Zinc modulates airway epithelium susceptibility to death receptor-mediated apoptosis

The accelerated loss of lung epithelium through activation of extrinsic apoptosis is believed to play a causative role in lung pathogenesis. Previous investigations have shown that zinc is required to sustain lung epithelial cell viability under stress conditions and that depletion of intracellular zinc predisposes cells to apoptosis. In this investigation, we determined whether intracellular zinc deficiency enhanced the susceptibility of primary, differentiated cultures of human lung epithelium to death receptor-mediated apoptosis, leading to barrier dysfunction. Cultures obtained from multiple donors were exposed to stimuli that provoke death receptor-mediated apoptosis and depleted of intracellular zinc with a zinc-specific chelating agent. Transepithelial resistance, paracellular transport, caspase-8 and caspase-3 activity, and apoptosis were measured. Activation of extrinsic apoptosis or zinc chelation alone resulted in a nominal increase in caspase function and apoptosis without major evidence of barrier disruption. Activation of extrinsic apoptosis in addition to zinc depletion resulted in an abrupt decrease in transepithelial resistance, a substantial increase in apoptosis, and an increased paracellular leak. Cultures were rescued by supplementation with zinc sulfate. Further analysis revealed that exogenous zinc facilitates cell survival through activation of the phosphatidylinositol 3-kinase/Akt signaling pathway. We conclude that intracellular zinc is a vital factor in lung epithelium that protects cells from death receptor-mediated apoptosis and barrier dysfunction.     

Intracellular serine protease inhibitor SERPINB4 inhibits granzyme M-induced cell death

Granzyme-mediated cell death is the major pathway for cytotoxic lymphocytes to kill virus-infected and tumor cells. In humans, five different granzymes (i.e. GrA, GrB, GrH, GrK, and GrM) are known that all induce cell death. Expression of intracellular serine protease inhibitors (serpins) is one of the mechanisms by which tumor cells evade cytotoxic lymphocyte-mediated killing. Intracellular expression of SERPINB9 by tumor cells renders them resistant to GrB-induced apoptosis. In contrast to GrB, however, no physiological intracellular inhibitors are known for the other four human granzymes. In the present study, we show that SERPINB4 formed a typical serpin-protease SDS-stable complex with both recombinant and native human GrM. Mutation of the P2-P1-P1' triplet in the SERPINB4 reactive center loop completely abolished complex formation with GrM and N-terminal sequencing revealed that GrM cleaves SERPINB4 after P1-Leu. SERPINB4 inhibited GrM activity with a stoichiometry of inhibition of 1.6 and an apparent second order rate constant of 1.3×10(4) M(-1) s(-1). SERPINB4 abolished cleavage of the macromolecular GrM substrates α-tubulin and nucleophosmin. Overexpression of SERPINB4 in tumor cells inhibited recombinant GrM-induced as well as NK cell-mediated cell death and this inhibition depended on the reactive center loop of the serpin. As SERPINB4 is highly expressed by squamous cell carcinomas, our results may represent a novel mechanism by which these tumor cells evade cytotoxic lymphocyte-induced GrM-mediated cell death.     

HtrA2 promotes cell death through its serine protease activity and its ability to antagonize inhibitor of apoptosis proteins

Inhibitor of apoptosis (IAP) proteins inhibit caspases, a function counteracted by IAP antagonists, insect Grim, HID, and Reaper and mammalian DIABLO/Smac. We now demonstrate that HtrA2, a mammalian homologue of the Escherichia coli heat shock-inducible protein HtrA, can bind to MIHA/XIAP, MIHB, and baculoviral OpIAP but not survivin. Although produced as a 50-kDa protein, HtrA2 is processed to yield an active serine protease with an N terminus similar to that of Grim, Reaper, HID, and DIABLO/Smac that mediates its interaction with XIAP. HtrA2 is largely membrane-associated in healthy cells, with a significant proportion observed within the mitochondria, but in response to UV irradiation, HtrA2 shifts into the cytosol, where it can interact with IAPs. HtrA2 can, like DIABLO/Smac, prevent XIAP inhibition of active caspase 3 in vitro and is able to counteract XIAP protection of mammalian NT2 cells against UV-induced cell death. The proapoptotic activity of HtrA2 in vivo involves both IAP binding and serine protease activity. Mutations of either the N-terminal alanine of mature HtrA2 essential for IAP interaction or the catalytic serine residue reduces the ability of HtrA2 to promote cell death, whereas a complete loss in proapoptotic activity is observed when both sites are mutated.     

