Inert and seed-competent tau monomers suggest structural origins of aggregation.

Tauopathies feature progressive accumulation of tau amyloids. Pathology may begin when these amplify from a protein template, or seed, whose structure is unknown. We have purified and characterized distinct forms of tau monomer-inert (M) and seed-competent (M).

Designing well-defined photopolymerized synthetic matrices for three-dimensional culture and differentiation of induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are of interest for the study of disease, where these cells can be derived from patients and have the potential to be differentiated into any cell type; however, three-dimensional (3D) culture and differentiation of iPSCs within well-defined synthetic matrices for these applications remains limited. Here, we aimed to establish synthetic cell-degradable hydrogels that allow precise presentation of specific biochemical cues for 3D culture of iPSCs with relevance for hypothesis testing and lineage-specific differentiation. We synthesized poly(ethylene glycol)-(PEG)-peptide-based hydrogels by photoinitiated step growth polymerization and used them to test the hypothesis that the viability of iPSCs within these matrices could be rescued with appropriate biochemical cues inspired by proteins and integrins important for iPSC culture on Matrigel.

The N-terminus of the prion protein is a toxic effector regulated by the C-terminus.

PrP, the cellular isoform of the prion protein, serves to transduce the neurotoxic effects of PrP, the infectious isoform, but how this occurs is mysterious. Here, using a combination of electrophysiological, cellular, and biophysical techniques, we show that the flexible, N-terminal domain of PrP functions as a powerful toxicity-transducing effector whose activity is tightly regulated by the globular C-terminal domain. Ligands binding to the N-terminal domain abolish the spontaneous ionic currents associated with neurotoxic mutants of PrP, and the isolated N-terminal domain induces currents when expressed in the absence of the C-terminal domain.

Prefibrillar huntingtin oligomers isolated from HD brain potently seed amyloid formation.

Many neurodegenerative diseases are associated with deposits of aggregated protein in the brain. The molecular pathways through which soluble proteins misfold to form amyloids and large protein aggregates often include diverse oligomeric species, only some of which progress to the amyloid state. Here we show that prefibrillar huntingtin (HTT) oligomers, isolated from Huntington's disease (HD) affected human brain samples or mouse models, stimulate polyglutamine amyloid formation.

Conformation-dependent epitopes recognized by prion protein antibodies probed using mutational scanning and deep sequencing.

Prion diseases are caused by a structural rearrangement of the cellular prion protein, PrP(C), into a disease-associated conformation, PrP(Sc), which may be distinguished from one another using conformation-specific antibodies. We used mutational scanning by cell-surface display to screen 1341 PrP single point mutants for attenuated interaction with four anti-PrP antibodies, including several with conformational specificity. Single-molecule real-time gene sequencing was used to quantify enrichment of mutants, returning 26,000 high-quality full-length reads for each screened population on average.

Comparative study of naturally occurring huntingtin fragments in Drosophila points to exon 1 as the most pathogenic species in Huntington's disease.

Although Huntington's disease is caused by the expansion of a CAG triplet repeat within the context of the 3144-amino acid huntingtin protein (HTT), studies reveal that N-terminal fragments of HTT containing the expanded PolyQ region can be produced by proteolytic processing and/or aberrant splicing. N-terminal HTT fragments are also prevalent in postmortem tissue, and expression of some of these fragments in model organisms can cause pathology. This has led to the hypothesis that N-terminal peptides may be critical modulators of disease pathology, raising the possibility that targeting aberrant splicing or proteolytic processing may present attractive therapeutic targets.

Conformational features of tau fibrils from Alzheimer's disease brain are faithfully propagated by unmodified recombinant protein.

Fibrils composed of tau protein are a pathological hallmark of several neurodegenerative disorders including Alzheimer's disease (AD). Here we show that when recombinant tau protein is seeded with paired helical filaments (PHFs) isolated from AD brain, the amyloid formed shares many of the structural features of AD PHFs. In contrast, tau amyloids formed with heparin as an inducing agent-a common biochemical model of tau misfolding-are structurally distinct from brain-derived PHFs.

Convergent replication of mouse synthetic prion strains.

Prion diseases are neurodegenerative disorders characterized by the aberrant folding of endogenous proteins into self-propagating pathogenic conformers. Prion disease can be initiated in animal models by inoculation with amyloid fibrils formed from bacterially derived recombinant prion protein. The synthetic prions that accumulated in infected organisms are structurally distinct from the amyloid preparations used to initiate their formation and change conformationally on repeated passage.

Protein misfolding detected early in pathogenesis of transgenic mouse model of Huntington disease using amyloid seeding assay.

Huntington disease (HD) is one of several fatal neurodegenerative disorders associated with misfolded proteins. Here, we report a novel method for the sensitive detection of misfolded huntingtin (HTT) isolated from the brains of transgenic (Tg) mouse models of HD and humans with HD using an amyloid seeding assay (ASA), which is based on the propensity of misfolded proteins to act as a seed and shorten the nucleation-associated lag phase in the kinetics of amyloid formation in vitro. Using synthetic polyglutamine peptides as the substrate for amyloid formation, we found that partially purified misfolded HTT obtained from end-stage brain tissue of two Tg HD mouse models and brain tissue of post-mortem human HD patients was capable of specifically accelerating polyglutamine amyloid formation compared with unseeded reactions and controls.

