Granzyme M is a regulatory protease that inactivates proteinase inhibitor 9, an endogenous inhibitor of granzyme B

Granzyme M is a trypsin-fold serine protease that is specifically found in the granules of natural killer cells. This enzyme has been implicated recently in the induction of target cell death by cytotoxic lymphocytes, but unlike granzymes A and B, the molecular mechanism of action of granzyme M is unknown. We have characterized the extended substrate specificity of human granzyme M by using purified recombinant enzyme, several positional scanning libraries of coumarin substrates, and a panel of individual p-nitroanilide and coumarin substrates. In contrast to previous studies conducted using thiobenzyl ester substrates (Smyth, M. J., O'Connor, M. D., Trapani, J. A., Kershaw, M. H., and Brinkworth, R. I. (1996) J. Immunol. 156, 4174-4181), a strong preference for leucine at P1 over methionine was demonstrated. The extended substrate specificity was determined to be lysine = norleucine at P4, broad at P3, proline > alanine at P2, and leucine > norleucine > methionine at P1. The enzyme activity was found to be highly dependent on the length and sequence of substrates, indicative of a regulatory function for human granzyme M. Finally, the interaction between granzyme M and the serpins alpha(1)-antichymotrypsin, alpha(1)-proteinase inhibitor, and proteinase inhibitor 9 was characterized by using a candidate-based approach to identify potential endogenous inhibitors. Proteinase inhibitor 9 was effectively hydrolyzed and inactivated by human granzyme M, raising the possibility that this orphan granzyme bypasses proteinase inhibitor 9 inhibition of granzyme B.     

Antiviral cytokines induce hepatic expression of the granzyme B inhibitors, proteinase inhibitor 9 and serine proteinase inhibitor 6

Expression of the granzyme B inhibitors, human proteinase inhibitor 9 (PI-9), or the murine orthologue, serine proteinase inhibitor 6 (SPI-6), confers resistance to CTL or NK killing by perforin- and granzyme-dependent effector mechanisms. In light of prior studies indicating that virally infected hepatocytes are selectively resistant to this CTL effector mechanism, the present studies investigated PI-9 and SPI-6 expression in hepatocytes and hepatoma cells in response to adenoviral infection and to cytokines produced during antiviral immune responses. Neither PI-9 nor SPI-6 expression was detected by immunoblotting in uninfected murine or human hepatocytes. Similarly, human Huh-7 hepatoma cells were found to express only very low levels of PI-9 relative to levels detected in perforin- and granzyme-resistant CTL or lymphokine-activated killer cells. Following in vivo adenoviral infection or in vitro culture with IFN-alphabeta or IFN-gamma, SPI-6 expression was induced in murine hepatocytes. Similarly, after culture with IFN-alpha, induction of PI-9 mRNA and protein expression was observed in human hepatocytes and Huh-7 cells. IFN-gamma and TNF-alpha also induced 4- to 10-fold higher levels of PI-9 mRNA expression in Huh-7 cells, whereas levels of mRNA encoding a related serine proteinase inhibitor, proteinase inhibitor 8, were unaffected by culture of Huh-7 cells with IFN-alpha, IFN-gamma, or TNF-alpha. These findings indicate that cytokines that promote antiviral cytopathic responses also regulate expression of the cytoprotective molecules, PI-9 and SPI-6, in hepatocytes that are potential targets of CTL and NK effector mechanisms.     

The granzyme B inhibitor, protease inhibitor 9, is mainly expressed by dendritic cells and at immune-privileged sites

Granzyme B is released from CTLs and NK cells and an important mediator of CTL/NK-induced apoptosis in target cells. The human intracellular serpin proteinase inhibitor (PI)9 is the only human protein able to inhibit the activity of granzyme B. As a first step to elucidate the physiological role of PI9, PI9 protein expression in various human tissues was studied. A mAb directed against human PI9 was developed, which specifically stained PI9-transfected COS-7 cells, and was used for immunohistochemistry. Both in primary lymphoid organs and in inflammatory infiltrates, PI9 was present in different subsets of dendritic cells. Also T-lymphocytes in primary and organ-associated lymphoid tissues were PI9 positive. Endothelial cells of small vessels in most organs tested as well as the endothelial layer of large veins and arteries showed strong PI9 staining. Surprisingly, high PI9 protein expression was also found at immune-privileged sites like the placenta, the testis, the ovary, and the eye. These data fit with the hypothesis that PI9 is expressed at sites where degranulation of CTL or NK cells is potentially deleterious.     

