Serine Protease Inhibitor AEBSF Research Articles

Exploring the “Cascade Effect” of AEBSF on Reduced Glioblastoma Proliferation

Glioblastomas (GBMs) are one of the deadliest cancers, and yet still lack treatment. In this study, serine protease inhibitor AEBSF is shown to decrease GBM vi-ability and proliferation through a series of intertwined steps. By decreasing the expres-sion of ATP6V1A--the first step of the “cascade effect”--AEBSF creates an unfavorable environment for protease activity. Such “unfavorability” is proven by a decline in expres-sion of proteases MMP9 and ELANE upon GBM treatment with AEBSF. Increasingly higher concentrations of AEBSF are shown to decrease the percentage of cell viability, proliferation, and metastasis. This is explained through biological process enrichment analyses, which indicate that MMP9 and ELANE play a role in the disassembly of the extracellular protein network. This disassembly of the extracellular environment enables tumorigenic spread and viability. The results of the viability and proliferation assays, in combination with those of the indirect ELISAs, enrichment analyses, and scratch assays, underscore an intricate and profound sequence of events that constitute the “cascade effect”.

Cathepsin Release from Lysosomes Promotes Endocytosis of Clostridium perfringens Iota-Toxin.

Iota-toxin from Clostridium perfringens type E is a binary toxin composed of two independent proteins: actin-ADP-ribosylating enzyme component, iota-a (Ia), and binding component, iota-b (Ib). Ib binds to target cell receptors and mediates the internalization of Ia into the cytoplasm. Extracellular lysosomal enzyme acid sphingomyelinase (ASMase) was previously shown to facilitate the internalization of iota-toxin. In this study, we investigated how lysosomal cathepsin promotes the internalization of iota-toxin into target cells. Cysteine protease inhibitor E64 prevented the cytotoxicity caused by iota-toxin, but aspartate protease inhibitor pepstatin-A and serine protease inhibitor AEBSF did not. Knockdown of lysosomal cysteine protease cathepsins B and L decreased the toxin-induced cytotoxicity. E64 suppressed the Ib-induced ASMase activity in extracellular fluid, showing that the proteases play a role in ASMase activation. These results indicate that cathepsin B and L facilitate entry of iota-toxin via activation of ASMase.

Clinical implications of differential antivenom efficacy in neutralising coagulotoxicity produced by venoms from species within the arboreal viperid snake genus Trimeresurus

Snake envenomation globally is attributed to an ever-increasing human population encroaching into snake territories. Responsible for many bites in Asia is the widespread genus Trimeresurus. While bites lead to haemorrhage, only a few species have had their venoms examined in detail. We found that Trimeresurus venom causes haemorrhaging by cleaving fibrinogen in a pseudo-procoagulation manner to produce weak, unstable, short-lived fibrin clots ultimately resulting in an overall anticoagulant effect due to fibrinogen depletion. The monovalent antivenom ‘Thai Red Cross Green Pit Viper antivenin’, varied in efficacy ranging from excellent neutralisation of T. albolabris venom through to T. gumprechti and T. mcgregori being poorly neutralised and T. hageni being unrecognised by the antivenom. While the results showing excellent neutralisation of some non-T. albolabris venoms (such as T. flavomaculaturs, T. fucatus, and T. macrops) needs to be confirmed with in vivo tests, conversely the antivenom failure T. hageni, and the very poor results against T. gumprechti and T. mcgregori, despite being conducted in the ideal scenario of preincubation of antivenom:venom, indicates that the likelihood of clinically relevant cross-reactivity for these species is low (T. gumprechti and T. mcgregori) to non-existent (T. hageni). These same latter three species were also not inhibited by the serine protease inhibitor AEBSF, suggesting that the toxins leading to a coagulotoxic effect in these species are non-serine proteases while in contrast T. albolabris coagulotoxicity was completely impeded by AEBSF, and thus driven by kallikrein-type serine proteases. There was a conspicuous lack of phylogenetic pattern in venom variation, with the most potent venoms (T. albolabris and T. hageni) being distant to each other on the organismal tree, and with the three most divergent and poorly neutralised venoms (T. gumprechti, T. hageni, and T. mcgregori) were also not each others closest relatives. This reinforces the paradigm that the fundamental dynamic evolution of venom results in organismal phylogeny being a poor predictor of venom potency or antivenom efficacy. This study provides a robust investigation on the differential venom effects from a wide range of Trimeresurus species on coagulation, highlighting differential fibrinogenolytic effects, while also investigating the relative antivenom neutralisation capabilities of the widely available Thai Red Cross Green Pit Viper antivenom. These results therefore have immediate, real-world implications for patients envenomed by Trimeresurus species.

