Elevated levels of iodide promote peroxidase-mediated protein iodination and inhibit protein chlorination.

At inflammatory sites, immune cells generate oxidants including H2O2. Myeloperoxidase (MPO), released by activated leukocytes employs H2O2 and halide/pseudohalides to form hypohalous acids that mediate pathogen killing. Hypochlorous acid (HOCl) is a major species formed. Excessive or misplaced HOCl formation damages host tissues with this linked to multiple inflammatory diseases. Previously (Redox Biology, 2020, 28, 101331) we reported that iodide (I⁻) modulates MPO-mediated protein damage by decreasing HOCl generation with concomitant hypoiodous acid (HOI) formation. HOI may however impact on protein structure, so in this study we examined whether and how HOI, from peroxidase/H2O2/I⁻ systems ± Cl⁻, modifies proteins. Experiments employed MPO and lactoperoxidase (LPO) and multiple proteins (serum albumins, anastellin), with both chemical (intact protein and peptide mass mapping, LC-MS) and structural (SDS-PAGE) changes assessed. LC-MS analyses revealed dose-dependent iodination of anastellin and albumins by LPO/H2O2 with increasing I⁻. Incubation of BSA with MPO/H2O2/Cl⁻ revealed modest chlorination (Tyr286, Tyr475, ∼4 %) and Met modification. Lower levels of these species, and extensive iodination at specific Tyr and His residues (>20 % modification with ≥10 µM I⁻) were detected with increasing I⁻. Anastellin dimerization was inhibited by increasing I⁻, but less marked changes were observed with albumins. These data confirm that I⁻ competes with Cl⁻ for MPO and is an efficient HOCl scavenger. These processes decrease protein chlorination and oxidation, but result in extensive iodination. This is consistent with published data on the presence of iodinated Tyr on neutrophil proteins. The biological implications of protein iodination relative to chlorination require further clarification.

The structure of model and peptide disulfides markedly affects their reactivity and products formed with singlet oxygen.

Disulfide bonds are critical structural elements in proteins and stabilize folded structures. Modification of these linkages is associated with a loss of structure and function. Previous studies have reported large variations in the rate of disulfide oxidation by hypohalous acids, due to stabilization of reaction intermediates. In this study we hypothesized that considerable variation (and hence selective oxidation) would occur with singlet oxygen (1O2), a key intermediate in photo-oxidation reactions. The kinetics of disulfide-mediated 1O2 removal were monitored using the time-resolved 1270 nm phosphorescence of 1O2. Stern-Volmer plots of these data showed a large variation (∼103) in the quenching rate constants kq (from 2 × 107 for α-lipoic acid to 3.6 × 104 M-1s-1 for cystamine). The time course of disulfide loss and product formation (determined by LC-MS) support a role for 1O2, with mono- and di-oxygenated products detected. Elevated levels of these latter species were generated in D2O- compared to H2O buffers, which is consistent with solvent effects on the 1O2 lifetime. These data are interpreted in terms of the intermediacy of a zwitterion [-S+(OO-)-S-], which either isomerizes to a thiosulfonate [-S(O)2-S-] or reacts with another parent molecule to give two thiosulfinates [-S(O)-S-]. The variation in quenching rates and product formation are ascribed to zwitterion stabilization by neighboring, or remote, lone pairs of electrons. These data suggest that some disulfides, including some present within or attached to proteins (e.g., α-lipoic acid), may be selectively modified, and undergo subsequent cleavage, with adverse effects on protein structure and function.

Identification of bile acid-CoA:amino acid N-acyltransferase as the hepatic N-acyl taurine synthase for polyunsaturated fatty acids.

