The effect of mutation on an aggregation-prone protein: An in vivo, in vitro, and in silico analysis.

Aggregation of initially stably structured proteins is involved in more than 20 human amyloid diseases. Despite intense research, however, how this class of proteins assembles into amyloid fibrils remains poorly understood, principally because of the complex effects of amino acid substitutions on protein stability, solubility, and aggregation propensity. We address this question using ß2-microglobulin (ß2m) as a model system, focusing on D76N-ß2m that is involved in hereditary amyloidosis. This amino acid substitution causes the aggregation-resilient wild-type protein to become highly aggregation prone in vitro, although the mechanism by which this occurs remained elusive. Here, we identify the residues key to protecting ß2m from aggregation by coupling aggregation with antibiotic resistance in E. coli using a tripartite ß-lactamase assay (TPBLA). By performing saturation mutagenesis at three different sites (D53X-, D76X-, and D98X-ß2m) we show that residue 76 has a unique ability to drive ß2m aggregation in vivo and in vitro. Using a randomly mutated D76N-ß2m variant library, we show that all of the mutations found to improve protein behavior involve residues in a single aggregation-prone region (APR) (residues 60 to 66). Surprisingly, no correlation was found between protein stability and protein aggregation rate or yield, with several mutations in the APR decreasing aggregation without affecting stability. Together, the results demonstrate the power of the TPBLA to develop proteins that are resilient to aggregation and suggest a model for D76N-ß2m aggregation involving the formation of long-range couplings between the APR and Asn76 in a nonnative state.

Dimers of D76N-ß2-microglobulin display potent antiamyloid aggregation activity.

Self-association of WT ß2-microglobulin (WT-ß2m) into amyloid fibrils is associated with the disorder dialysis related amyloidosis. In the familial variant D76N-ß2m, the single amino acid substitution enhances the aggregation propensity of the protein dramatically and gives rise to a disorder that is independent of renal dysfunction. Numerous biophysical and structural studies on WT- and D76N-ß2m have been performed in order to better understand the structure and dynamics of the native proteins and their different potentials to aggregate into amyloid. However, the structural properties of transient D76N-ß2m oligomers and their role(s) in assembly remained uncharted. Here, we have utilized NMR methods, combined with photo-induced crosslinking, to detect, trap, and structurally characterize transient dimers of D76N-ß2m. We show that the crosslinked D76N-ß2m dimers have different structures from those previously characterized for the on-pathway dimers of ΔN6-ß2m and are unable to assemble into amyloid. Instead, the crosslinked D76N-ß2m dimers are potent inhibitors of amyloid formation, preventing primary nucleation and elongation/secondary nucleation when added in substoichiometric amounts with D76N-ß2m monomers. The results highlight the specificity of early protein-protein interactions in amyloid formation and show how mapping these interfaces can inform new strategies to inhibit amyloid assembly.

Strong acids induce amyloid fibril formation of ß2-microglobulin via an anion-binding mechanism.

Amyloid fibrils, crystal-like fibrillar aggregates of proteins associated with various amyloidoses, have the potential to propagate via a prion-like mechanism. Among known methodologies to dissolve preformed amyloid fibrils, acid treatment has been used with the expectation that the acids will degrade amyloid fibrils similar to acid inactivation of protein functions. Contrary to our expectation, treatment with strong acids, such as HCl or H2SO4, of ß2-microglobulin (ß2m) or insulin actually promoted amyloid fibril formation, proportionally to the concentration of acid used. A similar promotion was observed at pH 2.0 upon the addition of salts, such as NaCl or Na2SO4. Although trichloroacetic acid, another strong acid, promoted amyloid fibril formation of ß2m, formic acid, a weak acid, did not, suggesting the dominant role of anions in promoting fibril formation of this protein. Comparison of the effects of acids and salts confirmed the critical role of anions, indicating that strong acids likely induce amyloid fibril formation via an anion-binding mechanism. The results suggest that although the addition of strong acids decreases pH, it is not useful for degrading amyloid fibrils, but rather induces or stabilizes amyloid fibrils via an anion-binding mechanism.

