Large proteins have a great tendency to aggregate but a low propensity to form amyloid fibrils

The assembly of soluble proteins into ordered fibrillar aggregates with cross-β structure is an essential event of many human diseases. The polypeptides undergoing aggregation are generally small in size. To explore if the small size is a primary determinant for the formation of amyloids under pathological conditions we have created two databases of proteins, forming amyloid-related and non-amyloid deposits in human diseases, respectively. The size distributions of the two protein populations are well separated, with the systems forming non-amyloid deposits appearing significantly larger. We have then investigated the propensity of the 486-residue hexokinase-B from Saccharomyces cerevisiae (YHKB) to form amyloid-like fibrils in vitro. This size is intermediate between the size distributions of amyloid and non-amyloid forming proteins. Aggregation was induced under conditions known to be most effective for amyloid formation by normally globular proteins: (i) low pH with salts, (ii) pH 5.5 with trifluoroethanol. In both situations YHKB aggregated very rapidly into species with significant β-sheet structure, as detected using circular dichroism and X-ray diffraction, but a weak Thioflavin T and Congo red binding. Moreover, atomic force microscopy indicated a morphology distinct from typical amyloid fibrils. Both types of aggregates were cytotoxic to human neuroblastoma cells, as indicated by the MTT assay. This analysis indicates that large proteins have a high tendency to form toxic aggregates, but low propensity to form regular amyloid in vivo and that such a behavior is intrinsically determined by the size of the protein, as suggested by the in vitro analysis of our sample protein.     

The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis

RIP1 and RIP3 kinases are central players in TNF-induced programmed necrosis. Here, we report that?the RIP homotypic interaction motifs (RHIMs) of RIP1 and RIP3 mediate the assembly of heterodimeric filamentous structures. The fibrils exhibit classical characteristics of β-amyloids, as shown by Thioflavin T (ThT) and Congo red (CR) binding, circular dichroism, infrared spectroscopy, X-ray diffraction, and solid-state NMR. Structured amyloid cores are mapped in RIP1 and RIP3 that are flanked?by regions of mobility. The endogenous RIP1/RIP3 complex isolated from necrotic cells binds ThT, is ultrastable, and has a fibrillar core structure, whereas necrosis is partially inhibited by ThT, CR, and another amyloid dye, HBX. Mutations in the RHIMs of RIP1 and RIP3 that are defective in the interaction compromise cluster formation, kinase activation, and programmed necrosis in?vivo. The current study provides insight into the structural changes that occur when RIP kinases are triggered to execute different signaling outcomes and expands the realm of amyloids to complex formation and signaling.     

Amyloid-Like Fibril Formation by Tachykinin Neuropeptides and Its Relevance to Amyloid β-Protein Aggregation and Toxicity

Protein aggregation and amyloid formation are associated with both pathological conditions in humans such as Alzheimer's disease and native functions such as peptide hormone storage in the pituitary secretory granules in mammals. Here, we studied amyloid fibrils formation by three neuropeptides namely physalaemin, kassinin and substance P of tachykinin family using biophysical techniques including circular dichroism, thioflavin T, congo red binding and microscopy. All these neuropeptides under study have significant sequence similarity with Aβ(25-35) that is known to form neurotoxic amyloids. We found that all these peptides formed amyloid-like fibrils in vitro in the presence of heparin, and these amyloids were found to be nontoxic in neuronal cells. However, the extent of amyloid formation, structural transition, and morphology were different depending on the primary sequences of peptide. When Aβ(25-35) and Aβ40 were incubated with each of these neuropeptides in 1:1 ratio, a drastic increase in amyloid growths were observed compared to that of individual peptides suggesting that co-aggregation of Aβ and these neuropeptides. The electron micrographs of these co-aggregates were dissimilar when compared with individual peptide fibrils further supporting the possible incorporation of these neuropeptides in Aβ amyloid fibrils. Further, the fibrils of these neuropeptides can seed the fibrils formation of Aβ40 and reduced the toxicity of preformed Aβ fibrils. The present study of amyloid formation by tachykinin neuropeptides is not only providing an understanding of the mechanism of amyloid fibril formation in general, but also offering plausible explanation that why these neuropeptide might reduce the cytotoxicity associated with Alzheimer's disease related amyloids.     

