Formation and Maturation of Phase-Separated Liquid Droplets by RNA-Binding Proteins.

Eukaryotic cells possess numerous dynamic membrane-less organelles, RNP granules, enriched in RNA and RNA-binding proteins containing disordered regions. We demonstrate that the disordered regions of key RNP granule components and the full-length granule protein hnRNPA1 can phase separate in vitro, producing dynamic liquid droplets. Phase separation is promoted by low salt concentrations or RNA. Over time, the droplets mature to more stable states, as assessed by slowed fluorescence recovery after photobleaching and resistance to salt. Maturation often coincides with formation of fibrous structures. Different disordered domains can co-assemble into phase-separated droplets. These biophysical properties demonstrate a plausible mechanism by which interactions between disordered regions, coupled with RNA binding, could contribute to RNP granule assembly in vivo through promoting phase separation. Progression from dynamic liquids to stable fibers may be regulated to produce cellular structures with diverse physiochemical properties and functions. Misregulation could contribute to diseases involving aberrant RNA granules.

Probing initial transient oligomerization events facilitating Huntingtin fibril nucleation at atomic resolution by relaxation-based NMR.

The N-terminal region of the huntingtin protein, encoded by exon-1, comprises an amphiphilic domain (httNT), a polyglutamine (Q n ) tract, and a proline-rich sequence. Polyglutamine expansion results in an aggregation-prone protein responsible for Huntington's disease. Here, we study the earliest events involved in oligomerization of a minimalistic construct, httNTQ7, which remains largely monomeric over a sufficiently long period of time to permit detailed quantitative NMR analysis of the kinetics and structure of sparsely populated [Formula: see text] oligomeric states, yet still eventually forms fibrils. Global fitting of concentration-dependent relaxation dispersion, transverse relaxation in the rotating frame, and exchange-induced chemical shift data reveals a bifurcated assembly mechanism in which the NMR observable monomeric species either self-associates to form a productive dimer (τex ∼ 30 µs, Kdiss ∼ 0.1 M) that goes on to form a tetramer ([Formula: see text] µs; Kdiss ∼ 22 µM), or exchanges with a "nonproductive" dimer that does not oligomerize further (τex ∼ 400 µs; Kdiss ∼ 0.3 M). The excited state backbone chemical shifts are indicative of a contiguous helix (residues 3-17) in the productive dimer/tetramer, with only partial helical character in the nonproductive dimer. A structural model of the productive dimer/tetramer was obtained by simulated annealing driven by intermolecular paramagnetic relaxation enhancement data. The tetramer comprises a D2 symmetric dimer of dimers with largely hydrophobic packing between the helical subunits. The structural model, validated by EPR distance measurements, illuminates the role of the httNT domain in the earliest stages of prenucleation and oligomerization, before fibril formation.

Amyloidogenic Mutation Promotes Fibril Formation of the N-terminal Apolipoprotein A-I on Lipid Membranes.

The N-terminal amino acid 1-83 fragment of apolipoprotein A-I (apoA-I) has a strong propensity to form amyloid fibrils at physiological neutral pH. Because apoA-I has an ability to bind to lipid membranes, we examined the effects of the lipid environment on fibril-forming properties of the N-terminal fragment of apoA-I variants. Thioflavin T fluorescence assay as well as fluorescence and transmission microscopies revealed that upon lipid binding, fibril formation by apoA-I 1-83 is strongly inhibited, whereas the G26R mutant still retains the ability to form fibrils. Such distinct effects of lipid binding on fibril formation were also observed for the amyloidogenic prone region-containing peptides, apoA-I 8-33 and 8-33/G26R. This amyloidogenic region shifts from random coil to α-helical structure upon lipid binding. The G26R mutation appears to prevent this helix transition because lower helical propensity and more solvent-exposed conformation of the G26R variant upon lipid binding were observed in the apoA-I 1-83 fragment and 8-33 peptide. With a partially α-helical conformation induced by the presence of 2,2,2-trifluoroethanol, fibril formation by apoA-I 1-83 was strongly inhibited, whereas the G26R variant can form amyloid fibrils. These findings suggest a new possible pathway for amyloid fibril formation by the N-terminal fragment of apoA-I variants: the amyloidogenic mutations partially destabilize the α-helical structure formed upon association with lipid membranes, resulting in physiologically relevant conformations that allow fibril formation.

Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy.

The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ~57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin.

Role of force-sensitive amyloid-like interactions in fungal catch bonding and biofilms.

