Β2m Amyloid Research Articles

The extracellular chaperone clusterin prevents primary and secondary nucleation of an amyloidogenic variant of β2-microglobulin

Amyloid fibril formation by the extracellular protein β2-microglobulin (β2m) and its subsequent accumulation in periarticular tissues have been linked to dialysis-related amyloidosis. A natural variant of human β2m responsible for aggressive systemic amyloidosis contains an aspartate to asparagine mutation at residue 76 (i.e. D76N β2m), which readily forms amyloid fibrils in vitro under physiological conditions. In this study, we examined the role of the extracellular molecular chaperone clusterin in modulating D76N β2m fibril formation in vitro under physiological conditions. The presence of extrinsic charged amino acids modulated D76N β2m fibril formation, implying that electrostatic interactions are involved in the protein’s aggregation. Thioflavin T (ThT) and 1-anilinonaphthalene-8-sulfonate fluorescence assays indicated that clusterin interacts via hydrophobic and electrostatic forces with the monomeric, prefibrillar and fibrillar species of D76N β2m. As a result, clusterin was incorporated into D76N β2m aggregates during the latter’s fibril formation, as indicated by SDS-PAGE of depolymerised fibrils. SYPRO Orange and ThT fluorescence assays suggested that, compared to pure D76N β2m fibrils, those formed in the presence of clusterin are chemically more stable with a reduced ability to act as nucleation seeds. Detailed 15N NMR relaxation studies of mixtures of 15N-labelled β2m with clusterin confirmed that the chaperone interacts transiently and non-specifically with monomeric β2m. Clusterin inhibits both primary and secondary nucleation of D76N β2m fibril formation. In doing so, clusterin binds to D76N β2m fibrils and stabilises them to prevent possible fragmentation. In vivo, the multifaceted chaperone action of clusterin may delay, if not prevent, β2m amyloid proliferation and deposition in tissues.

Macromolecular crowding and supersaturation protect hemodialysis patients from the onset of dialysis-related amyloidosis

Dialysis-related amyloidosis (DRA), a serious complication among long-term hemodialysis patients, is caused by amyloid fibrils of β2-microglobulin (β2m). Although high serum β2m levels and a long dialysis vintage are the primary and secondary risk factors for the onset of DRA, respectively, patients with these do not always develop DRA, indicating that there are additional risk factors. To clarify these unknown factors, we investigate the effects of human sera on β2m amyloid fibril formation, revealing that sera markedly inhibit amyloid fibril formation. Results from over 100 sera indicate that, although the inhibitory effects of sera deteriorate in long-term dialysis patients, they are ameliorated by maintenance dialysis treatments in the short term. Serum albumin prevents amyloid fibril formation based on macromolecular crowding effects, and decreased serum albumin concentration in dialysis patients is a tertiary risk factor for the onset of DRA. We construct a theoretical model assuming cumulative effects of the three risk factors, suggesting the importance of monitoring temporary and accumulated risks to prevent the development of amyloidosis, which occurs based on supersaturation-limited amyloid fibril formation in a crowded milieu.

A novel substitution of proline (P32L) destabilises β2-microglobulin inducing hereditary systemic amyloidosis

Background β2-microglobulin amyloidosis was first described in the 1980s as a protein deposition disease associated with long-term haemodialysis. More recently, two inherited forms resulting from separate point mutations in the β2-microglobulin gene have been identified. In this report, we detail a novel β2M variant, P32L, caused by a unique dinucleotide mutation that is linked to systemic hereditary β2-microglobulin amyloidosis. Methods Three family members from a Portuguese kinship featured cardiomyopathy, requiring organ transplantation in one case, along with soft tissue involvement; other involvements included gastrointestinal, neuropathic and sicca syndrome. In vitro studies with recombinant P32L, P32G, D76N and wild-type β2-microglobulin were undertaken to compare the biophysical properties of the proteins. Results The P32L variant was caused by the unique heterozygous dinucleotide mutation c.154_155delinsTT. Amyloid disease featured lowered serum β2-microglobulin levels with near equal amounts of circulating P32L and wild-type proteins; amyloid deposits were composed exclusively of P32L variant protein. In vitro studies of P32L demonstrated thermodynamic and chemical instability and enhanced susceptibility to proteolysis with rapid formation of pre-fibrillar oligomeric structures by N- and C-terminally truncated species under physiological conditions. Conclusions This work provides both clinical and experimental evidence supporting the critical role of P32 residue replacement in β2M amyloid fibrillogenesis.

