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1.
Immunol Cell Biol ; 100(7): 529-546, 2022 08.
Article in English | MEDLINE | ID: mdl-35471730

ABSTRACT

To control infections phagocytes can directly kill invading microbes. Macrophage-expressed gene 1 (Mpeg1), a pore-forming protein sometimes known as perforin-2, is reported to be essential for bacterial killing following phagocytosis. Mice homozygous for the mutant allele Mpeg1tm1Pod succumb to bacterial infection and exhibit deficiencies in bacterial killing in vitro. Here we describe a new Mpeg mutant allele Mpeg1tm1.1Pib on the C57BL/6J background. Mice homozygous for the new allele are not abnormally susceptible to bacterial or viral infection, and irrespective of genetic background show no perturbation in bacterial killing in vitro. Potential reasons for these conflicting findings are discussed. In further work, we show that cytokine responses to inflammatory mediators, as well as antibody generation, are also normal in Mpeg1tm1.1Pib/tm1.1Pib mice. We also show that Mpeg1 is localized to a CD68-positive endolysosomal compartment, and that it exists predominantly as a processed, two-chain disulfide-linked molecule. It is abundant in conventional dendritic cells 1, and mice lacking Mpeg1 do not present the model antigen ovalbumin efficiently. We conclude that Mpeg1 is not essential for innate antibacterial protection or antiviral immunity, but may play a focused role early in the adaptive immune response.


Subject(s)
Antigen Presentation , Pore Forming Cytotoxic Proteins , Animals , Bacterial Infections/immunology , Immunity, Innate , Mice , Mice, Inbred C57BL , Pore Forming Cytotoxic Proteins/immunology , Virus Diseases/immunology
2.
Immunol Cell Biol ; 95(10): 884-894, 2017 11.
Article in English | MEDLINE | ID: mdl-28722018

ABSTRACT

Intracellular serpins are proposed to inactivate proteases released from lysosome-related organelles into the host cell interior, preventing cell death. Serpinb9 opposes the immune cytotoxic protease, granzyme B, and in a number of settings protects cells against granzyme B-mediated cell death. Using a knockout mouse line engineered to express green fluorescent protein under the serpbinb9 promoter, we demonstrate that serpinb9 is vital for host survival during Ectromelia virus infection by maintaining both mature natural killer NK) cells, and activated CD8+ T cells. Serpinb9 expression parallels granzyme B expression within both populations during infection. Maturing serpinb9-null NK cells exhibit higher levels of granzyme B-mediated apoptosis during infection; hence there are fewer mature NK cells, and these cells also have lower cytotoxic potential. Thus the serpinb9-granzyme B axis is important for homeostasis of both major cytotoxic effector cell populations.


Subject(s)
Granzymes/antagonists & inhibitors , Killer Cells, Natural/immunology , Membrane Proteins/pharmacology , Poxviridae Infections/immunology , Poxviridae/immunology , Serpins/pharmacology , Animals , Cell Death , Cell Survival , Homeostasis , Humans , Intracellular Space , Mice , Mice, Inbred C57BL , Mice, Knockout
3.
Immunol Cell Biol ; 95(8): 676-683, 2017 09.
Article in English | MEDLINE | ID: mdl-28428612

ABSTRACT

The biological role of granzyme K, a serine protease of cytotoxic T lymphocytes (CTL), is controversial. It has been reported to induce perforin-mediated cell death in vitro, but is also reported to be non-cytotoxic and to operate in inflammatory processes. To elucidate the biological role of this protease, we have deleted the granzyme K gene in mice (mutant allele: Gzmktm1.1Pib; MGI:5636646). Gzmk -/- mice are healthy, anatomically normal, fecund and show normal hematopoietic development. Gzmk -/- mice readily recover from lymphocytic choriomeningitis virus and mouse pox Ectromelia virus infection. Ex vivo, virus-specific granzyme K-deficient CTL are indistinguishable from those of wild-type mice in apoptosis induction of target cells. These data suggest that granzyme K does not play an essential role in viral immunity or cytotoxicity. Our granzyme K knockout line completes the collection of mouse models for the human granzymes, and will further our understanding of their biological roles and relationships.