The Candida albicans Als adhesin Als5p has an amyloid-forming sequence that is required for aggregation and formation of model biofilms on polystyrene. Because amyloid formation can be triggered by force, we investigated whether laminar flow could activate amyloid formation and increase binding to surfaces. Shearing Saccharomyces cerevisiae cells expressing Als5p or C. albicans at 0.8 dyne/cm(2) increased the quantity and strength of cell-to-surface and cell-to-cell binding compared to that at 0.02 dyne/cm(2). Thioflavin T fluorescence showed that the laminar flow also induced adhesin aggregation into surface amyloid nanodomains in Als5p-expressing cells. Inhibitory concentrations of the amyloid dyes thioflavin S and Congo red or a sequence-specific anti-amyloid peptide decreased binding and biofilm formation under flow. Shear-induced binding also led to formation of robust biofilms. There was less shear-activated increase in adhesion, thioflavin fluorescence, and biofilm formation in cells expressing the amyloid-impaired V326N-substituted Als5p. Similarly, S. cerevisiae cells expressing Flo1p or Flo11p flocculins also showed shear-dependent binding, amyloid formation, biofilm formation, and inhibition by anti-amyloid compounds. Together, these results show that laminar flow activated amyloid formation and led to enhanced adhesion of yeast cells to surfaces and to biofilm formation.

A yeast toxic mutant of HET-s amyloid disrupts membrane integrity.

Many studies have pointed out the interaction between amyloids and membranes, and their potential involvement in amyloid toxicity. Previously, we generated a yeast toxic amyloid mutant (M8) from the harmless amyloid protein by changing a few residues of the Prion Forming Domain of HET-s (PFD HET-s(218-289)) and clearly demonstrated the complete different behaviors of the non-toxic Wild Type (WT) and toxic amyloid (called M8) in terms of fiber morphology, aggregation kinetics and secondary structure. In this study, we compared the interaction of both proteins (WT and M8) with membrane models, as liposomes or supported bilayers. We first demonstrated that the toxic protein (M8) induces a significant leakage of liposomes formed with negatively charged lipids and promotes the formation of microdomains inside the lipid bilayer (as potential "amyloid raft"), whereas the non-toxic amyloid (WT) only binds to the membrane without further perturbations. The secondary structure of both amyloids interacting with membrane is preserved, but the anti-symmetric PO(2)(-) vibration is strongly shifted in the presence of M8. Secondly, we established that the presence of membrane models catalyzes the amyloidogenesis of both proteins. Cryo-TEM (cryo-transmission electron microscopy) images show the formation of long HET-s fibers attached to liposomes, whereas a large aggregation of the toxic M8 seems to promote a membrane disruption. This study allows us to conclude that the toxicity of the M8 mutant could be due to its high propensity to interact and disrupt lipid membranes.

Distinct region-specific alpha-synuclein oligomers in A53T transgenic mice: implications for neurodegeneration.

Aggregation of alpha-synuclein (alpha-syn), a process that generates oligomeric intermediates, is a common pathological feature of several neurodegenerative disorders. Despite the potential importance of the oligomeric alpha-syn intermediates in neuron function, their biochemical properties and pathobiological functions in vivo remain vastly unknown. Here we used two-dimensional analytical separation and an array of biochemical and cell-based assays to characterize alpha-syn oligomers that are present in the nervous system of A53T alpha-syn transgenic mice. The most prominent species identified were 53 A detergent-soluble oligomers, which preceded neurological symptom onset, and were found at equivalent amounts in regions containing alpha-syn inclusions as well as histologically unaffected regions. These oligomers were resistant to SDS, heat, and urea but were sensitive to proteinase-K digestion. Although the oligomers shared similar basic biochemical properties, those obtained from inclusion-bearing regions were prominently reactive to antibodies that recognize oxidized alpha-syn oligomers, significantly accelerated aggregation of alpha-syn in vitro, and caused primary cortical neuron degeneration. In contrast, oligomers obtained from non-inclusion-bearing regions were not toxic and delayed the in vitro formation of alpha-syn fibrils. These data indicate that specific conformations of alpha-syn oligomers are present in distinct brain regions of A53T alpha-syn transgenic mice. The contribution of these oligomers to the development of neuron dysfunction appears to be independent of their absolute quantities and basic biochemical properties but is dictated by the composition and conformation of the intermediates as well as unrecognized brain-region-specific intrinsic factors.

