Granzyme K initiates IL-6 and IL-8 release from epithelial cells by activating protease-activated receptor 2.

Granzyme K (GzmK) is a tryptic member of the granzyme family of chymotrypsin-like serine proteases produced by cells of the immune system. Previous studies have indicated that GzmK activates protease-activated receptor 1 (PAR1) enhancing activation of monocytes and wound healing in endothelial cells. Here, we show using peptides and full length proteins that GzmK and, to a lesser extent the related protease GzmA, are capable of activating PAR1 and PAR2. These cleavage events occur at the canonical arginine P1 residue and involve exosite interactions between protease and receptor. Despite cleaving PAR2 at the same point as trypsin, GzmK does not induce a classical Ca2+ flux but instead activates a distinct signalling cascade, involving recruitment of ß-arrestin and phosphorylation of ERK. In epithelial A549 cells, PAR2 activation by GzmK results in the release of inflammatory cytokines IL-6 and IL-8. These data suggest that during an immune response GzmK acts as a pro-inflammatory regulator, rather than as a cytotoxin.

Granule Leakage Induces Cell-Intrinsic, Granzyme B-Mediated Apoptosis in Mast Cells.

Mast cells are multifunctional immune cells scattered in tissues near blood vessels and mucosal surfaces where they mediate important reactions against parasites and contribute to the pathogenesis of allergic reactions. Serine proteases released from secretory granules upon mast cell activation contribute to these functions by modulating cytokine activity, platelet activation and proteolytic neutralization of toxins. The forced release of granule proteases into the cytosol of mast cells to induce cell suicide has recently been proposed as a therapeutic approach to reduce mast cell numbers in allergic diseases, but the molecular pathways involved in granule-mediated mast cell suicide are incompletely defined. To identify intrinsic granule proteases that can cause mast cell death, we used mice deficient in cytosolic serine protease inhibitors and their respective target proteases. We found that deficiency in Serpinb1a, Serpinb6a, and Serpinb9a or in their target proteases did not alter the kinetics of apoptosis induced by growth factor deprivation in vitro or the number of peritoneal mast cells in vivo. The serine protease cathepsin G induced marginal cell death upon mast cell granule permeabilization only when its inhibitors Serpinb1a or Serpinb6a were deleted. In contrast, the serine protease granzyme B was essential for driving apoptosis in mast cells. On granule permeabilization, granzyme B was required for caspase-3 processing and cell death. Moreover, cytosolic granzyme B inhibitor Serpinb9a prevented caspase-3 processing and mast cell death in a granzyme B-dependent manner. Together, our findings demonstrate that cytosolic serpins provide an inhibitory shield preventing granule protease-induced mast cell apoptosis, and that the granzyme B-Serpinb9a-caspase-3 axis is critical in mast cell survival and could be targeted in the context of allergic diseases.

Cathepsin G Inhibition by Serpinb1 and Serpinb6 Prevents Programmed Necrosis in Neutrophils and Monocytes and Reduces GSDMD-Driven Inflammation.

Neutrophil granule serine proteases contribute to immune responses through cleavage of microbial toxins and structural proteins. They induce tissue damage and modulate inflammation if levels exceed their inhibitors. Here, we show that the intracellular protease inhibitors Serpinb1a and Serpinb6a contribute to monocyte and neutrophil survival in steady-state and inflammatory settings by inhibiting cathepsin G (CatG). Importantly, we found that CatG efficiently cleaved gasdermin D (GSDMD) to generate the signature N-terminal domain GSDMD-p30 known to induce pyroptosis. Yet GSDMD deletion did not rescue neutrophil survival in Sb1a.Sb6a-/- mice. Furthermore, Sb1a.Sb6a-/- mice released high levels of pro-inflammatory cytokines upon endotoxin challenge in vivo in a CatG-dependent manner. Canonical inflammasome activation in Sb1a.Sb6a-/- macrophages showed increased IL-1ß release that was dependent on CatG and GSDMD. Together, our findings demonstrate that cytosolic serpins expressed in myeloid cells prevent cell death and regulate inflammatory responses by inhibiting CatG and alternative activation of GSDMD.

Analysis of the evolution of granule associated serine proteases of immune defence (GASPIDs) suggests a revised nomenclature.

GASPIDs (granule associated serine protease of immune defence) are a family of serine proteases intimately involved with the function of the vertebrate immune system. With the availability of a large and growing set of assembled genomes, we undertook an evolutionary analysis to plot the development of this protein family from a single precursor to the modern mammalian cohort of 12 genes, in an attempt to define and systematically classify subgroups or clades within this family, which are implied by the conventional gene designations. We identified a primordial GASPID gene as either GzmA or GzmK in cartilaginous fish and reconstructed an evolutionary path through to humans. Apart from historic value, the current sub-designations (granzymes, mast cell proteases and neutrophil serine proteases) serve no useful purpose and are increasingly misleading. We therefore used our phylogenetic and point mutation analyses to separate GASPIDs into three clades. These could form the basis of a simple nomenclature that allows effective classification of GASPIDs without implying functional roles.

Detection of human and mouse granzyme B activity in cell extracts.

The serine protease granzyme B (GrB) is a key effector molecule in cell-mediated immunity, released by cytotoxic lymphocytes (CLs) to induce cell death in neoplastic or virus-infected cells. The ability to detect and measure GrB activity is important for understanding CLs. Unfortunately, such analyses are complicated by significant differences in the substrate specificities of human and mouse GrB, which is reflected by their different activities on commonly used peptide substrates. Here, we present methods for the detection of active human and mouse GrB in extracts from primary cells, and evaluate the sensitivity of the various substrates and inhibitors. Mouse splenocytes produce approximately 120-fold more GrB than similarly activated human cells, which allows the use of the hGrB substrate IETD-AFC to follow mouse GrB activity despite its unfavourable kinetic properties.

The effects of exosite occupancy on the substrate specificity of thrombin.

Thrombin (EC 3.4.4.13) has two exosites that mediate interactions between the enzyme and its substrates and cofactors. The binding of ligands to the exosites alters the functions of the protease, for example, when the cofactor thrombomodulin binds to both exosites I and II, it converts the enzyme from a procoagulant to an anticoagulant factor. It is unknown whether ligand binding to a thrombin exosite will alter the substrate specificity of the enzyme and thus contribute to the changed substrate repertoire of the enzyme upon engagement with cofactors. We first examined whether binding of ligands to exosites I and II altered the activity of the enzyme against fluorogenic peptide substrates. The efficiency of cleavage of substrates by thrombin did change when thrombomodulin or hirugen was present, indicating that exosite I occupancy changed the active site of the protease. The presence of heparin did not change the activity of the enzyme, indicating that exosite II occupancy had little effect on active site function. Investigation of the effects of exosite I occupancy by hirugen on thrombin specificity using phage display substrate libraries revealed that the ligand only changed the specificity of the enzyme to a small degree. Occupancy of both exosites by thrombomodulin induced greater changes to the specificity of the enzyme, with the prime side showing broader changes in amino acid frequencies. Thus, exosite I ligands do affect the activity and specificity of thrombin, but not greatly enough to explain the altered substrate profile of the enzyme when complexed with thrombomodulin.