Dialysis-related carpal tunnel syndrome in the past 40\xa0years

Purpose and methodPatients on hemodialysis develop carpal tunnel syndrome (CTS) due to an accumulation of dialysis-related β2 microglobulin (β2m) amyloid (DRA). In Japan, dialysis technology has progressed remarkably in the past 40 years and has increased the time until patients require surgery for CTS. However, unclear is whether the time from the start of hemodialysis to the first surgery for CTS is associated with β2m clearance by the different hemodialysis techniques. Therefore, we retrospectively evaluated β2m clearance, serum β2m levels, and the change in the length of this period in patients across 4 periods according to the year that first surgery for CTS was performed: period 1, 1982–1989; period 2, 1990–1999; period 3, 2000–2009; and period 4, 2010–2019.ResultA total of 222 patients who met the selection criteria were included. Mean β2m clearance was −1.8 ± 16.7% in period 1, and improved to 65.4 ± 8.6% in period 3. Accordingly, the serum β2m value after hemodialysis decreased significantly. The time from the start of hemodialysis to the first surgery for CTS was 12.4 ± 2.9 years in period 1 but increased to 21.8 ± 6.3 years in period 3. In multivariable linear regression analysis, the significant factors contributing to β2m clearance were periods 2, 3, and 4. In particular, the relation between removal of β2m and the extension of the dialysis vintage in period 1 and 2 was remarkable compared with periods 3 and 4.ConclusionOur findings indicate that improvement of β2m clearance via advances in dialysis technology might result in a significant extension in the time between starting HD and the first surgery for CTS.

β2-microglobulin triggers NLRP3 inflammasome activation in tumor-associated macrophages to promote multiple myeloma progression

As substantial constituents of the multiple myeloma (MM) microenvironment, pro-inflammatory macrophages have emerged as key promoters of disease progression, bone destruction, and immune impairment. We identify beta-2-microglobulin (β2m) as a driver in initiating inflammation in myeloma-associated macrophages (MAMs). Lysosomal accumulation of phagocytosed β2m promotes β2m amyloid aggregation in MAMs, resulting in lysosomal rupture and ultimately production of active interleukin-1β (IL-1β) and IL-18. This process depends on activation of the NLRP3 inflammasome after β2m accumulation, as macrophages from NLRP3-deficient mice lack efficient β2m-induced IL-1β production. Moreover, depletion or silencing of β2m in MM cells abrogates inflammasome activation in a murine MM model. Finally, we demonstrate that disruption of NLRP3 or IL-18 diminishes tumor growth and osteolytic bone destruction normally promoted by β2m-induced inflammasome signaling. Our results provide mechanistic evidence for β2m's role as an NLRP3 inflammasome activator during MM pathogenesis. Moreover, inhibition of NLRP3 represents a potential therapeutic approach in MM.

β <sub>2</sub>-Microglobulin - A Trigger for NLRP3 Inflammasome Activation in Tumor-Associated Macrophages Promoting Multiple Myeloma Progression

As significant constituents of the tumor microenvironment in multiple Myeloma (MM), pro-inflammatory macrophages are key promoters of disease progression, bone destruction, and immune-impairment. Consequently, the identification of endogenous mediators of these inflammatory processes can open novel therapeutic avenues against major pathological features of MM. Here, we identify beta-2-microglobulin (β2m) as an important driver in the initiation of inflammation in myeloma-associated macrophages (MAMs). Lysosomal accumulation of phagocytosed β2m in patient-derived MAMs promoted β2m amyloid aggregation, resulting in lysosomal rupture and ultimately in the production of active interleukin (IL)-1β and IL-18. Interestingly, this process strictly depended on the activation of the NLRP3 inflammasome after β2m accumulation, as macrophages from NLRP3-deficient mice lacked efficient β2m-induced IL-1β production. Moreover, depletion or silencing of β2m in MM cells abrogated inflammasome activation in a murine MM model. Finally, we demonstrate that specific disruption of NLRP3 or IL-18 diminished tumor growth and osteolytic bone destruction normally promoted by β2m-induced inflammasome signaling. Taken together our results provide novel mechanistic evidence for β2m’s role as an NLRP3 inflammasome activator during MM pathogenesis. Moreover, inhibition of NLRP3 is a potential novel therapeutic approach to combat this severe malignancy.

