Research article summary (published 29 Sep 2009):

Time-dependent insulin oligomer reaction pathway prior to fibril formation: cooling and seeding.

Full Abstract

The difficulty in identifying the toxic agents in all amyloid-related diseases is likely due to the complicated kinetics and thermodynamics of the nucleation process and subsequent fibril formation. The slow progression of these diseases suggests that the formation, incorporation, and/or action of toxic agents are possibly rate limiting. Candidate toxic agents include precursors (some at very low concentrations), also called oligomers and protofibrils, and the fibrils. Here, we investigate the kinetic and thermodynamic behavior of human insulin oligomers (imaged by cryo-EM) under fibril-forming conditions (pH 1.6 and 65 degrees C) by probing the reaction pathway to insulin fibril formation using two different types of experiments-cooling and seeding-and confirm the validity of the nucleation model and its effect on fibril growth. The results from both the cooling and seeding studies confirm the existence of a time-changing oligomer reaction process prior to fibril formation that likely involves a rate-limiting nucleation process followed by structural rearrangements of intermediates (into beta-sheet rich entities) to form oligomers that then form fibrils. The latter structural rearrangement step occurs even in the absence of nuclei (i.e., with added heterologous seeds). Nuclei are formed at the fibrillation conditions (pH 1.6 and 65 degrees C) but are also continuously formed during cooling at pH 1.6 and 25 degrees C. Within the time-scale of the experiments, only after increasing the temperature to 65 degrees C are the trapped insulin nuclei and resultant structures able to induce the structural rearrangement step and overcome the energy barrier to form fibrils. This delay in fibrillation and accumulation of nuclei at low temperature (25 degrees C) result in a decrease in the mean length of the fibers when placed at 65 degrees C. Fits of an empirical model to the data provide quantitative measures of the delay in the lag-time during the nucleation process and subsequent reduction in fibril growth rate resulting from the cooling. Also, the seeding experiments, within the time-scale of the measurements, demonstrate that fibers can initiate fast fibrillation with dissolved insulin (fresh or taken during the lag-period) but not with other fibers. Qualitatively this is explained with a conjectual free-energy space plot.

Author information

Author/s: Sorci, Mirco (M); Grassucci, Robert A (RA); Hahn, Ingrid (I); Frank, Joachim (J); Belfort, Georges (G);

Affiliation: Howard P. Isermann Department of Chemical and Biological Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180-3590, USA.

Grants: R37 GM29169 (Agency:NIGMS NIH HHS) ; (Agency:Howard Hughes Medical Institute)

Journal and publication information

Publication Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.

Journal: Proteins (Proteins), published in United States. (Language: eng)

Reference: 2009-Oct; vol 77 (issue 1) : pp 62-73

Dates: Created 2009/08/24; Completed 2009/10/29;

PMID: 19408310, status: MEDLINE (last retrieval date: 10/29/2009, IMS Date: )

Sourced from the National Library of Medicine. Abstract text and other information may be subject to copyright.

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MeSH headings (categories)

This article was linked to the MESH Headings shown below.

Amyloid - chemistry; ultrastructure Cryoelectron Microscopy Humans Hydrogen-Ion Concentration Insulin - chemistry

Kinetics Protein Folding Protein Multimerization Temperature Thermodynamics

Associated Chemicals: Amyloid (0) ; Insulin (11061-68-0)

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