Abstract
Insulin is susceptible to fibrillation, a misfolding process leading to well ordered cross-beta assembly. Protection from fibrillation in beta cells is provided by sequestration of the susceptible monomer within zinc hexamers. We demonstrate that proinsulin is refractory to fibrillation under conditions that promote the rapid fibrillation of zinc-free insulin. Proinsulin fibrils, as probed by Raman microscopy, are nonetheless similar in structure to insulin fibrils. The connecting peptide, although not well ordered in native proinsulin, participates in a fibril-specific beta-sheet. Native insulin and proinsulin exhibit similar free energies of unfolding as inferred from guanidine denaturation studies: relative amyloidogenicities are thus not correlated with global stability. Strikingly, the susceptibility of proinsulin to fibrillation is increased by scission of the connecting peptide at single sites. We thus propose that the connecting peptide constrains a large scale conformational change in the misfolded protein. A tethering mechanism is proposed based on a model of an insulin protofilament derived from electron-microscopic image reconstruction. The proposed relationship between cross-beta assembly and protein topology is supported by studies of single-chain analogs (mini-proinsulin and insulin-like growth factor I) in which foreshortened connecting peptides further retard fibrillation. In addition to its classic function to facilitate disulfide pairing, the connecting peptide may protect beta cells from toxic protein misfolding in the endoplasmic reticulum.
Highlights
Insulin is a small globular protein containing two chains, A (21 residues) and B (30 residues)
We demonstrate that proinsulin is refractory to fibrillation under conditions that promote the rapid fibrillation of zinc-free insulin
In part I Raman spectroscopy is employed to compare the structures of insulin and proinsulin in respective native states and as fibrils
Summary
Insulin is a small globular protein containing two chains, A (21 residues) and B (30 residues). Products of single-site scission within the connecting peptide, are more susceptible to fibrillation than is the intact protein; by contrast, foreshortening of the connecting peptide (green segments, A and 1B) (18 –20) markedly retards the fibrillation of mini-proinsulin [10] Such effects are likewise uncorrelated with stabilities. Enhanced fibrillation is observed on cleavage of insulin-like growth factor I (IGF-I, an homologous single-chain protein), suggesting a general relationship between the kinetics of fibrillation and protein topology Together, these studies suggest that the connecting region hinders fibril formation by functioning as a topological tether. These studies suggest that the connecting region hinders fibril formation by functioning as a topological tether We propose that such tethering interferes with a critical step in the mechanism of cross- assembly and so protects the  cell from proinsulin misfolding and its associated proteotoxicity [21]
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