The serine protease inhibitor TLCK attenuates intrinsic death pathways in neurons upstream of mitochondrial demise

It is well-established that activation of proteases, such as caspases, calpains and cathepsins are essential components in signaling pathways of programmed cell death (PCD). Although these proteases have also been linked to mechanisms of neuronal cell death, they are dispensable in paradigms of intrinsic death pathways, e.g. induced by oxidative stress. However, emerging evidence implicated a particular role for serine proteases in mechanisms of PCD in neurons. Here, we investigated the role of trypsin-like serine proteases in a model of glutamate toxicity in HT-22 cells. In these cells glutamate induces oxytosis, a form of caspase-independent cell death that involves activation of the pro-apoptotic protein BH3 interacting-domain death agonist (Bid), leading to mitochondrial demise and ensuing cell death. In this model system, the trypsin-like serine protease inhibitor Nα-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK) inhibited mitochondrial damage and cell death. Mitochondrial morphology alterations, the impairment of the mitochondrial membrane potential and ATP depletion were prevented and, moreover, lipid peroxidation induced by glutamate was completely abolished. Strikingly, truncated Bid-induced cell death was not affected by TLCK, suggesting a detrimental activity of serine proteases upstream of Bid activation and mitochondrial demise. In summary, this study demonstrates the protective effect of serine protease inhibition by TLCK against oxytosis-induced mitochondrial damage and cell death. These findings indicate that TLCK-sensitive serine proteases play a crucial role in cell death mechanisms upstream of mitochondrial demise and thus, may serve as therapeutic targets in diseases, where oxidative stress and intrinsic pathways of PCD mediate neuronal cell death.     

Dimers initiate and propagate serine protease inhibitor polymerisation

The serine protease inhibitor (serpin) family can readily form long-chain polymers by a process that underlies a variety of diseases. We show here that monomers of plasma serpins α₁-antitrypsin and antithrombin are stable on incubation with the rate-limiting step in their polymerisation being the formation of the initial dimer. Once formed, the dimers readily interlink to form tetramers and can bind monomers to form trimers and longer oligomers. Cleavage of the only exposed reactive loop, in unit I of the dimers, prevents their interlinkage, but these cleaved dimers can still link to monomers. The rapid binding by the cleaved dimers of a peptide specific to the lower half of β-sheet A of the molecule indicates the ready opening of this β-sheet in unit II of the dimers. The failure of the cleaved dimers to bind peptide-complexed monomers, together with the relative inaccessibility of the P14 hinge residue in the oligomers, is evidence that partial insertion of the reactive loop into its own A-sheet is required for polymer formation. We propose that serpin dimers initiate and propagate polymerisation by having one exposed loop with an optimal conformation as a β-strand donor and a readily opened β-sheet as an acceptor. The sequential reformation of these activated β-interfaces as the oligomer extends, molecule by molecule, provides a model for the fibril and amyloid formation of conformational diseases in general as well as for the infectivity of prion encephalopathies.     

Specificity of the serine protease inhibitor, phenylmethylsulfonyl fluoride.

Clarified cell-free extracts were prepared from rapidly dividing $\text{Bacillus subtilis}$ cells and from rabbit liver cells. These extracts were treated with [³H]-phenylmethylsulfonyl fluoride (PMSF) and analyzed by electrophoresis in isoelectric focusing polyacrylamide gels or detergent gels. Not less than 14 proteins in the $\text{B. subtilis}$ extracts and not less than 15 proteins in rabbit liver extracts reacted covalently with PMSF. These results suggest that PMSF is not as specific for serine proteases as sometimes supposed, and its effects in physiological experiments should be interpreted with caution.     

Modulation of death receptor-mediated apoptosis by CK2

Protein kinase CK2 has long been known to be involved in cell growth and proliferation. Recent work has also implicated its role in the suppression of apoptosis. We originally documented that removal of survival or growth stimuli resulted in rapid loss of CK2 from the nuclear matrix and chromatin which preceded induction of apoptosis. Further, we demonstrated that overexpression of CK2 in cells promotes suppression of drug-mediated apoptosis. In the present work, we have extended these observations to demonstrate that CK2 can influence apoptosis mediated via the death receptors. Overexpression of CK2 resulted in suppression of apoptosis mediated by TNF-α, TRAIL, and Fas-L in cells responsive to these ligands, whereas downregulation of CK2 resulted in augmentation of apoptosis mediated by these ligands. To our knowledge, this is the first report to show that receptor-mediated apoptosis can be modulated by changes in CK2 in prostate cancer cells. Based on our previous observations together with the evidence presented here, we propose that CK2 has an impact on the process of apoptosis mediated by diverse type of mechanisms thus playing a global role in regulation of apoptotic activity in cells.     

