Parallel Β-sheet Research Articles

Type II Ice-Binding Proteins Isolated from an Arctic Microalga Are Similar to Adhesin-Like Proteins and Increase Freezing Tolerance in Transgenic Plants.

Microalgal ice-binding proteins (IBPs) in the polar region are poorly understood at the genome-wide level, although they are important for cold adaptation. Through the transcriptome study with the Arctic green alga Chloromonas sp. KNF0032, we identified six Chloromonas IBP genes (CmIBPs), homologous with the previously reported IBPs from Antarctic snow alga CCMP681 and Antarctic Chloromonas sp. They were organized with multiple exon/intron structures and low-temperature-responsive cis-elements in their promoters and abundantly expressed at low temperature. The biological functions of three representative CmIBPs (CmIBP1, CmIBP2 and CmIBP3) were tested using in vitro analysis and transgenic plant system. CmIBP1 had the most effective ice recrystallization inhibition (IRI) activities in both in vitro and transgenic plants, and CmIBP2 and CmIBP3 had followed. All transgenic plants grown under nonacclimated condition were freezing tolerant, and especially 35S::CmIBP1 plants were most effective. After cold acclimation, only 35S::CmIBP2 plants showed slightly increased freezing tolerance. Structurally, the CmIBPs were predicted to have β-solenoid forms with parallel β-sheets and repeated TXT motifs. The repeated TXT structure of CmIBPs appears similar to the AidA domain-containing adhesin-like proteins from methanogens. We have shown that the AidA domain has IRI activity as CmIBPs and phylogenetic analysis also supported that the AidA domains are monophyletic with ice-binding domain of CmIBPs, and these results suggest that CmIBPs are a type of modified adhesins.

The Functional Mammalian CRES (Cystatin-Related Epididymal Spermatogenic) Amyloid is Antiparallel \u03b2-Sheet Rich and Forms a Metastable Oligomer During Assembly

An amyloid matrix composed of several family 2 cystatins, including the reproductive cystatin CRES, is an integral structure in the mouse epididymal lumen and has proposed functions in sperm maturation and protection. Understanding how CRES amyloid assembles in vitro may provide clues on how the epididymal amyloid matrix forms in vivo. We therefore purified full-length CRES under nondenaturing conditions and followed its aggregation from monomer to amyloid under conditions that may approximate those in the epididymal lumen. CRES transitioned into a metastable oligomer that was resistant to aggregation and only over extended time formed higher-ordered amyloids. High protein concentrations facilitated oligomer assembly and also were required to maintain the metastable state since following dilution the oligomer was no longer detected. Similar to other amyloid precursors, the formation of CRES amyloids correlated with a loss of α-helix and a gain of β-sheet content. However, CRES is unique in that its amyloids are rich in antiparallel β-sheets instead of the more common parallel β-sheets. Taken together, our studies suggest that early metastable oligomers may serve as building blocks for functional amyloid assembly and further reveal that antiparallel β-sheet-rich amyloids can be functional forms.

Aggregation of amylin: Spectroscopic investigation

Apart from its relevance to pathology, protein misfolding disease like Type-II Diabetes Mellitus, caused by amyloids of amylin protein has attracted more attention due to structural changes occurring during the aggregation process. We report extensive spectroscopy data of amylin during fibril formation through Raman, FTIR, CD, UV–vis absorption and photoluminescence (PL) spectroscopy. UV–vis and PL spectrum showed the sigmoidal growth of fibril with a lag time of ~2 days, which is consistent with earlier reported work using dynamic light scattering (DLS). Raman spectra revealed the formation of parallel and anti-parallel β-sheet from 0% to 20% with ageing (1st day to 21st day) at pH 6.5 ± 0.1. The results are corroborated by CD and FTIR data. These show the change in β-sheet by 23% at pH 6.5 ± 0.1, 26% at pH = 1.0 ± 0.1 and 30% at pH = 12 ± 0.1. It is also shown that the formation and conversion of other secondary structures into β-sheet is very sensitive towards the pH and ageing. The study may be used for the development of therapeutic strategies that could inhibit or even reverse the process of aggregation.

