Ultrasonication-dependent acceleration of amyloid fibril formation

Amyloid fibrils, similar to crystals, form through nucleation and growth. Because of the high free-energy barrier of nucleation, the spontaneous formation of amyloid fibrils occurs only after a long lag phase. Ultrasonication is useful for inducing amyloid nucleation and thus for forming fibrils, while the use of a microplate reader with thioflavin T fluorescence is suitable for detecting fibrils in many samples simultaneously. Combining the use of ultrasonication and microplate reader, we propose an efficient approach to studying the potential of proteins to form amyloid fibrils. With β₂-microglobulin, an amyloidogenic protein responsible for dialysis-related amyloidosis, fibrils formed within a few minutes at pH 2.5. Even under neutral pH conditions, fibrils formed after a lag time of 1.5 h. The results propose that fibril formation is a physical reaction that is largely limited by the high free-energy barrier, which can be effectively reduced by ultrasonication. This approach will be useful for developing a high-throughput assay of the amyloidogenicity of proteins.     

Thiol compounds inhibit the formation of amyloid fibrils by beta 2-microglobulin at neutral pH

Dialysis-related amyloidosis frequently develops in patients undergoing long-term hemodialysis, in which the major component of fibrils is β₂-microglobulin (β2-m). To prevent the disease, it is important to stop the formation of fibrils. β2-m has one disulfide bond, which stabilizes the native structure, and amyloid fibrils. Here, the effects of reductants (i.e., dithiothreitol and cysteine) on the formation of β2-m amyloid fibrils were examined at neutral pH. Fibrils were generated by three methods: seed-dependent, ultrasonication-induced, and salt-and-heat-induced fibrillation. Thioflavin T fluorescence, electron microscopy, and far-UV circular dichroism revealed that the addition of reductants significantly inhibits seed-dependent and ultrasonication-induced fibrillation. For salt-and-heat-induced fibrillation, where the solution of β2-m was strongly agitated, formation of amyloid fibrils was markedly reduced in the presence of reductants, although a small number of fibrils formed even after the reduction of the disulfide bond. The results suggest that reductants such as cysteine and dithiothreitol would be useful for preventing the formation of β2-m amyloid fibrils under physiological conditions.     

A Generic Mechanism of β2-Microglobulin Amyloid Assembly at Neutral pH Involving a Specific Proline Switch

Although numerous measurements of amyloid assembly of different proteins under distinct conditions in vitro have been performed, the molecular mechanisms underlying the specific self-association of proteins into amyloid fibrils remain obscure. Elucidating the nature of the events that initiate amyloid formation remains a particularly difficult challenge because of the heterogeneity and transient nature of the species involved. Here, we have used site-directed mutagenesis to create five proline to glycine variants in the naturally amyloidogenic protein β₂-microglobulin (β₂m). One of these variants, P5G, allowed us to isolate and characterise an intermediate containing a non-native trans Pro32 backbone conformation, a feature that is known to be required for amyloid elongation at neutral pH. By analysing oligomerisation and amyloid formation using analytical size-exclusion chromatography, multi-angle static light-scattering, analytical ultracentrifugation, circular dichroism and thioflavin T fluorescence we reveal a pathway for β₂m amyloid assembly at pH 7.5 that does not require the addition of metal ions, detergents, co-solvents or other co-factors that have been used to facilitate amyloid formation at physiological pH and temperature. Assembly is shown to involve the transient formation of a non-native monomer containing a trans P32 backbone conformation. This is followed by the formation of dimeric species and higher molecular mass oligomers that accumulate before the development of amyloid fibrils. On the basis of these results, we propose a generic mechanism for β₂m fibrillogenesis at neutral pH that is consistent with the wide range of published studies of this protein. In this mechanism, amyloid formation is initiated by a specific cis to trans proline switch, the rate of which we show to be controlled by the amino acid sequence proximal to P32 and to the applied solution conditions.     