N-acyl taurines (NATs) are bioactive lipids with emerging roles in glucose homeostasis and lipid metabolism. The acyl chains of hepatic and biliary NATs are enriched in polyunsaturated fatty acids (PUFAs). Dietary supplementation with a class of PUFAs, the omega-3 fatty acids, increases their cognate NATs in mice and humans. However, the synthesis pathway of the PUFA-containing NATs remains undiscovered. Here, we report that human livers synthesize NATs and that the acyl-chain preference is similar in murine liver homogenates. In the mouse, we found that hepatic NAT synthase activity localizes to the peroxisome and depends upon an active-site cysteine. Using unbiased metabolomics and proteomics, we identified bile acid-CoA:amino acid N-acyltransferase (BAAT) as the likely hepatic NAT synthase in vitro. Subsequently, we confirmed that BAAT knockout livers lack up to 90% of NAT synthase activity and that biliary PUFA-containing NATs are significantly reduced compared with wildtype. In conclusion, we identified the in vivo PUFA-NAT synthase in the murine liver and expanded the known substrates of the bile acid-conjugating enzyme, BAAT, beyond classic bile acids to the synthesis of a novel class of bioactive lipids.

Identification of galectin-1 and other cellular targets of alpha,beta-unsaturated carbonyl compounds, including dimethylfumarate, by use of click-chemistry probes.

α,ß-Unsaturated carbonyls are a common motif in environmental toxins (e.g. acrolein) as well as therapeutic drugs, including dimethylfumarate (DMFU) and monomethylfumarate (MMFU), which are used to treat multiple sclerosis and psoriasis. These compounds form adducts with protein Cys residues as well as other nucleophiles. The specific targets ('adductome') that give rise to their therapeutic or toxic activities are poorly understood. This is due, at least in part, to the absence of antigens or chromophores/fluorophores in these compounds. We have recently reported click-chemistry probes of DMFU and MMFU (Redox Biol., 2022, 52, 102299) that allow adducted proteins to be visualized and enriched for further characterization. In the current study, we hypothesized that adducted proteins could be 'clicked' to agarose beads and thereby isolated for LC-MS analysis of DMFU/MMFU targets in primary human coronary artery smooth muscle cells. We show that the probes react with thiols with similar rate constants to the parent drugs, and give rise to comparable patterns of gene induction, confirming similar biological actions. LC-MS proteomic analysis identified ∼2970 cellular targets of DMFU, ∼1440 for MMFU, and ∼140 for the control (succinate-probe) treated samples. The most extensively modified proteins were galectin-1, annexin-A2, voltage dependent anion channel-2 and vimentin. Other previously postulated DMFU targets, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cofilin, p65 (RELA) and Keap1 were also identified as adducted species, though at lower levels with the exception of GAPDH. These data demonstrate the utility of the click-chemistry approach to the identification of cellular protein targets of both exogenous and endogenous compounds.

Aryl Fluorosulfate Based Inhibitors That Covalently Target the SIRT5 Lysine Deacylase.

The sirtuin enzymes are a family of lysine deacylases that regulate gene transcription and metabolism. Sirtuin 5 (SIRT5) hydrolyzes malonyl, succinyl, and glutaryl ϵ-N-carboxyacyllysine posttranslational modifications and has recently emerged as a vulnerability in certain cancers. However, chemical probes to illuminate its potential as a pharmacological target have been lacking. Here we report the harnessing of aryl fluorosulfate-based electrophiles as an avenue to furnish covalent inhibitors that target SIRT5. Alkyne-tagged affinity-labeling agents recognize and capture overexpressed SIRT5 in cultured HEK293T cells and can label SIRT5 in the hearts of mice upon intravenous injection of the compound. This work demonstrates the utility of aryl fluorosulfate electrophiles for targeting of SIRT5 and suggests this as a means for the development of potential covalent drug candidates. It is our hope that these results will serve as inspiration for future studies investigating SIRT5 and general sirtuin biology in the mitochondria.

Influence of plasma halide, pseudohalide and nitrite ions on myeloperoxidase-mediated protein and extracellular matrix damage.