Modulators of inflammation use nuclear factor-kappa B and activator protein-1 sites to induce the caspase-1 and granzyme B inhibitor,proteinase inhibitor 9

Proteinase inhibitor 9 (PI-9) inhibits caspase-1 (interleukin (IL)-1beta-converting enzyme) and granzyme B, thereby regulating production of the pro-inflammatory cytokine IL-1beta and susceptibility to granzyme B-induced apoptosis. We show that cellular PI-9 mRNA and protein are induced by IL-1beta, lipopolysaccharide, and 12-O-tetradecanoylphorbol-13-acetate. We identified functional imperfect nuclear factor-kappaB (NF-kappaB) sites at -135 and -88 and a consensus activator protein-1 (AP-1) site at -308 in the PI-9 promoter region. Using transient transfections in HepG2 cells to assay PI-9 promoter mutations, we find that mutational ablation of the AP-1 site or of either NF-kappaB site reduces IL-1beta-induced expression of PI-9 by approximately 60%. Mutational ablation of the two NF-kappaB sites and of the AP-1 site nearly abolishes both basal and IL-1beta-induced expression of PI-9. Nuclear extracts from IL-1beta-treated HepG2 cells exhibited strong, IL-1beta-inducible binding to the NF-kappaB sites and to the AP-1 site. Electrophoretic mobility shift assays show that after IL-1beta treatment c-Jun/c-Fos and JunD bind to the AP-1 site, whereas the p50/p65 heterodimer binds to the two NF-kappaB sites. Estrogens induce PI-9, but induction of PI-9 by estrogens and IL-1beta is not synergistic. In transiently transfected, estrogen receptor-positive HepG2ER7 cells, estrogens do not interfere with IL-1beta induction, whereas IL-1beta exhibits dose-dependent repression of estrogen-inducible PI-9 expression. Our surprising finding that the pro-inflammatory cytokine IL-1beta strongly induces PI-9 suggests a novel mechanism for regulating inflammation and apoptosis through a negative feedback loop controlling expression of the anti-inflammatory and anti-apoptotic protein, PI-9.     

The granzyme B inhibitor, PI-9, is present in endothelial and mesothelial cells, suggesting that it protects bystander cells during immune responses

Proteinase inhibitor 9 (PI-9) is a 42-kDa human intracellular serpin present in cytotoxic lymphocytes (CLs). PI-9 is an extremely efficient inhibitor of the pro-apoptotic CL granule proteinase granzyme B and is thought to function in the cytosol of CLs to protect against apoptosis induced by endogenously expressed or released granzyme B, particularly during target cell killing. Here we show by immunohistochemistry that PI-9 is also present in endothelial cells, in every tissue examined. Cultured endothelial cells express functional PI-9 (as assessed by binding to recombinant granzyme B) localized to the cytoplasm and nucleus. Immunohistochemistry also showed PI-9 in mesothelial cells, and this was confirmed by analysis of primary cells cultured from pleural and serous effusions. Granzyme B expression was not detected in either endothelial or mesothelial cells. In both cell types, PI-9 is up-regulated at the mRNA and protein level by exposure to the phorbol ester PMA, consistent with a response to inflammatory stimuli. We postulate that PI-9 is present in these lining cell types to protect against misdirected, free granzyme B released during a local immune response.     

Tumor suppressor NM23-H1 is a granzyme A-activated DNase during CTL-mediated apoptosis,and the nucleosome assembly protein SET is its inhibitor

Granzyme A (GzmA) induces a caspase-independent cell death pathway characterized by single-stranded DNA nicks and other features of apoptosis. A GzmA-activated DNase (GAAD) is in an ER associated complex containing pp32 and the GzmA substrates SET, HMG-2, and Ape1. We show that GAAD is NM23-H1, a nucleoside diphosphate kinase implicated in suppression of tumor metastasis, and its specific inhibitor (IGAAD) is SET. NM23-H1 binds to SET and is released from inhibition by GzmA cleavage of SET. After GzmA loading or CTL attack, SET and NM23-H1 translocate to the nucleus and SET is degraded, allowing NM23-H1 to nick chromosomal DNA. GzmA-treated cells with silenced NM23-H1 expression are resistant to GzmA-mediated DNA damage and cytolysis, while cells overexpressing NM23-H1 are more sensitive.     