Structure of interacting segments in the growing amyloid fibril of beta2-microglobulin probed with IR spectroscopy

Inter-segmental interaction at the growing tip of the amyloid fibril of beta2-microglobulin (beta2m) was investigated using IR microscopy. Cross-seeded fibril formation was implemented, in which the amyloid fibril of the #21-31 fragment of beta2m (fA[#21-31]) was generated on the beta2m amyloid fibril (fA[beta2m]) as a seed. Differences between the IR spectra of the cross-seeded fibril and those of the seed were attributed to the contribution from the tip, whose structure is discussed. The results indicated that 6.5 +/- 1.0 out of 11 residues of the fA[#21-31] tip on fA[beta2m] are contained in a beta-sheet at pH 2.5, which was smaller than the corresponding value (7.5 +/- 1.1 residues) of the spontaneous fA[#21-31] at pH 2.5. The tip was suggested to have a planar structure, indicating the planarity of the interacting segment. The N-terminal region of fA[#21-31] in the fibril is more exposed to the solvent than that in the tip, and vice versa for the C-terminal region. This is consistent with the different protonation levels of these regions, and the direction of peptide in the fibrils is determined from these results.     

Kinetic Control in Protein Folding for Light Chain Amyloidosis and the Differential Effects of Somatic Mutations

Light chain amyloidosis is a devastating disease where immunoglobulin light chains form amyloid fibrils, resulting in organ dysfunction and death. Previous studies have shown a direct correlation between the protein thermodynamic stability and the propensity for amyloid formation for some proteins involved in light chain amyloidosis. Here we investigate the effect of somatic mutations on protein stability and in vitro fibril formation of single and double restorative mutants of the protein AL-103 compared to the wild-type germline control protein. A scan rate dependence and hysteresis in the thermal unfolding and refolding was observed for all proteins. This indicates that the unfolding/refolding reaction is kinetically determined with different kinetic constants for unfolding and refolding even though the process remains experimentally reversible. Our structural analysis of AL-103 and AL-103 delP95aIns suggests a kinetic coupling of the unfolding/refolding process with cis–trans prolyl isomerization. Our data reveal that the deletion of proline 95a (AL-103 delP95aIns), which removes the trans–cis di-proline motif present in the patient protein AL-103, results in a dramatic increment in the thermodynamic stability and a significant delay in fibril formation kinetics with respect to AL-103. Fibril formation is pH dependent; all proteins form fibrils at pH 2; reactions become slower and more stochastic as the pH increases up to pH 7. Based on these results, we propose that, in addition to thermodynamic stability, kinetic stability (possibly influenced by the presence of cis proline 95a) plays a major role in the AL-103 amyloid fibril formation process.     

Fibril growth kinetics reveal a region of beta2-microglobulin important for nucleation and elongation of aggregation

Amyloid is a highly ordered form of aggregate comprising long, straight and unbranched proteinaceous fibrils that are formed with characteristic nucleation-dependent kinetics in vitro. Currently, the structural molecular mechanism of fibril nucleation and elongation is poorly understood. Here, we investigate the role of the sequence and structure of the initial monomeric precursor in determining the rates of nucleation and elongation of human β₂-microglobulin (β₂m). We describe the kinetics of seeded and spontaneous (unseeded) fibril growth of wild-type β₂m and 12 variants at pH 2.5, targeting specifically an aromatic-rich region of the polypeptide chain (residues 62-70) that has been predicted to be highly amyloidogenic. The results reveal the importance of aromatic residues in this part of the β₂m sequence in fibril formation under the conditions explored and show that this region of the polypeptide chain is involved in both the nucleation and the elongation phases of fibril formation. Structural analysis of the conformational properties of the unfolded monomer for each variant using NMR relaxation methods revealed that all variants contain significant non-random structure involving two hydrophobic clusters comprising regions 29-51 and 58-79, the extent of which is critically dependent on the sequence. No direct correlation was observed, however, between the extent of non-random structure in the unfolded state and the rates of fibril nucleation and elongation, suggesting that the early stages of aggregation involve significant conformational changes from the initial unfolded state. Together, the data suggest a model for β₂m amyloid formation in which structurally specific interactions involving the highly hydrophobic and aromatic-rich region comprising residues 62-70 provide a complementary interface that is key to the generation of amyloid fibrils for this protein at acidic pH.     