Epigallocatechin-3-gallate Inhibits Cu(II)-Induced β-2-Microglobulin Amyloid Formation by Binding to the Edge of Its β-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 β-2-microglobulin (β2m), 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 β2m 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 β2m monomer on the edge of two β-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 β2m amyloid formation. EGCG's binding at the edge of the β-sheets in β2m is consistent with a previous hypothesis that EGCG generally prevents amyloid formation by binding cross-β-sheet aggregation intermediates.

Collagen I Weakly Interacts with the β-Sheets of β2-Microglobulin and Enhances Conformational Exchange To Induce Amyloid Formation.

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.

Structural Features of Amyloid Fibrils Formed from the Full-Length and Truncated Forms of Beta-2-Microglobulin Probed by Fluorescent Dye Thioflavin T.

The persistence of high concentrations of beta-2-microglobulin (β2M) in the blood of patients with acute renal failure leads to the development of the dialysis-related amyloidosis. This disease manifests in the deposition of amyloid fibrils formed from the various forms of β2M in the tissues and biological fluids of patients. In this paper, the amyloid fibrils formed from the full-length β2M (β2m) and its variants that lack the 6 and 10 N-terminal amino acids of the protein polypeptide chain (ΔN6β2m and ΔN10β2m, respectively) were probed by using the fluorescent dye thioflavin T (ThT). For this aim, the tested solutions were prepared via the equilibrium microdialysis approach. Spectroscopic analysis of the obtained samples allowed us to detect one binding mode (type) of ThT interaction with all the studied variants of β2M amyloid fibrils with affinity ~104 M−1. This interaction can be explained by the dye molecules incorporation into the grooves that were formed by the amino acids side chains of amyloid protofibrils along the long axis of the fibrils. The decrease in the affinity and stoichiometry of the dye interaction with β2M fibrils, as well as in the fluorescence quantum yield and lifetime of the bound dye upon the shortening of the protein amino acid sequence were shown. The observed differences in the ThT-β2M fibrils binding parameters and characteristics of the bound dye allowed to prove not only the difference of the ΔN10β2m fibrils from other β2M fibrils (that can be detected visually, for example, by transmission electron microscopy (TEM), but also the differences between β2m and ΔN6β2m fibrils (that can not be unequivocally confirmed by other approaches). These results prove an essential role of N-terminal amino acids of the protein in the formation of the β2M amyloid fibrils. Information about amyloidogenic protein sequences can be claimed in the development of ways to inhibit β2M fibrillogenesis for the treatment of dialysis-related amyloidosis.

Using Covalent Labeling and Mass Spectrometry To Study Protein Binding Sites of Amyloid Inhibiting Molecules.

Amyloid aggregates are associated with several debilitating diseases, and there are numerous efforts to develop small molecule treatments against these diseases. One challenge associated with these efforts is determining protein binding site information for potential therapeutics because amyloid-forming proteins rapidly form oligomers and aggregates, making traditional protein structural analysis techniques challenging. Using β-2-microglobulin (β2m) as a model amyloid-forming protein along with two recently identified small molecule amyloid inhibitors (i.e., rifamycin SV and doxycycline), we demonstrate that covalent labeling and mass spectrometry (MS) can be used to map small-molecule binding sites for a rapidly aggregating protein. Specifically, three different covalent labeling reagents, namely diethylpyrocarbonate, 2,3-butanedione, and the reagent pair EDC/GEE, are used together to pinpoint the binding sites of rifamycin SV, doxycycline, and another molecule, suramin, which binds but does not inhibit Cu(II)-induced β2m amyloid formation. The labeling results reveal binding sites that are consistent with the known effects of these molecules on β2m amyloid formation and are in general agreement with molecular docking results. We expect that this combined covalent labeling approach will be applicable to other protein/small molecule systems that are difficult to study by traditional means.