Intrahepatic lymphocyte expression of dipeptidyl peptidase I-processed granzyme B and perforin induces hepatocyte expression of serine proteinase inhibitor 6 (Serpinb9/SPI-6)

Human proteinase inhibitor 9 (PI-9/serpinB9) and the murine ortholog, serine proteinase inhibitor 6 (SPI-6/serpinb9) are members of a family of intracellular serine proteinase inhibitors (serpins). PI-9 and SPI-6 expression in immune-privileged cells, APCs, and CTLs protects these cells against the actions of granzyme B, and when expressed in tumor cells or virally infected hepatocytes, confers resistance to killing by CTL and NK cells. The present studies were designed to assess the existence of any correlation between granzyme B activity in intrahepatic lymphocytes and induction of hepatic SPI-6 expression. To this end, SPI-6, PI-9, and serpinB9 homolog expression was examined in response to IFN-alpha treatment and during in vivo adenoviral infection of the liver. SPI-6 mRNA expression increased 10- to 100-fold in the liver after IFN-alpha stimulation and during the course of viral infection, whereas no significant up-regulation of SPI-8 and <5-fold increases in other PI-9/serpinB9 homolog mRNAs was observed. Increased SPI-6 gene expression during viral infection correlated with influxes of NK cells and CTL. Moreover, IFN-alpha-induced up-regulation of hepatocyte SPI-6 mRNA expression was not observed in NK cell-depleted mice. Additional experiments using genetically altered mice either deficient in perforin or unable to process or express granzyme B indicated that SPI-6 is selectively up-regulated in hepatocytes in response to infiltration of the liver by NK cells that express perforin and enzymatically active granzyme B.     

Importance of the P4' residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop

The cytotoxic lymphocyte serine proteinase granzyme B induces apoptosis of abnormal cells by cleaving intracellular proteins at sites similar to those cleaved by caspases. Understanding the substrate specificity of granzyme B will help to identify natural targets and develop better inhibitors or substrates. Here we have used the interaction of human granzyme B with a cognate serpin, proteinase inhibitor 9 (PI-9), to examine its substrate sequence requirements. Cleavage and sequencing experiments demonstrated that Glu(340) is the P1 residue in the PI-9 RCL, consistent with the preference of granzyme B for acidic P1 residues. Ala-scanning mutagenesis demonstrated that the P4-P4' region of the PI-9 RCL is important for interaction with granzyme B, and that the P4' residue (Glu(344)) is required for efficient serpin-proteinase binding. Peptide substrates based on the P4-P4' PI-9 RCL sequence and containing either P1 Glu or P1 Asp were cleaved by granzyme B (k(cat)/K(m) 9.5 x 10(3) and 1.2 x 10(5) s(-1) M(-1), respectively) but were not recognized by caspases. A substrate containing P1 Asp but lacking P4' Glu was cleaved less efficiently (k(cat)/K(m) 5.3 x 10(4) s(-1) M(-1)). An idealized substrate comprising the previously described optimal P4-P1 sequence (Ile-Glu-Pro-Asp) fused to the PI-9 P1'-P4' sequence was efficiently cleaved by granzyme B (k(cat)/K(m) 7.5 x 10(5) s(-1) M(-1)) and was also recognized by caspases. This contrasts with the literature value for a tetrapeptide comprising the same P4-P1 sequence (k(cat)/K(m) 6.7 x 10(4) s(-1) M(-1)) and confirms that P' residues promote efficient interaction of granzyme B with substrates. Finally, molecular modeling predicted that PI-9 Glu(344) forms a salt bridge with Lys(27) of granzyme B, and we showed that a K27A mutant of granzyme B binds less efficiently to PI-9 and to substrates containing a P4' Glu. We conclude that granzyme B requires an extended substrate sequence for specific and efficient binding and propose that an acidic P4' substrate residue allows discrimination between early (high affinity) and late (lower affinity) targets during the induction of apoptosis.     

The potency and specificity of the interaction between the IA3 inhibitor and its target aspartic proteinase from Saccharomyces cerevisiae

The yeast IA3 polypeptide consists of only 68 residues, and the free inhibitor has little intrinsic secondary structure. IA3 showed subnanomolar potency toward its target, proteinase A from Saccharomyces cerevisiae, and did not inhibit any of a large number of aspartic proteinases with similar sequences/structures from a wide variety of other species. Systematic truncation and mutagenesis of the IA3 polypeptide revealed that the inhibitory activity is located in the N-terminal half of the sequence. Crystal structures of different forms of IA3 complexed with proteinase A showed that residues in the N-terminal half of the IA3 sequence became ordered and formed an almost perfect alpha-helix in the active site of the enzyme. This potent, specific interaction was directed primarily by hydrophobic interactions made by three key features in the inhibitory sequence. Whereas IA3 was cut as a substrate by the nontarget aspartic proteinases, it was not cleaved by proteinase A. The random coil IA3 polypeptide escapes cleavage by being stabilized in a helical conformation upon interaction with the active site of proteinase A. This results, paradoxically, in potent selective inhibition of the target enzyme.     

