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Scientific Reports Dec 2019The availability of a genetic model organism with which to study key molecular events underlying amyloidogenesis is crucial for elucidating the mechanism of the disease...
The availability of a genetic model organism with which to study key molecular events underlying amyloidogenesis is crucial for elucidating the mechanism of the disease and the exploration of new therapeutic avenues. The natural human variant of β-microglobulin (D76N β-m) is associated with a fatal familial form of systemic amyloidosis. Hitherto, no animal model has been available for studying in vivo the pathogenicity of this protein. We have established a transgenic C. elegans line, expressing the human D76N β-m variant. Using the INVertebrate Automated Phenotyping Platform (INVAPP) and the algorithm Paragon, we were able to detect growth and motility impairment in D76N β-m expressing worms. We also demonstrated the specificity of the β-m variant in determining the pathological phenotype by rescuing the wild type phenotype when β-m expression was inhibited by RNA interference (RNAi). Using this model, we have confirmed the efficacy of doxycycline, an inhibitor of the aggregation of amyloidogenic proteins, in rescuing the phenotype. In future, this C. elegans model, in conjunction with the INVAPP/Paragon system, offers the prospect of high-throughput chemical screening in the search for new drug candidates.
Topics: Amyloid; Amyloidosis; Animals; Animals, Genetically Modified; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Disease Models, Animal; Drug Evaluation, Preclinical; Mutation, Missense; Phenotype; Protein Aggregation, Pathological; Protein Folding; beta 2-Microglobulin
PubMed: 31882874
DOI: 10.1038/s41598-019-56498-5 -
The Journal of Experimental Medicine Jul 1976We have used a rabbit antiserum prepared against purified rat beta2-microglobulin to immunoprecipitate molecules from lysates of radioiodinated murine thymocytes and...
We have used a rabbit antiserum prepared against purified rat beta2-microglobulin to immunoprecipitate molecules from lysates of radioiodinated murine thymocytes and splenocytes. All the molecules that are reactive with this serum have subunits of 44,000 and 12,000 and can be identified as H-2 and TL antigens. Thus, the anti-beta2mu serum can deplete lysates of the majority of the TL and H-2 atigens which can be subsequently recognized by alloantisera. If TL and H-2 are precipitated from the lysates before the addition of anti-beta2mu, no beta2mu-reactive molecules remain. Our results indicate that Ia antigens cannot be depleted from the lysates with anti-beta2mu. The studies also suggest that TL and H-2 heavy chains can exist as both monomers and dimers. These observations are discussed with regard to previous studies concerning the native structure of H-2 and TL antigens.
Topics: Animals; Antigens, Neoplasm; Beta-Globulins; Histocompatibility Antigens; Isoantigens; Leukemia; Mice; Mice, Inbred A; Molecular Weight; Protein Conformation; Spleen; Surface Properties; Thymus Gland; beta 2-Microglobulin
PubMed: 58956
DOI: 10.1084/jem.144.1.179 -
Proceedings of the National Academy of... May 1994The technique of single-strand conformation polymorphism (SSCP) was used to screen a series of 37 established colorectal cell lines, 22 fresh tumor samples, and 22...
The technique of single-strand conformation polymorphism (SSCP) was used to screen a series of 37 established colorectal cell lines, 22 fresh tumor samples, and 22 normal DNA samples for mutations in the beta 2-microglobulin gene. Exon 1 (including the leader peptide sequence) and exon 2 were screened separately. Six of 37 colorectal cell lines and 1 of 22 fresh tumors were shown to contain mutations, whereas no mutations were detected in the normal DNA samples. Sequencing of these mutations showed that an 8-bp CT repeat in the leader peptide sequence was particularly variable, since 3 of the cell lines and one fresh tumor sample have deletions in this region. In the related cell lines, DLD-1 and HCT-15, two similar mutations were identified, a C-->A substitution in codon 10 and a G-->T mutation in the splice sequence of intron 1. Expression of beta 2-microglobulin was examined using a series of monoclonal antibodies in an ELISA system. Reduced expression correlated with a mutation in one allele of beta 2-microglobulin, whereas loss of expression was seen in instances where a line was homozygous for a mutation or heterozygous for two mutations.
Topics: Amino Acid Sequence; Base Sequence; Colorectal Neoplasms; DNA, Neoplasm; Exons; Humans; Molecular Sequence Data; Mutation; Polymerase Chain Reaction; Polymorphism, Genetic; Tumor Cells, Cultured; beta 2-Microglobulin
PubMed: 8197130
DOI: 10.1073/pnas.91.11.4751 -
Clinical and Experimental Immunology Nov 1991Soluble CD8, soluble CD4, soluble CD25 (IL-2 receptor), beta 2-microglobulin and the cytokine tumour necrosis factor-alpha (TNF-alpha) were measured in sera from...
