Abstract
SummaryAge‐related cataractogenesis is associated with disulfide‐linked high molecular weight (HMW) crystallin aggregates. We recently found that the lens crystallin disulfidome was evolutionarily conserved in human and glutathione‐depleted mouse (LEGSKO) cataracts and that it could be mimicked by oxidation in vitro (Mol. Cell Proteomics, 14, 3211‐23 (2015)). To obtain a comprehensive blueprint of the oxidized key regulatory and cytoskeletal proteins underlying cataractogenesis, we have now used the same approach to determine, in the same specimens, all the disulfide‐forming noncrystallin proteins identified by ICAT proteomics. Seventy‐four, 50, and 54 disulfide‐forming proteins were identified in the human and mouse cataracts and the in vitro oxidation model, respectively, of which 17 were common to all three groups. Enzymes with oxidized cysteine at critical sites include GAPDH (hGAPDH, Cys247), glutathione synthase (hGSS, Cys294), aldehyde dehydrogenase (hALDH1A1, Cys126 and Cys186), sorbitol dehydrogenase (hSORD, Cys140, Cys165, and Cys179), and PARK7 (hPARK7, Cys46 and Cys53). Extensive oxidation was also present in lens‐specific intermediate filament proteins, such as BFSP1 and BFSP12 (hBFSP1 and hBFSP12, Cys167, Cys65, and Cys326), vimentin (mVim, Cys328), and cytokeratins, as well as microfilament and microtubule filament proteins, such as tubulin and actins. While the biological impact of these modifications for lens physiology remains to be determined, many of these oxidation sites have already been associated with either impaired metabolism or cytoskeletal architecture, strongly suggesting that they have a pathogenic role in cataractogenesis. By extrapolation, these findings may be of broader significance for age‐ and disease‐related dysfunctions associated with oxidant stress.
Highlights
The mammalian genome encodes 214 000 cysteine residues (Go & Jones, 2013)
It is important to understand that each peptide reported below represents the ratio of the peptide labeled with the heavy chain (C13 Isotope-coded Affinity Tag labeling Experiment (ICAT) stable isotope) that is present in either aged normal human lens, aged cataractous human lens, LEGSKO or hydrogen peroxideoxidized mouse lens protein extract, vs. the identical peptide labeled with the light chain (C12 ICAT isotope) present in extract from young human lens, age-matched WT mouse lens, and mouse lens extract without hydrogen peroxide oxidation
The ratio of ICAT heavy/ICAT light reflects the relative degree of disulfide bonding occurring in the aged normal and aged cataractous human lens peptides vs. the degree of disulfide bonding occurring in the same peptides from young control lenses
Summary
Some of the cysteine residue pairs are playing a key role in high-ordered protein structure and conformation through intradisulfide bonds (Borges & Sherma, 2014). These cysteine pairs tend to be evolutionarily conserved (Thornton, 1981), and any disturbance of this precise disulfide bond formation will likely have a detrimental effect on protein function. It has been estimated that 10–20% of cysteine residues are redox-active (Go & Jones, 2013) These cysteine residues, together with other redox regulatory components such as glutathione (GSH), oxidized glutathione (GSSG), cysteine, thioredoxin (Trx), and thioredoxin reductase (TrxR), constitute a powerful force in maintaining intracellular and extracellular redox status. Several groundbreaking methods and techniques have offered researchers great opportunities to systematically study protein cysteine oxidation, including cysteine sulfenylation, sulfinylation, sulfonylation, nitrosylation, and disulfide formation (Paulsen & Carroll, 2013)
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