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β2-Microglobulin amyloid fibril-induced membrane disruption is enhanced by endosomal lipids and acidic pH.

ABSTRACT: Although the molecular mechanisms underlying the pathology of amyloidoses are not well understood, the interaction between amyloid proteins and cell membranes is thought to play a role in several amyloid diseases. Amyloid fibrils of β2-microglobulin (β2m), associated with dialysis-related amyloidosis (DRA), have been shown to cause disruption of anionic lipid bilayers in vitro. However, the effect of lipid composition and the chemical environment in which β2m-lipid interactions occur have not been investigated previously. Here we examine membrane damage resulting from the interaction of β2m monomers and fibrils with lipid bilayers. Using dye release, tryptophan fluorescence quenching and fluorescence confocal microscopy assays we investigate the effect of anionic lipid composition and pH on the susceptibility of liposomes to fibril-induced membrane damage. We show that β2m fibril-induced membrane disruption is modulated by anionic lipid composition and is enhanced by acidic pH. Most strikingly, the greatest degree of membrane disruption is observed for liposomes containing bis(monoacylglycero)phosphate (BMP) at acidic pH, conditions likely to reflect those encountered in the endocytic pathway. The results suggest that the interaction between β2m fibrils and membranes of endosomal origin may play a role in the molecular mechanism of β2m amyloid-associated osteoarticular tissue destruction in DRA.

SUBMITTER: Goodchild SC

PROVIDER: S-EPMC4123989 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The role of conformational flexibility in β2-microglobulin amyloid fibril formation at neutral pH.

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

Polymorphism of β2-microglobulin amyloid fibrils manifested by ultrasonication-enhanced fibril formation in trifluoroethanol.

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

Initiating Fibril Breakdown♦ : Effect of Tetracyclines on the Dynamics of Formation and Destructuration of β2-Microglobulin Amyloid Fibrils

Project description: Not available

| S-EPMC3023532 | biostudies-literature

Understanding the complex mechanisms of β2-microglobulin amyloid assembly.

Project description:Several protein misfolding diseases are associated with the conversion of native proteins into ordered protein aggregates known as amyloid. Studies of amyloid assemblies have indicated that non-native proteins are responsible for initiating aggregation in vitro and in vivo. Despite the importance of these species for understanding amyloid disease, the structural and dynamic features of amyloidogenic intermediates and the molecular details of how they aggregate remain elusive. This review focuses on recent advances in developing a molecular description of the folding and aggregation mechanisms of the human amyloidogenic protein β(2)-microglobulin under physiologically relevant conditions. In particular, the structural and dynamic properties of the non-native folding intermediate I(T) and its role in the initiation of fibrillation and the development of dialysis-related amyloidosis are discussed.

| S-EPMC3229708 | biostudies-literature

Inhibition of beta2-microglobulin amyloid fibril formation by alpha2-macroglobulin.

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

Heating during agitation of ?2-microglobulin reveals that supersaturation breakdown is required for amyloid fibril formation at neutral pH.

Project description:Amyloidosis-associated amyloid fibrils are formed by denatured proteins when supersaturation of denatured proteins is broken. ?2-Microglobulin (?2m) forms amyloid fibrils and causes dialysis-related amyloidosis in patients receiving long-term hemodialysis. Although amyloid fibrils of ?2m in patients are observed at neutral pH, formation of ?2m 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 ?2m 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 ?2m 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.

| S-EPMC6816083 | biostudies-literature

Nicking and fragmentation are responsible for α-lactalbumin amyloid fibril formation at acidic pH and elevated temperature.

Project description:Amyloid fibrils are ordered aggregates that may be formed from disordered, partially unfolded, and fragments of proteins and peptides. There are several diseases, which are due to the formation and deposition of insoluble β-sheet protein aggregates in various tissue, collectively known as amyloidosis. Here, we have used bovine α-lactalbumin as a model protein to understand the mechanism of amyloid fibril formation at pH 1.6 and 65°C under non-reducing conditions. Amyloid fibril formation is confirmed by Thioflavin T fluorescence and atomic force microscopy (AFM). Our finding demonstrates that hydrolysis of peptide bonds occurs under these conditions, which results in nicking and fragmentation. The nicking and fragmentation have been confirmed on non-reducing and reducing gel. We have identified the fragments by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The fragmentation may initiate nucleation as it coincides with AFM images. Conformational changes associated with monomer resulting in fibrillation are shown by circular dichroism and Raman spectroscopy. The current study highlights the importance of nicking and fragmentation in amyloid fibril formation, which may help understand the role of acidic pH and proteolysis under in vivo conditions in the initiation of amyloid fibril formation.

| S-EPMC8376409 | biostudies-literature

Small molecule-mediated inhibition of β-2-microglobulin-based amyloid fibril formation.

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.

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Insights into Insulin Fibril Assembly at Physiological and Acidic pH and Related Amyloid Intrinsic Fluorescence.

Project description:Human insulin is a widely used model protein for the study of amyloid formation as both associated to insulin injection amyloidosis in type II diabetes and highly prone to form amyloid fibrils in vitro. In this study, we aim to gain new structural insights into insulin fibril formation under two different aggregating conditions at neutral and acidic pH, using a combination of fluorescence, circular dichroism, Fourier-transform infrared spectroscopy, and transmission electron miscroscopy. We reveal that fibrils formed at neutral pH are morphologically different from those obtained at lower pH. Moreover, differences in FTIR spectra were also detected. In addition, only insulin fibrils formed at neutral pH showed the characteristic blue-green fluorescence generally associated to amyloid fibrils. So far, the molecular origin of this fluorescence phenomenon has not been clarified and different hypotheses have been proposed. In this respect, our data provide experimental evidence that allow identifying the molecular origin of such intrinsic property.

| S-EPMC5751154 | biostudies-other

Possible mechanisms of polyphosphate-induced amyloid fibril formation of β<sub>2</sub>-microglobulin.

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

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