MOLECULAR CHARACTERIZATION OF FUNCTIONAL AMYLOID HETEROFILAMENTS FORMED BY NEUROENDOCRINE PEPTIDES

Amyloids are highly ordered protein aggregates commonly associated with several neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases. Many proteins can form amyloids under specific conditions, suggesting that amyloid formation is an intrinsic property of polypeptide chains. Recent studies have also demonstrated that amyloids play important roles in a variety of biological functions. One such example occurs in the neuroendocrine system, where neuropeptides such as β-endorphin (β-end) and adrenocorticotropic hormone (ACTH) are stored within secretory granules as reversible amyloid aggregates, enabling dense and stable packaging while allowing their coordinated release. Neuropeptides are small signaling peptides produced and secreted by neurons through the regulated secretory pathway, regulating neuronal activity and intercellular communication. In this project, ACTH and β-end are investigated because they are derived from a common precursor, proopiomelanocortin (POMC), and are co-localized within secretory granules. The amyloid structure provides semi-autonomous properties that facilitate protein sorting, inert storage, and regulated release, thereby reducing the need for highly specific cellular machinery. Although the individual roles of these hormones have been studied, the potential for cross-interactions between β-end and ACTH remains largely unexplored. The hypothesis that these peptides may assemble into amyloid heterofilaments represents a conceptual frontier that has received little attention in the literature, particularly in the context of functional amyloids. The ACTH peptide was produced by heterologous protein expression, whereas β-endorphin was obtained by solid-phase peptide synthesis. The individual neuropeptides were first characterized using circular dichroism (CD) spectroscopy and thioflavin T (ThT) fluorescence assays. Based on these initial results, additional CD, ThT, sodium dodecyl sulfate (SDS) assays, and transmission electron microscopy (TEM) analyses were performed to begin investigating the heterofilamentation of the two peptides. Similar experiments are also being conducted in the presence and absence of heparin to assess whether it can promote or assist heterofilament formation, as suggested in the literature. Preliminary results indicate that the presence of β-end and heparin, used here to promote amyloid formation, influences the aggregation kinetics of ACTH, suggesting a possible cross-interaction between these peptides during amyloid formation.