Amyloid fibril formation from full-length and fragments of amylin

Amyloiddeposits of fibrillar human amylin (hA) in the pancreas may be a causative factor in type-2 diabetes. A detailed comparison of in vitro fibril formation by full-length hA(1-37) versus fragments of this peptide-hA(8-37) and hA(20-29)-is presented. Circular dichroism spectroscopy revealed that fibril formation was accompanied by a conformational change: random coil to beta-sheet/alpha-helical structure. Fibril morphologies were visualized by electron microscopy and displayed a remarkable diversity. hA(20-29) formed flat ribbons consisting of numerous 3. 6-nm-wide protofibrils. In contrast, hA(1-37) and hA(8-37) formed polymorphic higher order fibrils by lateral association and/or coiling together of 5.0-nm-wide protofibril subunits. For full-length hA(1-37), the predominant fibril type contained three protofibrils and for hA(8-37), the predominant type contained two protofibrils. Polymerization was also monitored with the thioflavin-T binding assay, which revealed different kinetics of assembly for hA(1-37) and hA(8-37) fibrils. hA(20-29) fibrils did not bind thioflavin-T. Together the results demonstrate that the N-terminal region of the hA peptide influences the relative frequencies of the various higher order fibril types and thereby the overall kinetics of fibril formation. Furthermore, while residues 20-29 contribute to the fibrils' beta-sheet core, the flanking C- and N-terminal regions of the hA peptide determine the interactions involved in the formation of higher order coiled polymorphic superstructures.     

Nanomedicine: Action of Metal Nanoparticles on Neuronal Nitric Oxide Synthase—Fluorimetric Analysis on the Mechanism for Fibrillogenesis

The incubation of neuronal nitric oxide synthase with the five amyloid peptide fragments [Aβ_17–21; Aβ_25–29; Aβ_29–33; Aβ_33–37; Aβ_25–37] catalyzed the formation of fibrils. The role of neuronal isomer (nNOS) involved the entrapment of free monomers and seed aggregates to initiate the events of nucleation and elongation, critical for the formation of fibrils. It was evident that the hydrophobic nature of Aβ_17–21, the three glycine zipper peptides [Aβ_25–29; Aβ_29–33; Aβ_33–37] and Aβ_25–37 was a trigger in the formation of fibrils and was a force critical in the association of the peptides with the enzyme. Gold and silver nanoparticles (average 4.0 nm) inhibited fibril formation when added to the induced fibrils from nNOS-Aβ incubation. The addition of nNOS and/or Aβ to co-incubated solutions of nanoparticle-Aβ or nanoparticle-nNOS respectively did not prevent fibril formation but reversed it. Three mechanisms for this reversal were proposed: (1) depletion of free Aβ monomer in solution and blocking potential aggregation sites on the nNOS molecule due to large surface area of the nanoparticle (2) hydrophobic interaction between the Aβ peptide and nanoparticle (3) disruption of binary adducts between Aβ-peptides and nNOS by nanoparticles.     

Beta-amyloid peptides 1-40betaA and 25-35betaA suppress human amylin-mediated death of RINm5F islet beta-cells with distinct actions on fibril formation

Amyloid deposition is a common feature of Alzheimer's disease and type 2 diabetes related to beta-amyloid peptides (betaA) and human amylin (hA), respectively. Both betaA and hA form aggregates and fibrils and kill cultured cells. To investigate whether betaA and hA display peptide-specific toxicity on cultured islet beta-cells, we examined the effects of (1-40)betaA and (25-35)betaA peptides on hA-mediated cell death and [(125)I-Tyr(37)]hA precipitation. Synthetic hA aggregated in solution and evoked both conformation- and sequence-dependent cell death. While neither (1-40)betaA nor (25-35)betaA was toxic to islet beta-cells, they suppressed hA-evoked cell death in a concentration-dependent and saturable manner. Only (1-40)betaA, but not (25-35)betaA, showed trophic effects on cultured islet beta-cells and inhibited the precipitation of [(125)I]hA caused by hA. These results suggest that (25-35)betaA does not interfere with hA-mediated fibril formation. Suppression of hA-evoked death of cultured pancreatic islet beta-cells by the betaA peptides is likely to occur through a competing interaction at these cells.     

