Formation of protein cross-links by singlet oxygen-mediated disulfide oxidation

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Cross-links formed within and between proteins are a major cause of protein dysfunction, and are postulated to drive the accumulation of protein aggregates in some human pathologies. Cross-links can be formed from multiple residues and can be reversible (usually sulfur-sulfur bonds) or irreversible (typically carbon-carbon or carbon-heteroatom bonds). Disulfides formed from oxidation of two Cys residues are widespread, with these formed both deliberately, via enzymatic reactions, or as a result of unintended oxidation reactions. We have recently demonstrated that new protein-glutathione mixed disulfides can be formed through oxidation of a protein disulfide to a thiosulfinate, and subsequent reaction of this species with glutathione. Here we investigate whether similar reactions occur between an oxidized protein disulfide, and a Cys residues on a second protein, to give novel protein cross-links. Singlet oxygen (1O2)-mediated oxidation of multiple proteins (?-lactalbumin, lysozyme, beta-2-microglobulin, C-reactive protein), and subsequent incubation with the Cys-containing protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH), generates inter-protein cross-links as detected by SDSPAGE, immunoblotting and mass spectrometry (MS). The cross-link yield is dependent on the 1O2 concentration, the presence of the original protein disulfide bond, and the free Cys on GAPDH. MS with 18O-labeling has allowed identification of the residues involved in some cases (e.g. Cys25 from the Cys25-Cys80 disulfide in beta2-microglobulin, with Cys149 or Cys244 of GAPDH). The formation of these cross-links results in a loss of GAPDH enzymatic activity. These data provide ?proof-of-concept? for a novel mechanism of protein cross-link formation which may help rationalize the accumulation of cross-linked proteins in multiple human pathologies.

Original languageEnglish
Article number101874
JournalRedox Biology
Number of pages16
Publication statusPublished - 2021

    Research areas

  • Disulfide, Singlet oxygen, Protein cross-links, Protein aggregation, Thiol-disulfide exchange, Post-translational modification, Photo-oxidation, INDUCED ELECTROSTATIC STABILIZATION, GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE, MOLECULAR-OXYGEN, SULFENIC ACIDS, MECHANISMS, PHOTOOXIDATION, TYROSINE, BONDS, BETA(2)-MICROGLOBULIN, INACTIVATION

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