Characterization of hydrophobic residue requirements for alpha-synuclein fibrillization.

alpha-Synuclein is the major component of pathological inclusions characteristic of diseases like Parkinson's disease, dementia with Lewy bodies, and multiple-system atrophy. A role for alpha-synuclein in neurodegenerative diseases is further supported by point mutations and duplication and triplication of the alpha-synuclein gene (SNCA) that are causative of these disorders. The middle hydrophobic region of the alpha-synuclein protein, also termed the "non-Abeta component of Alzheimer's disease amyloid plaque (NAC)" domain, is required for alpha-synuclein to polymerize into amyloid filaments, which are the major components of alpha-synuclein pathological inclusions. In this study, we assessed the importance of specific stretches of hydrophobic residues in driving the intrinsic ability of alpha-synuclein to polymerize. Several small deletions, even one with as few as two amino acid residues (A76 and V77), dramatically impaired the ability of alpha-synuclein to polymerize into mature amyloidogenic fibrils, and instead, it preferentially formed oligomers. However, this inhibition of filament assembly was clearly dependent on the spatial context, since similar and larger hydrophobic deletions in other parts of the NAC domain reduced only the rate of fibril formation, without abrogating filament assembly. Further, mutation of residue E83 to an A rescued the ability of mutant Delta76-77 alpha-synuclein to polymerize. These findings support the notion that while both the location and hydrophobicity of protein segments are important elements that affect the propensity to form amyloid fibrils, the intrinsic ability of a polypeptide to fold structurally into amyloid is also critical.

Characterization of Apin, a secreted protein highly expressed in tooth-associated epithelia.

We previously reported expression of a protein by enamel organ (EO) cells in rat incisors, originally isolated from the amyloid of Pindborg odontogenic tumors called Apin. The aim of the present study was to further characterize the Apin gene and its protein in various species, assess tissue specificity, and clarify its localization within the EO. Northern blotting and RT-PCR revealed that expression of Apin was highest in the EO and gingiva, moderate in nasal and salivary glands, and lowest in the epididymis. The protein sequences deduced from the cloned cDNA for rat, mouse, pig, and human were aligned together with those obtained from four other mammal genomes. Apin is highly conserved in mammals but is absent in fish, birds, and amphibians. Comparative SDS-PAGE analyses of the protein obtained from bacteria, transfected cells, and extracted from EOs all indicated that Apin is post-translationally modified, a finding consistent with the presence of predicted sites for phosphorylation and O-linked glycosylation. In rodent incisors, Apin was detected only in the ameloblast layer of the EO, starting at post-secretory transition and extending throughout the maturation stage. Intense labeling was visible over the Golgi region as well as on the apices of ameloblasts abutting the enamel matrix. Apin was also immunodetected in epithelial cells of the gingiva which bind it to the tooth surface (junctional epithelium). The presence of Apin at cell-tooth interfaces suggests involvement in adhesive mechanisms active at these sites, but its presence among other epithelial tissues indicates Apin likely possesses broader physiological roles.

Multifunctional LUV liposomes decorated for BBB and amyloid targeting - B. In vivo brain targeting potential in wild-type and APP/PS1 mice.

Multifunctional liposomes (mf-LIPs) having a curcumin-lipid ligand (to target amyloids) together with two ligands to target the transferrin, and the low-density apolipoprotein receptor of the blood-brain-barrier (BBB) on their surface, were previously studied (in vitro) as potential theranostic systems for Alzheimer's disease (AD) (Papadia et al., 2017, Eur. J. Pharm. Sciences; 101:140-148). Herein, the targeting potential of mf-LIPs was compared to that of BBB-LIPs (liposomes having only the two BBB-specific ligands) in FVB mice (normal), as well as in double transgenic mice (APP/PS1) and their corresponding littermates (WT), by live-animal (in vivo) and explanted organ (ex vivo) imaging. In FVB mice, the head-signals of mf-LIPs and BBB-LIPs are either similar, or signals from mf-LIP are higher, suggesting that the co-presence of the curcumin derivative on the liposome surface does not disturb the functionality of the BBB-specific ligands. Higher brain/liver+spleen ratios (ex vivo) were calculated post-injection of mf-LIP, compared to those found after BBB-LIP injection, due to the reduced distribution of mf-LIPs in the liver and spleen; showing that the curcumin ligand increases the stealth properties of liposomes by reducing their uptake by liver and spleen. The later effect is more pronounced when the density of the BBB-specific ligands on the mf-LIPs is 0.1mol%, compared to 0.2%, highlighting the importance of this parameter. When a high lipid dose (4mg/mouse) is injected in WT and APP/PS1 mice, the head-signals of mf-LIPs are significantly higher than those of BBB-LIPs, but no differences are observed between WT and APP/PS1 mice. However, after administration of a low liposome dose (0.05mg/mouse) of mf-LIPs, significant differences in the head-signals are found between WT and transgenic mice, highlighting the AD theranostic potential of the multifunctional liposomes, as well as the importance of the experimental parameters used in such in vivo screening studies.

