A range of human degenerative conditions, including Alzheimer's 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.

The deposition of proteins in the form of amyloid fibrils and plaques is the characteristic feature of more than 20 degenerative conditions affecting either the central nervous system or a variety of peripheral tissues. As these conditions include Alzheimer's, Parkinson's and the prion diseases, several forms of fatal systemic amyloidosis, and at least one condition associated with medical intervention (haemodialysis), they are of enormous importance in the context of present-day human health and welfare. Much remains to be learned about the mechanism by which the proteins associated with these diseases aggregate and form amyloid structures, and how the latter affect the functions of the organs with which they are associated. A great deal of information concerning these diseases has emerged, however, during the past 5 years, much of it causing a number of fundamental assumptions about the amyloid diseases to be re-examined. For example, it is now apparent that the ability to form amyloid structures is not an unusual feature of the small number of proteins associated with these diseases but is instead a general property of polypeptide chains. It has also been found recently that aggregates of proteins not associated with amyloid diseases can impair the ability of cells to function to a similar extent as aggregates of proteins linked with specific neurodegenerative conditions. Moreover, the mature amyloid fibrils or plaques appear to be substantially less toxic than the pre-fibrillar aggregates that are their precursors. The toxicity of these early aggregates appears to result from an intrinsic ability to impair fundamental cellular processes by interacting with cellular membranes, causing oxidative stress and increases in free Ca2+ that eventually lead to apoptotic or necrotic cell death. The 'new view' of these diseases also suggests that other degenerative conditions could have similar underlying origins to those of the amyloidoses. In addition, cellular protection mechanisms, such as molecular chaperones and the protein degradation machinery, appear to be crucial in the prevention of disease in normally functioning living organisms. It also suggests some intriguing new factors that could be of great significance in the evolution of biological molecules and the mechanisms that regulate their behaviour.

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 Alzheimer's and Parkinson's 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.

We have been able to convert a small ␣͞␤ protein, acylphosphatase, from its soluble and native form into insoluble amyloid fibrils of the type observed in a range of pathological conditions. This was achieved by allowing slow growth in a solution containing moderate concentrations of trif luoroethanol. When analyzed with electron microscopy, the protein aggregate present in the sample after long incubation times consisted of extended, unbranched filaments of 30-50 Å in width that assemble subsequently into higher order structures. This fibrillar material possesses extensive ␤-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit Congo red birefringence, increased f luorescence with thiof lavine T and cause a redshift of the Congo red absorption spectrum. All of these characteristics are typical of amyloid fibrils. The results indicate that formation of amyloid occurs when the native fold of a protein is destabilized under conditions in which noncovalent interactions, and in particular hydrogen bonding, within the polypeptide chain remain favorable. We suggest that amyloid formation is not restricted to a small number of protein sequences but is a property common to many, if not all, natural polypeptide chains under appropriate conditions.A range of human disorders is associated with the extracellular deposition of insoluble protein aggregates known as amyloid fibrils (1). These include Alzheimer's disease, type II diabetes, primary and secondary systemic amyloidosis, and the spongiform encephalopathies such as Creutzfeldt-Jakob disease. Although it is still debatable as to whether amyloid fibril deposition is itself the primary origin of the pathological condition with which it is associated, a variety of experimental observations suggests that there is a strong causal link between fibril formation and the onset of pathological symptoms (2, 3). Electron microscopy shows that the amyloid fibrils associated with the various diseases all appear straight and unbranched, and are 40-120 Å in diameter (4). X-ray fiber diffraction studies indicate a characteristic cross-␤ structure in which the polypeptide chains form ␤-strands oriented perpendicular to the long axis of the fibril, resulting in ␤-sheets propagating in the direction of the fibril (4). This overall structure is common to all amyloid fibrils despite the lack of sequence homologies among the amyloidogenic proteins and the absence of structural similarities in the folds that these proteins adopt under normal nonpathological conditions. This observation of a common structure for amyloid indicates that it is stabilized by interactions associated with the common covalent structure of proteins, such as backbone hydrogen bonding or hydrophobic interactions, rather than through specific interactions of the different side chains (1, 4). Recently, the SH3 domain of the p85␣ subunit of phosphatidylinositol 3-kinase, a protein that is not associated with any of the known amyloid diseases, was found readily to form amylo...

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We have systematically studied the effects of 40 single point mutations on the conversion of the denatured form of the alpha/beta 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 beta-sheet structure. The measured changes in aggregation rate upon mutation correlate with changes in the hydrophobicity and beta-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.

The intracellular free Ca 2؉ concentration and redox status of murine fibroblasts exposed to prefibrillar aggregates of the HypF N-terminal domain have been investigated in vitro and in vivo using a range of fluorescent probes. Aggregate entrance into the cytoplasm is followed by an early rise of reactive oxygen species and free Ca 2؉ levels and eventually by cell death. Such changes correlate directly with the viability of the cells and are not observed when cell are cultured in the presence of reducing agents or in Ca 2؉ -free media. In addition, moderate cell stress following exposure to the aggregates was found to be fully reversible. The results show that the cytotoxicity of prefibrillar aggregates of HypF-N, a protein not associated with clinical disease, has the same fundamental origin as that produced by similar types of aggregates of proteins linked with specific amyloidoses. These findings suggest that misfolded proteinaceous aggregates stimulate generic cellular responses as a result of the exposure of regions of the structure (such as hydrophobic residues and the polypeptide main chain) that are buried in the normally folded proteins. They also support the idea that a higher number of degenerative pathologies than previously known might be considered as protein deposition diseases.

The data reported in the past 5 years have highlighted new aspects of protein misfolding and aggregation. Firstly, it appears that protein aggregation may be a generic property of polypeptide chains possibly linked to their common peptide backbone that does not depend on specific amino acid sequences. In addition, it has been shown that even the toxic effects of protein aggregates, mainly in their pre-fibrillar organization, result from common structural features rather than from specific sequences of side chains. These data lead to hypothesize that every polypeptide chain, in itself, possesses a previously unsuspected hidden dark side leading it to transform into a generic toxin to cells in the presence of suitable destabilizing conditions. This new view of protein biology underscores the key importance, in protein evolution, of the negative selection against molecules with significant tendency to aggregate as well as, in biological evolution, of the development of the complex molecular machineries aimed at hindering the appearance of misfolded proteins and their toxic early aggregates. These data also suggest that, in addition to the well-known amyloidoses, a number of degenerative diseases whose molecular basis are presently unknown might be determined by the intra- or extracellular deposition of aggregates of presently unsuspected proteins. From these considerations one could also envisage the possibility that protein aggregation may be exploited by nature to perform specific physiological functions in differing biological contexts. The present review focuses the most recent reports supporting these ideas and discusses their clinical and biological significance.