Subject(s)
Ectromelia virus/immunology , Ectromelia, Infectious/immunology , Granzymes/genetics , Lymphocytic Choriomeningitis/immunology , Lymphocytic choriomeningitis virus/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Antigens, Viral/immunology , Cells, Cultured , Cytotoxicity, Immunologic , Granzymes/metabolism , Hematopoiesis/genetics , Lymphocyte Activation/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout
4.
J Biol Chem ; 291(7): 3626-38, 2016 Feb 12.
Article in English | MEDLINE | ID: mdl-26670609

ABSTRACT

The intracellular protease inhibitor Sb9 (SerpinB9) is a regulator of the cytotoxic lymphocyte protease GzmB (granzyme B). Although GzmB is primarily involved in the destruction of compromised cells, recent evidence suggests that it is also involved in lysosome-mediated death of the cytotoxic lymphocyte itself. Sb9 protects the cell from GzmB released from lysosomes into the cytosol. Here we show that reactive oxygen species (ROS) generated within cytotoxic lymphocytes by receptor stimulation are required for lyososomal permeabilization and release of GzmB into the cytosol. Importantly, ROS also inactivate Sb9 by oxidizing a highly conserved cysteine pair (P1-P1' in rodents and P1'-P2' in other mammals) in the reactive center loop to form a vicinal disulfide bond. Replacement of the P4-P3' reactive center loop residues of the prototype serpin, SERPINA1, with the P4-P5' residues of Sb9 containing the cysteine pair is sufficient to convert SERPINA1 into a ROS-sensitive GzmB inhibitor. Conversion of the cysteine pair to serines in either human or mouse Sb9 results in a functional serpin that inhibits GzmB and resists ROS inactivation. We conclude that ROS sensitivity of Sb9 allows the threshold for GzmB-mediated suicide to be lowered, as part of a conserved post-translational homeostatic mechanism regulating lymphocyte numbers or activity. It follows, for example, that antioxidants may improve NK cell viability in adoptive immunotherapy applications by stabilizing Sb9.


Subject(s)
CD8-Positive T-Lymphocytes/metabolism , Granzymes/metabolism , Killer Cells, Natural/metabolism , Membrane Proteins/metabolism , Reactive Oxygen Species/metabolism , Serpins/metabolism , Animals , Apoptosis , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Cell Line , Cells, Cultured , Cystine/chemistry , Granzymes/antagonists & inhibitors , Granzymes/chemistry , Granzymes/genetics , Humans , Jurkat Cells , Killer Cells, Natural/cytology , Killer Cells, Natural/immunology , Lysosomes/enzymology , Lysosomes/metabolism , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutant Proteins , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Serpins/chemistry , Serpins/genetics
5.
J Biol Chem ; 290(52): 31101-12, 2015 Dec 25.
Article in English | MEDLINE | ID: mdl-26542805

ABSTRACT

Perforin is an essential component in the cytotoxic lymphocyte-mediated cell death pathway. The traditional view holds that perforin monomers assemble into pores in the target cell membrane via a calcium-dependent process and facilitate translocation of cytotoxic proteases into the cytoplasm to induce apoptosis. Although many studies have examined the structure and role of perforin, the mechanics of pore assembly and granzyme delivery remain unclear. Here we have employed quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate binding and assembly of perforin on lipid membranes, and show that perforin monomers bind to the membrane in a cooperative manner. We also found that cholesterol influences perforin binding and activity on intact cells and model membranes. Finally, contrary to current thinking, perforin efficiently binds membranes in the absence of calcium. When calcium is added to perforin already on the membrane, the QCM-D response changes significantly, indicating that perforin becomes membranolytic only after calcium binding.


