Niccolò Taddei

Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases.

A range of human degenerative conditions, including Alzheimers disease, light-chain amyloidosis and the spongiform encephalopathies, is associated with the deposition in tissue of proteinaceous aggregates known as amyloid fibrils or plaques. It has been shown previously that fibrillar aggregates that are closely similar to those associated with clinical amyloidoses can be formed in vitro from proteins not connected with these diseases, including the SH3 domain from bovine phosphatidyl-inositol-3′-kinase and the amino-terminal domain of the Escherichia coli HypF protein. Here we show that species formed early in the aggregation of these non-disease-associated proteins can be inherently highly cytotoxic. This finding provides added evidence that avoidance of protein aggregation is crucial for the preservation of biological function and suggests common features in the origins of this family of protein deposition diseases.

Rationalization of the effects of mutations on peptide and protein aggregation rates.

In order for any biological system to function effectively, it is essential to avoid the inherent tendency of proteins to aggregate and form potentially harmful deposits. In each of the various pathological conditions associated with protein deposition, such as Alzheimers and Parkinsons diseases, a specific peptide or protein that is normally soluble is deposited as insoluble aggregates generally referred to as amyloid. It is clear that the aggregation process is generally initiated from partially or completely unfolded forms of the peptides and proteins associated with each disease. Here we show that the intrinsic effects of specific mutations on the rates of aggregation of unfolded polypeptide chains can be correlated to a remarkable extent with changes in simple physicochemical properties such as hydrophobicity, secondary structure propensity and charge. This approach allows the pathogenic effects of mutations associated with known familial forms of protein deposition diseases to be rationalized, and more generally enables prediction of the effects of mutations on the aggregation propensity of any polypeptide chain.

Kinetic partitioning of protein folding and aggregation

We have systematically studied the effects of 40 single point mutations on the conversion of the denatured form of the α/β protein acylphosphatase (AcP) into insoluble aggregates. All the mutations that significantly perturb the rate of aggregation are located in two regions of the protein sequence, residues 16–31 and 87–98, each of which has a relatively high hydrophobicity and propensity to form β-sheet structure. The measured changes in aggregation rate upon mutation correlate with changes in the hydrophobicity and β-sheet propensity of the regions of the protein in which the mutations are located. The two regions of the protein sequence that determine the aggregation rate are distinct from those parts of the sequence that determine the rate of protein folding. Dissection of the protein into six peptides corresponding to different regions of the sequence indicates that the kinetic partitioning between aggregation and folding can be attributed to the intrinsic conformational preferences of the denatured polypeptide chain.

Mutational analysis of acylphosphatase suggests the importance of topology and contact order in protein folding

Muscle acylphosphatase (AcP) is a small protein that folds very slowly with two-state behavior. The conformational stability and the rates of folding and unfolding have been determined for a number of mutants of AcP in order to characterize the structure of the folding transition state. The results show that the transition state is an expanded version of the native protein, where most of the native interactions are partially established. The transition state of AcP turns out to be remarkably similar in structure to that of the activation domain of procarboxypeptidase A2 (ADA2h), a protein having the same overall topology but sharing only 13% sequence identity with AcP. This suggests that transition states are conserved between proteins with the same native fold. Comparison of the rates of folding of AcP and four other proteins with the same topology, including ADA2h, supports the concept that the average distance in sequence between interacting residues (that is, the contact order) is an important determinant of the rate of protein folding.

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

Protein aggregation and the formation of highly insoluble amyloid structures is associated with a range of debilitating human conditions, which include Alzheimers disease, Parkinsons 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.

