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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 25 January 2011, Vol.108(4), pp.1314-9
    Description: Atomic-level structural investigation of the key conformational intermediates of amyloidogenesis remains a challenge. Here we demonstrate the utility of nanobodies to trap and characterize intermediates of β2-microglobulin (β2m) amyloidogenesis by X-ray crystallography. For this purpose, we selected five single domain antibodies that block the fibrillogenesis of a proteolytic amyloidogenic fragment of β2m (ΔN6β2m). The crystal structure of ΔN6β2m in complex with one of these nanobodies (Nb24) identifies domain swapping as a plausible mechanism of self-association of this amyloidogenic protein. In the swapped dimer, two extended hinge loops--corresponding to the heptapetide NHVTLSQ that forms amyloid in isolation--are unmasked and fold into a new two-stranded antiparallel β-sheet. The β-strands of this sheet are prone to self-associate and stack perpendicular to the direction of the strands to build large intermolecular β-sheets that run parallel to the axis of growing oligomers, providing an elongation mechanism by self-templated growth.
    Keywords: Protein Multimerization ; Amyloid -- Chemistry ; Antibodies -- Immunology ; Beta 2-Microglobulin -- Chemistry
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    Language: English
    In: Journal of the American Chemical Society, 28 April 2010, Vol.132(16), pp.5556-7
    Description: Elucidating the fine structure of amyloid fibrils as well as understanding their processes of nucleation and growth remains a difficult yet essential challenge, directly linked to our current poor insight into protein misfolding and aggregation diseases. Here we consider beta-2-microglobulin (beta2m), the MHC-1 light chain component responsible for dialysis-related amyloidosis, which can give rise to amyloid fibrils in vitro under various experimental conditions, including low and neutral pH. We have used solid-state NMR to probe the structural features of fibrils formed by full-length beta2m (99 residues) at pH 2.5 and pH 7.4. A close comparison of 2D (13)C-(13)C and (15)N-(13)C correlation experiments performed on beta2m, in both the crystalline and fibrillar states, suggests that, in spite of structural changes affecting the protein loops linking the protein beta-strands, the protein chain retains a substantial share of its native secondary structure in the fibril assembly. Moreover, variations in the chemical shifts of the key Pro32 residue suggest the involvement of a cis-trans isomerization in the process of beta2m fibril formation. Lastly, the analogy of the spectra recorded on beta2m fibrils grown at different pH values hints at a conserved architecture of the amyloid species thus obtained.
    Keywords: Beta 2-Microglobulin -- Chemistry
    ISSN: 00027863
    E-ISSN: 1520-5126
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  • 3
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 28 January 2014, Vol.111(4), pp.1539-44
    Description: The Ser52Pro variant of transthyretin (TTR) produces aggressive, highly penetrant, autosomal-dominant systemic amyloidosis in persons heterozygous for the causative mutation. Together with a minor quantity of full-length wild-type and variant TTR, the main component of the ex vivo fibrils was the residue 49-127 fragment of the TTR variant, the portion of the TTR sequence that previously has been reported to be the principal constituent of type A, cardiac amyloid fibrils formed from wild-type TTR and other TTR variants [Bergstrom J, et al. (2005) J Pathol 206(2):224-232]. This specific truncation of Ser52Pro TTR was generated readily in vitro by limited proteolysis. In physiological conditions and under agitation the residue 49-127 proteolytic fragment rapidly and completely self-aggregates into typical amyloid fibrils. The remarkable susceptibility to such cleavage is likely caused by localized destabilization of the β-turn linking strands C and D caused by loss of the wild-type hydrogen-bonding network between the side chains of residues Ser52, Glu54, Ser50, and a water molecule, as revealed by the high-resolution crystallographic structure of Ser52Pro TTR. We thus provide a structural basis for the recently hypothesized, crucial pathogenic role of proteolytic cleavage in TTR amyloid fibrillogenesis. Binding of the natural ligands thyroxine or retinol-binding protein (RBP) by Ser52Pro variant TTR stabilizes the native tetrameric assembly, but neither protected the variant from proteolysis. However, binding of RBP, but not thyroxine, inhibited subsequent fibrillogenesis.