Enhanced accessibility and hydrophobicity of amyloidogenic intermediates of the ß2-microglobulin D76N mutant revealed by high-pressure experiments.

ß2-Microglobulin (ß2m) is the causative protein of dialysis-related amyloidosis. Its unfolding mainly proceeds along the pathway of NC →UC ⇄ UT, whereas refolding follows the UT → IT (→NT) →NC pathway, in which N, I, and U are the native, intermediate, and unfolded states, respectively, with the Pro32 peptidyl-prolyl bond in cis or trans conformation as indicated by the subscript. It is noted that the IT state is a putative amyloidogenic precursor state. Several aggregation-prone variants of ß2m have been reported to date. One of these variants is D76N ß2m, which is a naturally occurring amyloidogenic mutant. To elucidate the molecular mechanisms contributing to the enhanced amyloidogenicity of the mutant, we investigated the equilibrium and kinetic transitions of pressure-induced folding/unfolding equilibria in the wild type and D76N mutant by monitoring intrinsic tryptophan and 1-anilino-8-naphthalene sulfonate fluorescence. An analysis of kinetic data revealed that the different folding/unfolding behaviors of the wild type and D76N mutant were due to differences in the activation energy between the unfolded and the intermediate states as well as stability of the native state, leading to more rapid accumulation of IT state for D76N in the refolding process. In addition, the IT state was found to assume more hydrophobic nature. These changes induced the enhanced amyloidogenicity of the D76N mutant and the distinct pathogenic symptoms of patients. Our results suggest that the stabilization of the native state will be an effective approach for suppressing amyloid fibril formation of this mutant.

The corona of protein-gold nanoparticle systems: the role of ionic strength.

The nature of the nanoparticle-protein corona is emerging as a key aspect in determining the impact of nanomaterials on proteins and in general on the biological response. We previously demonstrated that citrate-stabilized gold nanoparticles (Cit-AuNPs) interact with ß2-microglobulin (ß2m) preserving the protein native structure. Moreover, Cit-AuNPs are able to hinder in vitro fibrillogenesis of a ß2m pathologic variant, namely D76N, by reducing the oligomeric association of the protein in solution. Here, we clarify the characteristics of the interaction between ß2m and Cit-AuNPs by means of different techniques, i.e. surface enhanced Raman spectroscopy, NMR and quartz crystal microbalance with dissipation monitoring. All the results obtained clearly show that by simply changing the ionic strength of the medium it is possible to switch from a labile and transient nature of the protein-NP adduct featuring the so-called soft corona, to a more "hard" interaction with a layer of proteins having a longer residence time on the NP surface. This confirms that the interaction between ß2m and Cit-AuNPs is dominated by electrostatic forces which can be tuned by modifying the ionic strength.

Visualization of transient protein-protein interactions that promote or inhibit amyloid assembly.

In the early stages of amyloid formation, heterogeneous populations of oligomeric species are generated, the affinity, specificity, and nature of which may promote, inhibit, or define the course of assembly. Despite the importance of the intermolecular interactions that initiate amyloid assembly, our understanding of these events remains poor. Here, using amyloidogenic and nonamyloidogenic variants of ß2-microglobulin, we identify the interactions that inhibit or promote fibril formation in atomic detail. The results reveal that different outcomes of assembly result from biomolecular interactions involving similar surfaces. Specifically, inhibition occurs via rigid body docking of monomers in a head-to-head orientation to form kinetically trapped dimers. By contrast, the promotion of fibrillation involves relatively weak protein association in a similar orientation, which results in conformational changes in the initially nonfibrillogenic partner. The results highlight the complexity of interactions early in amyloid assembly and reveal atomic-level information about species barriers in amyloid formation.

