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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Proteases play a well-recognized role in the emergence of highly aggregation-prone protein fragments in vivo, while in vitro limited proteolysis is often employed to probe different phases of amyloidogenic pathways. Here we show that addition of moderate amounts of pepsin to acidified bovine insulin at close-to-physiological temperature results in an abrupt self-assembly of amyloid-like fibrils from partially digested insulin fragments. Biochemical analysis of the pepsin-induced fibrils implicates peptide fragments (named H) comprising of the 13 or 15 N-terminal residues of the A-chain and 11 or 13 N-terminal residues of the B-chain linked by disulfide bond between Cys7A-Cys7B as the main constituents. There are up to 8 pepsin-cleavage sites remaining within the double chain peptide which become protected upon fast fibrillation unless concentration of the enzyme is increased resulting in complete digestion of insulin. Controlled re-association of H-peptides leads to explosive fibrillation only under non-reducing conditions implying the key role of the disulfide bond in their amyloidogenicity. Such re-assembled amyloid is similar in terms of morphology and infrared features to typical bovine insulin fibrils although it lacks the ability to seed the intact protein. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 01/2015; DOI:10.1074/jbc.M114.608844 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Formation of amyloid fibrils is often facilitated in the presence of specific charge-compensating ions. Dissolved sodium chloride is known to accelerate insulin fibrillation at low pH which has been attributed to the shielding of electrostatic repulsion between positively charged insulin molecules by chloride ions. However, the subsequent fate of Cl¯ anions - i.e. possible entrapment within elongating fibrils or escape into the bulk solvent - remains unclear. Here we show that while the presence of NaCl at the onset of insulin aggregation induces structural variants of amyloid with distinct fingerprint infrared features, a delayed addition of salt to fibrils that have been already formed in its absence and under quiescent conditions triggers "condensation effect": amyloid superstructures with strong chiroptical properties are formed. Chloride ions appear to stabilize these superstructures in a manner similar to stabilization of DNA condensates by polyvalent cations. Concentration of residual chloride ions trapped within bovine insulin fibrils grown in 0.1 M NaCl, at pD 1.9, and rinsed extensively with water afterwards, is less than one anion per 16 insulin monomers (as estimated using ion chromatography) implying absence of defined solvent-sequestered nesting sites for chloride counterions. Our results have been discussed in the context of mechanisms of insulin aggregation.
    Langmuir 01/2015; 31(7). DOI:10.1021/la5048694 · 4.38 Impact Factor

Full-text (2 Sources)

Available from
Jul 3, 2014