How an inhibitor of the HIV-I protease modulates proteasome activity

The human immunodeficiency virus, type I protease inhibitor Ritonavir has been used successfully in AIDS therapy for 4 years. Clinical observations suggested that Ritonavir may exert a direct effect on the immune system unrelated to inhibition of the human immunodeficiency virus, type I protease. In fact, Ritonavir inhibited the major histocompatibility complex class I restricted presentation of several viral antigens at therapeutically relevant concentrations (5 microM). In search of a molecular target we found that Ritonavir inhibited the chymotrypsin-like activity of the proteasome whereas the tryptic activity was enhanced. In this study we kinetically analyzed how Ritonavir modulates proteasome activity and what consequences this has on cellular functions of the proteasome. Ritonavir is a reversible effector of proteasome activity that protected the subunits MB-1 (X) and/or LMP7 from covalent active site modification with the vinyl sulfone inhibitor(125)I-NLVS, suggesting that they are the prime targets for competitive inhibition by Ritonavir. At low concentrations of Ritonavir (5 microM) cells were more sensitive to canavanine but proliferated normally whereas at higher concentrations (50 microM) protein degradation was affected, and the cell cycle was arrested in the G(1)/S phase. Ritonavir thus modulates antigen processing at concentrations at which vital cellular functions of the proteasome are not yet severely impeded. Proteasome modulators may hence qualify as therapeutics for the control of the cytotoxic immune response.     

柞蚕Kazal型丝氨酸蛋白酶抑制剂ApKTSPI基因克隆及表达特征分析

【目的】克隆柞蚕(Antheraea pernyi)Kazal型丝氨酸蛋白酶抑制剂(ApKTSPI)基因的cDNA序列并进行序列分析,研究ApKTSPI基因的组织表达分布及病原物免疫刺激后的表达模式,原核表达ApKTSPI。【方法】利用RACE-PCR方法扩增柞蚕ApKTSPI基因全长cDNA,生物信息学软件进行序列分析,利用实时定量PCR检测柞蚕ApKTSPI基因的组织分布及免疫刺激后的表达模式,利用pET-28a载体在大肠杆菌BL21中融合表达ApKTSPI。【结果】柞蚕ApKTSPI基因的cDNA全长568 bp,开放阅读框编码96个氨基酸,含一个Kazal结构域。ApKTSPI基因在柞蚕5龄幼虫脂肪体中特异性高表达,在核型多角体病毒、大肠杆菌和白僵菌免疫刺激后表达量都能上调,但上调的程度和时间都不同。ApKTSPI在大肠杆菌中成功诱导表达。【结论】获得了柞蚕ApKTSPI基因的cDNA全长,并研究了ApKTSPI基因的表达模式,为进一步研究其在柞蚕免疫中的功能及作用机理奠定了基础。     

NMR three-dimensional solution structure of the serine protease inhibitor cyclotheonamide A

The nmr solution conformation of cyclotheonamide A (CtA) was determined in aqueous media. The data produced 15 distance and 10 torsional constraints which were used to generate conformations using restrained simulated annealing (SA) and distance geometry/simulated annealing (DG/SA) calculations. Two different calculation protocols were performed to ensure proper sampling of conformational space and even though the torsional restraints were input differently, both calculation methods yielded the same conformation of CtA. In the structure calculations, all solutions of the Karplus equation were sampled simultaneously using the restrained SA protocol and large ranges were used for the dihedral restraints in the DG/SA protocol because all solutions to the Karplus equation could not be sampled simultaneously. The solution conformation was also compared to the solid state x-ray conformations of CtA bound to thrombin and trypsin. The conformation of the residues important for active site binding (d -Phe, h-Arg, and Pro) are nearly identical in aqueous solution and solid state with largest differences at the a-Ala and v-Tyr residues. CtA appears to be preordered in structure and does not undergo a significant conformational change upon binding to the enzyme active site. © 1997 John Wiley & Sons, Inc.     

A novel serine protease inhibitor from Bungarus fasciatus venom

By Sephadex G-50 gel filtration, cation-exchange CM-Sephadex C-25 chromatography and reversed phase high-performance liquid chromatography (HPLC), a novel serine protease inhibitor named bungaruskunin was purified and characterized from venom of Bungarus fasciatus. Its cDNA was also cloned from the cDNA library of B. fasciatus venomous glands. The predicted precursor is composed of 83 amino acid (aa) residues including a 24-aa signal peptide and a 59-aa mature bungaruskunin. Bungaruskunin showed maximal similarity (64%) with the predicted serine protease inhibitor blackelin deduced from the cDNA sequence of the red-bellied black snake Pseudechis porphyriacus. Bungaruskunin is a Kunitz protease inhibitor with a conserved Kunitz domain and could exert inhibitory activity against trypsin, chymotrypsin, and elastase. By screening the cDNA library, two new B chains of beta-bungarotoxin are also identified. The overall structures of bungaruskunin and beta-bungarotoxin B chains are similar; especially they have highly conserved signal peptide sequences. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor.     

Neuroserpin, an axonally secreted serine protease inhibitor.

