Mechanism of interaction of betanin and indicaxanthin with human myeloperoxidase and hypochlorous acid

Production of nitrogen dioxide by the activity of myeloperoxidase (MPO) in the presence of nitrite is now considered a key step in the pathophysiology of low-density lipoprotein (LDL) oxidation. This study shows that betanin, a phytochemical of the betalain class, inhibits the production of lipid hydroperoxides in human LDL submitted to a MPO/nitrite-induced oxidation. Kinetic measurements including time-course of particle oxidation and betanin consumption, either in the presence or in the absence of nitrite, suggest that the antioxidant effect is possibly the result of various actions. Betanin scavenges the initiator radical nitrogen dioxide and can also act as a lipoperoxyl radical-scavenger. In addition, unidentified oxidation product(s) of betanin by MPO/nitrite inhibit(s) the MPO/nitrite-induced LDL oxidation as effectively as the parent compound. In the light of betanin bioavailability and post-absorbtion distribution in humans, present findings may suggest favourable in vivo activity of this phytochemical

Structure-function analysis of peroxidasin provides insight into the mechanism of collagen IV crosslinking

Basement membranes provide structural support and convey regulatory signals to cells in diverse tissues. Assembly of collagen IV into a sheet-like network is a fundamental mechanism during the formation of basement membranes. Peroxidasin (PXDN) was recently described to catalyze crosslinking of collagen IV through the formation of sulfilimine bonds. Despite the significance of this pathway in tissue genesis, our understanding of PXDN function is far from complete. In this work we demonstrate that collagen IV crosslinking is a physiological function of mammalian PXDN. Moreover, we carried out structure-function analysis of PXDN to gain a better insight into its role in collagen IV synthesis. We identify conserved cysteines in PXDN that mediate the oligomerization of the protein into a trimeric complex. We also demonstrate that oligomerization is not an absolute requirement for enzymatic activity, but optimal collagen IV coupling is only catalyzed by the PXDN trimers. Localization experiments of different PXDN mutants in two different cell models revealed that PXDN oligomers, but not monomers, adhere on the cell surface in "hot spots," which represent previously unknown locations of collagen IV crosslinking. ©2015 Published by Elsevier Inc

Isoniazid as a substrate and inhibitor of myeloperoxidase: Identification of amine adducts and the influence of superoxide dismutase on their formation

Neutrophils ingest Mycobacteria tuberculosis (Mtb) in the lungs of infected individuals. During phagocytosis they use myeloperoxidase (MPO) to catalyze production of hypochlorous acid (HOCl), their most potent antimicrobial agent. Isoniazid (INH),the foremost antibiotic in the treatment oftuberculosis, is oxidized by MPO. It rapidly reduced compound I of MPO [k = (1.22 0.05) 106 M 1 s 1 ] but reacted less favorably with compound II [(9.8 0.6) 102 M 1 s 1 ]. Oxidation of INH by MPO and hydrogen peroxide was unaffected by chloride,the physiological substrate for compound I, and the enzyme was partially converted to compound III. This indicates that INH is oxidized outside the classical peroxidation cycle. In combination with superoxide dismutase (SOD), MPO oxidized INH without exogenous hydrogen peroxide. SOD must favor reduction of oxygen by the INH radical to give superoxide and ultimately hydrogen peroxide. In both oxidation systems, an adduct with methionine was formed and it was a major product with MPO and SOD. We show that it is a conjugate of an acyldiimide with amines. INH substantially inhibited HOCl production by MPO and neutrophils below pharmacological concentrations. The reversible inhibition is explained by diversion of MPO to its ferrous and compound III forms during oxidation of INH. MPO, along with SOD released by Mtb, will oxidize INH at sites of infection and their interactions are likely to limit the efficacy of the drug, promote adverse drug reactions via formation of protein adducts, and impair a major bacterial killing mechanism of neutrophils

The myeloperoxidase-derived oxidant HOSCN inhibits protein tyrosine phosphatases and modulates cell signalling via the mitogen-activated protein kinase (MAPK) pathway in macrophages

