Submitted to: Investigative Ophthalmology and Visual Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/23/2009
Publication Date: 2/6/2010
Citation: Dudek, E.J., Lampi, K.J., Lampi, J., Shang, F., King, J., Wang, Y., Taylor, A. 2010. Ubiquitin proteasome pathway-mediated degradation of proteins: effects due to site-specific substrate deamidation. Investigative Ophthalmology and Visual Science. 51(8):4164-4173. Interpretive Summary: The stability of many molecules change upon aging, and proteins in cells are not exceptions. Gaining an understanding of how proteins change and what happens to them when they do change will allow us to adjust cellular metabolism to prolong function. In this work, we showed for the first time that proteins loose nitrogen upon aging, a process specifically called deamidation. These deamidated proteins are treated differently in cells as compared with the fully functional "younger" forms of the protein. Gaining this understanding also provided insight into the aging process and information about dietary and pharmacologic means to prolong youthful function.
Technical Abstract: The accumulation, aggregation, and precipitation of proteins are 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 12**5I-radiolabeled. Ubiquitination and degradation were monitored separately. For BetaB2 crystallins, rates of ubiquitination and adenosine triphosphate–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 only limited degradation. Supplementation with proteasome failed to enhance degradation. Rates of ubiquitination and degradation of WT and deamidated BetaB1 crystallins were rapid and showed little relationship to the site of deamidation using N157D and Q204E mutants. GammaD-Crystallins were not degraded by the UPP. Deamidation altered amine reactivity, circular dichroism spectra, surface hydrophobicity, and thermal stability. These data demonstrates 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 executeubiquitination and degrade the ubiquitin-conjugates may explain their accumulation on aging and in cataractogenesis.