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In the cell cytochrome, c performs different functions depending on the environment in which it acts; therefore, it has been classified as a multifunction protein. When anchored to the outer side of the inner mitochondrial membrane, native cytochrome c acts as a Schweitzer-StennerSchweitzer-Stenner that transfers electrons from cytochrome c reductase to cytochrome c oxidase in the respiratory chain. On the other hand, to interact with cardiolipin (one of the phospholipids making up the mitochondrial membrane) and form the cytochrome c/cardiolipin complex in the apoptotic process, the protein reorganizes its structure into a non-native state characterized by different asymmetry. The formation of the cytochrome c/cardiolipin complex is a fundamental step of the apoptotic pathway, since the structural rearrangement induces peroxidase activity in cytochrome c, the subsequent permeabilization of the membrane, and the release of the free protein into the cytoplasm, where cytochrome c activates the apoptotic process. Apoptosis is closely related to the pathogenesis of neoplastic, neurodegenerative and cardiovascular diseases; in this contest, the biosynthesis and remodeling of cardiolipin are crucial for the regulation of the apoptotic process. Since the role of cytochrome c as a promoter of apoptosis strictly depends on the non-native conformation(s) that the protein acquires when bound to the cardiolipin and such event leads to cytochrome c traslocation into the cytosol, the structural and functional properties of the cytochrome c/cardiolipin complex in cell fate will be the focus of the present review.

The binding of lipids (free fatty acids as well as acidic phospholipids) to cytochrome c (cyt c) induces conformational changes and partial unfolding of the protein, strongly influencing cyt c oxidase/peroxidase activity. ATP is unique among the nucleotides in being able to turn non-native states of cyt c back to the native conformation. The peroxidase activity acquired by lipid-bound cyt c turns out to be very critical in the early stages of apoptosis. Nucleotide specificity is observed for apoptosome formation and caspase activation, the cleavage occurring only in the presence of dATP or ATP. In this study, we demonstrate the connection between peroxidase activity and oleic acid-induced conformational transitions of cyt c and show how ATP is capable of modulating such interplay. By NMR measurement, we have demonstrated that ATP interacts with a site (S 1) formed by K88, R91, and E62 and such interaction was weakened by mutation of E62, suggesting the selective role in the interaction played by the base moiety. Interestingly, the interactions of ATP and GTP with cyt c are significantly different at low nucleotide concentrations, with GTP being less effective in perturbing the SI site and in eliciting apoptotic activity. To gain insights into the structural features of cyt c required for its pro-apoptotic activity and to demonstrate a regulatory role for ATP (compared to the effect of GTP), we have performed experiments on cell lysates by using cyt c proteins mutated on amino acid residues that, as suggested by NMR measurements, belong to S1. Thus, we provide evidence that ATP acts as an allosteric effector, regulating structural transitions among different conformations and different oxidation states of cyt c, which are endowed with apoptotic activity or not. On this basis, we suggest a previously unrecognized role for ATP binding to cyt c at low millimolar concentrations in the cytosol, beyond the known regulatory role during the oxidative phosphorylation in mitochondria.

Although the tertiary structures of mitochondrial cytochromes c (cyts c) seem to be remarkably similar, there are variations in their amino acid sequences, stability and functional properties. GdnHCl-induced unfolding experiments on engineered yeast and horse cyt c were carried out with the aim to to clarify, at molecular level, some aspects concerning the stability of this class of proteins. The results obtained are discussed in the light of the three-dimensional structures of the two proteins.

An increasing body of evidence ascribes to misfolded forms of cytochrome c (cyt c) a role in pathophysiological events such as apoptosis and disease. Here, we examine the conformational changes induced by lipid binding to horse heart cyt c at pH 7 and study the ability of ATP (and other nucleotides) to refold several forms of unfolded cyt c such as oleic acid-bound cyt c, nicked cyt c, and acid denatured cyt c. The CD and fluorescence spectra demonstrate that cyt c unfolded by oleic acid has an intact secondary structure, and a disrupted tertiary structure and heme environment. Furthermore, evidence from the Soret CD, electronic absorption, and resonance Raman spectra indicates the presence of an equilibrium of at least two low-spin species having distinct heme-iron(III) coordination. As a whole, the data indicate that binding of cyt c to oleic acid leads to a partially unfolded conformation of the protein, resembling that typical of the molten globule state. Interestingly, the native conformation is almost fully recovered in the presence of ATP or dATP, while other nucleotides, such as GTP, are ineffective. Molecular modeling of ATP binding to cyt c and mutagenesis experiments show the interactions of phosphate groups with Lys88 and Arg91, with adenosine ring interaction with Glu62 explaining the unfavorable binding of GTP: The finding that ATP and dATP are unique among the nucleotides in being able to turn non-native states of cyt c back to native conformation is discussed in the light of cyt c involvement in cell apoptosis.