RNA recognition motifs of disease-linked RNA-binding proteins contribute to amyloid formation

Aberrant expression, dysfunction and particularly aggregation of a group of RNA-binding proteins, including TDP-43, FUS and RBM45, are associated with neurological disorders. These three disease-linked RNA-binding proteins all contain at least one RNA recognition motif (RRM). However, it is not clear if these RRMs contribute to their aggregation-prone character. Here, we compare the biophysical and fibril formation properties of five RRMs from disease-linked RNA-binding proteins and five RRMs from non-disease-associated proteins to determine if disease-linked RRMs share specific features making them prone to self-assembly. We found that most of the disease-linked RRMs exhibit reversible thermal unfolding and refolding, and have a slightly lower average thermal melting point compared to that of normal RRMs. The full domain of TDP-43 RRM1 and FUS RRM, as well as the β-peptides from these two RRMs, could self-assemble into fibril-like aggregates which are amyloids of parallel β-sheets as verified by X-ray diffraction and FT-IR spectroscopy. Our results suggest that some disease-linked RRMs indeed play important roles in amyloid formation and shed light on why RNA-binding proteins with RRMs are frequently identified in the cellular inclusions of neurodegenerative diseases.

Characterization of Hidden Chirality: Two-Fold Helicity in β-Strands

A β-strand is a component of a β-sheet and is an important structural motif in biomolecules. An α-helix has clear helicity, while chirality of a β-strand had been discussed on the basis of molecular twists generated by forming hydrogen bonds in parallel or non-parallel β-sheets. Herein we describe handedness determination of two-fold helicity in a zig-zag β-strand structure. Left- (M) and right-handedness (P) of the two-fold helicity was defined by application of two concepts: tilt-chirality and multi-point approximation. We call the two-fold helicity in a β-strand, whose handedness has been unrecognized and unclarified, as hidden chirality. Such hidden chirality enables us to clarify precise chiral characteristics of biopolymers. It is also noteworthy that characterization of chirality of high dimensional structures like a β-strand and α-helix, referred to as high dimensional chirality (HDC) in the present study, will contribute to elucidation of the possible origins of chirality and homochirality in nature because such HDC originates from not only asymmetric centers but also conformations in a polypeptide chain.

Microstructural and conformational changes of gluten proteins in wheat-rye sourdough

Changes in the microstructure and secondary structure of sourdough gluten proteins were studied. Four strains of lactic acid bacteria (LAB) were used in fermentations, Lactobacillus delbrueckii subsp. bulgaricus (LB), Lactobacillus fermentum (LF), Lactobacillus plantarum (LP), and Pediococcus pentosaceus (PP). LAB fermentative profiles were performed by potenciometric measurements, microstructural analyses by scanning electron microscopy (SEM), and conformational changes by Raman spectroscopy. Gluten proteins experienced different degree of depolymerisation into defined microstructures, as fibril networks and laminar-like structures, and their occurrence was associated to an increase in β-sheets structures. These changes were dependent upon the distinctive acidifying kinetics of each strain. Gluten proteins of LF and LP sourdough were preferably arranged into laminar structures, and, in both cases, parallel β-sheet was the predominant conformation. Meanwhile for LB and PP sourdough, the development of three-dimensional fibril networks prevailed and it was accompanied by an increase of antiparallel β-sheets. LAB fermentative activity also lead to modifications in disulphide bonds and in the microenvironment concerning aromatic amino acids.

