Prion disease susceptibility is affected by beta-structure folding propensity and local side-chain interactions in PrP.

Prion diseases occur when the normally α-helical prion protein (PrP) converts to a pathological ß-structured state with prion infectivity (PrP(Sc)). Exposure to PrP(Sc) from other mammals can catalyze this conversion. Evidence from experimental and accidental transmission of prions suggests that mammals vary in their prion disease susceptibility: Hamsters and mice show relatively high susceptibility, whereas rabbits, horses, and dogs show low susceptibility. Using a novel approach to quantify conformational states of PrP by circular dichroism (CD), we find that prion susceptibility tracks with the intrinsic propensity of mammalian PrP to convert from the native, α-helical state to a cytotoxic ß-structured state, which exists in a monomer-octamer equilibrium. It has been controversial whether ß-structured monomers exist at acidic pH; sedimentation equilibrium and dual-wavelength CD evidence is presented for an equilibrium between a ß-structured monomer and octamer in some acidic pH conditions. Our X-ray crystallographic structure of rabbit PrP has identified a key helix-capping motif implicated in the low prion disease susceptibility of rabbits. Removal of this capping motif increases the ß-structure folding propensity of rabbit PrP to match that of PrP from mouse, a species more susceptible to prion disease.

Probing Alzheimer amyloid peptide aggregation using a cell-free fluorescent protein refolding method.

Fibrillation of the Alzheimer beta-amyloid peptide (Abeta) and (or) formation of toxic oligomers are key pathological events in Alzheimer's disease. Several strategies have been introduced to identify small molecule aggregation inhibitors, and based on these methods, a number of aggregation inhibitors have been identified. However, most of these methods use chemically synthesized Abeta42 peptides, which are difficult to maintain in a monomeric state at neutral pH where anti-aggregation screening is usually carried out. We have developed a cell-free Abeta42 aggregation assay based on fluorescence protein refolding. This assay utilizes nanomolar concentrations of protein. We genetically fused Abeta42 to the N-terminus of vYFP, a variant of of GFP, and expressed and purified the folded fusion protein. The refolding efficiency of Abeta42-vYFP fusion was inversely correlated with the solubility of Abeta42. Using fluorescence to monitor refolding of Abeta42-vYFP, we confirmed that Zn2+ binds to Abeta42 and increases its aggregation. The IC50 value estimated for Zn binding is 3.03 +/- 0.65 micromol/L. We also show that this technique is capable of monitoring the aggregation of chemically synthesized Abeta42.

Substoichiometric inhibition of transthyretin misfolding by immune-targeting sparsely populated misfolding intermediates: a potential diagnostic and therapeutic for TTR amyloidoses.

Wild-type and mutant transthyretin (TTR) can misfold and deposit in the heart, peripheral nerves, and other sites causing amyloid disease. Pharmacological chaperones, Tafamidis(®) and diflunisal, inhibit TTR misfolding by stabilizing native tetrameric TTR; however, their minimal effective concentration is in the micromolar range. By immune-targeting sparsely populated TTR misfolding intermediates (i.e. monomers), we achieved fibril inhibition at substoichiometric concentrations. We developed an antibody (misTTR) that targets TTR residues 89-97, an epitope buried in the tetramer but exposed in the monomer. Nanomolar misTTR inhibits fibrillogenesis of misfolded TTR under micromolar concentrations. Pan-specific TTR antibodies do not possess such fibril inhibiting properties. We show that selective targeting of misfolding intermediates is an alternative to native state stabilization and requires substoichiometric concentrations. MisTTR or its derivative may have both diagnostic and therapeutic potential.

Early steps in oxidation-induced SOD1 misfolding: implications for non-amyloid protein aggregation in familial ALS.

Among the diseases of protein misfolding, amyotrophic lateral sclerosis (ALS) is unusual in that the proteinaceous neuronal inclusions that are the hallmark of the disease have neither the classic fibrillar appearance of amyloid by transmission electron microscopy nor the affinity for the dye Congo red that is a defining feature of amyloid. Mutations in the Cu, Zn superoxide dismutase (SOD1) cause the largest subset of inherited ALS cases. The mechanism by which this highly stable enzyme misfolds to form non-amyloid aggregates is currently poorly understood, as are the stresses that initiate misfolding. The oxidative damage hypothesis proposes that SOD1's normal free radical scavenger role puts it at risk of oxidative damage and that it is this damage that triggers the misfolding primed by mutation. Here, we present evidence that hydrogen peroxide treatment, which generates free radical species at the SOD1 active site, causes oxidative damage to active-site histidine residues, leading to major structural changes and non-amyloid aggregation similar to that seen in ALS. Time-resolved measurements of release of bound metal ligands, exposure of hydrophobic surface area, and alterations in the SOD1 proton NMR spectrum have allowed us to model the early structural changes occurring as SOD1 misfolds, prior to aggregation. ALS-causing SOD1 mutations apparently alter this pathway by increasing exposure of buried epitopes in misfolded species populated at endpoint. We have identified a well-populated early misfolding intermediate that could serve as a target for therapies designed to block downstream misfolding and aggregation events and thereby treat SOD1-associated ALS.

Conversion of Abeta42 into a folded soluble native-like protein using a semi-random library of amphipathic helices.

The amyloid cascade model hypothesizes that neurotoxic oligomers or aggregates formed by the Alzheimer amyloid peptide (Abeta) cause disease pathology in Alzheimer's disease. Attempted treatment strategies for Alzheimer's disease have involved either inhibiting Abeta oligomerization or aggregation, or dissolving existing aggregates. Blocking such downhill processes, however, has proved daunting. We have used a different approach that targets Abeta before the oligomerization cascade begins. We predicted that an amphipathic helix could convert Abeta into a native-like protein and inhibit initiation of oligomerization and aggregation. This idea was tested with a designed library and genetic screen. We exhaustively screened a library of semi-randomized amphipathic helical sequences, each expressed as a fusion protein with an Abeta42-yellow fluorescent protein sequence serving as a reporter for folding and solubilization. This yielded an amphipathic helix capable of initiating native-like folding in Abeta42 and preventing aggregation. This amphipathic helix has direct application to Alzheimer's disease therapy development.