Myeloperoxidase (MPO) mediates pathogen destruction by generating the bactericidal oxidant hypochlorous acid (HOCl). Formation of this oxidant is however associated with host tissue damage and disease. MPO also utilizes H2O2 to oxidize other substrates, and we hypothesized that mixtures of other plasma anions, including bromide (Br-), iodide (I-), thiocyanate (SCN-) and nitrite (NO2-), at normal or supplemented concentrations, might modulate MPO-mediated HOCl damage. For the (pseudo)halide anions, only SCN- significantly modulated HOCl formation (IC50 ∼33 µM), which is within the normal physiological range, as judged by damage to human plasma fibronectin or extracellular matrix preparations detected by ELISA and LC-MS. NO2- modulated HOCl-mediated damage, in a dose-dependent manner, at physiologically-attainable anion concentrations. However, this was accompanied by increased tyrosine and tryptophan nitration (detected by ELISA and LC-MS), and the overall extent of damage remained approximately constant. Increasing NO2- concentrations (0.5-20 µM) diminished HOCl-mediated modification of tyrosine and methionine, whereas tryptophan loss was enhanced. At higher NO2- concentrations, enhanced tyrosine and methionine loss was detected. These analytical data were confirmed in studies of cell adhesion and metabolic activity. Together, these data indicate that endogenous plasma levels of SCN- (but not Br- or I-) can modulate protein modification induced by MPO, including the extent of chlorination. In contrast, NO2- alters the type of modification, but does not markedly decrease its extent, with chlorination replaced by nitration. These data also indicate that MPO could be a major source of nitration in vivo, and particularly at inflammatory sites where NO2- levels are often elevated.

Synthesis and cellular evaluation of click-chemistry probes to study the biological effects of alpha, beta-unsaturated carbonyls.

Humans are commonly exposed to α,ß-unsaturated carbonyls as both environmental toxins (e.g. acrolein) and therapeutic drugs (e.g. dimethylfumarate, DMFU, a front-line drug for the treatment of multiple sclerosis and psoriasis). These compounds undergo rapid Michael addition reactions with amine, imidazole and thiol groups on biological targets, with reaction at protein Cys residues being a major reaction pathway. However, the cellular targets of these species (the 'adductome') are poorly understood due to the absence of readily identifiable tags or reporter groups (chromophores/fluorophores or antigens) on many α,ß-unsaturated carbonyls. Here we report a 'proof of concept' study in which we synthesize novel α,ß-unsaturated carbonyls containing an alkyne function introduced at remote sites on the α,ß-unsaturated carbonyl compounds (e.g. one of the methyl groups of dimethylfumarate). The presence of this tag allows 'click-chemistry' to be used to visualize, isolate, enrich and characterize the cellular targets of such compounds. The probes show similar selectivity and reactivity to the parent compounds, and compete for cellular targets, yielding long-lived (stable) adducts that can be visualized in intact cells (such as primary human coronary artery smooth muscle cells), and extracted and enriched for subsequent target analysis. It is shown using this approach that dimethylfumarate forms adducts with multiple intracellular targets including cytoskeletal, organelle and nuclear species, with these including the rate-limiting glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This approach should be amenable to use with multiple α,ß-unsaturated carbonyls and a wide variety of targets containing nucleophilic sites.

Effect of macromolecular crowding on protein oxidation: Consequences on the rate, extent and oxidation pathways.

Biological systems are heterogeneous and crowded environments. Such packed milieus are expected to modulate reactions both inside and outside the cell, including protein oxidation. In this work, we explored the effect of macromolecular crowding on the rate and extent of oxidation of Trp and Tyr, in free amino acids, peptides and proteins. These species were chosen as they are readily oxidized and contribute to damage propagation. Dextran was employed as an inert crowding agent, as this polymer decreases the fraction of volume available to other (macro)molecules. Kinetic analysis demonstrated that dextran enhanced the rate of oxidation of free Trp, and peptide Trp, elicited by AAPH-derived peroxyl radicals. For free Trp, the rates of oxidation were 15.0 ± 2.1 and 30.5 ± 3.4 µM min-1 without and with dextran (60 mg mL-1) respectively. Significant increases were also detected for peptide-incorporated Trp. Dextran increased the extent of Trp consumption (up to 2-fold) and induced short chain reactions. In contrast, Tyr oxidation was not affected by the presence of dextran. Studies on proteins, using SDS-PAGE and LC-MS, indicated that oxidation was also affected by crowding, with enhanced amino acid loss (45% for casein), chain reactions and altered extents of oligomer formation. The overall effects of dextran-mediated crowding were however dependent on the protein structure. Overall, these data indicate that molecular crowding, as commonly encountered in biological systems affect the rates, and extents of oxidation, and particularly of Trp residues, illustrating the importance of appropriate choice of in vitro systems to study biological oxidations.