Assessing the effect of D59P mutation in the DE loop region in amyloid aggregation propensity of β2‐microglobulin: A molecular dynamics simulation study

Dialysis-related amyloidosis (DRA) is a severe condition characterized by the accumulation of amyloidogenic β2-microglobulin (β2m) protein around skeletal joints and bones. The recent studies highlighted a critical role of the DE loop region for β2m stability and amyloid aggregation propensity. Despite significant efforts, the molecular mechanism of enhanced aggregation due to D59P mutation in the DE loop region remain elusive. In the present study, explicit-solvent molecular dynamics (MD) simulations were performed to examine the key changes in the structural and dynamic properties of wild type (wt) β2m upon D59P mutation. MD simulations reveal a decrease in the average number of hydrogen bonds in the loop regions on D59P mutation that enhances conformational flexibility, which lead to higher aggregation propensity of D59P as compare to wt β2m. The principal component analysis (PCA) highlight that D59P covers a larger region of phase space and display a higher trace value than wt β2m, which suggest an overall enhancement in the conformational flexibility. D59P display two minimum energy basins in the free energy landscape (FEL) that are associated with thermodynamically less stable conformational states as compare to single minimum energy basin in wt β2m. The present study provides theoretical insights into the molecular mechanism behind the higher aggregation propensity of D59P as compare to wt β2m.

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

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.

Increased β-Sheet Dynamics and D-E Loop Repositioning Are Necessary for Cu(II)-Induced Amyloid Formation by β-2-Microglobulin.

β-2-Microglobulin (β2m) forms amyloid fibrils in the joints of patients undergoing dialysis treatment as a result of kidney failure. One of the ways in which β2m can be induced to form amyloid fibrils in vitro is via incubation with stoichiometric amounts of Cu(II). To better understand the structural changes caused by Cu(II) binding that allow β2m to form amyloid fibrils, we compared the effect of Ni(II) and Zn(II) binding, which are two similarly sized divalent metal ions that do not induce β2m amyloid formation. Using hydrogen/deuterium exchange mass spectrometry (HDX/MS) and covalent labeling MS, we find that Ni(II) has little effect on β2m structure, despite binding in the same region of the protein as Cu(II). This observation indicates that subtle differences in the organization of residues around Cu(II) cause distant changes that are necessary for oligomerization and eventual amyloid formation. One key difference that we find is that only Cu(II), not Ni(II) or Zn(II), is able to cause the cis-trans isomerization of Pro32 that is an important conformational switch that initiates β2m amyloid formation. By comparing HDX/MS data from the three metal-β2m complexes, we also discover that increased dynamics in the β-sheet formed by the A, B, D, and E β strands of the protein and repositioning of residues in the D-E loop are necessary aspects of β2m forming an amyloid-competent dimer. Altogether, our results reveal new structural insights into the unique effect of Cu(II) in the metal-induced amyloid formation of β2m.

Endocytosed β2-Microglobulin Amyloid Fibrils Induce Necrosis and Apoptosis of Rabbit Synovial Fibroblasts by Disrupting Endosomal/Lysosomal Membranes: A Novel Mechanism on the Cytotoxicity of Amyloid Fibrils

Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients. In dialysis-related amyloidosis, β2-microglobulin (β2-m) amyloid fibrils deposit in the osteoarticular tissue, leading to carpal tunnel syndrome and destructive arthropathy with cystic bone lesions, but the mechanism by which these amyloid fibrils destruct bone and joint tissue is not fully understood. In this study, we assessed the cytotoxic effect of β2-m amyloid fibrils on the cultured rabbit synovial fibroblasts. Under light microscopy, the cells treated with amyloid fibrils exhibited both necrotic and apoptotic changes, while the cells treated with β2-m monomers and vehicle buffer exhibited no morphological changes. As compared to β2-m monomers and vehicle buffer, β2-m amyloid fibrils significantly reduced cellular viability as measured by the lactate dehydrogenase release assay and the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay and significantly increased the percentage of apoptotic cells as measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. β2-m amyloid fibrils added to the medium adhered to cell surfaces, but did not disrupt artificial plasma membranes as measured by the liposome dye release assay. Interestingly, when the cells were incubated with amyloid fibrils for several hours, many endosomes/lysosomes filled with amyloid fibrils were observed under confocal laser microscopy and electron microscopy, Moreover, some endosomal/lysosomal membranes were disrupted by intravesicular fibrils, leading to the leakage of the fibrils into the cytosol and adjacent to mitochondria. Inhibition of actin-dependent endocytosis by cytochalasin D attenuated the toxicity of amyloid fibrils. These results suggest that endocytosed β2-m amyloid fibrils induce necrosis and apoptosis by disrupting endosomal/lysosomal membranes, and this novel mechanism on the cytotoxicity of amyloid fibrils is described.

