No abstract

Charged polyelectrolytes such as glycosaminoglycans and nucleic acids have frequently been found associated with the proteinaceous deposits in the tissues of patients with amyloid diseases. We have investigated the nature and generality of this phenomenon by studying the ability of different polyanions, including DNA, ATP, heparin, and heparan sulfate, to promote the aggregation of amyloidogenic proteins and to bind to the resulting aggregates. Preformed amyloid fibrils of human muscle acylphosphatase and human lysozyme, proteins with a net positive charge at physiological pH values, were found to bind tightly to the negatively charged DNA or ATP. The effects of the polyelectrolytes on the kinetics of aggregation were studied for acylphosphatase, and the presence of ATP, DNA, or heparin was found to increase its aggregation rate dramatically, with a degree dependent on the net charge and size of the polyanion. Magnesium or calcium ions were found to attenuate, and ultimately to suppress, these interactions, suggesting that they are electrostatic in nature. Moreover, heparin was found to stabilize the aggregated state of acylphosphatase through compensation of electrostatic repulsion. Noteworthy, differences in affinity between native and aggregated acylphosphatase with heparin suggest that amyloid fibrils can themselves behave as polyelectrolytes, interacting very strongly with other polyelectrolytes bearing the opposite charge. Within an in vivo context, the strengthening of the electrostatic interactions with other biological polyelectrolytes, as a consequence of protein misfolding and aggregation, could therefore result in depletion of essential molecular components and contribute to the known cytotoxicity of amyloid fibrils and their precursors.

Six glycine residues of human muscle acylphosphatase (AcP) are evolutionarily conserved across the three domains of life. We have generated six variants of AcP, each having a glycine substituted by an alanine (G15A, G19A, G37A, G45A, G53A, and G69A). Three additional variants had Gly45 replaced by serine, glutamate, and arginine, respectively. The mutational variants do not, on average, have a lower conformational stability than other variants with substitutions of nonconserved residues. In addition, only the G15A variant is enzymatically inactive. However, all variants, with the exception of the G15A mutant, form amyloid aggregates more rapidly than the wild-type. Dynamic light-scattering experiments carried out under conditions close to physiological confirm that aggregate formation is generally more pronounced for the glycine-substituted variants. Apart from the glycine at position 15, all other conserved glycine residues in this protein could have been maintained during evolution because of their ability to inhibit aggregation.

We have measured the intramolecular diffusion rate between distant residues in the aggregation-prone protein HypF-N under various denaturing conditions. Using the method of cysteine quenching of the tryptophan triplet state, we find that intramolecular diffusion remains roughly constant at high concentrations of denaturant (2-6 M GdnHCl) and slows down at low concentrations of denaturant, but the decrease is not uniform throughout the chain. Extrapolation of these measurements to 0 M GdnHCl gives D approximately 10(-7) cm(2) s(-1), about 1 order of magnitude lower than unstructured peptides and at least 2 orders of magnitude higher than well-behaved proteins. This suggests that there is a dynamic range of conformational reorganization within which partially unfolded states are prone to aggregation.