Is Congo red an amyloid-specific dye?

Congo red (CR) binding, monitored by characteristic yellow-green birefringence under crossed polarization has been used as a diagnostic test for the presence of amyloid in tissue sections for several decades. This assay is also widely used for the characterization of in vitro amyloid fibrils. In order to probe the structural specificity of Congo red binding to amyloid fibrils we have used an induced circular dichroism (CD) assay. Amyloid fibrils from insulin and the variable domain of Ig light chain demonstrate induced CD spectra upon binding to Congo red. Surprisingly, the native conformations of insulin and Ig light chain also induced Congo red circular dichroism, but with different spectral shapes than those from fibrils. In fact, a wide variety of native proteins exhibited induced CR circular dichroism indicating that CR bound to representative proteins from different classes of secondary structure such as alpha (citrate synthase), alpha + beta (lysozyme), beta (concavalin A), and parallel beta-helical proteins (pectate lyase). Partially folded intermediates of apomyoglobin induced different Congo red CD bands than the corresponding native conformation, however, no induced CD bands were observed with unfolded protein. Congo red was also found to induce oligomerization of native proteins, as demonstrated by covalent cross-linking and small angle x-ray scattering. Our data suggest that Congo red is sandwiched between two protein molecules causing protein oligomerization. The fact that Congo red binds to native, partially folded conformations and amyloid fibrils of several proteins shows that it must be used with caution as a diagnostic test for the presence of amyloid fibrils in vitro.     

A multimethod approach for analyzing FapC fibrillation and determining mass per length

Amyloid proteins are found in a wide range of organisms owing to the high stability of the β-sheet core of the amyloid fibrils. There are both pathological amyloids involved in various diseases and functional amyloids that play a beneficial role for the organism. The aggregation process is complex and often involves many different species. Full understanding of this process requires parallel acquisition of data by complementary techniques monitoring the time course of aggregation. This is not an easy task, given the often-stochastic nature of aggregation, which can lead to significant variations in lag time. Here, we investigate the aggregation process of the functional amyloid FapC by simultaneous use of four different techniques, namely dynamic light scattering, small-angle x-ray scattering (SAXS), circular dichroism, and Thioflavin T fluorescence. All these approaches are applied to the same FapC sample just after desalting. Our data allow us to construct a master time-course graph showing the same time-course of aggregation by all techniques. This allows us to integrate insights from approaches that report on different structural and length scales. During the lag phase, loosely aggregated oligomers with random-coil structure are formed, which subsequently transform to fibrils without accumulation of additional significant species. Subsequently, the loosely associated protofilaments/subfilaments, which form side by side, mature to more compact fibrils. Furthermore, we determine the mass per length of the mature fibrils, obtaining very similar results by SAXS (33 kDa/nm) and tilted-beam transmission electron microscopy (31 kDa/nm). Transmission electron microscopy showed that the fibrils consist of primarily two protofilaments and similar dimensions of the cross section of the fibrils as revealed by SAXS modeling when the number of protofilaments per fibril was taken into account. Mass per length information underscores the general usefulness of SAXS in fibrillation analysis and provides an important constraint for further modeling the fibril structures.     

Thermally induced fibrillar aggregation of hen egg white lysozyme

We study the effect of pH and temperature on fibril formation from hen egg white lysozyme. Fibril formation is promoted by low pH and temperatures close to the midpoint temperature for protein unfolding (detected using far-ultraviolet circular dichroism). At the optimal conditions for fibril formation (pH 2.0, T = 57 degrees C), on-line static light-scattering shows the formation of fibrils after a concentration-independent lag time of approximately 48 h. Nucleation presumably involves a change in the conformation of individual lysozyme molecules. Indeed, long-term circular dichroism measurements at pH 2.0, T = 57 degrees C show a marked change of the secondary structure of lysozyme molecules after approximately 48 h of heating. From atomic force microscopy we find that most of the fibrils have a thickness of approximately 4 nm. These fibrils have a coiled structure with a periodicity of approximately 30 nm and show characteristic defects after every four or five turns.     

