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International Journal of Molecular... Oct 2021Aggregation of β microglobulin (βm) into amyloid fibrils is associated with systemic amyloidosis, caused by the deposition of amyloid fibrils containing the wild-type...
Aggregation of β microglobulin (βm) into amyloid fibrils is associated with systemic amyloidosis, caused by the deposition of amyloid fibrils containing the wild-type protein and its truncated variant, ΔN6 βm, in haemo-dialysed patients. A second form of familial systemic amyloidosis caused by the βm variant, D76N, results in amyloid deposits in the viscera, without renal dysfunction. Although the folding and misfolding mechanisms of β microglobulin have been widely studied in vitro and in vivo, we lack a comparable understanding of the molecular mechanisms underlying toxicity in a cellular and organismal environment. Here, we established transgenic lines expressing wild-type (WT) human βm, or the two highly amyloidogenic naturally occurring variants, D76N βm and ΔN6 βm, in the bodywall muscle. Nematodes expressing the D76N βm and ΔN6 βm variants exhibit increased age-dependent and cell nonautonomous proteotoxicity associated with reduced motility, delayed development and shortened lifespan. Both βm variants cause widespread endogenous protein aggregation contributing to the increased toxicity in aged animals. We show that expression of βm reduces the capacity of to cope with heat and endoplasmic reticulum (ER) stress, correlating with a deficiency to upregulate BiP/ transcripts in response to ER stress in young adult animals. Interestingly, protein secretion in all βm variants is reduced, despite the presence of the natural signal sequence, suggesting a possible link between organismal βm toxicity and a disrupted ER secretory metabolism.
Topics: Animals; Caenorhabditis elegans; Endoplasmic Reticulum Stress; Heat-Shock Response; Humans; Longevity; Mutation; Protein Aggregates; Unfolded Protein Response; beta 2-Microglobulin
PubMed: 34639093
DOI: 10.3390/ijms221910752 -
Archives of Disease in Childhood Jan 1994Familial Mediterranean fever is characterised by recurrent and self limited attacks of fever and polyserositis and its devastating complication is the development of... (Comparative Study)
Comparative Study
Familial Mediterranean fever is characterised by recurrent and self limited attacks of fever and polyserositis and its devastating complication is the development of renal amyloidosis. In order to detect the presence of early glomerular and tubular damage in patients with familial Mediterranean fever and to assess the possible role of beta 2-microglobulin in the inflammatory attacks of this disease, serum and urine beta 2-microglobulin concentrations and microalbuminuria were evaluated in these patients. A total of 20 patients with familial Mediterranean fever were studied on and off colchicine treatment; seven of these patients developed a familial Mediterranean fever attack when they were off treatment. During the familial Mediterranean fever attacks serum beta 2-microglobulin concentrations decreased, whereas fractional excretion of beta 2-microglobulin, urine beta 2-microglobulin creatinine, and urine albumin/creatinine ratios increased. We conclude that glomerular and tubular functions deteriorate during the attacks. Further studies are needed to discover the effector(s) causing these transient glomerular and tubular disorders.
Topics: Adolescent; Albuminuria; Child; Colchicine; Creatinine; Familial Mediterranean Fever; Female; Humans; Male; beta 2-Microglobulin
PubMed: 8110003
DOI: 10.1136/adc.70.1.27 -
Kidney International Nov 1995beta 2 microglobulin (beta 2m) is classically known to have isoforms with isoelectric points (pI) 5.7 and 5.3. New isoforms of beta 2m with lower pI, probably due to...
beta 2 microglobulin (beta 2m) is classically known to have isoforms with isoelectric points (pI) 5.7 and 5.3. New isoforms of beta 2m with lower pI, probably due to modifications with advanced glycation end products, were found in the amyloid deposits of dialysis related amyloidosis (DRA), and they were proposed as the amyloidogenic forms of beta 2m. The other modifications in beta 2m from amyloid deposits are partial proteolysis and single amino acid replacement (Asn by ASp at position 17). However, there are no data on the sequence of the different isoforms of beta 2 m from amyloid deposits. Amyloid deposits surgically obtained from the carpal tunnel from 13 dialysis treated patients and urine from 10 healthy volunteers and 5 living-related kidney donors were analyzed for beta 2m content. Two-dimensional gel electrophoresis (2D-PAGE) of beta 2m from amyloid deposits showed the presence of four or more isoforms with pIs < 5.7. All the spots migrating at 12 kDa Mr region and between 4 and 6 pH reacted with rabbit anti-human beta 2m antibody by Western blotting, confirming that they were beta 2m isoforms. beta 2m isoforms from the amyloid deposits were then separately purified with an IEF column (PB94, Pharmacia) for analysis. Enough quantities of three pure beta 2m isoforms could be obtained in two cases. The sequence analysis showed an intact N-terminus in all the isoforms. There was Asn in the 17th residue in all the isoforms sequenced. 2D-PAGE of urine from 8 out of the 10 healthy volunteers showed the presence of beta 2m. In two of them beta 2m also displayed four different isoforms. At least four isoforms were observed in urine of all the kidney donors. The present study shows that the elution peaks of three different beta 2m isoforms in gel isoelectrofocusing contain beta 2m with intact N-terminus. None of them have deamidated their 17th residue. More importantly, the beta 2m isoforms with lower pI are not specific for amyloidosis as they were found in urine from kidney donors and in normal volunteers. These results bring into question the hypothesis that dialysis related amyloidosis is due to the known modifications on beta 2m. They suggest that the precipitation of beta 2m into amyloid fibrils should result from the interaction of beta 2m with other factors with amyloid enhancing activity.
