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Molecular interpretation of ACTH-β-endorphin coaggregation: relevance to secretory granule biogenesis.

Ranganathan S, Singh PK, Singh U, Singru PS, Padinhateeri R, Maji SK - PLoS ONE (2012)

Bottom Line: Using all atom molecular dynamics simulation we investigate the early molecular interaction events in the ACTH-β-endorphin system, β-endorphin-only system and ACTH-only system.We find that β-endorphin and ACTH formed an interacting unit, whereas negligible interactions were observed between ACTH molecules in ACTH-only system.Our data suggest that ACTH is not only involved in interaction with β-endorphin but also enhances the stability of mixed oligomers of the entire system.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences and Bioengineering, Wadhwani Research Centre for Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India.

ABSTRACT
Peptide/protein hormones could be stored as non-toxic amyloid-like structures in pituitary secretory granules. ACTH and β-endorphin are two of the important peptide hormones that get co-stored in the pituitary secretory granules. Here, we study molecular interactions between ACTH and β-endorphin and their colocalization in the form of amyloid aggregates. Although ACTH is known to be a part of ACTH-β-endorphin aggregate, ACTH alone cannot aggregate into amyloid under various plausible conditions. Using all atom molecular dynamics simulation we investigate the early molecular interaction events in the ACTH-β-endorphin system, β-endorphin-only system and ACTH-only system. We find that β-endorphin and ACTH formed an interacting unit, whereas negligible interactions were observed between ACTH molecules in ACTH-only system. Our data suggest that ACTH is not only involved in interaction with β-endorphin but also enhances the stability of mixed oligomers of the entire system.

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Related in: MedlinePlus

Structural transition and amyloid aggregation of ACTH and β-end.A) 2 mg/ml ACTH in presence of different concentrations of heparin were incubated for two months. Insignificant structural changes are observed at day 0 (top panel, left) and after two months (top panel, right) of incubation at 37°C with slight rotation at pH 5.5, suggesting that ACTH is highly soluble and non-amyloidogenic. Different symbols represent concentrations of heparin in mM. Similar experiments were also performed with various ACTH and heparin concentration ratios for two months (lower panel). No significant structural transformations were observed at day 0 (left, lower panel) and after two months of incubation (right, lower panel). B) ThT binding of ACTH and β-end samples incubated for two weeks either in PBS or in 5% D-mannitol at 37°C with slight rotation. Only β-end was able to bind ThT significantly, both in PBS and in 5% D-mannitol, 0.4 M heparin suggesting β-end is amyloidogenic. C) Conformational transition of 2 mg/ml ACTH (left) and β-end (right) incubated in PBS, pH 7.4, 0.01% sodium azide for two weeks. ACTH did not show significant conformational transition whereas β-end showed random coil to β-sheet transition after two weeks of incubation. D) TFE induced structural transition of ACTH and β-end. ACTH is unable to change its conformation even in presence of 80% TFE. Whereas, β-end changes its conformation from random coil to helix in presence of TFE concentration (>20% TFE v/v). Each symbol represents different concentrations of TFE (v/v).
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pone-0031924-g001: Structural transition and amyloid aggregation of ACTH and β-end.A) 2 mg/ml ACTH in presence of different concentrations of heparin were incubated for two months. Insignificant structural changes are observed at day 0 (top panel, left) and after two months (top panel, right) of incubation at 37°C with slight rotation at pH 5.5, suggesting that ACTH is highly soluble and non-amyloidogenic. Different symbols represent concentrations of heparin in mM. Similar experiments were also performed with various ACTH and heparin concentration ratios for two months (lower panel). No significant structural transformations were observed at day 0 (left, lower panel) and after two months of incubation (right, lower panel). B) ThT binding of ACTH and β-end samples incubated for two weeks either in PBS or in 5% D-mannitol at 37°C with slight rotation. Only β-end was able to bind ThT significantly, both in PBS and in 5% D-mannitol, 0.4 M heparin suggesting β-end is amyloidogenic. C) Conformational transition of 2 mg/ml ACTH (left) and β-end (right) incubated in PBS, pH 7.4, 0.01% sodium azide for two weeks. ACTH did not show significant conformational transition whereas β-end showed random coil to β-sheet transition after two weeks of incubation. D) TFE induced structural transition of ACTH and β-end. ACTH is unable to change its conformation even in presence of 80% TFE. Whereas, β-end changes its conformation from random coil to helix in presence of TFE concentration (>20% TFE v/v). Each symbol represents different concentrations of TFE (v/v).