The residues 4 to 6 at the N-terminus in particular modulate fibril propagation of ß-microglobulin.

The ΔN6 truncation is the main posttranslational modification of ß-microglobulin (ßM) found in dialysis-related amyloid. Investigation of the interaction of wild-type (WT) ßM with N-terminally truncated variants is therefore of medical relevance. However, it is unclear which residues among the six residues at the N-terminus are crucial to the interactions and the modulation of amyloid fibril propagation of ßM. We herein analyzed homo- and heterotypic seeding of amyloid fibrils of WT human ßM and its N-terminally-truncated variants ΔN1 to ΔN6, lacking up to six residues at the N-terminus. At acidic pH 2.5, we produced amyloid fibrils from recombinant, WT ßM and its six truncated variants, and found that ΔN6 ßM fibrils exhibit a significantly lower conformational stability than WT ßM fibrils. Importantly, under more physiological conditions (pH 6.2), we assembled amyloid fibrils only from recombinant, ΔN4, ΔN5, and ΔN6 ßM but not from WT ßM and its three truncated variants ΔN1 to ΔN3. Notably, the removal of the six, five or four residues at the N-terminus leads to enhanced fibril formation, and homo- and heterotypic seeding of ΔN6 fibrils strongly promotes amyloid fibril formation of WT ßM and its six truncated variants, including at more physiological pH 6.2. Collectively, these results demonstrated that the residues 4 to 6 at the N-terminus particularly modulate amyloid fibril propagation of ßM and the interactions of WT ßM with N-terminally truncated variants, potentially indicating the direct relevance to the involvement of the protein's aggregation in dialysis-related amyloidosis.

Possible mechanisms of polyphosphate-induced amyloid fibril formation of ß2-microglobulin.

Polyphosphate (polyP), which is found in various microorganisms and human cells, is an anionic biopolymer consisting of inorganic phosphates linked by high-energy phosphate bonds. Previous studies revealed that polyPs strongly promoted the amyloid formation of several amyloidogenic proteins; however, the mechanism of polyP-induced amyloid formation remains unclear. In the present study using ß2-microglobulin (ß2m), a protein responsible for dialysis-related amyloidosis, we investigated amyloid formation in the presence of various chain lengths of polyPs at different concentrations under both acidic (pH 2.0 to 2.5) and neutral pH (pH 7.0 to 7.5) conditions. We found that the amyloid formation of ß2m at acidic pH was significantly accelerated by the addition of polyPs at an optimal polyP concentration, which decreased with an increase in chain length. The results obtained indicated that electrostatic interactions between positively charged ß2m and negatively charged polyPs play a major role in amyloid formation. Under neutral pH conditions, long polyP with 60 to 70 phosphates induced the amyloid formation of ß2m at several micromoles per liter, a similar concentration range to that in vivo. Since ß2m with an isoelectric point of 6.4 has a slightly negative net charge at pH 7, polyPs were unlikely to interact with ß2m electrostatically. PolyPs appear to dehydrate water molecules around ß2m under the unfolded conformation, leading to the preferential stabilization of less water-exposed amyloid fibrils. These results not only revealed the pH-dependent mechanism of the amyloid formation of ß2m but also suggested that polyPs play an important role in the development of dialysis-related amyloidosis.

Supersaturation-Dependent Formation of Amyloid Fibrils.

The supersaturation of a solution refers to a non-equilibrium phase in which the solution is trapped in a soluble state, even though the solute's concentration is greater than its thermodynamic solubility. Upon breaking supersaturation, crystals form and the concentration of the solute decreases to its thermodynamic solubility. Soon after the discovery of the prion phenomena, it was recognized that prion disease transmission and propagation share some similarities with the process of crystallization. Subsequent studies exploring the structural and functional association between amyloid fibrils and amyloidoses solidified this paradigm. However, recent studies have not necessarily focused on supersaturation, possibly because of marked advancements in structural studies clarifying the atomic structures of amyloid fibrils. On the other hand, there is increasing evidence that supersaturation plays a critical role in the formation of amyloid fibrils and the onset of amyloidosis. Here, we review the recent evidence that supersaturation plays a role in linking unfolding/folding and amyloid fibril formation. We also introduce the HANABI (HANdai Amyloid Burst Inducer) system, which enables high-throughput analysis of amyloid fibril formation by the ultrasonication-triggered breakdown of supersaturation. In addition to structural studies, studies based on solubility and supersaturation are essential both to developing a comprehensive understanding of amyloid fibrils and their roles in amyloidosis, and to developing therapeutic strategies.

