title: Sodium 4-phenylbutyrate ameliorates the effects of cataract-causing mutant gammaD-crystallin in cultured cells.
abstract: OBJECTIVE
  gammaD-Crystallin (CRYGD) is a major structural lens crystallin and its mutations result in congenital cataract formation. In this study, we attempted to correct the altered protein features of G165fsX8 CRYGD protein with small chemical molecules.
  
  
  METHODS
  Recombinant FLAG-tagged mutants (R15C, R15S, P24T, R61C, and G165fsX8) of CRYGD were expressed in COS-7 cells and treated with small chemical molecules with reported protein chaperoning properties (sodium 4-phenylbutyrate [4-PBA], trimethylamine N-oxide [TMAO], and glycerol and DMSO [DMSO]). Protein solubility in 0.5% Triton X-100 and subcellular distribution was examined by western blotting and immunofluorescence, respectively. Apoptosis was assayed as the percentage of fragmented nuclei in transfected cells. Expression of heat-shock proteins (Hsp70 and Hsp90) was examined by reverse transcription-polymerase chain reaction analysis.
  
  
  RESULTS
  Unlike WT and most mutants (R15C, R15S, P24T, and R61C) of CRYGD, G165fsX8 CRYGD was significantly insoluble in 0.5% Triton X-100. This insolubility was alleviated by dose-dependent 4-PBA treatment. The treatment relieved the mislocalization of G165fsX8 CRYGD from the nuclear envelope. Also, 4-PBA treatment reduced cell apoptosis and caused an upregulation of Hsp70.
  
  
  CONCLUSIONS
  4-PBA treatment reduced the defective phenotype of mutant G165fsX8 CRYGD and rescued the affected cells from apoptosis. This could be a potential treatment for lens structural protein and prevent lens opacity in cataract formation.
authors.affiliation: Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Gong
authors.forename: Li-Yun
authors.initials: LY
authors.lastname: Zhang
authors.forename: Dennis Shun-Chiu
authors.initials: DS
authors.lastname: Lam
authors.forename: Chi-Pui
authors.initials: CP
authors.lastname: Pang
authors.forename: Gary Hin-Fai
authors.initials: GH
authors.lastname: Yam
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D019556:COS Cells / Q000378:metabolism / Q000502:physiology; D002386:Cataract / Q000151:congenital / Q000235:genetics* / Q000517:prevention & control; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D009154:Mutation; D009685:Nuclear Envelope / Q000378:metabolism; D010641:Phenotype; D010654:Phenylbutyrates / Q000008:administration & dosage / Q000494:pharmacology*; D012995:Solubility / Q000187:drug effects; D014018:Tissue Distribution / Q000187:drug effects; D014162:Transfection; D015854:Up-Regulation; D038222:gamma-Crystallins / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics*
chemical_list: D018840:HSP70 Heat-Shock Proteins; D010654:Phenylbutyrates; D038222:gamma-Crystallins; C075773:4-phenylbutyric acid