Role of cardiolipin in mitochondrial diseases and apoptosis.

Apoptosis is a highly programmed cell death strictly connected to the pathogenesis of many human diseases, including neoplastic, neurodegenerative or cardiovascular diseases. Mitochondria play a key role in the apoptotic process; their damage activates a series of events which provoke the release of cytochrome c and other pro-apoptotic factors from the mitochondrial intermembrane space, and culminate in cell death. This review provides an overview of the key role played by mitochondria in the activation of the apoptotic process. In particular, the interest is focused on the role played by cardiolipin, a phospholipid deeply involved in the first steps of the process culminating in cell apoptosis. Mitochondrial phospholipids are involved in several cellular functions, such as cell respiration, apoptosis, and autophagy. Therefore, any alteration in the production of phospholipids or in their structural properties causes deep effects on the cell behavior and induces the arising of different pathologies. The present review summarizes the most recent advances in the study of the role that CL, a phospholipid possessing a unique structure, plays in mitochondrial activity, in apoptosis, and in the onset of human diseases.

Apoptosis and human diseases: mitochondrion damage and lethal role of released cytochrome C as proapoptotic protein.

Apoptosis is strictly connected to the pathogenesis of many human diseases, including neoplastic, neurodegenerative or cardiovascular diseases. It is a highly programmed cell death which can be activated by various factors. Mitochondria play a key role in the apoptotic process; their damage, which involves permeabilization of the outer mitochondrial membrane, activates a series of events that lead to cell death. Of the two proposed signaling pathways of apoptosis, i.e. the 'extrinsic' and the 'intrinsic' pathway, the latter is assumed to initiate in mitochondria. Its activation involves release of cytochrome c and other pro-apoptotic factors from the mitochondrial intermembrane space. In the cytosol, cytochrome c exerts its pro-apoptotic action. It binds to the apoptosis protease activation factor (APAf-1) and forms a complex indicated as 'apoptosome'. The complex-induced activation of pro-caspase 9 initiates an enzymatic reaction cascade leading to the execution of apoptosis in cells. This review provides an overview of the key role played by mitochondria and cytochrome c in the activation of the apoptotic process.

Protein folding, unfolding and misfolding: role played by intermediate States.

Most proteins fold into their native structure through well defined pathways which involve a limited number of transient intermediates. Intermediates play a relevant role in the folding process; many diseases of genetic nature are in fact coupled with protein misfolding due to formation of stable, inactive intermediate species of the protein. This review deals with a number of diseases associated with protein misfolding and briefly describes the mechanism(s) responsible, at molecular level, for such pathologies. It is also considered the (native <--> molten globule) transition, recently observed for some proteins, in which a native protein converts into a stable compact intermediate state able to carry out distinct physiological functions inside the cell. A non-native compact form of cyt c, for example, appears to have a role in the programmed cell death (apoptosis) after that the protein is released from the mitochondrion, and non-native forms of the same protein appear involved in some of the disorders attributed to amyloid formation.

Mast cell tryptase, a still enigmatic enzyme.

Tryptases constitute a subfamily of trypsin-like proteinases, stored in the mast cell secretory granules of all mammalian organisms. These enzymes are released along with other mediators into the extracellular medium upon mast cell activation/degranulation. Among the trypsin-like enzymes, tryptases are unique: they are present as active enzymes in the mast cell granules, but display activity only extracellularly, and have a specificity which is much more restricted than trypsin. Tryptases are mostly tetrameric, and in only few organisms (not in humans) are they inhibited by endogenous inhibitors in vitro. The enzymatic and molecular properties of tryptases are far better characterized that any of their plausible biological functions. On the basis of its structural and functional features it could be predicted that tryptase would not degrade a large number of proteins in vivo due to low accessibility to the tetramer central pore where the active sites face inwards. Although their biological function has not yet been clarified, tryptases seem to be involved in a number of mast cell-mediated allergic and inflammatory diseases. In particular, the involvement of tryptase in asthma, an inflammatory disease of the airways often caused by allergy, has been proposed. Here we review the present knowledge on the structure-function relationship of tryptases from different organisms, with special emphasis on human enzymes, and on their role in a variety of pathophysiological processes.