Folding and Fibrillogenesis: Clues from β2-Microglobulin

Renal failure impairs the clearance of β₂-microglobulin from the serum, with the result that this protein accumulates in joints under the form of amyloid fibrils. While the molecular mechanism leading to deposition of amyloid in vivo is not totally understood, some organic compounds, such as trifluoroethanol (TFE), are commonly used to promote the elongation of amyloid fibrils in vitro. This article gives some insights into the structural properties and the conformational states of β₂-microglobulin in the presence of TFE, using both the wild-type protein and the mutant Trp60Gly. The structure of the native state of the protein is rather insensitive to the presence of the alcohol, but the stability of this state is lowered in comparison to some other conformational states. In particular, a native-like folding intermediate is observed in the presence of moderate concentrations of TFE. Instead, at higher concentrations of the alcohol, the population of a disordered native-unlike state is dominant and correlates with the ability to elongate fibrils.     

A systematic investigation into the effect of protein destabilisation on beta 2-microglobulin amyloid formation

Beta-2-microglobulin (beta(2)m) has been shown to form amyloid fibrils with distinct morphologies under acidic conditions in vitro. Short, curved fibrils (<600 nm in length), form rapidly without a lag phase, with a maximum rate at pH 3.5. By contrast, fibrils with a long (approximately 1 microm), straight morphology are produced by incubation of the protein at pH< or =3.0. Both fibril types display Congo red birefringence, bind Thioflavin-T and have X-ray fibre diffraction patterns consistent with a cross-beta structure. In order to investigate the role of different partially folded states in generating fibrils of each type, and to probe the effect of protein stability on amyloid formation, we have undertaken a detailed mutagenesis study of beta(2)m. Thirteen variants containing point mutations in different regions of the native protein were created and their structure, stability and fibril forming propensities were investigated as a function of pH. By altering the stability of the native protein in this manner, we show that whilst destabilisation of the native state is important in the generation of amyloid fibrils, population of specific denatured states is a pre-requisite for amyloid formation from this protein. Moreover, we demonstrate that the formation of fibrils with different morphologies in vitro correlates with the relative population of different precursor states.     

Heat-induced conversion of beta(2)-microglobulin and hen egg-white lysozyme into amyloid fibrils

Thermodynamic parameters characterizing protein stability can be obtained for a fully reversible folding/unfolding system directly by differential scanning calorimetry (DSC). However, the reversible DSC profile can be altered by an irreversible step causing aggregation. Here, to obtain insight into amyloid fibrils, ordered and fibrillar aggregates responsible for various amyloidoses, we studied the effects on human beta(2)-microglobulin and hen egg-white lysozyme of a combination of agitation and heating. Aggregates formed by mildly agitating protein solutions in the native state in the presence of NaCl were heated in the cell of the DSC instrument. For beta(2)-microglobulin, with an increase in the concentration of NaCl at neutral pH, the thermogram began to show an exothermic transition accompanied by a large decrease in heat capacity, followed by a kinetically controlled thermal response. Similarly, the aggregated lysozyme at a high concentration of NaCl revealed a similar distinct transition in the DSC thermogram over a wide pH range. Electron microscopy demonstrated the conformational change into amyloid fibrils. Taken together, the combined use of agitation and heating is a powerful way to generate amyloid fibrils from two proteins, beta(2)-microglobulin and hen egg-white lysozyme, and to evaluate the effects of heat on fibrillation, in which the heat capacity is crucial to characterizing the transition.     

Elongation of amyloid fibrils through lateral binding of monomers revealed by total internal reflection fluorescence microscopy

Amyloid fibrils are fibrillar aggregates of denatured proteins associated with a large number of amyloidoses. The formation of amyloid fibrils has been considered to occur by nucleation and elongation. Real-time imaging of the elongation as well as linear morphology of amyloid fibrils suggests that all elongation events occur at the growing ends of fibrils. On the other hand, we suggested that monomers also bind to the lateral sides of preformed fibrils during the seed-dependent elongation, diffuse to the growing ends, and finally make further conformation changes to the mature amyloid fibrils. To examine lateral binding during the elongation of fibrils, we used islet amyloid polypeptide (IAPP), which has been associated with type II diabetes, and prepared IAPP modified with the fluorescence dye, Alexa532. By monitoring the elongation process with amyloid specific thioflavin T and Alexa532 fluorescence, we obtained overlapping images of the two fluorescence probes, which indicated lateral binding. These results are similar to the surface diffusion-dependent growth of crystals, further supporting the similarities between amyloid fibrillation and the crystallization of substances.     

