Protein misfolding and deposition underlie an increasing number of debilitating human disorders. We have shown that model proteins unrelated to disease, such as the Src homology 3 (SH3) domain of the p58␣ subunit of bovine phosphatidyl-inositol-3-kinase (PI3-SH3), can be converted in vitro into assemblies with structural and cytotoxic properties similar to those of pathological aggregates. By contrast, homologous proteins, such as ␣-spectrin-SH3, lack the capability of forming amyloid fibrils at a measurable rate under any of the conditions we have so far examined. However, transplanting a small sequence stretch (6 aa) from PI3-SH3 to ␣-spectrin-SH3, comprising residues of the diverging turn and adjacent RT loop, creates an amyloidogenic protein closely similar in its behavior to the original PI3-SH3. Analysis of specific PI3-SH3 mutants further confirms the involvement of this region in conferring amyloidogenic properties to this domain. Moreover, the inclusion in this stretch of two consensus residues favored in SH3 sequences substantially inhibits aggregation. These findings show that short specific amino acid stretches can act as mediators or facilitators in the incorporation of globular proteins into amyloid structures, and they support the suggestion that natural protein sequences have evolved in part to code for structural characteristics other than those included in the native fold, such as avoidance of aggregation.protein misfolding ͉ protein aggregation ͉ protein evolution T he spontaneous conversion of soluble proteins or protein fragments into aggregates and amyloid fibrils is a challenging problem in biological and medical sciences. An increasing body of evidence supports the anomalous misassembly of proteins into insoluble deposits as the fundamental cause behind a growing number of debilitating human disorders, such as Alzheimer's disease and Parkinson's disease, type II diabetes, and the transmissible spongiform encephalopathies (1-4). Important clues to understand the molecular basis of amyloid diseases and, more generally, the biological significance of protein aggregation have emerged recently from observations made in proteins unrelated to human disease, which have been found to convert in vitro into aggregates with structural and cytotoxic properties indistinguishable from those exhibited by amyloid assemblies associated with pathological conditions (5-14).The Src homology 3 (SH3) domain of the p58␣ subunit of phosphatidyl-inositol-3Ј-kinase (PI3-SH3) is one of the best characterized examples of a small globular protein unrelated to any known pathological condition that can form amyloid fibrils in vitro (5,15,16). Aggregated species obtained from this protein have been found to be cytotoxic when added to cell cultures (13). This observation suggests that the protein aggregates underlying different human disorders could show similar mechanisms of cytotoxicity and more generally that during evolution nature had to develop strategies to avoid protein misassembly to preserve the viability of liv...

Alzheimer's disease (AD) is characterized by the accumulation of insoluble fibrillar aggregates of beta-amyloid peptides (Abeta), a 39-42 residue peptide, in the brain of AD patients. It is hypothesized that the disease causing form is not the fibrillar species but an oligomeric Abeta molecule, which is often referred to as the "critical oligomer" of Abeta. We show in this paper that Abeta(1-40) undergoes chemical exchange between a monomeric, soluble state and an oligomeric, aggregated state under physiological conditions. In circular dichroism spectroscopy, we observe for this intermediate an alpha-helical structure. The oligomer is assigned a molecular weight of >100 kDa by diffusion-ordered spectroscopy-solution-state NMR spectroscopy (NMR). We can show by saturation transfer difference NMR experiments that the oligomer is related to monomeric Abeta. This experiment also allows us to identify the chemical groups that are involved in interactions between mono- and oligomeric Abeta molecules. Variation of the anionic strength in the buffer induces a shift of equilibrium between mono- and oligomeric states and possibly allows for the stabilization of these intermediate structures.

Gamma-secretase is an intramembrane aspartyl protease complex that cleaves type I integral membrane proteins, including the amyloid beta-protein precursor and the Notch receptor, and is composed of presenilin, Pen-2, nicastrin, and Aph-1. Although all four of these membrane proteins are essential for assembly and activity, the stoichiometry of the complex is unknown, with the number of presenilin molecules present being especially controversial. Here we analyze functional gamma-secretase complexes, isolated by immunoprecipitation from solubilized membrane fractions and able to produce amyloid beta-peptides and amyloid beta-protein precursor intracellular domain. We show that the active isolated protease contains only one presenilin per complex, which excludes certain models of the active site that require aspartate dyads formed between two presenilin molecules. We also quantified components in the isolated complexes by Western blot using protein standards and found that the amounts of Pen-2 and nicastrin were the same as that of presenilin. Moreover, we found that one Aph-1 was not co-immunoprecipitated with another in active complexes, evidence that Aph-1 is likewise present as a monomer. Taken together, these results demonstrate that the stoichiometry of gamma-components presenilin:Pen-2:nicastrin:Aph-1 is 1:1:1:1.