Mentions: Previous studies of hACTH, rACTH and pACTH have suggested that none of the ACTHs self aggregated into amyloid-like fibrils in vitro[13]. However, ACTH co-aggregated with β-end into amyloid-like structure. Detailed studies were carried out to illustrate the self-aggregating behavior of hACTH at varying concentrations and also in presence of varying amounts of GAGs (heparin) in 5% D-mannitol, pH 5.5, 0.01% sodium azide (Figures 1A and B). Amyloid formations were monitored by CD, ThT binding and EM. CD studies indicated that ACTH did not undergo any structural transition even after two months of incubation (Figure 1A) under different conditions. Moreover, negligible ThT fluorescence of the two months incubated samples (Figure 1B) and absence of any amyloid-like morphology under EM suggest that ACTH is non-amyloidogenic (Figure 2B). We also examined whether a buffer with high salt concentration such as PBS might modulate the aggregation and amyloid formation of ACTH and β-end. The aggregation data in PBS, pH 7.4, 0.01% sodium azide suggested that β-end formed amyloid-like fibrils in PBS after two weeks of incubation (Figures 1B, 1C and 2B) as it showed random coil (RC) to β-sheet structural transition in CD, high ThT binding and appearance of fibrillar morphology under EM; whereas ACTH did not show any amyloid-like fibrils in identical experimental conditions. A decrease in CD signal at wavelength ∼198 nm was observed for ACTH after two weeks of incubation in PBS, indicating subtle change in secondary structure. However, CD signal in the wavelength range of 208–222 was mostly unchanged during incubation suggesting that major secondary structural transition was absent. To further evaluate whether ACTH has any intrinsic structural propensity, we initiated the experiment with TFE. TFE is known to destabilize hydrophobic interactions within the polypeptide chain and stabilizes local hydrogen bonds between residues close in the amino acid sequence [28]. It has long been suggested that TFE could induce helices in peptide/proteins in solution. To study the effect of TFE on secondary structures of ACTH and β-end, we performed CD experiments of 25 µM each of ACTH and β-end in PBS, pH 7.4 with increasing concentrations of TFE. The data suggested that ACTH is unable to change to any helical structure up to 80% of TFE concentration (v/v). Interestingly, β-end showed increased helicity in a TFE concentration dependent manner (Figure 1D). These results further suggest that ACTH does not possess any intrinsic tendency for structural change and/or aggregation by its own, but might change its structure in presence of β-end.


Molecular interpretation of ACTH-β-endorphin coaggregation: relevance to secretory granule biogenesis.

Ranganathan S, Singh PK, Singh U, Singru PS, Padinhateeri R, Maji SK - PLoS ONE (2012)

Structural transition and amyloid aggregation of ACTH and β-end.A) 2 mg/ml ACTH in presence of different concentrations of heparin were incubated for two months. Insignificant structural changes are observed at day 0 (top panel, left) and after two months (top panel, right) of incubation at 37°C with slight rotation at pH 5.5, suggesting that ACTH is highly soluble and non-amyloidogenic. Different symbols represent concentrations of heparin in mM. Similar experiments were also performed with various ACTH and heparin concentration ratios for two months (lower panel). No significant structural transformations were observed at day 0 (left, lower panel) and after two months of incubation (right, lower panel). B) ThT binding of ACTH and β-end samples incubated for two weeks either in PBS or in 5% D-mannitol at 37°C with slight rotation. Only β-end was able to bind ThT significantly, both in PBS and in 5% D-mannitol, 0.4 M heparin suggesting β-end is amyloidogenic. C) Conformational transition of 2 mg/ml ACTH (left) and β-end (right) incubated in PBS, pH 7.4, 0.01% sodium azide for two weeks. ACTH did not show significant conformational transition whereas β-end showed random coil to β-sheet transition after two weeks of incubation. D) TFE induced structural transition of ACTH and β-end. ACTH is unable to change its conformation even in presence of 80% TFE. Whereas, β-end changes its conformation from random coil to helix in presence of TFE concentration (>20% TFE v/v). Each symbol represents different concentrations of TFE (v/v).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293876&req=5

