Leukocyte Elastase Inhibitor, the precursor of L-DNase II, inhibits apoptosis by interfering with caspase-8 activation

LEI (Leukocyte Elastase Inhibitor), the precursor of the pro-apoptotic molecule L-DNase II, belongs to the ovalbumin subgroup of serpins. Several serpins can inhibit apoptosis: the viral serpin Crm A inhibits Fas or TNFalpha-induced apoptosis, and overexpression of PAI-2 or PI-9 protects cells from TNFalpha or granzyme B induced apoptosis. We have previously shown that LEI overexpression protects cells from etoposide-induced apoptosis. The molecular reason of this anti-apoptotic activity is now investigated. We show that, in BHK-21 and HeLa cells, LEI anti-protease activity is essential for its anti-apoptotic effect. The protease inhibited is cathepsin D, released from the lysosome during etoposide treatment. Cathepsin D enhances caspase activity in the cell by cleaving procaspase-8 and LEI overexpression slows down this cleavage, protecting cells from apoptosis. This let us presume that high expression of LEI in tumor cells may reduce the efficiency of etoposide as a chemotherapeutic agent.     

Comparison of human chromosome 6p25 with mouse chromosome 13 reveals a greatly expanded ov-serpin gene repertoire in the mouse

Ov-serpins are intracellular proteinase inhibitors implicated in the regulation of tumor progression, inflammation, and cell death. The 13 human ov-serpin genes are clustered at 6p25 (3 genes) and 18q21 (10 genes), and share common structures. We show here that a 1-Mb region on mouse chromosome 13 contains at least 15 ov-serpin genes compared with the three ov-serpin genes within 0.35 Mb at human 6p25 (SERPINB1 (MNEI), SERPINB6 (PI-6), SER-PINB9 (PI-9)). The mouse serpins have characteristics of functional inhibitors and fall into three groups on the basis of similarity to MNEI, PI-6, or PI-9. The genes map between the mouse orthologs of the Werner helicase interacting protein and NAD(P)H menadioine oxidoreductase 2 genes, in a region that contains the markers D13Mit136 and D13Mit116. They have the seven-exon structure typical of human 6p25 ov-serpin genes, with identical intron phasing. Most show restricted patterns of expression, with common sites of synthesis being the placenta and immune tissue. Compared with human, this larger mouse serpin repertoire probably reflects the need to regulate a larger proteinase repertoire arising from differing evolutionary pressures on the reproductive and immune systems.     

The intracellular granzyme B inhibitor,proteinase inhibitor 9, is up-regulated during accessory cell maturation and effector cell degranulation,and its overexpression enhances CTL potency

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.     

Crystal structure of the apoptotic suppressor CrmA in its cleaved form

BACKGROUND: Cowpox virus expresses the serpin CrmA (cytokine response modifier A) in order to avoid inflammatory and apoptotic responses of infected host cells. The targets of CrmA are members of the caspase family of proteases that either initiate the extrinsic pathway of apoptosis (caspases 8 and 10) or trigger activation of the pro-inflammatory cytokines interleukin-1beta and interleukin-18 (caspase 1). RESULTS: We have determined the structure of a cleaved form of CrmA to 2.26 A resolution. CrmA has the typical fold of a cleaved serpin, even though it lacks the N-terminal half of the A helix, the entire D helix, and a portion of the E helix that are present in all other known serpins. The reactive-site loop of CrmA was mutated to contain the optimal substrate recognition sequence for caspase 3; however, the mutation only marginally increased the ability of CrmA to inhibit caspase 3. Superposition of the reactive-site loop of alpha1-proteinase inhibitor on the cleaved CrmA structure provides a model for virgin CrmA that can be docked to caspase 1, but not to caspase 3. CONCLUSIONS: CrmA exemplifies viral economy, selective pressure having resulted in a 'minimal' serpin that lacks the regions not needed for structural integrity or inhibitory activity. The docking model provides an explanation for the selectivity of CrmA. Our demonstration that engineering optimal substrate recognition sequences into the CrmA reactive-site loop fails to generate a good caspase 3 inhibitor is consistent with the docking model.     