Serine protease inhibitor AEBSF reduces dengue virus infection via decreased cholesterol synthesis

Dengue virus (DENV) infection has evolved into a major global health menace and economic burden due to its intensity and geographic distribution. DENV infection in humans can cause a wide range of symptoms including dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). An antiviral agent that is effective against all four serotypes of DENV is urgently needed to prevent and to manage this condition. Reducing the viral load during the early phase of infection may minimize the chance of patients progressing to more severe DHF or DSS. In this study, we set forth to investigate the anti-viral effect of five commercially available protease inhibitors on DENV infection since both viral and host proteases can contribute to effective viral replication. Previously, the serine protease inhibitor AEBSF [4-(2-aminoethyl) benzene sulfonyl fluoride] has been shown to inhibit DENV NS3 protease activity. The results of the present study revealed that DENV genome replication and protein synthesis were significantly inhibited by AEBSF in a dose-dependent manner. AEBSF inhibited the expression of genes such as 3-hydroxy 3-methyl-glutaryl-CoA synthase (HMGCS), 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), and low-density lipoprotein receptor (LDLR). Moreover, AEBSF significantly inhibited HMGCR activity and intracellular cholesterol synthesis after DENV infection. The anti-DENV effect of AEBSF was confirmed in all four DENV serotypes and in three different cell lines. These results indicate that AEBSF reduces DENV infection via both viral and host protease activities.

Multifunctional Serine Protease Inhibitor-Coated Water-Soluble Gold Nanoparticles as a Novel Targeted Approach for the Treatment of Inflammatory Skin Diseases.

The overexpression and increased activity of the serine protease Kallikrein 5 (KLK5) is characteristic of inflammatory skin diseases such as Rosacea. The use of inhibitors of this enzyme-such as 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF·HCl) or the anti-human recombinant Kallikrein 5 (anti-KLK5) antibody-in the treatment of the disease has been limited due to their low bioavailability, for which their immobilization in drug delivery agents can contribute to making serine protease inhibitors clinically useful. In this work, we synthesized gold nanoparticles (GNP) coated with a mixture of hydroxyl- and carboxyl-terminated thiolates (GNP.OH/COOH), whose carboxyl groups were used to further functionalize the nanoparticles with the serine protease inhibitor AEBSF·HCl either electrostatically or covalently (GNP.COOH AEBSF and GNP.AEBSF, respectively), or with the anti-KLK5 antibody (GNP.antiKLK5). The synthesized and functionalized GNP were highly water-soluble, and they were extensively characterized using UV-vis absorption spectroscopy, Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), and Thermogravimetric Analysis (TGA). GNP.OH/COOH and their subsequent functionalizations effectively inhibited KLK5 in vitro. Internalization of fluorophore-coated GNP.OH/COOH in human keratinocytes (HaCaT cells) was proven using confocal fluorescence microscopy. Cell viability assays revealed that the cytotoxicity of free AEBSF is importantly decreased when it is incorporated in the nanoparticles, either ionically (GNP.COOH AEBSF) or, most importantly, covalently (GNP.AEBSF). The functionalized nanoparticles GNP.AEBSF and GNP.antiKLK5 inhibited intracellular KLK5 activity in HaCaT cells and diminished secretion of IL-8 under inflammatory conditions triggered by TLR-2 ligands. This study points to the great potential of these GNP as a new intracellular delivery strategy for both small drugs and antibodies in the treatment of skin diseases such as Rosacea.