Development of the Structural Core and of Conformational Heterogeneity during the Conversion of Oligomers of the Mouse Prion Protein to Worm-like Amyloid Fibrils

Understanding how structure develops during the course of amyloid fibril formation by the prion protein is important for understanding prion diseases. Determining how conformational heterogeneity manifests itself in the fibrillar and pre-fibrillar amyloid aggregates is critical for understanding prion strain phenotypes. In this study, the formation of worm-like amyloid fibrils by the mouse prion protein has been characterized structurally by hydrogen-deuterium exchange coupled to mass spectrometry. The structural cores of these fibrils and of the oligomer on the direct pathway of amyloid fibril formation have been defined, showing how structure develops during fibril formation. The structural core of the oligomer not on the direct pathway has also been defined, allowing the delineation of the structural features that make this off-pathway oligomer incompetent to directly form fibrils. Sequence segments that exhibit multiple local conformations in the three amyloid aggregates have been identified, and the development of structural heterogeneity during fibril formation has been characterized. It is shown that conformational heterogeneity is not restricted to only the C-terminal domain region, which forms the structural core of the aggregates; it manifests itself in the N-terminal domain of the protein as well. Importantly, all three amyloid aggregates are shown to be capable of disrupting lipid membrane structure, pointing to a mechanism by which they may be toxic.     

Ozone pollution influences soil carbon and nitrogen sequestration and aggregate composition in paddy soils

Background and aims

Much attention has focused on the effects of tropospheric ozone (O₃) on terrestrial ecosystems and plant growth. Since O₃ pollution is currently an issue in China and many parts of the world, understanding the effects of elevated O₃ on soil carbon (C) and nitrogen (N) sequestration is essential for efforts to predict C and N cycles in terrestrial ecosystems under predicted increases in O₃. Thus the main objective of this study was to determine whether an increases in atmospheric O₃ concentration influenced soil organic C (SOC) and N sequestration.

Methods

A free-air O₃ enrichment (O₃-FACE) experiment was started in 2007 and used continuous O₃ exposure from March to November each year during crop growth stage in a rice (Oryza sativa L.)—wheat (Triticum aestivum L.) rotation field in the Jiangsu Province, China. We investigated differences in SOC and N and soil aggregate composition in both elevated and ambient O₃ conditions.

Results

Elevated atmospheric O₃ (18–80 nmol mol^?1 or 50 % above the ambient) decreased the SOC and N concentration in the 0–20 cm soil layer after 5 years. Elevated O₃ significantly decreased the SOC concentration by 17 % and 5.6 % in the 0–3 cm and the 10–20 cm layers, respectively. Elevated O₃ significantly decreased the N concentration by 8.2–27.8 % in three layers at the 20 cm depth. In addition, elevated O₃ influenced the formation and transformation of soil aggregates and the distribution of SOC and N in the aggregates across soil layer classes. Elevated O₃ significantly decreased the macro-sized aggregate fraction (16.8 %) and associated C and N (0.5 g kg^?1 and 0.32 g kg^?1, respectively), and significantly increased the silt+ clay-sized aggregate fraction (61 %) and associated C (1.7 g kg^?1) in the 0–3 cm layer. Elevated O₃ significantly decreased the macro-sized aggregate fraction (9.6 %) and associated C and N (1.4 g kg^?1 and 0.35 g kg^?1, respectively), and significantly increased the silt+ clay-sized aggregate fraction (41.8 %) and decreased the corresponding associated N (0.14 g kg^?1) in the 3–10 cm layer. Elevated O₃ did not significantly effect the formation and transformation of aggregates in the 10–20 cm layer, yet it did significantly increase the C concentration in the macro-sized fraction (1 g kg^?1) and decrease the N concentration in the macro- and micro-sized fractions (0.24 g kg^?1 and 0.16 g kg^?1, respectively).