The role of the IT-state in D76N ß2-microglobulin amyloid assembly: A crucial intermediate or an innocuous bystander?

The D76N variant of human ß2-microglobulin (ß2m) is the causative agent of a hereditary amyloid disease. Interestingly, D76N-associated amyloidosis has a distinctive pathology compared with aggregation of WT-ß2m, which occurs in dialysis-related amyloidosis. A folding intermediate of WT-ß2m, known as the IT-state, which contains a nonnative trans Pro-32, has been shown to be a key precursor of WT-ß2m aggregation in vitro However, how a single amino acid substitution enhances the rate of aggregation of D76N-ß2m and gives rise to a different amyloid disease remained unclear. Using real-time refolding experiments monitored by CD and NMR, we show that the folding mechanisms of WT- and D76N-ß2m are conserved in that both proteins fold slowly via an IT-state that has similar structural properties. Surprisingly, however, direct measurement of the equilibrium population of IT using NMR showed no evidence for an increased population of the IT-state for D76N-ß2m, ruling out previous models suggesting that this could explain its enhanced aggregation propensity. Producing a kinetically trapped analog of IT by deleting the N-terminal six amino acids increases the aggregation rate of WT-ß2m but slows aggregation of D76N-ß2m, supporting the view that although the folding mechanisms of the two proteins are conserved, their aggregation mechanisms differ. The results exclude the IT-state as the origin of the rapid aggregation of D76N-ß2m, suggesting that other nonnative states must cause its high aggregation rate. The results highlight how a single substitution at a solvent-exposed site can affect the mechanism of aggregation and the resulting disease.

Protein secondary structure determines the temporal relationship between folding and disulfide formation.

How and when disulfide bonds form in proteins relative to the stage of their folding is a fundamental question in cell biology. Two models describe this relationship: the folded precursor model, in which a nascent structure forms before disulfides do, and the quasi-stochastic model, where disulfides form prior to folding. Here we investigated oxidative folding of three structurally diverse substrates, ß2-microglobulin, prolactin, and the disintegrin domain of ADAM metallopeptidase domain 10 (ADAM10), to understand how these mechanisms apply in a cellular context. We used a eukaryotic cell-free translation system in which we could identify disulfide isomers in stalled translation intermediates to characterize the timing of disulfide formation relative to translocation into the endoplasmic reticulum and the presence of non-native disulfides. Our results indicate that in a domain lacking secondary structure, disulfides form before conformational folding through a process prone to nonnative disulfide formation, whereas in proteins with defined secondary structure, native disulfide formation occurs after partial folding. These findings reveal that the nascent protein structure promotes correct disulfide formation during cotranslational folding.

The folding space of proteinß2-microglobulin is modulated by a single disulfide bridge.

