June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Synthesis and Characterization of Cell-penetrating Peptide Chaperone
Author Affiliations & Notes
  • K Krishna Sharma
    Ophthalmology , University of Missouri, Columbia, Missouri, United States
    Biochemistry, University of Missouri, Columbia, Missouri, United States
  • Murugesan Raju
    Ophthalmology , University of Missouri, Columbia, Missouri, United States
  • Puttur Santhoshkumar
    Ophthalmology , University of Missouri, Columbia, Missouri, United States
  • Footnotes
    Commercial Relationships   K Krishna Sharma, 62/352716 (P), Plex Pharmaceuticals (C); Murugesan Raju, 62/352716 (P); Puttur Santhoshkumar, 62/352716 (P)
  • Footnotes
    Support  EY 023219
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 5598. doi:
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    • Get Citation

      K Krishna Sharma, Murugesan Raju, Puttur Santhoshkumar; Synthesis and Characterization of Cell-penetrating Peptide Chaperone. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5598.

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

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Abstract

Purpose : Previous studies showed that the peptide chaperone, representing the chaperone site in αA-crystallin, can inhibit protein aggregation and offer protection to cells subjected to oxidative stress. The objective of the present study was to synthesize and test the properties of a peptide chaperone that can across the cell membrane more efficiently by incorporating a cell penetrating sequence to the peptide chaperone.

Methods : Synthetic mini-chaperone was prepared by fusing the VPTLK sequence to the N-terminus of mini-chaperone to get VPTLKFVIFLDVKHFSPEDLTVKGRD peptide (mini-chaperone-GRD). CD spectroscopy was used to determine the secondary structure and bis-ANS binding study was performed to determine the relative hydrophobicity of the peptide chaperone. The cell-penetration efficiency of mini-chaperone-GRD and αA-mini-chaperone where compared using FITC-labeled peptides. The chaperone-like function of the mini-chaperone GRD was measured using denaturing ADH and α-lactalbumin proteins and beta amyloid peptide. The anti-apoptotic action of the peptide chaperone was evaluated using H2O2 with Cos-7 and ARPE-19 cells in apoptosis assays.

Results : The mini-chaperone-GRD showed β-sheet structure and was found to be very hydrophobic in nature. The peptide chaperone entered the Cos-7 cells more readily than the mini-chaperone peptide characterized earlier. Addition of cell penetrating sequence did not affect the chaperone activity of the peptide chaperone. There was about 20-25 percent higher chaperone activity in the mini-chaperone-GRD peptide. Additionally, the mini-chaperone-GRD was found to prevent β-amyloid fibril formation and reduce cellular toxicity of β-amyloid when it was used in equimolar concentration. The mini-chaperone-GRD chaperone also showed anti-apoptotic activity when the Cos-7 and ARPE-19 cells were subjected to oxidative stress by H2O2.

Conclusions : The present study demonstrates that mini-chaperone-GRD peptide protects denaturing proteins from aggregation and suppresses cellular apoptosis by H2O2. Therefore, the mini-chaperone-GRD is a novel mini-chaperone with a potential to become a therapeutic agent in protein aggregation diseases.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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