Kinetic assessment of Michael addition reactions of alpha, beta-unsaturated carbonyl compounds to amino acid and protein thiols.

Humans have extensive adverse exposure to alpha,beta-unsaturated carbonyl compounds (ABuCs) as these are major toxins in smoke and exhaust fumes, as well as products of lipid peroxidation. In contrast, another ABuC, dimethylfumarate, is used to treat psoriasis and multiple sclerosis. ABuCs undergo Michael adduction with amine, imidazole and thiol groups, with reaction at Cys residues predominating. Here we report rate constants, k2, for ABuCs (acrolein, crotonaldehyde, dimethylfumarate, cyclohex-1-en-2-one, cyclopent-1-en-2-one) with Cys residues present on N-Ac-Cys, GSH, bovine serum albumin, creatine kinase, papain, glyceraldehyde-3-phosphate dehydrogenase, and both wild-type and the C151S mutant of Keap-1. k2 values for N-Ac-Cys and GSH vary by > 250-fold, indicating a marked ABuC structure dependence, with acrolein the most reactive. There is also considerable variation in k2 between protein Cys groups, with these significantly greater than for GSH. A linear inverse correlation for acrolein with the thiol pKa indicates that the thiolate anion is the reactive species. The modest k2 for GSH rationalizes the detection of protein adducts of ABuCs in cells. The k2 values for dimethylfumarate also vary markedly, with the Cys151 residue on Keap-1 being particularly reactive, with the C151S mutant giving a much lower k2 value. The data for crotonaldehyde, dimethylfumarate, and cyclohex-1-en-2-one show little correlation with the Cys pKa values, indicating that steric/electronic interactions, rather than Cys ionization are important. These data indicate that protein Cys residues, and particularly Cys151 on Keap-1, react readily with dimethylfumarate, and this may help rationalize the use of this compound as a therapeutic agent.

Peroxynitrous acid (ONOOH) modifies the structure of anastellin and influences its capacity to polymerize fibronectin.

Anastellin (AN), a fragment of the first type III module in fibronectin (FN), initiates formation of superfibronectin, a polymer which resembles the native cell-derived fibrillar FN found in the extracellular matrix of many tissues, but which displays remarkably different functional properties. Here we demonstrate that exposure of AN to the biologically-important inflammatory oxidant, peroxynitrous acid (ONOOH), either as a bolus or formed at low levels in a time-dependent manner from SIN-1, impairs the capability of AN to polymerize FN. In contrast, exposure of FN to ONOOH does not seem to affect superfibronectin formation to the same extent. This oxidant-induced loss-of-function in AN occurs in a dose-dependent manner, and correlates with structural perturbations, loss of the amino acid tyrosine and tryptophan, and dose-dependent formation of modified amino acid side-chains (3-nitrotyrosine, di-tyrosine and 6-nitrotryptophan). Reagent ONOOH also induces formation of oligomeric species which decrease in the presence of bicarbonate, whereas SIN-1 mainly generates dimers. Modifications were detected at sub-stoichiometric (0.1-fold), or greater, molar excesses of oxidant compared to AN. These species have been localized to specific sites by peptide mass mapping. With high levels of oxidant (>100 times molar excess), ONOOH also induces unfolding of the beta-sheet structure of AN, thermal destabilization, and formation of high molecular mass aggregates. These results have important implications for the understanding of FN fibrillogenesis in vivo, and indicates that AN is highly sensitive to pathophysiological levels of oxidants such as ONOOH.

Absolute quantitative analysis of intact and oxidized amino acids by LC-MS without prior derivatization.