Atomic force microscopy reveals defects within mica supported lipid bilayers induced by the amyloidogenic human amylin peptide.

To date, over 20 peptides or proteins have been identified that can form amyloid fibrils in the body and are thought to cause disease. The mechanism by which amyloid peptides cause the cytotoxicity observed and disease is not understood. However, one of the major hypotheses is that amyloid peptides cause membrane perturbation. Hence, we have studied the interaction between lipid bilayers and the 37 amino acid residue polypeptide amylin, which is the primary constituent of the pancreatic amyloid associated with type 2 diabetes. Using a dye release assay we confirmed that the amyloidogenic human amylin peptide causes membrane disruption; however, time-lapse atomic force microscopy revealed that this did not occur by the formation of defined pores. On the contrary, the peptide induced the formation of small defects spreading over the lipid surface. We also found that rat amylin, which has 84% identity with human amylin but cannot form amyloid fibrils, could also induce similar lesions to supported lipid bilayers. The effect, however, for rat amylin but not human amylin, was inhibited under high ionic conditions. These data provide an alternative theory to pore formation, and how amyloid peptides may cause membrane disruption and possibly cytotoxicity.

Engineering amyloid fibrils from ß-solenoid proteins for biomaterials applications.

Nature provides numerous examples of self-assembly that can potentially be implemented for materials applications. Considerable attention has been given to one-dimensional cross-ß or amyloid structures that can serve as templates for wire growth or strengthen materials such as glue or cement. Here, we demonstrate controlled amyloid self-assembly based on modifications of ß-solenoid proteins. They occur naturally in several contexts (e.g., antifreeze proteins, drug resistance proteins) but do not aggregate in vivo due to capping structures or distortions at their ends. Removal of these capping structures and regularization of the ends of the spruce budworm and rye grass antifreeze proteins yield micron length amyloid fibrils with predictable heights, which can be a platform for biomaterial-based self-assembly. The design process, including all-atom molecular dynamics simulations, purification, and self-assembly procedures are described. Fibril formation with the predicted characteristics is supported by evidence from thioflavin-T fluorescence, circular dichroism, dynamic light scattering, and atomic force microscopy. Additionally, we find evidence for lateral assembly of the modified spruce budworm antifreeze fibrils with sufficient incubation time. The kinetics of polymerization are consistent with those for other amyloid formation reactions and are relatively fast due to the preformed nature of the polymerization nucleus.

Spontaneous generation of infectious nucleating amyloids in the transmissible and nontransmissible cerebral amyloidoses.

The unconventional viruses of the transmissible subacute spongiform encephalopathies (kuru-CJD-GSS-FFI-scrapie-BSE) are nucleants spontaneously generated from host precursor proteins altered to beta-pleated sheet configuration that polymerize into insoluble infectious amyloid fibrils. The de novo conversion to infectious amyloids is facilitated or accelerated by many different point mutations causing amino acid changes, a stop codon, or octapeptide inserts that increase the likelihood of spontaneous conversion to infectious configuration by many orders of magnitude. Similar nucleating induction of configurational change to amyloid probably occurs in other amyloidoses of brain and in systemic amyloidoses. Thus, all amyloids, particularly so-called fibrillar amyloid enhancing factors, may be considered to be infectious scrapie-like agents. These events probably occur extracellularly, thus we are attempting to reproduce them in vitro, even from synthetic polypeptides.

[Clinical phenotype of Charcot-Marie-Tooth disease (CMT) and familial amyloid polyneuropathy (FAP)].