Improved enzymatic accessibility of peanut protein isolate pre-treated using thermosonication

Thermosonication pre-treatment was used to enhance the pancreatin-induced proteolysis of peanut protein isolate (PPI). Response surface methodology was applied to optimize the thermosonication conditions (including power-output and temperature), and the highest degree of hydrolysis (7.16%) was obtained at 475.0 W, 72 °C. SDS-PAGE analysis showed that at this optimized condition, the enzymatic accessibility of the major constitutive protein arachin in thermosonicated PPI (TS-PPI) was substantially improved compared to that in untreated PPI or sonicated PPI (475 W, 30 °C; S-PPI), resulting in a remarkable increase in protein solubility for the hydrolysates. Protein denaturation and conformation profiles of untreated PPI, S-PPI and TS-PPI were investigated using differential scanning calorimetry, intrinsic fluorescence emission spectroscopy, Fourier transform infra-red spectroscopy and thioflavin-T (ThT) fluorescence assay. It was found that heat could present a markedly additive effect to ultrasound on denaturing peanut proteins, leading to significant changes in protein conformation. TS-PPI was characterized by the appearance of high proportion of parallel intermolecular β-sheets and a strong fluorescence enhancement upon binding to ThT, suggesting that the protein unfolding and aggregation induced by thermosonication probably resulted in the formation of fibril protein aggregates in TS-PPI rather than spherical protein aggregates formed in S-PPI. As a result, the protein conformation of TS-PPI appeared to be more unfolded and flexible than that of untreated PPI or S-PPI, and therefore was more easily accessible to protease. This study shows that thermosonication pre-treatment could be a highly effective and feasible technique to improve the enzymatic accessibility of globular proteins, producing prominent functional benefits for the protein hydrolysates.

Mechanisms of Strain Diversity of Disease-Associated in-Register Parallel β-Sheet Amyloids and Implications About Prion Strains.

The mechanism of prion strain diversity remains unsolved. Investigation of inheritance and diversification of protein-based pathogenic information demands the identification of the detailed structures of abnormal isoforms of the prion protein (PrPSc); however, achieving purification is difficult without affecting infectivity. Similar prion-like properties are recognized also in other disease-associated in-register parallel β-sheet amyloids including Tau and α-synuclein (αSyn) amyloids. Investigations into structures of those amyloids via solid-state nuclear magnetic resonance spectroscopy and cryo-electron microscopy recently made remarkable advances due to their relatively small sizes and lack of post-translational modifications. Herein, we review advances regarding pathogenic amyloids, particularly Tau and αSyn, and discuss implications about strain diversity mechanisms of prion/PrPSc from the perspective that PrPSc is an in-register parallel β-sheet amyloid. Additionally, we present our recent data of molecular dynamics simulations of αSyn amyloid, which suggest significance of compatibility between β-sheet propensities of the substrate and local structures of the template for stability of amyloid structures. Detailed structures of αSyn and Tau amyloids are excellent models of pathogenic amyloids, including PrPSc, to elucidate strain diversity and pathogenic mechanisms.

Mechanistic insight into E22Q-mutation-induced antiparallel-to-parallel β-sheet transition of Aβ16-22 fibrils: an all-atom simulation study.

Alzheimer's disease is associated with the abnormal self-assembly of amyloid-β (Aβ) peptide into toxic oligomers and fibrils. Recent experiments reported that Aβ16-22, containing the central hydrophobic core (CHC) of Aβ, formed antiparallel β-sheet fibrils, while its E22Q mutant self-assembled into parallel β-sheet fibrils. However, the molecular mechanisms underlying E22Q-mutation-induced parallel β-sheet fibril formation are not well understood. Herein, we performed molecular dynamics (MD) simulations to study the dimerization processes of Aβ16-22 and Aβ16-22E22Q peptides. β-Sheet dimers with diverse hydrogen bond arrangements were observed and they exhibited highly dynamic and interconverting properties. An antiparallel-to-parallel β-sheet transition occurred in the assembly process of the E22Q mutant, but not in that of Aβ16-22. During this conformational transformation process, the inter-molecular Q22-Q22 hydrogen bonds were first formed and acted as a binder to facilitate the two chains forming a parallel orientation, then the hydrophobic interactions between residues in the CHC region consolidated this arrangement and drove the main-chain H-bond formation, hence resulting in parallel β-sheet formation. However, parallel β-sheets were less populated than antiparallel β-sheets of Aβ16-22E22Q dimers. In order to explore whether parallel β-sheets became dominant in larger size oligomers, we investigated the conformational ensembles of Aβ16-22 and Aβ16-22E22Q octamers by conducting replica exchange molecular dynamics (REMD) simulations. The REMD simulations revealed that the population of parallel β-strand alignment increased with an increase of the size of ordered Aβ16-22E22Q β-sheet oligomers, implying that the formation of full parallel β-sheets requires larger sized oligomers. Our findings provide a mechanistic explanation for the E22Q-mutation-induced formation of parallel β-sheet fibrils observed experimentally.