pone-0031924-g001: Structural transition and amyloid aggregation of ACTH and β-end.A) 2 mg/ml ACTH in presence of different concentrations of heparin were incubated for two months. Insignificant structural changes are observed at day 0 (top panel, left) and after two months (top panel, right) of incubation at 37°C with slight rotation at pH 5.5, suggesting that ACTH is highly soluble and non-amyloidogenic. Different symbols represent concentrations of heparin in mM. Similar experiments were also performed with various ACTH and heparin concentration ratios for two months (lower panel). No significant structural transformations were observed at day 0 (left, lower panel) and after two months of incubation (right, lower panel). B) ThT binding of ACTH and β-end samples incubated for two weeks either in PBS or in 5% D-mannitol at 37°C with slight rotation. Only β-end was able to bind ThT significantly, both in PBS and in 5% D-mannitol, 0.4 M heparin suggesting β-end is amyloidogenic. C) Conformational transition of 2 mg/ml ACTH (left) and β-end (right) incubated in PBS, pH 7.4, 0.01% sodium azide for two weeks. ACTH did not show significant conformational transition whereas β-end showed random coil to β-sheet transition after two weeks of incubation. D) TFE induced structural transition of ACTH and β-end. ACTH is unable to change its conformation even in presence of 80% TFE. Whereas, β-end changes its conformation from random coil to helix in presence of TFE concentration (>20% TFE v/v). Each symbol represents different concentrations of TFE (v/v).
Mentions: Previous studies of hACTH, rACTH and pACTH have suggested that none of the ACTHs self aggregated into amyloid-like fibrils in vitro[13]. However, ACTH co-aggregated with β-end into amyloid-like structure. Detailed studies were carried out to illustrate the self-aggregating behavior of hACTH at varying concentrations and also in presence of varying amounts of GAGs (heparin) in 5% D-mannitol, pH 5.5, 0.01% sodium azide (Figures 1A and B). Amyloid formations were monitored by CD, ThT binding and EM. CD studies indicated that ACTH did not undergo any structural transition even after two months of incubation (Figure 1A) under different conditions. Moreover, negligible ThT fluorescence of the two months incubated samples (Figure 1B) and absence of any amyloid-like morphology under EM suggest that ACTH is non-amyloidogenic (Figure 2B). We also examined whether a buffer with high salt concentration such as PBS might modulate the aggregation and amyloid formation of ACTH and β-end. The aggregation data in PBS, pH 7.4, 0.01% sodium azide suggested that β-end formed amyloid-like fibrils in PBS after two weeks of incubation (Figures 1B, 1C and 2B) as it showed random coil (RC) to β-sheet structural transition in CD, high ThT binding and appearance of fibrillar morphology under EM; whereas ACTH did not show any amyloid-like fibrils in identical experimental conditions. A decrease in CD signal at wavelength ∼198 nm was observed for ACTH after two weeks of incubation in PBS, indicating subtle change in secondary structure. However, CD signal in the wavelength range of 208–222 was mostly unchanged during incubation suggesting that major secondary structural transition was absent. To further evaluate whether ACTH has any intrinsic structural propensity, we initiated the experiment with TFE. TFE is known to destabilize hydrophobic interactions within the polypeptide chain and stabilizes local hydrogen bonds between residues close in the amino acid sequence [28]. It has long been suggested that TFE could induce helices in peptide/proteins in solution. To study the effect of TFE on secondary structures of ACTH and β-end, we performed CD experiments of 25 µM each of ACTH and β-end in PBS, pH 7.4 with increasing concentrations of TFE. The data suggested that ACTH is unable to change to any helical structure up to 80% of TFE concentration (v/v). Interestingly, β-end showed increased helicity in a TFE concentration dependent manner (Figure 1D). These results further suggest that ACTH does not possess any intrinsic tendency for structural change and/or aggregation by its own, but might change its structure in presence of β-end.

Bottom Line: Using all atom molecular dynamics simulation we investigate the early molecular interaction events in the ACTH-β-endorphin system, β-endorphin-only system and ACTH-only system.We find that β-endorphin and ACTH formed an interacting unit, whereas negligible interactions were observed between ACTH molecules in ACTH-only system.Our data suggest that ACTH is not only involved in interaction with β-endorphin but also enhances the stability of mixed oligomers of the entire system.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences and Bioengineering, Wadhwani Research Centre for Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India.

ABSTRACT
Peptide/protein hormones could be stored as non-toxic amyloid-like structures in pituitary secretory granules. ACTH and β-endorphin are two of the important peptide hormones that get co-stored in the pituitary secretory granules. Here, we study molecular interactions between ACTH and β-endorphin and their colocalization in the form of amyloid aggregates. Although ACTH is known to be a part of ACTH-β-endorphin aggregate, ACTH alone cannot aggregate into amyloid under various plausible conditions. Using all atom molecular dynamics simulation we investigate the early molecular interaction events in the ACTH-β-endorphin system, β-endorphin-only system and ACTH-only system. We find that β-endorphin and ACTH formed an interacting unit, whereas negligible interactions were observed between ACTH molecules in ACTH-only system. Our data suggest that ACTH is not only involved in interaction with β-endorphin but also enhances the stability of mixed oligomers of the entire system.

Show MeSH
Related in: MedlinePlus