pH Dependence of Amylin Fibrillization

Abstract

In type 2 diabetics, the hormone amylin misfolds into amyloid plaques implicated in the destruction of the pancreatic β-cells that make insulin and amylin. The aggregative misfolding of amylin is pH-dependent, and exposure of the hormone to acidic and basic environments could be physiologically important. Amylin has two ionizable residues between pH 3 and 9: the α-amino group and His18. Our approach to measuring the pKa values for these sites has been to look at the pH dependence of fibrillization in amylin variants that have only one of the two groups. The α-amino group at the unstructured N-terminus of amylin has a pKa near 8.0, similar to the value in random coil models. By contrast, His18, which is involved in the intermolecular β-sheet structure of the fibrils, has a pKa that is lowered to 5.0 in the fibrils compared to the random coil value of 6.5. The lowered pKa of His18 is due to the hydrophobic environment of the residue, and electrostatic repulsion between positively charged His18 residues on neighboring amylin molecules in the fibril. His18 acts as an electrostatic switch inhibiting fibrillization in its charged state. The presence of a charged side chain at position 18 also affects fibril morphology and lowers amylin cytotoxicity toward a MIN6 mouse model of pancreatic β-cells. In addition to the two expected pKa values, we detected an apparent pKa of ~4.0 for the amylin-derived peptide NAc-SNNFGAILSS-NH2, which has no titratable groups. This pKa is due to the pH-induced ionization of the dye thioflavin T. By using alternative methods to follow fibrillization such as the dye Nile Red or turbidimetry, we were able to distinguish between the titration of the dye and groups on the peptide. Large differences in reaction kinetics were observed between the different methods at acidic pH, because of charges on the ThT dye, which hinder fibril formation much like the charges on the protein.

Evolutionary Adaptation and Amyloid Formation: Does the Reduced Amyloidogenicity and Cytotoxicity of Ursine Amylin Contribute to the Metabolic Adaption of Bears and Polar Bears?

“…In contrast, the other substitutions are expected to slow amyloid formation by h‐amylin. Amyloid formation by h‐amylin is slower when His‐18 is positively charged and the His to Arg substitution is thus expected to reduce amyloidogenicity, since Arg will be fully charged at physiological pH . In addition, the His‐18 to Arg replacement is one of the substitutions that helps to render rat amylin nonamyloidogenic, and this substitution has been exploited to help design soluble nontoxic analogs of amylin ,,.…”

Implications of peptide assemblies in amyloid diseases

“…At low pH, an increase in the lag time and a decrease in the growth rate of IAPP fibrillization was observed. 174, 175 The electrostatic repulsion between IAPPs with protonated histidine18 (His18) is responsible for inhibiting the self-association of IAPP at low pH, 176 supported by DMD simulations of IAPP dimerization with and without protonation of His18. 177 However, since the pH value inside β-cell granules is close to the isoelectric point of His18 174, 178 and a significant portion of IAPP is still unprotonated, interactions of IAPP with other granule components are necessary for natively inhibiting the peptide amyloid aggregation at high concentrations.…”

The redox environment triggers conformational changes and aggregation of hIAPP in Type II Diabetes

“…In vivo , hIAPP is soluble at concentrations of 1–4 mM678, while in vitro hIAPP aggregates at 1000 times lower concentrations. Protein aggregation is influenced by concentration and pH9. In T2D, the redox state of the ER is shifted towards more reducing conditions, resulting in protein secretion and folding deficiencies101112.…”