The precise characterization and quantification of oxidative protein damage is a significant challenge due to the low abundance, large variety, and heterogeneity of modifications. Mass spectrometry (MS)-based techniques at the peptide level (proteomics) provide a detailed but limited picture due to incomplete sequence coverage and imperfect enzymatic digestion. This is particularly problematic with oxidatively modified and cross-linked/aggregated proteins. There is a pressing need for methods that can quantify large numbers of modified amino acids, which are often present in low abundance compared to the high background of non-damaged amino acids, in a rapid and reliable fashion. We have developed a protocol using zwitterionic ion-exchange chromatography coupled with LC-MS to simultaneously quantify both parent amino acids and their respective oxidation products. Proteins are hydrolyzed with methanesulfonic acid in the presence of tryptamine and purified by strong cation exchange solid phase extraction. The method was validated for the common amino acids (excluding Gln, Asn, Cys) and the oxidation products 3-chlorotyrosine (3-ClTyr), 3-nitrotyrosine (3-NO2Tyr), di-tyrosine, Nε-(1-carboxymethyl)-l-lysine, o,o'-di-tyrosine, 3,4,-dihydroxyphenylalanine, hydroxy-tryptophan and kynurenine. Linear standard curves were observed over ~3 orders of magnitude dynamic range (2-1000 pmol for parent amino acids, 80 fmol-20 pmol for oxidation products) with limit-of-quantification values as low as 200 fmol (o,o'-di-tyrosine). The validated method was used to quantify Tyr and Trp loss, and formation of 3-NO2Tyr on the isolated protein anastellin treated with peroxynitrous acid, and for 3-ClTyr formation (over a 2 orders of magnitude range) in cell lysates and complex protein mixtures treated with hypochlorous acid.

Iodide modulates protein damage induced by the inflammation-associated heme enzyme myeloperoxidase.

Iodide ions (I-) are an essential dietary mineral, and crucial for mental and physical development, fertility and thyroid function. I- is also a high affinity substrate for the heme enzyme myeloperoxidase (MPO), which is involved in bacterial cell killing during the immune response, and also host tissue damage during inflammation. In the presence of H2O2 and Cl-, MPO generates the powerful oxidant hypochlorous acid (HOCl), with excessive formation of this species linked to multiple inflammatory diseases. In this study, we have examined the hypothesis that elevated levels of I- would decrease HOCl formation and thereby protein damage induced by a MPO/Cl-/H2O2 system, by acting as a competitive substrate. The presence of increasing I- concentrations (0.1-10 µM; i.e. within the range readily achievable by oral supplementation in humans), decreased damage to both model proteins and extracellular matrix components as assessed by gross structural changes (SDS-PAGE), antibody recognition of parent and modified protein epitopes (ELISA), and quantification of both parent amino acid loss (UPLC) and formation of the HOCl-biomarker 3-chlorotyrosine (LC-MS) (reduced by ca. 50% at 10 µM I-). Elevated levels of I- ( > 1 µM) also protected against functional changes as assessed by a decreased loss of adhesion (eg. 40% vs. < 22% with >1 µM I-) of primary human coronary artery endothelial cells (HCAECs), to MPO-modified human plasma fibronectin. These data indicate that low micromolar concentrations of I-, which can be readily achieved in humans and are readily tolerated, may afford protection against cell and tissue damage induced by MPO.

3-Hydroxykynurenine bound to eye lens proteins induces oxidative modifications in crystalline proteins through a type I photosensitizing mechanism.

Photosensitized reactions mediated by endogenous chromophores have been associated with the etiology of age-related cataract disease. Endogenous chromophores such as 3-hydroxykynurenine (3OHKN) can be found in both free form, and bound to crystallin proteins. However, their efficiency in generating photo-induced oxidative modifications on eye lens proteins is not completely understood. In this work, the efficiency and photodynamic activity of 3OHKN bound to both lysine (3OHKN-Lys) and bovine lens proteins (3OHKN-BLP) was assessed and compared with the photosensitizing activity of the major chromophore arising from glucose degradation (GDC). The photosensitizing activity of 3OHKN-Lys, 3OHKN-BLP and GDC was characterized by measurement of singlet oxygen quantum yields, O2 consumption, SDS-PAGE and amino acid analysis of the photo-oxidized proteins. Singlet oxygen quantum yields under 20% O2 atmosphere were 0.02, 0.01, and 0.27 for 3OHKN-Lys, 3OHKN-BLP and GDC, respectively. O2 consumption by photosensitized reactions was more efficient for 3OHKN-BLP, with the extent of O2 consumption being ∼28% higher than for 3OHKN-Lys and GDC under both 5 and 20% O2. SDS-PAGE showed that protein crosslinking is dependent on the O2 concentration, and more extensive at 5 than 20% O2. GDC and 3OHKN-Lys were the most efficient crosslinkers at 20 and 5% O2, respectively. Amino acid analysis of the irradiated proteins showed consumption of Trp, His, Tyr and Phe, and formation of kynurenine (from Trp), methionine sulfoxide (from Met) and DOPA (from Tyr). Kynurenine formation was dependent on the O2 concentration with higher amounts detected at 5 than 20% O2 for 3OHKN-BLP and 3OHKN-Lys, with 3OHKN-BLP the most efficient sensitizer. Our results suggest that 3OHKN-BLP can elicit photo-oxidative damage mainly by a type I photosensitizing mechanism, with this likely to be the most prevalent pathway at the low physiologic O2 concentrations in the eye lens.

