Lability landscape and protease resistance of human insulin amyloid: a new insight into its molecular properties.
Amyloid formation is a universal behavior of proteins central to many important human pathologies and industrial processes. The extreme stability of amyloids towards chemical and proteolytic degradation is an acquired property compared to the precursor proteins and is a major prerequisite for their accumulation. Here, we report a study on the lability of human insulin amyloid as a function of pH and amyloid ageing. Using a range of methods such as atomic force microscopy, thioflavin T fluorescence, circular dichroism, and gas-phase electrophoretic mobility macromolecule analysis, we probed the propensity of human insulin amyloid to propagate or dissociate in a wide span of pH values and ageing in a low concentration regime. We generated a three-dimensional amyloid lability landscape in coordinates of pH and amyloid ageing, which displays three distinctive features: (i) a maximum propensity to grow near pH 3.8 and an age corresponding to the inflection point of the growth phase, (ii) an abrupt cutoff between growth and disaggregation at pH 8-10, and (iii) isoclines shifted towards older age during the amyloid growth phase at pH 4-9, reflecting the greater stability of aged amyloid. Thus, lability of amyloid strongly depends on the ionization state of insulin and on the structure and maturity of amyloid fibrils. The stability of insulin amyloid towards protease K was assessed by using real-time atomic force microscopy and thioflavin T fluorescence. We estimated that amyloid fibrils can be digested both from the free ends and within the length of the fibril with a rate of ca 4 nm/min. Our results highlight that amyloid structures, depending on solution conditions, can be less stable than commonly perceived. These results have wide implications for understanding the propagation of amyloids via a seeding mechanism as well as for understanding their natural clearance and dissociation under solution conditions unfavorable for amyloid formation in biological systems and industrial applications.
Malisauskas
Mantas
M
Department of Medical Biochemistry and Biophysics, Umeå University, Umeå SE 90187, Sweden.
Weise
Christoph
C
Yanamandra
Kiran
K
Wolf-Watz
Magnus
M
Morozova-Roche
Ludmilla
L
D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D052160:Benzothiazoles; D002942:Circular Dichroism; D004586:Electrophoresis; D019286:Endopeptidase K / Q000378:metabolism*; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D012996:Solutions; D013057:Spectrum Analysis; D013844:Thiazoles / Q000378:metabolism; D013997:Time Factors
D000682:Amyloid; D052160:Benzothiazoles; D007328:Insulin; D012996:Solutions; D013844:Thiazoles; C009462:thioflavin T; D019286:Endopeptidase K