Protein beta-2-microglobulin (ß2m) is classically considered the causative agent of dialysis related amyloidosis, a conformational disorder that affects patients undergoing long-term hemodialysis. The wild type (WT) form, the ΔN6 structural variant, and the D76N mutant have been extensively used as model systems ofß2m aggregation. In all of them, the native structure is stabilized by a disulfide bridge between the sulphur atoms of the cysteine residues 25 (at B strand) and 80 (at F strand), which has been considered fundamental inß2m fibrillogenesis. Here, we use extensive discrete molecular dynamics simulations of a full atomistic structure-based model to explore the role of this disulfide bridge as a modulator of the folding space ofß2m. In particular, by considering different models for the disulfide bridge, we explore the thermodynamics of the folding transition, and the formation of intermediate states that may have the potential to trigger the aggregation cascade. Our results show that the dissulfide bridge affects folding transition and folding thermodynamics of the considered model systems, although to different extents. In particular, when the interaction between the sulphur atoms is stabilized relative to the other intramolecular interactions, or even locked (i.e. permanently established), the WT form populates an intermediate state featuring a well preserved core and two unstructured termini, which was previously detected only for the D76N mutant. The formation of this intermediate state may have important implications in our understanding ofß2m fibrillogenesis.

Structural and Functional Basis for LILRB Immune Checkpoint Receptor Recognition of HLA-G Isoforms.

Human leukocyte Ig-like receptors (LILR) LILRB1 and LILRB2 are immune checkpoint receptors that regulate a wide range of physiological responses by binding to diverse ligands, including HLA-G. HLA-G is exclusively expressed in the placenta, some immunoregulatory cells, and tumors and has several unique isoforms. However, the recognition of HLA-G isoforms by LILRs is poorly understood. In this study, we characterized LILR binding to the ß2-microglobulin (ß2m)-free HLA-G1 isoform, which is synthesized by placental trophoblast cells and tends to dimerize and multimerize. The multimerized ß2m-free HLA-G1 dimer lacked detectable affinity for LILRB1, but bound strongly to LILRB2. We also determined the crystal structure of the LILRB1 and HLA-G1 complex, which adopted the typical structure of a classical HLA class I complex. LILRB1 exhibits flexible binding modes with the α3 domain, but maintains tight contacts with ß2m, thus accounting for ß2m-dependent binding. Notably, both LILRB1 and B2 are oriented at suitable angles to permit efficient signaling upon complex formation with HLA-G1 dimers. These structural and functional features of ligand recognition by LILRs provide novel insights into their important roles in the biological regulations.

Uncovering Structural and Molecular Dynamics of ESAT-6:ß2M Interaction: Asp53 of Human ß2-Microglobulin Is Critical for the ESAT-6:ß2M Complexation.

ESAT-6 is a small secreted protein of Mycobacterium tuberculosis involved in the ESAT-6 secretion system (ESX-1)-mediated virulence and pathogenesis. The protein interacts with ß2M, causing downregulation of MHC class I Ag presentation, which could be one of the mechanisms by which it favors increased survival of the bacilli inside the host. In an earlier study, we have shown that the C-terminal region of ESAT-6 is crucial for its interaction with ß2M. However, the interface of ß2M involved in interaction with ESAT-6 and detailed physicochemical changes associated with ESAT-6:ß2M complexation are not fully defined. In this study, using computational and site-directed mutagenesis studies, we demonstrate the presence of strong noncovalent hydrophobic interactions between ESAT-6 and ß2M in addition to the vital hydrogen bonding between the aspartate residue (Asp53) of ß2M and methionine (Met93) of ESAT-6. Docking-based high-throughput virtual screening followed by 16-point screening on microscale thermophoresis resulted in the identification of two potent inhibitors (SM09 and SM15) that mask the critical Met93 residue of ESAT-6 that is required for ESAT-6:ß2M interaction and could rescue cell surface expression of ß2M and HLA in human macrophages as well as MHC class I Ag presentation suppressed by ESAT-6 in peritoneal macrophages isolated from C57BL/6 mice. Both SM09 and SM15 significantly inhibited intracellular survival of M. tuberculosis in human macrophages. Further, we characterized the physicochemical properties involved in the ESAT-6:ß2M complexation, which may help in understanding host-pathogen interactions.

Proline isomerization effects in the amyloidogenic protein ß2-microglobulin.