Conclusion

Long-term exposure to elevated atmospheric O₃ negatively affected the physical structure of the soil and impaired soil C and N sequestration.     

Fibrillogenesis and cytotoxic activity of the amyloid-forming apomyoglobin mutant W7FW14F

The apomyoglobin mutant W7FW14F forms amyloid-like fibrils at physiological pH. We examined the kinetics of fibrillogenesis using three techniques: the time dependence of the fluorescence emission of thioflavin T and 1-anilino-8-naphthalenesulfonate, circular dichroism measurements, and electron microscopy. We found that in the early stage of fibril formation, non-native apomyoglobin molecules containing beta-structure elements aggregate to form a nucleus. Subsequently, more molecules aggregate around the nucleus, thereby resulting in fibril elongation. We evaluated by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) the cytotoxicity of these aggregates at the early stage of fibril elongation versus mature fibrils and the wild-type protein. Similar to other amyloid-forming proteins, cell toxicity was not due to insoluble mature fibrils but rather to early pre-fibrillar aggregates. Propidium iodide uptake showed that cell toxicity is the result of altered membrane permeability. Phalloidin staining showed that membrane damage is not associated to an altered cell shape caused by changes in the cytoskeleton.     

Full-length rat amylin forms fibrils following substitution of single residues from human amylin

Pancreatic amyloid deposits, composed of the 37 amino acid residue peptide amylin, represent an integral part of type 2 diabetes mellitus pathology. Human amylin (hA) forms fibrils in vitro and is toxic to cultured pancreatic islet beta-cells. In contrast, rat amylin (rA) which differs from hA by only six amino acid residues in the central region of the peptide, residues 18-29, does not form fibrils and is not cytotoxic. To elucidate the role of individual residues in fibril formation, we have generated a series of full-length rA variants and examined their ability to form fibrils in vitro. Single-residue substitutions with amino acids from corresponding positions of the hA sequence, i.e. R18H, L23F, or V26I, were sufficient to render rA competent for fibril formation albeit at a small yield. Combining two or three of these substitutions generally increased the ability to produce fibrils. Variant rA fibril morphologies were examined by negative stain electron microscopy and found to be similar to those generated by hA itself. Bulk assays, i.e. involving thioflavin-T fluorescence and sedimentation, showed that the amount of fibril formation was relatively small for these rA variants when compared to hA under the same conditions. Fibril growth was demonstrated by time-lapse atomic force microscopy, and MALDI-TOF mass spectrometry was used to verify that fibrils consisted of full-length peptide. Our observations confirm previous reports that the three proline residues play a dominant negative role in fibril formation. However, their presence is not sufficient to completely abolish the ability of rA to form fibrils, as each of the other three implicated residues (i.e. R18, L23 and V26) also has a dominant modulating effect.     

Impact of N-glycosylation site variants during human PrP aggregation and fibril nucleation