Abstract P239: (Pro)Renin Receptor Contributes to Albumin Overload Nephropathy: Role of Site-1 Protease-Derived Soluble (Pro) Renin Receptor

(Pro) renin receptor (PRR) is a new member of the renin-angiotensin system (RAS) and its role in chronic kidney disease largely remains elusive. We tested the role of PRR in albumin overload (OA) nephropathy. Uninephretomized Sprague-Dawley rats were treated for 7 weeks with vehicle, bovine serum albumin (BSA) (5 g/kg/d via a single i.p. injection) or in combination with PRO20 (500 μg/kg/d via i.p. 3 times a day). The OA rat exhibited severe proteinuria (23-fold), interstitial fibrosis, and oxidative stress, accompanied with increases in urinary renin activity (3.6-fold), urinary angiotensinogen (AGT, 3.6-fold), urinary AngII(7.8-fold), renal ACE mRNA (2.4-fold), all of which were significantly attenuated by PRO20. Urinary soluble PRR (sPRR) as assessed by ELISA was increased 13.6-fold in the OA model. In cultured HK-2 cells, BSA treatment for 24 h decreased full-length PRR (76% reduction) and increased sPRR (8-fold). BSA also increased medium renin activity (3.0-fold), IL6 (2.9-fold for mRNA and 4.1-fold for medium protein) and IL8 (5.5-fold for mRNA and 2.6-fold for medium protein), all of which were attenuated by PRO20 or PRR siRNA. Although furin or ADAM19 was previously shown to mediate the generation of sPRR, inhibition of either one of these had no effect on BSA-induced cleavage of PRR. In contrast, a serine protease inhibitor AEBSF significantly suppressed the production of sPRR. Of serine proteases, proprotein convertases (PCs) mediates post-translational processing of proprotein to active protein and are of particular interest. Screening of 9 PCs led to the identification of site-1 protease (S1P) as the predominant cleavage enzyme. S1P inhibition with siRNA or PF-429242 reduced the sPRR production by 47% and 87%, respectively. PF-429242 also reduced renin activity by 40%, in parallel with suppressed IL6 and IL8 levels. Administration of a recombinant sPRR reversed all the effects of S1P inhibition. Mutagenesis of the S1P but not furin cleavage site in PRR blocked the cleavage. Together, these results suggest that PRR mediates OA-induced renal injury at least in part through S1P-derived sPRR.

Respiratory syncytial virus (RSV) entry is inhibited by serine protease inhibitor AEBSF when present during an early stage of infection

BackgroundHost proteases have been shown to play important roles in many viral activities such as entry, uncoating, viral protein production and disease induction. Therefore, these cellular proteases are putative targets for the development of antivirals that inhibit their activity. Host proteases have been described to play essential roles in Ebola, HCV, HIV and influenza, such that specific protease inhibitors are able to reduce infection. RSV utilizes a host protease in its replication cycle but its potential as antiviral target is unknown. Therefore, we evaluated the effect of protease inhibitors on RSV infection.MethodsTo measure the sensitivity of RSV infection to protease inhibitors, cells were infected with RSV and incubated for 18 h in the presence or absence of the inhibitors. Cells were fixed, stained and studied using fluorescence microscopy.ResultsSeveral protease inhibitors, representing different classes of proteases (AEBSF, Pepstatin A, E-64, TPCK, PMSF and aprotinin), were tested for inhibitory effects on an RSV A2 infection of HEp-2 cells. Different treatment durations, ranging from 1 h prior to inoculation and continuing for 18 h during the assay, were evaluated. Of all the inhibitors tested, AEBSF and TPCK significantly decreased RSV infection. To ascertain that the observed effect of AEBSF was not a specific feature related to HEp-2 cells, A549 and BEAS-2B cells were also used. Similar to HEp-2, an almost complete block in the number of RSV infected cells after 18 h of incubation was observed and the effect was dose-dependent. To gain insight into the mechanism of this inhibition, AEBSF treatment was applied during different phases of an infection cycle (pre-, peri- and post-inoculation treatment). The results from these experiments indicate that AEBSF is mainly active during the early entry phase of RSV. The inhibitory effect was also observed with other RSV isolates A1998/3–2 and A2000/3–4, suggesting that this is a general feature of RSV.ConclusionRSV infection can be inhibited by broad serine protease inhibitors, AEBSF and TPCK. We confirmed that AEBSF inhibition is independent of the cell line used or RSV strain. The time point at which treatment with the inhibitor was most potent, was found to coincide with the expected moment of entry of the virion with the host cell.

