Formation Of N-formylkynurenine Research Articles

Elucidation of molecular interactions between human γD-crystallin and quercetin, an inhibitor against tryptophan oxidation

Different post-translational changes in eye lens crystallin proteins contribute towards the development of cataract. We have studied in vitro oxidative modification of tryptophan (Trp) residues of human γD-crystallin (HGD) towards formation of N-formylkynurenine (NFK) associated with cataractogenesis. This oxidation was found to be inhibited by quercetin at relatively low concentration. Interactions between quercetin and HGD were further studied using fluorescence techniques. Binding and quenching constants were determined as ∼104 M −1. Static quenching of fluorescence due to HGD–quercetin complex formation at ground state was confirmed by finding excited state life time of Trp residues. Energy transfer occurred between the protein and quercetin. Hydrogen bonding and/or van der Waals interactions were involved between HGD and quercetin. Synchronous and three-dimensional fluorescence along with far-UV CD studies suggested no major conformational alterations occurred in HGD due to quercetin binding. Experimental observations were supported by the docking results. Communicated by Ramaswamy H. Sarma

Global Protein Oxidation Profiling Suggests Efficient Mitochondrial Proteome Homeostasis During Aging

The free radical theory of aging is based on the idea that reactive oxygen species (ROS) may lead to the accumulation of age-related protein oxidation. Because themajority of cellular ROS is generated at the respiratory electron transport chain, this study focuses on the mitochondrial proteome of the aging model Podospora anserina as target for ROS-induced damage. To ensure the detection of even low abundant modified peptides, separation by long gradient nLC-ESI-MS/MS and an appropriate statistical workflow for iTRAQ quantification was developed. Artificial protein oxidation was minimized by establishing gel-free sample preparation in the presence of reducing and iron-chelating agents. This first large scale, oxidative modification-centric study for P. anserina allowed the comprehensive quantification of 22 different oxidative amino acid modifications, and notably the quantitative comparison of oxidized and nonoxidized protein species. In total 2341 proteins were quantified. For 746 both protein species (unmodified and oxidatively modified) were detected and the modification sites determined. The data revealed that methionine residues are preferably oxidized. Further prominent identified modifications in decreasing order of occurrence were carbonylation as well as formation of N-formylkynurenine and pyrrolidinone. Interestingly, for the majority of proteins a positive correlation of changes in protein amount and oxidative damage were noticed, and a general decrease in protein amounts at late age. However, it was discovered that few proteins changed in oxidative damage in accordance with former reports. Our data suggest that P. anserina is efficiently capable to counteract ROS-induced protein damage during aging as long as protein de novo synthesis is functioning, ultimately leading to an overall constant relationship between damaged and undamaged protein species. These findings contradict a massive increase in protein oxidation during aging and rather suggest a protein damage homeostasis mechanism even at late age.

The mechanism of substrate inhibition in human indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase catalyzes the O2-dependent oxidation of l-tryptophan (l-Trp) to N-formylkynurenine (NFK) as part of the kynurenine pathway. Inhibition of enzyme activity at high l-Trp concentrations was first noted more than 30 years ago, but the mechanism of inhibition has not been established. Using a combination of kinetic and reduction potential measurements, we present evidence showing that inhibition of enzyme activity in human indoleamine 2,3-dioxygenase (hIDO) and a number of site-directed variants during turnover with l-tryptophan (l-Trp) can be accounted for by the sequential, ordered binding of O2 and l-Trp. Analysis of the data shows that at low concentrations of l-Trp, O2 binds first followed by the binding of l-Trp; at higher concentrations of l-Trp, the order of binding is reversed. In addition, we show that the heme reduction potential (Em0) has a regulatory role in controlling the overall rate of catalysis (and hence the extent of inhibition) because there is a quantifiable correlation between Em0 (that increases in the presence of l-Trp) and the rate constant for O2 binding. This means that the initial formation of ferric superoxide (Fe3+–O2•–) from Fe2+-O2 becomes thermodynamically less favorable as substrate binds, and we propose that it is the slowing down of this oxidation step at higher concentrations of substrate that is the origin of the inhibition. In contrast, we show that regeneration of the ferrous enzyme (and formation of NFK) in the final step of the mechanism, which formally requires reduction of the heme, is facilitated by the higher reduction potential in the substrate-bound enzyme and the two constants (kcat and Em0) are shown also to be correlated. Thus, the overall catalytic activity is balanced between the equal and opposite dependencies of the initial and final steps of the mechanism on the heme reduction potential. This tuning of the reduction potential provides a simple mechanism for regulation of the reactivity, which may be used more widely across this family of enzymes.