Subject(s)
Calcium/chemistry , Cholesterol/chemistry , Membranes, Artificial , Perforin/chemistry , Quartz Crystal Microbalance Techniques/methods , Animals , Calcium/metabolism , Cholesterol/metabolism , Mice , Perforin/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
6.
Nat Commun ; 5: 3164, 2014.
Article in English | MEDLINE | ID: mdl-24445777

ABSTRACT

Maspin (SERPINB5) is accepted as an important tumour suppressor lost in many cancers. Consistent with a critical role in development or differentiation maspin knockout mice die during early embryogenesis, yet clinical data conflict on the prognostic utility of maspin expression. Here to reconcile these findings we made conditional knockout mice. Surprisingly, maspin knockout embryos develop into overtly normal animals. Contrary to original reports, maspin re-expression does not inhibit tumour growth or metastasis in vivo, or influence cell migration, invasion or survival in vitro. Bioinformatic analyses reveal that maspin is not commonly under-expressed in cancer, and that perturbation of genes near maspin may in fact explain poor survival in certain patient cohorts with low maspin expression.


Subject(s)
Embryonic Development/physiology , Neoplasms/physiopathology , Serpins/physiology , Animals , Cell Line, Tumor , Humans , Mice , Mice, Knockout
7.
Blood ; 121(14): 2659-68, 2013 Apr 04.
Article in English | MEDLINE | ID: mdl-23377437

ABSTRACT

Cytotoxic lymphocytes serve a key role in immune homeostasis by eliminating virus-infected and transformed target cells through the perforin-dependent delivery of proapoptotic granzymes. However, the mechanism of granzyme entry into cells remains unresolved. Using biochemical approaches combined with time-lapse microscopy of human primary cytotoxic lymphocytes engaging their respective targets, we defined the time course of perforin pore formation in the context of the physiological immune synapse. We show that, on recognition of targets, calcium influx into the lymphocyte led to perforin exocytosis and target cell permeabilization in as little as 30 seconds. Within the synaptic cleft, target cell permeabilization by perforin resulted in the rapid diffusion of extracellular milieu-derived granzymes. Repair of these pores was initiated within 20 seconds and was completed within 80 seconds, thus limiting granzyme diffusion. Remarkably, even such a short time frame was sufficient for the delivery of lethal amounts of granzymes into the target cell. Rapid initiation of apoptosis was evident from caspase-dependent target cell rounding within 2 minutes of perforin permeabilization. This study defines the final sequence of events controlling cytotoxic lymphocyte immune defense, in which perforin pores assemble on the target cell plasma membrane, ensuring efficient delivery of lethal granzymes.


Subject(s)
Apoptosis/immunology , Cell Membrane/immunology , Granzymes/immunology , Killer Cells, Natural/immunology , Pore Forming Cytotoxic Proteins/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Cell Membrane/metabolism , Complement Membrane Attack Complex/immunology , Complement Membrane Attack Complex/metabolism , Endocytosis/immunology , Exocytosis/immunology , Granzymes/metabolism , HeLa Cells , Humans , Jurkat Cells , Killer Cells, Natural/cytology , Killer Cells, Natural/metabolism , Mice , Perforin , Pore Forming Cytotoxic Proteins/metabolism , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/metabolism , Time Factors
8.
Immunol Cell Biol ; 90(9): 841-51, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22801574

ABSTRACT

Serpinb9 (Sb9, also called Spi6) is an intracellular inhibitor of granzyme B (GrB) that protects activated cytotoxic lymphocytes from apoptosis. We show here that the CD8(+) subset of splenic dendritic cells (DC), specialized in major histocompatibility complex class I (MHC I) presentation of exogenous antigens (cross-presentation), produce high levels of Sb9. Mice deficient in Sb9 are unable to generate a cytotoxic T-cell response against cell-associated antigen by cross-presentation, but maintain normal MHC-II presentation to helper T cells. This impaired cross-priming ability is autonomous to DC and is evident in animals deficient in both Sb9 and GrB, indicating that this role of Sb9 in DC is GrB-independent. In Sb9-deficient mice, CD8(+) DC develop normally, survive as well as wild-type DC after antigenic challenge, and exhibit unimpaired capacity to take up antigen. Although the core processing machinery is unaffected, Sb9-deficient DC appear to process antigen faster. Our results point to a novel, GrB-independent role for Sb9 in DC cross-priming.