Structural and Thermodynamic Characteristics of Amyloidogenic Intermediates of β-2-Microglobulin

β-2-microglobulin (β2m) self-aggregates to form amyloid fibril in renal patients taking long-term dialysis treatment. Despite the extensive structural and mutation studies carried out so far, the molecular details on the factors that dictate amyloidogenic potential of β2m remain elusive. Here we report molecular dynamics simulations followed by the solvation thermodynamic analyses on the wild-type β2m and D76N, D59P, and W60C mutants at the native (N) and so-called aggregation-prone intermediate (IT) states, which are distinguished by the native cis- and non-native trans-Pro32 backbone conformations. Three major structural and thermodynamic characteristics of the IT-state relative to the N-state in β2m protein are detected that contribute to the increased amyloidogenic potential: (i) the disruption of the edge D-strand, (ii) the increased solvent-exposed hydrophobic interface, and (iii) the increased solvation free energy (less affinity toward solvent water). Mutation effects on these three factors are shown to exhibit a good correlation with the experimentally observed distinct amyloidogenic propensity of the D76N (+), D59P (+), and W60C (−) mutants (+/− for enhanced/decreased). Our analyses thus identify the structural and thermodynamic characteristics of the amyloidogenic intermediates, which will serve to uncover molecular mechanisms and driving forces in β2m amyloid fibril formation.

Wild type beta-2 microglobulin and DE loop mutants display a common fibrillar architecture.

Beta-2 microglobulin (β2m) is the protein responsible for a pathologic condition known as dialysis related amyloidosis. In recent years an important role has been assigned to the peptide loop linking strands D and E (DE loop) in determining β2m stability and amyloid propensity. Several mutants of the DE loop have been studied, showing a good correlation between DE loop geometrical strain, protein stability and aggregation propensity. However, it remains unclear whether the aggregates formed by wild type (wt) β2m and by the DE loop variants are of the same kind, or whether the mutations open new aggregation pathways. In order to address this question, fibrillar samples of wt and mutated β2m variants have been analysed by means of atomic force microscopy and infrared spectroscopy. The data here reported indicate that the DE loop mutants form aggregates with morphology and structural organisation very similar to the wt protein. Therefore, the main effect of β2m DE loop mutations is proposed to stem from the different stabilities of the native fold. Considerations on the structural role of the DE loop in the free monomeric β2m and as part of the Major Histocompatibility Complex are also presented.

Substitution of proline32 by α-methylproline preorganizes β2-microglobulin for oligomerization but not for aggregation into amyloids.

Conversion of soluble folded proteins into insoluble amyloids generally proceeds in three distinct mechanistic stages: (1) initial protein misfolding into aggregation-competent conformers, (2) subsequent formation of oligomeric species and, finally, (3) self-assembly into extended amyloid fibrils. In the work reported herein, we interrogated the amyloidogenesis mechanism of human β2-microglobulin (β2m), which is thought to be triggered by a pivotal cis-trans isomerization of a proline residue at position 32 in the polypeptide, with nonstandard amino acids. Using chemical protein synthesis we prepared a β2m analogue in which Pro32 was replaced by the conformationally constrained amino acid α-methylproline (MePro). The strong propensity of MePro to adopt a trans prolyl bond led to enhanced population of a non-native [trans-MePro32]β2m protein conformer, which readily formed oligomers at neutral pH. In the presence of the antibiotic rifamycin SV, which inhibits amyloid growth of wild-type β2m, [MePro32]β2m was nearly quantitatively converted into different spherical oligomeric species. Self-assembly into amyloid fibrils was not observed in the absence of seeding, however, even at low pH (<3), where wild-type β2m spontaneously forms amyloids. Nevertheless, we found that aggregation-preorganized [MePro32]β2m can act in a prion-like fashion, templating misfolded conformations in a natively folded protein. Overall, these results provide detailed insight into the role of cis-trans isomerization of Pro32 and ensuing structural rearrangements that lead to initial β2m misfolding and aggregation. They corroborate the view that conformational protein dynamics enabled by reversible Pro32 cis-trans interconversion rather than simple population of the trans conformer is critical for both nucleation and subsequent growth of β2m amyloid structures.

