MSRC Schey Lab

Hotspots of Age-Related Protein Degradation. The Importance of Neighbouring Residues for the Formation of Non-Disulfide Crosslinks derived from Cysteine.

AUTHORS

ABSTRACT

Over time, the long-lived proteins that are present throughout the human body deteriorate. Typically, they become racemised, truncated and covalently crosslinked. One reaction responsible for age-related protein crosslinking in the lens was elucidated recently and shown to involve spontaneous formation of dehydroalanine (DHA) intermediates from phosphoserine. Cys residues are another potential source of DHA, and evidence was found for this in a number of lens crystallins. In the human lens, some sites were more prone to forming non-disulfide covalent crosslinks than others. Foremost among them was Cys 5 in βA4 crystallin. The reason for this enhanced reactivity was investigated using peptides. Oxidation of Cys to cystine was a pre-requisite for DHA formation and DHA production was accelerated markedly by the presence of a Lys, one residue separated from Cys 5. Modelling and direct investigation of the N-terminal sequence of βA4 crystallin, as well as a variety of homologous peptides, showed that the epsilon amino group of Lys can promote DHA production by nucleophilic attack on the alpha proton of cystine. Once a DHA residue was generated, it could form inter-molecular crosslinks with Lys and Cys. In the lens, the most abundant crosslink involved Cys 5 of βA4 crystallin attached via a thioether bond to glutathione. These findings illustrate the potential of Cys and disulfide bonds to act as precursors for irreversible covalent crosslinks and the role of nearby amino acids in creating “hotpsots” for the spontaneous processes responsible for protein degradation in aged tissues.