Mutational analysis of the propensity for amyloid formation by a globular protein

Acylphosphatase can be converted in vitro, by addition of trifluoroethanol (TFE), into amyloid fibrils of the type observed in a range of human diseases. The propensity to form fibrils has been investigated for a series of mutants of acylphosphatase by monitoring the range of TFE concentrations that result in aggregation. We have found that the tendency to aggregate correlates inversely with the conformational stability of the native state of the protein in the different mutants. In accord with this, the most strongly destabilized acylphosphatase variant forms amyloid fibrils in aqueous solution in the absence of TFE. These results show that the aggregation process that leads to amyloid deposition takes place from an ensemble of denatured conformations under conditions in which non‐covalent interactions are still favoured. These results support the hypothesis that the stability of the native state of globular proteins is a major factor preventing the in vivo conversion of natural proteins into amyloid fibrils under non‐pathological conditions. They also suggest that stabilizing the native states of amyloidogenic proteins could aid prevention of amyloidotic diseases.

Solution conditions can promote formation of either amyloid protofilaments or mature fibrils from the HypF N-terminal domain

The HypF N‐terminal domain has been found to convert readily from its native globular conformation into protein aggregates with the characteristics of amyloid fibrils associated with a variety of human diseases. This conversion was achieved by incubation at acidic pH or in the presence of moderate concentrations of trifluoroethanol. Electron microscopy showed that the fibrils grown in the presence of trifluoroethanol were predominantly 3–5 nm and 7–9 nm in width, whereas fibrils of 7–9 nm and 12–20 nm in width prevailed in samples incubated at acidic pH. These results indicate that the assembly of protofilaments or narrow fibrils into mature amyloid fibrils is guided by interactions between hydrophobic residues that may remain exposed on the surface of individual protofilaments. Therefore, formation and isolation of individual protofilaments appears facilitated under conditions that favor the destabilization of hydrophobic interactions, such as in the presence of trifluoroethanol.

Acceleration of the folding of acylphosphatase by stabilization of local secondary structure.

The addition of trifluoroethanol or hexafluoroisopropanol converts the apparent two-state folding of acylphosphatase, a small α/β protein, into a multistate mechanism where secondary structure accumulates significantly in the denatured state before folding to the native state. This results in a marked acceleration of folding as revealed by following the intrinsic fluorescence and circular dichroism changes upon folding. The folding rate is at a maximum when the secondary-structure content of the denatured state corresponds to that of the native state, while further stabilization of secondary structure decreases the folding rate. These findings indicate that stabilization of intermediate structure can either enhance or retard folding depending on its nature and content of native-like interactions.

Assessing the role of aromatic residues in the amyloid aggregation of human muscle acylphosphatase

Among the many parameters that have been proposed to promote amyloid fibril formation is the π‐stacking of aromatic residues. We have studied the amyloid aggregation of several mutants of human muscle acylphosphatase in which an aromatic residue was substituted with a non‐aromatic one. The aggregation rate was determined using the Thioflavin T test under conditions in which the variants populated initially an ensemble of partially unfolded conformations. Substitutions in aggregation‐promoting fragments of the sequence result in a dramatically decreased aggregation rate of the protein, confirming the propensity of aromatic residues to promote this process. Nevertheless, a statistical analysis shows that the measured decrease of aggregation rate following mutation arises predominantly from a reduction of hydrophobicity and intrinsic β‐sheet propensity. This suggests that aromatic residues favor aggregation because of these factors rather than for their aromaticity.

Changes in Na+,K-ATPase, Ca2-ATPase and some soluble enzymes related to energy metabolism in brains of patients with Alzheimer's disease

Hexokinase, lactate dehydrogenase, acylphosphatase, (Na+,K+)-ATPase and Ca2(+)-ATPase of selected areas from postmortem Alzheimers disease brains were studied. Hexokinase and lactate dehydrogenase were significantly changed in all the examined subcortical nuclei. (Na+,K+)-ATPase activity was altered in several areas of Alzheimers disease brains. No changes in Ca2(+)-ATPase and acylphosphatase were observed. The main alterations of the assayed enzymes were observed in subcortical areas but not in cortical areas of Alzheimers disease brains.