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Oligomerization with wt ?A- and ?B-crystallins reduces proteasome-mediated degradation of C-terminally truncated ?A-crystallin.

ABSTRACT: We previously demonstrated that the ubiquitin-proteasome pathway (UPP) is a general protein quality control system that selectively degrades damaged or abnormal lens proteins, including C-terminally truncated ?A-crystallin. The objective of this work was to determine the effects of wt ?A- and ?B-crystallins on the degradation of C-terminally truncated ?A-crystallin (?A(1-162)) and vice versa.Recombinant wt ?A, ?B, and ?A(1-162) were expressed in Escherichia coli and purified to homogeneity by chromatography. Subunit exchange and oligomerization were detected by fluorescence resonance energy transfer (FRET), multiangle-light scattering and coprecipitation assays. Protein substrates were labeled with (125)I and lens epithelial cell lysates were used as the source of the UPP for degradation assays.FRET, multiangle light scattering, and coprecipitation assays showed that ?A(1-162) exchanged subunits with wt ?A- or wt ?B- crystallin to form hetero-oligomers. ?A(1-162) was more susceptible than wt ?A-crystallin to degradation by the UPP. When mixed with wt ?A-crystallin at 1:1 or 1:4 (?A(1-162) : wt) ratios to form hetero-oligomers, the degradation of ?A(1-162) was significantly decreased. Conversely, formation of hetero-oligomers with ?A(1-162) enhanced the degradation of wt ?A-crystallin. The presence of ?A(1-162), but not wt ?A-crystallin, decreased the degradation of wt ?B-crystallin.?A(1-162) forms hetero-oligomers with wt ?A- and ?B-crystallins. Oligomerization with wt ?A- or ?B-crystallins reduces the susceptibility of ?A(1-162) to degradation by the UPP. In addition, the presence of ?A(1-162) in the hetero-oligomers also affects the degradation of wt ?A- and ?B-crystallins.

SUBMITTER: Wu M

PROVIDER: S-EPMC3358126 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Oligomerization with wt αA- and αB-crystallins reduces proteasome-mediated degradation of C-terminally truncated αA-crystallin.

Wu Mingxing M Zhang Xinyu X Bian Qingning Q Taylor Allen A Liang Jack J JJ Ding Linlin L Horwitz Joseph J Shang Fu F

Investigative ophthalmology & visual science 20120504 6

<h4>Purpose</h4>We previously demonstrated that the ubiquitin-proteasome pathway (UPP) is a general protein quality control system that selectively degrades damaged or abnormal lens proteins, including C-terminally truncated αA-crystallin. The objective of this work was to determine the effects of wt αA- and αB-crystallins on the degradation of C-terminally truncated αA-crystallin (αA(1-162)) and vice versa.<h4>Methods</h4>Recombinant wt αA, αB, and αA(1-162) were expressed in Escherichia coli a ...[more]

PMID: 22427585

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Project description:Interaction among crystallins is required for the maintenance of lens transparency. Deamidation is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the ?- and ?-crystallins. Here, we investigated the effects of the deamidation of ?A- and ?B-crystallins on their interaction with ?A3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT ?A- and WT ?B-crystallins and their deamidated mutants with ?A3-crystallin. The deamidated mutants of ?A-crystallin (?A N101D and ?A N123D) displayed lower adherence propensity for ?A3-crystallin relative to the binding affinity shown by WT ?A-crystallin. Among ?B-crystallin mutants, ?B N78D displayed higher adherence propensity whereas ?B N146D mutant showed slightly lower binding affinity for ?A3-crystallin relative to that shown by WT ?B-crystallin. Under the in vivo condition (FLIM-FRET), both ?A-deamidated mutants (?A N101D and ?A N123D) exhibited strong interaction with ?A3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT ?A-crystallin (18±4%). Similarly, the ?B N78D and ?B N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with ?A3-crystallin compared to 18±4% FRET efficiency of WT ?B-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of ?A- and ?B-crystallins with ?A3-crystallin suggested that interaction sites most likely reside in the ?A CTE and ?B NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT ?A- and WT?B-crystallins, the deamidated mutants showed strong interactionfor ?A3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant's large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of ?-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.

Dataset Information

Oligomerization with wt ?A- and ?B-crystallins reduces proteasome-mediated degradation of C-terminally truncated ?A-crystallin.

Publications

Oligomerization with wt αA- and αB-crystallins reduces proteasome-mediated degradation of C-terminally truncated αA-crystallin.

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