Molecular Interpretation of ACTH-β-Endorphin Coaggregation: Relevance to Secretory Granule Biogenesis

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

MD simulation of β-end system.

<p>All-atom MD simulation for the duration of 20 ns was performed in explicit solvent using four β-end. A) Snapshots indicating the initial (left) and final (right) states of the β-end system. B) Plot of distances between center of masses (Dij) of peptides within the β-end simulation against time. C) Contact map showing various residues in contact between peptides at t = 20 ns. Significant contacts are observed between amino acid residues in β-end A, β-end B and β-end D. D) A schematic depicting the various h-bonds observed between the peptides during the β-end simulation. The schematic showing C-terminus of β-end D is in contact with N-terminus of β-end A whereas C-terminus of β-end B is in contact with C-terminus β-end A. A salt bridge between E31 of β-end A and K28 of β-end B is represented by red dotted line.</p

Aggregation and colocalization of ACTH and β-end.

<p>A) Fluorescence photomicrographs of the anterior (A–C) and intermediate (D–F) lobes of the pituitary gland showing ACTH (A, D), β-end (B, E), and ACTH-β-end colocalized (C, F) cells. Note that presence of several double-labeled cells (arrows) in anterior as well as intermediate lobes. Scale bar = 50 µm. B) EM images of ACTH and β-end samples incubated for 14 days. The aggregations of the hormones were followed at 37°C at a concentration of 2 mg/ml in the presence of 0.4 mM LMW heparin in 5% D-mannitol (pH 5.5) and PBS under slight agitation. TEM of negative stained samples was performed. Scale bars, 500 nm.</p

MD simulation of ACTH system.

<p>A) Snapshots at the left-hand side indicating initial state (t = 0 ns) and at the right-hand side indicating final state (t = 20 ns) of the simulation. B) Plot of distances between center of masses (Dij) of peptides within the ACTH simulation against time showing large interpeptide distances during simulation. C) Contact map at t = 20 ns snapshot showing residues in contact. Negligible interpeptide contacts are observed suggesting that peptides are not self-assembling <i>in silico</i>.</p

Structural transition and amyloid aggregation of ACTH and β-end.

<p>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).</p

Snapshots at various time steps of ACTH-β-end simulation system showing hetero-oligomerization.

<p>The hetero-trimer consisting of two β-end and one ACTH is stable up to end of 20 ns simulation. β-end A, β-end B, ACTH C and ACTH D are represented by blue, yellow, green and purple color, respectively.</p