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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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Effect of NK9 binding over secondary structure of insulin during insulin fibrillation monitored by circular dichroism.Selected buffer subtracted far-UV CD spectra of (A) Insulin (B) Insulin in presence of NK9 (NK9 spectra at these selected time points were subtracted from insulin NK9 spectra). (C) Far UV-CD spectra of NK9 in presence and absence of insulin (Insulin spectra was subtracted from NK9 insulin spectra). The concentration of insulin and NK9 both were 350 μM (insulin:NK9 = 1∶1). During the measurement the samples were diluted to 50 μM.
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pone-0072318-g003: Effect of NK9 binding over secondary structure of insulin during insulin fibrillation monitored by circular dichroism.Selected buffer subtracted far-UV CD spectra of (A) Insulin (B) Insulin in presence of NK9 (NK9 spectra at these selected time points were subtracted from insulin NK9 spectra). (C) Far UV-CD spectra of NK9 in presence and absence of insulin (Insulin spectra was subtracted from NK9 insulin spectra). The concentration of insulin and NK9 both were 350 μM (insulin:NK9 = 1∶1). During the measurement the samples were diluted to 50 μM.

Mentions: A change in the secondary structure of proteins to cross β-sheet structure is the hall mark of fibril formation [49]. We investigated the changes in the secondary structure of insulin in the absence and presence of NK9 with the help of circular dichroism (CD) spectroscopy. Insulin, a helical protein, shows dual minima at 222 nm and 208 nm that are the characteristics of all major helical proteins. Figure 3A and 3B are the Far UV-CD spectra of insulin at different time points of incubation in the absence as well as presence of NK9, respectively. With the increase in the time of incubation, the negative ellipticity values at 222 nm and 208 nm also decrease, which indicates increased order in the secondary structure of insulin. In order to have a quantitative analysis of the change in secondary structure of insulin during fibrillation process, we have de-convoluted the far UV-CD data with the help of CDNN software. After 100 min of incubation, the helix content of insulin decreased to 39% with little increase in β-sheet structure. Whereas, the secondary structure of insulin in presence of NK9, remained almost unchanged for 180 min of incubation. At 240 min of incubation, helix content of insulin in presence of NK9 decreased only to 42%. This implies that NK9 helps insulin to retain its secondary structure for a prolonged period of time. Appearance of insoluble aggregates makes it difficult to continue the CD measurement beyond 100 min of incubation for insulin alone and 240 min of incubation, for insulin in presence of NK9. Nevertheless, NK9 does not have a well-defined structure even in the presence of insulin (Figure 3C).


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)

Effect of NK9 binding over secondary structure of insulin during insulin fibrillation monitored by circular dichroism.Selected buffer subtracted far-UV CD spectra of (A) Insulin (B) Insulin in presence of NK9 (NK9 spectra at these selected time points were subtracted from insulin NK9 spectra). (C) Far UV-CD spectra of NK9 in presence and absence of insulin (Insulin spectra was subtracted from NK9 insulin spectra). The concentration of insulin and NK9 both were 350 μM (insulin:NK9 = 1∶1). During the measurement the samples were diluted to 50 μM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756998&req=5

pone-0072318-g003: Effect of NK9 binding over secondary structure of insulin during insulin fibrillation monitored by circular dichroism.Selected buffer subtracted far-UV CD spectra of (A) Insulin (B) Insulin in presence of NK9 (NK9 spectra at these selected time points were subtracted from insulin NK9 spectra). (C) Far UV-CD spectra of NK9 in presence and absence of insulin (Insulin spectra was subtracted from NK9 insulin spectra). The concentration of insulin and NK9 both were 350 μM (insulin:NK9 = 1∶1). During the measurement the samples were diluted to 50 μM.
Mentions: A change in the secondary structure of proteins to cross β-sheet structure is the hall mark of fibril formation [49]. We investigated the changes in the secondary structure of insulin in the absence and presence of NK9 with the help of circular dichroism (CD) spectroscopy. Insulin, a helical protein, shows dual minima at 222 nm and 208 nm that are the characteristics of all major helical proteins. Figure 3A and 3B are the Far UV-CD spectra of insulin at different time points of incubation in the absence as well as presence of NK9, respectively. With the increase in the time of incubation, the negative ellipticity values at 222 nm and 208 nm also decrease, which indicates increased order in the secondary structure of insulin. In order to have a quantitative analysis of the change in secondary structure of insulin during fibrillation process, we have de-convoluted the far UV-CD data with the help of CDNN software. After 100 min of incubation, the helix content of insulin decreased to 39% with little increase in β-sheet structure. Whereas, the secondary structure of insulin in presence of NK9, remained almost unchanged for 180 min of incubation. At 240 min of incubation, helix content of insulin in presence of NK9 decreased only to 42%. This implies that NK9 helps insulin to retain its secondary structure for a prolonged period of time. Appearance of insoluble aggregates makes it difficult to continue the CD measurement beyond 100 min of incubation for insulin alone and 240 min of incubation, for insulin in presence of NK9. Nevertheless, NK9 does not have a well-defined structure even in the presence of insulin (Figure 3C).

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