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Annals of the Rheumatic Diseases Apr 1981
Comparative Study
Topics: Adult; Aged; Arthritis, Rheumatoid; Beta-Globulins; Female; Humans; Male; Middle Aged; beta 2-Microglobulin
PubMed: 6164345
DOI: 10.1136/ard.40.2.211-b -
Scientific Reports Apr 2017Systemic amyloidosis is caused by misfolding and aggregation of globular proteins in vivo for which effective treatments are urgently needed. Inhibition of protein...
Systemic amyloidosis is caused by misfolding and aggregation of globular proteins in vivo for which effective treatments are urgently needed. Inhibition of protein self-aggregation represents an attractive therapeutic strategy. Studies on the amyloidogenic variant of β-microglobulin, D76N, causing hereditary systemic amyloidosis, have become particularly relevant since fibrils are formed in vitro in physiologically relevant conditions. Here we compare the potency of two previously described inhibitors of wild type β-microglobulin fibrillogenesis, doxycycline and single domain antibodies (nanobodies). The β-microglobulin -binding nanobody, Nb24, more potently inhibits D76N β-microglobulin fibrillogenesis than doxycycline with complete abrogation of fibril formation. In β-microglobulin knock out mice, the D76N β-microglobulin/ Nb24 pre-formed complex, is cleared from the circulation at the same rate as the uncomplexed protein; however, the analysis of tissue distribution reveals that the interaction with the antibody reduces the concentration of the variant protein in the heart but does not modify the tissue distribution of wild type β-microglobulin. These findings strongly support the potential therapeutic use of this antibody in the treatment of systemic amyloidosis.
Topics: Amyloid; Amyloidosis; Animals; Cell Line, Tumor; Doxycycline; Humans; Mice, 129 Strain; Mice, Knockout; Mutation, Missense; Protein Aggregates; Protein Aggregation, Pathological; Single-Domain Antibodies; Tissue Distribution; beta 2-Microglobulin
PubMed: 28429761
DOI: 10.1038/srep46711 -
Proceedings of the National Academy of... Mar 2006Identifying sequence determinants of fibril-forming proteins is crucial for understanding the processes causing >20 proteins to form pathological amyloid depositions....
Identifying sequence determinants of fibril-forming proteins is crucial for understanding the processes causing >20 proteins to form pathological amyloid depositions. Our approach to identifying which sequences form amyloid-like fibrils is to screen the amyloid-forming proteins human insulin and beta(2)-microglobulin for segments that form fibrils. Our screen is of 60 sequentially overlapping peptides, 59 being six residues in length and 1 being five residues, covering every noncysteine-containing segment in these two proteins. Each peptide was characterized as amyloid-like or nonfibril-forming. Amyloid-like peptides formed fibrils visible in electron micrographs or needle-like microcrystals showing a cross-beta diffraction pattern. Eight of the 60 peptides (three from insulin and five from beta(2)-microglobulin) were identified as amyloid-like. The results of the screen were used to assess the computational method, and good agreement between prediction and experiments was found. This agreement suggests that the pair-of-sheets, zipper spine model on which the computational method is based is at least approximately correct for the structure of the fibrils and suggests the nature of the sequence signal for formation of amyloid-like fibrils.
Topics: Amino Acid Sequence; Amyloid; Benzothiazoles; Biophysical Phenomena; Biophysics; Congo Red; Crystallography, X-Ray; Humans; Insulin; Microscopy, Electron; Models, Molecular; Molecular Sequence Data; Oligopeptides; Peptide Fragments; Protein Structure, Quaternary; Protein Structure, Secondary; Thiazoles; beta 2-Microglobulin
PubMed: 16537488
DOI: 10.1073/pnas.0511298103 -
Kidney International Oct 1996The primary structure of beta 2-microglobulin (beta 2m), the major constituent protein of beta 2-microglobulin amyloidosis (A beta 2m) or dialysis-amyloidosis, was...
The primary structure of beta 2-microglobulin (beta 2m), the major constituent protein of beta 2-microglobulin amyloidosis (A beta 2m) or dialysis-amyloidosis, was initially shown to be identical to serum beta 2m, thereby strongly suggesting the polymerization of intact beta 2m in tissues. Recent biochemical data have been controversial, showing beta 2m acidic isoforms, fragmentation and amino acid sequence alteration of deposited beta 2m. The aim of this study was to reinvestigate beta 2m amyloid deposits for the presence of beta 2m fragments and/or amino acid sequence alteration. Four amyloid-laden tissues (3 femoral bone amyloid cysts and 1 heart tissue) from dialysis patients were used to isolate amyloidogenic beta 2m. Amyloid fibrils were isolated using the classic water extraction method, and purified in 6 M guanidine on a gel-filtration column. The protein was further purified on 17% SDS-PAGE gel, and transferred to a nitrocellulose membrane for immunostaining with antihuman beta 2m. beta 2m samples were microsequenced using the standard 03RPTH program on a 470A gas-phase sequencer, and HPLC was performed after digestion with trypsin. Two peaks were obtained with the gel filtration column, the second corresponding by molecular weight to beta 2m. SDS-PAGE analysis of this peak under reducing conditions, demonstrated one major band at 12,000 Da and a minor band at 25,000 Da (monomer and dimer), and no lower molecular weight bands were observed. The 12 kDa band was micro-sequenced and the amino acid sequence corresponded to that of normal beta 2m through the 40th residue. Amino acid sequence analysis showed no difference from normal beta 2m in any of the beta 2m proteins contained in the amyloid deposits isolated from the four studied tissues. Also, the HPLC profile of the four protein samples were strictly normal and identical to a commercial preparation of beta 2m. The present study demonstrates that beta 2m molecules polymerized in amyloid fibrils and deposits are intact and have a normal amino acid sequence, and produced by a specific and unique fibrillogenetic mechanism, which does not require proteolytic processing from the precursor protein to the amyloid fibrils.