Toxic Amyloid Tape: A Novel Mixed Antiparallel/Parallel β-Sheet Structure Formed by Amyloid β-Protein on GM1 Clusters.

The abnormal aggregation of amyloid β-protein (Aβ) is considered central in the pathogenesis of Alzheimer's disease. We focused on membrane-mediated amyloidogenesis and found that amyloid fibrils formed on monosialoganglioside GM1 clusters were more toxic than those formed in aqueous solution. In this study, we investigated the structure of the toxic fibrils by Aβ-(1-40) in detail in comparison with less-toxic fibrils formed in aqueous solution. The less-toxic fibrils contain in-resister parallel β-sheets, whereas the structure of the toxic fibrils is unknown. Atomic force microscopy revealed that the toxic fibrils had a flat, tape-like morphology composed of a single β-sheet layer. Isotope-edited infrared spectroscopy indicated that almost the entire sequence of Aβ is included in the β-sheet. Chemical cross-linking experiments using Cys-substituted Aβs suggested that the fibrils mainly contained both in-resister parallel and two-residue-shifted antiparallel β-sheet structures. Solid-state NMR experiments also supported this conclusion. Thus, the toxic fibrils were found to possess a novel unique structure.

FTIR study of Achilles tendinopathy: protein secondary structure changes in tendon post injury

Abstract Introduction Tendinopathy, a disease involving tendon inflammation and damage, results in considerable productivity and economic losses for the patient and the society. Currently available diagnosis/ treatment monitoring modalities are less efficacious and highly subjective, underlying the need for better tools. Previously, we have shown that Fourier Transform Infrared (FTIR) spectroscopy has potential in tendinopathy diagnosis/ monitoring, when combined with multivariate statistical analysis. The current study explores the spectral range that gave the best results in statistical analysis, to discover protein secondary structures associated with tendon injury and recovery that can act as markers of disease. Methods Animals (n=60) underwent a surgery in which Achilles tendon were injured by dropping a 20g weight. Rats were divided into three groups (n=20) – control (C), Achilles tendon injury (I) and Achilles tendon injury treated with amniotic membrane fragment treated (T). FTIR spectra were obtained from each group 3, 7, 14, and 28 days post injury/ treatment. Results Triple helix, β-turn, and disordered structure levels differ between control, injured and treated tendons over the time period studied. Parallel β-sheets increase steadily over time in treated tendons compared to control and injured. Conclusion Combined analysis of Triple helix, β- sheets, β-turn, and disordered structure levels may be useful for tendinopathy diagnosis and treatment monitoring. However, further studies in this area are required to confirm the findings.

Structural Analyses of Alanine Trimer and Tetramer Crystals with Antiparallel and Parallel β-Sheet Structures Using Solid-State 1H Spin-Diffusion 2D Correlation NMR Spectroscopy.

Poly-l-alanine (PLA) sequences are key elements of the crystalline domains of spider dragline and wild silkworm silks. In the present work, 1H spin-diffusion two-dimensional (2D) correlation NMR spectra were observed for selectively deuterated (Ala)3 and (Ala)4 crystals to develop the analytical method for the structure of PLA sequences. The build-up curves of the cross peaks for three kinds of 1H pairs in selectively deuterated (Ala)3 and (Ala)4 crystals were observed to obtain spin-diffusion rate constant k j, k from relaxation master equations P i, j(τm). The k j, k values subsequently lead to effective interproton distance r j, keff (obs) values for individual proton-proton pairs, which include intra- and intermolecular contributions. The r j, keff (obs) values were compared to r j, keff (calc) values obtained from the experimentally determined atomic coordinates of antiparallel (AP) β-sheet (Ala)3 and (Ala)4 and parallel (P) β-sheet of (Ala)3 and (Ala)4 crystals. The agreement between the r j, keff (obs) and r j, keff (calc) values was good for AP β-sheet (Ala)3 and (Ala)4 crystals but poor for P β-sheet (Ala)3 and (Ala)4 crystals. These deviations were obtained from the interproton distances of the interchain contributions due to different packing arrangements. The packing arrangements of the PLA region are important when considering the relevant structure and the mechanical properties of silks.

