgenic control of Perforin gene expression
Journal of Immunology, 1995Co-Authors: Mathias G. Lichtenheld, Eckhard R. Podack, Robert B. LevyAbstract:Perforin is a pore-forming effector molecule of CTL and NK cells. To characterize Perforin gene expression and its transcriptional control mechanisms in vivo, expression of a cell surface tag, i.e., human CD4, was driven by 5.1 kb of the murin Perforin 5{prime} flanking and promoter region in transgenic mice. Six out of seven transgenic lines expressed the Perforin-tag hybrid gene at low to intermediate levels, depending on the integration site. Transgene expression occurred in all cells that physiologically are able to express Perforin. At the whole organ level, significant amounts of transgenic mRNA and endogenous Perforin mRNA were co-expressed in the lymphoid organs, as well as in the lung, the ileum, the oviduct/uterus, and the bone marrow. At the single cell level, the Perforin tag was present on NK cells and on CD8{sup +}, as well as on CD4{sup +} cells. Also targeted were Thy-1.2{sup +} {gamma}{delta} T cells, but not Thy-1.2{sup -} {gamma}{delta} T cells, B cells, nor monocytes. During thymic T cell development, transgene expression occurred in double negative (CD4{sup -}CD8{sup -}) thymocytes and was detected at all subsequent stages, but exceeded the expression levels of the endogenous gene in the thymus. In conclusion, the analyzed Perforin 5{prime}more » flanking and promoter region contains important cis-acting sequences that restrict Perforin expression to T cells and NK cells, and therefore provides a unique tool for manipulating T cell and/or Nk cell-mediated immune responses in transgenic mice. On the other hand, the normal control of Perforin gene expression involves at least one additional negative control mechanism that was not mediated by the transgenic promoter and upstream region. This control restricts Perforin gene expression in thymically developing T cells and in most resting peripheral T cells, but can be released upon T cell activation. 43 refs., 7 figs., 1 tab.« less
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Perforin expression in endometrium during the menstrual cycle.
International journal of gynecological pathology : official journal of the International Society of Gynecological Pathologists, 1995Co-Authors: Arif Hameed, William M. Fox, Robert J. Kurman, Ralph H. Hruban, Eckhard R. PodackAbstract:Perforin is a marker of functionally active cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. We examined Perforin expression in endometrium throughout the menstrual cycle. In addition, Perforin expression was studied in endometrial leukocytes in women with anovulatory cycles with or without progesterone therapy and in postmenopausal women. In the endometrium, Perforin-positive endometrial lymphocytes increased in numbers in the middle through late secretory phases. In women with anovulatory cycles, the proliferative endometrium, before estrogen withdrawal endometrial breakdown, contained less Perforin-positive lymphocytes compared with the premenstrual endometrium of the normal menstrual cycle. The progestin-induced endometrial decidualization was accompanied by an extensive recruitment of Perforin-positive cells. In contrast, the postmenopausal atrophic endometrium showed complete absence of Perforin-positive cells. The lymphocytes isolated from secretory endometrium were CD3-, CD56+ (approximately 80%) or CD3+, CD8+ T-cell receptor (TCR)-gamma delta- (approximately 20%). Almost 90% +/- 5.5 of the CD3-, CD56+, and up to 25% +/- 7% of CD8+ T lymphocytes contained Perforin. Perforin expression was further confirmed in endometrial lymphocytes by Northern blot analysis. In vitro, isolated endometrial lymphocytes exhibited NK-like cytotoxicity. After cyclic hormone withdrawal during normal menstrual cycle, these Perforin-positive cytotoxic cells may be involved in endometrial stromal breakdown during menstruation.
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Structure and function of the murine Perforin promoter and upstream region. Reciprocal gene activation or silencing in Perforin positive and negative cells.
Journal of immunology (Baltimore Md. : 1950), 1992Co-Authors: Mathias G. Lichtenheld, Eckhard R. PodackAbstract:Gene expression of the cytolytic protein Perforin is restricted to and tightly regulated in cytolytic lymphocytes. To begin to understand the molecular basis of Perforin gene transcription, we cloned and analyzed 5.1 kb of the genuine murine Perforin promoter and upstream region. The murine Perforin promoter is located approximately 2.1 kb upstream of the translation start codon in the genomic DNA due to an intron in the 5' untranslated sequence. Although the sequenced murine promoter and upstream region was found to be quite homologous to that of the human gene, most of the interspecies conserved sequences lacked obvious consensus to known regulatory elements. Functional analysis of this region, however, indicated that it contains regulatory elements that may determine the cell-type-specific expression of this killer protein. After transient transfection into several cell lines, the Perforin promoter and upstream region was used to drive the expression of the chloramphenicol acetyltransferase (CAT) reporter gene. High levels of CAT activities, exceeding 110 times the expression of a promoterless reporter gene construct, were expressed in CTL. In contrast, in Perforin-negative cell types the Perforin promoter and upstream region mediated barely detectable transcription of the CAT gene. Analysis of the immediate proximal Perforin promoter, -120 to +2, revealed that it was ubiquitously active and that it expressed in all cells tested 20- to 50-fold higher CAT activity than the promoterless reporter gene construct. The cell-type restricted transcriptional activity of the Perforin promoter and upstream region, however, was controlled by at least four negative and positive cis-acting upstream regions that spread over the entire 5 kb of the cloned DNA and acted reciprocally in different cells. Thus, in Perforin-negative cells, the transcriptional activity of the immediate proximal Perforin promoter was dominantly suppressed by several upstream negative regulatory elements, whereas in Perforin-positive cells, the promoter activity was enhanced more than fivefold by several upstream regulatory elements.