Topics: Amino Acid Sequence; Amyloidosis; Blotting, Western; Chromatography, High Pressure Liquid; Dialysis; Electrophoresis, Polyacrylamide Gel; Female; Humans; Male; Middle Aged; Molecular Sequence Data; Sequence Homology, Amino Acid; beta 2-Microglobulin
PubMed: 8887286
DOI: 10.1038/ki.1996.436 -
Kidney International. Supplement Dec 1999The major clinical manifestations of dialysis-associated A beta 2M amyloidosis are chronic arthralgias, destructive arthropathy and the carpal tunnel syndrome. For... (Review)
Review
The major clinical manifestations of dialysis-associated A beta 2M amyloidosis are chronic arthralgias, destructive arthropathy and the carpal tunnel syndrome. For dialysis patients who have been maintained on renal replacement therapy for more than 10-15 years, this complication may become a major physical handicap. It may even be life-threatening in some instances due to cervical cord compression. Amyloid deposits in joint areas precede clinical symptoms and signs by several years. Systemic deposits may also occur but their clinical manifestations are infrequent. The diagnosis of dialysis arthropathy associated with beta 2-microglobulin-associated (A beta 2M) amyloidosis mostly relies on indirect clinical and radiological evidence. Histologic proof is rarely obtained in vivo. The pathogenesis of the disease is complex. It includes reduced elimination of beta 2M and potentially also as impaired degradation of A beta 2M as well as enhanced production of A beta 2M amyloid fibrils. Non enzymatic modifications of beta 2M probably play a role, including beta 2M protein modification with advanced glycation end-products (AGE) and advanced oxidation protein products. Modified beta 2M, collagen and proteoglycans appear actively involved in the induction of a local inflammatory response and beta 2M amyloid formation. There is also evidence in favor of treatment-related factors such as the type of hemodialysis membrane and the purity of dialysis water. Hopefully, the translation of our improving knowledge of all the factors involved will lead to a better treatment and eventually to the prevention of this dramatic complication of dialysis.
Topics: Amyloidosis; Humans; Kidney Failure, Chronic; Renal Dialysis; beta 2-Microglobulin
PubMed: 10633472
DOI: 10.1046/j.1523-1755.1999.07315.x -
The Journal of Biological Chemistry Jun 2017In dialysis patients, β-2 microglobulin (β2m) can aggregate and eventually form amyloid fibrils in a condition known as dialysis-related amyloidosis, which...
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 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 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 .
Topics: Amyloid; Amyloidosis; Copper; Doxycycline; Humans; Protein Aggregates; Renal Dialysis; Rifampin; beta 2-Microglobulin
PubMed: 28468825
DOI: 10.1074/jbc.M116.774083 -
American Journal of Physiology. Lung... May 2017β-Microglobulin (βM), the light chain of the major histocompatibility complex class I (MHC I), has been identified as a proaging factor and is involved in the...
β-Microglobulin (βM), the light chain of the major histocompatibility complex class I (MHC I), has been identified as a proaging factor and is involved in the pathogenesis of neurodegenerative disorders by driving cognitive and regenerative impairments. However, little attention has focused on the effect of βM in the development of lung emphysema. Here, we found that concentrations of βM in plasma were significantly elevated in patients with lung emphysema than those in normal control subjects (1.89 ± 0.12 vs. 1.42 ± 0.06 mg/l, < 0.01). Moreover, the expression of βM was significantly higher in lung tissue of emphysema (39.90 ± 1.97 vs. 23.94 ± 2.11%, < 0.01). Immunofluorescence showed that βM was mainly expressed in prosurfactant protein C-positive (pro-SPC) alveolar epithelial cells and CD14 macrophages. Exposure to recombinant human βM and cigarette smoke extract (CSE) in vitro enhanced cellular senescence and inhibited proliferation of A549 cells, which was partially reversed by the presence of anti-βM antibody. However, anti-βM antibody did not attenuate the elevated production of IL-1β, IL-6, and TNF-α in A549 cells that were exposed to CSE. Immunofluorescence showed that colocalization of βM, and the hemochromatosis gene (HFE) protein was observed on A549 cells. These data suggest βM might participate in the development of lung emphysema through induction of lung epithelial cell senescence and inhibition.
