June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Elastin-like polypeptide (ELP) fused to an αB crystallin mini-chaperone protects RPE cells from oxidant injury
Author Affiliations & Notes
  • Parameswaran Sreekumar
    Arnold and Mabel Beckman Macular reserach Center, Doheny Eye Institute, Los Angeles, CA
  • WAN WANG
    Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA
  • John MacKay
    Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA
  • Christine Spee
    Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, CA
  • Stephen Ryan
    Arnold and Mabel Beckman Macular reserach Center, Doheny Eye Institute, Los Angeles, CA
    Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, CA
  • Ram Kannan
    Arnold and Mabel Beckman Macular reserach Center, Doheny Eye Institute, Los Angeles, CA
    Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, CA
  • David Hinton
    Arnold and Mabel Beckman Macular reserach Center, Doheny Eye Institute, Los Angeles, CA
    Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, CA
  • Footnotes
    Commercial Relationships Parameswaran Sreekumar, University of Southern California (P); WAN WANG, University of Southern California (P); John MacKay, University of Southern California (P); Christine Spee, None; Stephen Ryan, Alergan (S); Ram Kannan, University of Southern California (P); David Hinton, RPT (I), RPT (P)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 6075. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Parameswaran Sreekumar, WAN WANG, John MacKay, Christine Spee, Stephen Ryan, Ram Kannan, David Hinton; Elastin-like polypeptide (ELP) fused to an αB crystallin mini-chaperone protects RPE cells from oxidant injury. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6075.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: In addition to being a chaperon, αB crystallin has multiple functions including inhibition of apoptosis. The purpose of this study was to investigate the protective effect of protein polymer nanoparticles decorated with αB crystallin minipeptide (residues 73-92) that protects against oxidative injury to RPE. Derived from human tropoelastin, these elastin-like polypeptide (ELP) polymers assemble nanostructures, which we hypothesized can modulate the RPE uptake, retention, and efficacy of the mini-peptide.

Methods: ELP-αB crystallin mini-chaperone (ELP- αB crystallin) fusion protein was expressed and purified. Chaperone activity of the purified ELP-αB crystallin was measured by the alcohol dehydrogenase assay. To study the protective effect of ELP-αB crystallin, primary human RPE cells were co-incubated with varying doses of ELP - αB crystallin or ELP alone and 200 µM H2O2 for 24 h. RPE cell death and caspase-3 activation induced by oxidative stress were determined by TUNEL assay and Western blot analysis. Cellular uptake was determined in ELP particles after conjugation with rhodamine and incubation of cells with 200 µM H2O2 and 5 µM labeled ELP for 24 h and analysis of confocal microscopic images. Subcellular localization of uptake was studied by transmission electron microscopy (TEM).

Results: ELP-αB crystallin mini-chaperone (ELP- αB crystallin) fusion protein was expressed and purified. Chaperone activity of the purified ELP-αB crystallin was measured by the alcohol dehydrogenase assay. To study the protective effect of ELP-αB crystallin, primary human RPE cells were co-incubated with varying doses of ELP - αB crystallin or ELP alone and 200 µM H2O2 for 24 h. RPE cell death and caspase-3 activation induced by oxidative stress were determined by TUNEL assay and Western blot analysis. Cellular uptake was determined in ELP particles after conjugation with rhodamine and incubation of cells with 200 µM H2O2 and 5 µM labeled ELP for 24 h and analysis of confocal microscopic images. Subcellular localization of uptake was studied by transmission electron microscopy (TEM).

Conclusions: ELP-αB crystallin fusion proteins are taken up efficiently by RPE cells and offer cellular protection through their intracellular actions by inhibiting caspase-3 activation.

Keywords: 488 crystallins • 426 apoptosis/cell death • 450 chaperones  
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×