Interaction between whey proteins and lipids during light-induced oxidation

Dairy products are a suitable group for polyunsaturated fatty acid (PUFA) fortification, but are very susceptible to light-induced oxidation. In this study, the combined effect of light and PUFA fortification on the stability of milk proteins and on the formation of protein-lipid complexes was studied. Therefore, emulsions were prepared containing whey proteins and lipids with a different degree of unsaturation (olive, soybean, fish and algae oil). PUFA stimulated the protein oxidation process, even after complete degradation of the photosensitizer. First order reaction kinetics were established for riboflavin and tryptophan degradation, as well as the formation of N-formylkynurenine, a tryptophan degradation product. Oxidation was further characterised by degradation of the essential amino acids lysine, histidine, methionine and tyrosine. Protein-lipid adduct formation, measured fluorometrically and by the protein-bound carbonyl groups, could be linked with a decreased extractability of the unsaturated lipids from the emulsion, but also caused the formation of protein aggregates of high molecular mass.

Age-dependent posttranslational modifications of voltage-dependent anion channel 1

The accumulation of oxidative damage in mitochondrial proteins, membranes and DNA during ageing is supposed to lead to mitochondrial inactivation, downstream molecular impairments and subsequent decline of biological systems.In a quantitative study investigating the age-related changes of mitochondrial proteins on the level of oxidative posttranslational modifications, we previously found a set of conserved biomarkers across ageing models in five species with consistent oxidative break-up of tryptophan residues and formation of N-formyl kynurenine. In an additional proteomic profiling of a long-living Drosophila mutant overexpressing mitochondrial Hsp22 and controls, we found age-related redundant isoforms of voltage-dependent anion channel 1 (VDAC-1). A re-examination of data from human umbilical vein endothelial cells (with normal and chemically accelerated in vitro ageing), revealed similar age-dependent alterations of voltage-dependent anion channel isoforms.Building on these results, we examined the expression of VDAC-1 in an in vitro model of excitotoxicity. We show that glutamate-induced calcium toxicity in neurons induces changes of voltage-dependent anion channel 1 related to downstream events of mitochondrial apoptosis like poly-ADP-ribosylation.

Tryptophan Oxidation by Singlet Molecular Oxygen [O2(1Δg)]: Mechanistic Studies Using18O-Labeled Hydroperoxides, Mass Spectrometry, and Light Emission Measurements

Proteins have been considered important targets for reactive oxygen species. Indeed, tryptophan (W) has been shown to be a highly susceptible amino acid to many oxidizing agents, including singlet molecular oxygen [O2(1Deltag)]. In this study, two cis- and trans-tryptophan hydroperoxide (WOOH) isomers were completely characterized by HPLC/mass spectrometry and NMR analyses as the major W-oxidation photoproducts. These photoproducts underwent thermal decay into the corresponding alcohols. Additionally, WOOHs were shown to decompose under heating or basification, leading to the formation of N-formylkynurenine (FMK). Using 18O-labeled hydroperoxides (W18O18OH), it was possible to confirm the formation of two oxygen-labeled FMK molecules derived from W18O18OH decomposition. This result demonstrates that both oxygen atoms in FMK are derived from the hydroperoxide group. In addition, these reactions are chemiluminescent (CL), indicating a dioxetane cleavage pathway. This mechanism was confirmed since the CL spectrum of the WOOH decomposition matched the FMK fluorescence spectrum, unequivocally identifying FMK as the emitting species.

Protein oxidation in plant mitochondria detected as oxidized tryptophan

The formation of N-formylkynurenine by dioxygenation of tryptophan was detected in peptides from rice leaf and potato tuber mitochondria. Proteins in matrix and membrane fractions were separated by two-dimensional gel electrophoresis and identified using a Q-TOF mass spectrometer. N-Formylkynurenine was detected in 29 peptides representing 17 different proteins. With one exception, the oxidation-sensitive aconitase, all of these proteins were either redox active themselves or subunits in redox-active enzyme complexes. The same site was modified in (i) several adjacent spots containing the P protein of the glycine decarboxylase complex, (ii) two different isoforms of the mitochondrial processing peptidase in complex III, and (iii) the same tryptophan residues in Mn–superoxide dismutase in both rice and potato mitochondria. This indicates that Trp oxidation is a selective process.

HCrO 4− photosensitized oxidation of tryptophan exposed to near-UV-visible light

The oxidation of tryptophan photosensitized by HCrO 4 −1 is reported. On irradiation at 365 or 436 nm, the reduction of chromium(VI) to chromium(II) occurs and the formation of N-formylkynurenine is observed as the only photoproduct. Provided that HCrO 4 − is the absorbing species, the overall phenomenon is not affected by the presence or absence of oxygen. The quantum yield of reduction is low and is dependent on pH and tryptophan concentration. A mechanism involving two-electron oxidation of tryptophan is proposed.