(361g) Protein Folding, Misfolding and Aggregation in Amyloid Disease

“Amyloid” is a general term describing protein aggregates with several physicochemical features in common: fibrillar morphology, predominantly cross b-sheet secondary structure, and protease resistance. Deposits of aggregated protein are linked to several neurodegenerative disorders including Alzheimer’s, Huntington’s, and Parkinson’s diseases. Numerous in vitro and animal studies provide support for the ‘amyloid cascade hypothesis’ – that aggregation is causally linked to cellular toxicity.

In Alzheimer’s disease (AD), for example, the peptide β-amyloid (Aβ) spontaneously self-associates into soluble oligomers and insoluble fibrillar aggregates. Aβ is intrinsically disordered as a monomer but partially folds in parallel with its self-assembly. On the other hand, some proteins with a stable native fold undergo partial unfolding prior to aggregation. Transthyretin (TTR) is an example of a folded homotetrameric protein that must partially unfold by losing quaternary structure before reassembly into the amyloid deposits that characterize Senile Systemic Amyloidosis. The predilection for amyloidogenesis may be attributable to a too-small difference between the stability of the native state (whether intrinsically disordered or folded) and the stability of the alternatively folded amyloid aggregate state.

Amyloidogenic proteins and peptides are conformationally diverse in their native state, but their aggregates are conformationally convergent. These shared structural features arise during the course of self-assembly of amyloidogenic proteins and are not present in the native unaggregated state. Thus, homotypic (self) association of amyloidogenic proteins may be based on structural rather than sequence recognition. Furthermore, since structure rather than sequence is the common feature shared by amyloid aggregates, it seems reasonable to imagine that heterotypic (non-self) association of amyloidogenic proteins is possible.

Since the amyloid assembly process is believed to lead to neuronal dysfunction and death, great efforts have been made to search for compounds that selectively bind to and inhibit fibrillization. In this talk I will present two examples where potentially amyloidogenic proteins serve as effective inhibitors of Aβ aggregation through heterotypic interactions. (1) Transthyretin (TTR) was discovered as an anti-amyloid agent in transgenic animal studies. Our studies show that Aβ binds to TTR and triggers destabilization of the protein quaternary structure. Although destabilization would be expected to lead to self-association into TTR amyloid fibrils, in this case Aβ aggregation is inhibited via heterotypic interaction. We will describe our efforts to create peptide mimics of the natural Aβ-binding domain, which interact with Aβ specifically and protect against Aβ-induced neurotoxicity. (2) Cystatin C monomers are metastable and the protein readily forms domain-swapped dimers and, under mildly denaturation conditions, will self-assembly into soluble oligomers or into amyloid fibrils. These observations led to the proposal that cystatin C fibrils grow via a propagated domain-swapping mechanism. We show that this is not the case and furthermore, cystatin C is a potent inhibitor of Aβ aggregation. Further complicating the cystatin C-Aβ interaction is that cystatin C is a potent inhibitor of cysteine proteases such as cathepsin B, and cathepsin B degrades Aβ. We present a kinetic model that illustrates how Ab aggregation and degradation are regulated by cathepsin B and cystatin C.