Sodium 4-phenylbutyrate acts as a chemical chaperone on misfolded myocilin to rescue cells from endoplasmic reticulum stress and apoptosis.
OBJECTIVE
To evaluate the effect of chemical chaperones on the trafficking of secretion-incompetent primary open-angle glaucoma-associated mutant myocilin and the possibility to rescue cells coexpressing mutant and wild-type myocilin from endoplasmic reticulum (ER) stress and apoptosis.


METHODS
CHO-K1, HEK293 and human trabecular meshwork cells were transfected to express wild-type or mutant (C245Y, G364V, P370L, Y437H) myocilin-green fluorescent protein fusion protein and were treated or not with various chemical chaperones (glycerol, dimethylsulfoxide, or sodium 4-phenylbutyrate) for different time periods. The secretion, Triton X-100 solubility, and intracellular distribution of wild-type and mutant myocilin were analyzed by immunoprecipitation, Western blotting, and confocal double immunofluorescence. The effect of sodium 4-phenylbutyrate on ER stress proteins and apoptosis was examined in cells coexpressing mutant and wild-type myocilin.


RESULTS
Treatment with sodium 4-phenylbutyrate, but not with glycerol or dimethylsulfoxide, reduced the amount of detergent-insoluble myocilin aggregates, diminished myocilin interaction with calreticulin, and restored the secretion of mutant myocilin. Heteromeric complexes formed by mutant and wild-type myocilin induced the ER stress-associated phosphorylated form of ER-localized eukaryotic initiation factor (eIF)-2alpha kinase and the active form of caspase 3, which resulted in an increased rate of apoptosis. Sodium 4-phenylbutyrate treatment of cells coexpressing mutant and wild-type myocilin relieved ER stress and significantly reduced the rate of apoptosis.


CONCLUSIONS
These findings indicate that sodium 4-phenylbutyrate protects cells from the deleterious effects of ER-retained aggregated mutant myocilin. These data point to the possibility of a chemical chaperone treatment for myocilin-caused primary open-angle glaucoma.
Yam
Gary Hin-Fai
GH
Division of Cell and Molecular Pathology, Department of Pathology, University of Zurich, Schmelzbergstrasse 12, CH-8091 Zurich, Switzerland.
Gaplovska-Kysela
Katarina
K
Zuber
Christian
C
Roth
Jürgen
J
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D016466:CHO Cells / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000187:drug effects*; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000196:embryology; D018613:Microscopy, Confocal; D018832:Molecular Chaperones / Q000494:pharmacology*; D010654:Phenylbutyrates / Q000494:pharmacology*; D010957:Plasmids; D017510:Protein Folding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000378:metabolism; D014162:Transfection; D019892:eIF-2 Kinase / Q000378:metabolism; D000099179:Myocilin
D053148:Caspase 3; D003598:Cytoskeletal Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D018832:Molecular Chaperones; D010654:Phenylbutyrates; D019892:eIF-2 Kinase; D000099179:Myocilin; C075773:4-phenylbutyric acid