The molecular species responsible for α1 -antitrypsin deficiency are suppressed by a small molecule chaperone.

The formation of ordered Z (Glu342Lys) α1 -antitrypsin polymers in hepatocytes is central to liver disease in α1 -antitrypsin deficiency. In vitro experiments have identified an intermediate conformational state (M*) that precedes polymer formation, but this has yet to be identified in vivo. Moreover, the mechanism of polymer formation and their fate in cells have been incompletely characterised. We have used cell models of disease in conjunction with conformation-selective monoclonal antibodies and a small molecule inhibitor of polymerisation to define the dynamics of polymer formation, accumulation and secretion. Pulse-chase experiments demonstrate that Z α1 -antitrypsin accumulates as short-chain polymers that partition with soluble cellular components and are partially secreted by cells. These precede the formation of larger, insoluble polymers with a longer half-life (10.9 ± 1.7 h and 20.9 ± 7.4 h for soluble and insoluble polymers, respectively). The M* intermediate (or a by-product thereof) was identified in the cells by a conformation-specific monoclonal antibody. This was completely abrogated by treatment with the small molecule, which also blocked the formation of intracellular polymers. These data allow us to conclude that the M* conformation is central to polymerisation of Z α1 -antitrypsin in vivo; preventing its accumulation represents a tractable approach for pharmacological treatment of this condition; polymers are partially secreted; and polymers exist as two distinct populations in cells whose different dynamics have likely consequences for the aetiology of the disease.

Small-molecule peptides inhibit Z alpha1-antitrypsin polymerization.

The Z variant of 1-antitrypsin (AT) polymerizes within the liver and gives rise to liver cirrhosis and the associated plasma deficiency leads to emphysema. In this work, a combinatorial approach based on the inhibitory mechanism of (alpha1)-AT was developed to arrest its pathogenic polymerization. One peptide, Ac-TTAI-NH(2), emerged as the most tight-binding ligand for Z (alpha1)-AT. Characterization of this tetrapeptide by gel electrophoresis and biosensor analysis revealed its markedly improved binding specificity and affinity compared with all previously reported peptide inhibitors. In addition, the peptide is not cytotoxic to lung cell lines. A model of the peptide-protein complex suggests that the peptide interacts with nearby residues by hydrogen bonds, hydrophobic interactions, and cavity-filling stabilization. The combinatorially selected peptide not only effectively blocks the polymerization but also promotes dissociation of the oligomerized (alpha1)-AT. These results are a significant step towards the potential treatment of Z (alpha1)-AT related diseases.

An analog of glibenclamide selectively enhances autophagic degradation of misfolded α1-antitrypsin Z.

The classical form of α1-antitrypsin deficiency (ATD) is characterized by intracellular accumulation of the misfolded variant α1-antitrypsin Z (ATZ) and severe liver disease in some of the affected individuals. In this study, we investigated the possibility of discovering novel therapeutic agents that would reduce ATZ accumulation by interrogating a C. elegans model of ATD with high-content genome-wide RNAi screening and computational systems pharmacology strategies. The RNAi screening was utilized to identify genes that modify the intracellular accumulation of ATZ and a novel computational pipeline was developed to make high confidence predictions on repurposable drugs. This approach identified glibenclamide (GLB), a sulfonylurea drug that has been used broadly in clinical medicine as an oral hypoglycemic agent. Here we show that GLB promotes autophagic degradation of misfolded ATZ in mammalian cell line models of ATD. Furthermore, an analog of GLB reduces hepatic ATZ accumulation and hepatic fibrosis in a mouse model in vivo without affecting blood glucose or insulin levels. These results provide support for a drug discovery strategy using simple organisms as human disease models combined with genetic and computational screening methods. They also show that GLB and/or at least one of its analogs can be immediately tested to arrest the progression of human ATD liver disease.

Sugar and alcohol molecules provide a therapeutic strategy for the serpinopathies that cause dementia and cirrhosis.

