Amyloid Aggregation Behavior of Human Calcitonin

Abstract

Under appropriate conditions, certain peptides and proteins, both intrinsically disordered and misfolded from their native state, can self-associate to form long proteinaceous fibrils known as amyloids. This transition forms the molecular basis of several pathologies, through both losses of native functions and cytotoxic effects. Calcitonin (CT) is a 32 amino acid therapeutic hormone peptide that can be considered a molecular paradigm for the central events associated with amyloid misfolding. CT’s biological activity is limited by its aggregation along the canonical amyloid aggregation pathway. A better understanding of the misfolding process would not only provide a structural basis to improve CT’s long-term stability and activity as a therapeutic, but also provide valuable insights into the pathological aggregation of other amyloids. As such, the aggregation of human CT (hCT) has been studied in this dissertation using a range of biophysical techniques, with a particular focus on native modulators of kinetic behavior. A direct relationship between human calcitonin (hCT) concentration and aggregation lag time was observed for the first time, contrary to the conventional understanding of amyloid aggregation. This kinetic trend was found to persist over a range of aggregation conditions, as confirmed by Thioflavin-T kinetics assays, CD spectroscopy, and transmission EM. On the basis of kinetics modeling and experimental results, a mechanism whereby structural conversion of hCT monomers is needed before incorporation into the fibril was proposed. Comparative studies of hCT and the canonically aggregating salmon CT (sCT) using experimental and computational techniques suggested that alpha-helical monomers represent a growth-competent species, whereas unstructured random coil monomers represent a growth-incompetent species. The kinetic mechanism proposed represents a novel mechanism in amyloid aggregation, and synthesizes several previously disparate amyloid behaviors. The determinants of hCT lag time were further investigated in a membrane environment, providing the first systematic study of the effect of membranes on CT aggregation. The direct relationship between peptide concentration and lag phase was found to persist in the presence of large unilamellar vesicles (LUVs), and was shown to be dependent on membrane composition. Specifically, lipid compositions encouraging stronger surface interactions increased the concentration dependent differences in lag time. CD experiments suggested adsorption and sequestration of growth-competent helical monomers to play a role in this behavior. An apparent reformatting of mature hCT fibrils was also observed, in a process which appears dependent on not only lipid composition but also the peptide to lipid ratio. The ability of LUVs to remodel fibers grown in solution suggests that interactions between mature fibrils and lipid bilayers are causative in the behavior, rather than membrane-peptide interactions during fiber growth. The results of this thesis, particularly as they relate to monomer growth competence, represent significant contributions to the amyloid field and CT therapy. The novel kinetic mechanism proposed reveals that intramolecular interactions in disordered monomers, while often transient and weak compared to intermolecular interactions, can play crucial roles in mediating amyloid aggregation. Additionally, the elucidated effects of monomer structure and lipid interactions on hCT aggregation kinetics present possible means by which aggregation kinetics can be modulating while maintaining peptide sequence and thus therapeutic efficacy, a key goal in hCT therapies. Such results present a richer picture of hCT aggregation than had previously been available, and potentially provide novel insights as to more general mechanisms of amyloid aggregation.