Misfolding and aggregation of the human prion protein (PrP) cause neurodegenerative transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease. Mature native PrP is composed of 209 residues and is folded into a C-terminal globular domain (residues 125–209) comprising a small two-stranded β-sheet and three α-helices. The N-terminal domain (residues 23–124) is intrinsically disordered. Expression of truncated PrP (residues 90–231) is sufficient to cause prion disease and residues 90/100–231 is comprising the amyloid-like fibril core of misfolded infectious PrP. During PrP fibril formation under native conditions in vitro, the disordered N-terminal domain slows down fibril formation likely due to a mechanism of initial aggregation forming morphologically disordered aggregates. The morphological disordered aggregate is a transient phase. Nucleation of fibrils occurs from this initial aggregate. The aggregate phase is largely circumvented by seeding with preformed PrP fibrils. In vivo PrP is N-glycosylated at positions Asn181 and Asn197. Little is known about the importance of these positions and their glycans for PrP stability, aggregation and fibril formation. We have in this study taken a step towards that goal by mutating residues 181 and 197 for cysteines to study the positional impact on these processes. We have further by organic synthetic chemistry and chemical modification generated synthetic glycosylations in these positions. Our data shows that residue 181 when mutated to a cysteine is a key residue for self-chaperoning, rendering a trap in the initial aggregate preventing conformational changes towards amyloid fibril formation. Position 197 is less involved in the aggregate trapping and is more geared towards β-sheet structure conversion within amyloid fibrils. As expected, synthetic glycosylated 197 is less affected towards fibril formation compared to glycosylated 181. Our data are rather compatible with the parallel in-register intermolecular β-sheet model structure of the PrP90–231 fibril and sheds light on the misfolding transitions of PrP in vitro. We hypothesize that glycosylation of position 181 is a key site for prion strain differentiation in vivo.     

Antifibrillizing agents catalyze the formation of unstable intermediate aggregates of beta‐amyloid

Although Alzheimer's disease (AD) is characterized by the extracellular deposition of fibrillar aggregates of beta‐amyloid (Aβ), transient oligomeric species of Aβ are increasingly implicated in the pathogenesis of AD. Natively unfolded monomeric Aβ can misfold and progressively assemble into fibrillar aggregates, following a well‐established “on pathway” seeded‐nucleation mechanism. Here, we show that three simple saccharides, mannose, sucrose, and raffinose, alter Aβ aggregation kinetics and morphology. The saccharides inhibit formation of Aβ fibrils but promote formation of various oligomeric aggregate species through different “off pathway” aggregation mechanisms at 37°C but not at 60°C. The various oligomeric Aβ aggregates formed when coincubated with the different saccharides are morphologically distinct but all are toxic toward SH‐SY5Y human neuroblastoma cells, increasing the level of toxicity and greatly prolonging toxicity compared with Aβ alone. As a wide variety of anti‐Aβ aggregation strategies are being actively pursued as potential therapeutics for AD, these studies suggest that care must be taken to ensure that the therapeutic agents also block toxic oligomeric Aβ assembly as well as inhibit fibril formation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Simultaneous monitoring of peptide aggregate distributions, structure, and kinetics using amide hydrogen exchange: application to Abeta(1-40) fibrillogenesis

Increasing evidence indicates that soluble aggregates of amyloid beta protein (Abeta) are neurotoxic. However, difficulty in isolating these unstable, dynamic species impedes studies of Abeta and other aggregating peptides and proteins. In this study, hydrogen-deuterium exchange (HX) detected by mass spectrometry (MS) was used to measure Abeta(1-40) aggregate distributions without purification or modification that might alter the aggregate structure or distribution. Different peaks in the mass spectra were assigned to monomer, low molecular weight oligomer, intermediate, and fibril based on HX labeling behavior and complementary assays. After 1 h labeling, the intermediates incorporated approximately ten more deuterons relative to fibrils, indicating a more solvent exposed structure of such intermediates. HX-MS also showed that the intermediate species dissociated much more slowly to monomer than did the very low molecular weight oligomers that were formed at very early times in Abeta aggregation. Atomic force microscopy (AFM) measurements revealed the intermediates were roughly spherical with relatively homogenous diameters of 30-50 nm. Quantitative analysis of the HX mass spectra showed that the amount of intermediate species was correlated with Abeta toxicity patterns reported in a previous study under the same conditions. This study also demonstrates the potential of the HX-MS approach to characterizing complex, multi-component oligomer distributions of aggregating peptides and proteins.     