Subject(s)
Antigen Presentation/immunology , Cross-Priming/immunology , Dendritic Cells/immunology , Membrane Proteins/immunology , Serine Endopeptidases/immunology , Serpins/immunology , Animals , Antigen Presentation/genetics , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Survival/genetics , Cell Survival/immunology , Cells, Cultured , Cross-Priming/genetics , Dendritic Cells/metabolism , Female , Flow Cytometry , Granzymes/genetics , Granzymes/immunology , Granzymes/metabolism , H-2 Antigens/immunology , Immunoblotting , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microscopy, Confocal , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Serpins/genetics , Serpins/metabolism , Spleen/immunology , Spleen/metabolism , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism
9.
Methods Mol Biol ; 844: 237-50, 2012.
Article in English | MEDLINE | ID: mdl-22262447

ABSTRACT

Investigation of Granzyme B (GrB) function and pathophysiology in both human settings and rodent models increasingly involve the use of indirect immunofluorescence imaging and fluorescence-activated cell sorting, which requires reliable GrB antibodies that do not recognise other closely related granzymes. Here, we describe the validation (using a set of recombinant granzymes, and GrB-deficient cells) and application of widely available monoclonal antibodies to specifically monitor GrB in human or mouse cells.


Subject(s)
Granzymes/metabolism , Microscopy, Fluorescence/methods , Animals , Blotting, Western , Cell Culture Techniques , Flow Cytometry/methods , Granzymes/immunology , Humans , Immunomagnetic Separation/methods , Lymphocytes/immunology , Lymphocytes/metabolism , Mice , Protein Transport , Tissue Distribution
10.
Biol Chem ; 391(8): 999-1004, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20536389

ABSTRACT

Reporter proteins comprising granzyme B (GrB) fused to eGFP, ecliptic pHluorin or mCherry, were generated and used to study granule (lysosome) distribution and properties in COS-1 cells and natural killer cells. The reporters resembled native GrB in biosynthesis and localization, and accumulated in granules. In live cells both the eGFP and pHluorin reporters were dark in lysosomes, but fluoresced when the granule integrity or pH was perturbed by Leu-Leu methyl ester, hydrogen peroxide, naphthazarin, or sphingosine treatment. By contrast, fluorescence of the mCherry reporter was not pH-dependent. The quenching of eGFP within granules indicates that this commonly-used fluorescent protein is not appropriate as a vital intra-lysosomal marker.


Subject(s)
Genes, Reporter , Granzymes/metabolism , Luminescent Proteins/metabolism , Lysosomes/enzymology , Protein Transport , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Enzyme Precursors/genetics , Enzyme Precursors/metabolism , Granzymes/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Hydrogen-Ion Concentration , Killer Cells, Natural , Luminescent Proteins/genetics , Microscopy, Fluorescence , Protein Sorting Signals , Recombinant Fusion Proteins/metabolism , Red Fluorescent Protein
11.
Immunol Cell Biol ; 87(3): 249-54, 2009.
Article in English | MEDLINE | ID: mdl-19079360

ABSTRACT

Granzyme B (GrB) plays a well-established intracellular role in cytotoxic lymphocyte (CL)-mediated killing of abnormal cells; however, emerging evidence suggests that it participates in extracellular matrix remodeling and target cell destruction through anoikis. As these processes require the release of GrB from the CL into the extracellular environment, we examined the secretion of GrB from natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). We found that a proportion of GrB is constitutively secreted by both CTLs and NK cells in the absence of target cell engagement. In NK cells, the protease is primarily released in an active form through secretory granules. By contrast, T lymphocytes primarily secrete inactive GrB zymogen, bypassing the granules. The release of GrB through two routes from unconjugated CLs suggests that it functions outside the cell and may contribute to pathology in cases of immune dysregulation, such as familial hemophagocytic lymphohistiocytosis (FHL). Our findings also predict the existence of an extracellular activator of GrB.