Pre-Steady-State Kinetic Analysis of the Elongation of Amyloid Fibrils of β2-Microglobulin with Tryptophan Mutagenesis

Amyloid fibrils elongate seed dependently, with preformed fibrils providing a template for propagation of amyloidogenic conformation. Most seeding experiments use relatively few seed fibrils in comparison with monomers, resembling steady-state enzyme kinetics. Pre-steady-state kinetics should also be useful for characterizing the elongation process. With β₂-microglobulin (β₂-m), a protein responsible for dialysis-related amyloidosis, we measured the pre-steady-state kinetics of fibril elongation at pH 2.5, conditions under which the monomer is largely unfolded. β₂-m has Trp residues at positions 60 and 95. We used three single Trp mutants and fluorescence spectroscopy to study structural change upon fibril elongation. To focus on conformational change in monomers, we prepared seeds with a mutant without a Trp residue. At a fixed concentration of monomeric β₂-m, the apparent rate of fibril elongation increased with an increase in the concentration of seeds and then saturated, suggesting the accumulation of a rate-limiting intermediate. Importantly, saturation occurred at a seed/monomer ratio of around 10, as expressed by the concentration of the monomer. Because the number of monomers constituting the seed fibrils is much larger than 10, the results suggest that the elongation process is limited by “non-active-site binding.” Spectral analysis indicated that, upon this non-active-site binding, both Trp60 and Trp95 are exposed to the solvent, and then only Trp60 is buried upon transition to the fibrils. We propose a new model of fibril elongation in which non-active-site binding plays a major role.     

Thermodynamic and Kinetic Characterization of a Germ Line Human λ6 Light-Chain Protein: The Relation between Unfolding and Fibrillogenesis

Proteins encoded by the gene segment 6a of the λ variable light-chain repertoire are strongly associated with amyloid deposition. 6aJL2 is a model protein constructed with the predicted sequences encoded by the 6a and JL2 germ line genes. In this work, we characterized the urea- and temperature-induced unfolding of 6aJL2. In the short time scale, spectroscopic, hydrodynamic and calorimetric experiments were compatible with a two-state transition. Furthermore, ΔG, m and the midpoint urea concentration obtained from equilibrium experiments were compatible with those obtained from kinetic experiments. Since fibril formation is a slow process, samples were also incubated for longer times. After incubation for several hours at 37 °C, spectroscopic, hydrodynamic and calorimetric experiments revealed the presence of a partially unfolded off-pathway intermediate around the midpoint urea concentration (1.5-3.0 M urea). In vitro fibrillogenesis assays show that the maximum growth rate for fibril formation and the minimum lag time were obtained at urea concentrations where the partially unfolded state was populated (2.5 M urea at 37 °C). This indicates that this partially unfolded state is critical for in vitro fibril formation. Concentration-dependent kinetics and hydrodynamic properties of the intermediate were consistent with a soluble oligomeric state. The intermediate is formed around the midpoint urea concentration, where the native and unfolded states are equally populated and their rate of interconversion is the slowest. This situation may promote the slow accumulation of an intermediate state that is prone to aggregate.     