Cytochrome c reconstituted from two peptide fragments displays native-like redox properties.

Recombination of two fragments of horse cytochrome c (the heme-containing N-fragment, residues 1-56, and the C-fragment, residues 57-104), which are substantially unstructured at neutral pH, gives rise to a 1:1 fragment complex with a compact conformation, in which the alpha helical structure and the native Met80-Fe(III) axial bond are recovered. With respect to the native protein, the ferric complex shows a less rigid atomic packing and a decreased stability [Delta(DeltaG(o))D = 14.7 kJ.mol(-1)], ascribed to perturbations involving the Trp59 microenvironment and, to a lower extent, the heme pocket region. The redox potential, E1/2 = 234 +/- 5 mV vs. normal hydrogen electrode at 25 degrees C, is close to that of the intact protein, consistent with recovery of the native Met80-heme Fe(III) axial bond. Furthermore, the fragment complex shows reactivity similar to intact cytochrome c, in the reaction with cytochrome c oxidase. We conclude that the absence in the complex of some native cross-links and interlocked packing important for protein rigidity and stability is not as relevant for maintaining the native redox properties of the protein, provided that some structural requirements (i.e. recovering of the native-like alpha helical structure) are fulfilled and coordination of Met80 to the heme-iron is restored.

Selective inhibition of human mast cell tryptase by gabexate mesylate, an antiproteinase drug.

Gabexate mesylate is a non-antigenic synthetic inhibitor of trypsin-like serine proteinases that is therapeutically used in the treatment of pancreatitis and disseminated intravascular coagulation and as a regional anticoagulant for hemodialysis. Considering the structural similarity between gabexate mesylate and arginine-based inhibitors of trypsin-like serine proteinases, the effect of gabexate mesylate on human and bovine mast cell tryptase action was investigated. Values of the inhibition constant (K(i)) for gabexate mesylate binding to human and bovine tryptase were 3.4 x 10(-9) M and 1.8 x 10(-7) M (at pH 7.4 and 37.0 degrees ), respectively. Furthermore, gabexate mesylate inhibited the fibrinogenolytic activity of human tryptase. On the basis of the available x-ray crystal structure of human tryptase, the possible binding mode of gabexate mesylate to human and bovine tryptase was analyzed. Human tryptase inhibition by gabexate mesylate may account for the reported prevention of inflammation, erosion, and ulceration of skin and mucosae.

The heme-containing N-fragment (residues 1-56) of cytochrome c is a bis-histidine functional system.

The structural and redox properties of a heme-containing fragment (1-56 residues) of cytochrome c have been investigated by spectroscopic (circular dichroism, electronic absorption, and EPR) and voltammetric techniques. The results indicate that the N-fragment lacks ordered secondary structure and has two histidines axially bound to the heme-iron (the native His18 and a misligated His26 or His33). Despite the absence of ordered secondary structure, the peptide chain shields the heme group from solvent, as shown by (i) the pK(a) of protonation of the nonnative histidine ligand (5.18 +/- 0.05), lower than that of the bis-histidine guanidine-unfolded cytochrome c (5.58 +/- 0.05), and (ii) the redox potential, E(o) = 0 +/- 5 mV versus NHE, close to that of bis-histidine cytochrome c mutants but less negative than that of bis-histidine complexes of microperoxidase with short peptides. The electroactive N-fragment may be taken as a "minichrome c" model, with interesting potential for application to biosensor technology; further, the system provides useful information for a deeper understanding of cytochrome c folding and structural/functional organization.

Human mast cells take up and hydrolyze anandamide under the control of 5-lipoxygenase and do not express cannabinoid receptors.

