Holger Wille

Structural studies of the scrapie prion protein by electron crystallography

Because the insolubility of the scrapie prion protein (PrPSc) has frustrated structural studies by x-ray crystallography or NMR spectroscopy, we used electron crystallography to characterize the structure of two infectious variants of the prion protein. Isomorphous two-dimensional crystals of the N-terminally truncated PrPSc (PrP 27-30) and a miniprion (PrPSc106) were identified by negative stain electron microscopy. Image processing allowed the extraction of limited structural information to 7 Å resolution. By comparing projection maps of PrP 27-30 and PrPSc106, we visualized the 36-residue internal deletion of the miniprion and localized the N-linked sugars. The dimensions of the monomer and the locations of the deleted segment and sugars were used as constraints in the construction of models for PrPSc. Only models featuring parallel β-helices as the key element could satisfy the constraints. These low-resolution projection maps and models have implications for understanding prion propagation and the pathogenesis of neurodegeneration.

Prion Protein of 106 Residues Creates an Artificial Transmission Barrier for Prion Replication in Transgenic Mice

A redacted prion protein (PrP) of 106 amino acids with two large deletions was expressed in transgenic (Tg) mice deficient for wild-type (wt) PrP (Prnp0/0) and supported prion propagation. RML prions containing full-length PrP(Sc)produced disease in Tg(PrP106)Prnp0/0 mice after approximately 300 days, while transmission of RML106 prions containing PrP(Sc)106 created disease in Tg(PrP106) Prnp0/0 mice after only approximately 66 days on repeated passage. This artificial transmission barrier for the passage of RML prions was diminished by the coexpression of wt MoPrPc in Tg(PrP106)Prnp+/0 mice that developed scrapie in approximately 165 days, suggesting that wt MoPrP acts in trans to accelerate replication of RML106 prions. Purified PrP(Sc)106 was protease resistant, formed filaments, and was insoluble in nondenaturing detergents. The unique features of RML106 prions offer insights into the mechanism of prion replication, and the small size of PrP(Sc)106 should facilitate structural analysis.

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. The colloids localized to preformed fibers and prevented new fiber formation in electron micrographs. They also blocked infection of yeast cells with Sup35 prions, which suggests that colloidal inhibition may be relevant in more biological milieus.

Branched Polyamines Cure Prion-Infected Neuroblastoma Cells

ABSTRACT Branched polyamines, including polyamidoamine and polypropyleneimine (PPI) dendrimers, are able to purge PrPSc, the disease-causing isoform of the prion protein, from scrapie-infected neuroblastoma (ScN2a) cells in culture (S. Supattapone, H.-O. B. Nguyen, F. E. Cohen, S. B. Prusiner, and M. R. Scott, Proc. Natl. Acad. Sci. USA 96:14529–14534, 1999). We now demonstrate that exposure of ScN2a cells to 3 μg of PPI generation 4.0/ml for 4 weeks not only reduced PrPSc to a level undetectable by Western blot but also eradicated prion infectivity as determined by a bioassay in mice. Exposure of purified RML prions to branched polyamines in vitro disaggregated the prion rods, reduced the β-sheet content of PrP 27-30, and rendered PrP 27-30 susceptible to proteolysis. The susceptibility of PrPSc to proteolytic digestion induced by branched polyamines in vitro was strain dependent. Notably, PrPSc from bovine spongiform encephalopathy-infected brain was susceptible to PPI-mediated denaturation in vitro, whereas PrPSc from natural sheep scrapie-infected brain was resistant. Fluorescein-labeled PPI accumulated specifically in lysosomes, suggesting that branched polyamines act within this acidic compartment to mediate PrPSc clearance. Branched polyamines are the first class of compounds shown to cure prion infection in living cells and may prove useful as therapeutic, disinfecting, and strain-typing reagents for prion diseases.

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. The direct relationship between stability and incubation time of prion strains suggests that labile prions are more fit, in that they accumulate more rapidly and thus kill the host faster. Although incubation times can be changed by altering the PrP expression level, PrP sequence, prion dose, or route of inoculation, we report here the ability to modify the incubation time predictably in mice by modulating the prion conformation.

Natural and synthetic prion structure from X-ray fiber diffraction

A conformational isoform of the mammalian prion protein (PrPSc) 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-β diffraction: meridional intensity at 4.8 Å resolution, indicating the presence of β strands running approximately at right angles to the filament axis and characteristic of amyloid structure. Some of the patterns also indicated the presence of a repeating unit along the fiber axis, corresponding to four β-strands. We found that recombinant (rec) PrP amyloid differs substantially from highly infectious brain-derived prions, both in structure as demonstrated by the diffraction data, and in heterogeneity as shown by electron microscopy. In addition to the strong 4.8 Å meridional reflection, the recPrP amyloid diffraction is characterized by strong equatorial intensity at approximately 10.5 Å, absent from brain-derived prions, and indicating the presence of stacked β-sheets. Synthetic prions recovered from transgenic mice inoculated with recPrP amyloid displayed structural characteristics and homogeneity similar to those of naturally occurring prions. The relationship between the structural differences and prion infectivity is uncertain, but might be explained by any of several hypotheses: only a minority of recPrP amyloid possesses a replication-competent conformation, the majority of recPrP amyloid has to undergo a conformational maturation to acquire replication competency, or inhibitory forms of recPrP amyloid interfere with replication during the initial transmission.

