Radiation- and Photo-Induced Oxidation Pathways of Methionine in Model Peptide Backbone under Anoxic Conditions

Within the reactive oxygen species (ROS) generated by cellular metabolisms, hydroxyl radicals (HO^•) play an important role, being the most aggressive towards biomolecules. The reactions of HO^• with methionine residues (Met) in peptides and proteins have been intensively studied, but some fundamental aspects remain unsolved. In the present study we examined the biomimetic model made of Ac-Met-OMe, as the simplest model peptide backbone, and of HO^• generated by ionizing radiation in aqueous solutions under anoxic conditions. We performed the identification and quantification of transient species by pulse radiolysis and of final products by LC-MS and high-resolution MS/MS after γ-radiolysis. By parallel photochemical experiments, using 3-carboxybenzophenone (CB) triplet with the model peptide, we compared the outcomes in terms of short-lived intermediates and stable product identification. The result is a detailed mechanistic scheme of Met oxidation by HO^•, and by CB triplets allowed for assigning transient species to the pathways of products formation.     

The reaction of hydrogen atoms with methionine residues: A model of reductive radical stress causing tandem protein-lipid damage

The occurrence of tandem damage, due to reductive radical stress involving proteins and lipids, is shown by using a biomimetic model. It is made of unsaturated lipid vesicle suspensions in phosphate buffer in the presence of methionine, either as a single amino acid or as part of a protein such as RNase A, which contains four methionine residues. The radical process starts with the formation of H(.) atoms by reaction of solvated electrons with dihydrogen phosphate anions, which selectively attack the thioether function of methionine. The modification of methionine to alpha-aminobutyric acid is accompanied by the formation of thiyl radicals, which in turn cause the isomerization of the cis fatty acid residues to the trans isomers. The relationship between methionine modification and lipid damage and some details of the reductive radical stress obtained by proteomic analysis of irradiated RNase A are presented.     

<Emphasis Type="Italic">Trans</Emphasis> fatty acids and atopic eczema/dermatitis syndrome: The relationship with a free radical <Emphasis Type="Italic">cis-trans</Emphasis> isomerization of membrane lipids

The formation of trans FA residues in membrane phospholipids may be due to a free radical-catalyzed isomerization process occurring to the cis unsaturated FA moieties. Radical stress is well documented in inflammatory processes of atopic diseases, but no data are yet available about a possible association with trans FA detected in these patients. We investigated the presence of trans lipid isomers in the erythrocyte and T-lymphocyte membranes of 26 children affected by atopic eczema/dermatitis syndrome (AEDS). Trans lipid isomers were found in both cell membranes, up to a total content of 2.7 and 4.9% of the FA composition, respectively. By using the geometrical trans lipid library derived from in vitro models of thiyl radical-catalyzed isomerization, oleic and arachidonic acid isomers were detected. The statistical significance was evaluated by comparison with an age-matched control group. These results suggest the role of an endogenous free radical isomerization path occurring to membrane unsaturated lipids, complementary to the dietary contribution, which can be involved in the lipid impairment in AEDS. This study contributes to lipidomic research regarding the double bond structure and the influence of a geometrical change of membrane lipids in physiology and diseases.     

Geometrical trans lipid isomers: a new target for lipidomics

Evidence that lipids play different roles in the biological environment, particularly in dealing with metabolic regulation and cell signaling, has led to a growing interest in these molecules, and nowadays the research field of lipid structures and functions is called lipidomics. The term describes diverse research areas, from mapping the entire spectrum of lipids in organisms to describing the function and metabolism of individual lipids. Recent investigations on geometrical trans isomers of fatty acid derivatives, which have the double bonds in the same position as the natural compounds but with the trans instead of the naturally occurring cis geometry, highlighted these compounds as a new target for lipidomics. In addition to the identification of their structures and functions, research in a multidisciplinary context aims at understanding the biochemical significance of cis and trans lipid geometry, and a chemical biology approach can be envisaged to explore the role of the geometry change as either an alteration or a signal that can perturb a biological system and induce a cellular response.     

Trans lipids: the free radical path

Free radical-catalyzed cis-trans isomerization of unsaturated lipids and its outcome in biomimetic conditions are reviewed. They provided indications for an endogenous origin of trans unsaturated fatty acids in humans by the attack of thiyl radicals. A multidisciplinary approach highlights the relevance of this topic. The role of cis lipid geometry and its maintenance in the biological environment is an emerging question, with future developments in the fields of lipidomics, biology, and medicine.     

Biomimetic models of radical stress and related biomarkers

The biological consequences of free radical production is the central subject of a very lively scientific debate, focusing on the estimation of the type and extent of damage, as well as the efficiency of the protective and repair systems. When studying free radical based chemical mechanisms, it is very important to establish biomimetic models, which allow the experiments to be performed in a simplified environment, but suitably designed to be in strict connection with cellular conditions. The biomimetic modeling approach has been coupled with physical organic chemistry methodologies and knowledge of free radical reactivity. Molecular basis of important processes have been identified, building up molecular libraries of products concerning unsaturated lipids, sulfur-containing proteins and nucleic acids, to be developed as biomarkers. Ongoing projects in our group deal with lipidomics, genomics and proteomics of free radical stress and some examples will be described.     

Thiyl radical-catalyzed isomerization of oils: An entry to the trans lipid library

The unsaturated fatty acyl moieties of TAG present in natural oils of borage, olive, and rice were converted to their corresponding geometrical trans isomers by thiyl radical-catalyzed isomerization. Thiyl radicals were generated from 2-mercaptoethanol under photolytic or thermal conditions. A relevant feature of this method is the absence of double-bond shifts, so that no positional trans isomers or conjugated polyenes are formed. Oils obtained after the isomerization were winterized to further increase their trans fatty acid content. Methanolysis and hydrolysis of the trans oil mixtures using an enzymatic method (lipase B from Candida antarctica) gave good conversions to the corresponding trans FAME and fatty acids, respectively. These results are relevant for the studies of lipid isomerism and trans fatty acid recognition, which is a growing concern in biochemistry and nutrition, and open new perspectives for the synthesis of glycerides and studies of their structure-activity relationships.     

Biomimetic Thiyl Radical Chemistry by γ-Irradiation of Micelles and Vesicles Containing Unsaturated Fatty Acids

Different types of lipid aggregations, such as micelles and liposomes, can be used as biomimetic models. The uses of γ-irradiation as a valid methodology for simulating the biological generation of thiyl radicals in these models are summarized and, in particular, thiyl radical catalyzed cis trans isomerization of unsaturated lipids is underlined. The efficiency of antioxidants against lipid isomerization assayed by biomimetic models and the importance of trans lipids as novel biomarkers of radical stress are also described.