A Computational Module Assembled from Different Protease Family Motifs Identifies PI PLC from Bacillus cereus as a Putative Prolyl Peptidase with a Serine Protease Scaffold

Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

Functional dissection of the alphavirus capsid protease: sequence requirements for activity

BackgroundThe alphavirus capsid is multifunctional and plays a key role in the viral life cycle. The nucleocapsid domain is released by the self-cleavage activity of the serine protease domain within the capsid. All alphaviruses analyzed to date show this autocatalytic cleavage. Here we have analyzed the sequence requirements for the cleavage activity of Chikungunya virus capsid protease of genus alphavirus.ResultsAmongst alphaviruses, the C-terminal amino acid tryptophan (W261) is conserved and found to be important for the cleavage. Mutating tryptophan to alanine (W261A) completely inactivated the protease. Other amino acids near W261 were not having any effect on the activity of this protease. However, serine protease inhibitor AEBSF did not inhibit the activity. Through error-prone PCR we found that isoleucine 227 is important for the effective activity. The loss of activity was analyzed further by molecular modelling and comparison of WT and mutant structures. It was found that lysine introduced at position 227 is spatially very close to the catalytic triad and may disrupt electrostatic interactions in the catalytic site and thus inactivate the enzyme. We are also examining other sequence requirements for this protease activity.ConclusionsWe analyzed various amino acid sequence requirements for the activity of ChikV capsid protease and found that amino acids outside the catalytic triads are important for the activity.

Proinflammatory Impact of Staphylococcus epidermidis on the Nasal Epithelium Quantified by IL-8 and GRO-α Responses in Primary Human Nasal Epithelial Cells

Background: Bacterial etiology of chronic rhinosinusitis (CRS) still remains controversial. Whereas Staphylococcus aureus enterotoxins have been detected in CRS, the impact of Staphylococcus epidermidis, a major commensal inhabitant of the nose, has not been studied. Among others, serine and cysteine proteases have been identified as factors of virulence in S. epidermidis. Methods:S. epidermidis was examined in tissue biopsies of 30 CRS patients (16 with nasal polyposis) using standard procedures. Primary human nasal epithelial cells from inferior nasal turbinates (HNECs), from nasal polyps (NPECs) and A549 airway epithelial cells were stimulated with S. epidermidis supernatants DSM20044 or ATCC35984 and the IL-8 and GRO-α response was quanti- fied by ELISA. Protease-triggered chemokine responses and involvement of NF-ĸB were investigated by addition of protease or NF-ĸB inhibitors. Activation of NF-ĸB was demonstrated by quantitative DNA binding assay. Results:S. epidermidis was the most frequently isolated bacteria in the majority of CRS patients. HNECs and NPECs revealed no different IL-6 and IL-8 synthesis following stimulation with DSM20044 or ATCC35984. Stimulation of HNECs and A549 cells with S. epidermidis supernatants resulted in increased IL-8 and GRO-α expression which could be suppressed by the serine protease inhibitor AEBSF and the NF-ĸB inhibitor BAY 11 but not by the cysteine protease inhibitor E64. Results obtained for A549 cells were similar to HNECs. Conclusion:S. epidermidis was present in the majority of CRS specimens. Proinflammatory impact of S. epidermidis supernatants on nasal epithelial cells was demonstrated by serine protease-triggered and NF-ĸB-dependent chemokine responses.

OOKINETE SECRETION OF A HIGH-MOLECULAR-WEIGHT COMPLEX-MEDIATING SERINE PROTEASE ACTIVATION OF A MALARIA PARASITE PROCHITINASE CRITICAL FOR MALARIA TRANSMISSION.

