Inhibition of cathepsins and related proteases by amino acid, peptide, and protein hydroperoxides

Reaction of radicals in the presence of O2, and singlet oxygen, with some amino acids, peptides, and proteins yields hydroperoxides. These species are key intermediates in chain reactions and protein damage. Previously we have shown that peptide and protein hydroperoxides react rapidly with thiols, and that this can result in inactivation of thiol-dependent enzymes. The major route for the cellular removal of damaged proteins is via catabolism mediated by proteosomal and lysosomal pathways; cysteine proteases (cathepsins) play a key role in the latter system. We hypothesized that inactivation of cysteine proteases by hydroperoxide-containing oxidised proteins may contribute to the accumulation of modified proteins within cells. We show here that thiol-dependent cathepsins, either isolated or in cell lysates, are rapidly and efficiently inactivated by amino acid, peptide, and protein hydroperoxides in a time- and concentration-dependent manner; this occurs with similar efficacy to equimolar H2O2. Inactivation involves reaction of the hydroperoxide with Cys residues as evidenced by thiol loss and formation of sulfenic acid intermediates. Structurally related, non-thiol-dependent cathepsins are less readily inactivated by these hydroperoxides. This inhibition, by oxidized proteins, of the system designed to remove modified proteins, may contribute to the accumulation of damaged proteins in cells subject to oxidative stress. © 2006 Elsevier Inc. All rights reserved

Association between both lipid and protein oxidation and the risk of fatal or non-fatal coronary heart disease in a human population.

The role of oxidative damage in the aetiology of coronary disease remains controversial, as clinical trials investigating the effect of antioxidants have not generally been positive. In the present study, 227 coronary cases, identified from a cohort study, were matched, by age and gender, with 420 controls in a nested case-control design. Stored plasma samples were analysed for F2-isoprostanes by stable isotope dilution MS, and specifically oxidized forms of apoA-I (apolipoprotein A-I) by HPLC of HDL (high-density lipoprotein). Median values of F2-isoprostanes were higher in plasma samples that contained oxidized apoA-I compared with samples with undetectable oxidized apoA-I (1542 compared with 1165 pmol/l). F2-Isoprostanes were significantly correlated with variants of non-oxidized apoA-II (r=-0.15) and were associated with HDL-cholesterol (P<0.0001). F2-Isoprostanes in cases (median, 1146 pmol/l) were not different from controls (1250 pmol/l); the odds ratio (95% confidence interval) for a 1 S.D. increase in F2-isoprostanes was 1.08 (0.91-1.29). Similarly, there was no independent association between the presence of oxidized apoA-I, detected in approx. 20% of the samples, and coronary risk. In conclusion, we found no evidence of associations between markers of lipid (F2-isoprostanes) and protein (oxidized apoA-I) oxidation and the risk of fatal or non-fatal coronary heart disease in a general population. This may be due to a true lack of association or insufficient power

Interpretation of the Temperature-Dependence of the Epr-Spectrum of Cu2+-Doped (nh4)(2)[cd(nh3)(2)(cro4)(2)] and Crystal-Structures of the High-Temperature and Low-Temperature Forms of the Host Lattice

The crystal structure of (NH4)(2)[Cd(NH3)(2)(CrO4)(2)] is reported. Below about 300 K the compound changes from a monoclinic cell (space group C2/m, Z = 2, a = 12.8380(11) Angstrom, b = 6.0308(6) Angstrom, c = 7.5890(6) Angstrom, beta = 110.154(14)degrees) in which all four Cd-O bonds of the trans-Cd(NH3)(2)O-4 coordination sphere are crystallographically equivalent to a triclinic cell (space group P ($) over bar 1, Z = 1, a = 6.0210(4) Angstrom, b = 7.0363(4) Angstrom, c = 7.5714(8) Angstrom, alpha = 106.802(18); beta = 93.032(12); gamma = 114.079(11)degrees) in which the only symmetry element of the Cd complex is an inversion center. It is shown that the previously reported temperature dependence of the EPR spectrum of similar to 0.3% Cu2+ doped into this compound is consistent with the change in crystal structure. The spectra may be explained using a model of dynamic vibronic coupling in which the effects of Jahn-Teller coupling and a ''strain'' due to the inequivalence of the ligands are applied to the e(g) vibrational and E(g) electronic wave functions of the Cu2+ ion. The balance between the ligand field asymmetry and the natural tendency of Cu2+ to adopt a tetragonally elongated octahedral coordination geometry results in a complex with an orthorhombic coordination geometry having short bonds to the ammine groups and intermediate and long bonds to the chromate oxygen atoms. However, the long Cu-O bonds may occur to either pair of trans chromate oxygen atoms. In the high-temperature monoclinic unit cell, the EPR spectrum confirms that these two conformations are energetically equivalent, but in the low-temperature triclinic cell this is no longer the case, and the EPR spectrum is consistent with a temperature-dependent equilibrium between the two possible structural isomers. However, the model suggests that significant delocalization of the vibronic wave functions may occur, so that it is difficult to define precisely the bond lengths and electronic wave function parameters of the guest copper(II) complexes

Singlet molecular oxygen regulates vascular tone and blood pressure in inflammation

Singlet molecular oxygen (O-1(2)) has well-established roles in photosynthetic plants, bacteria and fungi(1-3), but not in mammals. Chemically generated O-1(2) oxidizes the amino acid tryptophan to precursors of a key metabolite called N-formylkynurenine(4), whereas enzymatic oxidation of tryptophan to N-formylkynurenine is catalysed by a family of dioxygenases, including indoleamine 2,3-dioxygenase 1(5). Under inflammatory conditions, this haem-containing enzyme is expressed in arterial endothelial cells, where it contributes to the regulation of blood pressure(6). However, whether indoleamine 2,3-dioxygenase 1 forms O-1(2) and whether this contributes to blood pressure control have remained unknown. Here we show that arterial indoleamine 2,3-dioxygenase 1 regulates blood pressure via formation of O-1(2). We observed that in the presence of hydrogen peroxide, the enzyme generates O-1(2) and that this is associated with the stereoselective oxidation of L-tryptophan to a tricyclic hydroperoxide via a previously unrecognized oxidative activation of the dioxygenase activity. The tryptophan-derived hydroperoxide acts in vivo as a signalling molecule, inducing arterial relaxation and decreasing blood pressure; this activity is dependent on Cys42 of protein kinase G1 alpha. Our findings demonstrate a pathophysiological role for O-1(2) in mammals through formation of an amino acid-derived hydroperoxide that regulates vascular tone and blood pressure under inflammatory conditions