Studies of the Nucleation and Growth Reactions of Selected Types of Insulin Fibrils

Insulin is susceptible to fibrillation, a misfolding process leading to well ordered cross-␤ assembly. Protection from fibrillation in ␤ 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 ␤-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-␤ 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 ␤ cells from toxic protein misfolding in the endoplasmic reticulum.

mentioning

confidence: 73%

Section: Overviewmentioning

confidence: 99%

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Proinsulin Is Refractory to Protein Fibrillation

Huang

Dong

Phillips

et al. 2005

“…The protein is hexameric in the presence of zinc at neutral pH, monomeric in 20% acetic acid (pH 2), and dimeric in 20 mM HCl (pH 2.0). Insulin forms amyloid-like fibrils (29,30) that pose a variety of problems in its biomedical and biotechnological applications (especially insulin pumps). Amyloid deposits of insulin have been observed both in patients with type II diabetes and in normal aging, as well as after subcutaneous insulin infusion and after repeated injection (31,32).…”

mentioning

confidence: 99%

Stimulation of Insulin Fibrillation by Urea-induced Intermediates

Ahmad

Millett

Doniach

et al. 2004

106

105

Fibrillar deposits of insulin cause serious problems in implantable insulin pumps, commercial production of insulin, and for some diabetics. We performed a systematic investigation of the effect of urea-induced structural perturbations on the mechanism of fibrillation of insulin. The addition of as little as 0.5 M urea to zincbound hexameric insulin led to dissociation into dimers. Moderate concentrations of urea led to accumulation of a partially unfolded dimer state, which dissociates into an expanded, partially folded monomeric state. Very high concentrations of urea resulted in an unfolded monomer with some residual structure. The addition of even very low concentrations of urea resulted in increased fibrillation. Accelerated fibrillation correlated with population of the partially folded intermediates, which existed at up to 8 M urea, accounting for the formation of substantial amounts of fibrils under such conditions. Under monomeric conditions the addition of low concentrations of urea slowed down the rate of fibrillation, e.g. 5-fold at 0.75 M urea. The decreased fibrillation of the monomer was due to an induced non-native conformation with significantly increased ␣-helical content compared with the native conformation. The data indicate a close-knit relationship between insulin conformation and propensity to fibrillate. The correlation between fibrillation and the partially unfolded monomer indicates that the latter is a critical amyloidogenic intermediate in insulin fibrillation.

“…Accordingly, the susceptibility of the Cha B24 analogs to fibrillation was probed on gentle agitation at 37°C in relation to WT insulin, KP-insulin, [Orn B29 ]insulin, and [Met (Table 3); the lag times were defined on the basis of a 2-fold enhancement of thioflavin T fluorescence (34). Because of the stochastic nature of a nucleation-propagation reaction (60), results are shown for each individual vial (Fig. 8B) ]insulin was similar to that described previously in rat studies of KP-insulin (62), whereas the negligible activity of the Pro B24 analog was similar to that seen on injection of a buffer alone (the gradual linear fall in mean blood glucose concentration is likely to reflect progressive fasting).…”

Section: B24mentioning

confidence: 60%

Aromatic Anchor at an Invariant Hormone-Receptor Interface

Pandyarajan¹,

Smith

Phillips³

et al. 2014

Background: Invariant insulin residue Phe B24 (a site of diabetes-associated mutation) contacts the insulin receptor. Results: Hormonal function requires hydrophobicity rather than aromaticity at this site. Conclusion: The B24 side chain provides a nonpolar anchor at the receptor interface. Significance: Nonstandard aliphatic modification of residue B24 may enhance therapeutic properties of insulin analogs.