Mutations in neuroserpin and alpha1-antitrypsin cause these proteins to form ordered polymers that are retained within the endoplasmic reticulum of neurones and hepatocytes, respectively. The resulting inclusions underlie the dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) and Z alpha1-antitrypsin-associated cirrhosis. Polymers form by a sequential linkage between the reactive centre loop of one molecule and beta-sheet A of another, and strategies that block polymer formation are likely to be successful in treating the associated disease. We show here that glycerol, the sugar alcohol erythritol, the disaccharide trehalose and its breakdown product glucose reduce the rate of polymerization of wild-type neuroserpin and the Ser49Pro mutant that causes dementia. They also attenuate the polymerization of the Z variant of alpha1-antitrypsin. The effect on polymerization was apparent even when these agents had been removed from the buffer. None of these agents had any detectable effect on the structure or inhibitory activity of neuroserpin or alpha1-antitrypsin. These data demonstrate that sugar and alcohol molecules can reduce the polymerization of serpin mutants that cause disease, possibly by binding to and stabilizing beta-sheet A.

Inactivation of α1-proteinase inhibitor by Candida albicans aspartic proteases favors the epithelial and endothelial cell colonization in the presence of neutrophil extracellular traps.

Candida albicans, a causative agent of opportunistic fungal infections in immunocompromised patients, uses ten secreted aspartic proteases (SAPs) to deregulate the homeostasis of the host organism on many levels. One of these deregulation mechanisms involves a SAP-dependent disturbance of the control over proteolytic enzymes of the host by a system of dedicated proteinase inhibitors, with one important example being the neutrophil elastase and alpha1-proteinase inhibitor (A1PI). In this study, we found that soluble SAPs 1-4 and the cell membrane-anchored SAP9 efficiently cleaved A1PI, with the major cleavage points located at the C-terminal part of A1PI in a close vicinity to the reactive-site loop that plays a critical role in the inhibition mechanism. Elastase is released by neutrophils to the environment during fungal infection through two major processes, a degranulation or formation of neutrophil extracellular traps (NET). Both, free and NET-embedded elastase forms, were found to be controlled by A1PI. A local acidosis, resulting from the neutrophil activity at the infection sites, favors A1PI degradation by SAPs. The deregulation of NET-connected elastase affected a NET-dependent damage of epithelial and endothelial cells, resulting in the increased susceptibility of these host cells to candidal colonization. Moreover, the SAP-catalyzed cleavage of A1PI was found to decrease its binding affinity to a proinflammatory cytokine, interleukin-8. The findings presented here suggest a novel strategy used by C. albicans for the colonization of host tissues and overcoming the host defense.

Local control of alpha1-proteinase inhibitor levels: regulation of alpha1-proteinase inhibitor in the human cornea by growth factors and cytokines.

Alpha 1-proteinase inhibitor is a major serine proteinase inhibitor in the human cornea involved in the protection of the avascular corneal tissue against proteolytic damage. This inhibitor is upregulated systemically during infection, inflammation and injury. Cytokines that mediate the acute phase response such as IL-1beta and IL-2 increased alpha1-proteinase inhibitor present in corneal organ culture media. This released inhibitor represented mainly newly synthesized protein. However, IL-6, a general inducer of the acute phase response that upregulates alpha1-proteinase inhibitor in all other tissues and cells tested, failed to alter corneal alpha1-proteinase inhibitor levels over the tested period of 24 h. In addition to IL-1beta and IL-2, alpha1-proteinase inhibitor levels in the corneal organ culture medium increased following the addition of FGF-2 and IGF-I. The effect of the above growth factors and cytokines was relatively fast with maximal induction observed within the first 5 h. Among the tested growth factors and cytokines, IL-1beta was the most potent and increased total corneal alpha1-proteinase inhibitor levels approximately 2.4-fold in the cornea organ culture medium. Newly, synthesized alpha1-proteinase secreted into the medium increased 3.9-fold. In addition to the effect on corneal alpha1-proteinase inhibitor, IL-1beta also increased the amount of alpha1-proteinase inhibitor released by monocytes and macrophages but not by HepG2, CaCo2, and MCF-7 cells within 24 h. These results suggest that the cornea can locally control levels of alpha1-proteinase inhibitor in response to an inflammatory insult.

