No abstract

Insulin, a 51-residue peptide hormone, is an intrinsically amyloidogenic peptide, forming amyloid fibrils in vitro. In the secretory granules, insulin is densely packed together with Zn(II) into crystals of Zn(2)Insulin(6) hexamer, which assures osmotic stability of vesicles and prevents fibrillation of the peptide. However, after release from the pancreatic beta-cells, insulin dissociates into active monomers, which tend to fibrillize not only at acidic, but also at physiological, pH values. The effect of co-secreted Zn(II) ions on the fibrillation of monomeric insulin is unknown, however, it might prevent insulin fibrillation. We showed that Zn(II) inhibits fibrillation of monomeric insulin at physiological pH values by forming a soluble Zn(II)-insulin complex. The inhibitory effect of Zn(II) ions is very strong at pH 7.3 (IC(50)=3.5 microM), whereas at pH 5.5 it progressively weakens, pointing towards participation of the histidine residue(s) in complex formation. The results obtained indicate that Zn(II) ions might suppress fibrillation of insulin at its release sites and in circulation. It is hypothesized that misfolded oligomeric intermediates occurring in the insulin fibrillation pathway, especially in zinc-deficient conditions, might induce autoantibodies against insulin, which leads to beta-cell damage and autoimmune Type 1 diabetes.

Zinc is an essential trace element involved in the correct packing and storage of insulin. Total zinc content in the pancreatic β-cells is among the highest in the body and changes in the Zn(2+) levels have been found to be associated with diabetes. The most common form of the Zn-insulin complex is a hexamer containing two zinc ions. However, zinc can also form other complexes with insulin, whereas dissociation constants of these complexes are not known. We have determined that the dissociation constant value of the monomeric 1 : 1 Zn-insulin complex is equal to 0.40 μM. The apparent binding affinity decreases drastically at higher insulin concentrations where the peptide forms dimers. Cu(2+) ions also bind to monomeric insulin, whereas the apparent Cu(2+)-binding affinity depends on HEPES concentration. The conditional dissociation constant of the Cu(2+)-insulin complex is equal to 0.025 μM. The analysis demonstrates that insulin cannot form complexes with zinc ions in circulation due to the low concentration of free Zn(2+) in this environment.

The tight binding of Cu and Zn ions to superoxide dismutase 1 (SOD1) maintains the protein stability, associated with amyotrophic lateral sclerosis (ALS). Yet, the quantitative studies remain to be explored for the metal-binding affinity of wild-type SOD1 and its mutants. We have investigated the demetallation of Cu,Zn-SOD1 and its ALS-related G93A mutant in the presence of different standard metal ion chelators at varying temperatures by using an LC-ICP MS-based approach and fast size-exclusion chromatography. Our results showed that from the slow first-order kinetics both metal ions Zn2+ and Cu2+ were released simultaneously from the protein at elevated temperatures. The rate of the release depends on the concentration of chelating ligands but is almost independent of their metal-binding affinities. Similar studies with the G93A mutant of Cu,Zn-SOD1 revealed slightly faster metal-release. The demetallation of Cu,Zn-SOD1 comes always to completion, which hindered the calculation of the KD values. From the Arrhenius plots of the demetallation in the absence of chelators ΔH‡ = 173 kJ/mol for wt and 191 kJ/mol for G93A mutant Cu,Zn-SOD1 was estimated. Obtained high ΔH values are indicative of the occurrence of protein conformational changes before demetallation and we concluded that Cu,Zn-SOD1 complex is in native conditions kinetically inert. The fibrillization of both forms of SOD1 was similar.