A Biomimetic Model of Tandem Radical Damage Involving Sulfur‐Containing Proteins and Unsaturated Lipids

Abstract

Lipidomics research, which focuses on the global changes in lipid metabolites, has recently been concerned with the type and roles of unsaturated lipids in the biological environment. The structural change induced by their conversion from the naturally occurring cis fatty acid geometry to the more thermodynamically stable trans configuration can affect membrane arrangement as well as lipid metabolism.[1] In the biomimetic model of thiyl radical-catalyzed isomerization of cis phospholipids, it was shown that when thiyl radicals are generated in the aqueous compartment and are able to diffuse in the lipid bilayer, then the interaction with unsaturated fatty acyl chains efficiently produces trans double bonds.[2] These findings suggested that radical-based degradation of sulfur-containing amino acid residues that are known to release diffusible thiol molecules could be the primer for tandem radical damage involving protein and lipid domains. We modeled such damage using g irradiation of lipid vesicle suspensions containing bovine pancreatic ribonuclease A (RNase A). The reaction of this protein with HC atoms was studied, and the inactivation was connected to the specific damage of sulfur moieties with release of low-molecular-weight thiols. [3] Liposomes were prepared by using dioleoyl phosphatidyl choline (DOPC) in the form of large unilamellar vesicles (LUVET) of 100 nm diameter.[4] The protein was added to the LUVET suspension and saturated with N 2 O prior to irradiation at a dose rate of 14.5 Gy min À1. 100 mL aliquots of the suspension were withdrawn at different irradiation times, over the interval of 2-70 min, for lipid isolation and derivatization to the corresponding fatty acid methyl esters.[5] This was followed by GC analysis to determine the cis/trans ratio. The solid circles in Figure 1 show the percentage of trans isomers (elaidate residues) formed as a function of irradiation dose. Control experiments in the absence of RNase A or by replacing RNase A with a protein without sulfur-containing amino acids, such as histone H1 type IIA from calf thymus, did not show any isomerization.In parallel, we followed changes in the enzyme activity, [6] as well as the transformation of the sulfur moieties by Raman spectroscopy, using lyophylized samples of aqueous solutions of native and irradiated RNase A. As expected from the radiation-induced inactivation, [3] a residual enzymatic activity of 67 % was found after exposure to only 33.3 Gy, followed by a slower decrease of the activity, which reached 50 % after 500 Gy. In the Raman spectra, the SÀS and CÀS stretching bands are visible in the 420-780 cm À1 spectral range. Native RNase A has four disulfide bridges that give rise to two different disulfide bands (n S-S ) at 514 and 535 cm À1

Investigation of radical‐based damage of RNase A in aqueous solution and lipid vesicles

“…Control experiments in the absence of RNase A or by replacing RNase A with a protein without sulfur-containing amino acids, such as histone H1 type IIA from calf thymus, did not show any isomerization, suggesting the involvement of sulfur-containing residues of the protein in the radical damage of the unsaturated lipids. 17 The formation of trans lipids is likely due to the formation of diffusible thiyl radical species, derived from the methionine residues present in the RNase sequence. 35 Scheme 1 illustrates the mechanism of the proposed tandem protein-lipid damage; the process derives from the initial attack of H atoms to Met moieties of RNase, leading to a sulfuranyl-type radical which fragments to release methanethiol.…”

“…On the contrary, in a preliminary communication on parallel enzyme damage and lipid isomerization, RNase A was shown to have a stronger reactivity, involving in particular sulfurcontaining residues. 17 In the present work, a detailed investigation of both the protein and lipid behavior is presented.…”

Reductive Modification of a Methionine Residue in the Amyloid‐β Peptide

“…Hydrogen atoms are known to selectively attack Met residues, thereby resulting in the formation of a-aminobutyric acid and CH 3 SH. [7,8] We suggest that HC atoms react with Met35 of AbA C H T U N G T R E N N U N G (1-40) or AbA C H T U N G T R E N N U N G (40-1) to afford the diffusible thiyl radical that catalytically isomerizes the double bond of POPC (Scheme 1). [10] To address the Met35 modification in the Ab peptide by HC atoms, the reaction was studied in the absence of liposomes by HPLC and mass spectrometry.…”

“…[8] We were interested in applying a chemical biology approach to focus on radical damage of Ab, by replacement of Met35 with a-aminobutyric acid in the peptide sequence. The replacement of methionine by other amino acids in Ab is known to occur by genetic mutation, as in the case of valine, which causes the disease to appear at very early ages.…”

“…[8,10] Liposomes were prepared from 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), in the form of large unilamellar vesicles (LUVETs) with a 100 nm diameter; these vesicles were obtained by membrane extrusion with LiposoFast. [10,11] AbA C H T U N G T R E N N U N G (1-40) or AbA C H T U N G T R E N N U N G (40-1) was added to this suspension.…”