Limits...
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.

Show MeSH

Related in: MedlinePlus

Changes in the association state of insulin in presence and absence of NK9.Absorbance profiles at 220 nm from size exclusion chromatography experiments with insulin (350 μM): (A) Absence and (B) Presence of 350 µM NK9 at different time of incubation. Molar ratio of NK9 to insulin is 1∶1. Panel C shows SEC profile of insulin in presence of NK9 at 280 nm wavelength to distinguish insulin peak from the NK9.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3756998&req=5

pone-0072318-g005: Changes in the association state of insulin in presence and absence of NK9.Absorbance profiles at 220 nm from size exclusion chromatography experiments with insulin (350 μM): (A) Absence and (B) Presence of 350 µM NK9 at different time of incubation. Molar ratio of NK9 to insulin is 1∶1. Panel C shows SEC profile of insulin in presence of NK9 at 280 nm wavelength to distinguish insulin peak from the NK9.

Mentions: The association states of insulin in the presence and absence of NK9 were determined by the size exclusion chromatography using TSKgel SuperSW2000 HPLC column. The column was pre-calibrated using size exclusion marker proteins β-amylase, ADH, BSA, carbonic anhydrase, lysozyme, and ribonuclease. The calibration data fitted nicely into a linear equation (correlation coefficient, R2 = 0.98). Since incubation of insulin samples for fibrillation experiment were done using citrate phosphate buffer of pH 2.6, the HPLC column was equilibrated with this buffer for experiment with insulin. It was confirmed by using BSA and lysozyme that both proteins retained their globular shape as retention time at this acidic pH did not significantly change from those at pH 7.0 (data not shown). Aliquots of insulin solution at different time points of incubation were centrifuged at 4000×g force to remove visible turbidity. Supernatant was loaded on to the HPLC column. Prior incubation of insulin in citrate phosphate buffer showed a retention time of 16.9 ml (Figure 5A). This retention time corresponds to the trimeric structure of insulin that corroborates well with earlier findings by Banga and co-workers [39]. Retention time of insulin remained unchanged for 1.5 hr with concomitant decrease in its absorbance value. The increase in incubation time up to 2 hr, shifted the retention time of insulin to 17.9 min that corresponds to the monomeric insulin. After 2 hr of incubation, fluorescence emission intensity just started to increase (Figure 1) implying that active nucleus of insulin fibrils was formed just after 2 hr of incubation. The size exclusion chromatography data shows that the building block of this active nucleus is a monomer of insulin. This conclusion is in agreement with the literature data as well [55].


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)

Changes in the association state of insulin in presence and absence of NK9.Absorbance profiles at 220 nm from size exclusion chromatography experiments with insulin (350 μM): (A) Absence and (B) Presence of 350 µM NK9 at different time of incubation. Molar ratio of NK9 to insulin is 1∶1. Panel C shows SEC profile of insulin in presence of NK9 at 280 nm wavelength to distinguish insulin peak from the NK9.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0072318-g005: Changes in the association state of insulin in presence and absence of NK9.Absorbance profiles at 220 nm from size exclusion chromatography experiments with insulin (350 μM): (A) Absence and (B) Presence of 350 µM NK9 at different time of incubation. Molar ratio of NK9 to insulin is 1∶1. Panel C shows SEC profile of insulin in presence of NK9 at 280 nm wavelength to distinguish insulin peak from the NK9.
Mentions: The association states of insulin in the presence and absence of NK9 were determined by the size exclusion chromatography using TSKgel SuperSW2000 HPLC column. The column was pre-calibrated using size exclusion marker proteins β-amylase, ADH, BSA, carbonic anhydrase, lysozyme, and ribonuclease. The calibration data fitted nicely into a linear equation (correlation coefficient, R2 = 0.98). Since incubation of insulin samples for fibrillation experiment were done using citrate phosphate buffer of pH 2.6, the HPLC column was equilibrated with this buffer for experiment with insulin. It was confirmed by using BSA and lysozyme that both proteins retained their globular shape as retention time at this acidic pH did not significantly change from those at pH 7.0 (data not shown). Aliquots of insulin solution at different time points of incubation were centrifuged at 4000×g force to remove visible turbidity. Supernatant was loaded on to the HPLC column. Prior incubation of insulin in citrate phosphate buffer showed a retention time of 16.9 ml (Figure 5A). This retention time corresponds to the trimeric structure of insulin that corroborates well with earlier findings by Banga and co-workers [39]. Retention time of insulin remained unchanged for 1.5 hr with concomitant decrease in its absorbance value. The increase in incubation time up to 2 hr, shifted the retention time of insulin to 17.9 min that corresponds to the monomeric insulin. After 2 hr of incubation, fluorescence emission intensity just started to increase (Figure 1) implying that active nucleus of insulin fibrils was formed just after 2 hr of incubation. The size exclusion chromatography data shows that the building block of this active nucleus is a monomer of insulin. This conclusion is in agreement with the literature data as well [55].

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