Article

Molecular basis for insulin fibril assembly

Howard Hughes Medical Institute, UCLA-DOE Institute for Genomics and Proteomics, Los Angeles CA 90095-1570, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 11/2009; 106(45):18990-5. DOI: 10.1073/pnas.0910080106
Source: PubMed

ABSTRACT In the rare medical condition termed injection amyloidosis, extracellular fibrils of insulin are observed. We found that the segment of the insulin B-chain with sequence LVEALYL is the smallest segment that both nucleates and inhibits the fibrillation of full-length insulin in a molar ratio-dependent manner, suggesting that this segment is central to the cross-beta spine of the insulin fibril. In isolation from the rest of the protein, LVEALYL forms microcrystalline aggregates with fibrillar morphology, the structure of which we determined to 1 A resolution. The LVEALYL segments are stacked into pairs of tightly interdigitated beta-sheets, each pair displaying the dry steric zipper interface typical of amyloid-like fibrils. This structure leads to a model for fibrils of human insulin consistent with electron microscopic, x-ray fiber diffraction, and biochemical studies.

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Available from: Michael R Sawaya, Jun 23, 2014
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    • "So far, most of the 3D structures solved for amyloids correspond to assemblies of peptide fragments (Eisenberg and Jucker, 2012), such as Alzheimer's Ab (1-40/42) (Fä ndrich et al., 2011). However, fibers built on fulllength protein molecules are also amenable to structural analyses , as for insulin (Ivanova et al., 2009), b2-microglobulin (Liu et al., 2011), and SOD1 (Elam et al., 2003), or the HypF-N (Campioni et al., 2012) domain. In those cases in which the amyloidogenic proteins have stable 3D folds, a relevant issue is whether the protein molecules acting as building blocks in the crossed-b sheets undergo a radical unfolding or not, i.e., they partially and "
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    ABSTRACT: Most available structures of amyloids correspond to peptide fragments that self-assemble in extended cross β sheets. However, structures in which a whole protein domain acts as building block of an amyloid fiber are scarce, in spite of their relevance to understand amyloidogenesis. Here, we use electron microscopy (EM) and atomic force microscopy (AFM) to analyze the structure of amyloid filaments assembled by RepA-WH1, a winged-helix domain from a DNA replication initiator in bacterial plasmids. RepA-WH1 functions as a cytotoxic bacterial prionoid that recapitulates features of mammalian amyloid proteinopathies. RepA are dimers that monomerize at the origin to initiate replication, and we find that RepA-WH1 reproduces this transition to form amyloids. RepA-WH1 assembles double helical filaments by lateral association of a single-stranded precursor built by monomers. Double filaments then associate in mature fibers. The intracellular and cytotoxic RepA-WH1 aggregates might reproduce the hierarchical assembly of human amyloidogenic proteins. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Structure 12/2014; DOI:10.1016/j.str.2014.11.007 · 6.79 Impact Factor
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    • "The exact location or sequence identity of such hydrophobic ''patches'' or hot spots has been experimentally determined for many polypeptides [12] [13] [14] [15], but only for a few folded proteins [16] [17] [18], and for monoclonal antibodies has only been speculated upon based on analogies with smaller polypeptide systems [19] [20] [21]. Hydrophobic patches have been identified on the surface of some MAbs, and in some cases mutations to those regions have resulted in reduced aggregation rates [22] [23]. "
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    ABSTRACT: Aggregation is mediated by local unfolding to allow aggregation "hot spot(s)" to become solvent exposed and available to associate with a hot spot on another partially unfolded protein. Historically, the unfolding of either the Fc or the Fab regions of a given MAb has been implicated in aggregation, with differing results across different proteins. The present work focuses on separately quantifying the aggregation kinetics of isolated Fc, isolated Fab, and intact MAb as a function of pH under accelerated (high temperature) conditions. The results show that both Fab and Fc are aggregation prone and compete within the same MAb.
    FEBS letters 02/2014; 588(6). DOI:10.1016/j.febslet.2014.01.051 · 3.34 Impact Factor
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    • "The VEALYL segment is proposed to play an important role in full-length insulin misfolding [34] [35] [18] [36] and to participate in the β-strand core region of mature insulin fibrils [37] [38]. Here, we analyzed the structures of different aggregation forms (fibrillar, microcrystal-like, oligomeric aggregates) of the VEALYL steric zipper peptide by solid-state nuclear magnetic resonance (ssNMR). "
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    ABSTRACT: Recently, several short peptides have been shown to self-assemble into amyloid fibrils with generic cross-β spines, so-called steric zippers, suggesting common underlying structural features and aggregation mechanisms. Understanding these mechanisms is a prerequisite for designing fibril-binding compounds and inhibitors of fibril formation. The hexapeptide VEALYL, corresponding to the residues B12-17 of full-length insulin, has been identified as one of these short segments. Here, we analyzed the structures of multiple, morphologically different (fibrillar, microcrystal-like, oligomeric) [13C,15N]VEALYL samples by solid-state nuclear magnetic resonance complemented with results from molecular dynamics simulations. By performing NHHC/CHHC experiments, we could determine that the β-strands within a given sheet of the amyloid-like fibrils formed by the insulin hexapeptide VEALYL are stacked in an antiparallel manner, whereas the sheet-to-sheet packing arrangement was found to be parallel. Experimentally observed secondary chemical shifts for all aggregate forms, as well as ∅ and ψ backbone torsion angles calculated with TALOS, are indicative of β-strand conformation, consistent with the published crystal structure (PDB ID: 2OMQ). Thus, we could demonstrate that the structural features of all the observed VEALYL aggregates are in agreement with the previously observed homosteric zipper spine packing in the crystalline state, suggesting that several distinct aggregate morphologies share the same molecular architecture.
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