Human mast cells (HMC-1) take up anandamide (arachidonoyl-ethanolamide, AEA) with a saturable process (K(m)=200+/-20 nM, V(max)=25+/-3 pmol min(-1) mg protein(-1)), enhanced two-fold over control by nitric oxide-donors. Internalized AEA was hydrolyzed by a fatty acid amide hydrolase (FAAH), whose activity became measurable only in the presence of 5-lipoxygenase, but not cyclooxygenase, inhibitors. FAAH (K(m)=5.0+/-0.5 microM, V(max)=160+/-15 pmol min(-1) mg protein(-1)) was competitively inhibited by palmitoylethanolamide. HMC-1 cells did not display a functional cannabinoid receptor on their surface and neither AEA nor palmitoylethanolamide affected tryptase release from these cells.

Kinetic and thermodynamic analysis of leech-derived tryptase inhibitor interaction with bovine tryptase and bovine trypsin.

The interaction of leech-derived tryptase inhibitor (LDTI) with bovine liver capsule tryptase (BLCT) and bovine trypsin has been studied using both thermodynamic and kinetic approaches. Several differences were detected: (i) the equilibrium affinity of LDTI for BLCT (Ka = 8.9 x 10(5) M(-1)) is about 600-fold lower than that for bovine trypsin (Ka = 5.1 x 10(8) M(-1)); (ii) LDTI behaves as a purely non-competitive inhibitor of BLCT, while it is a purely competitive inhibitor of bovine trypsin. These functional data are compared with those previously reported for the LDTI binding to human tryptase, where tight inhibition occurs at two of the four active sites of the tetramer (Ka = 7.1 x 10(8) M(-1)). Amino acid sequence alignment of BLCT, human betaII-tryptase and bovine trypsin allows us to infer some possible structural basis for the observed functional differences.

Structural and functional properties of Bos taurus tryptase: a search for a possible propeptide processing role.

Some structural features of bovine tryptase were discussed based on spectroscopic analysis. The far UV-CD spectrum of the enzymatically active bovine tryptase is consistent with a structure containing very little, if any alpha-helix, as found for other serine proteases. The analysis of near UV-CD and UV absorption spectra reveals the presence of a high number of Trp residues arranged probably in strong structural motifs. At variance with other tryptases, the bovine enzyme shows an electrophoretic behaviour in native and denaturating conditions compatible with an association state larger than a tetramer (probably a dodecamer). From a biochemical point of view, the bovine tryptase shares with the human counterpart, the preference for cleaving substrates bearing dibasic cleavage sites. Thus, it is hypothesized that tryptase may be involved in some proprotein processing mechanism(s).

Bovine mast cell tryptase inactivation: effect of temperature.

The thermal stability of bovine tryptase, a serine proteinase present in the bovine mast cell secretory granules, has been studied by circular dichroism and catalytic activity measurements. Bovine tryptase shows a peculiar dichroic negative band centered at 230 nm. The decrease of this band in a temperature dependent fashion represents a good marker to monitor the native conformation of the enzyme. Bovine tryptase inactivation has been followed in the temperature range between 10 degrees C and 80 degrees C, and reversibility of the process has been also studied. The results obtained show that the temperature dependent loss of activity and the conformational change, as monitored by circular dichroism, are both fully reversible between 10 degrees C and 40 degrees C, while only the CD change displays reversibility in going from 60 degrees C to 10 degrees C. Moreover, a functional analysis of the temperature-dependent enzymatic activity of bovine tryptase toward peptide substrates in the 10 degrees C - 40 degrees C range is reported and compared with the temperature dependence of the enzymatic activity of trypsin.

Histone-tryptase interaction: H2A N-terminal tail removal and inhibitory activity.