Amyloid fibril formation by macrophage migration inhibitory factor

We demonstrate herein that human macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine expressed in the brain and not previously considered to be amyloidogenic, forms amyloid fibrils similar to those derived from the disease associated amyloidogenic proteins beta-amyloid and alpha-synuclein. Acid denaturing conditions were found to readily induce MIF to undergo amyloid fibril formation. MIF aggregates to form amyloid-like structures with a morphology that is highly dependent on pH. The mechanism of MIF amyloid formation was probed by electron microscopy, turbidity, Thioflavin T binding, circular dichroism spectroscopy, and analytical ultracentrifugation. The fibrillar structures formed by MIF bind Congo red and exhibit the characteristic green birefringence under polarized light. These results are consistent with the notion that amyloid fibril formation is not an exclusive property of a select group of amyloidogenic proteins, and contribute to a better understanding of the factors which govern protein conformational changes and amyloid fibril formation in vivo.     

Quantification of amyloid fibrils using size exclusion chromatography coupled with online fluorescence and ultraviolet detection

An amyloid fibrils investigation within biofilm samples requires distinguishing the amyloid β-sheet structure of these proteins and quantifying them. In this study, the property of amyloids to incorporate the fluorescent dye Thioflavin T has been exploited to propose a method of quantification. The experimental protocol includes the preparation of amyloids from commercial κ-casein (κCN) and their fractionation through size exclusion chromatography (SEC) to provide calibration curves from fluorescence and absorbance signals. Finally, a bacterial biofilm extract was injected into SEC, and the amyloid fibrils could be expressed as equivalent κCN, representing approximately 21% of the total proteins.     

Template-directed self-assembly and growth of insulin amyloid fibrils

The formation of amyloid aggregates in tissue is a pathological feature of many neurodegenerative diseases and type II diabetes. Amyloid deposition, the process of amyloid growth by the association of individual soluble amyloid molecules with a pre-existing amyloid template (i.e., plaque), is known to be critical for amyloid formation in vivo. The requirement for a natural amyloid template, however, has made amyloid deposition study difficult and cumbersome. In the present work, we developed a novel, synthetic amyloid template by attaching amyloid seeds covalently onto an N-hydroxysuccinimide-activated surface, where insulin was chosen as a model amyloidogenic protein. According to ex situ atomic force microscopy observations, insulin monomers in solution were deposited onto the synthetic amyloid template to form fibrils, like hair growth. The fibril formation on the template occurred without lag time, and its rate was highly accelerated than in the solution. The fibrils were long, over 2 mum, and much thinner than those in the solution, which was caused by limited nucleation sites on the template surface and lack of lateral twisting between fibrils. According to our investigations using thioflavin T-induced fluorescence, birefringent Congo red binding, and circular dichroism, fibrils grown on the template were identified to be amyloids that formed through a conformational rearrangement of insulin monomers upon interaction with the template. The amyloid deposition rate followed saturation kinetics with respect to insulin concentration in the solution. The characteristics of amyloid deposition on the synthetic template were in agreement with previous studies performed with human amyloid plaques. It is demonstrated that the synthetic amyloid template can be used for the screening of inhibitors on amyloid deposition in vitro.     

Cyclin-dependent Kinase 5 Phosphorylation of Familial Prion Protein Mutants Exacerbates Conversion into Amyloid Structure

