Jijun Dong

Engineering metal ion coordination to regulate amyloid fibril assembly and toxicity

Protein and peptide assembly into amyloid has been implicated in functions that range from beneficial epigenetic controls to pathological etiologies. However, the exact structures of the assemblies that regulate biological activity remain poorly defined. We have previously used Zn2+ to modulate the assembly kinetics and morphology of congeners of the amyloid β peptide (Aβ) associated with Alzheimers disease. We now reveal a correlation among Aβ-Cu2+ coordination, peptide self-assembly, and neuronal viability. By using the central segment of Aβ, HHQKLVFFA or Aβ(13–21), which contains residues H13 and H14 implicated in Aβ-metal ion binding, we show that Cu2+ forms complexes with Aβ(13–21) and its K16A mutant and that the complexes, which do not self-assemble into fibrils, have structures similar to those found for the human prion protein, PrP. N-terminal acetylation and H14A substitution, Ac-Aβ(13–21)H14A, alters metal coordination, allowing Cu2+ to accelerate assembly into neurotoxic fibrils. These results establish that the N-terminal region of Aβ can access different metal-ion-coordination environments and that different complexes can lead to profound changes in Aβ self-assembly kinetics, morphology, and toxicity. Related metal-ion coordination may be critical to the etiology of other neurodegenerative diseases.

Prion induction involves an ancient system for the sequestration of aggregated proteins and heritable changes in prion fragmentation

When the translation termination factor Sup35 adopts the prion state, [PSI+], the read-through of stop codons increases, uncovering hidden genetic variation and giving rise to new, often beneficial, phenotypes. Evidence suggests that prion induction involves a process of maturation, but this has never been studied in detail. To do so, we used a visually tractable prion model consisting of the Sup35 prion domain fused to GFP (PrD-GFP) and overexpressed it to achieve induction in many cells simultaneously. PrD-GFP first assembled into Rings as previously described. Rings propagated for many generations before the protein transitioned into a Dot structure. Dots transmitted the [PSI+] phenotype through mating and meiosis, but Rings did not. Surprisingly, the underlying amyloid conformation of PrD-GFP was identical in Rings and Dots. However, by electron microscopy, Rings consisted of very long uninterrupted bundles of fibers, whereas Dot fibers were highly fragmented. Both forms were deposited at the IPOD, a biologically ancient compartment for the deposition of irreversibly aggregated proteins that we propose is the site of de novo prion induction. We find that oxidatively damaged proteins are also localized there, helping to explain how proteotoxic stresses increase the rate of prion induction. Curing PrD-GFP prions, by inhibiting Hsp104’s fragmentation activity, reversed the induction process: Dot cells produced Rings before PrD-GFP reverted to the soluble state. Thus, formation of the genetically transmissible prion state is a two-step process that involves an ancient system for the asymmetric inheritance of damaged proteins and heritable changes in the extent of prion fragmentation.

Optical trapping with high forces reveals unexpected behaviors of prion fibrils

Amyloid fibrils are important in diverse cellular functions, feature in many human diseases and have potential applications in nanotechnology. Here we describe methods that combine optical trapping and fluorescent imaging to characterize the forces that govern the integrity of amyloid fibrils formed by a yeast prion protein. A crucial advance was to use the self-templating properties of amyloidogenic proteins to tether prion fibrils, enabling their manipulation in the optical trap. At normal pulling forces the fibrils were impervious to disruption. At much higher forces (up to 250 pN), discontinuities occurred in force-extension traces before fibril rupture. Experiments with selective amyloid-disrupting agents and mutations demonstrated that such discontinuities were caused by the unfolding of individual subdomains. Thus, our results reveal unusually strong noncovalent intermolecular contacts that maintain fibril integrity even when individual monomers partially unfold and extend fibril length.

Conversion of a yeast prion protein to an infectious form in bacteria

Prions are infectious, self-propagating protein aggregates that have been identified in evolutionarily divergent members of the eukaryotic domain of life. Nevertheless, it is not yet known whether prokaryotes can support the formation of prion aggregates. Here we demonstrate that the yeast prion protein Sup35 can access an infectious conformation in Escherichia coli cells and that formation of this material is greatly stimulated by the presence of a transplanted [PSI+] inducibility factor, a distinct prion that is required for Sup35 to undergo spontaneous conversion to the prion form in yeast. Our results establish that the bacterial cytoplasm can support the formation of infectious prion aggregates, providing a heterologous system in which to study prion biology.

Probing the Role of PrP Repeats in Conformational Conversion and Amyloid Assembly of Chimeric Yeast Prions

Oligopeptide repeats appear in many proteins that undergo conformational conversions to form amyloid, including the mammalian prion protein PrP and the yeast prion protein Sup35. Whereas the repeats in PrP have been studied more exhaustively, interpretation of these studies is confounded by the fact that many details of the PrP prion conformational conversion are not well understood. On the other hand, there is now a relatively good understanding of the factors that guide the conformational conversion of the Sup35 prion protein. To provide a general model for studying the role of oligopeptide repeats in prion conformational conversion and amyloid formation, we have substituted various numbers of the PrP octarepeats for the endogenous Sup35 repeats. The resulting chimeric proteins can adopt the [PSI+] prion state in yeast, and the stability of the prion state depends on the number of repeats. In vitro, these chimeric proteins form amyloid fibers, with more repeats leading to shorter lag phases and faster assembly rates. Both pH and the presence of metal ions modulate assembly kinetics of the chimeric proteins, and the extent of modulation is highly sensitive to the number of PrP repeats. This work offers new insight into the properties of the PrP octarepeats in amyloid assembly and prion formation. It also reveals new features of the yeast prion protein, and provides a level of control over yeast prion assembly that will be useful for future structural studies and for creating amyloid-based biomaterials.

Controlling amyloid growth in multiple dimensions

The great progress made in defining the structure of protein and peptide amyloid assemblies, particularly the arrangement of peptides in β-sheets, is counterbalanced by the still poor understanding of the higher organization of β-sheets within the fibril and overall fibril/fibril associations. The assembly pathway and basis of amyloid toxicity may well depend on these higher-order structural features. For example, significant evidence points to association between sheets as the rate limiting step in fibril assembly, and a critical metal binding site has now been identified that involves residues from different individual sheets. Here we review experiments that are identifying some of the issues associated with sheet–sheet association by investigating simple model peptides derived from the central core of the Aβ peptide implicated in Alzheimers disease. These peptides transit between fibril/ribbon/nanotube morphologies in response to assembly conditions, laying the foundation for understanding the folding landscape for these higher order assemblies, revealing potential targets for therapeutic intervention, and opening strategies for the design of highly ordered peptide self-assembled microscale morphologies.