The present study evidenced the critical levels of six major polyhalogenated hydrocarbons (PHH's), namely chloroform, carbon tetrachloride, 1,1,1-trichloroethane, 1,2-dibromoethane, perchloro-ethylene, hexachlorobutadiene, over which significant inhibitory effects of the mitochondrial respiratory chain take place in vitro. At these critical levels, even in PB-induced animals only a very little fraction of cytochrome P-450 is saturated by the compounds and therefore the microsomal metabolism plays no effective role either in decreasing the levels of the test chemicals under the threshold of clear direct adverse effects in mitochondria, nor to the formation of toxic metabolites. Our data show also that phenobarbital not only enhances both the direct and metabolism-mediated interaction of most tested PHH with microsomal cytochrome P-450, but also increases the affinity of hexachloro butadiene, chloroform and carbon tetrachloride for the mitochondrial sites resulting in respiration inhibition.

Previous studies have shown that bovine spleen contains, besides aprotinin (bovine pancreatic trypsin inhibitor, BPTI), three iso-inhibitors (I-III), which are structurally and functionally very similar to BPTI. The primary structure of inhibitors I and II indicates that they are not post-translational products of BPTI, in agreement with independent findings on two homologous genes reported to be present in the bovine genome, which encode BPTI and inhibitors I and II, respectively. A careful reinvestigation on the occurrence of these inhibitors in various bovine organs is presented in this paper, based on their isolation with standard biochemical techniques and on functional and structural studies. All data indicate that the same isoinhibitors found in spleen are also present, besides BPTI, in organs such as lung, liver and pituitary gland, where they were never found before. BPTI and inhibitor II always prevail.

Cytochrome c, a protein that belongs to class 1 of the c-type cytochrome family, exerts different functions depending on its cellular localization and the conditions in which it operates; therefore, it can be defined as 'extreme multifunctional' protein. It mediates electron-transfer in the respiratory chain and acts as a detoxifying agent to dispose of ROS. In addition, cytochrome c plays a role in cell apoptosis. After its release into the cytosol, the protein binds to APAF-1, activates pro-caspase 9, and triggers an enzymatic cascade leading to cell death. The interaction with cardiolipin, one of the phospholipids making up the mitochondrial membrane, is essential to start apoptosis; the binding partially unfolds cytochrome c, alters the heme pocket region, and facilitates detachment of Met80 from the sixth coordination position of the heme iron. These events change the function of cytochrome c from an electron-transfer shuttle to a peroxidase-like hemoprotein, capable to trigger the process that leads to cell death. This review provides an overview of the key role played by the cytochrome c-cardiolipin interaction in apoptosis. This is not only important per se, it provides interesting perspectives for applications in clinical diagnostics that use the protein as a biomarker.

A further function of cytochrome c (cyt c), beyond respiration, is realized outside mitochondria in the apoptotic program. In the early events of apoptosis, the interaction of cyt c with a mitochondrion-specific phospholipid, cardiolipin (CL), brings about a conformational transition of the protein and acquirement of peroxidase activity. The hallmark of cyt c with peroxidase activity is its partial unfolding accompanied by loosening of the Fe sixth axial bond and an enhanced access of the heme catalytic site to small molecules like H2O2. To investigate the peroxidase activity of non-native cyt c, different forms of the protein were analyzed with the aim to correlate their structural features with the acquired enzymatic activity and apoptogenic properties (wt cyt c/CL complex and two single cyt c variants. H26Y and Y67H, free and bound to CL). The results suggest that cyt c may respond to different environments by changing its fold thus favouring the exertion of different biological functions in different pathophysiological cell conditions. Transitions among different conformations are regulated by endogenous molecules such as ATP and may be affected by synthetic molecules such as minocycline, thus suggesting a mechanism explaining its use as therapeutic agent impacting on disease-associated oxidative and apoptotic mechanisms. (C) 2012 Elsevier Inc. All rights reserved.