Conformational Diversity of Wild-type Tau Fibrils Specified by Templated Conformation Change

Tauopathies are sporadic and genetic neurodegenerative diseases characterized by aggregation of the microtubule-associated protein Tau. Tau pathology occurs in over 20 phenotypically distinct neurodegenerative diseases, including Alzheimer disease and frontotemporal dementia. The molecular basis of this diversity among sporadic tauopathies is unknown, but distinct fibrillar wild-type (WT) Tau conformations could play a role. Using Fourier transform infrared spectroscopy, circular dichroism, and electron microscopy, we show that WT Tau fibrils and P301L/V337M Tau fibrils have distinct secondary structures, fragilities, and morphologies. Furthermore, P301L/V337M fibrillar seeds induce WT Tau monomer to form a novel fibrillar conformation, termed WT*, that is maintained over multiple seeding reactions. WT* has secondary structure, fragility, and morphology that are similar to P301L/V337M fibrils and distinct from WT fibrils. WT Tau is thus capable of conformational diversity that arises via templated conformation change, as has been described for amyloid β, β2-microglobulin, and prion proteins.

Mechanisms of prion protein assembly into amyloid

The conversion of the α-helical, cellular isoform of the prion protein (PrPC) to the insoluble, β-sheet-rich, infectious, disease-causing isoform (PrPSc) is the key event in prion diseases. In an earlier study, several forms of PrP were converted into a fibrillar state by using an in vitro conversion system consisting of low concentrations of SDS and 250 mM NaCl. Here, we characterize the structure of the fibril precursor state, that is, the soluble state under fibrillization conditions. CD spectroscopy, analytical ultracentrifugation, and chemical cross-linking indicate that the precursor state exists in a monomer-dimer equilibrium of partially denatured, α-helical PrP, with a well defined contact site of the subunits in the dimer. Using fluorescence with thioflavin T, we monitored and quantitatively described the kinetics of seeded fibril formation, including dependence of the reaction on substrate and seed concentrations. Exponential, seed-enhanced growth can be achieved in homogeneous solution, which can be enhanced by sonication. From these data, we propose a mechanistic model of fibrillization, including the presence of several intermediate structures. These studies also provide a simplified amplification system for prions.

Evolutionary descent of prion genes from the ZIP family of metal ion transporters.

In the more than twenty years since its discovery, both the phylogenetic origin and cellular function of the prion protein (PrP) have remained enigmatic. Insights into a possible function of PrP may be obtained through the characterization of its molecular neighborhood in cells. Quantitative interactome data demonstrated the spatial proximity of two metal ion transporters of the ZIP family, ZIP6 and ZIP10, to mammalian prion proteins in vivo. A subsequent bioinformatic analysis revealed the unexpected presence of a PrP-like amino acid sequence within the N-terminal, extracellular domain of a distinct sub-branch of the ZIP protein family that includes ZIP5, ZIP6 and ZIP10. Additional structural threading and orthologous sequence alignment analyses argued that the prion gene family is phylogenetically derived from a ZIP-like ancestral molecule. The level of sequence homology and the presence of prion protein genes in most chordate species place the split from the ZIP-like ancestor gene at the base of the chordate lineage. This relationship explains structural and functional features found within mammalian prion proteins as elements of an ancient involvement in the transmembrane transport of divalent cations. The phylogenetic and spatial connection to ZIP proteins is expected to open new avenues of research to elucidate the biology of the prion protein in health and disease.

Rapid acquisition of beta-sheet structure in the prion protein prior to multimer formation.

The N-terminally truncated form of the prion protein, PrP 27-30, and the corresponding recombinant protein, rPrP, were solubilized in 0.2% SDS, and the transitions induced by changing the conditions from 0.2% SDS to physiological conditions, i.e. removing SDS, were characterized with respect to solubility, resistance to proteolysis, secondary structure and multimerization. Circular dichroism, electron microscopy and fluorescence correlation spectroscopy were used to study the structural transitions of PrP. Within one minute the alpha-helical structure of PrP was transformed into one that was enriched in beta-sheets and consisted mainly of dimers. Larger oligomers were found after 20 minutes and larger multimers exhibiting resistance to proteolysis were found after several hours. It was concluded that the monomeric alpha-helical conformation was stable in SDS or when attached to the membrane; however, the state of lowest free energy in aqueous solution at neutral pH seems to be the multimeric, beta-sheet enriched conformation.