The Brucella abortus CcrM DNA methyltransferase is essential for viability, and its overexpression attenuates intracellular replication in murine macrophages

The CcrM DNA methyltransferase of the alpha-proteobacteria catalyzes the methylation of the adenine in the sequence GAnTC. Like Dam in the enterobacteria, CcrM plays a regulatory role in Caulobacter crescentus and Rhizobium meliloti. CcrM is essential for viability in both of these organisms, and we show here that it is also essential in Brucella abortus. Further, increased copy number of the ccrM gene results in striking changes in B. abortus morphology, DNA replication, and growth in murine macrophages. We generated strains that carry ccrM either on a low-copy-number plasmid (strain GR131) or on a moderate-copy-number plasmid (strain GR132). Strain GR131 has wild-type morphology and chromosome number, as assessed by flow cytometry. In contrast, strain GR132 has abnormal branched morphology, suggesting aberrant cell division, and increased chromosome number. Although these strains exhibit different morphologies and DNA content, the replication of both strains in macrophages is attenuated. These data imply that the reduction in survival in host cells is not due solely to a cell division defect but is due to additional functions of CcrM. Because CcrM is essential in B. abortus and increased ccrM copy number attenuates survival in host cells, we propose that CcrM is an appropriate target for new antibiotics.     

B7-H1 signaling is integrated during CD8+ T cell priming and restrains effector differentiation

A promising strategy in tumor immunotherapy is the use of activated dendritic cells as vehicles for tumor vaccines with the goal of activating anti-tumor T cell responses. Current formulations for dendritic cell-based immunotherapies have limited effects on patient survival, providing motivation for further investigation of ways to enhance dendritic cell priming of anti-tumor T cell responses. Using a brief in vitro priming model, we have found that B7-H1 expressed by activated dendritic cells is integrated during priming of naïve CD8⁺ T cells and functions to limit the differentiation of effector T cell responses. CD8⁺ T cells primed by B7-H1-deficient dendritic cells exhibit increased production of IFN-γ, enhanced target cell killing, and improved anti-tumor activity. Additionally, enhanced memory populations arise from CD8⁺ T cells primed by B7-H1-deficient dendritic cells. Based on these findings, we suggest that early blockade of B7-H1 signaling should be investigated as a strategy to improve dendritic cell-based anti-tumor immunotherapy.     

A new renal mitochondrial carrier, KMCP1, is up-regulated during tubular cell regeneration and induction of antioxidant enzymes

The mitochondrial carrier family transports a variety of metabolites across the inner mitochondrial membrane. We identified and cloned a new member of this family, KMCP1 (kidney mitochondrial carrier protein-1), that is highly homologous to the previously identified protein BMCP1 (brain mitochondrial carrier protein-1). Western blotting and in situ experiments showed that this carrier is expressed predominantly within the kidney cortex in the proximal and distal tubules. KMCP1 was increased during fasting and during the regenerative phase of glycerol-induced renal failure. We show that both situations are associated with transiently increased expression of superoxide-generating enzymes, followed by increased mitochondrial metabolism and antioxidant defenses. Given that KMCP1 expression occurs simultaneously with these latter events, we propose that KMCP1 is involved in situations in which mitochondrial metabolism is increased, in particular when the cellular redox balance tends toward a pro-oxidant status.     

The RhoA effector mDia is induced during T cell activation and regulates actin polymerization and cell migration in T lymphocytes

Regulation of actin polymerization is critical for many different functions of T lymphocytes, including cell migration. Here we show that the RhoA effector mDia is induced in vitro in activated PBL and is highly expressed in vivo in diseased tissue-infiltrating activated lymphocytes. mDia localizes at the leading edge of polarized T lymphoblasts in an area immediately posterior to the leading lamella, in which its effector protein profilin is also concentrated. Overexpression of an activated mutant of mDia results in an inhibition of both spontaneous and chemokine-directed T cell motility. mDia does not regulate the shape of the cell, which involves another RhoA effector, p160 Rho-coiled coil kinase, and is not involved in integrin-mediated cell adhesion. However, mDia activation blocked CD3- and PMA-mediated cell spreading. mDia activation increased polymerized actin levels, which resulted in the blockade of chemokine-induced actin polymerization by depletion of monomeric actin. Moreover, mDia was shown to regulate the function of the small GTPase Rac1 through the control of actin availability. Together, our data demonstrate that RhoA is involved in the control of the filamentous actin/monomeric actin balance through mDia, and that this balance is critical for T cell responses.     

