Strong acids induce amyloid fibril formation of β2-microglobulin via an anion-binding mechanism.
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ABSTRACT:
SUBMITTER: Yamaguchi K
PROVIDER: S-EPMC8564678 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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 β2-microglobulin (β2m), 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 β2m 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 β2m 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 β2m at several micromoles per liter, a similar concentration range to that in vivo. Since β2m with an isoelectric point of 6.4 has a slightly negative net charge at pH 7, polyPs were unlikely to interact with β2m electrostatically. PolyPs appear to dehydrate water molecules around β2m 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 β2m but also suggested that polyPs play an important role in the development of dialysis-related amyloidosis.
| S-EPMC6600944 | biostudies-literature
Project description:The relationship between various amyloidoses and chaperones is gathering attention. In patients with dialysis-related amyloidosis, ?(2)-macroglobulin (?2M), an extracellular chaperone, forms a complex with ?(2)-microglobulin (?2-m), a major component of amyloid fibrils, but the molecular mechanisms and biological implications of the complex formation remain unclear. Here, we found that ?2M substoichiometrically inhibited the ?2-m fibril formation at a neutral pH in the presence of SDS, a model for anionic lipids. Binding analysis showed that the binding affinity between ?2M and ?2-m in the presence of SDS was higher than that in the absence of SDS. Importantly, SDS dissociated tetrameric ?2M into dimers with increased surface hydrophobicity. Western blot analysis revealed that both tetrameric and dimeric ?2M interacted with SDS-denatured ?2-m. At a physiologically relevant acidic pH and in the presence of heparin, ?2M was also dissociated into dimers, and both tetrameric and dimeric ?2M interacted with ?2-m, resulting in the inhibition of fibril growth reaction. These results suggest that under conditions where native ?2-m is denatured, tetrameric ?2M is also converted to dimeric form with exposed hydrophobic surfaces to favor the hydrophobic interaction with denatured ?2-m, thus dimeric ?2M as well as tetrameric ?2M may play an important role in controlling ?2-m amyloid fibril formation.
| S-EPMC3059021 | biostudies-literature
Project description:In dialysis patients, β-2 microglobulin (β2m) can aggregate and eventually form amyloid fibrils in a condition known as dialysis-related amyloidosis, which deleteriously affects joint and bone function. Recently, several small molecules have been identified as potential inhibitors of β2m amyloid formation in vitro Here we investigated whether these molecules are more broadly applicable inhibitors of β2m amyloid formation by studying their effect on Cu(II)-induced β2m amyloid formation. Using a variety of biophysical techniques, we also examined their inhibitory mechanisms. We found that two molecules, doxycycline and rifamycin SV, can inhibit β2m amyloid formation in vitro by causing the formation of amorphous, redissolvable aggregates. Rather than interfering with β2m amyloid formation at the monomer stage, we found that doxycycline and rifamycin SV exert their effect by binding to oligomeric species both in solution and in gas phase. Their binding results in a diversion of the expected Cu(II)-induced progression of oligomers toward a heterogeneous collection of oligomers, including trimers and pentamers, that ultimately matures into amorphous aggregates. Using ion mobility mass spectrometry, we show that both inhibitors promote the compaction of the initially formed β2m dimer, which causes the formation of other off-pathway and amyloid-incompetent oligomers that are isomeric with amyloid-competent oligomers in some cases. Overall, our results suggest that doxycycline and rifamycin are general inhibitors of Cu(II)-induced β2m amyloid formation. Interestingly, the putative mechanism of their activity is different depending on how amyloid formation is initiated with β2m, which underscores the complexity of how these structures assemble in vitro.
| S-EPMC5481568 | biostudies-literature
Project description:RationaleAmyloid formation is implicated in a number of human diseases. β(2)-Microglobulin (β(2)m) is the precursor protein in dialysis-related amyloidosis and it has been shown that partial, or more complete, unfolding is key to amyloid fibril formation in this pathology. Here the relationship between conformational flexibility and β(2)m amyloid formation at physiological pH has been investigated.MethodsHDX-ESI-MS was used to study the conformational dynamics of β(2)m. Protein engineering, or the addition of Cu(2+) ions, sodium dodecyl sulphate, trifluoroethanol, heparin, or protein stabilisers, was employed to perturb the conformational dynamics of β(2)m. The fibril-forming propensities of the protein variants and the wild-type protein in the presence of additives, which resulted in >5-fold increase in the EX1 rate of HDX, were investigated further.ResultsESI-MS revealed that HDX occurs via a mixed EX1/EX2 mechanism under all conditions. Urea denaturation and tryptophan fluorescence indicated that EX1 exchange occurred from a globally unfolded state in wild-type β(2)m. Although >30-fold increase in the HDX exchange rate was observed both for the protein variants and for the wild-type protein in the presence of specific additives, large increases in exchange rate did not necessarily result in extensive de novo fibril formation.ConclusionsThe conformational dynamics measured by the EX1 rate of HDX do not predict the ability of β(2)m to form amyloid fibrils de novo at neutral pH. This suggests that the formation of amyloid fibrils from β(2)m at neutral pH is dependent on the generation of one or more specific aggregation-competent species which facilitate self-assembly.
