Purchase this article with an account.
B. Gong, L. Zhang, D. Lam, C. Pang, G. Yam; Chemical Chaperones Reduced Aggregate Formation of Cataract-Causing Gly98arg Alpha-Crystallin. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2092.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To investigate the mechanism how chemical chaperones affect cataract-causing Gly98Arg mutant alphaA-crystallin.
Recombinant expression construct containing human full-length wildtype and Gly98Arg alphaA-crystallin were created, verified by sequencing and transfected to human lens epithelial B3 cells. The cells were treated with chemical chaperones (4-phenylbutyric acid, 4-PBA, 0.25 to 3mM or trimethylamine N-oxide, TMAO, 25 to 300mM) for 1 to 4 days. The effect on alphaA-crystallin degradation was studied by treatment with MG132 (inhibitor of ubiquitin-proteasome pathway) or 3-methyladenine (inhibitor of autophagy). Protein solubility and localization were examined by western blotting and immunofluorescence, respectively.
Gly98Arg alphaA-crystallin was predominantly insoluble upon 1% Triton X-100 (Tx) extraction, when compared to the soluble wildtype protein. By immunofluorescence, mutant-expressing cells had extensive intracellular aggregates, which were positive for myc (representing Gly98Arg alphaA-crystallin) and co-localized with the ER resident protein, protein disulfide isomerase. Treatment with chemical chaperones (4-PBA or TMAO) reduced Tx-insoluble Gly98Arg alphaA-crystallin in a dose-dependent manner. It also suppressed the ER aggregates, which were prone to be degraded. Simultaneous treatment with MG132 only moderately reduced Tx-insoluble mutant protein. Morphologically, ER aggregates containing Gly98Arg alphaA-crystallin remained after inhibition.
This PDF is available to Subscribers Only