Amyloid fibrils embodying distinctive yeast prion phenotypes exhibit diverse morphologies.

Yeast prions are self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that confer distinct phenotypes when introduced into cells that do not carry the prion. Here, we investigate the structure of NM fibrils templated into the prion conformation with cellular lysates. Our electron microscopy studies reveal that NM fibrils that confer either a strong or a weak prion phenotype are both mixtures of thin and thick fibrils that result from differences in packing of the M domain. Strong NM fibrils have more thin fibrils and weak NM fibrils have more thick fibrils. Interestingly, both mass per length and solid state NMR reveal that the thin and thick fibrils have different underlying molecular structures in the prion strain variants that do not interconvert.

Copper(II)-bis-histidine coordination structure in a fibrillar amyloid ß-peptide fragment and model complexes revealed by electron spin echo envelope modulation spectroscopy.

Truncated and mutated amyloid-ß (Aß) peptides are models for systematic study-in homogeneous preparations-of the molecular origins of metal ion effects on Aß aggregation rates, types of aggregate structures formed, and cytotoxicity. The 3D geometry of bis-histidine imidazole coordination of Cu(II) in fibrils of the nonapetide acetyl-Aß(13-21)H14A has been determined by powder (14) N electron spin echo envelope modulation (ESEEM) spectroscopy. The method of simulation of the anisotropic combination modulation is described and benchmarked for a Cu(II) -bis-cis-imidazole complex of known structure. The revealed bis-cis coordination mode, and the mutual orientation of the imidazole rings, for Cu(II) in Ac-Aß(13-21)H14A fibrils are consistent with the proposed ß-sheet structural model and pairwise peptide interaction with Cu(II) , with an alternating [-metal-vacancy-]n pattern, along the N-terminal edge. Metal coordination does not significantly distort the intra-ß-strand peptide interactions, which provides a possible explanation for the acceleration of Ac-Aß(13-21)H14A fibrillization by Cu(II) , through stabilization of the associated state and low-reorganization integration of ß-strand peptide pair precursors.

Physical properties of polymorphic yeast prion amyloid fibers.

Amyloid fibers play important roles in many human diseases and natural biological processes and have immense potential as novel nanomaterials. We explore the physical properties of polymorphic amyloid fibers formed by yeast prion protein Sup35. Amyloid fibers that conferred distinct prion phenotypes ([PSI(+)]), strong (S) versus weak (W) nonsense suppression, displayed different physical properties. Both S[PSI(+)] and W[PSI(+)] fibers contained structural inhomogeneities, specifically local regions of static curvature in S[PSI(+)] fibers and kinks and self-cross-linking in W[PSI(+)] fibers. Force-extension experiments with optical tweezers revealed persistence lengths of 1.5 µm and 3.3 µm and axial stiffness of 5600 pN and 9100 pN for S[PSI(+)] and W[PSI(+)] fibers, respectively. Thermal fluctuation analysis confirmed the twofold difference in persistence length between S[PSI(+)] and W[PSI(+)] fibers and revealed a torsional stiffness of kinks and cross-links of ~100-200 pN·nm/rad.

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.

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.

Facial symmetry in protein self-assembly.

Amyloids are self-assembled protein architectures implicated in dozens of misfolding diseases. These assemblies appear to emerge through a "selection" of specific conformational "strains" which nucleate and propagate within cells to cause disease. The short Abeta(16-22) peptide, which includes the central core of the Alzheimer's disease Abeta peptide, generates an amyloid fiber which is morphologically indistinguishable from the full-length peptide fiber, but it can also form other morphologies under distinct conditions. Here we combine spectroscopic and microscopy analyses that reveal the subtle atomic-level differences that dictate assembly of two conformationally pure Abeta(16-22) assemblies, amyloid fibers and nanotubes, and define the minimal repeating unit for each assembly.

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.

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 Zn(2+) to modulate the assembly kinetics and morphology of congeners of the amyloid beta peptide (Abeta) associated with Alzheimer's disease. We now reveal a correlation among Abeta-Cu(2+) coordination, peptide self-assembly, and neuronal viability. By using the central segment of Abeta, HHQKLVFFA or Abeta(13-21), which contains residues H13 and H14 implicated in Abeta-metal ion binding, we show that Cu(2+) forms complexes with Abeta(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-Abeta(13-21)H14A, alters metal coordination, allowing Cu(2+) to accelerate assembly into neurotoxic fibrils. These results establish that the N-terminal region of Abeta can access different metal-ion-coordination environments and that different complexes can lead to profound changes in Abeta self-assembly kinetics, morphology, and toxicity. Related metal-ion coordination may be critical to the etiology of other neurodegenerative diseases.

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 beta-sheets, is counterbalanced by the still poor understanding of the higher organization of beta-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 Abeta peptide implicated in Alzheimer's 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.

Modulating amyloid self-assembly and fibril morphology with Zn(II).