Amyloid-β fibrils assembled on ganglioside-enriched membranes contain both parallel β-sheets and turns

Some protein and peptide aggregates, such as those of amyloid-β protein (Aβ), are neurotoxic and have been implicated in several neurodegenerative diseases. Aβ accumulates at nanoclusters enriched in neuronal lipids called gangliosides in the presynaptic neuronal membrane, and the resulting oligomeric and/or fibrous forms accelerate the development of Alzheimer's disease. Although the presence of Aβ deposits at such nanoclusters is known, the mechanism of their assembly and the relationship between Aβ secondary structure and topography are still unclear. Here, we first confirmed by atomic force microscopy that Aβ40 fibrils can be obtained by incubating seed-free Aβ40 monomers with a membrane composed of sphingomyelin, cholesterol, and the ganglioside GM1. Using Fourier transform infrared (FTIR) reflection-absorption spectroscopy, we then found that these lipid-associated fibrils contained parallel β-sheets, whereas self-assembled Aβ40 molecules formed antiparallel β-sheets. We also found that the fibrils obtained at GM1-rich nanoclusters were generated from turn Aβ40 Our findings indicate that Aβ generally self-assembles into antiparallel β-structures but can also form protofibrils with parallel β-sheets by interacting with ganglioside-bound Aβ. We concluded that by promoting the formation of parallel β-sheets, highly ganglioside-enriched nanoclusters help accelerate the elongation of Aβ fibrils. These results advance our understanding of ganglioside-induced Aβ fibril formation in neuronal membranes and may help inform the development of additional therapies for Alzheimer's disease.

Effect of Differences in the Primary Structure of the A-Chain on the Aggregation of Insulin Fragments.

Bovine and human insulin have similar primary structures. In this article, the region of the insulin A-chain of bovine and human insulin where the amino acid composition is different was studied. Bovine insulin fragment (BIF) and human insulin fragment (HIF) were synthesized in solid-phase peptide synthesis. The effects of pH, temperature, urea, ionic strength, and stirring on the formation of fibrils were studied using a fractional factorial resolution III experimental design. Fibrillation was monitored by fluorescence and infrared spectroscopy and optical microscopy. Both fragments formed fibrils at pH 1.6 and a temperature of 60 °C. The lag time and apparent aggregation growth rate constant were determined using a two-parameter kinetic model. It was found that the bovine insulin fragment has a shorter lag time than the human insulin one, whereas the exponential phase rate was faster for HIF than for BIF. An increase in β-sheets content with time was observed in both fragments. The increase in β-sheets was preceded by an initial decrease in α-helices followed by an intermediate increase during the transition from the lag phase to elongation phase. Temperature and ionic strength are among the most important experimental factors during the lag phase, whereas ionic strength is replaced by pH during the elongation phase for both the fragments. Congo red binding confirmed the presence of ringlike oligomer structures rich in antiparallel β-sheets, which tend to form fibrils rich in parallel β-sheets.

