Addition of αA-crystallin sequence 164-173 to a mini-chaperone DFVIFLDVKHFSPEDLT alters the conformation but not the chaperone-like activity.
It has been shown that αA-mini-chaperone, a peptide representing the chaperone binding site in αA-crystallin, prevents destabilized protein aggregation. αA-Mini-chaperone has been shown to form amyloid fibrils. This study was undertaken to improve the stability of αA-mini-chaperone while preserving its anti-aggregation activity by fusing the flexible and solvent-exposed C-terminal 164-173 region of αA-crystallin to the mini-chaperone sequence DFVIFLDVKHFSPEDLT. The resulting chimeric chaperone peptide, DFVIFLDVKHFSPEDLTEEKPTSAPSS (designated CP1), was characterized. Circular dichroism studies showed that unlike αA-mini-chaperone with its β-sheet structure, the CP1 peptide exhibited a random structure. Transmission electron microscopy (TEM) examination of the CP1 peptide incubated in a shaker at 37 °C for 72 h did not reveal amyloid fibrils, whereas αA-mini-chaperone showed distinct fibrils. Consistent with TEM observation, the thioflavin T binding assay showed an increased level of dye binding in the mini-chaperone incubated at 37 °C and subjected to shaking but not of the CP1 peptide incubated under similar conditions. The chaperone activity of the CP1 peptide was comparable to that of αA-mini-chaperone against denaturing alcohol dehydrogenase, citrate synthase, and α-lactalbumin. Transduction of both peptide chaperones to COS-7 cells showed no cytotoxic effects. The antioxidation assay involving the H2O2 treatment of COS-7 cells revealed that αA-mini-chaperone and the CP1 peptide have comparable cytoprotective properties against H2O2-induced oxidative damage in COS-7 cells. This study therefore shows that the addition of C-terminal sequence 164-173 of αA-crystallin to αA-mini-chaperone influences the conformation of αA-mini-chaperone without affecting its chaperone function or cytoprotective activity.
Department of Ophthalmology, University of Missouri-Columbia School of Medicine , Columbia, Missouri 65212, United States.
Murugesan
M
Raju
Puttur
P
Santhoshkumar
Leike
L
Xie
K Krishna
KK
Sharma
D000595:Amino Acid Sequence; D000818:Animals; D052160:Benzothiazoles; D019556:COS Cells / Q000187:drug effects / Q000378:metabolism; D002522:Chlorocebus aethiops; D002942:Circular Dichroism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D046529:Microscopy, Electron, Transmission; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D038202:alpha-Crystallin A Chain / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D052160:Benzothiazoles; D018832:Molecular Chaperones; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013844:Thiazoles; D038202:alpha-Crystallin A Chain; C009462:thioflavin T; D006861:Hydrogen Peroxide