Metal ions (Zn(II)) are demonstrated as probes of amyloid structure in simple segments of the Abeta peptide, Abeta(13-21). By restricting the possible metal binding sites to His13/His14 dyad, we show that Zn2+ can specifically control the rate of self-assembly and dramatically regulate amyloid morphology via distinct coordination environments as characterized by X-ray absorption spectroscopy. The data establish that the single His13 is sufficient to coordinate Zn2+ productively for typical amyloid fiber formation, while a distinct Zn2+ coordination environment can be accessed in the presence of His13/Hi14 dyad to stabilize sheet/sheet associations and the transition to a ribbon/tube morphology.

Expression of urokinase-type plasminogen activator receptor is correlated with metastases of lingual squamous cell carcinoma.

Lingual squamous cell carcinoma is common and the survival rate is relatively low. The invasion of cancer cells from the primary tumour into the surrounding tissue is an early step in the process of metastasis and urokinase-type plasminogen activator receptor (uPAR) is a vital mediator of cellular migration in some carcinomas. By binding urokinase-type plasminogen activator, uPAR localises proteolytic activity to the leading edge of the cells, thereby facilitating cellular migration and penetration through tissue boundaries. uPAR also binds directly to vitronectin and associates with integrins within the plasma membrane, which alters the strength of cellular adhesion. In this study we used reverse transcription polymerase chain reaction, immunocytochemistry, and Western-blot to examine the expression of uPAR mRNA and protein in Ts and Tca 8113 cell lines of lingual squamous cell carcinoma and in normal oral mucosal cells. uPAR mRNA and protein were expressed in Ts cells, but not in Tca 8113 cells or in normal oral epithelial cells. Ts cells have higher metastatic potential than Tca 8113 cells. The results suggest that uPAR has an important role in the aggressiveness of lingual squamous cell carcinoma.

[Effects of 17 beta-estradiol on the adhesion, invasion and motility potential of salivary mucoepidermoid carcinoma Mc3 cells].

OBJECTIVE: To study the effects of 17 beta-estradiol on the adhesion, invasion and motility potential of salivary mucoepidermoid carcinoma Mc3 cells. METHODS: The effects of 17 beta-estradiol on adhesion, invasion and motility potential of salivary mucoepidermoid carcinoma Mc3 cells were investigated with cell attachment assay on fibronectin (FN), wound assay, chemotaxis assay, and gelatin-incorporated SDS-PAGE electrophoresis. The expression of estrogen receptor in Mc3 cells was determined by immunohistochemistry assay. RESULTS: Attachment rates of Mc3 cells treated with E2 at 10(-9), 10(-8), 10(-7), 10(-6) mol/L were 38.3%, 50.4%, 69.2% and 91.1% respectively, and the rate in control was 25.0%. When exposed to 17 beta-estradiol at 10(-9), 10(-8), 10(-7) and 10(-6) mol/L for 48 h, motility of Mc3 cells on FN increased by 16.9%, 40.9%, 36.4% and 38.8% respectively. When at 10(-6) mol/l, 17 beta-estradiol increased chemotaxis potential of Mc3 cells to FN by 60.3%. The activity of 68 000 matrix metalloproteinase (MMP-2) of Mc3 cells was enhanced at different levels by 10(-9), 10(-8), 10(-7), 10(-6) mol/L of 17 beta-estradiol, and estrogen receptor was also detected in nucleus of Mc3 cells by immunohistochemistry assay. CONCLUSIONS: 17 beta-estradiol at physiological concentration may enhance the adhesion, invasion and motility potential of salivary mucoepidermoid carcinoma Mc3 cells.

Imaging amyloid beta peptide oligomeric particles in solution.

While all protein misfolding diseases are characterized by fibrous amyloid deposits, the favorable free energy and strongly cooperative nature of the self-assembly have complicated the development of therapeutic strategies aimed at preventing their formation. As structural models for the amyloid fibrils approach atomic resolution, increasing evidence suggests that early folding intermediates, rather than the final structure, are more strongly associated with the loss of neuronal function. For that reason we now demonstrate the use of cryo-etch high-resolution scanning electron microscopy (cryo-HRSEM) for the direct observation of pathway intermediates in amyloid assembly. A congener of the Abeta peptide of Alzheimer's disease, Abeta(13-21), samples a variety of time-dependent self-assembles in a manner similar to those seen for larger proteins. A morphological description of these intermediates is the first step towards their structural characterization and the definition of their role in both amyloid assembly and neurotoxicity.

Metal switch for amyloid formation: insight into the structure of the nucleus.

The role of Zn2+ in pre-organizing Abeta(10-21) amyloid formation is shown to preferentially alter the relative rate of fibril nucleation and to have little influence on fibril propagation. Fibril morphology, as determined by small angle neutron scattering (SANS) and transmission electron microscopy (TEM), was unchanged in the presence and absence of Zn2+ in Abeta(10-21), as well as in a series of site-specifically altered variants. The metal-independence of the Abeta(10-21)H13Q peptide suggested that the increase in nucleation rate in Abeta(10-21) is due to Zn2+-mediated inter-sheet interactions, involving both histidine 13 and histidine 14.