Local structural motifs in proteins: Detection and characterization of fragments inserted in helices

The global/local fold of protein structures is stabilized by a variety of specific interactions. A primary role in this context is played by hydrogen bonds. In order to identify novel motifs in proteins, we searched Protein Data Bank structures looking for backbone H-bonds formed by NH groups of two (or more) consecutive residues with consecutive CO groups of distant residues in the sequence. The present analysis unravels the occurrence of recurrent structural motifs that, to the best of our knowledge, had not been characterized in literature. Indeed, these H-bonding patterns are found (i) in a specific parallel β-sheet capping, (ii) in linking of β-hairpins to α-helices, and (iii) in α-helix insertions. Interestingly, structural analyses of these motifs indicate that Gly residues frequently occupy prominent positions. The formation of these motifs is likely favored by the limited propensity of Gly to be embodied in helices/sheets. Of particular interest is the motif corresponding to insertions in helices that was detected in 1% of analyzed structures. Inserted fragments may assume different structures and aminoacid compositions and usually display diversified evolutionary conservation. Since inserted regions are physically separated from the rest of the protein structure, they represent hot spots for ad-hoc protein functionalization.

Monitoring Unfolding of Titin I27 Single and Bi Domain with High-Pressure NMR Spectroscopy

A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the folding energy landscape. Simulations, when corroborated by experimental data yielding global information on the folding process, can provide this level of insight. Molecular dynamics (MD) has often been combined with force spectroscopy experiments to decipher the unfolding mechanism of titin immunoglobulin-like single or multidomain, the giant multimodular protein from sarcomeres, yielding information on the sequential events during titin unfolding under stretching. Here, we used high-pressure NMR to monitor the unfolding of titin I27 Ig-like single domain and tandem. Because this method brings residue-specific information on the folding process, it can provide quasiatomic details on this process without the help of MD simulations. Globally, the results of our high-pressure analysis are in agreement with previous results obtained by the combination of experimental measurements and MD simulation and/or protein engineering, although the intermediate folding state caused by the early detachment of the AB β-sheet, often reported in previous works based on MD or force spectroscopy, cannot be detected. On the other hand, the A′G parallel β-sheet of the β-sandwich has been confirmed as the Achilles heel of the three-dimensional scaffold: its disruption yields complete unfolding with very similar characteristics (free energy, unfolding volume, kinetics rate constants) for the two constructs.

Simulations and Experiments Delineate Amyloid Fibrilization by Peptides Derived from Glaucoma-Associated Myocilin.

Mutant myocilin aggregation is associated with inherited open angle glaucoma, a prevalent optic neuropathy leading to blindness. Comprehension of mutant myocilin aggregation is of fundamental importance to glaucoma pathogenesis and ties glaucoma to amyloid diseases such as Alzheimer's. Here, we probe the aggregation properties of peptides derived from the myocilin olfactomedin domain. Peptides P1 (residues 326-337) and P3 (residues 426-442) were identified previously to form amyloids. Coarse-grained discontinuous molecular dynamics simulations using the PRIME20 force field (DMD/PRIME20) predict that P1 and P3 are aggregation-prone; P1 consistently forms fibrillar aggregates with parallel in-register β-sheets, whereas P3 forms β-sheet-containing aggregates without distinct order. Natural abundance 13C solid-state NMR spectra validate that aggregated P1 exhibits amyloid signatures and is more homogeneous than aggregated P3. DMD/PRIME20 simulations provide a viable method to predict peptide aggregation propensities and aggregate structure/order which cannot be accessed by bioinformatics or readily attained experimentally.

Elucidating the Structures of Amyloid Oligomers with Macrocyclic β-Hairpin Peptides: Insights into Alzheimer's Disease and Other Amyloid Diseases.

