Protein Dimerization via Tyr Residues: Highlight of a Slow Process with Co-Existence of Numerous Intermediates and Final Products

Abstract

Protein dimerization via tyrosine residues is a crucial process in response to an oxidative attack, which has been identified in many ageing-related pathologies. Recently, it has been found that for isolated tyrosine amino acid, dimerization occurs through three types of tyrosine-tyrosine crosslinks and leads to at least four final products. Herein, considering two protected tyrosine residues, tyrosine-containing peptides and finally proteins, we investigate the dimerization behavior of tyrosine when embedded in a peptidic sequence. After azide radical oxidation and by combining UPLC-MS and H/D exchange analyzes, we were able to evidence: (i) the slow kinetics of Michael Addition Dimers (MAD) formation, i.e., more than 48 h; (ii) the co-existence of intermediates and final cyclized dimer products; and (iii) the probable involvement of amide functions to achieve Michael additions even in proteins. This raises the question of the possible in vivo existence of both intermediates and final entities as well as their toxicity and the potential consequences on protein structure and/or function.

Keywords: oxidative stress; proteins; radicals; tyrosine dimers.

Scheme 1

Representation of the tyrosine dimer cross-links and evolved structures composing the MAD family.

Figure 1

Oxidation of N-acetyl-L-tyrosine (Ac-Y) at 40 Gy in N₃^• production conditions. Extracted ion chromatograms m/z 445.16 ± 0.05 ([M + H]⁺) after oxidation in H₂O at (a) T0 (0 h); (b) T48 (48 h). Full scan MS spectra extracted at RT of dimers after oxidation in D₂O at (c) T0 (0 h) and (d) T48 (48 h). Minor peaks in mass spectra arise from isotopic profiles.

Figure 2

Oxidation of peptide KTSLY at 40 Gy in N₃^• production conditions. Extracted ion chromatograms at m/z 407.22 ± 0.05 for [M + 3H]³⁺ after oxidation in H₂O at (a) T0 (0 h), (b) T5 (5 h) and (c) T48 (48 h). Full scan MS spectra for [M + 3H]³⁺ extracted at RT of dimers after oxidation in D₂O at (d) T0 (0 h), (e) T5 (5 h) and (f) T48 (48 h). Minor peaks in mass spectra arise from isotopic profiles.

Figure 3

HsCen2 oligomerization at 40 Gy in N₃^• production conditions. (a) SDS-PAGE to evidence covalent oligomers; (b) mass analysis of tryptic fragment before and after a 48 h incubation at 37 °C, extracted ion chromatograms (m/z 610.83 ± 0.05) corresponding to KTSLY-^CYLSTK^N dimer; (c) full scan MS of dimer for [M + 2H]²⁺.

Figure 4

Oxidation of N-Acetyl-L-tyrosine ethyl ester (Ac-Y-Et) at 40 Gy in N₃^• production conditions. Extracted ion chromatograms m/z 501.23 ± 0.05 [M + H]⁺ after oxidation in H₂O at (a) T0 (0 h) and (b) T48 (48 h). Full scan MS spectra extracted at RT of dimers after oxidation in D₂O at (c) T0 (0 h) and (d) T48 (48 h). Minor peaks in mass spectra arise from isotopic profiles.

Figure 5

Oxidation of peptide KTSLYG at 40 Gy in the presence of N₃^•. Extracted ion chromatograms m/z 445.31 [M + 3H]³⁺ after oxidation in H₂O at (a) T0 (0 h) and (b) T72 (72 h). Full scan MS spectra extracted at RT of dimers obtained in D₂O at (c) T0 (0 h) and (d) T72 (72 h). (*) P6 intensity was multiplied by a factor of 10. Minor peaks in mass spectra arise from isotopic profiles.

Figure 6

CaM oligomerization at 40 Gy in the presence of N₃^•. (a) Local representation of Tyr residues of CaM (from [25], 3cln PDB structure) and (b) SDS-PAGE of CaM after radiolytic oxidation by N₃^• for doses up to 112 Gy.

Figure 7

Tryptic digestion of oxidized CaM at 40 Gy in N₃^• production conditions. Extracted ion chromatograms (m/z 702.14 ± 0.05, [M + 5H]⁵⁺) for (a) the homodimer ⁹⁹Y-⁹⁹Y in fragment ⁹²V-R¹⁰⁷ and (b) (m/z 849.18 ± 0.05, [M + 5H]⁵⁺) for the heterodimer ⁹⁹Y-¹³⁸Y. Full scan MS spectra extracted at RT of dimers for (c) ⁹⁹Y-⁹⁹Y, (d) ⁹⁹Y-¹³⁸Y.