Effects of Protein-Derived Amino Acid Modification Products Present in Infant Formula on Metabolic Function, Oxidative Stress, and Intestinal Permeability in Cell Models.

Proteins present in infant formulas are modified by oxidation and glycation during processing. Modified amino acid residues released from proteins may be absorbed in the gastrointestinal tract, and pose a health risk to infants. In this study, the markers of glycation furosine (1.7-3.5 µg per milligram of protein) and Nε-(carboxymethyl)lysine (28-81 ng per milligram of protein) were quantitated in infant formulas. The effects of these species, and other amino acid modifications, at the levels detected in infant formulas, on 3T3-L1 (murine preadipocyte) and Caco-2 (human intestinal epithelial) cells were assessed. Incubation of 3T3-L1 cells for 48 h with amino acid side chain oxidation and glycation products (1 and 10 µM) resulted in a loss (up to 40%, p < 0.05) of cell thiols and decreased metabolic activity compared with those of the controls. In contrast, Caco-2 cells showed a stimulation (10-50%, p < 0.05) of cellular metabolism on exposure to these products for 24 or 48 h. A 28% ( p < 0.05) increase in protein carbonyls was detected upon incubation with 200 µM modified amino acids for 48 h, although no alteration in transepithelial electrical resistance was detected. Oxidation products were detected in the basolateral compartments of Caco-2 monolayers when modified amino acids were applied to the apical side, consistent with limited permeability (up to 3.4%) across the monolayer. These data indicate that modified amino acids present in infant formulas can induce effects on different cell types, with evidence of bioavailability and induction of cellular stress. This may lead to potential health risks for infants consistently exposed to high levels of infant formulas.

Chlorination and oxidation of the extracellular matrix protein laminin and basement membrane extracts by hypochlorous acid and myeloperoxidase.

Basement membranes are specialized extracellular matrices that underlie arterial wall endothelial cells, with laminin being a key structural and biologically-active component. Hypochlorous acid (HOCl), a potent oxidizing and chlorinating agent, is formed in vivo at sites of inflammation via the enzymatic action of myeloperoxidase (MPO), released by activated leukocytes. Considerable data supports a role for MPO-derived oxidants in cardiovascular disease and particularly atherosclerosis. These effects may be mediated via extracellular matrix damage to which MPO binds. Herein we detect and quantify sites of oxidation and chlorination on isolated laminin-111, and laminin in basement membrane extracts (BME), by use of mass spectrometry. Increased modification was detected with increasing oxidant exposure. Mass mapping indicated selectivity in the sites and extent of damage; Met residues were most heavily modified. Fewer modifications were detected with BME, possibly due to the shielding effects. HOCl oxidised 30 (of 56 total) Met and 7 (of 24) Trp residues, and chlorinated 33 (of 99) Tyr residues; 3 Tyr were dichlorinated. An additional 8 Met and 10 Trp oxidations, 14 chlorinations, and 18 dichlorinations were detected with the MPO/H2O2/Cl- system when compared to reagent HOCl. Interestingly, chlorination was detected at Tyr2415 in the integrin-binding region; this may decrease cellular adhesion. Co-localization of MPO-damaged epitopes and laminin was detected in human atherosclerotic lesions. These data indicate that laminin is extensively modified by MPO-derived oxidants, with structural and functional changes. These modifications, and compromised cell-matrix interactions, may promote endothelial cell dysfunction, weaken the structure of atherosclerotic lesions, and enhance lesion rupture.

