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

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.

Advanced methodology combining UPLC-MS, isotopic labelling and H/D exchanges reveals three tyrosine-tyrosine cross-links induced by oxidative radicals evolving to at least four dimeric structures.

Di-tyrosine is one of the major protein cross-links involved in a large number of neurodegenerative or ageing-related diseases. Recently, no less than four different di-tyrosine bridge isomers have been highlighted while only two structures are characterized at the moment in the literature. In this study, the four dimers were produced by radiolytical-induced oxidation. Although the abundance of these additional dimers precluded the use of NMR or other structural characterization methods, we propose a new methodology combining UPLC-MS analysis, specific deuterium labelling and isotopic (H/D) exchanges with the solvent. Thus, we were able to identify three different covalent cross-links and propose different new original di-tyrosine structures based on double Michael additions, leading to tetracyclic products. Absorption and fluorescence characterizations of the four species were performed and consolidate our proposal.

Oxidative radicals (HO• or N3•) induce several di-tyrosine bridge isomers at the protein scale.

Among protein oxidative damages, di-tyrosine bridges formation has been evidenced in many neuropathological diseases. Combining oxidative radical production by gamma radiolysis with very performant chromatographic separation coupled to mass spectrometry detection, we brought into light new insights of tyrosine dimerization. Hydroxyl and azide radical tyrosine oxidation leading to di-tyrosine bridges formation was studied for different biological compounds: a full-length protein (Δ25-centrin 2), a five amino acid peptide (KTSLY) and free tyrosine. We highlighted that both radicals generate high proportion of dimers even for low doses. Surprisingly, no less than five different di-tyrosine isomers were evidenced for the protein and the peptide. For tyrosine alone, at least four distinct dimers were evidenced. These results raise some questions about their respective role in vivo and hence their relative toxicity. Also, as di-tyrosine is often used as a biomarker, a better knowledge of the type of dimer detected in vivo is now required.

From Protein Corona to Colloidal Self-Assembly: The Importance of Protein Size in Protein-Nanoparticle Interactions.

Protein adsorption on nanoparticles is an important field of study, particularly with regard to nanomedicine and nanotoxicology. Many factors can influence the composition and structure of the layer(s) of adsorbed proteins, the so-called protein corona. However, the role of protein size has not been specifically investigated, although some evidence has indicated its potential important role in corona composition and structure. To assess the role of protein size, we studied the interactions of hemoproteins (spanning a large size range) with monodisperse silica nanoparticles. We combined various techniques-adsorption isotherms, isothermal titration calorimetry, circular dichroism, and transmission electron cryomicroscopy-to address this issue. Overall, the results show that small proteins behaved as typical model proteins, forming homogeneous monolayers on the nanoparticle surface (protein corona). Their adsorption is purely enthalpy-driven, with subtle structural changes. In contrast, large proteins interact with nanoparticles via entropy-driven mechanisms. Their structure is completely preserved during adsorption, and any given protein can directly bind to several nanoparticles, forming bridges in these newly formed protein-nanoparticle assemblies. Protein size is clearly an overlooked factor that should be integrated into proteomics and toxicological studies.