Background The mosquito invasive form of the malaria parasite, the ookinete, secretes chitinases critical for mosquito invasion and continuation of the parasite life cycle. This class of enzyme is validated as a target for interrupting malaria transmission. A recombinant zymogen form of the P. gallinaceum chitinase rPgCHT1 was previously shown to be activated in vitro by the serine protease Endoproteinase Lys-C. Objective To test the hypothesis that an ookinete-produced serine protease activates pro-PgCHT1, we sought to characterize this enzyme toward the goal of validating this protease as a target of blocking malaria transmission. Methods Conditioned ookinete culture medium was subjected to a benzamidine (serine protease inhibitor) affinity column. The ability of the eluate to activate pro-rPgCHT1 in the presence and absence of the serine protease inhibitor AEBSF and the aspartic acid protease inhibitor pepstatin A was assessed using a microfluorimetry assay, which measured chitinase activity expressed as relative fluorescence units (RFU)/minute. Results Eluate containing 11.1 μg of protein increased the activity of 11.1 μg of rPgCHT1 from 30 RFU/minute to 488 RFU/minute (p = .002). This effect was diminished by 21% following the addition of the serine protease inhibitor AEBSF (p = .046) but not by the aspartic protease inhibitor pepstatin A. Eluate without rPgCHT1 had 477 RFU/minute of chitinase activity, significantly higher than rPgCHT1 by itself (p = .003) but not significantly different from eluate and rPgCHT1 together (p = .64). The addition of AEBSF to eluate decreased activity by 23% (p = .017), with no effect seen following the addition of pepstatin A. Discussion Benzamidine-column eluate of P. gallinaceum ookinete culture supernatants proteolytically activated pro-rPgCHT1. This eluate also possessed intrinsic chitinase activity, suggesting the cosecretion of PgCHT1 and serine protease activity in a complex. These findings are novel for apicomplexan parasites and have practical application toward developing novel approaches to blocking malaria transmission.

SARS Coronavirus, but Not Human Coronavirus NL63, Utilizes Cathepsin L to Infect ACE2-expressing Cells

Viruses require specific cellular receptors to infect their target cells. Angiotensin-converting enzyme 2 (ACE2) is a cellular receptor for two divergent coronaviruses, SARS coronavirus (SARS-CoV) and human coronavirus NL63 (HCoV-NL63). In addition to hostcell receptors, lysosomal cysteine proteases are required for productive infection by some viruses. Here we show that SARS-CoV, but not HCoV-NL63, utilizes the enzymatic activity of the cysteine protease cathepsin L to infect ACE2-expressing cells. Inhibitors of cathepsin L blocked infection by SARS-CoV and by a retrovirus pseudotyped with the SARS-CoV spike (S) protein but not infection by HCoV-NL63 or a retrovirus pseudotyped with the HCoV-NL63 S protein. Expression of exogenous cathepsin L substantially enhanced infection mediated by the SARS-CoV S protein and by filovirus GP proteins but not by the HCoV-NL63 S protein or the vesicular stomatitis virus G protein. Finally, an inhibitor of endosomal acidification had substantially less effect on infection mediated by the HCoV-NL63 S protein than on that mediated by the SARS-CoV S protein. Our data indicate that two coronaviruses that utilize a common receptor nonetheless enter cells through distinct mechanisms.

Characterization of a novel protein from Proatheris superciliaris venom: Proatherocytin, a 34-kDa platelet receptor PAR1 agonist

Many toxins from viperid venoms have been characterised as powerful activators of platelets. Here, the venom from the East African Lowland viper, Proatheris superciliaris, was investigated for its effect on platelets and the coagulation system. Whole P. superciliaris venom stimulated platelet shape change and aggregation; however, the stimulation of platelet activation was unaffected by the structurally distinct Src family kinase inhibitors PP1 and PD0173952, suggesting that platelet activation was mediated by a G protein-coupled receptor. A platelet reactive 34-kDa protein was isolated from P. superciliaris venom which we have designated proatherocytin. This protein induced Src kinase-independent aggregation of both human and mouse platelets that was inhibited by the serine protease inhibitor AEBSF. Proatherocytin did not clot bovine or human fibrinogen, degrade fibrinogen or hydrolyse the serine protease substrate benzoyl-FVR-pNA. It activated human PAR1 on stably transfected rat kidney epithelial cells, whereas no activation of the trypsin receptor PAR2 was shown. Surprisingly, Edman degradation of proatherocytin revealed sequence identity with existing disintegrin-like domains of snake venom metalloproteinases. These results suggest that proatherocytin is a highly selective PAR1 agonist that also causes mouse platelet aggregation, probably through cleavage of PAR4.