    Keywords: Misfolding ; Protein Aggregation ; Amyloid -- Metabolism ; Prealbumin -- Metabolism ; Proline -- Metabolism ; Serine -- Metabolism
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 4
    Language: English
    In: The Journal of biological chemistry, 29 April 2016, Vol.291(18), pp.9678-89
    Description: The amyloidogenic variant of β2-microglobulin, D76N, can readily convert into genuine fibrils under physiological conditions and primes in vitro the fibrillogenesis of the wild-type β2-microglobulin. By Fourier transformed infrared spectroscopy, we have demonstrated that the amyloid transformation of wild-type β2-microglobulin can be induced by the variant only after its complete fibrillar conversion. Our current findings are consistent with preliminary data in which we have shown a seeding effect of fibrils formed from D76N or the natural truncated form of β2-microglobulin lacking the first six N-terminal residues. Interestingly, the hybrid wild-type/variant fibrillar material acquired a thermodynamic stability similar to that of homogenous D76N β2-microglobulin fibrils and significantly higher than the wild-type homogeneous fibrils prepared at neutral pH in the presence of 20% trifluoroethanol. These results suggest that the surface of D76N β2-microglobulin fibrils can favor the transition of the wild-type protein into an amyloid conformation leading to a rapid integration into fibrils. The chaperone crystallin, which is a mild modulator of the lag phase of the variant fibrillogenesis, potently inhibits fibril elongation of the wild-type even once it is absorbed on D76N β2-microglobulin fibrils.
    Keywords: Fourier Transform IR (Ftir) ; Amyloid ; Fibril ; Protein Aggregation ; Protein Misfolding ; Β2-Microglobulin ; Mutation, Missense ; Protein Aggregation, Pathological ; Amyloid -- Chemistry ; Beta 2-Microglobulin -- Chemistry
    ISSN: 00219258
    E-ISSN: 1083-351X
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  • 5
    In: Chemical Communications, 2018, Vol.54(43), pp.5422-5425
    Description: Protein fibrillation is involved in many serious diseases, and protein oligomers are proved to be precursors of amyloid fibrils. NMR and QCMD experiments allowed us to establish that the interaction between citrate-stabilized gold nanoparticles and a paradigmatic amyloidogenic protein, 2-microglobulin, is able to interfere with protein association into oligomers.
    Keywords: Citric Acid -- Chemistry ; Gold -- Chemistry ; Metal Nanoparticles -- Chemistry ; Beta 2-Microglobulin -- Chemistry;
    ISSN: 1359-7345
    E-ISSN: 1364-548X
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  • 6
    In: PLoS ONE, 2012, Vol.7(12)
    Description: Availability of living organisms to mimic key step of amyloidogenesis of human protein has become an indispensable tool for our translation approach aiming at filling the deep gap existing between the biophysical and biochemical data obtained in vitro and the pathological features observed in patients. Human β 2 -microglobulin (β 2 -m) causes systemic amyloidosis in haemodialysed patients. The structure, misfolding propensity, kinetics of fibrillogenesis and cytotoxicity of this protein, in vitro , have been studied more extensively than for any other globular protein. However, no suitable animal model for β 2 -m amyloidosis has been so far reported. We have now established and characterized three new transgenic C. elegans strains expressing wild type human β 2 -m and two highly amyloidogenic isoforms: P32G variant and the truncated form ΔN6 lacking of the 6 N-terminal residues. The expression of human β 2 -m affects the larval growth of C. elegans and the severity of the damage correlates with the intrinsic propensity to self-aggregate that has been reported in previous in vitro studies. We have no evidence of the formation of amyloid deposits in the body-wall muscles of worms. However, we discovered a strict correlation between the pathological phenotype and the presence of oligomeric species recognized by the A11 antibody. The strains expressing human β 2 -m exhibit a locomotory defect quantified with the body bends assay. Here we show that tetracyclines can correct this abnormality confirming that these compounds are able to protect a living organism from the proteotoxicity of human β 2 -m.