Subject(s)
Enzyme Precursors/metabolism , Granzymes/metabolism , Killer Cells, Natural/immunology , T-Lymphocytes, Cytotoxic/immunology , Cell Line , Humans , Killer Cells, Natural/enzymology , Secretory Vesicles/enzymology , T-Lymphocytes, Cytotoxic/enzymology
12.
Science ; 317(5844): 1548-51, 2007 Sep 14.
Article in English | MEDLINE | ID: mdl-17717151

ABSTRACT

Proteins containing membrane attack complex/perforin (MACPF) domains play important roles in vertebrate immunity, embryonic development, and neural-cell migration. In vertebrates, the ninth component of complement and perforin form oligomeric pores that lyse bacteria and kill virus-infected cells, respectively. However, the mechanism of MACPF function is unknown. We determined the crystal structure of a bacterial MACPF protein, Plu-MACPF from Photorhabdus luminescens, to 2.0 angstrom resolution. The MACPF domain reveals structural similarity with poreforming cholesterol-dependent cytolysins (CDCs) from Gram-positive bacteria. This suggests that lytic MACPF proteins may use a CDC-like mechanism to form pores and disrupt cell membranes. Sequence similarity between bacterial and vertebrate MACPF domains suggests that the fold of the CDCs, a family of proteins important for bacterial pathogenesis, is probably used by vertebrates for defense against infection.


Subject(s)
Bacterial Proteins/chemistry , Photorhabdus/chemistry , Protein Conformation , Protein Folding , Amino Acid Motifs , Amino Acid Sequence , Animals , Bacterial Proteins/metabolism , Complement Membrane Attack Complex/chemistry , Complement Membrane Attack Complex/metabolism , Crystallography, X-Ray , Cytotoxins/chemistry , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Molecular Sequence Data , Perforin , Pore Forming Cytotoxic Proteins/chemistry , Pore Forming Cytotoxic Proteins/genetics , Pore Forming Cytotoxic Proteins/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Vertebrates
13.
J Cell Biol ; 175(4): 619-30, 2006 Nov 20.
Article in English | MEDLINE | ID: mdl-17116752

ABSTRACT

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.


Subject(s)
Genetic Variation , Granzymes/metabolism , Amino Acid Sequence , Animals , BH3 Interacting Domain Death Agonist Protein/metabolism , Cell Death , Conserved Sequence , Cytotoxicity, Immunologic/immunology , Glycine/metabolism , Granzymes/chemistry , Humans , Jurkat Cells , Membrane Glycoproteins/metabolism , Mice , Molecular Sequence Data , Peptide Library , Peptides/metabolism , Perforin , Pore Forming Cytotoxic Proteins/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Serpins/metabolism , Substrate Specificity
14.
Mol Cell Biol ; 25(17): 7854-67, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16107729

ABSTRACT

Granzyme B (GrB) is a key effector of cytotoxic lymphocyte-mediated cell death. It is delivered to target cells bound to the proteoglycan serglycin, but how it crosses the plasma membrane and accesses substrates in the cytoplasm is poorly understood. Here we identify two cationic sequences on GrB that facilitate its binding and uptake. Mutation of cationic sequence 1 (cs1) prevents accumulation of GrB in a distinctive intracellular compartment and reduces cytotoxicity 20-fold. Mutation of cs2 reduces accumulation in this intracellular compartment and cytotoxicity two- to threefold. We also show that GrB-mediated cytotoxicity is abrogated by heparin and that target cells deficient in cell surface sulfate or glycosaminoglycans resist GrB. However, heparin does not completely prevent GrB internalization and chondroitin 4-sulfate does not inhibit cytotoxicity, suggesting that glycosaminoglycans are not essential GrB receptors. We propose that GrB enters cells by nonselective adsorptive pinocytosis, exchanging from chondroitin sulfate on serglycin to anionic components of the cell surface. In this electrostatic "exchange-adsorption" model, cs1 and cs2 participate in binding of GrB to the cell surface, thereby promoting its uptake and eventual release into the cytoplasm.