Abrogation of IFN-γ mediated epithelial barrier disruption by serine protease inhibition

The intestinal barrier function is often impaired in a variety of diseases including chronic inflammatory bowel disease. Increased intestinal permeability during episodes of active disease correlates with destruction or rearrangement of the tight junction protein complex. IFN-gamma has been widely studied for its effect on barrier function and tight junction structures but its mode of action remains unclear. Since the claudin family of tight junction proteins is proposed to be involved in barrier maintenance we studied the effect of IFN-gamma on claudin expression in relation to epithelial barrier function. Cycloheximide and protease inhibitors were used to study mechanisms of IFN-gamma mediated barrier disruption. Intestinal epithelial cells were exposed to IFN-gamma and permeability was evaluated by horse radish peroxidase (HRP) and 4 kD FITC-dextran fluxes. Occludin and claudin-1, -2, -3, and -4 tight junction protein expression was determined by Western blotting. Occludin and claudin-2 protein expression was dramatically reduced after IFN-gamma exposure, which correlated with increased permeability for HRP and FITC-dextran. Interestingly, cleavage of claudin-2 was observed after incubation with IFN-gamma. Serine protease inhibitor AEBSF completely abrogated IFN-gamma mediated barrier disruption which was associated with preservation of claudin-2 expression. Moreover, IFN-gamma induced loss of barrier integrity was found to affect claudin-2 and occludin expression through different mechanisms. Since inhibition of serine protease activity abrogates IFN-gamma mediated barrier disruption this may be an important target for therapeutic intervention.

Shedding of the Germinal Angiotensin I-Converting Enzyme (gACE) Involves a Serine Protease and Is Activated by Epididymal Fluid1

The present report describes how the soluble germinal angiotensin I-converting enzyme (gACE) appears in the epididymal fluid, where it has been identified in some laboratory rodents and domestic ungulates. We showed that this gACE results from an active proteolytic process that releases the enzyme's extracellular domain from sperm in a precise spatiotemporal location during epididymal transit and that this process involves serine protease activity. Using polyclonal antibodies against the C-terminal intracellular sequence of ACE, a fragment of approximately 10 kDa was detected on the sperm extract only in the epididymal region, where the gACE release occurs. The fluid enzyme was purified, and the cleavage site was determined by mass spectrometry to be between Arg622 and Leu623 of the mature sheep gACE sequence (equivalent to Arg627 and Arg1203 of the human mature gACE and somatic ACE sequences, respectively). Thereafter, the C-terminal Arg was removed, leaving Ala621 as a C-terminal. Using an in vitro assay, gACE cleavage from sperm was strongly increased by the presence of epididymal fluid from the release zone, and this increase was inhibited specifically by the serine protease-inhibitor AEBSF but not by para-aminobenzamidine. None of the other inhibitors tested, such as metallo- or cystein-protease inhibitors, had a similar effect on release. It was also found that this process did not involve changes in gACE phosphorylation.

Inhibition of the MAPK and PI3K pathways enhances UDCA-induced apoptosis in primary rodent hepatocytes

The mechanisms by which bile acids induce apoptosis in hepatocytes and the signaling pathways involved in the control of cell death are not understood fully. Here, we examined the impact of mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase (PI3K) signaling on the survival of primary hepatocytes exposed to bile acids. Treatment of hepatocytes with deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) or ursodeoxycholic acid (UDCA) caused sustained MAPK activation that was dependent on activation of the epidermal growth factor receptor (EGFR). Activation of MAPK was partially blocked by inhibitors of PI3K. Inhibition of DCA-, CDCA-, and UDCA-stimulated MAPK activation resulted in ∼20%, ∼35%, and ∼55% apoptosis, respectively. The potentiation of DCA- and CDCA-induced apoptosis by MEK1/2 inhibitors correlated with cleavage of procaspase 3, which was blocked by inhibitors of caspase 8 (ile-Glu-Thr-Asp-p-nitroanilide [IETD]) and caspase 3 (DEVD). In contrast, the potentiation of UDCA-induced apoptosis weakly correlated with procaspase 3 cleavage, yet this effect was also blocked by IETD and DEVD. Incubation of hepatocytes with the serine protease inhibitor AEBSF reduced the death response of cells treated with UDCA and MEK1/2 inhibitor to that observed for DCA and MEK1/2 inhibitor. The apoptotic response was FAS receptor– and neutral sphingomyelinase–dependent and independent of FAS ligand expression, and neither chelation of intracellular and extracellular Ca2+ nor down-regulation of PKC expression altered the apoptotic effects of bile acids. In conclusion, bile acid apoptosis is dependent on the production of ceramide and is counteracted by activation of the MAPK and PI3K pathways. (HEPATOLOGY 2002;35;779-789.)

