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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)
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.
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.
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.
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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