Abstract
Purpose: :
To determine the effect of small molecule chemical chaperone (4–phenylbutyric acid, PBA) on the trafficking defects of glaucoma–causing mutants of myocilin (MYOC).
Methods: :
FLAG–tagged wild–type (WT) MYOC was transiently transfected to clonal cells stably expressing green fluorescent protein–tagged Cys245Tyr (GenBank Accession No. AY599652) and other disease–causing mutants (Gly364Val, Pro370Leu and Tyr437His) of MYOC. The cells were treated or not with 1 mM PBA for different duration. The secretion pattern, Triton X–100 solubility and intracellular distribution of these proteins were characterized by immunoprecipitation, western blotting and confocal double–labeled immunofluorescence. The effect of PBA on apoptosis and unfolded protein response was examined.
Results: :
Cys245Tyr MYOC mutant was retained in the endoplasmic reticulum (ER) via preferential interaction with calreticulin, which resulted in a secretion blockade. The mutant Cys245Tyr MYOC caused a gain–of–function effect by interacting with WT and the resulting heterodimers formed insoluble, intracellularly retained protein aggregates. The presence of combined WT and mutant MYOC protein aggregates caused apoptosis. The existence of different apoptosis rates among disease–causing mutants suggested a genotype–phenotype relationship. PBA treatment significantly increased the soluble fraction of mutant MYOC and thus, induced its secretion. In heterozygous MYOC expression, treatment enhanced the solubility of both WT and mutant MYOC and led to reduced apoptosis rates. This was accompanied by suppression of the unfolded protein response.
Conclusions: :
Defective trafficking of glaucoma–causing mutants of MYOC was corrected by PBA treatment. The use of small molecule chemical chaperone offers a strategy for the therapy of myocilin glaucoma.
Keywords: protein modifications-post translational • chaperones • cell survival