Soluble CD8, soluble CD4, soluble CD25 (IL-2 receptor), beta 2-microglobulin and the cytokine tumour necrosis factor-alpha (TNF-alpha) were measured in sera from patients with common variable immunodeficiency (CVI). Levels of soluble CD8, soluble CD25 and beta 2-microglobulin but not of soluble CD4 and TNF-alpha were raised significantly above levels in normal sera. Sera from patients with X-linked agammaglobulinaemia, who are also antibody deficient, did not show this marked elevation. The raised levels of soluble CD8, soluble CD25 and beta 2-microglobulin in CVI, correlated with the extent of the defects in the B lymphocytes assessed in vitro, as well as with the clinical severity of the disease. The selective release of these molecules into sera may indicate that abnormal cellular activation occurs in most CVI patients. It is also possible that the raised levels of these soluble molecules play a part in the immunodeficiency.
Topics: Acquired Immunodeficiency Syndrome; CD8 Antigens; Humans; Lymphocyte Activation; Receptors, Interleukin-2; Solubility; Tumor Necrosis Factor-alpha; beta 2-Microglobulin
PubMed: 1934593
DOI: 10.1111/j.1365-2249.1991.tb05805.x -
Biochemistry Oct 2009Beta-2-microglobulin (beta2m) deposits as amyloid fibrils in the musculoskeletal system of patients undergoing long-term dialysis treatment as a result of kidney...
Beta-2-microglobulin (beta2m) deposits as amyloid fibrils in the musculoskeletal system of patients undergoing long-term dialysis treatment as a result of kidney failure. Previous work has shown that Cu(II) binding causes beta2m to organize into nativelike dimers and tetramers that precede amyloid formation. Cu(II) is then released from higher-order oligomers before mature Cu(II)-free amyloid fibrils are formed. While some of the Cu(II)-induced structural changes that enable beta2m self-assembly are starting to be revealed, the details of how the Cu(II) binding site evolves from the monomer to the dimers and tetramers are not known. Here, we report results from three mass spectrometry (MS)-based methods that provide insight into the changing Cu-beta2m interactions. We find that monomeric beta2m binds Cu(II) via the N-terminal amine, the amide of Gln2, His31, and Asp59. In the dimer and tetramer, Asp59 is no longer bound to Cu(II), but the other residues still comprise a well-defined albeit weaker binding site that is better able to release Cu(II). Consistent with this is the observation that a fraction of the tetrameric species no longer binds Cu(II) at this weakened binding site, which agrees with a previous report that suggested the tetramer as the first Cu(II)-free oligomer. Our results also provide some insight into structural changes caused by Cu(II) binding that facilitate oligomer formation. Specifically, binding by Asp59 in the monomer requires significant movement of this residue, and we propose that this repositioning is important for establishing a pair of dimer-stabilizing salt bridges between this residue and Lys19. We also find evidence that Cu(II) binding in the N-terminal region of the monomer repels Arg3, which likely allows this residue to form a pair of dimer-stabilizing salt bridges with Glu16. Overall, our measurements suggest that the previously proposed conformational switch caused by Cu(II) binding includes not only a cis-trans isomerization at Pro32 but also the repositioning of residues that are critical for the formation of new electrostatic interactions.
Topics: Binding Sites; Chromatography, Liquid; Copper; Dimerization; Humans; Isomerism; Kinetics; Mass Spectrometry; Models, Molecular; Musculoskeletal Diseases; Oxidation-Reduction; Protein Conformation; Renal Insufficiency; Static Electricity; beta 2-Microglobulin
PubMed: 19754160
DOI: 10.1021/bi901172y -
International Journal of Molecular... May 2021Proteolytic enzymes are known to be involved in the formation and degradation of various monomeric proteins, but the effect of proteases on the ordered protein...
Proteolytic enzymes are known to be involved in the formation and degradation of various monomeric proteins, but the effect of proteases on the ordered protein aggregates, amyloid fibrils, which are considered to be extremely stable, remains poorly understood. In this work we study resistance to proteolytic degradation of lysozyme amyloid fibrils with two different types of morphology and beta-2-microglobulun amyloids. We showed that the proteolytic enzyme of the pancreas, trypsin, induced degradation of amyloid fibrils, and the mechanism of this process was qualitatively the same for all investigated amyloids. At the same time, we found a dependence of efficiency and rate of fibril degradation on the structure of the amyloid-forming protein as well as on the morphology and clustering of amyloid fibrils. It was assumed that the discovered relationship between fibrils structure and the efficiency of their degradation by trypsin can become the basis of a new express method for the analysis of amyloids polymorphism. Unexpectedly lower resistance of both types of lysozyme amyloids to trypsin exposure compared to the native monomeric protein (which is not susceptible to hydrolysis) was attributed to the higher availability of cleavage sites in studied fibrils. Another intriguing result of the work is that the cytotoxicity of amyloids treated with trypsin was not only failing to decline, but even increasing in the case of beta-2-microglobulin fibrils.
Topics: Amyloid; Amyloid beta-Peptides; Anilino Naphthalenesulfonates; Benzothiazoles; Fluorescent Dyes; HeLa Cells; Humans; Hydrogen-Ion Concentration; Hydrolysis; Muramidase; Proteolysis; Trypsin; beta 2-Microglobulin
PubMed: 34063223
DOI: 10.3390/ijms22094828 -
The Journal of Veterinary Medical... Feb 2009Uterine natural killer (uNK) cells have roles for immune responses at the feto-maternal interface in mice. We studied the effects of beta(2)-microglobulin (beta(2)m) and...