Topics: A549 Cells; Antibodies; Cell Proliferation; Cellular Senescence; Demography; Epithelial Cells; Female; Humans; Lung; Male; Middle Aged; Phenotype; Pulmonary Emphysema; Smoking; beta 2-Microglobulin
PubMed: 28213472
DOI: 10.1152/ajplung.00516.2016 -
Biomolecules Sep 2019β-Microglobulin (βm) is the causative protein of dialysis-related amyloidosis, and its D76N variant is less stable and more prone to aggregation. Since their crystal...
Loosening of Side-Chain Packing Associated with Perturbations in Peripheral Dynamics Induced by the D76N Mutation of β-Microglobulin Revealed by Pressure-NMR and Molecular Dynamic Simulations.
β-Microglobulin (βm) is the causative protein of dialysis-related amyloidosis, and its D76N variant is less stable and more prone to aggregation. Since their crystal structures are indistinguishable from each other, enhanced amyloidogenicity induced by the mutation may be attributed to changes in the structural dynamics of the molecule. We examined pressure and mutation effects on the βm molecule by NMR and MD simulations, and found that the mutation induced the loosening of the inter-sheet packing of βm, which is relevant to destabilization and subsequent amyloidogenicity. On the other hand, this loosening was coupled with perturbed dynamics at some peripheral regions. The key result for this conclusion was that both the mutation and pressure induced similar reductions in the mobility of these residues, suggesting that there is a common mechanism underlying the suppression of inherent fluctuations in the βm molecule. Analyses of data obtained under high pressure conditions suggested that the network of dynamically correlated residues included not only the mutation site, but also distal residues, such as those of the C- and D-strands. Reductions in these local dynamics correlated with the loosening of inter-sheet packing.
Topics: Amino Acid Substitution; Crystallography, X-Ray; Humans; Models, Molecular; Molecular Dynamics Simulation; Nuclear Magnetic Resonance, Biomolecular; Pressure; Protein Aggregates; Protein Conformation, beta-Strand; Protein Folding; beta 2-Microglobulin
PubMed: 31527472
DOI: 10.3390/biom9090491 -
Human Immunology Dec 1996The light chain of HLA class I protein (beta 2m) has been expressed in Aspergillus nidulans. The cDNA of beta 2m was modified using the polymerase chain reaction to...
The light chain of HLA class I protein (beta 2m) has been expressed in Aspergillus nidulans. The cDNA of beta 2m was modified using the polymerase chain reaction to include overlapping extensions for its subsequent fusion into an Aspergillus vector. This fusion resulted in beta 2m cDNA being flanked by the Aspergillus awamori glucoamylase promoter and the Aspergillus niger glucoamylase terminator. Expression of beta 2m was induced by the addition of starch to the culture medium. In preliminary mass culture trials, 177 micrograms/liter of f beta 2m were obtained in 60-liter fermentations. N-terminal sequencing of purified human beta 2m produced in fungi (f beta 2m) revealed that 28% of the purified protein was of proper sequence and 61% of the protein had an additional serine and lysine residue derived from the C-terminus of the fungal leader. Purified f beta 2m from culture supernatants appeared biochemically similar to beta 2m obtained from human urine (u beta 2m) as seen in immunoblot analysis. Functionally, f beta 2m effectively interacted as a subunit of class I MHC molecules. This was seen both in a sandwich ELISA for detecting properly folded HLA class I heavy chain and in assays showing cell-surface beta 2m exchange into the mouse class I MHC H-2Kd. In these experiments the biological activity of f beta 2m was indistinguishable from u beta 2m. The successful expression of biologically active beta 2m in A. nidulans suggests that fungal systems might be useful for the production of other active components of the HLA class I MHC complex.
Topics: Aspergillus nidulans; Biological Transport; Enzyme-Linked Immunosorbent Assay; Genetic Vectors; Humans; Recombinant Fusion Proteins; beta 2-Microglobulin
PubMed: 8960907
DOI: 10.1016/s0198-8859(96)00224-8 -
Nature Communications Oct 2018All amyloid fibrils contain a cross-β fold. How this structure differs in fibrils formed from proteins associated with different diseases remains unclear. Here, we...
All amyloid fibrils contain a cross-β fold. How this structure differs in fibrils formed from proteins associated with different diseases remains unclear. Here, we combine cryo-EM and MAS-NMR to determine the structure of an amyloid fibril formed in vitro from β-microglobulin (βm), the culprit protein of dialysis-related amyloidosis. The fibril is composed of two identical protofilaments assembled from subunits that do not share βm's native tertiary fold, but are formed from similar β-strands. The fibrils share motifs with other amyloid fibrils, but also contain unique features including π-stacking interactions perpendicular to the fibril axis and an intramolecular disulfide that stabilises the subunit fold. We also describe a structural model for a second fibril morphology and show that it is built from the same subunit fold. The results provide insights into the mechanisms of fibril formation and the commonalities and differences within the amyloid fold in different protein sequences.
Topics: Amyloid; Amyloidosis; Cryoelectron Microscopy; Humans; Magnetic Resonance Spectroscopy; Protein Folding; Protein Structure, Tertiary; Renal Dialysis; beta 2-Microglobulin
PubMed: 30375379
DOI: 10.1038/s41467-018-06761-6