title: Antiapoptotic properties of α-crystallin-derived peptide chaperones and characterization of their uptake transporters in human RPE cells.
abstract: OBJECTIVE
  The chaperone proteins, α-crystallins, also possess antiapoptotic properties. The purpose of the present study was to investigate whether 19 to 20-mer α-crystallin-derived mini-chaperone peptides (α-crystallin mini-chaperone) are antiapoptotic, and to identify their putative transporters in human fetal RPE (hfRPE) cells.
  
  
  METHODS
  Cell death and caspase-3 activation induced by oxidative stress were quantified in early passage hfRPE cells in the presence of 19 to 20-mer αA- or αB-crystallin-derived or scrambled peptides. Cellular uptake of fluorescein-labeled, α-crystallin-derived mini-peptides and recombinant full-length αB-crystallin was determined in confluent hfRPE. The entry mechanism in hfRPE cells for α-crystallin mini-peptides was investigated. The protective role of polycaprolactone (PCL) nanoparticle encapsulated αB-crystallin mini-chaperone peptides from H2O2-induced cell death was studied.
  
  
  RESULTS
  Primary hfRPE cells exposed to oxidative stress and either αA- or αB-crystallin mini-chaperones remained viable and showed marked inhibition of both cell death and activation of caspase-3. Uptake of full-length αB-crystallin was minimal while a time-dependent uptake of αB-crystallin-derived peptide was observed. The mini-peptides entered the hfRPE cells via the sodium-coupled oligopeptide transporters 1 and 2 (SOPT1, SOPT2). PCL nanoparticles containing αB-crystallin mini-chaperone were also taken up and protected hfRPE from H2O2-induced cell death at significantly lower concentrations than free αB-crystallin mini-chaperone peptide.
  
  
  CONCLUSIONS
  αA- and αB-crystallin mini-chaperones offer protection to hfRPE cells and inhibit caspase-3 activation. The oligopeptide transporters SOPT1 and SOPT2 mediate the uptake of these peptides in RPE cells. Nanodelivery of αB-crystallin-derived mini-chaperone peptide offers an alternative approach for protection of hfRPE cells from oxidant injury.
authors.affiliation: Arnold and Mabel Beckman Macular Research Center, Doheny Eye Institute, Los Angeles, California, USA.
authors.forename: Parameswaran G
authors.initials: PG
authors.lastname: Sreekumar
authors.forename: Paresh
authors.initials: P
authors.lastname: Chothe
authors.forename: Krishna K
authors.initials: KK
authors.lastname: Sharma
authors.forename: Rinku
authors.initials: R
authors.lastname: Baid
authors.forename: Uday
authors.initials: U
authors.lastname: Kompella
authors.forename: Christine
authors.initials: C
authors.lastname: Spee
authors.forename: Nandini
authors.initials: N
authors.lastname: Kannan
authors.forename: Christina
authors.initials: C
authors.lastname: Manh
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Ryan
authors.forename: Vadivel
authors.initials: V
authors.lastname: Ganapathy
authors.forename: Ram
authors.initials: R
authors.lastname: Kannan
authors.forename: David R
authors.initials: DR
authors.lastname: Hinton
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000502:physiology; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D026901:Membrane Transport Proteins / Q000378:metabolism*; D018832:Molecular Chaperones / Q000502:physiology*; D016877:Oxidants / Q000633:toxicity; D018384:Oxidative Stress / Q000187:drug effects; D010455:Peptides / Q000502:physiology*; D011994:Recombinant Proteins; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000378:metabolism; D013997:Time Factors; D038202:alpha-Crystallin A Chain / Q000502:physiology*; D038203:alpha-Crystallin B Chain / Q000502:physiology*
chemical_list: D051017:Apoptosis Regulatory Proteins; D026901:Membrane Transport Proteins; D018832:Molecular Chaperones; D016877:Oxidants; D010455:Peptides; D011994:Recombinant Proteins; D038202:alpha-Crystallin A Chain; D038203:alpha-Crystallin B Chain; D006861:Hydrogen Peroxide; D053148:Caspase 3