Unravelling molecular mechanisms in atherosclerosis using cellular models and omics technologies.

Despite the discovery and prevalent clinical use of potent lipid-lowering therapies, including statins and PCSK9 inhibitors, cardiovascular diseases (CVD) caused by atherosclerosis remain a large unmet clinical need, accounting for frequent deaths worldwide. The pathogenesis of atherosclerosis is a complex process underlying the presence of modifiable and non-modifiable risk factors affecting several cell types including endothelial cells (ECs), monocytes/macrophages, smooth muscle cells (SMCs) and T cells. Heterogeneous composition of the plaque and its morphology could lead to rupture or erosion causing thrombosis, even a sudden death.

Mapping of oxidative modifications on the alpha-keto glutarate dehydrogenase complex induced by singlet oxygen: Effects on structure and activity.

The large multi-subunit mitochondrial alpha-keto glutarate dehydrogenase (KGDH) complex plays a key, rate-determining, role in the tricarboxylic acid (Krebs) cycle, catalyzing the conversion of alpha-keto glutarate to succinyl-CoA. This complex is both a source and target of oxidants, but the sites of modification and association with structural changes and activity loss are poorly understood. We report here oxidative modifications induced by Rose Bengal (RB) in the presence of O, a source of singlet oxygen (O).

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.

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.

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.

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

α,β-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.

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.

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 HO to oxidize other substrates, and we hypothesized that mixtures of other plasma anions, including bromide (Br), iodide (I), thiocyanate (SCN) and nitrite (NO), at normal or supplemented concentrations, might modulate MPO-mediated HOCl damage.

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

Humans are commonly exposed to α,β-unsaturated carbonyls as both environmental toxins (e.g. acrolein) and therapeutic drugs (e.

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.

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.

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.

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.

The leucine-rich repeat domain of human peroxidasin 1 promotes binding to laminin in basement membranes.

Human peroxidasin 1 (PXDN) is a homotrimeric multidomain heme peroxidase and essential for tissue development and architecture. It has a biosynthetic function and catalyses the hypobromous acid-mediated formation of specific covalent sulfilimine (SN) bonds, which cross-link type IV collagen chains in basement membranes. Currently, it is unknown whether and which domain(s) [i.

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 HO and Cl, MPO generates the powerful oxidant hypochlorous acid (HOCl), with excessive formation of this species linked to multiple inflammatory diseases.

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.

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.

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.

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.

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.

Oxidative Damage of Biomolecules by the Environmental Pollutants NO and NO

Air pollution is responsible for the premature death of about 7 million people every year. Ozone (O) and nitrogen dioxide (NO) 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.

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.

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.