Uterine natural killer (uNK) cells have roles for immune responses at the feto-maternal interface in mice. We studied the effects of beta(2)-microglobulin (beta(2)m) and perforin on proliferation and differentiation of uNK cells in pregnancy, using beta(2)-microglobulin-deficient (beta(2)m(-/-)) mice and perforin-deficient (P(-/-)) mice. The cell population of uNK cells in the metrial gland of P(-/-) mice was tended to be higher than the control B6 mice. The cell population of uNK cells in the metrial gland of beta(2)m(-/-) mice was significantly increased at Day 12 of pregnancy comparing to B6 and P(-/-) mice. On the other hand, the cell population of uNK cells in the decidua basalis of beta(2)m(-/-) mice was tended to be lower than B6 and P(-/-) mice. These results indicate that beta(2)m may be involved in proliferation of uNK cells in the metrial gland, and that beta(2)m may affect the maturation of uNK cells in the decidua basalis.
Topics: Animals; Cell Differentiation; Cytotoxins; Female; Killer Cells, Natural; Major Histocompatibility Complex; Metrial Gland; Mice; Mice, Inbred C57BL; Perforin; Pregnancy; Uterus; beta 2-Microglobulin
PubMed: 19262044
DOI: 10.1292/jvms.71.251 -
Biophysical Journal Apr 2012β-2 microglobulin (β2m) is an amyloidogenic protein involved in dialysis-related amyloidosis. We report here the study of the structural properties of the protein in...
β-2 microglobulin (β2m) is an amyloidogenic protein involved in dialysis-related amyloidosis. We report here the study of the structural properties of the protein in solution and in the form of single crystals by Fourier transform infrared (FTIR) spectroscopy and microspectroscopy. The investigation has been extended to four β2m mutants previously characterized by x-ray crystallography: Asp(53)Pro, Asp(59)Pro, Trp(60)Gly, and Trp(60)Val. These variants displayed very similar three-dimensional structures but different thermal stability and aggregation propensity, investigated here by FTIR spectroscopy. For each variant, appreciable spectral differences were found between the protein in solution and in single crystals, consisting in a downshift of the main β-sheet band and in better resolved turn and loop bands, indicative of reduced protein secondary structure dynamics in the crystalline state. Notably, the well-resolved spectra of the β2m crystalline variants enabled us to identify structural differences induced by the single amino acid mutations. Such differences encompass turn and loop structures that might affect the stability and aggregation propensity of the investigated β2m variants. This study highlights the potential of FTIR microspectroscopy to acquire useful structural information on protein crystals, complementary to the crystallographic analyses.
Topics: Crystallography, X-Ray; Kinetics; Models, Molecular; Mutant Proteins; Mutation; Protein Multimerization; Protein Stability; Protein Structure, Quaternary; Solutions; Spectroscopy, Fourier Transform Infrared; Temperature; beta 2-Microglobulin
PubMed: 22500768
DOI: 10.1016/j.bpj.2012.02.045 -
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 -
Nephrology, Dialysis, Transplantation :... Oct 2018Aggressive removal of middle molecules or larger low-molecular-weight proteins (LMWPs) has been a growing concern following studies on their harmful effects on the... (Review)
Review
Aggressive removal of middle molecules or larger low-molecular-weight proteins (LMWPs) has been a growing concern following studies on their harmful effects on the mortality and morbidity of chronic dialysis patients. To remove larger LMWPs and some protein-bound uremic toxins (PBUTs), high- and medium-cutoff (HCOs and MCOs, respectively) membranes, convective therapy and protein adsorptive membranes are available. When we use HCO or MCO membranes for convective therapy, we have to take care to avoid massive albumin leakage during a dialysis session. Convection volume is an important element to increase middle molecule removal; however, a larger convection volume has a risk of larger leakage of albumin. Predilution hemodiafiltration is a useful measurement to increase larger LMWPs without massive albumin leakage. β2-microglobulin (B2M), α1-microglobulin (A1M) and albumin leakage during a dialysis session are useful parameters for assessing middle-molecule removal. Reduction ratios of B2M >80% and of A1M >35% are favorable to improve severe dialysis-related symptoms. The efficacy of middle molecule removal should be evaluated in comparison with clinical outcomes, mortality, morbidity and the improvement of dialysis-related symptoms. Recently some dialysis-related symptoms such as sleep disturbance, skin itchiness and dialysis hypotension have been recognized as good surrogate makers for mortality. Further studies to evaluate the relationship between middle molecule or PBUTs removal and the improvement of patient symptoms should be performed in well-designed randomized controlled trials.
Topics: Albumins; Animals; Hemodiafiltration; Humans; Membranes, Artificial; Molecular Weight; Renal Dialysis; Toxins, Biological; beta 2-Microglobulin
PubMed: 30281129
DOI: 10.1093/ndt/gfy224