The expression of a mammalian proteinase inhibitor,bovine spleen trypsin inhibitor in tobacco and its effects on Helicoverpa armigera larvae

The cDNA for bovine spleen trypsin inhibitor (SI), a homologue of bovine pancreatic trypsin inhibitor (BPTI), including the natural mammalian presequence was expressed in tobacco using Agrobacterium tumefaciens-mediated transformation. Stable expression required the N-terminal targeting signal presequence although subcellular localization was not proven. SI was found to exist as two forms, one coinciding with authentic BPTI on western blots and the second marginally larger due to retention of the C-terminal peptide. Both were retained on a trypsin-agarose affinity gel and had inhibitory activity. Newly emergent leaves contained predominantly the large form whereas senescent leaves had little except the fully processed form present. Intermediate-aged leaves showed a gradual change indicating that a slow processing of the inhibitor peptide was occurring. The stability of SI was shown by the presence of protein at high levels in completely senescent leaves. Modifications to the cDNA (3 and 5 changes and minor codon changes) resulted in a 20-fold variation in expression. Expression of modified SI in transgenic tobacco leaves at 0.5% total soluble protein reduced both survival and growth of Helicoverpa armigera larvae feeding on leaves from the late first instar. In larvae surviving for 8 days, midgut trypsin activity was reduced in SI-tobacco fed larvae, while chymotrypsin activity was increased. Activities of leucine aminopeptidase and elastase-like chymotrypsin remained unaltered. The use of SI as an insect resistance factor is discussed.     

Adenosine enhances progenitor cell recruitment and nerve growth via its A2B receptor during adult fin regeneration

A major issue in regenerative medicine is the control of progenitor cell mobilisation. Apoptosis has been reported as playing a role in cell plasticity, and it has been recently shown that apoptosis is necessary for organ and appendage regeneration. In this context, we explore its possible mode of action in progenitor cell recruitment during adult regeneration in zebrafish. Here, we show that apoptosis inhibition impairs blastema formation and nerve growth, both of which can be restored by exogenous adenosine acting through its A2B receptor. Moreover, adenosine increases the number of progenitor cells. Purinergic signalling is therefore an early and essential event in the pathway from lesion to blastema formation and provides new targets for manipulating cell plasticity in the adult.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9420-9) contains supplementary material, which is available to authorized users.     

The change of antizyme inhibitor expression and its possible role during mammalian cell cycle

Antizyme inhibitor (AIn), a homolog of ODC, binds to antizyme and inactivates it. We report here that AIn increased at the G1 phase of the cell cycle, preceding the peak of ODC activity in HTC cells in culture. During interphase AIn was present mainly in the cytoplasm and turned over rapidly with the half-life of 10 to 20 min, while antizyme was localized in the nucleus. The level of AIn increased again at the G2/M phase along with ODC, and the rate of turn-over of AIn in mitotic cells decreased with the half-life of approximately 40 min. AIn was colocalized with antizyme at centrosomes during the period from prophase through late anaphase and at the midzone/midbody during telophase. Thereafter, AIn and antizyme were separated and present at different regions on the midbody at late telophase. AIn disappeared at late cytokinesis, whereas antizyme remained at the cytokinesis remnant. Reduction of AIn by RNA interference caused the increase in the number of binucleated cells in HTC cells in culture. These findings suggested that AIn contributed to a rapid increase in ODC at the G1 phase and also played a role in facilitating cells to complete mitosis during the cell cycle.     

The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila

The initiation factor 4E for eukaryotic translation (eIF4E) binds the messenger RNA 5'-cap structure and is important in the regulation of protein synthesis. Mammalian eIF4E activity is inhibited when the initiation factor binds to the translational repressors, the 4E-binding proteins (4E-BPS). Here we show that the Drosophila melanogaster 4E-BP (d4E-BP) is a downstream target of the phosphatidylinositol-3-OH kinase (PI(3)K) signal-transduction cascade, which affects the interaction of d4E-BP with eIF4E. Ectopic expression of a highly active d4E-BP mutant in wing-imaginal discs causes a reduction of wing size, brought about by a decrease in cell size and number. A marked reduction in cell size was also observed in post-mitotic cells. Expression of d4E-BP in the eye and wing together with PI(3)K or dAkt1, the serine/threonine kinase downstream of PI(3)K, resulted in suppression of the growth phenotype elicited by these kinases. Our results support a role for d4E-BP as an effector of cell growth.