Familial prion protein (PrP) mutants undergo conversion from soluble and protease-sensitive to insoluble and partially protease-resistant proteins. Cyclin-dependent kinase 5 (Cdk5) phosphorylation of wild type PrP (pPrP) at serine 43 induces a conversion of PrP into aggregates and fibrils. Here, we investigated whether familial PrP mutants are predisposed to Cdk5 phosphorylation and whether phosphorylation of familial PrP mutants increases conversion. PrP mutants representing three major familial PrP diseases and different PrP structural domains were studied. We developed a novel in vitro kinase reaction coupled with Thioflavin T binding to amyloid structure assay to monitor phosphorylation-dependent amyloid conversion. Although non-phosphorylated full-length wild type or PrP mutants did not convert into amyloid, Cdk5 phosphorylation rapidly converted these into Thioflavin T-positive structures following first order kinetics. Dephosphorylation partially reversed conversion. Phosphorylation-dependent conversion of PrP from α-helical structures into β-sheet structures was confirmed by circular dichroism. Relative to wild type pPrP, most PrP mutants showed increased rate constants of conversion. In contrast, non-phosphorylated truncated PrP Y145X (where X represents a stop codon) and Q160X mutants converted spontaneously into Thioflavin T-positive fibrils after a lag phase of over 20 h, indicating nucleation-dependent polymerization. Phosphorylation reduced the lag phase by over 50% and thus accelerated the formation of the nucleating event. Consistently, phosphorylated Y145X and phosphorylated Q160X exacerbated conversion in a homologous seeding reaction, whereas WT pPrP could not seed WT PrP. These results demonstrate an influence of both the N terminus and the C terminus of PrP on conversion. We conclude that post-translational modifications of the flexible N terminus of PrP can cause or exacerbate PrP mutant conversion.     

Mechanism of thioflavin T binding to amyloid fibrils

Thioflavin T is a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils and is commonly used to diagnose amyloid fibrils, both ex vivo and in vitro. In aqueous solutions, thioflavin T was found to exist as micelles at concentrations commonly used to monitor fibrils by fluorescence assay ( approximately 10-20 microM). Specific conductivity changes were measured at varying concentration of thioflavin T and the critical micellar concentration was calculated to be 4.0+/-0.5 microM. Interestingly, changes in the fluorescence excitation and emission of thioflavin T were also dependent on the micelle formation. The thioflavin T micelles of 3 nm diameter were directly visualized using atomic force microscopy, and bound thioflavin T micelles were observed along the fibril length for representative fibrils. Increasing concentration of thioflavin T above the critical micellar concentration shows increased numbers of micelles bound along the length of the amyloid fibrils. Thioflavin T micelles were disrupted at low pH as observed by atomic force microscopy and fluorescence enhancement upon binding of thioflavin T to amyloid fibrils also reduced by several-fold upon decreasing the pH to below 3. This suggests that positive charge on the thioflavin T molecule has a role in its micelle formation that then bind the amyloid fibrils. Our data suggests that the micelles of thioflavin T bind amyloid fibrils leading to enhancement of fluorescence emission.     

Simultaneous Measurement of Amyloid Fibril Formation by Dynamic Light Scattering and Fluorescence Reveals Complex Aggregation Kinetics

An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit associated with Huntington''s disease, in vitro. Huntington''s disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer''s and Parkinson''s disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, β-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides associated with these diseases, the resulting amyloids display strikingly similar physical structure, an observation which suggests a physical basis for amyloid fibril formation. Thioflavin T fluorescence reports β-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation.     

Amyloid fibrils formation and amorphous aggregation in concanavalin A

We here report an experimental study on the thermal aggregation process of concanavalin A, a protein belonging to the legume lectins family. The aggregation process and the involved conformational changes of the protein molecules were followed by means of fluorescence techniques, light scattering, circular dichroism, zeta potential measurements and atomic force microscopy. Our results show that the aggregation process of concanavalin A may evolve through two distinct pathways leading, respectively, to the formation of amyloids or amorphous aggregates. The relative extent of the two pathways is determined by pH, as amyloid aggregation is favored at high pH values ( approximately 9), while the formation of amorphous aggregates is favored at low pH ( approximately 5). At difference from amorphous aggregation, the formation of amyloid fibrils requires significant conformational changes on the protein, both at secondary and tertiary structural level. To our knowledge, this is the first observation of amyloid fibrils from concanavalin A.     