The controlling roles of Trp60 and Trp95 in beta2-microglobulin function, folding and amyloid aggregation properties

Amyloidosis associated to hemodialysis is caused by persistently high β₂-microglobulin (β₂m) serum levels. β₂m is an intrinsically amyloidogenic protein whose capacity to assemble into amyloid fibrils in vitro and in vivo is concentration dependent; no β₂m genetic variant is known in the human population. We investigated the roles of two evolutionary conserved Trp residues in relation to β₂m structure, function and folding/misfolding by means of a combined biophysical and functional approach. We show that Trp60 plays a functional role in promoting the association of β₂m in class I major histocompatibility complex; it is exposed to the solvent at the apex of a protein loop in order to accomplish such function. The Trp60 → Gly mutation has a threefold effect: it stabilizes β₂m, inhibits β₂m amyloidogenic propensity and weakens the interaction with the class I major histocompatibility complex heavy chain. On the contrary, Trp95 is buried in the β₂m core; the Trp95 → Gly mutation destabilizes the protein, which is unfolded in solution, yielding nonfibrillar β₂m aggregates. Trp60 and Trp95 therefore play differential and complementary roles in β₂m, being relevant for function (Trp60) and for maintenance of a properly folded structure (Trp95) while affecting in distinct ways the intrinsic propensity of wild-type β₂m towards self-aggregation into amyloid fibrils.     

Association of MR1 protein, an MHC class I-related molecule, with beta(2)-microglobulin.

MR1 is a major histocompatibility complex (MHC) class I-related gene conserved among mammals, and its predicted amino acid sequence is relatively closer to the classical MHC class I molecules among several divergent class I molecules. However, as its molecular nature and function have not yet been clarified, we set out in this study to establish transfected P388 murine cell lines that stably produce a large number of MR1 proteins and conducted analyses to investigate the molecular nature of MR1. Immunoprecipitation and Western blot analyses with specific antisera revealed that the MR1 protein can associate with beta(2)-microglobulin, suggesting its molecular form of a typical class I heterodimer composed of a heavy and a light chain (beta(2)-microglobulin), like the classical MHC class I molecules.     

Identification of an amyloidogenic region on keratoepithelin via synthetic peptides

Mutations of keratoepithelin (KE) gene in human chromosome 5q31 have been linked with corneal epithelial or stromal dystrophies characterized by the abnormal deposits of amyloid fibrils and/or non-amyloid aggregations in corneal tissue. We report herein that synthetic peptide containing amino acid (a.a.) residues of 515-532 of native KE protein can readily form beta-sheet-containing amyloid fibrils in vitro. Amyloid fibrils formed in various conditions from short synthetic peptides (containing a.a. 515-532 and 515-525, respectively) were characterized by thioflavin T (ThT) fluorescence assay, Congo red staining, electron microscopy (EM) and circular dichroism (CD). Triple-N-methylation of the synthetic peptides prevented the beta-sheet polymerization and related amyloid fibril formation. Comparison study with ThT fluorescence further demonstrated that synthetic peptides containing corneal dystrophy-related mutations within this region formed amyloid fibrils to various extents. Our results suggest that each individual dystrophy-related mutation by itself does not necessarily potentiate amyloid fibril formation of KE. Roles of these intrinsically amyloidogenic foci in abnormal KE aggregations and amyloid deposits of stromal corneal dystrophies await further investigation.     

A β2-microglobulin cleavage variant fibrillates at near-physiological pH

β₂-microglobulin (β₂m) deposits as amyloid in dialysis-related amyloidosis (DRA), predominantly in joints. The molecular mechanisms underlying the amyloidogenicity of β₂m are still largely unknown. In vitro, acidic conditions, pH < 4.5, induce amyloid fibrillation of native β₂m within several days. Here, we show that amyloid fibrils are generated in less than an hour when a cleavage variant of β₂m—found in the circulation of many dialysis patients—is exposed to pH levels (pH 6.6) occurring in joints during inflammation. Aggregation and fibrillation, including seeding effects with intact, native β₂m were studied by Thioflavin T fluorescence spectroscopy, turbidimetry, capillary electrophoresis, and electron microscopy. We conclude that a biologically relevant variant of β₂m is amyloidogenic at slightly acidic pH. Also, only a very small amount of preformed fibrils of this variant is required to induce fibrillation of native β₂m. This may explain the apparent lack of detectable amounts of the variant β₂m in extracts of amyloid from DRA patients.     