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.

Cytochrome c undergoes structural variations during the apoptotic process; such changes have been related to modifications occurring in the protein when it forms a complex with cardiolipin, one of the phospholipids constituting the mitochondrial membrane. Although several studies have been performed to identify the site(s) of the protein involved in the cytochrome c-cardiolipin interaction, to date the location of this hosting region(s) remains unidentified and is a matter of debate. To gain deeper insight into the reaction mechanism, we investigate the role that the Lys72, Lys73, and Lys79 residues play in the cytochrome c-cardiolipin interaction, as these side chains appear to be critical for cytochrome c-cardiolipin recognition. The Lys72Asn, Lys73Asn, Lys79Asn, Lys72/73Asn, and Lys72/73/79Asn mutants of horse heart cytochrome c were produced and characterized by circular dichroism, ultraviolet-visible, and resonance Raman spectroscopies, and the effects of the mutations on the interaction of the variants with cardiolipin have been investigated. The mutants are characterized by a subpopulation with non-native axial coordination and are less stable than the wild-type protein. Furthermore, the mutants lacking Lys72 and/or Lys79 do not bind cardiolipin, and those lacking Lys73, although they form a complex with the phospholipid, do not show any peroxidase activity. These observations indicate that the Lys72, Lys73, and Lys79 residues stabilize the native axial Met80-Fe(III) coordination as well as the tertiary structure of cytochrome c. Moreover, while Lys72 and Lys79 are critical for cytochrome c-cardiolipin recognition, the simultaneous presence of Lys72, Lys73, and Lys79 is necessary for the peroxidase activity of cardiolipin-bound cytochrome c.

""Acetylpolyamine and spermine oxidases are involved in the catabolism of polyamines. The discovery of selective. inhibitors of these enzymes represents an important tool for the development of novel anti-neoplastic drugs. Here, a. comparative study on acetylpolyamine and spermine oxidases inhibition by the polyamine analogue chlorhexidine. is reported. Chlorhexidine is an antiseptic diamide, commonly used as a bactericidal and bacteriostatic agent.. Docking simulations indicate that chlorhexidine binding to these enzymes is compatible with the stereochemical. properties of both acetylpolyamine oxidase and spermine oxidase active sites. In fact, chlorhexidine is predicted. to establish several polar and hydrophobic interactions with the active site residues of both enzymes, with binding. energy values ranging from −7.6 to −10.6 kcal\\\/mol. In agreement with this hypothesis, inhibition studies indicate that. chlorhexidine behaves as a strong competitive inhibitor of both enzymes, values of Ki being 0.10 μM and 0.55 μM for. acetylpolyamine oxidase and spermine oxidase, respectively.""

A complementary DNA encoding a new bovine tryptase isoform (here named BLT) was cloned and sequenced from lung tissue. Analysis of sequence indicates the presence of a 26-amino acid prepro-sequence and a 245 amino acid catalytic domain. It contains six different residues when compared with the previously characterized tryptase from bovine liver capsule (BLCT), with the most significant difference residing at the primary specificity S1 pocket. In BLT, the canonical residues Asp-Ser are present at positions 188-189, while in BLCT these positions are occupied by residues Asn-Phe. This finding was confirmed by mass fingerprinting of the peptide mixture obtained upon in-gel tryptic digestion of BLT. Analysis by gel filtration of the purified protein shows that BLT is probably tetrameric, similar to the previously identified tryptases from other species, with monomer migrating as 35-40 kDa multiple bands in SDS/PAGE. As expected, the catalytic abilities of the two bovine tryptases are different. The specificity constant values (k c a t /K m ) assayed with model substrates are 10- to 60-fold higher in the case of BLT. The tissue-specific expression of the two tryptases was evaluated at the RNA level by analysis of their different restriction patterns. In lung, only BLT was found to be expressed, while in liver capsule only BLCT is present. Both isoforms are distributed in similar amounts in heart and spleen. Analysis of the two gene sequences reveals the presence of several recognition sequences in the promoter regions and suggest a role for hormones in governing the mechanism of tissue expression of bovine tryptases.

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 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.