Interaction of the nuclear localizing cytolytic granule serine protease granzyme B with importin alpha or beta: modulation by the serpin inhibitor PI-9

Conditional on perforin-dependent delivery to the nucleus of target cells, the cytolytic granule serine protease granzyme B (GrB) plays a central role in eliciting the nuclear events of apoptosis, as shown by the fact that reducing GrB nuclear entry prevents nuclear apoptosis. Apart from a requirement for cytosolic factors and lack of dependence on the guanine-nucleotide-binding protein Ran, little is known regarding the nuclear import pathway of GrB. In this study we use quantitative yeast two-hybrid and direct binding assays to show that GrB can be recognized independently by either of the nuclear import receptor family members importin (IMP) alpha and beta1, but that these proteins either alone or in combination cannot replace exogenous cytosol to reconstitute GrB nuclear import in vitro. Whereas antibodies to IMP(alpha) inhibit transport, indicating that IMP(alpha) is required for GrB nuclear import, those to IMP(beta) enhance transport, implying that IMP(beta) inhibits GrB nuclear import; consistent with this, the addition of recombinant IMP(beta) but not IMP(alpha) reduces maximal nuclear accumulation in the presence of cytosol. Intriguingly, complexation of GrB with its specific serpin inhibitor PI-9 was found to prevent recognition by IMP(beta) but not by IMP(alpha), and eliminate the apparent requirement for IMP(alpha) for nuclear import. We conclude that GrB nuclear import exhibits complex regulation by IMPs; that heterodimerization with PI-9 can modulate the interaction has implications for protection against apoptosis.     

Targeted disruption of SPI3/Serpinb6 does not result in developmental or growth defects, leukocyte dysfunction, or susceptibility to stroke

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.     

Viral Cross-Class Serpin Inhibits Vascular Inflammation and T Lymphocyte Fratricide; A Study in Rodent Models In Vivo and Human Cell Lines In Vitro

Poxviruses express highly active inhibitors, including serine proteinase inhibitors (serpins), designed to target host immune defense pathways. Recent work has demonstrated clinical efficacy for a secreted, myxomaviral serpin, Serp-1, which targets the thrombotic and thrombolytic proteases, suggesting that other viral serpins may have therapeutic application. Serp-2 and CrmA are intracellular cross-class poxviral serpins, with entirely distinct functions from the Serp-1 protein. Serp-2 and CrmA block the serine protease granzyme B (GzmB) and cysteine proteases, caspases 1 and 8, in apoptotic pathways, but have not been examined for extracellular anti-inflammatory activity. We examined the ability of these cross-class serpins to inhibit plaque growth after arterial damage or transplant and to reduce leukocyte apoptosis. We observed that purified Serp-2, but not CrmA, given as a systemic infusion after angioplasty, transplant, or cuff-compression injury markedly reduced plaque growth in mouse and rat models in vivo. Plaque growth was inhibited both locally at sites of surgical trauma, angioplasty or transplant, and systemically at non-injured sites in ApoE-deficient hyperlipidemic mice. With analysis in vitro of human cells in culture, Serp-2 selectively inhibited T cell caspase activity and blocked cytotoxic T cell (CTL) mediated killing of T lymphocytes (termed fratricide). Conversely, both Serp-2 and CrmA inhibited monocyte apoptosis. Serp-2 inhibitory activity was significantly compromised either in vitro with GzmB antibody or in vivo in ApoE/GzmB double knockout mice. Conclusions The viral cross-class serpin, Serp-2, that targets both apoptotic and inflammatory pathways, reduces vascular inflammation in a GzmB-dependent fashion in vivo, and inhibits human T cell apoptosis in vitro. These findings indicate that therapies targeting Granzyme B and/or T cell apoptosis may be used to inhibit T lymphocyte apoptosis and inflammation in response to arterial injury.     

