Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 293, Issue 46, Pages 17997-18009Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA118.004551
Keywords
lens; crystallin; cataract; disulfide; protein aggregation; protein misfolding; conformational intermediates; disulfide exchange; oxidoreductase; redox mechanism
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Funding
- National Institutes of Health [R01GM111955, F32GM126651]
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Increased light scattering in the eye lens due to aggregation of the long-lived lens proteins, crystallins, is the cause of cataract disease. Several mutations in the gene encoding human D-crystallin (HD) cause misfolding and aggregation. Cataract-associated substitutions at Trp(42) cause the protein to aggregate in vitro from a partially unfolded intermediate locked by an internal disulfide bridge, and proteomic evidence suggests a similar aggregation precursor is involved in age-onset cataract. Surprisingly, WT HD can promote aggregation of the W42Q variant while itself remaining soluble. Here, a search for a biochemical mechanism for this interaction has revealed a previously unknown oxidoreductase activity in HD. Using in vitro oxidation, mutational analysis, cysteine labeling, and MS, we have assigned this activity to a redox-active internal disulfide bond that is dynamically exchanged among HD molecules. The W42Q variant acts as a disulfide sink, reducing oxidized WT and forming a distinct internal disulfide that kinetically traps the aggregation-prone intermediate. Our findings suggest a redox hot potato competition among WT and mutant or modified polypeptides wherein variants with the lowest kinetic stability are trapped in aggregation-prone intermediate states upon accepting disulfides from more stable variants. Such reactions may occur in other long-lived proteins that function in oxidizing environments. In these cases, aggregation may be forestalled by inhibiting disulfide flow toward mutant or damaged polypeptides.
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