Degradation of moist soil aggregates by rapid temperature rise under low intensity fire

Background and aims

Soil structure degradation by fire is usually attributed to qualitative and quantitative change of organic and inorganic binding agents, especially in high severity burns (>300 °C) that last for prolonged periods (> 1 hour). In contrast, controlled burns are typically managed to be low in intensity and severity. Such burns are considered benign to soil structural stability because organic matter and inorganic binding agents (e.g., gypsum) are relatively stable at such low temperatures. Recent observations at a controlled burn site in the eastern Great Basin (Nevada) showed soil aggregate breakdown found in shrub canopies where soil temperatures briefly exceeded 300 °C as well as interspaces between shrubs, where the temperatures were likely lower than beneath shrubs because of less surface biomass. These alterations cannot be explained in terms of thermal alteration of binding agents. This study was designed to test whether pressure created by rapidly vaporized pore water can cause aggregate breakdown.

Methods

We subjected three different sizes of aggregates (0.25–1, 1–2 and 2–4 mm) of soils derived from the eastern Great Basin burn site as well as from a forest and urban garden in California to rapid and slow (3 °C/min) heating rates. These treatments were conducted at 5 peak temperatures (75, 100, 125, 150 and 175 °C).

Results

Post-burn water stability of the aggregates showed that rapid heating rate caused more pronounced degradation of aggregate stability than slow heating. Moreover, the heating-rate dependent structural degradation increased with peak temperature. For the majority of the aggregates, the effect also increased with initial water content. In all the soils tested, there was no preferential loss of organic matter in the rapid-heating treatment that can explain the observed enhanced breakdown of aggregates.

Conclusions

Our observations indicate that soil structural degradation under low-intensity fire occurs as a result of mechanical stresses extorted by rapidly escaping steam from soil pores under rapid heating rate.     

The Antioxidant Activity of Copper(II) (3,5-Diisopropyl Salicylate)4 and Its Protective Effect Against Streptozotocin-Induced Diabetes Mellitus in Rats

Oxidative stress has been suggested as a potential contributor to the development of diabetic complications. In this study, we investigated the protective effect of a strong antioxidant copper complex against streptozotocin (STZ)-induced diabetes in animals. Out of four copper complexes used, copper(II) (3,5-diisopropyl salicylate)₄ (Cu(II)DIPS) was found to be the most potent antioxidant–copper complex. Pretreatment with Cu(II)DIPS (5 mg/kg) twice a week prior to the injection of streptozotocin (50 mg/kg) has reduced the level of hyperglycemia by 34 % and the mortality rate by 29 %. Injection of the same dosage of the ligand 3,5-diisopropyl salicylate has no effect on streptozotocin-induced hyperglycemia. The same copper complex has neither hypoglycemic activity when injected in normal rats nor antidiabetic activity when injected in STZ-induced diabetic rats. The protective effect of Cu(II)DIPS could be related to its strong antioxidant activity compared to other copper complexes median effective concentration (MEC)?=?23.84 μg/ml and to Trolox MEC?=?29.30 μg/ml. In addition, it reduced serum 8-hydroxy-2′-deoxyguanosine, a biomarker of oxidative DNA damage, by 29 %. This effect may explain why it was not effective against diabetic rats, when β Langerhans cells were already destroyed. Similar protective activities were reported by other antioxidants like Trolox.     

Converse modulation of toxic α-synuclein oligomers in living cells by N′-benzylidene-benzohydrazide derivates and ferric iron