The protein ß2-microglobulin (ß2-m) can aggregate in insoluble amyloid fibrils, which deposit in the skeletal muscle system of patients undergoing long-term haemodialysis. The molecular mechanisms of such amyloidogenesis are still not fully understood. A potential, although debated, triggering factor is the cis to trans isomerization of a specific proline (Pro32) in ß2-m. Here we investigate this process in the native protein and in the aggregation-prone mutant D76N by means of molecular dynamics and the enhanced sampling method metadynamics. Our simulations, including the estimation of the free energy difference between the cis and trans isomers, are in good agreement with in vitro experiments and highlight the importance of the hydrogen bond and hydrophobic interaction network around the critical Pro32 in stabilizing and de-stabilizing the two isomers.

The Double-Edged Sword of Beta2-Microglobulin in Antibacterial Properties and Amyloid Fibril-Mediated Cytotoxicity.

Beta2-microglobulin (B2M) a key component of major histocompatibility complex class I molecules, which aid cytotoxic T-lymphocyte (CTL) immune response. However, the majority of studies of B2M have focused only on amyloid fibrils in pathogenesis to the neglect of its role of antimicrobial activity. Indeed, B2M also plays an important role in innate defense and does not only function as an adjuvant for CTL response. A previous study discovered that human aggregated B2M binds the surface protein structure in Streptococci, and a similar study revealed that sB2M-9, derived from native B2M, functions as an antibacterial chemokine that binds Staphylococcus aureus. An investigation of sB2M-9 exhibiting an early lymphocyte recruitment in the human respiratory epithelium with bacterial challenge may uncover previously unrecognized aspects of B2M in the body's innate defense against Mycobactrium tuberculosis. B2M possesses antimicrobial activity that operates primarily under pH-dependent acidic conditions at which B2M and fragmented B2M may become a nucleus seed that triggers self-aggregation into distinct states, such as oligomers and amyloid fibrils. Modified B2M can act as an antimicrobial peptide (AMP) against a wide range of microbes. Specifically, these AMPs disrupt microbe membranes, a feature similar to that of amyloid fibril mediated cytotoxicity toward eukaryotes. This study investigated two similar but nonidentical effects of B2M: the physiological role of B2M, in which it potentially acts against microbes in innate defense and the role of B2M in amyloid fibrils, in which it disrupts the membrane of pathological cells. Moreover, we explored the pH-governing antibacterial activity of B2M and acidic pH mediated B2M amyloid fibrils underlying such cytotoxicity.

Trypsin Induced Degradation of Amyloid Fibrils.

Proteolytic enzymes are known to be involved in the formation and degradation of various monomeric proteins, but the effect of proteases on the ordered protein aggregates, amyloid fibrils, which are considered to be extremely stable, remains poorly understood. In this work we study resistance to proteolytic degradation of lysozyme amyloid fibrils with two different types of morphology and beta-2-microglobulun amyloids. We showed that the proteolytic enzyme of the pancreas, trypsin, induced degradation of amyloid fibrils, and the mechanism of this process was qualitatively the same for all investigated amyloids. At the same time, we found a dependence of efficiency and rate of fibril degradation on the structure of the amyloid-forming protein as well as on the morphology and clustering of amyloid fibrils. It was assumed that the discovered relationship between fibrils structure and the efficiency of their degradation by trypsin can become the basis of a new express method for the analysis of amyloids polymorphism. Unexpectedly lower resistance of both types of lysozyme amyloids to trypsin exposure compared to the native monomeric protein (which is not susceptible to hydrolysis) was attributed to the higher availability of cleavage sites in studied fibrils. Another intriguing result of the work is that the cytotoxicity of amyloids treated with trypsin was not only failing to decline, but even increasing in the case of beta-2-microglobulin fibrils.

Computational Evolution of Beta-2-Microglobulin Binding Peptides for Nanopatterned Surface Sensors.