The involvement of tryptase, the trypsin-like serine proteinase of mast cell granules, in many (patho)physiological conditions is now recognized. In vitro this enzyme is known to act as a potent growth factor for fibroblasts and epithelial cells. Moreover, a role in inflammatory diseases and in dermatological disorders characterized by increased cell turnover has been suggested for this protease. In an attempt to understand the molecular basis of tryptase activity, we have investigated the interaction in vitro between bovine tryptase and histones. Here we show that tryptase cleaves histone H2A at a specific site (Arg20-Ala21), resulting in the removal of the N-terminal flexible fragment of the molecule. Furthermore, we demonstrate that the H2A major fragment (H2A*, 109 residues) generated by hydrolysis and lacking the N-terminal domain, is a noncompetitive, reversible and highly specific inhibitor (Ki = 29 nM) of tryptase enzymatic activity. H2A* is able to inhibit the hydrolysis of a small substrate as well as the cleavage of fibronectin, a high-molecular-weight substrate of tryptase.

pH dependence of bovine mast cell tryptase catalytic activity and of its inhibition by 4',6-diamidino-2-phenylindole.

Tryptases are oligomeric enzymes localized in the secretory granules of mast cells. Their role(s) in vivo has yet to be clarified and the lack of powerful and specific inhibitors has hampered the comprehension of the biological functions of these enzymes. In this paper, we identify 4',6-diamidino-2-phenylindole as a potent inhibitor for bovine tryptase. This inhibitory effect and the enzyme catalyzed hydrolysis of the synthetic substrate Boc-Phe-Ser-Arg-methyl-coumarin were investigated in the pH range of 6.0-9.0. On the basis of the pK shifts occurring upon formation of the inhibitor(substrate)/enzyme complexes, some aminoacidic groups are proposed to play a role in such interactions.

cDNA cloning and primary structure of tryptase from bovine mast cells, and evidence for the expression of bovine pancreatic trypsin inhibitor mRNA in the same cells.

A partial cDNA encoding bovine tryptase, an oligomeric serine proteinase previously isolated from bovine mast cells, was obtained by reverse transcription/polymerase chain reaction of mast cell mRNA, using combinations of primers designed on the basis of information obtained from partial sequencing of the purified protein. The complete amino acid sequence of bovine tryptase (245 residues) was deduced from a 711-bp nucleotide sequence and from Edman degradation of the protein. Bovine tryptase primary structure has an identity of about 75% with tryptases from other species and includes all the essential residues of the active-site regions; sequence data in the region of the putative substrate binding pocket suggest a rearrangement capable of maintaining the specificity of trypsin-like proteinases. From the same mast cell mRNA, cDNA encoding bovine trypsin protease inhibitor (BPTI) was obtained and amplified with specific primers, confirming the synthesis of BPTI in these cells. Results are consistent with previous data on the presence of BPTI and bovine tryptase in the same granules of bovine mast cells and with their interaction in vitro.

Localization and interaction of bovine pancreatic trypsin inhibitor and tryptase in the granules of bovine mast cells.

The interaction of bovine pancreatic trypsin inhibitor and bovine tryptase, isolated from liver capsule mast cells, was investigated. They form a complex in vitro with a Ki of 5.6 nM at pH 8.0 and are localized within the mast cell granules, as shown by immunogold staining at the electron microscope level. In addition, double immunogold electron microscopy revealed that the inhibitor and the enzyme are present in the same granules, where they occur in clusters; this may be taken as an indication of their interaction in vivo and suggests a physiological role for bovine pancreatic trypsin inhibitor in the regulation of tryptase proteolytic activity.

Evidence for multiple interacting binding sites in bovine tryptase.

The interaction of bovine pancreatic trypsin inhibitor (BPTI) and bovine tryptase, which are co-localized in the same granules of bovine mast cells, has been analyzed at 30 degrees C in 0.1 M Tris-HCl, pH 8.0. The analysis has unravelled that the functional unit of bovine tryptase is formed of (at least) four binding sites for this inhibitor. These interaction sites display a simple binding behaviour for small inhibitors (and substrates), whereas heterogeneous properties have been observed in the binding of BPTI. Furthermore, in the presence of BPTI, a positive functional interaction can be detected among the binding sites also for a small synthetic inhibitor, like benzamidine. Such features indicate the existence of a complex functional interplay among the sites of the functional unit which is transmitted through the secondary specificity sites.