Subject(s)
Serine Endopeptidases/chemistry , Serine Endopeptidases/toxicity , Amino Acid Sequence , Animals , Apoptosis/drug effects , Binding Sites , Catalysis , Cations/chemistry , Cell Line, Tumor , Gene Products, gag/metabolism , Glycosaminoglycans/metabolism , Granzymes , Heparin/pharmacology , Humans , Mannosephosphates/chemistry , Mannosephosphates/pharmacology , Membrane Glycoproteins/metabolism , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Perforin , Pore Forming Cytotoxic Proteins , Protein Structure, Tertiary , Protein Transport , Sequence Alignment , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Static Electricity
15.
J Biol Chem ; 280(25): 23549-58, 2005 Jun 24.
Article in English | MEDLINE | ID: mdl-15843372

ABSTRACT

Human granzyme B (GrB) released from cytotoxic lymphocytes plays a key role in the induction of target cell apoptosis when internalized in the presence of perforin. Here we demonstrate that GrB also possesses a potent extracellular matrix remodeling activity. Both native and recombinant GrB caused detachment of immortalized and transformed cell lines, primary endothelial cells, and chondrocytes. Cell detachment by GrB induced endothelial cell death (anoikis). GrB also inhibited tumor cell spreading, migration, and invasion in vitro. Investigation into the underlying mechanism revealed that GrB efficiently cleaves three proteins involved in extracellular matrix structure and function: vitronectin, fibronectin, and laminin. In vitronectin, GrB cleaves after an Arg-Lys-Asp (RGD) motif, which is part of the integrin-binding site found in matrix proteins. We propose that targeting of the integrin-extracellular matrix interface by GrB may allow perforin-independent killing of target cells via anoikis, restrict motility of tumor cells, facilitate lymphocyte migration, or directly reduce virus infectivity. It may also contribute to tissue destruction in diseases in which extracellular GrB is evident, such as rheumatoid arthritis and atherosclerosis.


Subject(s)
Extracellular Matrix/metabolism , Fibronectins/metabolism , Laminin/metabolism , Serine Endopeptidases/metabolism , Vitronectin/metabolism , Arteriosclerosis/metabolism , Arteriosclerosis/pathology , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/pathology , Cell Adhesion , Cell Line, Tumor , Granzymes , Humans , Hydrolysis , Recombinant Proteins/metabolism
16.
Mol Cell Biol ; 24(9): 4075-82, 2004 May.
Article in English | MEDLINE | ID: mdl-15082799

ABSTRACT

Protease inhibitor 6 (PI-6/SERPINB6) is a widely expressed nucleocytoplasmic serpin. It inhibits granulocyte cathepsin G and neuronal neuropsin, and it is thought to protect cells from death caused by ectopic release or internalization of protease during stress such as infection or cerebral ischemia. To probe the biological functions of PI-6, we generated mice lacking its ortholog (SPI3/Serpinb6). SPI3-deficient mice developed normally and were fertile, and no abnormal pathology or increased sensitivity to cerebral ischemia was observed. There were no perturbations in leukocyte development or numbers, and recruitment of leukocytes to the peritoneal cavity was normal. SPI3-deficient mice were equally susceptible as wild-type mice to systemic Candida albicans infection, although there was a slight decrease in the ability of neutrophils from SPI3-deficient mice to kill C. albicans in vitro. Increased levels of a related inhibitor Serpinb1 (monocyte/neutrophil elastase inhibitor) in the tissues of targeted mice suggests that compensation by other serpins reduces the impact of SPI3 deficiency in these animals and may explain the lack of a more obvious phenotype.


Subject(s)
Leukocytes/physiology , Serpins/genetics , Serpins/metabolism , Stroke/metabolism , Animals , Brain Ischemia/metabolism , Candidiasis/metabolism , Disease Susceptibility , Gene Targeting , Humans , Mice , Mice, Knockout , Peritoneum/cytology , Recombinant Fusion Proteins/metabolism , Stroke/genetics , Tissue Distribution
17.
J Biol Chem ; 279(17): 16907-11, 2004 Apr 23.
Article in English | MEDLINE | ID: mdl-14752093

ABSTRACT

A key function of human granzyme B (GrB) is to induce apoptosis of target cells in conjunction with perforin. The RAH allele is the first documented variant of the human GrB gene, occurs at a frequency of 25-30%, and encodes three amino acid substitutions (Q48R, P88A, and Y245H). It was initially reported that RAH GrB is incapable of inducing apoptosis, but here we show that it has essentially identical proteolytic and cytotoxic properties to wild type GrB. Recombinant RAH and wild type GrB cleave peptide substrates with similar kinetics, are both capable of cleaving Bid and procaspase 3, and are equally inhibited by proteinase inhibitor 9, an endogenous regulator of GrB. Furthermore, cytotoxic lymphocytes from RAH heterozygotes and homozygotes have no defect in target cell killing, and in vitro RAH GrB and wild type GrB kill cells equally well in the presence of perforin. We conclude that the RAH allele represents a neutral polymorphism in the GrB gene.