Chlorination and oxidation of human plasma fibronectin by myeloperoxidase-derived oxidants, and its consequences for smooth muscle cell function.

Fibronectin (FN) occurs as both a soluble form, in plasma and at sites of tissue injury, and a cellular form in tissue extracellular matrices (ECM). FN is critical to wound repair, ECM structure and assembly, cell adhesion and proliferation. FN is reported to play a critical role in the development, progression and stability of cardiovascular atherosclerotic lesions, with high FN levels associated with a thick fibrotic cap, stable disease and a low risk of rupture. Evidence has been presented for FN modification by inflammatory oxidants, and particularly myeloperoxidase (MPO)-derived species including hypochlorous acid (HOCl). The targets and consequences of FN modification are poorly understood. Here we show, using a newly-developed MS protocol, that HOCl and an enzymatic MPO system, generate site-specific dose-dependent Tyr chlorination and dichlorination (up to 16 of 100 residues modified), and oxidation of Trp (7 of 39 residues), Met (3 of 26) and His (1 of 55) within selected FN domains, and particularly the heparin- and cell-binding regions. These alterations increase FN binding to heparin-containing columns. Studies using primary human coronary artery smooth muscle cells (HCASMC) show that exposure to HOCl-modified FN, results in decreased adherence, increased proliferation and altered expression of genes involved in ECM synthesis and remodelling. These findings indicate that the presence of modified fibronectin may play a major role in the formation, development and stabilisation of fibrous caps in atherosclerotic lesions and may play a key role in the switching of quiescent contractile smooth muscle cells to a migratory, synthetic and proliferative phenotype.

Amide Neighbouring-Group Effects in Peptides: Phenylalanine as Relay Amino Acid in Long-Distance Electron Transfer.

In nature, proteins serve as media for long-distance electron transfer (ET) to carry out redox reactions in distant compartments. This ET occurs either by a single-step superexchange or through a multi-step charge hopping process, which uses side chains of amino acids as stepping stones. In this study we demonstrate that Phe can act as a relay amino acid for long-distance electron hole transfer through peptides. The considerably increased susceptibility of the aromatic ring to oxidation is caused by the lone pairs of neighbouring amide carbonyl groups, which stabilise the Phe radical cation. This neighbouring-amide-group effect helps improve understanding of the mechanism of extracellular electron transfer through conductive protein filaments (pili) of anaerobic bacteria during mineral respiration.

Oxidative Damage of Biomolecules by the Environmental Pollutants NO2• and NO3•.

Air pollution is responsible for the premature death of about 7 million people every year. Ozone (O3) and nitrogen dioxide (NO2•) are the key gaseous pollutants in the troposphere, which predominantly result from combustion processes. Their inhalation leads to reactions with constituents in the airway surface fluids (ASF) of the respiratory tract and/or lungs. ASF contain small molecular-weight antioxidants, which protect the underlying epithelial cells against oxidative damage. When this defense system is overwhelmed, proteins and lipids present on cell surfaces or within the ASF become vulnerable to attack. The resulting highly reactive protein and lipid oxidation products could subsequently damage the epithelial cells through secondary reactions, thereby causing inflammation. While reactions of NO2• with biological molecules are considered to proceed through radical pathways, the biological effect of O3 is attributed to its high reactivity with π systems. Because O3 and NO2• always coexist in the polluted ambient atmosphere, synergistic effects resulting from in situ formed strongly oxidizing nitrate radicals (NO3•) may also require consideration. For example, in vitro product studies revealed that phenylalanine, which is inert not only to oxidants produced through biochemical processes, but also to NO2• or O3 in isolation, is damaged by NO3•. The reaction is initiated by oxidation of the aromatic ring and, depending on the availability of NO2•, leads to formation of nitrophenylalanine or ß-nitrooxyphenylalanine, which could serve as marker for NO3•-induced oxidative damage in peptides. More easily oxidizable aromatic amino acids are directly attacked by NO2• and are converted to the same products independent of whether O3 is also present. Remarkably, NO2•-induced oxidative damage in peptides occurs not only through the well-established radical oxidation of peptide side chains, but also through an unprecedented fragmentation/rearrangement of the peptide backbone. This process is initiated by a nonradical N-nitrosation of a peptide bond involving the dimer of NO2•, i.e., N2O4, and contracts the peptide chain in the N → C direction by expelling one amino acid residue with simultaneous fusion of the remaining molecular termini, thereby forming a new peptide bond. This peptide cleavage could potentially be highly relevant for peptide segments with "nonvulnerable" side chains closer to the terminus that are not tied up in complex secondary and tertiary structures and therefore accessible for environmental oxidants. Likewise, NO2• reacts with cholesterol at the C═C moiety through an ionic mechanism, which leads to formation of 6-nitrocholesterol in the presence of moisture. Contrary to common belief, this clearly shows that ionic chemistry, in particular nitrosation reactions by intermediately formed NO+, requires consideration when assessing NO2• toxicity. This conclusion is supported by recent work by Colussi et al. (Enami, S.; Hoffmann, M. R.; Colussi, A. J. Absorption of inhaled NO2. J. Phys. Chem. B. 2009, 113, 7977-7981), who showed that anions in the airway surfaces fluids mediate NO2• absorption by catalyzing its hydrolytic disproportionation into NO2-/HNO2 and NO3-. These findings could be the key to our understanding why NO2•, despite its low water solubility, has such pronounced biological effects in vivo.