β2-Microglobulin Amyloid Fibrils Are Nanoparticles That Disrupt Lysosomal Membrane Protein Trafficking and Inhibit Protein Degradation by Lysosomes

Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of β2-microglobulin (β2m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented β2m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented β2m fibrils did not, however, cause cell death. Instead, fragmented β2m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway.

Sonographic features suggestive of amyloidosis in hemodialysis patients: Relations to serum beta2-microglobulin

Aim of the workTo determine sonographic features suggestive of amyloidosis in hemodialysis patients complaining of shoulder pain, and to study their relations to serum beta2-microglobulin (β2M). Patients and methodsClinical examination, skeletal survey, musculoskeletal ultrasonography of the shoulder joints, and serum β2M were done for 32 patients with end stage renal disease, who were regular on hemodialysis. ResultsSerum β2M levels were markedly raised in all patients, and increased with increasing duration of dialysis (r=0.91, p<0.001). Twenty-five patients had a non homogeneous thickening of the supraspinatus tendon >7mm, and thickening of the biceps tendon >4mm, 30 had synovial deposits, 27 had subdeltoid bursa effusion, 25 had thickened subacromial bursa, 7 had supraspinatus tendon tear, and 17 had bony erosions. Serum β2M levels significantly correlated with thickened supraspinatus tendon (>7mm) and supraspinatus tendon tear (r=0.41, p=0.03 and r=0.42, p=0.01 respectively). Long time on hemodialysis was the significant independent determinant for supraspinatus tendon tear and humeral head erosions (p=0.001 for each). ConclusionElevated serum β2M levels and sonographic features suggestive of dialysis-related amyloidosis (DRA) were found in all hemodialyzed patients complaining of shoulder pain either with or without clinical and/or radiological features suggestive of DRA. So, for diagnosis of DRA, sonographic features should correspond to the presence of clinically or radiologically evident β2M amyloid, and we should exclude other causes of non-amyloid changes.

PH‐Dependent structural ensembles of haptoglobin suppress β2‐microglobulin amyloidosis: role in lysosomal localization and extracellular protein quality control (752.7)

Several diseases involve extracellular deposition of misfolded proteins, which is associated with inflammation and local acidosis. We have investigated the chaperone‐like activity of haptoglobin towards the amyloid aggregation of β2‐microglobulin (β2m) and of α‐synuclein and its role in clearing the amyloid deposits from the extracellular space. At neutral pH, haptoglobin prevents the formation of β2m amyloid fibrils but not that of α‐synuclein. Under conditions of local acidosis, pH 5.5‐6.5, it prevents β2m fibril formation with relatively decreased efficiency. We have investigated the structural and stability aspects of haptoglobin as a function of pH. Between pH 5.5‐8.0, it exists in a continuum of conformational states with subtle perturbation in tertiary structure, moderately exposed hydrophobic surfaces and distinct latent heat of unfolding. Below pH 5.0 it exists in a molten‐globule‐like conformation with no chaperone activity. Live cell imaging and flow cytometry studies show that the species of β2m formed in the presence of haptoglobin are not cytotoxic and readily undergo lysosomal degradation. Analytical ultracentrifugation and dot blot studies reveal that haptoglobin interacts with prefibrillar oligomeric species formed during β2m fibril formation. Thus, haptoglobin is an extracellular chaperone with promising therapeutic implications in extracellular amyloid diseases.