Intracellular α-synuclein (α-syn) aggregates are the pathological hallmark in several neurodegenerative diseases including Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy. Recent evidence suggests that small oligomeric aggregates rather than large amyloid fibrils represent the main toxic particle species in these diseases. We recently characterized iron-dependent toxic α-syn oligomer species by confocal single molecule fluorescence techniques and used this aggregation model to identify several N′-benzylidene-benzohydrazide (NBB) derivatives inhibiting oligomer formation in vitro. In our current work, we used the bioluminescent protein-fragment complementation assay (BPCA) to directly analyze the formation of toxic α-syn oligomers in cell culture and to investigate the effect of iron and potential drug-like compounds in living cells. Similar to our previous findings in vitro, we found a converse modulation of toxic α-syn oligomers by NBB derivates and ferric iron, which was characterized by an increase in aggregate formation by iron and an inhibitory effect of certain NBB compounds. Inhibition of α-syn oligomer formation by the NBB compound 293G02 was paralleled by a reduction in cytotoxicity indicating that toxic α-syn oligomers are present in the BPCA cell culture model and that pharmacological inhibition of oligomer formation can reduce toxicity. Thus, this approach provides a suitable model system for the development of new disease-modifying drugs targeting toxic oligomer species. Moreover, NBB compounds such as 293G02 may provide useful tool compounds to dissect the functional role of toxic oligomer species in cell culture models and in vivo.     

Surfactant-mediated amyloidogenesis behavior of stem bromelain; a biophysical insight

Neurodegenerative disorders are mainly associated with amyloid fibril formation of different proteins. Stem bromelain (SB), a cysteine protease, is known to exist as a molten globule state at pH 10.0. It passes through the identical surrounding (pH 10.0) in the gut epithelium of intestine upon oral administration. Protein–surfactant complexes are widely employed as drug carriers, so the nature of surfactant toward protein is of great interest. The present work describes the effect of cationic surfactants (CTAB & DTAB) and their hydrophobic behavior toward amyloidogenesis behavior of SB at pH 10.0. Multiple approaches including light scattering, far UV-CD, turbidity measurements, and dye binding assay (ThT, Congo red and ANS) were performed to measure the aggregation propensity of SB. Further, we monitored the hydrodynamic radii of aggregates formed using dynamic light scattering technique. Structure of fibrils was also visualized through fluorescence microscopy as well as TEM. At pH 10.0, low concentration of CTAB (0–200 μM) induced amyloid formation in SB as evident from a prominent increase in turbidity and light scattering, gain in β-sheet content, and enhanced ThT fluorescence intensity. However, further increase in CTAB concentration suppressed the fibrillation phenomenon. In contrast, DTAB did not induce fibril formation at any concentration used (0–500 μM) due to lower hydrophobicity. Net negative charge developed on protein at high pH (10.0) might have facilitated amyloid formation at low concentration of cationic surfactant (CTAB) due to electrostatic and hydrophobic interactions.     

Turn nucleation perturbs amyloid β self-assembly and cytotoxicity

The accumulation of senile plaques composed of amyloid β (Aβ) fibrils is a hallmark of Alzheimer's disease, although prefibrillar oligomeric species are believed to be the primary neurotoxic congeners in the pathogenesis of Alzheimer's disease. Uncertainty regarding the mechanistic relationship between Aβ oligomer and fibril formation and the cytotoxicity of these aggregate species persists. β-Turn formation has been proposed to be a potential rate-limiting step during Aβ fibrillogenesis. The effect of turn nucleation on Aβ self-assembly was probed by systematically replacing amino acid pairs in the putative turn region of Aβ (residues 24-27) with d-ProGly ((D)PG), an effective turn-nucleating motif. The kinetic, thermodynamic, and cytotoxic effects of these mutations were characterized. It was found that turn formation dramatically accelerated Aβ fibril self-assembly dependent on the site of turn nucleation. The cytotoxicity of the three (D)PG-containing Aβ variants was significantly lower than that of wild-type Aβ40, presumably due to decreased oligomer populations as a function of a more rapid progression to mature fibrils; oligomer populations were not eliminated, however, suggesting that turn formation is also a feature of oligomer structures. These results indicate that turn nucleation is a critical step in Aβ40 fibril formation.     

