Ditryptophan Cross-Links as Novel Products of Protein Oxidation

Abstract

Protein oxidation is an unavoidable consequence of aerobic metabolism. The oxidation of most proteins residues is non-repairable and may affect protein structure and function. In particular, protein cross-links arising from oxidative modifications are presumably toxic to cells because they may accumulate and induce protein aggregation. However, most of these irreversible protein cross-links remain partially characterized. Up to very recently, ditryptophan cross-links (Trp-Trp), in particular, have been largely disregarded in the literature. Here, we briefly review studies showing that Trp-Trp cross-links can be formed in proteins exposed to a variety of oxidants. The challenges to fully characterize Trp-Trp cross-links are discussed as well as their potential roles in protein dysfunction and aggregation.Keywords: ditryptophan, cross-links, protein oxidation, free radicals Protein OxidationFree radicals and other oxidants are formed in living organisms by oxidation-reduction processes, which occur continuously during metabolism and through interactions with the environment. Organisms evolved enzymatic antioxidant defenses and the capability to use antioxidants from the diet to control the levels of these species. 1,2 Nevertheless, under certain circumstances, the levels of radicals and oxidants increase, promoting the oxidation of biomolecules. Among them, proteins are the major targets, due to their high biological abundance and reactivity towards one-and two-electron oxidants. [3][4][5] Protein residues most susceptible to oxidation are the sulfur-containing residues Cys and Met, and the aromatic residues His, Phe, Tyr, and Trp. In vivo, the oxidation of Cys and Met residues can be reversed by biological reductants with the assistance of enzymatic systems. In fact, the reversible oxidation of protein-Cys residues is emerging as a fundamental cell regulatory mechanism. 2,[6][7][8] The oxidation of all the other protein residues is irreversible, and includes several covalent modifications, such as protein cleavage, carbonylation, nitration, hydroxylation, halogenation and protein cross-linking with other proteins, lipids, carbohydrates and nucleic acids. [3][4][5][9][10][11] These modifications result in protein fragmentation, loss of protein function, protein aggregation and/or altered protein turnover, leading to cell and tissue dysfunction and various human pathologies. To maintain cellular homeostasis, proteins irreversibly oxidized are targeted for degradation by the proteasomal and lysosomal degradation pathways.11,12 During aging and pathological conditions associated with oxidative stress, however, the levels of over-oxidized and cross-linked proteins accumulate because these species are poor substrates for the intracellular proteolytic systems. This may lead to protein aggregation, which is a hallmark of age-related diseases, such as neurodegenerative diseases, atherosclerosis and cataract.11,12 Therefore, the study of irreversible protein oxidation in vitro and in vivo is relevant to the understanding o...

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“…It was shown that the hydroxylation of tryptophan by Fenton's reaction and photolysis oxidation occurs in different positions of the molecule yielding many possible isomers of hydroxytryptophans and monohydroxytryptophan dimers. 21,22 However, none of these studies have ever reported the formation of nanoparticles 21,22 . It is well known that sonication of an aqueous solution results in the formation of H and OH radicals due to acoustic cavitation 15 .…”

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“…Notably, the ditryptophan dimer in superoxide dismutase or lysozyme can cleave under MS/MS conditions [ 47 ], suggesting that it is not as stable as a dityrosine link and thus is similar to the labile crosslinks we have detected in the current study. Paviani et al have suggested that the susceptibility to cleavage of ditryptophan is more consistent with a C–N bond than a C–C bond [ 47 ]. Accordingly, the crosslink that is reversed by ascorbate in the current study is likely a C–N bond.…”

“…The nature of ditryptophan bonds is not yet fully elucidated [ 47 ]. Available data suggest that C and N participate in ditryptophan crosslinking [ 47 ], giving the possibility of either C–C or C–N crosslinks.…”

“…The nature of ditryptophan bonds is not yet fully elucidated [ 47 ]. Available data suggest that C and N participate in ditryptophan crosslinking [ 47 ], giving the possibility of either C–C or C–N crosslinks. Notably, the ditryptophan dimer in superoxide dismutase or lysozyme can cleave under MS/MS conditions [ 47 ], suggesting that it is not as stable as a dityrosine link and thus is similar to the labile crosslinks we have detected in the current study.…”

“…Available data suggest that C and N participate in ditryptophan crosslinking [ 47 ], giving the possibility of either C–C or C–N crosslinks. Notably, the ditryptophan dimer in superoxide dismutase or lysozyme can cleave under MS/MS conditions [ 47 ], suggesting that it is not as stable as a dityrosine link and thus is similar to the labile crosslinks we have detected in the current study. Paviani et al have suggested that the susceptibility to cleavage of ditryptophan is more consistent with a C–N bond than a C–C bond [ 47 ].…”