TY - JOUR
T1 - Ubiquitin proteasome pathway-mediated degradation of proteins
T2 - Effects due to site-specific substrate deamidation
AU - Dudek, Edward J.
AU - Lampi, Kirsten J.
AU - Lampi, Jason A.
AU - Shang, Fu
AU - King, Jonathan
AU - Wang, Yongting
AU - Taylor, Allen
PY - 2010/8
Y1 - 2010/8
N2 - PURPOSE. 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. METHODS. Wild-type (WT) and deamidated crystallins were expressed and 125I-radiolabeled. Ubiquitination and degradation were monitored separately. RESULTS. For β2 crystallins, rates of ubiquitination and adenosine triphosphate-dependent degradation, both indicators of active UPP, occurred in the order Q70E/Q162E>Q162E>Q70E=WT>B2 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 β1 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. CONCLUSIONS. 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.
AB - PURPOSE. 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. METHODS. Wild-type (WT) and deamidated crystallins were expressed and 125I-radiolabeled. Ubiquitination and degradation were monitored separately. RESULTS. For β2 crystallins, rates of ubiquitination and adenosine triphosphate-dependent degradation, both indicators of active UPP, occurred in the order Q70E/Q162E>Q162E>Q70E=WT>B2 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 β1 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. CONCLUSIONS. 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.
UR - http://www.scopus.com/inward/record.url?scp=77955866089&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=77955866089&partnerID=8YFLogxK
U2 - 10.1167/iovs.09-4087
DO - 10.1167/iovs.09-4087
M3 - Article
C2 - 20592226
AN - SCOPUS:77955866089
SN - 0146-0404
VL - 51
SP - 4164
EP - 4173
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
IS - 8
ER -