Importin-beta family members mediate alpharetrovirus gag nuclear entry via interactions with matrix and nucleocapsid

The retroviral Gag polyprotein orchestrates the assembly and release of virus particles from infected cells. We previously reported that nuclear transport of the Rous sarcoma virus (RSV) Gag protein is intrinsic to the virus assembly pathway. To identify cis- and trans-acting factors governing nucleocytoplasmic trafficking, we developed novel vectors to express regions of Gag in Saccharomyces cerevisiae. The localization of Gag proteins was examined in the wild type and in mutant strains deficient in members of the importin-beta family. We confirmed the Crm1p dependence of the previously identified Gag p10 nuclear export signal. The known nuclear localization signal (NLS) in MA (matrix) was also functional in S. cerevisiae, and additionally we discovered a novel NLS within the NC (nucleocapsid) domain of Gag. MA utilizes Kap120p and Mtr10p import receptors while nuclear entry of NC involves the classical importin-alpha/beta (Kap60p/95p) pathway. NC also possesses nuclear targeting activity in avian cells and contains the primary signal for the import of the Gag polyprotein. Thus, the nucleocytoplasmic dynamics of RSV Gag depend upon the counterbalance of Crm1p-mediated export with two independent NLSs, each interacting with distinct nuclear import factors.     

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.     

Nuclear export of LEI/L-DNase II by Crm1 is essential for cell survival

LEI/L-DNase II is the key protein of a caspase-independent pathway activated by serine proteases. LEI (Leukocyte elastase inhibitor), L-DNase II precursor, is a member of the clade B serpins (also called serpin b1). In its native conformation it inhibits several intracellular proteases and has an anti-apoptotic activity. Following a metabolic stress and the increase of protease activity in the cell, LEI is cleaved and transformed into L-DNase II (LEI-derived DNase II). This transformation is due to a conformational modification that exposes a nuclear localization signal and an endonuclease active site. In this paper we show that LEI can bind the exportin Crm1, and we identify on LEI a nuclear export signal involved in the control of LEI/L-DNase II nuclearization in healthy cells. Point mutation of this site increases the accumulation of the molecule in the nucleus and triggers cell death.     

Distribution of the human intracellular serpin protease inhibitor 8 in human tissues.

Ovalbumin-like serine protease inhibitors are mainly localized intracellularly and their in vivo functions are largely unknown. To elucidate their physiological role(s), we studied the expression of one of these inhibitors, protease inhibitor 8 (PI-8), in normal human tissues by immunohistochemistry using a PI-8-specific monoclonal antibody. PI-8 was strongly expressed in the nuclei of squamous epithelium of mouth, pharynx, esophagus, and epidermis, and by the epithelial layer of skin appendages, particularly by more differentiated epithelial cells. PI-8 was also expressed by monocytes and by neuroendocrine cells in the pituitary gland, pancreas, and digestive tract. Monocytes showed nuclear and cytoplasmic localization of PI-8, whereas neuroendocrine cells showed only cytoplasmic staining. In vitro nuclear localization of PI-8 was confirmed by confocal analysis using serpin-transfected HeLa cells. Furthermore, mutation of the P(1) residue did not affect the subcellular distribution pattern of PI-8, indicating that its nuclear localization is independent of the interaction with its target protease. We conclude that PI-8 has a unique distribution pattern in human tissues compared to the distribution patterns of other intracellular serpins. Additional studies must be performed to elucidate its physiological role.     

The human serpin proteinase inhibitor-9 self-associates at physiological temperatures

The metastable serpin architecture is perturbed by extremes of temperature, pH, or changes in primary sequence resulting in the formation of inactive, polymeric conformations. Polymerization of a number of human serpins in vivo leads to diseases such as emphysema, thrombosis, and dementia, and in these cases mutations are present within the gene encoding the aggregating protein. Here we show that aggregation of the human serpin, proteinase inhibitor-9 (PI-9), occurs under physiological conditions, and forms aggregates that are morphologically distinct from previously characterized serpin polymers. Incubation of monomeric PI-9 at 37 degrees C leads to the rapid formation of aggregated PI-9. Using a variety of spectroscopic methods we analyzed the nature of the structures formed after incubation at 37 degrees C. Electron microscopy showed that PI-9 forms ordered circular and elongated-type aggregates, which also bind the fluorescent dye Thioflavin T. Our data show that in vitro wild-type PI-9 forms aggregates at physiological temperatures. The biological implications of PI-9 aggregates at physiological temperatures are discussed.     

