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Addition of αA-crystallin sequence 164-173 to a mini-chaperone DFVIFLDVKHFSPEDLT alters the conformation but not the chaperone-like activity.

Raju M, Santhoshkumar P, Xie L, Sharma KK - Biochemistry (2014)

Bottom Line: The resulting chimeric chaperone peptide, DFVIFLDVKHFSPEDLTEEKPTSAPSS (designated CP1), was characterized.Transduction of both peptide chaperones to COS-7 cells showed no cytotoxic effects.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Missouri-Columbia School of Medicine , Columbia, Missouri 65212, United States.

ABSTRACT
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.

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Suppressionof oxidant-induced cell death by αA-mini-chaperoneor CP1-chaperone. For the data displayed in the left panel, COS-7cells were treated with 150 μM H2O2 and0–20 μg of either mini-αA or chimeric mini-αAfor 3 h. Apoptosis was assessed by TUNEL staining, and the percentageof dead cells was quantified by counting grids. Asterisks indicate P < 0.01 for mini-αA or CP1 at 10–20 μgcompared to control. The right panel shows TUNEL staining of the samples:(A) control, (B) CP1 (20 μg/mL), (C) αA-mini-chaperone(20 μg/mL), and (D) wild-type αA (100 μg/mL).
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fig8: Suppressionof oxidant-induced cell death by αA-mini-chaperoneor CP1-chaperone. For the data displayed in the left panel, COS-7cells were treated with 150 μM H2O2 and0–20 μg of either mini-αA or chimeric mini-αAfor 3 h. Apoptosis was assessed by TUNEL staining, and the percentageof dead cells was quantified by counting grids. Asterisks indicate P < 0.01 for mini-αA or CP1 at 10–20 μgcompared to control. The right panel shows TUNEL staining of the samples:(A) control, (B) CP1 (20 μg/mL), (C) αA-mini-chaperone(20 μg/mL), and (D) wild-type αA (100 μg/mL).

Mentions: To studywhether αA-mini-chaperone and CP1 peptide offer protection againstoxidative stress in mammalian cells, we incubated COS-7 cells with0–20 μg/mL chaperone peptides for 4 h in Bio-Porter,followed by 150 μM H2O2. In another setof experiments, 25–100 μg/mL αA-crystallin wasused in place of chaperone peptides. We selected this concentrationfor mini-chaperones because other studies have shown that αA-mini-chaperoneexerts protection against oxidative injury to RPE cells at this concentration.9 Apoptotic cell death was assessed by the TUNELassay. Our data show that both αA-mini-chaperone and CP1 protectedCOS-7 cells from apoptotic cell death (Figure 8). This protection was highly significant (P <0.01) when compared to the cells treated with H2O2 and without chaperone peptides. The dose-dependent effect of thepeptide chaperone against H2O2 treatment isalso shown in Figure 8. The CP1 peptide wasless effective than the αA-mini-chaperone, although it was protective.


Addition of αA-crystallin sequence 164-173 to a mini-chaperone DFVIFLDVKHFSPEDLT alters the conformation but not the chaperone-like activity.

Raju M, Santhoshkumar P, Xie L, Sharma KK - Biochemistry (2014)

Suppressionof oxidant-induced cell death by αA-mini-chaperoneor CP1-chaperone. For the data displayed in the left panel, COS-7cells were treated with 150 μM H2O2 and0–20 μg of either mini-αA or chimeric mini-αAfor 3 h. Apoptosis was assessed by TUNEL staining, and the percentageof dead cells was quantified by counting grids. Asterisks indicate P < 0.01 for mini-αA or CP1 at 10–20 μgcompared to control. The right panel shows TUNEL staining of the samples:(A) control, (B) CP1 (20 μg/mL), (C) αA-mini-chaperone(20 μg/mL), and (D) wild-type αA (100 μg/mL).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4007981&req=5

fig8: Suppressionof oxidant-induced cell death by αA-mini-chaperoneor CP1-chaperone. For the data displayed in the left panel, COS-7cells were treated with 150 μM H2O2 and0–20 μg of either mini-αA or chimeric mini-αAfor 3 h. Apoptosis was assessed by TUNEL staining, and the percentageof dead cells was quantified by counting grids. Asterisks indicate P < 0.01 for mini-αA or CP1 at 10–20 μgcompared to control. The right panel shows TUNEL staining of the samples:(A) control, (B) CP1 (20 μg/mL), (C) αA-mini-chaperone(20 μg/mL), and (D) wild-type αA (100 μg/mL).
Mentions: To studywhether αA-mini-chaperone and CP1 peptide offer protection againstoxidative stress in mammalian cells, we incubated COS-7 cells with0–20 μg/mL chaperone peptides for 4 h in Bio-Porter,followed by 150 μM H2O2. In another setof experiments, 25–100 μg/mL αA-crystallin wasused in place of chaperone peptides. We selected this concentrationfor mini-chaperones because other studies have shown that αA-mini-chaperoneexerts protection against oxidative injury to RPE cells at this concentration.9 Apoptotic cell death was assessed by the TUNELassay. Our data show that both αA-mini-chaperone and CP1 protectedCOS-7 cells from apoptotic cell death (Figure 8). This protection was highly significant (P <0.01) when compared to the cells treated with H2O2 and without chaperone peptides. The dose-dependent effect of thepeptide chaperone against H2O2 treatment isalso shown in Figure 8. The CP1 peptide wasless effective than the αA-mini-chaperone, although it was protective.

Bottom Line: The resulting chimeric chaperone peptide, DFVIFLDVKHFSPEDLTEEKPTSAPSS (designated CP1), was characterized.Transduction of both peptide chaperones to COS-7 cells showed no cytotoxic effects.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Missouri-Columbia School of Medicine , Columbia, Missouri 65212, United States.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus