Characterization of a possible amyloidogenic precursor in glutamine-repeat neurodegenerative diseases

Armen, Roger S.; Bernard, Brady; Day, Ryan; Alonso, Darwin O. V.; Daggett, Valerie

doi:10.1073/pnas.0502068102

Cited by 70 publications

(83 citation statements)

References 46 publications

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“…Previous studies have noted that these chemical shifts are indicative of β-sheet rather than α-helical or random coil structure. However, some argue that polyQ aggregation may involve a nonstandard secondary structure, known as α-sheet, which may be indistinguishable from β-sheet by its NMR shifts (20,21). To test this assertion, we did chemical shift-independent torsion angle measurements (22) and found unambiguous evidence for a β-sheet conformation.…”

Section: Resultsmentioning

confidence: 99%

Huntingtin exon 1 fibrils feature an interdigitated β-hairpin–based polyglutamine core

Hoop

Lin

Kar

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

158

278

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Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington's disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid-state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intramolecular and intermolecular contacts, backbone and side-chain torsion angles, relaxation measurements, and calculations of chemical shifts. These experiments reveal the presence of β-hairpin-containing β-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical β-strand-based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are coassembled from differently structured monomers, which we describe as a type of "intrinsic" polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. We show that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms.solid-state NMR | Huntington's disease | amyloid disease | protein aggregation | amyloid

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Section: Resultsmentioning

confidence: 99%

Huntingtin exon 1 fibrils feature an interdigitated β-hairpin–based polyglutamine core

Hoop

Lin

Kar

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

158

278

View full text Add to dashboard Cite

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“…Simulations were performed using the Levitt et al force field (42) and the microcanonical (number of molecules, volume, and total energy held fixed) ensemble using the in lucem molecular mechanics modeling package (43). In all 498-K simulations, an 8-Å force-shifted nonbonded cutoff was used and the nonbonded list was updated every two steps; a 10-Å nonbonded cutoff was used at 298 K. Nonbonded interactions between charge groups separated by three bonds were included and scaled to 0.4 (44). A 2-fs time step was used, and structures were saved 0.2-1 ps for analysis.…”

Section: Methodsmentioning

confidence: 99%

Malleability of folding intermediates in the homeodomain superfamily

Banachewicz

Religa

Schaeffer

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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Members of the homeodomain superfamily are three-helix bundle proteins whose second and third helices form a helix-turn-helix motif (HTH). Their folding mechanism slides from the ultrafast, three-state framework mechanism for the engrailed homeodomain (EnHD), in which the HTH motif is independently stable, to an apparent two-state nucleation-condensation model for family members with an unstable HTH motif. The folding intermediate of EnHD has nearly native HTH structure, but it is not docked with helix1. The determinant of whether two-or three-state folding was hypothesized to be the stability of the HTH substructure. Here, we describe a detailed Φ-value analysis of the folding of the Pit1 homeodomain, which has similar ultrafast kinetics to that of EnHD. Formation of helix1 was strongly coupled with formation of HTH, which was initially surprising because they are uncoupled in the EnHD folding intermediate. However, we found a key difference between Pit1 and EnHD: The isolated peptide corresponding to the HTH motif in Pit1 was not folded in the absence of H1. Independent molecular dynamics simulations of Pit1 unfolding found an intermediate with H1 misfolded onto the HTH motif. The Pit1 folding pathway is the connection between that of EnHD and the slower folding homeodomains and provides a link in the transition of mechanisms from two-to three-state folding in this superfamily. The malleability of folding intermediates can lead to unstable substructures being stabilized by a variety of nonnative interactions, adding to the continuum of folding mechanisms.protein folding | temperature jump | diffusion collision | transition state T he nucleation-condensation mechanism was proposed from a Φ-value analysis of the folding of the protein chymotrypsin inhibitor 2 (CI2) (1, 2). The individual elements of secondary structure in CI2 are not independently stable, leading to highly cooperative formation of structure in the transition state (TS) for folding in which the helix is the best-formed region, stabilized by long-range interactions. No element of secondary structure is fully formed in the TS and its folding is kinetically two-state (3). It was hypothesized that multistate folding requires independently stable elements of structure or an unstable element of structure being stabilized by interacting with other elements. At the other extreme, the three-helix engrailed homeodomain, EnHD, is a small protein that folds ultrafast via a stable intermediate in line with the framework mechanism, whereby secondary structure forms first followed by docking of the helices (4, 5). Its folding intermediate consists of helix1 (H1) being helical but not docked on to the helix2-turn-helix3 (HTH) DNA binding motif (5, 6). The HTH motif is independently stable in the on-pathway intermediate (7). Studies of other members of the homoedomain superfamily show a slide from this multistate framework folding mechanism to two-state folding and nucleation condensation for members with less stable HTH motifs, consistent with our earlier hypoth...

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“…87 A number of computational studies have also provided insights on the aggregation mechanism of Q/N rich sequences. [88][89][90] The crystal structures of two short peptides NNQQNY and GNNQQNY have been solved. 66 These structures elucidated a 'steric zipper' motif implicated in fibril formation.…”

Section: Prediction Of Potential Aggregation-prone Regionsmentioning

confidence: 99%

Potential aggregation prone regions in biotherapeutics

Wang

Das

Singh

et al. 2009

mAbs

185

139

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Aggregation of a biotherapeutic is of significant concern and judicious process and formulation development is required to minimize aggregate levels in the final product. Aggregation of a protein in solution is driven by intrinsic and extrinsic factors. In this work we have focused on aggregation as an intrinsic property of the molecule. We have studied the sequences and Fab structures of commercial and non-commercial antibody sequences for their vulnerability towards aggregation by using sequence based computational tools to identify potential aggregation-prone motifs or regions. The mAbs in our dataset contain 2 to 8 aggregation-prone motifs per heavy and light chain pair. Some of these motifs are located in variable domains, primarily in CDRs. Most aggregation-prone motifs are rich in β branched aliphatic and aromatic residues. Hydroxyl-containing Ser/Thr residues are also found in several aggregation-prone motifs while charged residues are rare. The motifs found in light chain CDR3 are glutamine (Q)/asparagine (N) rich. These motifs are similar to the reported aggregation promoting regions found in prion and amyloidogenic proteins that are also rich in Q/N, aliphatic and aromatic residues. The implication is that one possible mechanism for aggregation of mAbs may be through formation of cross-β structures and fibrils. Mapping on the available Fab-receptor/ antigen complex structures reveals that these motifs in CDRs might also contribute significantly towards receptor/antigen binding. Our analysis identifies the opportunity and tools for simultaneous optimization of the therapeutic protein sequence for potency and specificity while reducing vulnerability towards aggregation.

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Characterization of a possible amyloidogenic precursor in glutamine-repeat neurodegenerative diseases

Cited by 70 publications

References 46 publications

Huntingtin exon 1 fibrils feature an interdigitated β-hairpin–based polyglutamine core

Huntingtin exon 1 fibrils feature an interdigitated β-hairpin–based polyglutamine core

Malleability of folding intermediates in the homeodomain superfamily

Potential aggregation prone regions in biotherapeutics

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