Topics: Adult; Amino Acid Sequence; Amyloid; Animals; Female; Humans; Isomerism; Male; Middle Aged; Molecular Sequence Data; Rabbits; Reference Values; beta 2-Microglobulin
PubMed: 8544395
DOI: 10.1038/ki.1995.428 -
International Journal of Molecular... Sep 2018The 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...
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. 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.
Topics: Amyloid; Amyloidosis; Benzothiazoles; Circular Dichroism; Fluorescent Dyes; Humans; Kinetics; Mass Spectrometry; Molecular Imaging; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Spectrophotometry, Ultraviolet; beta 2-Microglobulin
PubMed: 30223436
DOI: 10.3390/ijms19092762 -
The Journal of Biological Chemistry Jun 2019Amyloid deposition of WT human β-microglobulin (WT-hβm) in the joints of long-term hemodialysis patients is the hallmark of dialysis-related amyloidosis. , WT-hβm...
Amyloid deposition of WT human β-microglobulin (WT-hβm) in the joints of long-term hemodialysis patients is the hallmark of dialysis-related amyloidosis. , WT-hβm 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-hβm 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-hβm 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-hβm assembly. Our results revealed that LMW-heparin strongly promotes WT-hβm fibrillogenesis during all stages of aggregation. However, collagen I affected WT-hβm 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-hβm amyloid fibrils attenuates surface-mediated growth of WT-hβm fibrils, demonstrating a key role of secondary nucleation in WT-hβm amyloid formation. Interestingly, collagen I fibrils did not suppress surface-mediated assembly of WT-hβm monomers when cross-seeded with fibrils formed from the N-terminally truncated variant ΔN6-hβm. Together, these results provide detailed insights into how collagen I and LMW-heparin impact different stages in the aggregation of WT-hβm into amyloid, which lead to dramatic effects on the time course of assembly.
Topics: Amyloid; Amyloidosis; Anticoagulants; Collagen Type I; Extracellular Matrix; Heparin, Low-Molecular-Weight; Humans; Mutation; beta 2-Microglobulin
PubMed: 30996004
DOI: 10.1074/jbc.RA119.008300 -
The FEBS Journal Feb 2020The molecular bases of amyloid aggregation propensity are still poorly understood, especially for proteins that display a stable folded native structure. A prototypic...
The molecular bases of amyloid aggregation propensity are still poorly understood, especially for proteins that display a stable folded native structure. A prototypic example is human beta-2 microglobulin (β2m), which, when accumulated in patients, gives rise to dialysis-related amyloidosis. Interestingly, although the physiologic concentration of β2m in mice is five times higher than that found in human patients, no amyloid deposits are observed in mice. Moreover, murine β2m (mβ2m) not only displays a lower amyloid propensity both in vivo and in vitro but also inhibits the aggregation of human β2m in vitro. Here, we compared human and mβ2m for their aggregation propensity, ability to form soluble oligomers, stability, three-dimensional structure and dynamics. Our results indicate that mβ2m low-aggregation propensity is due to two concomitant aspects: the low-aggregation propensity of its primary sequence combined with the absence of high-energy amyloid-competent conformations under native conditions. The identification of the specific properties determining the low-aggregation propensity of mouse β2m will help delineate the molecular risk factors which cause a folded protein to aggregate.
Topics: Amyloid; Animals; Humans; Mice; Molecular Dynamics Simulation; Protein Folding; Protein Multimerization; Protein Stability; beta 2-Microglobulin
PubMed: 31420997
DOI: 10.1111/febs.15046 -
Cellular Immunology Sep 2012Cellular immunity is dependent on major histocompatibility complex (MHC) class I molecules enabling cytotoxic T cell recognition of malignant and infected cells. Loading...
