May 2003
Volume 44, Issue 13
ARVO Annual Meeting Abstract  |   May 2003
Inhibition of Amyloid Fibrillogenesis by a Peptide Chaperone Derived from A-crystallin
Author Affiliations & Notes
  • P. Santhoshkumar
    Ophthalmology, University of Missouri, Columbia, MO, United States
  • K. Sharma
    Ophthalmology and Biochemistry, University of Missouri, Columbia, MO, United States
  • Footnotes
    Commercial Relationships  P. Santhoshkumar, None; K. Sharma, None.
  • Footnotes
    Support  EY11981
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 2375. doi:
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      P. Santhoshkumar, K. Sharma; Inhibition of Amyloid Fibrillogenesis by a Peptide Chaperone Derived from A-crystallin . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2375.

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

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Abstract: : Purpose: To test the ability of a peptide chaperone (DFVIFLDVKHFSPEDLTVK), representing the functional site in αA-crystallin, to prevent amyloid fibrillogenesis and toxicity. Methods Human beta amyloid (ßA) peptides (100 µg) with sequence 1-40 and 22-35 were incubated with various amounts of αA-crystallin peptide in betaine buffer at 37oC. Aliquots were drawn at various time intervals and observed under transmission electron microscopy after negatively staining with 1.3% phosophotungstic acid. To determine the extent of amyloid formation, aliquots were withdrawn from the incubation mixture and treated with thioflavin T (ThT) dye. The sample was excited at 437 nm and the ThT fluorescence emission was measured at 485 nm. The toxic effect of peptide aggregates was determined using rat pheochromocytoma (PC12) cells. PC12 cells cultured in DME medium on a 96 well plate were treated with various concentrations of peptide mixtures and incubated for 24 hr. It was then treated with 3 – (4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and incubated further for 6 hr. The cellular MTT reduction was measured by colorimetric determination of MTT formazan product formation at 570 nm. Results At half the molar concentration, αA-crystallin peptide suppress ßA1-40 and 22-35 fibril formation and at 1:1 concentration it totally inhibits amyloid fibrillogenesis. This is also indicated by decrease in ThT fluorescence of ßA –αA-crystallin peptide incubations. PC12 cells incubated with ßA peptide showed a significant decrease in MTT reduction when compared to those incubated with ßA and αA-peptide mixture. Conclusions The αA-crystallin peptide chaperone inhibits amyloid fibrillogenesis and decreases the toxicity in vitro. The data suggests that the chaperone region in αA-crystallin is involved in the prevention of amyloid fibrillogenesis. This work was supported by NIH grant EY11981 and Research to Prevent Blindness.

Keywords: chaperones • crystallins • neuroprotection 

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