| S-EPMC3568905 | biostudies-literature
Project description:The polymorphic property of amyloid structures has been focused on as a molecular basis of the presence and propagation of different phenotypes of amyloid diseases, although little is known about the molecular mechanism for expressing diverse structures from only one protein sequence. Here, we have found that, in combination with an enhancing effect of ultrasonication on nucleation, β(2)-microglobulin, a protein responsible for dialysis-related amyloidosis, generates distinct fibril conformations in a concentration-dependent manner in the presence of 2,2,2-trifluoroethanol (TFE). Although the newly formed fibrils all exhibited a similar needle-like morphology with an extensive cross-β core, as suggested by Fourier transform infrared absorption spectra, they differed in thioflavin T intensity, extension kinetics, and tryptophan fluorescence spectra even in the same solvents, representing polymorphic structures. The hydrophobic residues seemed to be more exposed in the fibrils originating at higher concentrations of TFE, as indicated by the increased binding of 1-anilinonaphthalene-8-sulfonic acid, suggesting that the modulation of hydrophobic interactions is critical to the production of polymorphic amyloid structures. Interestingly, the fibrils formed at higher TFE concentrations showed significantly higher stability against guanidium hydrochloride, the perturbation of ionic strength, and, furthermore, pressurization. The cross-β structure inside the fibrils seems to have been more idealized, resulting in increased stability when nucleation occurred in the presence of the alcohol, indicating that a weaker contribution of hydrophobic interactions is intrinsically more amenable to the formation of a non-defective amyloid structure.
| S-EPMC3391109 | biostudies-literature
Project description:The fibril formation of the neurodegenerative peptide amyloid β (Aβ42) is sensitive to solution conditions, and several proteins and peptides have been found to retard the process. Aβ42 fibril formation was followed with ThT fluorescence in the presence of polyamino acids (poly-glutamic acid, poly-lysine, and poly-threonine) and other polymers (poly(acrylic acid), poly(ethylenimine), and poly(diallyldimethylammonium chloride). An accelerating effect on the Aβ42 aggregation process is observed from all positively charged polymers, while no effect is seen from the negative or neutral polymers. The accelerating effect is dependent on the concentration of positive polymer in a highly reproducible manner. Acceleration is observed from a 1:500 polymer to Aβ42 weight ratio and up. Polyamino acids and the other polymers exert quantitatively the same effect at the same concentrations based on weight. Fibrils are formed in all cases as verified by transmission electron microscopy. The concentrations of polymers required for acceleration are too low to affect the Aβ42 aggregation process through increased ionic strength or molecular crowding effects. Instead, the acceleration seems to arise from the locally increased Aβ42 concentration near the polymers, which favors association and affects the electrostatic environment of the peptide.
| S-EPMC4191606 | biostudies-literature
Project description:Amyloid deposition of WT human ?2-microglobulin (WT-h?2m) in the joints of long-term hemodialysis patients is the hallmark of dialysis-related amyloidosis. In vitro, WT-h?2m does not form amyloid fibrils at physiological pH and temperature unless co-solvents or other reagents are added. Therefore, understanding how fibril formation is initiated and maintained in the joint space is important for elucidating WT-h?2m aggregation and dialysis-related amyloidosis onset. Here, we investigated the roles of collagen I and the commonly administered anticoagulant, low-molecular-weight (LMW) heparin, in the initiation and subsequent aggregation phases of WT-h?2m in physiologically relevant conditions. Using thioflavin T fluorescence to study the kinetics of amyloid formation, we analyzed how these two agents affect specific stages of WT-h?2m assembly. Our results revealed that LMW-heparin strongly promotes WT-h?2m fibrillogenesis during all stages of aggregation. However, collagen I affected WT-h?2m amyloid formation in contrasting ways: decreasing the lag time of fibril formation in the presence of LMW-heparin and slowing the rate at higher concentrations. We found that in self-seeded reactions, interaction of collagen I with WT-h?2m amyloid fibrils attenuates surface-mediated growth of WT-h?2m fibrils, demonstrating a key role of secondary nucleation in WT-h?2m amyloid formation. Interestingly, collagen I fibrils did not suppress surface-mediated assembly of WT-h?2m monomers when cross-seeded with fibrils formed from the N-terminally truncated variant ?N6-h?2m. Together, these results provide detailed insights into how collagen I and LMW-heparin impact different stages in the aggregation of WT-h?2m into amyloid, which lead to dramatic effects on the time course of assembly.