The bottom-up design of smart nanodevices largely depends on the accuracy by which each of the inherent nanometric components can be functionally designed with predictive methods. Here, we present a rationally designed, self-assembled nanochip capable of capturing a target protein by means of pre-selected binding sites. The sensing elements comprise computationally evolved peptides, designed to target an arbitrarily selected binding site on the surface of beta-2-Microglobulin (ß2m), a globular protein that lacks well-defined pockets. The nanopatterned surface was generated by an atomic force microscopy (AFM)-based, tip force-driven nanolithography technique termed nanografting to construct laterally confined self-assembled nanopatches of single stranded (ss)DNA. These were subsequently associated with an ssDNA-peptide conjugate by means of DNA-directed immobilization, therefore allowing control of the peptide's spatial orientation. We characterized the sensitivity of such peptide-containing systems against ß2m in solution by means of AFM-based differential topographic imaging and surface plasmon resonance (SPR) spectroscopy. Our results show that the confined peptides are capable of specifically capturing ß2m from the surface-liquid interface with micromolar affinity, hence providing a viable proof-of-concept for our approach to peptide design.

Epigallocatechin-3-gallate Inhibits Cu(II)-Induced ß-2-Microglobulin Amyloid Formation by Binding to the Edge of Its ß-Sheets.

Epigallocatechin-3-gallate (EGCG) is a catechin found in green tea that can inhibit the amyloid formation of a wide variety of proteins. EGCG's ability to prevent or redirect the amyloid formation of so many proteins may reflect a common mechanism of action, and thus, greater molecular-level insight into how it exerts its effect could have broad implications. Here, we investigate the molecular details of EGCG's inhibition of the protein ß-2-microglobulin (ß2m), which forms amyloids in patients undergoing long-term dialysis treatment. Using size-exclusion chromatography and a collection of mass spectrometry-based techniques, we find that EGCG prevents Cu(II)-induced ß2m amyloid formation by diverting the normal progression of preamyloid oligomers toward the formation of spherical, redissolvable aggregates. EGCG exerts its effect by binding with a micromolar affinity (Kd ≈ 5 µM) to the ß2m monomer on the edge of two ß-sheets near the N-terminus. This interaction destabilizes the preamyloid dimer and prevents the formation of a tetramer species previously shown to be essential for Cu(II)-induced ß2m amyloid formation. EGCG's binding at the edge of the ß-sheets in ß2m is consistent with a previous hypothesis that EGCG generally prevents amyloid formation by binding cross-ß-sheet aggregation intermediates.

Heating during agitation of ß2-microglobulin reveals that supersaturation breakdown is required for amyloid fibril formation at neutral pH.

Amyloidosis-associated amyloid fibrils are formed by denatured proteins when supersaturation of denatured proteins is broken. ß2-Microglobulin (ß2m) forms amyloid fibrils and causes dialysis-related amyloidosis in patients receiving long-term hemodialysis. Although amyloid fibrils of ß2m in patients are observed at neutral pH, formation of ß2m amyloids in vitro has been difficult to discern at neutral pH because of the amyloid-resistant native structure. Here, to further understand the mechanism underlying in vivo amyloid formation, we investigated the relationship between protein folding/unfolding and misfolding leading to amyloid formation. Using thioflavin T assays, CD spectroscopy, and transmission EM analyses, we found that ß2m efficiently forms amyloid fibrils even at neutral pH by heating with agitation at high-salt conditions. We constructed temperature- and NaCl concentration-dependent conformational phase diagrams in the presence or absence of agitation, revealing how amyloid formation under neutral pH conditions is related to thermal unfolding and breakdown of supersaturation. Of note, after supersaturation breakdown and following the law of mass action, the ß2m monomer equilibrium shifted to the unfolded state, destabilizing the native state and thereby enabling amyloid formation even under physiological conditions with a low amount of unfolded precursor. The amyloid fibrils depolymerized at both lower and higher temperatures, resembling cold- or heat-induced denaturation of globular proteins. Our results suggest an important role for heating in the onset of dialysis-related amyloidosis and related amyloidoses.