In the more than a century since its identification, Alzheimer's disease has become the archetype of amyloid diseases. The first glimpses of the chemical basis of Alzheimer's disease began with the identification of "amyloid" plaques in the brain in 1892 and extended to the identification of proteinaceous fibrils with "cross-β" structure in 1968. Further efforts led to the discovery of the β-amyloid peptide, Aβ, as a 40- or 42-amino acid peptide that is responsible for the plaques and fibrils. At this point, a three-decade-long marathon began to elucidate the structure of the fibrils and identify the molecular basis of Alzheimer's disease. Along the way, an alternative model began to emerge in which small aggregates of Aβ, called "oligomers", rather than fibrils, are the culprits that lead to neurodegeneration in Alzheimer's disease. This Account describes what is known about the structures of the fibrils and details our research group's efforts to understand the structural, biophysical, and biological properties of the oligomers in amyloid diseases. β-Sheets are the building blocks of amyloid fibrils and oligomers. Amyloid fibrils generally consist of extended networks of parallel β-sheets. Amyloid oligomers appear to be more compact enclosed structures, some of which are thought to be composed of antiparallel β-sheets comprising β-hairpins. β-Hairpins are special because their twisted shape, hydrophobic surfaces, and exposed hydrogen-bonding edges impart a unique propensity to form compact assemblies. Our laboratory has developed macrocyclic β-sheets that are designed to mimic β-hairpins formed by amyloidogenic peptides and proteins. The β-hairpin mimics contain two β-strand peptide fragments linked together at their N- and C-termini by two δ-linked ornithine turn mimics to create a macrocycle. An N-methyl group is installed on one of the β-strands to prevent uncontrolled aggregation. These design features facilitate crystallization of the β-hairpin mimics and determination of the X-ray crystallographic structures of the oligomers that they form. During the past few years, our laboratory has elucidated the X-ray crystallographic structures of oligomers formed by β-hairpin mimics derived from Aβ, α-synuclein, and β2-microglobulin. Out of these three amyloidogenic peptides and proteins, the Aβ β-hairpin mimics have provided the most insight into amyloid oligomers. Our studies have revealed a previously undiscovered mode of self-assembly, whereby three Aβ β-hairpin mimics assemble to form a triangular trimer. The triangular trimers are remarkable, because they contain two largely hydrophobic surfaces that pack together with other triangular trimers to form higher-order oligomers, such as hexamers and dodecamers. Some of the dodecamers pack in the crystal lattice to form annular porelike assemblies. Some of the β-hairpin mimics and triangular trimers assemble in solution to form oligomers that recapitulate the crystallographically observed oligomers. These oligomers exhibit toxicity toward neuronally derived cells, recapitulating the toxicity of the oligomers formed by full-length amyloidogenic peptides and proteins. These findings are significant, because they address a gap in understanding the molecular basis of amyloid diseases. We anticipate that these studies will pave the way for developing diagnostics and therapeutics to combat Alzheimer's disease, Parkinson's disease, and other amyloid diseases.

Anti-Prion Systems in Yeast and Inositol Polyphosphates.

The amyloid-based yeast prions are folded in-register parallel β-sheet polymers. Each prion can exist in a wide array of variants, with different biological properties resulting from different self-propagating amyloid conformations. Yeast has several anti-prion systems, acting in normal cells (without protein overexpression or deficiency). Some anti-prion proteins partially block prion formation (Ssb1,2p, ribosome-associated Hsp70s); others cure a large portion of prion variants that arise [Btn2p, Cur1p, Hsp104 (a disaggregase), Siw14p, and Upf1,2,3p, nonsense-mediated decay proteins], and others prevent prion-induced pathology (Sis1p, essential cytoplasmic Hsp40). Study of the anti-prion activity of Siw14p, a pyrophosphatase specific for 5-diphosphoinositol pentakisphosphate (5PP-IP5), led to the discovery that inositol polyphosphates, signal transduction molecules, are involved in [PSI+] prion propagation. Either inositol hexakisphosphate or 5PP-IP4 (or 5PP-IP5) can supply a function that is needed by nearly all [PSI+] variants. Because yeast prions are informative models for mammalian prion diseases and other amyloidoses, detailed examination of the anti-prion systems, some of which have close mammalian homologues, will be important for the development of therapeutic measures.

Conversion between parallel and antiparallel β-sheets in wild-type and Iowa mutant Aβ40 fibrils.

Using a variant of Hamilton-replica-exchange, we study for wild type and Iowa mutant Aβ40 the conversion between fibrils with antiparallel β-sheets and such with parallel β-sheets. We show that wild type and mutant form distinct salt bridges that in turn stabilize different fibril organizations. The conversion between the two fibril forms leads to the release of small aggregates that in the Iowa mutant may shift the equilibrium from fibrils to more toxic oligomers.