We have identified and chromatographically purified an axonally secreted glycoprotein of CNS and PNS neurons. Several peptides derived from it were microsequenced. Based on these sequences, a fragment of the corresponding cDNA was amplified and used as a probe to isolate a full length cDNA from a chicken brain cDNA library. Because the deduced amino acid sequence qualified the protein as a novel member of the serpin family of serine protease inhibitors, we called it neuroserpin. Analysis of the primary structural features further characterized neuroserpin as a heparin-independent, functional inhibitor of a trypsin-like serine protease. In situ hybridization revealed a predominantly neuronal expression during the late stages of neurogenesis and in the adult brain in regions which exhibit synaptic plasticity. Thus, neuroserpin might function as an axonally secreted regulator of the local extracellular proteolysis involved in the reorganization of the synaptic connectivity during development and synapse plasticity in the adult.     

Insights into the serine protease mechanism based on structural observations of the conversion of a peptidyl serine protease inhibitor to a substrate

BackgroundSerine proteases are one of the most studied group of enzymes. Despite the extensive mechanistic studies, some crucial details remain controversial, for example, how the cleaved product is released in the catalysis reaction. A cyclic peptidyl inhibitor (CSWRGLENHRMC, upain-1) of a serine protease, urokinase-type plasminogen activator (uPA), was found to become a slow substrate and cleaved slowly upon the replacement of single residue (W3A).MethodsBy taking advantage of the unique property of this peptide, we report the high-resolution structures of uPA in complex with upain-1-W3A peptide at four different pH values by X-ray crystallography.ResultsIn the structures obtained at low pH (pH 4.6 and 5.5), the cyclic peptide upain-1-W3A was found to be intact and remained in the active site of uPA. At 7.4, the scissile bond of the peptide was found cleaved, showing that the peptide became a uPA substrate. At pH 9.0, the C-terminal part of the substrate was no longer visible, and only the P1 residue occupying the S1 pocket was identified.ConclusionsThe analysis of these structures provides explanations why the upain-1-W3A is a slow substrate. In addition, we clearly identified the cleaved fragments of the peptide at both sides of the scissile bond in the active site of the enzyme, showing a slow release of the cleaved peptide.General significanceThis work indicates that the quick release of the cleaved P′ fragment after the first step of hydrolysis may not always be needed for the second hydrolysis.     

Inhibition of kallikrein-related peptidases by the serine protease inhibitor of Kazal-type 6

Kallikrein-related peptidases (KLKs) are a group of serine proteases, expressed in several tissues. Their activity is regulated by inhibitors including members of the serine protease of Kazal-type (SPINK) family. Recently, we discovered that SPINK6 is expressed in human skin and inhibits KLK5, KLK7, KLK14 but not KLK8. In this study we tested whether SPINK6 inhibits other members of the KLK family and caspase-14. Using chromogenic substrates, SPINK6 exhibited inhibitory activity against KLK12 and KLK13 with K_i around 1 nM, KLK4 with K_i = 27.3 nM, KLK6 with K_i = 140 nM, caspase-14 with a K_i approximating 1 μM and no activity against KLK1, KLK3 and KLK11. Taken together, SPINK6 is a potent inhibitor of distinct KLKs members.     

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.     

家蚕丝氨酸蛋白酶抑制剂20的表达特征分析

【目的】原核表达家蚕Bombyx mori丝氨酸蛋白酶抑制剂20(Serine proteinase inhibitor 20,Bm Serpin-20),分析Bm Serpin-20基因和蛋白组织表达分布特点,以及不同外源病原物刺激家蚕后Bm Serpin-20基因的表达模式。【方法】利用p ET-28a表达载体在大肠杆菌Transetta(DE3)中融合表达Bm Serpin-20蛋白,利用纯化的重组蛋白作为抗原免疫新西兰大白兔制备多克隆抗体,利用实时定量PCR技术及Western blot方法分别分析Bm Serpin-20基因和蛋白的组织表达水平,以及病原物免疫刺激后的表达模式。【结果】在大肠杆菌中正确表达和纯化了融合Bm Serpin-20蛋白,并制备了多克隆抗体;Bm Serpin-20基因和蛋白在脂肪体、中肠、血细胞、马氏管、表皮、睾丸、卵巢中均有表达,且在表皮中表达量最高;家蚕5龄幼虫经核型多角体病毒(Nuclear polyhedrosis viruses)、藤黄微球菌Micrococcus luteus、大肠杆菌Escherichia coli、白僵菌Beauveria bassiana处理后,Bm Serpin-20基因表达量先下调而后上调,但不同病原物在不同时间的影响不尽相同。【结论】研究了Bm Serpin-20基因和蛋白的表达模式,为下一步研究其在家蚕免疫系统中的作用奠定了基础。