A disulfide-free single-domain V(L) intrabody with blocking activity towards huntingtin reveals a novel mode of epitope recognition.

We present the crystal structure and biophysical characterization of a human V(L) [variable domain immunoglobulin (Ig) light chain] single-domain intrabody that binds to the huntingtin (Htt) protein and has been engineered for antigen recognition in the absence of its intradomain disulfide bond, otherwise conserved in the Ig fold. Analytical ultracentrifugation demonstrated that the αHtt-V(L) 12.3 domain is a stable monomer under physiological conditions even at concentrations >20 μM.

De novo generation of prion strains.

Prions are self-replicating proteins that can cause neurodegenerative disorders such as bovine spongiform encephalopathy (also known as mad cow disease). Aberrant conformations of prion proteins accumulate in the central nervous system, causing spongiform changes in the brain and eventually death. Since the inception of the prion hypothesis - which states that misfolded proteins are the infectious agents that cause these diseases - researchers have sought to generate infectious proteins from defined components in the laboratory with varying degrees of success.

Prions.

The discovery of infectious proteins, denoted prions, was unexpected. After much debate over the chemical basis of heredity, resolution of this issue began with the discovery that DNA, not protein, from pneumococcus was capable of genetically transforming bacteria (Avery et al. 1944).

Protease-sensitive synthetic prions.

Prions arise when the cellular prion protein (PrP(C)) undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrP(Sc). Frequently, PrP(Sc) is protease-resistant but protease-sensitive (s) prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec) PrP into amyloid fibers.

Design and construction of diverse mammalian prion strains.

Prions are infectious proteins that encipher biological information within their conformations; variations in these conformations dictate different prion strains. Toward elucidating the molecular language of prion protein (PrP) conformations, we produced an array of recombinant PrP amyloids with varying conformational stabilities. In mice, the most stable amyloids produced the most stable prion strains that exhibited the longest incubation times, whereas more labile amyloids generated less stable strains and shorter incubation times.

Natural and synthetic prion structure from X-ray fiber diffraction.

A conformational isoform of the mammalian prion protein (PrP(Sc)) is the sole component of the infectious pathogen that causes the prion diseases. We have obtained X-ray fiber diffraction patterns from infectious prions that show cross-beta diffraction: meridional intensity at 4.8 A resolution, indicating the presence of beta strands running approximately at right angles to the filament axis and characteristic of amyloid structure.

Small-molecule aggregates inhibit amyloid polymerization.

Many amyloid inhibitors resemble molecules that form chemical aggregates, which are known to inhibit many proteins. Eight known chemical aggregators inhibited amyloid formation of the yeast and mouse prion proteins Sup35 and recMoPrP in a manner characteristic of colloidal inhibition. Similarly, three known anti-amyloid molecules inhibited beta-lactamase in a detergent-dependent manner, which suggests that they too form colloidal aggregates.

Prion detection by an amyloid seeding assay.

Polymerization of recombinant prion protein (recPrP), which was produced in bacteria, into amyloid fibers was accompanied by the acquisition of prion infectivity. We report here that partially purified preparations of prions seed the polymerization of recPrP into amyloid as detected by a fluorescence shift in the dye Thioflavin T. Our amyloid seeding assay (ASA) detected PrP(Sc), the sole component of the prion, in brain samples from humans with sporadic Creutzfeldt-Jakob disease, as well as in rodents with experimental prion disease.

Stochastic kinetics of intracellular huntingtin aggregate formation.

Neurodegeneration in Huntington disease is described by neuronal loss in which the probability of cell death remains constant with time. However, the quantitative connection between the kinetics of cell death and the molecular mechanism initiating neurodegeneration remains unclear. One hypothesis is that nucleation of protein aggregates containing exon I fragments of the mutant huntingtin protein (mhttex1), which contains an expanded polyglutamine region in patients with the disease, is the explanation for the infrequent but steady occurrence of neuronal death, resulting in adult onset of the disease.

Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion in the number of polyglutamine-encoding CAG repeats in the gene that encodes the huntingtin (htt) protein. A property of the mutant protein that is intimately involved in the development of the disease is the propensity of the glutamine-expanded protein to misfold and generate an N-terminal proteolytic htt fragment that is toxic and prone to aggregation. Intracellular antibodies (intrabodies) against htt have been shown to reduce htt aggregation by binding to the toxic fragment and inactivating it or preventing its misfolding.

Development of a human light chain variable domain (V(L)) intracellular antibody specific for the amino terminus of huntingtin via yeast surface display.

Intracellular antibodies (intrabodies) provide an attractive means for manipulating intracellular protein function, both for research and potentially for therapy. A challenge in the isolation of effective intrabodies is the ability to find molecules that exhibit sufficient binding affinity and stability when expressed in the reducing environment of the cytoplasm. Here, we have used yeast surface display of proteins to isolate novel scFv clones against huntingtin from a non-immune human antibody library.