Thiols and disulphides can aggravate peroxynitrite-dependent inactivation of alpha1-antiproteinase.

Peroxynitrite (ONOO-) is a cytotoxic species formed in vivo. There is considerable interest in the development of ONOO- 'scavengers' as therapeutic agents; several thiols have been suggested to fulfil this role. One protein inactivated by ONOO- is alpha1-antiproteinase (alpha1AP), the major inhibitor of serine proteinases in human body fluids. At low thiol:ONOO- concentration ratios, several thiols (captopril, penicillamine, cysteine, cystine and penicillamine disulphide) aggravated inactivation of alpha1AP by ONOO- , whereas GSH, GSSG, homocysteine, ergothioneine, N-acetylcysteine, lipoate and dihydrolipoate did not. We suggest that sulphur-containing radicals are produced by reaction of certain thiols/disulphides with ONOO- or ONOO- -derived products and could mediate biological damage, including inactivation of alpha1AP. This must be considered in attempts to use thiols as 'peroxynitrite scavengers'.

Oncostatin M, but not interleukin-6 or leukemia inhibitory factor, stimulates expression of alpha1-proteinase inhibitor in A549 human alveolar epithelial cells.

Alpha-1 proteinase inhibitor (A1-Pi) is the main serine proteinase inhibitor found in human plasma and is a potent elastase inhibitor in various tissues, including lung. A1-Pi is expressed and induced in liver during inflammatory responses but can also be produced by epithelial cells. Since hepatocyte A1-Pi production is stimulated by interleukin-6 (IL-6) and other gp130-cytokines, such as leukemia inhibitory factor (LIF) and oncostatin M (OM), we investigated the role of these cytokines in regulating A1-Pi in lung epithelial cells. We show that OM, a monocyte and T cell product, can specifically and potently induce A1-Pi production in lung-derived A549 alveolar (epithelial) cells, as well as in liver-derived HepG2 cells. Both A1-Pi protein (as detected by ELISA and Western blots) and mRNA levels were enhanced 20-fold to 30-fold in A549 cells. OM was also able to stimulate the expression of tissue inhibitor of metalloproteinase-1 in these cells. Interestingly, other members of the IL-6 family (IL-6 and LIF) had little or no effect on A549 cells, and proinflammatory cytokines, such as IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) also had no stimulatory effect on A1-Pi synthesis in A549 cells. Costimulation with IL-1 beta resulted in a decrease in A1-Pi production from OM-stimulated A549 cells. However, IL-6 production was synergistically enhanced. OM was also able to stimulate A1-Pi production from a bronchial epithelial primary cell line, whereas an intestinal epithelial cell line HT29 responded to IL-6 but not OM. These results suggest that lung levels A1-Pi could be derived not only from liver and inflammatory cells but also from epithelial cells, which can be upregulated on stimulation by OM. This may have implications for regulation of local activity of human neutrophil elastase (HNE) in such diseases as emphysema and cystic fibrosis.

Modulation of peroxynitrite- and hypochlorous acid-induced inactivation of alpha1-antiproteinase by mercaptoethylguanidine.

