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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 -
International Journal of Nephrology and... 2017Although advancements have been made in the management of thalassemic patients, many unrecognized complications have emerged, such as renal abnormalities.
BACKGROUND
Although advancements have been made in the management of thalassemic patients, many unrecognized complications have emerged, such as renal abnormalities.
AIM
To measure serum levels of cystatin-C and β-2 microglobulin in children with beta-thalassemia major (β-TM) and investigate their significance as early markers of glomerular and tubular dysfunctions.
SUBJECTS AND METHODS
The study was performed on 70 children with (β-TM) and 20 apparently healthy children matched for age and sex as a control group. For all the enrolled children, a comprehensive medical history was obtained and complete physical examination was performed, blood urea, serum creatinine, serum ferritin, estimated glomerular filtration rate (eGFR) by Schwartz formula and creatinine clearance, albumin/creatinine ratio in urine, serum cystatin-C levels and β-2 microglobulin were measured.
RESULTS
Thalassemic children had significantly higher cystatin-C and β-2 microglobulin levels compared with control. In addition, serum cystatin-C and β-2 microglobulin were positively correlated with urea, creatinine, serum ferritin, albumin/creatinine ratio, duration of chelation therapy and frequency of blood transfusion/year and negatively correlated with creatinine clearance, hemoglobin, and eGFR. Our data demonstrated that cystatin-C and β-2 microglobulin had higher sensitivity and specificity (91.4%, 90.0%, and 85.7%, 100%, respectively) than serum creatinine and creatinine clearance (83.0%, 100% and 81.4%, 100%, respectively) for small changes in GFR.
CONCLUSION
Cystatin-C and β-2 microglobulin are specific and sensitive early biomarkers for monitoring glomerular and tubular dysfunction in children with β-TM.
PubMed: 28979155
DOI: 10.2147/IJNRD.S142824 -
Scientific Reports Sep 2015β-2-microglobulin (β2m) self-aggregates to form amyloid fibril in renal patients taking long-term dialysis treatment. Despite the extensive structural and mutation...
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.
Topics: Amyloidogenic Proteins; Humans; Hydrophobic and Hydrophilic Interactions; Molecular Dynamics Simulation; Mutation; Protein Conformation; Protein Stability; Solvents; Thermodynamics; beta 2-Microglobulin
PubMed: 26348154
DOI: 10.1038/srep13631 -
Protein Science : a Publication of the... May 2017Amyloid fibrils are fibrillar deposits of denatured proteins associated with amyloidosis and are formed by a nucleation and growth mechanism. We revisited an alternative...
Amyloid fibrils are fibrillar deposits of denatured proteins associated with amyloidosis and are formed by a nucleation and growth mechanism. We revisited an alternative and classical view of amyloid fibrillation: amyloid fibrils are crystal-like precipitates of denatured proteins formed above solubility upon breaking supersaturation. Various additives accelerate and then inhibit amyloid fibrillation in a concentration-dependent manner, suggesting that the combined effects of stabilizing and destabilizing forces affect fibrillation. Heparin, a glycosaminoglycan and anticoagulant, is an accelerator of fibrillation for various amyloidogenic proteins. By using β -microglobulin, a protein responsible for dialysis-related amyloidosis, we herein examined the effects of various concentrations of heparin on fibrillation at pH 2. In contrast to previous studies that focused on accelerating effects, higher concentrations of heparin inhibited fibrillation, and this was accompanied by amorphous aggregation. The two-step effects of acceleration and inhibition were similar to those observed for various salts. The results indicate that the anion effects caused by sulfate groups are one of the dominant factors influencing heparin-dependent fibrillation, although the exact structures of fibrils and amorphous aggregates might differ between those formed by simple salts and matrix-forming heparin. We propose that a conformational phase diagram, accommodating crystal-like amyloid fibrils and glass-like amorphous aggregates, is important for understanding the effects of various additives.
Topics: Amyloid; Amyloidosis; Heparin; Hydrogen-Ion Concentration; Models, Chemical; Protein Structure, Quaternary; Renal Dialysis; beta 2-Microglobulin
PubMed: 28249361
DOI: 10.1002/pro.3149 -
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 -
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 -
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 -
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 -
Proceedings of the National Academy of... Nov 1979Human histocompatibility antigens HLA-A, HLA-B, and HLA-C are a complex of two noncovalently associated subunits: a heavy chain glycoprotein (alpha) carrying the genetic...
Human histocompatibility antigens HLA-A, HLA-B, and HLA-C are a complex of two noncovalently associated subunits: a heavy chain glycoprotein (alpha) carrying the genetic polymorphism and an invariant light chain, beta 2-microglobulin (beta 2m). Upon incubation of papain-solubilized HLA with radiolabeled urinary beta 2m, the latter is incorporated into HLA, where it substitutes for the preexisting beta 2m that has dissociated from the complex. The association-dissociation equilibrium that governs this beta 2m exchange reaction was investigated and found to be characterized by a long lifetime of the complex (half-life of 80 min at 37 degrees C) and a relatively low Kd (4 nM). The beta 2m exchange was used as the basis of a radioimmunoassay for HLA antigens with radiolabeled beta 2m as a unique label for all HLA specificities. In a similar fashion, radiolabeled beta 2m can be incorporated into HLA at the cell surface. Although the process is slower and less extensive than in solution, it can be used as a means to tag cells with specific probes for HLA antigens.
Topics: Beta-Globulins; Cell Line; Cell Membrane; HLA Antigens; Humans; Kinetics; Macromolecular Substances; Membrane Proteins; Protein Binding; Radioimmunoassay; Thermodynamics; beta 2-Microglobulin
PubMed: 93282
DOI: 10.1073/pnas.76.11.5834 -
Blood Oct 1999Major histocompatibility complex (MHC) molecules play an important role in antigen presentation for induction of tumor as well as cellular and humoral immunities. Recent... (Comparative Study)
Comparative Study
Major histocompatibility complex (MHC) molecules play an important role in antigen presentation for induction of tumor as well as cellular and humoral immunities. Recent studies using anti-MHC antibodies demonstrated that antibodies specific for HLA class I molecules induced cellular activation and a type of apoptosis that may be distinct from Fas-dependent or TNFR (tumor necrosis factor-alpha receptor)-dependent processes. We purified a previously untested apoptosis-inducing factor from HL-60 human leukemic cell-conditioned media to homogeneity and sequenced it. It was identified as beta(2)-microglobulin (beta(2)m), which has been previously known as thymotaxin and is a part of the HLA class I antigen complex. beta(2)m acts on both T-leukemic cells and myeloid leukemic cells to induce apoptosis, which then activates caspase 1 and 3. Cross-linking studies showed that biotinilated beta(2)m recognized an epitope distinct from those recognized by the anti-HLA class I antibody, as reported previously. We demonstrated that beta(2)m plays a previously unrecognized and important role in regulating the elimination of tumor cells, which occurs as a result of the action of beta(2)m as an apoptosis-inducing factor.
Topics: Amino Acid Chloromethyl Ketones; Amino Acid Sequence; Animals; Antibodies, Monoclonal; Apoptosis; Culture Media, Conditioned; Cysteine Proteinase Inhibitors; Fas Ligand Protein; HL-60 Cells; Humans; K562 Cells; Membrane Glycoproteins; Mice; Molecular Sequence Data; Neoplasm Proteins; Oligopeptides; Receptors, Tumor Necrosis Factor; Sequence Alignment; Sequence Homology, Amino Acid; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; U937 Cells; beta 2-Microglobulin; fas Receptor
PubMed: 10515878
DOI: No ID Found