Subject(s)
Alleles , Apoptosis , Serine Endopeptidases/biosynthesis , Amino Acids , Animals , BH3 Interacting Domain Death Agonist Protein , Carrier Proteins/chemistry , Caspase 3 , Caspases/metabolism , Dose-Response Relationship, Drug , Granzymes , Heterozygote , Homozygote , Humans , K562 Cells , Killer Cells, Lymphokine-Activated/metabolism , Kinetics , Lymphocytes/metabolism , Mice , Pichia/metabolism , Polymorphism, Genetic , Polymorphism, Single-Stranded Conformational , Protease Inhibitors/pharmacology , Recombinant Proteins/chemistry , Serine Endopeptidases/genetics , Time Factors
18.
J Immunol ; 170(2): 805-15, 2003 Jan 15.
Article in English | MEDLINE | ID: mdl-12517944

ABSTRACT

Granzyme B (grB) is a serine proteinase released by cytotoxic lymphocytes (CLs) to kill abnormal cells. GrB-mediated apoptotic pathways are conserved in nucleated cells; hence, CLs require mechanisms to protect against ectopic or misdirected grB. The nucleocytoplasmic serpin, proteinase inhibitor 9 (PI-9), is a potent inhibitor of grB that protects cells from grB-mediated apoptosis in model systems. Here we show that PI-9 is present in CD4(+) cells, CD8(+) T cells, NK cells, and at lower levels in B cells and myeloid cells. PI-9 is up-regulated in response to grB production and degranulation, and associates with grB-containing granules in activated CTLs and NK cells. Intracellular complexes of PI-9 and grB are evident in NK cells, and overexpression of PI-9 enhances CTL potency, suggesting that cytoplasmic grB, which may threaten CL viability, is rapidly inactivated by PI-9. Because dendritic cells (DCs) acquire characteristics similar to those of target cells to activate naive CD8(+) T cells and therefore may also require protection against grB, we investigated the expression of PI-9 in DCs. PI-9 is evident in thymic DCs (CD3(-), CD4(+), CD8(-), CD45(+)), tonsillar DCs, and DC subsets purified from peripheral blood (CD16(+) monocytes and CD123(+) plasmacytoid DCs). Furthermore, PI-9 is expressed in monocyte-derived DCs and is up-regulated upon TNF-alpha-induced maturation of monocyte-derived DCs. In conclusion, the presence and subcellular localization of PI-9 in leukocytes and DCs are consistent with a protective role against ectopic or misdirected grB during an immune response.


Subject(s)
Antigen-Presenting Cells/cytology , Cell Degranulation/immunology , Cytotoxicity, Immunologic , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/biosynthesis , Serpins/biosynthesis , T-Lymphocyte Subsets/metabolism , T-Lymphocytes, Cytotoxic/immunology , Up-Regulation/immunology , Adjuvants, Immunologic/biosynthesis , Adjuvants, Immunologic/blood , Adjuvants, Immunologic/genetics , Adjuvants, Immunologic/physiology , Antigen-Presenting Cells/metabolism , Cell Differentiation/immunology , Cell Line , Cells, Cultured , Cytotoxicity, Immunologic/genetics , Dendritic Cells/classification , Dendritic Cells/metabolism , Granzymes , Humans , Intracellular Fluid/enzymology , Intracellular Fluid/metabolism , Leukocytes/metabolism , Serine Endopeptidases/biosynthesis , Serine Proteinase Inhibitors/blood , Serine Proteinase Inhibitors/genetics , Serine Proteinase Inhibitors/physiology , Serpins/blood , Serpins/genetics , Serpins/physiology , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Cytotoxic/enzymology , Up-Regulation/genetics
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