High pressure promotes circularly shaped insulin amyloid

Amyloids, initially associated with certain degenerative diseases, and recently with the prions and prion-based inheritance in yeasts, are linearly-ordered beta-sheet-rich protein aggregates, presently thought to represent a rather common generic trait of proteins as polymers. Regardless of genetic origins and properties of precursor protein molecules, amyloids share many physicochemical properties, including the linear fibrillar morphology. Here, we show that under high hydrostatic pressure insulin forms amyloids of a unique circular morphology. Despite a degree of size-distribution, the smallest forms of the approximate radius of 340-420 nm are most abundant among the ring-shaped structures. The circular amyloid is accompanied by bent 20-100 nm long fibrils. The pressure-enhancement of a ring-like supramolecular fold suggests an anisotropic distribution of void volumes in regular amyloid fibres. While the ability of high pressure to evoke such drastic perturbations on an amyloidogenic pathway may help tune conformation of amyloid templates (e.g. inducing the PrP(Sc)-type infectivity in amyloids grown in vitro from recombinant PrP), the very finding raises new questions concerning possible consequences for high-pressure food processing.     

Concanavalin A aggregation and toxicity on cell cultures

A number of neurodegenerative diseases are known to involve protein aggregation. Common mechanisms and structural properties of amyloids are thought to be involved in aggregation-related cytotoxicity. In this context we propose an experimental study on Concanavalin A (Con A) aggregation and use it as a model to study the relationship between cell toxicity and aggregation processes. Depending on solution conditions, Con A aggregation has been monitored by static and dynamic light scattering, Thioflavin T emission, and FTIR absorption. The morphology of different aggregate species was verified by means of Atomic Force Microscopy and Confocal Microscopy. During the aggregation pathway the native protein conformation is destabilized and as a consequence, the simultaneous occurrence of conformational changes and protein aggregation is observed in both conditions. The effects of the extracellular addition of native protein, oligomers and mature fibrils were tested on LAN5 neuroblastoma cells by MTS assay. Results showed the toxicity of the first two species while a negligible effect was detected for amyloid fibrils. Both native and oligomeric aggregates were found to be able to activate apoptosis exclusively by extrinsic pathway through caspase 8 activation. Those results suggest that cytotoxicity mechanisms arise from specific membrane interactions with reactive conformations of destabilized molecules occurring during the amyloidal aggregation pathway. Those conformations, populated when native or preformed oligomers are incubated, are unavailable to bind cell membrane proteins. This happens because they are recruited in the mature fibrillar structure which–as a consequence–turns out to be non-toxic.     

Structure analysis of an amyloid-forming model peptide by a systematic glycine and proline scan

The ability to adopt at least two different stable conformations is a common feature of proteins involved in many neurodegenerative diseases. The involved molecules undergo a conformational transition from native, mainly helical states to insoluble amyloid structures that have high β-sheet content. A detailed characterization of the molecular architecture of highly ordered amyloid structures, however, is still challenging. Their intrinsically low solubility and high tendency to aggregate often considerably limits the application of established high-resolution techniques such as NMR and X-ray crystallography. An alternative approach to elucidating the tertiary and quaternary organization within an amyloid fibril is the systematic replacement of residues with amino acids that exhibit special conformational characteristics, such as glycine and proline. Substitutions within the β-sheet-prone sequences of the molecules usually severely affect their ability to form fibrils, whereas incorporation at external loop- and bend-like positions often has only marginal effects. Here we present the characterization of the internal architecture of a de novo designed coiled-coil-based amyloid-forming model peptide by means of a series of systematic single glycine and proline replacements in combination with a set of simple low-resolution methods. The folding and assembly behavior of the substituted peptides was monitored simultaneously using circular dichroism spectroscopy, Thioflavin T fluorescence staining, and transmission electron microscopy. On the basis of the obtained data, we successfully identify characteristic bend and core positions within the peptide sequence and propose a detailed structural model of the internal fibrillar arrangement.     

Michler's hydrol blue: a sensitive probe for amyloid fibril detection

Michler's hydrol blue (MHB) is investigated with respect to photophysical properties in varied solvent environment and when bound to insulin and lysozyme fibrils. The MHB chromophore is shown to act like a molecular rotor and bind well to amyloid fibrils, where it exhibits a characteristic red-shift in its excitation spectrum and an increase in the emission quantum yield upon binding. MHB is more sensitive to environmental changes than Thioflavin T (ThT) and furthermore, in contrast to the latter amyloid probe, can differentiate between insulin and lysozyme fibrils by a more red-shifted excitation spectrum for insulin fibrils. To support the experimental observations, time-dependent density functional theory (TDDFT) calculations were performed on MHB at several levels of theory. The predicted changes of spectral properties as a function of the environment are in good agreement with the experimental results. Linear dichroism (LD) is used to determine the orientation of the MHB within the fibrils. It was shown through LD and molecular modeling that MHB aligns itself preferentially parallel with the amyloid fiber at an angle of 14°-22° to the fibril axis and along the grooves of the β-sheet.     