Structural factors affecting the choice between latency transition and polymerization in inhibitory serpins

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor (serpin) protein family, is unique among the serpins in its conformational lability. This lability allows spontaneous conversion of the active form to a more stable, latent conformation under physiological conditions. In other serpins, polymerization, rather than latency transition, is induced under pathological conditions or upon heat treatment. To identify specific factors promoting latency conversion in PAI-1, we mutated PAI-1 at various positions and compared the effects with those of equivalent mutations in alpha(1)-antitrypsin, the archetypal serpin. Mutations that improved interactions with the turn between helix F and the third strand of beta-sheet A (thFs3A) or the fifth strand of beta-sheet A (s5A), which are near the site of latency transition-associated insertion of the reactive center loop, retarded latency conversion but did not greatly increase structural stability. Mutations that decreased interactions with s2C facilitated conformational conversion, possibly by releasing the reactive center loop from beta-sheet C. Mutations of Thr93 that filled a hydrophobic surface pocket on s2A dramatically increased structural stability but had a negligible effect on the conformational transition. Our results suggest that the structural features controlling latency transition in PAI-1 are highly localized, whereas the conformational strain of the native forms of other inhibitory serpins is distributed throughout the molecule and induces polymerization.     

The C-D interhelical domain of the serpin plasminogen activator inhibitor-type 2 is required for protection from TNF-alpha induced apoptosis

The serine proteinase inhibitor (serpin), plasminogen activator inhibitor type 2 (PAI-2), has been reported to inhibit tumor necrosis factor-alpha (TNF) induced apoptosis. In order to begin to understand the molecular basis for this protection, we have investigated the importance of a structural domain within the PAI-2 molecule, the C-D interhelical region, in mediating the protective effect. The C-D interhelical region is a 33 amino acid insertion which is unique among serpins and has been implicated in transglutaminase catalyzed cross-linking of PAI-2 to cell membranes. We have constructed a mutant of PAI-2 wherein 23 amino acids are deleted from the C-D interhelical region generating a structure predicted to be homologous to the closely related, but non-inhibitory serpin, chicken ovalbumin. The PAI-2Delta65/87 deletion mutant retained inhibitory activity against its known serine proteinase target, urokinase-type plasminogen activator (uPA); however expression of this mutant in HeLa cells failed to protect from TNF-induced apoptosis. Analyses of the cellular distribution of PAI-2 showed that intracellular PAI-2, and not secreted or cell-surface PAI-2, was likely responsible for the observed protection from TNF-induced apoptosis. No evidence was found for specific cross-linking of PAI-2 to the plasma membrane in either control or TNF/cycloheximide treated cells. The data demonstrate that the PAI-2 C-D interhelical domain is functionally important in PAI-2 protection from TNF induced apoptosis and suggest a novel function for the C-D interhelical domain in the protective mechanism.     

Quantification of Nuclear Protein Transport using Induced Heterodimerization

Trafficking of proteins between the cytoplasm and nucleus occurs exclusively across the nuclear pore complex of eucaryotic cells. Fundamental aspects of this process affect temporal and spatial parameters, the latter carried out by specific import [nuclear localization sequence (NLS)] and export [nuclear export sequence (NES)] sequences. In this study, we focused on the adaptation of a protein heterodimerization assay to kinetically measure Crm1-mediated nuclear export in living cells using the rapalog AP21967, a heterodimerizing agent and NLS- and NES-containing fusion proteins equipped with distinct AP21967-specific binding motifs. In HeLa cells, we observed rapid nuclear export of the NLS-containing fusion protein in the presence of AP21967, with the extent of this process being a function of the number of AP21967-binding motifs. AP21967-induced nuclear export was specifically inhibited by the Crm1-binding molecule leptomycin B. Half maximal export was achieved after ∼ 10 min. We further applied protein heterodimerization in HeLa cells to study induced NLS-mediated nuclear import. Only in the presence of heterodimerizer AP21967 nuclear import of a cytoplasmically localizing fusion protein was observed. Induced protein heterodimerization is thus a valuable tool to quantitatively study nucleocytoplasmic protein trafficking in cultured cells, in a non-invasive, time-saving manner.     