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The mechanisms by which bile acids induce apoptosis in hepatocytes and the signaling pathways involved in the control of cell death are not understood fully. Here, we examined the impact of mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase (PI3K) signaling on the survival of primary hepatocytes exposed to bile acids. Treatment of hepatocytes with deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) or ursodeoxycholic acid (UDCA) caused sustained MAPK activation that was dependent on activation of the epidermal growth factor receptor (EGFR). Activation of MAPK was partially blocked by inhibitors of PI3K. Inhibition of DCA-, CDCA-, and UDCA-stimulated MAPK activation resulted in ∼20%, ∼35%, and ∼55% apoptosis, respectively. The potentiation of DCA- and CDCA-induced apoptosis by MEK1/2 inhibitors correlated with cleavage of procaspase 3, which was blocked by inhibitors of caspase 8 (ile-Glu-Thr-Asp-p-nitroanilide [IETD]) and caspase 3 (DEVD). In contrast, the potentiation of UDCA-induced apoptosis weakly correlated with procaspase 3 cleavage, yet this effect was also blocked by IETD and DEVD. Incubation of hepatocytes with the serine protease inhibitor AEBSF reduced the death response of cells treated with UDCA and MEK1/2 inhibitor to that observed for DCA and MEK1/2 inhibitor. The apoptotic response was FAS receptor– and neutral sphingomyelinase–dependent and independent of FAS ligand expression, and neither chelation of intracellular and extracellular Ca2+ nor down-regulation of PKC expression altered the apoptotic effects of bile acids. In conclusion, bile acid apoptosis is dependent on the production of ceramide and is counteracted by activation of the MAPK and PI3K pathways. (HEPATOLOGY 2002;35;779-789.)

Mutant p53 can provoke apoptosis in p53-deficient Hep3B cells with delayed kinetics relative to wild-type p53.

Wild-type (wt) p53 frequently induces apoptosis when expressed in tumor cells whereas mutant p53 acts as an oncoprotein and consequently, stimulates cell proliferation. We report here exceptions to that rule. p53 conformational mutant 175H and DNA contact mutant 273H provoke apoptosis in human p53-deficient Hep3B hepatoma cells with delayed kinetics relative to wt p53. Similarly, c-Myc strongly stimulates apoptosis in these cells. In contrast, viral oncoproteins E1A and E7, and the cellular oncoprotein MDM-2, fail to elicit cytocidal responses. Efficient apoptotic cell death by mutant p53 requires oligomerization as 175H and 273H with deletions between amino acid residues 326 and 347 of the oligomerization domain are nontoxic. Apoptosis by mutant or wt p53 was significantly inhibited by the serine protease inhibitor AEBSF but not by the inactive analog AEBSA. Together, these results suggest that a wt p53-independent control mechanism is operational in Hep3B cells that eliminates cells upon sensing illegitimate proliferation signals originating from certain oncoproteins, including mutant p53 and Myc. We suggest that some tumor cell types lack p53 altogether because they tolerate neither wild-type nor mutant forms of the protein.

Inhibition of Amyloid β-Protein Production in Neural Cells by the Serine Protease Inhibitor AEBSF

Cerebral deposition of amyloid β protein (Aβ) is an early and critical feature of Alzheimer's disease. Aβ production requires the proteolytic release of Aβ from the β-amyloid precursor protein (βAPP). Thus, inhibition of Aβ release is a prime therapeutic goal. Here, we show that the broad spectrum, irreversible serine protease inhibitor, AEBSF, inhibits the constitutive production of Aβ in five different human cell lines, both neural and nonneural. AEBSF also stabilizes full-length βAPP and enhances α-secretion, as shown by an increase in the proteolytic derivative, α-APPs. Further, we demonstrate that the inhibitory effect of AEBSF is specific for Aβ proteins starting at Aspartate 1, suggesting that AEBSF directly inhibits β-secretase, the Methionine–Aspartate (Met–Asp)-cleaving enzyme. These results indicate that specific inhibition of this Aβ-generating protease is possible in living human neural cells and provide information about the characteristics of this as yet unidentified enzyme.