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Use of a small peptide fragment as an inhibitor of insulin fibrillation process: a study by high and low resolution spectroscopy.

Banerjee V, Kar RK, Datta A, Parthasarathi K, Chatterjee S, Das KP, Bhunia A - PLoS ONE (2013)

Bottom Line: In vitro hemolytic activity of the peptide showed insignificant cytotoxicity against HT1080 cells.The insulin aggregation is probed due to the inter play of two key residues, Phe(B24) and Tyr(B26) monitored from molecular dynamics simulations studies.Further new peptide based leads may be developed from this nine residue peptide.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Bose Institute, Kolkata, India.

ABSTRACT
A non-toxic, nine residue peptide, NIVNVSLVK is shown to interfere with insulin fibrillation by various biophysical methods. Insulin undergoes conformational changes under certain stress conditions leading to amyloid fibrils. Fibrillation of insulin poses a problem in its long-term storage, reducing its efficacy in treating type II diabetes. The dissociation of insulin oligomer to monomer is the key step for the onset of fibrillation. The time course of insulin fibrillation at 62°C using Thioflavin T fluorescence shows an increase in the lag time from 120 min without peptide to 236 min with peptide. Transmission electron micrographs show branched insulin fibrils in its absence and less inter-fibril association in its presence. Upon incubation at 62°C and pH 2.6, insulin lost some α-helical structure as seen by Fourier transformed infra-red spectroscopy (FT-IR), but if the peptide is added, secondary structure is almost fully maintained for 3 h, though lost partially at 4 h. FT-IR spectroscopy also shows that insulin forms the cross beta structure indicative of fibrils beyond 2 h, but in the presence of the peptide, α-helix retention is seen till 4 h. Both size exclusion chromatography and dynamic light scattering show that insulin primarily exists as trimer, whose conversion to a monomer is resisted by the peptide. Saturation transfer difference nuclear magnetic resonance confirms that the hydrophobic residues in the peptide are in close contact with an insulin hydrophobic groove. Molecular dynamics simulations in conjunction with principal component analyses reveal how the peptide interrupts insulin fibrillation. In vitro hemolytic activity of the peptide showed insignificant cytotoxicity against HT1080 cells. The insulin aggregation is probed due to the inter play of two key residues, Phe(B24) and Tyr(B26) monitored from molecular dynamics simulations studies. Further new peptide based leads may be developed from this nine residue peptide.

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Conformational changes during insulin fibrillation as detected by FT-IR.Time dependent FT-IR spectra of insulin (2.0 mg/ml in D2O) in absence (A) and presence (B) of NK9 in 62°C and pD 2.6. Amide-I band were acquired in the 1700–1600 cm−1 range at a resolution of 2 cm−1. Each spectrum is an average of 32 scans and subtracted from solvent spectrum. Panel C shows the ratio of absorbance of β-sheet peak at 1628 cm−1 to helical peak at 1654 cm−1. Filled square represents insulin and open circle represents insulin in presence of NK9. Panel D shows percentage of α-helix and β-sheet present in native and fibrillar insulin in presence and absence of NK9. The molar ratio of insulin to NK9 is 1∶1.
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pone-0072318-g004: Conformational changes during insulin fibrillation as detected by FT-IR.Time dependent FT-IR spectra of insulin (2.0 mg/ml in D2O) in absence (A) and presence (B) of NK9 in 62°C and pD 2.6. Amide-I band were acquired in the 1700–1600 cm−1 range at a resolution of 2 cm−1. Each spectrum is an average of 32 scans and subtracted from solvent spectrum. Panel C shows the ratio of absorbance of β-sheet peak at 1628 cm−1 to helical peak at 1654 cm−1. Filled square represents insulin and open circle represents insulin in presence of NK9. Panel D shows percentage of α-helix and β-sheet present in native and fibrillar insulin in presence and absence of NK9. The molar ratio of insulin to NK9 is 1∶1.

Mentions: FT-IR has been used to study secondary structure of proteins for decades [47], [50]–[53]. It is particularly sensitive to changes of β-sheet content in proteins [43]. Being an α helical protein insulin has a characteristic peak at ∼1654 cm−1 in its amide I contour. On conversion into amyloid like fibrils, the peak shifts to 1628 cm−1, which is the hallmark of cross β-sheet structure formation [54]. Figure 3 shows FT-IR spectra of insulin measured at various intervals of incubation. Plot of A1628/A1654 against time of incubation (Figure 4) showed very small changes in the ratio and spectral shape take place for the first 2 h of incubation. The transition involving significant increases in the A1628/A1654 ratio was observed after 2 h of incubation, and it persisted for 4 h. This is the cause of steady increase in intensity at 1628 cm−1, with a concomitant decrease in intensity at 1654 cm−1 (Figure 4). This change reflects the formation of cross β sheet structure in insulin at the expense of its helical content. Insulin in presence of NK9 retains its secondary structure for 4 h of incubation (Figure 4). A1628/A1654 ratio at 4.5 h of incubation showed a much lower value as compared to the ratio in case of insulin alone after 4 h of incubation (Figure 4). This observation indicates that NK9 slowed down the stepwise changes in insulin secondary structure during the process of fibrillation. The amount of helical content left and β-sheet content increased after formation of mature fibril was calculated using the multi curve fitting program of origin 6.0 software (Figure S1). Curve fitting analysis showed that insulin in the presence and absence of NK9 has similar percentages of α-helix and β-sheet content (Figure 4). Although NK9 delays the fibrillation process of insulin, mature fibrils of insulin have ∼60% β-sheet content irrespective of NK9 presence.