Amyloidogenic properties of the artificial protein albebetin and its biologically active derivatives. The role of electrostatic interactions in fibril formation

The artificial protein albebetin (ABB) and its derivatives containing biologically active fragments of natural proteins form fibrils at physiological pH. The amyloid nature of the fibrils was confirmed by far UV circular dichroism spectra indicating for rich beta-structure, thioflavin T binding assays, and examination of the obtained polymers by atomic force microscopy. Fusing of short peptides--octapeptide of human alpha(2)-interferon (130-137) or hexapeptide HLDF-6 (41-46) of human leukemia differentiation factor--with the N-terminus of ABB led to increased amyloidogenicity of the protein: the rate of fibril formation increased and the morphology of fibrils became more complex. The presence of free hexapeptide HLDF-6 in the ABB solution had the same effect. Increasing ionic strength also activated the process of amyloid formation, but to less extent than did the peptides fused with ABB or added to the ABB solution. We suggest an important role of electrostatic interactions in formation of ABB fibrils. The foregoing ways (addition of salt or peptides) allow decrease in electrostatic repulsion between ABB molecules carrying large negative charge (-12) at neutral pH, thus promoting fibril formation.     

Stimulation of α-synuclein amyloid formation by phosphatidylglycerol micellar tubules

α-Synuclein (α-Syn) is a presynaptic protein that is accumulated in its amyloid form in the brains of Parkinson's patients. Although its biological function remains unclear, α-syn has been suggested to bind to synaptic vesicles and facilitate neurotransmitter release. Recently, studies have found that α-syn induces membrane tubulation, highlighting a potential mechanism for α-syn to stabilize highly curved membrane structures which could have both functional and dysfunctional consequences. To understand how membrane remodeling by α-syn affects amyloid formation, we have studied the α-syn aggregation process in the presence of phosphatidylglycerol (PG) micellar tubules, which were the first reported example of membrane tubulation by α-syn. Aggregation kinetics, β-sheet content, and macroscopic protein-lipid structures were observed by Thioflavin T fluorescence, circular dichroism spectroscopy and transmission electron microscopy, respectively. Collectively, the presence of PG micellar tubules formed at a stochiometric (L/P = 1) ratio was found to stimulate α-syn fibril formation. Moreover, transmission electron microscopy and solid-state nuclear magnetic resonance spectroscopy revealed the co-assembly of PG and α-syn into fibril structures. However, isolated micellar tubules do not form fibrils by themselves, suggesting an important role of free α-syn monomers during amyloid formation. In contrast, fibrils did not form in the presence of excess PG lipids (≥L/P = 50), where most of the α-syn molecules are in a membrane-bound α-helical form. Our results provide new mechanistic insights into how membrane tubules modulate α-syn amyloid formation and support a pivotal role of protein–lipid interaction in the dysfunction of α-syn.     

Spectroscopic characterization of Coomassie blue and its binding to amyloid fibrils

Coomassie brilliant blue G-250 (CB) is the dye used frequently in the Bradford assay for protein concentration determination. In this study, we investigated how the solvent polarity and viscosity affect the CB absorption and fluorescence spectra and apply this understanding to investigate the binding of CB to lysozyme and insulin in the native and amyloid fibril states. Coomassie blue binds both to the native protein and to amyloid fibrils but gives distinctly different spectral responses. The absorption and fluorescence spectra of CB indicate that binding sites in the fibrils are less polar and hold the CB dye more rigidly than in the native forms. The spectral comparison of CB bound to the two different fibrils showed that the binding sites are different, and this was most likely due to differences in secondary structure as monitored by circular dichroism. Finally, linear dichroism was used to show that the fibril-bound CB is oriented preferentially parallel to the insulin amyloid fibril axis.