What Are the Potential Sites of Protein Arylation by N-Acetyl-p-benzoquinone Imine (NAPQI)?

Acetaminophen (paracetamol, APAP) is a safe and widely used analgesic medication when taken at therapeutic doses. However, APAP can cause potentially fatal hepatotoxicity when taken in overdose or in patients with metabolic irregularities. The production of the electrophilic and putatively toxic compound N-acetyl-p-benzoquinone imine (NAPQI), which cannot be efficiently detoxicated at high doses, is implicated in APAP toxicity. Numerous studies have identified that excess NAPQI can form covalent linkages to the thiol side chains of cysteine residues in proteins; however, the reactivity of NAPQI toward other amino acid side chains is largely unexplored. Here, we report a survey of the reactivity of NAPQI toward 11 N-acetyl amino acid methyl esters and four peptides. (1)H NMR analysis reveals that NAPQI forms covalent bonds to the side-chain functional groups of cysteine, methionine, tyrosine, and tryptophan residues. Analogous reaction products were observed when NAPQI was reacted with synthetic model peptides GAIL-X-GAILR for X = Cys, Met, Tyr, and Trp. Tandem mass spectrometry peptide sequencing showed that the NAPQI modification sites are located on the "X" residue in each case. However, when APAP and the GAIL-X-GAILR peptide were incubated with rat liver microsomes that contain many metabolic enzymes, NAPQI formed by oxidative metabolism reacted with GAIL-C-GAILR exclusively. For the peptides where X = Met, Tyr, and Trp, competing reactions between NAPQI and alternative nucleophiles precluded arylation of the target peptide by NAPQI. Although Cys residues are favorably targeted under these conditions, these data suggest that NAPQI can, in principle, also damage proteins at Met, Tyr, and Trp residues.

Fragmentation-Rearrangement of Peptide Backbones Mediated by the Air Pollutant NO2 (.).

The fragmentation-rearrangement of peptide backbones mediated by nitrogen dioxide, NO2 (.) , was explored using di-, tri-, and tetrapeptides 8-18 as model systems. The reaction, which is initiated through nonradical N-nitrosation of the peptide bond, shortens the peptide chain by the expulsion of one amino acid moiety with simultaneous fusion of the remaining molecular termini through formation of a new peptide bond. The relative rate of the fragmentation-rearrangement depends on the nature of the amino acids and decreases with increasing steric bulk at the α carbon in the order Gly>Ala>Val. Peptides that possessed consecutive aromatic side chains only gave products that resulted from nitrosation of the sterically less congested N-terminal amide. Such backbone fragmentation-rearrangement occurs under physiologically relevant conditions and could be an important reaction pathway for peptides, in which sections without readily oxidizable side chains are exposed to the air pollutant NO2 (.) . In addition to NO2 (.) -induced radical oxidation processes, this outcome shows that ionic reaction pathways, in particular nitrosation, should be factored in when assessing NO2 (.) reactivity in biological systems.