A nationwide study of CMT and FAP has been performed. In FAP TTR Met30 families with late onset, neuropathy showed male preponderance, low penetrance, little relationship to endemic foci, sensorimotor symptoms beginning distally in the lower extremities with disturbance of both superficial and deep sensation, and relatively mild autonomic symptoms, consistent with pathological findings. In contrast, families with early onset showed higher penetrance, concentration in endemic foci, predominant loss of superficial sensation, severe autonomic dysfunction. Demyelinating versus axonal phenotypes are major issues in CMT. CMT1A duplication caused mainly demyelinating phenotype, while axonal features were variably present. In CMT1B, two distinctive phenotypic subgroups were present: one showed exclusively axonal features; and another was exclusively demyelinating. CMTX showed intermediate slowing of MCV, predominantly axonal features, and relatively mild demyelinating pathology. Differing from CMT1B, these axonal and demyelinating features were concomitantly present in individual patients in variable extent. Median nerve MCVs were well maintained independently of age, disease duration, and severity of clinical and pathologic abnormalities. Amplitude of CMAPs was correlated significantly with distal muscle strength, indicating that clinical weakness results from reduced numbers of functional large axons, not from demyelination. CMT patients with demyelinating and/or axonal features, together with FAP patients with axonal feature and scattered distribution, are supposed to increase according to the development of genetic diagnosis for hereditary neuropathy that verifies late-onset, de novo and asymptomatic patients.

ß2-Microglobulin amyloid fibril-induced membrane disruption is enhanced by endosomal lipids and acidic pH.

Although the molecular mechanisms underlying the pathology of amyloidoses are not well understood, the interaction between amyloid proteins and cell membranes is thought to play a role in several amyloid diseases. Amyloid fibrils of ß2-microglobulin (ß2m), associated with dialysis-related amyloidosis (DRA), have been shown to cause disruption of anionic lipid bilayers in vitro. However, the effect of lipid composition and the chemical environment in which ß2m-lipid interactions occur have not been investigated previously. Here we examine membrane damage resulting from the interaction of ß2m monomers and fibrils with lipid bilayers. Using dye release, tryptophan fluorescence quenching and fluorescence confocal microscopy assays we investigate the effect of anionic lipid composition and pH on the susceptibility of liposomes to fibril-induced membrane damage. We show that ß2m fibril-induced membrane disruption is modulated by anionic lipid composition and is enhanced by acidic pH. Most strikingly, the greatest degree of membrane disruption is observed for liposomes containing bis(monoacylglycero)phosphate (BMP) at acidic pH, conditions likely to reflect those encountered in the endocytic pathway. The results suggest that the interaction between ß2m fibrils and membranes of endosomal origin may play a role in the molecular mechanism of ß2m amyloid-associated osteoarticular tissue destruction in DRA.

Inhibiting the nucleation of amyloid structure in a huntingtin fragment by targeting α-helix-rich oligomeric intermediates.

Although oligomeric intermediates are transiently formed in almost all known amyloid assembly reactions, their mechanistic roles are poorly understood. Recently, we demonstrated a critical role for the 17-amino-acid N-terminus (htt(NT) segment) of huntingtin (htt) in the oligomer-mediated amyloid assembly of htt N-terminal fragments. In this mechanism, the htt(NT) segment forms the α-helix-rich core of the oligomers, leaving much of the polyglutamine (polyQ) segment disordered and solvent-exposed. Nucleation of amyloid structure occurs within this local high concentration of disordered polyQ. Here we demonstrate the kinetic importance of htt(NT) self-assembly by describing inhibitory htt(NT)-containing peptides that appear to work by targeting nucleation within the oligomer fraction. These molecules inhibit amyloid nucleation by forming mixed oligomers with the htt(NT) domains of polyQ-containing htt N-terminal fragments. In one class of inhibitors, nucleation is passively suppressed due to the reduced local concentration of polyQ within the mixed oligomer. In the other class, nucleation is actively suppressed by a proline-rich polyQ segment covalently attached to htt(NT). Studies with D-amino acid and scrambled sequence versions of htt(NT) suggest that inhibition activity is strongly linked to the propensity of inhibitory peptides to make amphipathic α-helices. Htt(NT) derivatives with C-terminal cell-penetrating peptide segments also exhibit excellent inhibitory activity. The htt(NT)-based peptides described here, especially those with protease-resistant d-amino acids and/or with cell-penetrating sequences, may prove useful as lead therapeutics for inhibiting the nucleation of amyloid formation in Huntington's disease.

Helix stabilization precedes aqueous and bilayer-catalyzed fiber formation in islet amyloid polypeptide.