1. Peroxynitrite is a cytotoxic species that can be formed, among other mechanisms, by the rapid reaction of superoxide with nitric oxide. Peroxynitrite formation has been implicated in a wide range of neurodegenerative and chronic inflammatory diseases, as has the formation of hypochlorous acid by myeloperoxidase. 2. There is considerable interest in the development of peroxynitrite scavengers as therapeutic agents. The thiol compound mercaptoethylguanidine has been suggested to fulfil this role since it has recently been shown to be not only a potent inhibitor of inducible nitric oxide synthase but also a scavenger of peroxynitrite. Indeed, it has been shown to be protective in some experimental models of circulatory shock and inflammation at plasma levels in the approximate range 100-300 microM. 3. One protein inactivated by peroxynitrite is the major inhibitor of serine proteinases in human body fluids, alpha1-antiproteinase. At high (250-1000 microM) concentrations, mercaptoethylguanidine was found to be effective in preventing peroxynitrite-mediated tyrosine nitration and alpha1-AP inactivation. 4. By contrast, lower concentrations of mercaptoethylguanidine (1-60 microM) enhanced the inactivation of alpha1-antiproteinase by peroxynitrite. 5. At all concentrations tested (1-1000 microM), mercaptoethylguanidine decreased the inactivation of alpha1-antiproteinase by hypochlorous acid. 6. We suggest that products of reaction of mercaptoethylguanidine with peroxynitrite or peroxynitrite-derived products could cause damage to alpha1-antiproteinase, and possibly other proteins in vivo, whereas scavenging of hypochlorous acid by mercaptoethylguanidine could contribute to its anti-inflammatory action in vivo.

Use of heparin in the treatment of protein-losing enteropathy after fontan operation for complex congenital heart disease.

Protein-losing enteropathy (PLE) is a serious complication of the Fontan operation and is associated with pronounced mortality. Medical management of PLE has been only partially successful. A recent report noted dramatic improvement in patients with PLE within 3 weeks of subcutaneous administration of heparin. We report a case of reversal of PLE with resolution of clinical symptoms and normalization of serum albumin, total protein, and fecal alpha1-antitrypsin values after several months of heparin treatment. Our findings substantiate those recently reported but suggest that reversal of PLE may necessitate more than a few weeks of heparin therapy.

Effect of beta propiolactone viral inactivation on alpha1 antitrypsin values.

AIMS: alpha1 Antitrypsin was undetectable in several patient samples treated with 0.5% beta propiolactone, which was used as a virucidal agent. This study was designed to confirm beta propiolactone as the cause and determine why it might have such an effect. METHODS: Volumes of 0, 5, 10, and 20 micro l of beta propiolactone were added to 2 ml aliquots of serum to make final concentrations of 0%, 0.25%, 0.5%, and 1% of beta propiolactone. alpha1 Antitrypsin concentrations and the pH were measured at different time intervals. The effects of adding buffer before the addition of beta propiolactone, NaOH after beta propiolactone, and 6M HCl instead of beta propiolactone were also measured. RESULTS: The addition of beta propiolactone to a volunteer's serum showed a fall in both alpha1 antitrypsin values and pH with increasing time and concentration of beta propiolactone. This effect was also seen when adding HCl, but was partially prevented by buffering the serum or adding NaOH. CONCLUSIONS: These results suggest that it is the acidity of the degradation products of beta propiolactone that is responsible for the fall in alpha1 antitrypsin values. This fall in alpha1 antitrypsin values was dependent on the concentration of beta propiolactone used and the length of time before the test was performed. The effect of beta propiolactone on laboratory tests should be re-evaluated, with attention being paid to sample pH, storage time, and storage temperature.

Protection against peroxynitrite-dependent tyrosine nitration and alpha 1-antiproteinase inactivation by ascorbic acid. A comparison with other biological antioxidants.

Peroxynitrite, formed by reaction of superoxide and nitric oxide, appears to be an important tissue-damaging species generated at sites of inflammation. In this paper, we compare the abilities of several biological antioxidants to protect against peroxynitrite-dependent inactivation of alpha 1-antiproteinase, and to inhibit tyrosine nitration upon addition of peroxynitrite. GSH and ascorbate protected efficiently in both systems. Uric acid inhibited tyrosine nitration but not alpha 1-antiproteinase inactivation. The possibility that ascorbic acid is an important scavenger of reactive nitrogen species in vivo is discussed.

Lipoic and dihydrolipoic acids as antioxidants. A critical evaluation.