Use of a small peptide fragment as an inhibitor of insulin fibrillation process: a study by high and low resolution spectroscopy.

Banerjee V, Kar RK, Datta A, Parthasarathi K, Chatterjee S, Das KP, Bhunia A - PLoS ONE (2013)

Conformational changes during insulin fibrillation as detected by FT-IR.Time dependent FT-IR spectra of insulin (2.0 mg/ml in D2O) in absence (A) and presence (B) of NK9 in 62°C and pD 2.6. Amide-I band were acquired in the 1700–1600 cm−1 range at a resolution of 2 cm−1. Each spectrum is an average of 32 scans and subtracted from solvent spectrum. Panel C shows the ratio of absorbance of β-sheet peak at 1628 cm−1 to helical peak at 1654 cm−1. Filled square represents insulin and open circle represents insulin in presence of NK9. Panel D shows percentage of α-helix and β-sheet present in native and fibrillar insulin in presence and absence of NK9. The molar ratio of insulin to NK9 is 1∶1.
© Copyright Policy
Related In: Results  -  Collection

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pone-0072318-g004: Conformational changes during insulin fibrillation as detected by FT-IR.Time dependent FT-IR spectra of insulin (2.0 mg/ml in D2O) in absence (A) and presence (B) of NK9 in 62°C and pD 2.6. Amide-I band were acquired in the 1700–1600 cm−1 range at a resolution of 2 cm−1. Each spectrum is an average of 32 scans and subtracted from solvent spectrum. Panel C shows the ratio of absorbance of β-sheet peak at 1628 cm−1 to helical peak at 1654 cm−1. Filled square represents insulin and open circle represents insulin in presence of NK9. Panel D shows percentage of α-helix and β-sheet present in native and fibrillar insulin in presence and absence of NK9. The molar ratio of insulin to NK9 is 1∶1.
Mentions: FT-IR has been used to study secondary structure of proteins for decades [47], [50]–[53]. It is particularly sensitive to changes of β-sheet content in proteins [43]. Being an α helical protein insulin has a characteristic peak at ∼1654 cm−1 in its amide I contour. On conversion into amyloid like fibrils, the peak shifts to 1628 cm−1, which is the hallmark of cross β-sheet structure formation [54]. Figure 3 shows FT-IR spectra of insulin measured at various intervals of incubation. Plot of A1628/A1654 against time of incubation (Figure 4) showed very small changes in the ratio and spectral shape take place for the first 2 h of incubation. The transition involving significant increases in the A1628/A1654 ratio was observed after 2 h of incubation, and it persisted for 4 h. This is the cause of steady increase in intensity at 1628 cm−1, with a concomitant decrease in intensity at 1654 cm−1 (Figure 4). This change reflects the formation of cross β sheet structure in insulin at the expense of its helical content. Insulin in presence of NK9 retains its secondary structure for 4 h of incubation (Figure 4). A1628/A1654 ratio at 4.5 h of incubation showed a much lower value as compared to the ratio in case of insulin alone after 4 h of incubation (Figure 4). This observation indicates that NK9 slowed down the stepwise changes in insulin secondary structure during the process of fibrillation. The amount of helical content left and β-sheet content increased after formation of mature fibril was calculated using the multi curve fitting program of origin 6.0 software (Figure S1). Curve fitting analysis showed that insulin in the presence and absence of NK9 has similar percentages of α-helix and β-sheet content (Figure 4). Although NK9 delays the fibrillation process of insulin, mature fibrils of insulin have ∼60% β-sheet content irrespective of NK9 presence.

Bottom Line: In vitro hemolytic activity of the peptide showed insignificant cytotoxicity against HT1080 cells.The insulin aggregation is probed due to the inter play of two key residues, Phe(B24) and Tyr(B26) monitored from molecular dynamics simulations studies.Further new peptide based leads may be developed from this nine residue peptide.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Bose Institute, Kolkata, India.

ABSTRACT
A non-toxic, nine residue peptide, NIVNVSLVK is shown to interfere with insulin fibrillation by various biophysical methods. Insulin undergoes conformational changes under certain stress conditions leading to amyloid fibrils. Fibrillation of insulin poses a problem in its long-term storage, reducing its efficacy in treating type II diabetes. The dissociation of insulin oligomer to monomer is the key step for the onset of fibrillation. The time course of insulin fibrillation at 62°C using Thioflavin T fluorescence shows an increase in the lag time from 120 min without peptide to 236 min with peptide. Transmission electron micrographs show branched insulin fibrils in its absence and less inter-fibril association in its presence. Upon incubation at 62°C and pH 2.6, insulin lost some α-helical structure as seen by Fourier transformed infra-red spectroscopy (FT-IR), but if the peptide is added, secondary structure is almost fully maintained for 3 h, though lost partially at 4 h. FT-IR spectroscopy also shows that insulin forms the cross beta structure indicative of fibrils beyond 2 h, but in the presence of the peptide, α-helix retention is seen till 4 h. Both size exclusion chromatography and dynamic light scattering show that insulin primarily exists as trimer, whose conversion to a monomer is resisted by the peptide. Saturation transfer difference nuclear magnetic resonance confirms that the hydrophobic residues in the peptide are in close contact with an insulin hydrophobic groove. Molecular dynamics simulations in conjunction with principal component analyses reveal how the peptide interrupts insulin fibrillation. In vitro hemolytic activity of the peptide showed insignificant cytotoxicity against HT1080 cells. The insulin aggregation is probed due to the inter play of two key residues, Phe(B24) and Tyr(B26) monitored from molecular dynamics simulations studies. Further new peptide based leads may be developed from this nine residue peptide.

Show MeSH
Related in: MedlinePlus