Islet amyloid polypeptide (IAPP) is an unstructured polypeptide hormone that is cosecreted with insulin. In patients with type 2 diabetes, IAPP undergoes a transition from its natively disordered state to a highly ordered, all-beta-strand amyloid fiber. Although predominantly disordered, IAPP transiently samples alpha-helical structure in solution. IAPP adopts a fully helical structure when bound to membrane surfaces in a process associated with catalysis of amyloid formation. Here, we use spectroscopic techniques to study the structure of full-length, monomeric IAPP under amyloidogenic conditions. We observe that the residues with helical propensity in solution (1-22) also form the membrane-associated helix. Additionally, reduction of the N-terminal disulfide bond (Cys2-Cys7) decreases the extent of helix formed throughout this region. Through manipulation of sample conditions to increase or decrease the amount of helix, we show that the degree of helix formed affects the rate of amyloid assembly. Formation of helical structure is directly correlated with enhanced amyloid formation both on the membrane surface and in solution. These observations support suggested mechanisms in which parallel helix associations bring together regions of the peptide that could nucleate beta-strand structure. Remarkably, stabilization of non-amyloid structure appears to be a key intermediate in assembly of IAPP amyloid.

The molecular basis of functional bacterial amyloid polymerization and nucleation.

Amyloid fibers are filamentous proteinaceous structures commonly associated with mammalian neurodegenerative diseases. Nucleation is the rate-limiting step of amyloid propagation, and its nature remains poorly understood. Escherichia coli assembles functional amyloid fibers called curli on the cell surface using an evolved biogenesis machine. In vivo, amyloidogenesis of the major curli subunit protein, CsgA, is dependent on the minor curli subunit protein, CsgB. Here, we directly demonstrated that CsgB(+) cells efficiently nucleated purified soluble CsgA into amyloid fibers on the cell surface. CsgA contains five imperfect repeating units that fulfill specific roles in directing amyloid formation. Deletion analysis revealed that the N- and C-terminal most repeating units were required for in vivo amyloid formation. We found that CsgA nucleation specificity is encoded by the N- and C-terminal most repeating units using a blend of genetic, biochemical, and electron microscopic analyses. In addition, we found that the C-terminal most repeat was most aggregation-prone and dramatically contributed to CsgA polymerization in vitro. This work defines the elegant molecular signatures of bacterial amyloid nucleation and polymerization, thereby revealing how nature directs amyloid formation to occur at the correct time and location.

Odontogenic ameloblast-associated protein nature of the amyloid found in calcifying epithelial odontogenic tumors and unerupted tooth follicles.

We have previously reported that the amyloid found in three patients with calcifying epithelial odontogenic tumors (CEOT) was composed of N-terminal fragments of a putative 153-residue protein specified by a gene designated FLJ20513 now known to represent exons 5 through 10 of the odontogenic ameloblast-associated protein (ODAM) locus that encodes a 279-residue polypeptide. Confirmation of the amyloidogenic potential of ODAM has resulted from analyses of four other cases where we found, in addition, a 74-residue segment specified by exon 4. Through preparation of ODAM-related synthetic peptides, it was possible to localize the fibril-forming region of this molecule, as well as generate a monoclonal antibody that reacted specifically with the amyloid associated with CEOT. Notably, we also detected green birefringent congophilic material in unerupted tooth follicles - a precursor of CEOT - and demonstrated through immunologic and chemical analyses the ODAM nature of the deposits. Our studies have provided further evidence for this unique form of odontogenic amyloid that we provisionally designate "AODAM".

Formation of toxic Abeta(1-40) fibrils on GM1 ganglioside-containing membranes mimicking lipid rafts: polymorphisms in Abeta(1-40) fibrils.

The abnormal aggregation and deposition of amyloid beta protein (Abeta) on neuronal cells are critical to the onset of Alzheimer's disease. The entity (oligomers or fibrils) of toxic Abeta species responsible for the pathogenesis of the disease has been controversial. We have reported that the Abeta aggregates on ganglioside-rich domains of neuronal PC12 cells as well as in raft-like model membranes. Here, we identified toxic Abeta(1-40) aggregates formed with GM1-ganglioside-containing membranes. Abeta(1-40) was incubated with raft-like liposomes composed of GM1/cholesterol/sphingomyelin at 1:2:2 and 37 degrees C. After a lag period, toxic amyloid fibrils with a width of 12 nm were formed and subsequently laterally assembled with slight changes in their secondary structure as confirmed by viability assay, thioflavin-T fluorescence, circular dichroism, and transmission electron microscopy. In striking contrast, Abeta fibrils formed without membranes were thinner (6.7 nm) and much less toxic because of weaker binding to cell membranes and a smaller surface hydrophobicity. This study suggests that toxic Abeta(1-40) species formed on membranes are not soluble oligomers but amyloid fibrils and that Abeta(1-40) fibrils exhibit polymorphisms.