Polymerization of plasminogen activator inhibitor-1

The activity of the serine proteinase inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) is controlled by the intramolecular incorporation of the reactive loop into beta-sheet A with the generation of an inactive latent species. Other members of the serpin superfamily can be pathologically inactivated by intermolecular linkage between the reactive loop of one molecule and beta-sheet A of a second to form chains of polymers associated with diverse diseases. It has long been believed that PAI-1 is unique among active serpins in that it does not form polymers. We show here that recombinant native and latent PAI-1 spontaneously form polymers in vitro at low pH although with distinctly different electrophoretic patterns of polymerization. The polymers of both the native and latent species differ from the typical loop-A-sheet polymers of other serpins in that they readily dissociate back to their original monomeric form. The findings with PAI-1 are compatible with different mechanisms of linkage, each involving beta-strand addition of the reactive loop to s7A in native PAI-1 and to s1C in latent PAI-1. Glycosylated native and latent PAI-1 can also form polymers under similar conditions, which may be of in vivo importance in the low pH environment of the platelet.     

Identification of a nuclear targeting domain in the insertion between helices C and D in protease inhibitor-10

Protease inhibitor 10 (PI-10), an intracellular ovalbumin-serpin, contains a series of basic amino acids in the loop between helices C and D that exhibit homology to known nuclear targeting signals. Transfection of HeLa cells with plasmids encoding enhanced green fluorescent protein (EGFP) coupled to PI-10 revealed an intense fluorescence of the nucleus. Immunoblotting demonstrated a single Mr 80,000 EGFP.PI-10 complex in isolated nuclei. Mutation of four basic amino acids in the interhelical loop to alanines (i.e. K74A, K75A, R76A, K77A) resulted in the fluorescent complex being confined to the cytoplasm. Further evidence for a nuclear targeting signal in this region was provided by localization of the fluorescent label to the nucleus in cells transfected with a plasmid encoding EGFP fused to the 25 amino acids comprising the interhelical loop of PI-10 (i.e. Arg-63 to Glu-87), whereas a cytoplasmic distribution was noted for the construct encoding EGFP coupled to the mutated interhelical loop. These data raise the possibility that PI-10 may play a role in regulating protease activity within the nucleus, a property unique in the field of serpin biology.     

The crystal structure of plasminogen activator inhibitor 2 at 2.0 A resolution: implications for serpin function.

BACKGROUND: Plasminogen activator inhibitor 2 (PAI-2) is a member of the serpin family of protease inhibitors that function via a dramatic structural change from a native, stressed state to a relaxed form. This transition is mediated by a segment of the serpin termed the reactive centre loop (RCL); the RCL is cleaved on interaction with the protease and becomes inserted into betasheet A of the serpin. Major questions remain as to what factors facilitate this transition and how they relate to protease inhibition. RESULTS: The crystal structure of a mutant form of human PAI-2 in the stressed state has been determined at 2.0 A resolution. The RCL is completely disordered in the structure. An examination of polar residues that are highly conserved across all serpins identifies functionally important regions. A buried polar cluster beneath betasheet A (the so-called 'shutter' region) is found to stabilise both the stressed and relaxed forms via a rearrangement of hydrogen bonds. CONCLUSIONS: A statistical analysis of interstrand interactions indicated that the shutter region can be used to discriminate between inhibitory and non-inhibitory serpins. This analysis implied that insertion of the RCL into betasheet A up to residue P8 is important for protease inhibition and hence the structure of the complex formed between the serpin and the target protease.     

Selective Regulation of Apoptosis: the Cytotoxic Lymphocyte Serpin Proteinase Inhibitor 9 Protects against Granzyme B-Mediated Apoptosis without Perturbing the Fas Cell Death Pathway

Cytotoxic lymphocytes (CLs) induce caspase activation and apoptosis of target cells either through Fas activation or through release of granule cytotoxins, particularly granzyme B. CLs themselves resist granule-mediated apoptosis but are eventually cleared via Fas-mediated apoptosis. Here we show that the CL cytoplasmic serpin proteinase inhibitor 9 (PI-9) can protect transfected cells against apoptosis induced by either purified granzyme B and perforin or intact CLs. A PI-9 P₁ mutant (Glu to Asp) is a 100-fold-less-efficient granzyme B inhibitor that no longer protects against granzyme B-mediated apoptosis. PI-9 is highly specific for granzyme B because it does not inhibit eight of the nine caspases tested or protect transfected cells against Fas-mediated apoptosis. In contrast, the P₁(Asp) mutant is an effective caspase inhibitor that protects against Fas-mediated apoptosis. We propose that PI-9 shields CLs specifically against misdirected granzyme B to prevent autolysis or fratricide, but it does not interfere with homeostatic deletion via Fas-mediated apoptosis.