Cellular immunity is dependent on major histocompatibility complex (MHC) class I molecules enabling cytotoxic T cell recognition of malignant and infected cells. Loading of antigenic peptides onto MHC class I is assisted by a peptide-loading protein complex including tapasin. We found that tapasin expression is enhanced by beta 2-microglobulin via both transcriptional and post-transcriptional mechanisms. In addition, using conditions which preserve the tapasin-ERp57 disulfide-bonded conjugate, we demonstrated that beta 2-microglobulin increases tapasin-containing protein complexes, and reduces the level of MHC class I/ERp57 complexes lacking tapasin. Overall, our results provide a new perspective on the regulation of tapasin expression and association.
Topics: Animals; Blotting, Western; Cell Line, Tumor; Disulfides; Gene Expression; Histocompatibility Antigens Class I; Humans; Membrane Transport Proteins; Mice; Protein Binding; Protein Disulfide-Isomerases; Reverse Transcriptase Polymerase Chain Reaction; beta 2-Microglobulin
PubMed: 23089196
DOI: 10.1016/j.cellimm.2012.09.010 -
Kidney International Oct 1994Enhanced extracorporeal removal of beta 2-microglobulin (beta 2m) may prevent the development of dialysis-related amyloidosis (DRA). One mechanism of beta 2m removal is...
Enhanced extracorporeal removal of beta 2-microglobulin (beta 2m) may prevent the development of dialysis-related amyloidosis (DRA). One mechanism of beta 2m removal is membrane adsorption. Therefore, we fundamentally characterized beta 2m adsorption to the highly permeable polyacrylonitrile (PAN) membrane. Porous and nonporous PAN fragments were incubated in buffer containing 125I-beta 2m. Over a concentration range of 8 to 60 mg/liter, the equilibrium adsorption isotherm was linear (r = 0.99) for porous PAN while the isotherm for nonporous PAN suggested either multilayer binding or adsorption of proteins with differing orientations. In kinetic analyses, the approach to equilibrium versus (time)1/2 was evaluated. For both porous and nonporous PAN, this relationship was linear (r = 0.99), consistent with a diffusion-controlled process. Adsorption reversibility was assessed by comparing the amount bound at varying residence times (0 to 4 hr) to the amount remaining adsorbed after a subsequent incubation in buffer. The fractions remaining bound at 60, 120, and 240 minutes (0.34 +/- 0.02, 0.36 +/- 0.06, and 0.44 +/- 0.03; mean +/- SEM) were significantly greater (P < 0.05) than the value at five minutes (0.23 +/- 0.01). This suggests membrane-induced conformational changes in adsorbed beta 2m. This investigation permits the comparison of beta 2m adsorptive properties of PAN to those of other membrane-based and nonmembrane-based therapies designed to prevent DRA.
Topics: Acrylic Resins; Adsorption; Amyloidosis; Humans; In Vitro Techniques; Kidneys, Artificial; Kinetics; Membranes, Artificial; Renal Dialysis; beta 2-Microglobulin
PubMed: 7861709
DOI: 10.1038/ki.1994.377 -
Biochemistry Feb 2017β-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...
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.
Topics: Amyloid; Copper; Models, Molecular; Protein Conformation, beta-Strand; Protein Multimerization; Zinc; beta 2-Microglobulin
PubMed: 28168880
DOI: 10.1021/acs.biochem.6b01198 -
Proceedings of the National Academy of... Apr 1983beta 2-Microglobulin (beta 2m) is expressed on the cell surface after introduction of a beta 2mb (C57BL/6N) genomic clone into thymidine kinase-deficient mouse L cells...
beta 2-Microglobulin (beta 2m) is expressed on the cell surface after introduction of a beta 2mb (C57BL/6N) genomic clone into thymidine kinase-deficient mouse L cells by cotransformation using the calcium phosphate precipitate method. Stable transformant cell lines were identified that express the beta 2mb allele, as determined by reaction of the cells with appropriate monoclonal antibodies and by two-dimensional gel electrophoresis of endogenously labeled immunoprecipitates of cell extracts. These beta 2mb transformants now express ly-m11.2, as detected by an indirect radioimmunoassay. A plasmid subclone of the beta 2mb gene that contains an 8.4-kilobase insert, after introduction into mouse L cells, similarly directs the synthesis of both the beta 2mb and the ly-m11.2 antigens. Thus, the beta 2mb and ly-m11.2 determinants most likely represent sites on the same protein structure.
Topics: Animals; Antibodies, Monoclonal; Antigens, Surface; Beta-Globulins; Cloning, Molecular; Epitopes; Genes; Genetic Linkage; Mice; Transformation, Genetic; beta 2-Microglobulin
PubMed: 6188162
DOI: 10.1073/pnas.80.8.2328