ABSTRACT
In type 2 diabetics, the hormone amylin misfolds into amyloid plaques implicated in the destruction of the pancreatic ß-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 ß-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 ß-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.
Subject(s)
Islet Amyloid Polypeptide/chemistry , Islet Amyloid Polypeptide/metabolism , Animals , Benzothiazoles , Cell Survival/drug effects , Fluorescent Dyes/chemistry , Humans , Hydrogen-Ion Concentration , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Islet Amyloid Polypeptide/pharmacology , Kinetics , Mice , Models, Animal , Models, Molecular , Molecular Structure , Protein Binding/drug effects , Structure-Activity Relationship , Thiazoles/chemistry , Tumor Cells, CulturedABSTRACT
Semen-derived enhancer of virus infection (SEVI), a naturally occurring peptide fragment of prostatic acid phosphatase, enhances HIV infectivity by forming cationic amyloid fibrils that aid the fusion of negatively charged virion and target cell membranes. Cu(II) and Zn(II) inhibit fibrillization of SEVI in a kinetic assay using the fibril-specific dye ThT. TEM suggests that the metals do not affect fibril morphology. NMR shows that the metals bind to histidines 3 and 23 in the SEVI sequence. ITC experiments indicate that SEVI forms oligomeric complexes with the metals. Dissociation constants are micromolar for Cu(II) and millimolar for Zn(II). Because the Cu(II) and Zn(II) concentrations that inhibit fibrillization are comparable with those found in seminal fluid the metals may modulate SEVI fibrillization under physiological conditions.