A detailed evaluation of the antioxidant and pro-oxidant properties of lipoic acid (LA) and dihydrolipoic acid (DHLA) was performed. Both compounds are powerful scavengers of hypochlorous acid, able to protect alpha 1-antiproteinase against inactivation by HOCl. LA was a powerful scavenger of hydroxyl radicals (OH.) and could inhibit both iron-dependent OH. generation and peroxidation of ox-brain phospholipid liposomes in the presence of FeCl3-ascorbate, presumably by binding iron ions and rendering them redox-inactive. By contrast, DHLA accelerated iron-dependent OH. generation and lipid peroxidation, probably by reducing Fe3+ to Fe2+. LA inhibited this pro-oxidant action of DHLA. However, DHLA did not accelerate DNA degradation by a ferric bleomycin complex and slightly inhibited peroxidation of arachidonic acid by the myoglobin-H2O2 system. Under certain circumstances, DHLA accelerated the loss of activity of alpha-antiproteinase exposed to ionizing radiation under a N2O/O2 atmosphere and also the loss of creatine kinase activity in human plasma exposed to gas-phase cigarette smoke. Neither LA nor DHLA reacted with superoxide radical (O.2-) or H2O2 at significant rates, but both were good scavengers of trichloromethylperoxyl radical (CCl3O2.). We conclude that LA and DHLA have powerful antioxidant properties. However, DHLA can also exert pro-oxidant properties, both by its iron ion-reducing ability and probably by its ability to generate reactive sulphur-containing radicals that can damage certain proteins, such as alpha 1-antiproteinase and creatine kinase.

Aqueous cigarette tar extracts damage human alpha-1-proteinase inhibitor.

The elastase inhibitory capacity (EIC) of human alpha-1-proteinase inhibitor (alpha 1PI) is severely compromised by aqueous cigarette tar extract (ACTE). An aqueous extract of the tar from two cigarettes causes a loss of EIC of at least 60% in 24 h at 37 degrees C (pH 7.4) and the damaging capability of the ACTE is retained for many hours. Hydrogen peroxide appears to be an essential component of the mechanism by which ACTE damages alpha 1 PI, since catalase substantially protects alpha 1PI from ACTE-mediated damage. Only mild protection is offered by 10 mM diethylenetriamine pentaacetic acid, indicating only a minor role for transition metal ions in the alpha 1PI-damaging process. Hydroxyl radicals are unlikely agents of alpha 1PI damage in the ACTE system, as judged from hydroxyl radical scavenger studies. Ascorbate and various thiols offer protection to different degrees, dependent on the incubation conditions. Of several amino acids tested, cysteine and methionine (but not methionine sulfoxide) are the only two that protect alpha 1PI. We suggest that components of cigarette smoke particulate matter extracted into the aqueous lung fluid environment may cause local deficiencies in alpha 1PI in smokers' lungs.

Changes of immunoreactivity in alpha 1-antitrypsin in patients with autoimmune diseases.

Recent studies from this laboratory have shown that a monoclonal antibody prepared against a specific epitope on alpha 1-antitrypsin is a valuable diagnostic marker for autoimmune conditions. In the present study we have further characterized this monoclonal antibody and reassessed its diagnostic value in screening samples from patients with various autoimmune conditions. alpha 1-Antitrypsin was micropurified from patients with selected autoimmune conditions and from normal donors. The purified alpha 1-antitrypsin isolated from patients with autoimmune conditions and normal donors was deglycosylated using both a mixture of exoglycosidases and endoglycosidase F. The immunoreactivity of the native and deglycosylated alpha 1-antitrypsin was examined using both a monoclonal antibody and a polyclonal antibody in enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), respectively. It was noted that alpha 1-antitrypsin isolated from patients with autoimmune diseases generated a displacement curve dissimilar to alpha 1-antitrypsin purified from normal donors or alpha 1-antitrypsin from patients with autoimmune diseases subjected to deglycosylation when these samples were examined by ELISA using the monoclonal antibody. However, when the polyclonal antibody was used for these studies, no difference was found between the native and deglycosylated alpha 1-antitrypsin suggesting that the monoclonal antibody recognized an epitope not detectable by the polyclonal antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of methanol and its metabolites on the activity of alpha1-antitrypsin.