Upregulation of SIRT1 deacetylase in phenylephrine-treated cardiomyoblasts

Deposition of amyloid fibrils consisting of amyloid β (Aβ) protein as senile plaques in the brain is a pathological hallmark of Alzheimer’s disease. However, a growing body of evidence shows that soluble Aβ oligomers correlate better with dementia than fibrils, suggesting that Aβ oligomers may be the primary toxic species. The structure and oligomerization mechanism of these Aβ oligomers are crucial for developing effective therapeutics. Here we investigated the oligomerization of Aβ42 in the context of a fusion protein containing GroES and ubiquitin fused to the N-terminus of Aβ sequence. The presence of fusion protein partners, in combination with a denaturing buffer containing 8 M urea at pH 10, is unfavorable for Aβ42 aggregation, thus allowing only the most stable structures to be observed. Transmission electron microscopy showed that Aβ42 fusion protein formed globular oligomers, which bound weakly to thioflavin T and Congo red. SDS–PAGE shows that Aβ42 fusion protein formed SDS-resistant hexamers and tetramers. In contrast, Aβ40 fusion protein remained as monomers on SDS gel, suggesting that the oligomerization of Aβ42 fusion protein is not due to the fusion protein partners. Cysteine scanning mutagenesis at 22 residue positions further revealed that single cysteine substitutions of the C-terminal hydrophobic residues (I31, I32, L34, V39, V40, and I41) led to disruption of hexamer and tetramer formation, suggesting that hydrophobic interactions between these residues are most critical for Aβ42 oligomerization.     

Relationships between the sequence of alpha-synuclein and its membrane affinity, fibrillization propensity, and yeast toxicity

To investigate the alpha-synuclein protein and its role in Parkinson's disease, we screened a library of random point mutants both in vitro and in yeast to find variants in an unbiased way that could help us understand the sequence-phenotype relationship. We developed a rapid purification method that allowed us to screen 59 synuclein mutants in vitro and discovered two double-point mutants that fibrillized slowly relative to wild-type, A30P, and A53T alpha-synucleins. The yeast toxicity of all of these proteins was measured, and we found no correlation with fibrillization rate, suggesting that fibrillization is not necessary for synuclein-induced yeast toxicity. We found that beta-synuclein was of intermediate toxicity to yeast, and gamma-synuclein was non-toxic. Co-expression of Parkinson's disease-related genes DJ-1, parkin, Pink1, UCH-L1, or synphilin, with synuclein, did not affect synuclein toxicity. A second screen, of several thousand library clones in yeast, identified 25 non-toxic alpha-synuclein sequence variants. Most of these contained a mutation to either proline or glutamic acid that caused a defect in membrane binding. We hypothesize that yeast toxicity is caused by synuclein binding directly to membranes at levels sufficient to non-specifically disrupt homeostasis.     

The amyloid fibrils of the constant domain of immunoglobulin light chain

Light chain-associated (AL) amyloidosis is characterized by dominant fibril deposition of the variable domain (VL) of an immunoglobulin light chain, and thus its constant domain (CL) has been considered not to be amyloidogenic. We examined the in vitro fibril formation of the isolated CL in comparison with β₂-microglobulin (β2-m), an immunoglobulin domain-like amyloidogenic protein responsible for dialysis-related amyloidosis. Two methods useful for β2-m at neutral pH also induced amyloid fibrils of CL, which were monitored by thioflavin-T binding and electron microscopy (EM). These results suggest that CL plays an important role, more than previously assumed, in the development of AL-amyloidosis.     

Globular Tetramers of β2-Microglobulin Assemble into Elaborate Amyloid Fibrils

Amyloid fibrils are ordered polymers in which constituent polypeptides adopt a non-native fold. Despite their importance in degenerative human diseases, the overall structure of amyloid fibrils remains unknown. High-resolution studies of model peptide assemblies have identified residues forming cross-β-strands and have revealed some details of local β-strand packing. However, little is known about the assembly contacts that define the fibril architecture. Here we present a set of three-dimensional structures of amyloid fibrils formed from full-length β₂-microglobulin, a 99-residue protein involved in clinical amyloidosis. Our cryo-electron microscopy maps reveal a hierarchical fibril structure built from tetrameric units of globular density, with at least three different subunit interfaces in this homopolymeric assembly. These findings suggest a more complex superstructure for amyloid than hitherto suspected and prompt a re-evaluation of the defining features of the amyloid fold.