MPO (myeloperoxidase) catalyses the oxidation of chloride, bromide and thiocyanate by hydrogen peroxide to HOCl (hypochlorous acid), HOBr (hypobromous acid) and HOSCN (hypothiocyanous acid) respectively. Specificity constants indicate that SCN− is a major substrate for MPO. HOSCN is also a major oxidant generated by other peroxidases including salivary, gastric and eosinophil peroxidases. While HOCl and HOBr are powerful oxidizing agents, HOSCN is a less reactive, but more specific, oxidant which targets thiols and especially low pKa species. In the present study we show that HOSCN targets cysteine residues present in PTPs (protein tyrosine phosphatases) with this resulting in a loss of PTP activity for the isolated enzyme, in cell lysates and intact J774A.1 macrophage-like cells. Inhibition also occurs with MPO-generated HOCl and HOBr, but is more marked with MPO-generated HOSCN, particularly at longer incubation times. This inhibition is reversed by dithiothreitol, particularly at early time points, consistent with the reversible oxidation of the active site cysteine residue to give either a cysteine–SCN adduct or a sulfenic acid. Inhibition of PTP activity is associated with increased phosphorylation of p38a and ERK2 (extracellular-signal-regulated kinase 2) as detected by Western blot analysis and phosphoprotein arrays, and results in altered MAPK (mitogen-activated protein kinase) signalling. These data indicate that the highly selective targeting of some protein thiols by HOSCN can result in perturbation of cellular phosphorylation and altered cell signalling. These changes occur with (patho)physiological concentrations of SCN− ions, and implicate HOSCN as an important mediator of inflammation-induced oxidative damage, particularly in smokers who have elevated plasma levels of SCN−

A study of the structure–activity relationship of GABAA–benzodiazepine receptor bivalent ligands by conformational analysis with low temperature NMR and X-ray analysis

The stable conformations of GABAA-benzodiazepine receptor bivalent ligands were determined by low temperature NMR spectroscopy and confirmed by single crystal X-ray analysis. The stable conformations in solution correlated well with those in the solid state. The linear conformation was important for these dimers to access the binding site and exhibit potent in vitro affinity and was illustrated for α5 subtype selective ligands. Bivalent ligands with an oxygen-containing linker folded back upon themselves both in solution and the solid state. Dimers which are folded do not bind to Bz receptors

Structure of the complex of cytochrome c with cardiolipin in non-polar environment

The complex of mitochondrial protein cytochrome c (CytC) with anionic phospholipid cardiolipin (CL) plays a crucial role in the initiation of apoptosis by catalyzing lipid peroxidation in mitochondrial membranes. In our previous papers, we found that CytC and CL mixed in millimolar concentrations form a sediment showing microcrystals composed of nanospheres (Cyt-CL) of 11–12 and 8 nm in diameter. The hypothesis was proposed that Cyt-CL, having hydrophobic shell, may appear inside the membrane lipid bilayer in mitochondria and peroxidase membrane phospholipids so initiating the apoptotic cascade. In this work, Cyt-CL complex dissolved in chloroform or hexane was investigated as a model of the complex in mitochondrial membranes. We used dynamic light scattering method to measure the size of the particles. The analysis of particles size distribution of Cyt-CL in chloroform allows to reveal three dominant diameters of 12.1 ± 1.4, 7.8 ± 1.0, and 4.7 ± 0.7 nm. The first two values are closed to those, earlier obtained with small-angle X-ray scattering method in Cyt-CL microcrystals, 11.1 ± 1.0 and 8.0 ± 0.7 nm. CL extracted in chloroform-methanol forms a real solution of particles with diameter of 0.7 ± 0.1 nm. In methanol-water phase, CL and CL + CytC mixture form particles of 83.7 ± 9.8 and 71.3 ± 11.6 nm, respectively. Apparently, cardiolipin in 50% methanol forms single-layer liposomes regardless of the presence of CytC in the medium. Partial unfolding of CytC in the complex was evidenced by (a) appearance of fluorescence of tyrosine and tryptophan residues and (b) disappearance of the absorption band at 699 nm due to breakdown of heme iron – methionine bond > F⋯S(Met80). In hydrophobic solvent Cyt-CL exhibited quasi-lipoperoxidase and lipoxygenase activity as was shown in kinetic measurements of chemiluminescence enhanced by coumarin C-525, a selective sensitizer of chemiluminescence, associated with reactions of lipid peroxyl radicals. Our data in this model system do not contradict the hypothesis (Vladimirov, Y.A. et al. Biochemistry (Mosc) 78, 1086–1097) that nanospheres of Cyt-CL complex, embedded into the lipid phase of mitochondrial membrane, catalyze lipid peroxidation, thereby initiating apoptosis

Earthquake Excited Base-Isolated Structures Protected by Tuned Liquid Column Dampers: Design Approach and Experimental Verification

In this contribution a direct approach for optimal design of a Tuned Liquid Column Damper (TLCD) device attached to the base slab of a base-isolated structure is presented, aiming at reducing the seismic displacement demand of the base-isolation subsystem. Assuming white noise base excitation, for a wide parameter range a direct optimization procedure yields design charts for optimal TLCD quantities. The performance of the base-isolated structure equipped with optimally tuned TLCD device in comparison to the simple base-isolated one is evaluated both numerically and experimentally. In a numerical study the system is subjected to the 44 records of the FEMA P-695 far-field ground motion set. The experimental studies are conducted on a three-story small-scale base-isolated shear frame model. From the results it can be concluded that a TLCD effectively controls the seismic response of earthquake excited low-damped base-isolated structures