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Edward J. Dudek, Kirsten J. Lampi, Jason A. Lampi, Fu Shang, Jonathan King, Yongting Wang, Allen Taylor; Ubiquitin Proteasome Pathway–Mediated Degradation of Proteins: Effects Due to Site-Specific Substrate Deamidation. Invest. Ophthalmol. Vis. Sci. 2010;51(8):4164-4173. doi: https://doi.org/10.1167/iovs.09-4087.
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The accumulation, aggregation, and precipitation of proteins is etiologic for age-related diseases, particularly cataract, because the precipitates cloud the lens. Deamidation of crystallins is associated with protein precipitation, aging, and cataract. Among the roles of the ubiquitin proteasome pathway (UPP) is protein surveillance and maintenance of protein quality. The purpose of this study was to determine whether deamidation can alter clearance of crystallins by the UPP.
Wild-type (WT) and deamidated crystallins were expressed and 125I-radiolabeled. Ubiquitination and degradation were monitored separately.
For βB2 crystallins, rates of ubiquitination and adenosine triphosphate–dependent degradation, both indicators of active UPP, occurred in the order Q70E/Q162E>Q162E> Q70E=WT βB2 using reticulocyte lysate as the source of degradation machinery. Human lens epithelial cell lysates and lens fiber cell lysates also catalyzed ubiquitination but only limited degradation. Supplementation with proteasome failed to enhance degradation. Rates of ubiquitination and degradation of WT and deamidated βB1 crystallins were rapid and showed little relationship to the site of deamidation using N157D and Q204E mutants. γD-Crystallins were not degraded by the UPP. Deamidation altered amine reactivity, circular dichroism spectra, surface hydrophobicity, and thermal stability.
These data demonstrate for the first time that, like mild oxidative stress, deamidation of some proteins makes them preferred substrates for ubiquitination and, in some cells, for UPP-dependent degradation. Failure to properly execute ubiquitination and degrade the ubiquitin-conjugates may explain their accumulation on aging and in cataractogenesis.
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