    Keywords: Research Article ; Biology ; Medicine ; Physics
    E-ISSN: 1932-6203
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  • 7
    Language: English
    In: PLoS ONE, Dec 1, 2015, Vol.10(12)
    Description: D76N is the first natural variant of human [beta]-2 microglobulin ([beta]2m) so far identified. Contrary to the wt protein, this mutant readily forms amyloid fibres in physiological conditions, leading to a systemic and severe amyloidosis. Although the Asp76Asn mutant has been extensively characterized, the molecular bases of its instability and aggregation propensity remain elusive. In this work all Asp residues of human [beta]2m were individually substituted to Asn; D-to-N mutants (D34N, D38N, D53N, D59N, D96N and D98N) were characterised in terms of thermodynamic stability and aggregation propensity. Moreover, crystal structures of the D38N, D53N, D59N and D98N variants were solved at high-resolution (1.24-1.70 #197;). Despite showing some significant variations in their thermal stabilities, none showed the dramatic drop in melting temperature (relative to the wt protein) as observed for the pathogenic mutant. Consistently, none of the variants here described displayed any increase in aggregation propensity under the experimental conditions tested. The crystal structures confirmed that D-to-N mutations are generally well tolerated, and lead only to minor reorganization of the side chains in close proximity of the mutated residue. D38N is the only exception, where backbone readjustments and a redistribution of the surface electrostatic charges are observed. Overall, our results suggest that neither removing negative charges at sites 34, 38, 53, 59, 96 and 98, nor the difference in [beta]2m pI, are the cause of the aggressive phenotype observed in D76N. We propose that the dramatic effects of the D76N natural mutation must be linked to effects related to the crucial location of this residue within the [beta]2m fold.
    Keywords: Crystal Structure -- Analysis ; Amyloidosis -- Risk Factors ; Amyloidosis -- Care And Treatment ; Beta Globulins -- Research
    ISSN: 1932-6203
    Source: Cengage Learning, Inc.
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  • 8
    Language: English
    In: PLoS ONE, 01 January 2015, Vol.10(12), p.e0144061
    Description: D76N is the first natural variant of human β-2 microglobulin (β2m) so far identified. Contrary to the wt protein, this mutant readily forms amyloid fibres in physiological conditions, leading to a systemic and severe amyloidosis. Although the Asp76Asn mutant has been extensively characterized, the molecular bases of its instability and aggregation propensity remain elusive. In this work all Asp residues of human β2m were individually substituted to Asn; D-to-N mutants (D34N, D38N, D53N, D59N, D96N and D98N) were characterised in terms of thermodynamic stability and aggregation propensity. Moreover, crystal structures of the D38N, D53N, D59N and D98N variants were solved at high-resolution (1.24-1.70 Å). Despite showing some significant variations in their thermal stabilities, none showed the dramatic drop in melting temperature (relative to the wt protein) as observed for the pathogenic mutant. Consistently, none of the variants here described displayed any increase in aggregation propensity under the experimental conditions tested. The crystal structures confirmed that D-to-N mutations are generally well tolerated, and lead only to minor reorganization of the side chains in close proximity of the mutated residue. D38N is the only exception, where backbone readjustments and a redistribution of the surface electrostatic charges are observed. Overall, our results suggest that neither removing negative charges at sites 34, 38, 53, 59, 96 and 98, nor the difference in β2m pI, are the cause of the aggressive phenotype observed in D76N. We propose that the dramatic effects of the D76N natural mutation must be linked to effects related to the crucial location of this residue within the β2m fold.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 9
    In: ELECTROPHORESIS, October 2015, Vol.36(19), pp.2465-2472
    Description: Free solution capillary electrophoresis with UV detection is here used to retrieve information on the conformational changes of wild‐type β‐microglobulin and a series of naturally and artificially created variants known to have different stability and amyloidogenic potential. Under nondenaturing conditions, the resolution of at least two folding conformers at equilibrium is obtained and a third species is detected for the less stable isoforms. Partial denaturation by using chaotropic agents such as acetonitrile or trifluoroethanol reveals that the separated peaks are at equilibrium, as the presence of less structured species is either enhanced or induced at the expenses of the native form. Reproducible CE data allow to obtain an interesting semiquantitative correlation between the peak areas observed and the protein stability. Thermal unfolding over the range 25–42°C is induced inside the capillary for the two pathogenic proteins (wtβ‐microglobulin and D76N variant): the large differences observed upon small temperature variation draw attention on the robustness of analytical methods when dealing with proteins prone to misfolding and aggregation.
    Keywords: Amyloidosis ; Β 2 ‐Microglobulin ; Capillary Electrophoresis ; Folding Conformers ; Stability
    ISSN: 0173-0835
    E-ISSN: 1522-2683
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  • 10
    Language: English
    In: ELECTROPHORESIS, 10/2015, Vol.36(19), pp.2465-2472
    ISSN: ELECTROPHORESIS
    E-ISSN: 01730835
    Source: Wiley (via CrossRef)
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