Human Amylin Oligomer Growth and Fibril Elongation Define Two Distinct Phases in Amyloid Formation

Abstract

Human amylin (hA), a 37-amino-acid polypeptide, is one of a number of peptides with the ability to form amyloid fibrils and cause disease. It is the main constituent of the pancreatic amyloid deposits associated with type 2 diabetes. Increasing interest in early assembly intermediates rather than the mature fibrils as the cytotoxic agent has led to this study in which the smallest hA oligomers have been captured by atomic force microscopy. These are 2.3 ؎ 1.9 nm in height, 23 ؎ 14 nm in length, and consist of an estimated 16 hA molecules. Oligomers first grow to a height of about 6 nm before they begin to significantly elongate into fibrils. Congo red inhibits elongation but not the growth in height of hA oligomers. Two distinct phases have thus been identified in hA fibrillogenesis: lateral growth of oligomers followed by longitudinal growth into mature fibrils. These observations suggest that mature fibrils are assembled directly via longitudinal growth of full-width oligomers, making assembly by lateral association of protofibrils appear less likely.

Is type 2 diabetes an amyloidosis and does it really matter (to patients)?

“…Human amylin's physiological conformation is one of random coil, but it undergoes rapid conformational change into β-conformers when placed in physiological solutions [35]. Based on the available evidence, many workers have concluded that small oligomers formed by aggregated human amylin [36,37] are probably the species that could ultimately cause diabetes [38,39]. Others, among them Zraika and colleagues, believe that the cytotoxicity of larger amyloid fibrils must be considered [11,15], as indeed it must.…”

“…Oligomers are now known to form spontaneously in human amylin solutions, among and often concomitantly with the larger, precipitable fibrillar structures that have typically been visualised by electron microscopy [26]. Amylin oligomers, which are usually at least two orders of magnitude smaller than fibrils, are initially a few nm in diameter [37]. Application of real-time atomic force microscopy (AFM) has enabled measurement of their dimensions, mass and time-dependent growth [36,37].…”

“…Amylin oligomers, which are usually at least two orders of magnitude smaller than fibrils, are initially a few nm in diameter [37]. Application of real-time atomic force microscopy (AFM) has enabled measurement of their dimensions, mass and time-dependent growth [36,37]. When amylin monomers first become visible in the AFM, they comprise structured assemblies of some 16 monomers, which grow over minutes by rapid bidirectional elongation through the addition of further monomers at a roughly constant rate [37].…”

“…Application of real-time atomic force microscopy (AFM) has enabled measurement of their dimensions, mass and time-dependent growth [36,37]. When amylin monomers first become visible in the AFM, they comprise structured assemblies of some 16 monomers, which grow over minutes by rapid bidirectional elongation through the addition of further monomers at a roughly constant rate [37]. Time-dependent growth is an intrinsic property of oligomers that might be related to their toxicity [40] and may well provide an explanation for the differing estimates of oligomer mass highlighted by Zraika et al In reality, AFM data indicate that there is no single oligomer, but rather a rapidly shifting population of related oligomeric nanoparticles, which shift seamlessly between each other over time.…”

“…Zraika et al have argued that mature amyloid fibrils, rather than small, soluble cytotoxic oligomers [37][38][39]43], could play the main role in beta cell degeneration and diabetes causation. As noted, they have pointed to the numerous weaknesses and shortcomings of the currently available evidence, and in so doing have indicated further lines of investigation that might improve our understanding.…”