Abstract
Oxidative stress in humans is related to various pathophysiological processes, which can manifest in numerous diseases including cancer, cardiovascular diseases, and Alzheimer’s disease. On the atomistic level, oxidative stress causes posttranslational modifications, thus inducing structural and functional changes into the proteins structure. This study focuses on fibrinogen, a blood plasma protein that is frequently targeted by reagents causing posttranslational modifications in proteins. Fibrinogen was in vitro modified by three reagents, namely sodium hypochlorite, malondialdehyde, and 3-morpholinosydnonimine that mimic the oxidative stress in diseases. Newly induced posttranslational modifications were detected via mass spectrometry. Electron microscopy was used to visualize changes in the fibrin networks, which highlight the extent of disturbances in fibrinogen behavior after exposure to reagents. We used molecular dynamics simulations to observe the impact of selected posttranslational modifications on the fibrinogen structure at the atomistic level. In total, 154 posttranslational modifications were identified, 84 of them were in fibrinogen treated with hypochlorite, 51 resulted from a reaction of fibrinogen with malondialdehyde, and 19 were caused by 3-morpholinosydnonimine. Our data reveal that the stronger reagents induce more posttranslational modifications in the fibrinogen structure than the weaker ones, and they extensively alter the architecture of the fibrin network. Molecular dynamics simulations revealed that the effect of posttranslational modifications on fibrinogen secondary structure varies from negligible alternations to serious disruptions. Among the serious disruptions is the oxidation of γR375 resulting in the release of Ca2+ ion that is necessary for appropriate fibrin fiber formation. Folding of amino acids γE72–γN77 into a short α-helix is a result of oxidation of γP76 to glutamic acid. The study describes behaviour of fibrinogen coiled-coil connecter in the vicinity of plasmin and hementin cleavage sites.
Highlights
Posttranslational modifications (PTMs) may lead to alterations in the protein secondary structure as well as their functional and binding sites by changing the charge and/or structure of their amino acid side-chains [1]
We have demonstrated that oxidation has a serious impact on the structure of fibrin clots and that the extent of changes increases with the oxidative strength of the reagent
We have showed that molecular dynamics (MD) simulations performed with Gromos force field are able to capture the impact of PTMs on fibrinogen structures for isolated fragments of fibrinogen in solution on timescale of 100ns
Summary
Posttranslational modifications (PTMs) may lead to alterations in the protein secondary structure as well as their functional and binding sites by changing the charge and/or structure of their amino acid side-chains [1]. PTMs can be introduced in the protein structure either enzymatically or by reactions of amino acid side chains with free radicals and other reactive species. Nonenzymatic PTMs are usually a result of protein interactions with various reactive oxygen, nitrogen, sulfur, carbonyl, selenium, chlorine, or bromine species under physiological conditions [9]. Higher concentrations of these elements may lead to an imbalance in oxidants and antioxidants in favor of the former, causing oxidative stress. Among the blood plasma proteins, fibrinogen is known be the most frequent target of PTM [10]
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