The amylin peptide implicated in type 2 diabetes stimulates copper-mediated carbonyl group and ascorbate radical formation

Human amylin (hA), which is toxic to islet β-cells, can self-generate H₂O₂, and this process is greatly enhanced in the presence of Cu(II) ions. Here we show that carbonyl groups, a marker of oxidative modification, were formed in hA incubated in the presence of Cu(II) ions or Cu(II) ions plus H₂O₂, but not in the presence of H₂O₂ alone. Furthermore, under similar conditions (i.e., in the presence of both Cu(II) ions and H₂O₂), hA also stimulated ascorbate radical formation. The same observations concerning carbonyl group formation were made when the histidine residue (at position 18) in hA was replaced by alanine, indicating that this residue does not play a key role. In complete contrast to hA, rodent amylin, which is nontoxic, does not generate H₂O₂, and binds Cu(II) ions only weakly, showed none of these properties. We conclude that the hA-Cu(II)/Cu(I) complex is redox active, with electron donation from the peptide reducing the oxidation state of the copper ions. The complex is capable of forming H₂O₂ from O₂ and can also generate ^•OH via Fenton chemistry. These redox properties of hA can explain its ability to stimulate copper-mediated carbonyl group and ascorbate radical formation. The formation of reactive oxygen species from hA in this way could hold the key to a better understanding of the damaging consequences of amyloid formation within the pancreatic islets of patients with type 2 diabetes mellitus.     

Toxic SOD1 trimers are off-pathway in the formation of amyloid-like fibrils in ALS

Accumulation of insoluble amyloid fibrils is widely studied as a critical factor in the pathology of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. Misfolded Cu, Zn superoxide dismutase (SOD1) was the first protein linked to ALS, and non-native SOD1 trimeric oligomers were recently linked to cytotoxicity, while larger oligomers were protective to cells. The balance between trimers and larger aggregates in the process of SOD1 aggregation is, thus, a critical determinant of potential therapeutic approaches to treat ALS. However, it is unknown whether these trimeric oligomers are a necessary intermediate for larger aggregate formation or a distinct off-pathway species competing with fibril formation. Depending on the on- or off-pathway scenario of trimer formation, we expect drastically different therapeutic approaches. Here, we show that the toxic SOD1 trimer is an off-pathway intermediate competing with protective fibril formation. We design mutant SOD1 constructs that remain in a trimeric state (super-stable trimers) and show that stabilizing the trimeric SOD1 prevents formation of fibrils in vitro and in a motor neuron-like cell model (NSC-34). Using size exclusion chromatography, we track the aggregation kinetics of purified SOD1 and show direct competition of trimeric SOD1 with larger oligomer and fibril formation. Finally, we show the trimer is structurally independent of both larger soluble oligomers and insoluble fibrils using circular dichroism spectroscopy and limited proteolysis.     

Interpreting the aggregation kinetics of amyloid peptides

Amyloid fibrils are insoluble mainly beta-sheet aggregates of proteins or peptides. The multi-step process of amyloid aggregation is one of the major research topics in structural biology and biophysics because of its relevance in protein misfolding diseases like Alzheimer's, Parkinson's, Creutzfeld-Jacob's, and type II diabetes. Yet, the detailed mechanism of oligomer formation and the influence of protein stability on the aggregation kinetics are still matters of debate. Here a coarse-grained model of an amphipathic polypeptide, characterized by a free energy profile with distinct amyloid-competent (i.e. beta-prone) and amyloid-protected states, is used to investigate the kinetics of aggregation and the pathways of fibril formation. The simulation results suggest that by simply increasing the relative stability of the beta-prone state of the polypeptide, disordered aggregation changes into fibrillogenesis with the presence of oligomeric on-pathway intermediates, and finally without intermediates in the case of a very stable beta-prone state. The minimal-size aggregate able to form a fibril is generated by collisions of oligomers or monomers for polypeptides with unstable or stable beta-prone state, respectively. The simulation results provide a basis for understanding the wide range of amyloid-aggregation mechanisms observed in peptides and proteins. Moreover, they allow us to interpret at a molecular level the much faster kinetics of assembly of a recently discovered functional amyloid with respect to the very slow pathological aggregation.