Among methanol and its metabolites, formaldehyde was found to have the strongest inactivating effect on the activity of alpha1-antitrypsin preparation and inhibitor existing in blood serum. The influence of formaldehyde on the activity of serum alpha1-antitrypsin is lower in comparison with purified inhibitor. alpha1-Antitrypsin modified by formaldehyde inactivates the trypsin in its action on the BAPA to a smaller degree than on the hemoglobin. The effective formaldehyde concentration in the case of the BAPA is about 64 mM and in the case of the hemoglobin is about 256 mM. The significant inhibitory effect of methanol on alpha1-antitrypsin appears only at a high concentration of this compound. Formate does not decrease alpha1-antitrypsin activity. In people intoxicated with methanol, alpha1-antitrypsin activity decreases, whereas the content of this inhibitor does not change.

The effect of methotrexate and azathioprine on the serum levels of IgA-alpha 1-antitrypsin complex in juvenile chronic arthritis.

In the present study we investigated the influence of methotrexate (MTX) and azathioprine (AZA) on the serum levels of the IgA-alpha 1-antitrypsin (IgA-AT) complex in patients with the systemic form of juvenile chronic arthritis (JCA). Fifty-six JCA patients (22 treated with MTX, 18 treated with AZA, and 16 not treated with any immunosuppressive agent) were enrolled in the study. MTX dosage ranged from 0.3 to 0.5 mg/kg-1 week-1, while AZA was given daily at an average dose of 1 mg/kg. MTX was given for 13 months (SD = 7 months) whereas AZA for 11 months (SD = 6 months). The average value of the complex was higher in JCA patients than in both control groups (0.74 +/- 0.73 U vs 0.37 +/- 0.13 U (control children), P < 0.001 and vs 0.23 +/- 0.12 U (control adults), P < 0.001). Values exceeding the normal range were found in twenty-two JCA patients (39.4%). Serum IgA-AT level was lowest in the MTX group compared to AZA and non-treated patients (0.56 +/- 0.24 U, 0.76 = 0.43 U, 0.95 +/- 0.52 U, respectively, P < 0.05). IgA values exceeding normal levels for age were found in 14% of the patients. A correlation between the levels of the IgA-AT complex and C-reactive protein (r = 0.43, P < 0.01), alpha 1-acid-glycoprotein (r = 0.45, P < 0.01), alpha 1-antichymotrypsin (r = 0.52, P < 0.01), alpha 1-antitrypsin (r = 0.40, P < 0.01) and IgA (r = 0.56, P < 0.01) was established.

[The joint action of nitrates and gamma radiation on the blood plasma proteinase-inhibiting and antioxidative systems in rats]. / Sochetannoe vozdeistvie nitratov i gamma-izlucheniia na proteinazoingibitornuiu i antiokislitel'nuiu sistemy plazmy krovi krys.

The separate and combine influence of nitrates (40 mg of natrium nitrate) 100 g of body mass during 60 days) and gamma radiation (3 Gy) on condition of proteolytic and antioxidative blood plasma systems of rats have been investigated. There are were increase of total proteolytic activity (TPA) on 3-day, adaptive increase of alpha-antiproteinase inhibitor (API) on 7- and 14-day after factors separate action and tendency to increase TPA together with the invariable content of API after combine influence. The decrease of alpha-2-macroglobulin level on 14-day after all influences and more significantly after combination of factors action have been also shown. The decrease of antioxidant system and system of free radicals inactivation have been observed.

[Restoration of the protease--alpha 1-protease inhibitor system after exposure of rats to cold]. / Vosstanovlenie sistemy proteinaza--al'fa-1-ingibitor proteinaz posle vozdeistviia kholoda u krys.

The trasilol injection and warming of rats were investigated for their influence on activities of proteinase and alpha-1-proteinase inhibitor (alpha-1PT) after two-hour- and a week-long cold stress. The one-hour return to normal temperature is the most effective method of proteinase-antiproteinase correction after two-hour cold stress. Nevertheless, the return of rats to the normal temperature even for 24 hours after a week cold stress is not enough for the proteinase and alpha-1-PI balancing normalization. The trasilol injection is a more effective method in the last case.