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K. J. Lampi, E. J. Dudek, F. Shang, J. King, Y. Wang, A. Taylor; Protein Ubiquitination and Ubiquitin-mediated Degradation Respond to Substrate Deamidation. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2098.
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© ARVO (1962-2015); The Authors (2016-present)
Removal of damaged and altered proteins, usually by the ubiquitin proteasome pathway (UPP) is critical to retain cellular homeostatic function, particularly upon aging. The accumulation, aggregation and precipitation of damaged proteins are etiologic for cataract. The purpose of this research was to determine if deamidation enhanced the degradation of crystallins by the UPP.
Wild-type (WT) and deamidated crystallins were expressed in E.coli, isolated, and 125I-radiolabeled. Ubiquitination and degradation were monitored using autoradiography and quantitation of TCA-soluble CPM.
For betaB2 crystallins, rates of ubiquitination and ATP-dependent degradation, both indicators of active UPP, occurred in the order Q70E/Q162E> Q162E> Q70E= WT betaB2 using reticulocyte lysate as the source of degradation machinery. Human lens epithelial cell lysates and lens fiber cell lysates also catalyzed ubiquitination, but they failed to execute degradation. Supplementation with proteasome failed to enhance degradation in the lens cell lysates, consistent with faulty ubiquitination in the lens systems. Rates of ubiquitination and degradation of WT and deamidated betaB1 crystallins were also UPP-dependent and much more rapid than for the betaB2 crystallins, but showed little relationship to the site of deamidation using N157D and Q204E variants. gammaD-crystallins were not degraded by the UPP. CD, surface hydrophobicity, thermal denaturation, and amine reactivity were not systematically associated with propensity for degradation.
These data demonstrate for the fist time, that like mild oxidative stress, deamidation of some, but not all proteins makes them preferred substrates for ubiquitination, and in some cells for UPP-dependent degradation. A failure to degrade the ubiquitin-conjugates that are formed may explain their accumulation upon aging and cataract formation.
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