Bovine tryptase: purification and characterization.

Bovine tryptase, a mast cell trypsin-like protease, was isolated from liver capsula and from mast cells obtained from the same tissue. The purification procedure which leads to an increase in tryptase activity of 850 fold, involves high salt extraction, hydrophobic interaction chromatography on octyl-Sepharose and affinity chromatography on heparin-Sepharose. The enzyme is oligomeric, with an apparent M(r) of 360,000 +/- 40,000 (as obtained by gel filtration in high salt). The constituent subunits with M(r) 39,000 and 41,000 Da are both labeled with [3H] diisopropyl fluorophosphate and cross-react with anti-rat tryptase immunoglobulins. Only a single N-terminal sequence was found, identical to that of human, dog and rat tryptases. Tripeptide fluorogenic substrates with basic residues in P1 and P2 positions are preferentially hydrolyzed by this enzyme, suggesting a possible processing role as proposed for other tryptases. Bovine tryptase activity is inhibited by NaCl and is insensitive to high molecular weight inhibitors, such as alpha 1 antitrypsin and soybean trypsin inhibitor, as for human and dog tryptases. However it is inhibited by low molecular weight serine protease inhibitors and, similarly to rat tryptase, by the bovine pancreatic trypsin inhibitor (BPTI or aprotinin), in a pH dependent fashion.

Monoclonal antibodies against protease inhibitors: the case of the bovine pancreatic trypsin inhibitor.

A monoclonal antibody (MoAb) directed against bovine basic pancreatic trypsin inhibitor (BPTI or aprotinin), a small protein made up by 58 aminoacids, has been produced. Mice were immunized with the native form of the protein and gave low antibody response. After somatic hybridization of spleen cells from immunized mice, few clones secreting antibodies against BPTI have been found and just two of them were stable. Both secreted IgM. One (ICI) produces a monoclonal antibody which binds to BPTI with an equilibrium dissociation constant, Kd, of 6.1 x 10(-7) M at pH 7.4. Competition experiments demonstrated that ICI recognizes an epitope close to the reactive site of BPTI. Furthermore, Kd of ICI with an isoinhibitor similar to BPTI (S.I. II) but with few differences at the active site is higher, confirming specificity of binding.

Identification and immunohistochemical localization of various bovine pancreatic trypsin inhibitor-isoforms in bovine pituitary gland.

Three isoinhibitors of bovine pancreatic trypsin inhibitor (BPTI) have been identified and isolated from bovine pituitary gland. The results of the purification process by affinity chromatography on immobilized trypsin, the electrophoretic mobility in non-denaturing conditions, the antiproteolytic activity and the immunochemical reactions indicate that these inhibitors correspond to those previously isolated from bovine spleen and lung. In addition, immunohistochemical experiments show that the isoinhibitors and BPTI are exclusively localized in the mast cells, and not in the endocrine cells, of the pars intermedia and posterior lobe (neurohypophysis) of the pituitary gland. The physiological implications of these findings are discussed.

Production and characterization of monoclonal antibodies against bovine pancreatic trypsin inhibitor.

Two monoclonal antibodies (MAbs) have been produced, without the use of a supporting carrier, against bovine basic pancreatic trypsin inhibitor (BPTI or aprotinin), a mini-protein composed of 58 amino acids. Both MAbs obtained were found to be IgM. One of them was purified and further characterized. This MAb (ICI) binds to the immunogen with an association constant of 1.6 X 10(6)M-1 at pH 7.4. Competition experiments with trypsin or inactivated trypsin demonstrate that ICI MAb interacts with BPTI at, or near, the proteinase-binding site. ICI MAb binds, with a much lower association constant (approximately 200M-1), to an isoinhibitor (spleen inhibitor II) which differs from BPTI in seven amino-acids; three of these substitutions are at the active site, in the contact area with the proteinase.