Studies of the aggregation of mutant proteins in vitro provide insights into the genetics of amyloid diseases - PubMed (original) (raw)

. 2002 Dec 10;99 Suppl 4(Suppl 4):16419-26.

doi: 10.1073/pnas.212527999. Epub 2002 Oct 8.

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Studies of the aggregation of mutant proteins in vitro provide insights into the genetics of amyloid diseases

Fabrizio Chiti et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Protein aggregation and the formation of highly insoluble amyloid structures is associated with a range of debilitating human conditions, which include Alzheimer's disease, Parkinson's disease, and the Creutzfeldt-Jakob disease. Muscle acylphosphatase (AcP) has already provided significant insights into mutational changes that modulate amyloid formation. In the present paper, we have used this system to investigate the effects of mutations that modify the charge state of a protein without affecting significantly the hydrophobicity or secondary structural propensities of the polypeptide chain. A highly significant inverse correlation was found to exist between the rates of aggregation of the protein variants under denaturing conditions and their overall net charge. This result indicates that aggregation is generally favored by mutations that bring the net charge of the protein closer to neutrality. In light of this finding, we have analyzed natural mutations associated with familial forms of amyloid diseases that involve alteration of the net charge of the proteins or protein fragments associated with the diseases. Sixteen mutations have been identified for which the mechanism of action that causes the pathological condition is not yet known or fully understood. Remarkably, 14 of these 16 mutations cause the net charge of the corresponding peptide or protein that converts into amyloid deposits to be reduced. This result suggests that charge has been a key parameter in molecular evolution to ensure the avoidance of protein aggregation and identifies reduction of the net charge as an important determinant in at least some forms of protein deposition diseases.

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Figures

Figure 1

Figure 1

Structure of AcP in its native state. Residues that have been mutated in the present study are labeled and their side chains shown. The various amino acid substitutions are listed in Table 1.

Figure 2

Figure 2

Urea denaturation curves of representative AcP variants in 50 mM acetate buffer, pH 5.5, 28°C. Curves are normalized to the fraction of folded protein and correspond to those of wild-type AcP (filled circles), K88Q (open circles), R23Q (filled squares), E90H (open squares), and S8H (open triangles) mutants. The solid lines through the data represent the best fits of the data points to the equation given by Santoro and Bolen (46). The resulting thermodynamic parameters for all protein variants are listed in Table 1.

Figure 3

Figure 3

(a) Aggregation of six representative AcP variants followed by ThT fluorescence. Aggregation was initiated in each case in 25% TFE/50 mM acetate buffer, pH 5.5, 25°C. Aliquots were withdrawn at regular time intervals for the ThT assay. The AcP variants shown are: wild-type (filled circles), R23Q (open triangles), E29Q (crosses), E29R (open circles), S21R (filled squares), and E90H (diamonds). The solid lines through the data points represent the best fits to single exponential functions. The resulting rate constant values are reported for all variants in Table 1. (b) Aggregation rate versus net charge constructed with the data points of the wild-type protein and the 15 mutants. Changes of net charge on mutation are calculated at pH 5.5 assuming standard pKa values for amino acid residues.

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