| S-EPMC6579475 | biostudies-literature
Project description:Amyloidogenesis is significant in both protein function and pathology. Amyloid formation of folded, globular proteins is commonly initiated by partial or complete unfolding. However, how this unfolding event is triggered for proteins that are otherwise stable in their native environments is not well understood. The accumulation of the immunoglobulin protein β2-microglobulin (β2m) into amyloid plaques in the joints of long-term hemodialysis patients is the hallmark of dialysis-related amyloidosis (DRA). While β2m does not form amyloid unassisted near neutral pH in vitro, the localization of β2m deposits to joint spaces suggests a role for the local extracellular matrix (ECM) proteins, specifically collagens, in promoting amyloid formation. Indeed, collagen and other ECM components have been observed to facilitate β2m amyloid formation, but the large size and anisotropy of the complex, combined with the low affinity of these interactions, have limited atomic-level elucidation of the amyloid-promoting mechanism(s) by these molecules. Using solution NMR approaches that uniquely probe weak interactions in large molecular weight complexes, we are able to map the binding interfaces on β2m for collagen I and detect collagen I-induced μs-ms time-scale dynamics in the β2m backbone. By combining solution NMR relaxation methods and 15N-dark-state exchange saturation transfer experiments, we propose a model in which weak, multimodal collagen I-β2m interactions promote exchange with a minor population of amyloid-competent species to induce fibrillogenesis. The results portray the intimate role of the environment in switching an innocuous protein into an amyloid-competent state, rationalizing the localization of amyloid deposits in DRA.
| S-EPMC7135851 | biostudies-literature
Project description:Serum amyloid A1 (SAA1) is an apolipoprotein that binds to the high-density lipoprotein (HDL) fraction of the serum and constitutes the fibril precursor protein in systemic AA amyloidosis. We here show that HDL binding blocks fibril formation from soluble SAA1 protein, whereas internalization into mononuclear phagocytes leads to the formation of amyloid. SAA1 aggregation in the cell model disturbs the integrity of vesicular membranes and leads to lysosomal leakage and apoptotic death. The formed amyloid becomes deposited outside the cell where it can seed the fibrillation of extracellular SAA1. Our data imply that cells are transiently required in the amyloidogenic cascade and promote the initial nucleation of the deposits. This mechanism reconciles previous evidence for the extracellular location of deposits and amyloid precursor protein with observations the cells are crucial for the formation of amyloid.
| S-EPMC5538634 | biostudies-literature
Project description:Polyphosphates (polyPs), chains of phosphate residues found in species across nature from bacteria to mammals, were recently reported to accelerate the amyloid fibril formation of many proteins. How polyPs facilitate this process, however, remains unknown. To gain insight into their mechanisms, we used various physicochemical approaches to examine the effects of polyPs of varying chain lengths on ultrasonication-dependent α-synuclein (α-syn) amyloid formation. Although orthophosphate and diphosphate exhibited a single optimal concentration of amyloid formation, triphosphate and longer-chain phosphates exhibited two optima, with the second at a concentration lower than that of orthophosphate or diphosphate. The second optimum decreased markedly as the polyP length increased. This suggested that although the optima at lower polyP concentrations were caused by interactions between negatively charged phosphate groups and the positive charges of α-syn, the optima at higher polyP concentrations were caused by the Hofmeister salting-out effects of phosphate groups, where the effects do not depend on the net charge. NMR titration experiments of α-syn with tetraphosphate combined with principal component analysis revealed that, at low tetraphosphate concentrations, negatively charged tetraphosphates interacted with positively charged "KTK" segments in four KTKEGV repeats located at the N-terminal region. At high concentrations, hydrated tetraphosphates affected the surface-exposed hydrophilic groups of compact α-syn. Taken together, our results suggest that long-chain polyPs consisting of 60 to 70 phosphates induce amyloid formation at sub-μM concentrations, which are comparable with the concentrations of polyPs in the blood or tissues. Thus, these findings may identify a role for polyPs in the pathogenesis of amyloid-related diseases.
| S-EPMC8059054 | biostudies-literature