Molecular basis for amyloid fibril formation and stability

Makin, O. Sumner; Atkins, E. D. T.; Sikorski, Pawel; Johansson, Jan; Serpell, Louise C.

doi:10.1073/pnas.0406847102

Cited by 632 publications

(624 citation statements)

References 42 publications

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“…Definitive proof of the molecular interactions of mAb 11-1F4 with misfolded LCs will require X-ray crystallographic analyses of F(ab′) 2 11-1F4 complexed with the Len (1-18) peptide, whereas biophysical characterization of the binding of mAb 11-1F4 to native Len (1-18) using CD, NMR, and FRET analyses could lead to further insight into the structural basis for the unique reactivity of this antibody. Future studies to determine the 3D nature of the epitope will contribute to our understanding of amyloid fibril assembly and provide a novel target for the development of antibody-based diagnostic and therapeutic agents for patients with AL amyloidosis.…”

Section: Discussionmentioning

confidence: 99%

Localization of a Conformational Epitope Common to Non-Native and Fibrillar Immunoglobulin Light Chains

et al. 2007

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Amyloid fibrils and partially unfolded intermediates may be distinguished serologically from native amyloidogenic precursor proteins or peptides. In this regard, we had previously reported that the IgG1 mAb 11-1F4, generated by immunizing mice with a thermally denatured variable region fragment of the human Igκ4 Bence Jones protein Len, reacted specifically with light chain (LC) fibrils, irrespective of κ or λ isotype but, notably, did not with native molecules (Hrncic, R. et al. (2000) Am. J. Pathol. 157, 1239Pathol. 157, -1246. To elucidate the molecular basis of this specificity, we have used a europium-linked fluorescent immunoassay, where it was demonstrated through epitope mapping that mAb 11-1F4 recognizes a conformational determinant contained within the first (Nterminal) 18 amino acids of misfolded LCs. The nature of this epitope was evidenced in competition studies where the peptide Len (1-18), but not the intact protein or other LCs, inhibited the binding of the antibody to fibrils. This unique reactivity was dependent on the structural integrity of this portion of the molecule, particularly the presence of a highly conserved prolyl residue at position 8. On the basis of our experimental data, we posit that the mAb 11-1F4 binding site found on partially denatured and fibrillar LCs involves an inducible N-terminal main chain reversal that results in the formation of a proline anchored β-turn. Our delineation of this LC fibril-associated epitope provides the rationale for the design of novel amyloid-reactive antibodies with diagnostic and therapeutic potential for patients with LC-associated and other forms of amyloidosis. Amyloidogenesis involves a process by which normally soluble proteins or peptides form nonnative intermediates that eventually assemble into fibrils (1). All types of amyloid, regardless of amino acid sequence, share common tertiary structural features, that is, they are comprised of extended β-sheets oriented perpendicularly to the long fibril axis (2,3) and possess sites that bind the dyes thioflavin T (ThT1) and Congo red (4,5). Additionally, these components and their assembly precursors, irrespective of primary structure, contain generic conformational epitopes not present on native molecules (6-12), further indicating their physical homogeneity.The common epitopes present on amyloid fibrils and their intermediates offer targets for the design of novel diagnostic and therapeutic reagents. In this regard, we have generated a murine monoclonal antibody (mAb), designated 11-1F4 (13), which reacts specifically with light chain (LC) fibrils, but not soluble proteins, and has been shown in an experimental in vivo model to accelerate the removal of amyloidomas composed of human LC fibrils, an effect independent of the κ or λ isotype or the V L subgroup (9). This mAb was generated using as immunogen a heat-aggregated suspension of the human κ4 variable domain (V L ) Len (13,14) To localize and determine the nature of the neo-antigen recognized on conformationally altered LCs by...

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Section: Discussionmentioning

confidence: 99%

Localization of a Conformational Epitope Common to Non-Native and Fibrillar Immunoglobulin Light Chains

et al. 2007

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“…However, during the lifetime of a protein these APRs may become exposed to a trigger aggregation. b-Aggregation involves nucleation via the formation of intermolecular b-sheets that gives rise to the core of an aggregate (Nelson et al, 2005;Makin et al, 2005). It is thought that intermolecular b-structures derive their great thermodynamic stability from the high degree of hydrogen bond formation of the backbone of the polypeptide chain, but the amino acid side chain configuration will strongly influence the rate of fibril formation (DuBay et al, 2004).…”

Section: Protein Misfolding and Aggregationmentioning

confidence: 99%

Protein aggregation in bacteria: the thin boundary between functionality and toxicity

et al. 2013

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“…The crystallization of peptides upon incubation in an aqueous phase has already been noted, for example for KFFEAAAKKFFE which crystallized after incubation of the peptide for several days in a phosphate buffer saline (PBS) aqueous solution. [14] Needle-shape crystals were observed, with lengths in the order of micrometers and diameters in the range ~(12-240) nm [14].…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

“…The sample was partially aligned by stretching the thread before the drying process started, and the resulting XRD pattern ( Figure 7) shows a well-defined "cross-β" pattern 1 with, unusually for amyloid fibrils, multiple orders of reflection. This facilitates determination of the unit cell, which was performed using the software CLEARER [14]. The results provided an orthorhombic unit cell, with unit axis a= 9.6 Å, b= 18.6 Å and c= 43.7 Å.…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

Self-assembly in aqueous solution of a modified amyloid beta peptide fragment

Castelletto

Hamley

Harris

2008

Biophysical Chemistry

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The self assembly in aqueous solution of a modified amyloid beta peptide fragment is studied. We focus on sequence , KLVFF, extended by two alanines at the N terminus to give AAKLVFF. Self-assembly into twisted ribbon fibrils is observed, as confirmed by transmission electron microscopy (TEM). Dynamic light scattering reveals the semi-flexible nature of the AAKLVFF fibrils, while polarized optical microscopy shows that the peptide fibrils crystallize after an aqueous solution of AAKLVFF is matured over 5 days.The secondary structure of the fibrils is studied by FTIR, circular dichroism and x-ray diffraction (XRD), which provide evidence for β-sheet structure in the fibril.From high resolution TEM it is concluded that the average width of an AAKLVFF fibril is (63±18) nm, indicating that these fibrils comprise beta-sheets with multiple repeats of the unit cell, determined by XRD to have have b and c dimensions 1.9 and 4.8 nm with an a axis 0.96nm, corresponding to twice the peptide backbone spacing in the antiparallel β-sheet.

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Molecular basis for amyloid fibril formation and stability

Cited by 632 publications

References 42 publications

Localization of a Conformational Epitope Common to Non-Native and Fibrillar Immunoglobulin Light Chains

Localization of a Conformational Epitope Common to Non-Native and Fibrillar Immunoglobulin Light Chains

Protein aggregation in bacteria: the thin boundary between functionality and toxicity

Self-assembly in aqueous solution of a modified amyloid beta peptide fragment

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