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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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Hemolytic and cytotoxity assay of NK9 peptide.(A) Percentage hemolysis of NK9 peptide on human red blood cells exhibiting less than 0.5% hemolysis for different concentrations (25, 50, 100, 150, 200 and 250 µM) of peptide been tested. Blank corresponds to untreated red blood cells (without peptide). The positive control for this experiment was 1% Triton X 100. (B) Percentage viability of HT1080 cells upon treatment with varying concentrations (5, 10, 25 and 50 µM) of NK9 peptide showing less than 5% cytotoxicity in each case, against untreated cells taken as blank. The positive control for this experiment was 0.5% Triton X 100.
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pone-0072318-g011: Hemolytic and cytotoxity assay of NK9 peptide.(A) Percentage hemolysis of NK9 peptide on human red blood cells exhibiting less than 0.5% hemolysis for different concentrations (25, 50, 100, 150, 200 and 250 µM) of peptide been tested. Blank corresponds to untreated red blood cells (without peptide). The positive control for this experiment was 1% Triton X 100. (B) Percentage viability of HT1080 cells upon treatment with varying concentrations (5, 10, 25 and 50 µM) of NK9 peptide showing less than 5% cytotoxicity in each case, against untreated cells taken as blank. The positive control for this experiment was 0.5% Triton X 100.

Mentions: In vitro hemolysis assay evaluates hemoglobin release in the plasma as an indicator of red blood cell lysis following exposure to the drug or agent under consideration. It is an accurate and sensitive method for predicting hemolytic activity of a drug. The study of hemolytic activity of NK9 peptide showed less than 0.5% hemolysis for the various concentrations of peptide assayed up to a final concentration of 250 µM (Figure 11). Percentage hemolysis was measured with respect to 1% Triton X 100 sample taken as a control, which was considered to exhibit 100% hemolysis. The experiment was conducted in three biological replicates and similar results were obtained in each case. Cytotoxicity assay of NK9 peptide on HT1080 cell line was also performed for an analysis of its effects on live cells post treatment. No significant (less than 5%) cytotoxicity was observed towards HT1080 cell line for the different concentrations being tested going up to a final concentration of 50 µM. 0.5% Triton X 100 was used as a control, which exhibited only 20% viability. Cytotoxicity was measured by calculating the percentage of viable cells at each concentration of NK9 peptide relative to the control sample without peptide, which was considered to have 100% viability. The experiment was repeated in 4 replicates and yielded similar results. These data clearly indicate that the peptide, NK9 is non-toxic and the lead compound(s) from this peptide sequence can pave way to develop of new generation anti-diabetic drugs.


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)

Hemolytic and cytotoxity assay of NK9 peptide.(A) Percentage hemolysis of NK9 peptide on human red blood cells exhibiting less than 0.5% hemolysis for different concentrations (25, 50, 100, 150, 200 and 250 µM) of peptide been tested. Blank corresponds to untreated red blood cells (without peptide). The positive control for this experiment was 1% Triton X 100. (B) Percentage viability of HT1080 cells upon treatment with varying concentrations (5, 10, 25 and 50 µM) of NK9 peptide showing less than 5% cytotoxicity in each case, against untreated cells taken as blank. The positive control for this experiment was 0.5% Triton X 100.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0072318-g011: Hemolytic and cytotoxity assay of NK9 peptide.(A) Percentage hemolysis of NK9 peptide on human red blood cells exhibiting less than 0.5% hemolysis for different concentrations (25, 50, 100, 150, 200 and 250 µM) of peptide been tested. Blank corresponds to untreated red blood cells (without peptide). The positive control for this experiment was 1% Triton X 100. (B) Percentage viability of HT1080 cells upon treatment with varying concentrations (5, 10, 25 and 50 µM) of NK9 peptide showing less than 5% cytotoxicity in each case, against untreated cells taken as blank. The positive control for this experiment was 0.5% Triton X 100.
Mentions: In vitro hemolysis assay evaluates hemoglobin release in the plasma as an indicator of red blood cell lysis following exposure to the drug or agent under consideration. It is an accurate and sensitive method for predicting hemolytic activity of a drug. The study of hemolytic activity of NK9 peptide showed less than 0.5% hemolysis for the various concentrations of peptide assayed up to a final concentration of 250 µM (Figure 11). Percentage hemolysis was measured with respect to 1% Triton X 100 sample taken as a control, which was considered to exhibit 100% hemolysis. The experiment was conducted in three biological replicates and similar results were obtained in each case. Cytotoxicity assay of NK9 peptide on HT1080 cell line was also performed for an analysis of its effects on live cells post treatment. No significant (less than 5%) cytotoxicity was observed towards HT1080 cell line for the different concentrations being tested going up to a final concentration of 50 µM. 0.5% Triton X 100 was used as a control, which exhibited only 20% viability. Cytotoxicity was measured by calculating the percentage of viable cells at each concentration of NK9 peptide relative to the control sample without peptide, which was considered to have 100% viability. The experiment was repeated in 4 replicates and yielded similar results. These data clearly indicate that the peptide, NK9 is non-toxic and the lead compound(s) from this peptide sequence can pave way to develop of new generation anti-diabetic drugs.

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