title: Mutations in the Corneal Endothelial Dystrophy-Associated Gene SLC4A11 Render the Cells More Vulnerable to Oxidative Insults.
abstract: OBJECTIVE
  To investigate the effect of mutations in SLC4A11 on cellular localization of the protein, mitochondrial function, and apoptosis due to oxidative stress. Mutations in SLC4A11 have been associated with 2 different forms of corneal endothelial dystrophy that lead to degeneration of the corneal endothelium, causing opacity of the cornea and gradual vision loss.
  
  
  METHODS
  HEK 293 cells were transfected with wild-type SLC4A11 or mutants, Ser213Leu, Arg233Cys, Gly418Asp, and Thr584Lys, and exposed to oxidative stress. Cellular localization of the proteins was detected by confocal microscopy, whereas mitochondrial dysfunction, reactive oxygen species (ROS) generation, and apoptosis were analyzed by flow cytometry and a colorimetric assay. Expressions of antioxidant genes were quantitated by real-time polymerase chain reaction.
  
  
  RESULTS
  Although wild-type SLC4A11 was localized on the cell membrane, mutant proteins were found diffused in the cytoplasm. Mutations in SLC4A11 caused an increase in generation of ROS and mitochondrial dysfunction due to oxidative stress. NRF2, HO-1, and NQO expression decreased significantly, and a higher rate of apoptosis was detected in cells with mutant proteins under oxidative stress.
  
  
  CONCLUSIONS
  Our data suggest that mutations in SLC4A11 cause retention of the protein in the cytoplasm and generate increased reactive oxygen species. We found that cells containing mutant SLC4A11 are more vulnerable to oxidative and mitochondrial damage, less able to overcome oxidative stress through the expression of sufficient levels of antioxidant genes, and are more prone to apoptotic death.
authors: Prof. Brien Holden Eye Research Center, Hyderabad Eye Research Foundation, LVPEI, Hyderabad, India.
authors: Sanhita
authors: S
authors: Roy
authors: D C
authors: DC
authors: Praneetha
authors: Venkata Pulla Rao
authors: VP
authors: Vendra
mesh_terms: D027321:Anion Transport Proteins / Q000235:genetics*; D017920:Antiporters / Q000235:genetics*; D017209:Apoptosis; D003124:Colorimetry; D003317:Corneal Dystrophies, Hereditary; D005434:Flow Cytometry; D005642:Fuchs' Endothelial Dystrophy / Q000235:genetics*; D005786:Gene Expression Regulation / Q000502:physiology*; D057809:HEK293 Cells / Q000187:drug effects / Q000473:pathology; D051547:Heme Oxygenase-1 / Q000235:genetics; D006801:Humans; D018613:Microscopy, Confocal; D028361:Mitochondrial Diseases / Q000235:genetics*; D020125:Mutation, Missense; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics; D051267:NF-E2-Related Factor 2 / Q000235:genetics; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D014162:Transfection; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
chemical_list: D027321:Anion Transport Proteins; D017920:Antiporters; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D017382:Reactive Oxygen Species; C425809:SLC4A11 protein, human; D020122:tert-Butylhydroperoxide; C496541:HMOX1 protein, human; D051547:Heme Oxygenase-1; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human