Time-dependent insulin oligomer reaction pathway prior to fibril formation: cooling and seeding.
Time-dependent insulin oligomer reaction pathway prior to fibril formation: cooling and seeding.
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DOI：
10.1002/prot.22417
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发表时间：
2009-10
影响因子：
2.9
通讯作者：
Belfort, Georges
中科院分区：
文献类型：
--
作者：
Sorci, Mirco;Grassucci, Robert A.;Hahn, Ingrid;Frank, Joachim;Belfort, Georges
在所有淀粉样蛋白相关疾病中识别毒性因子的困难可能是由于成核过程和随后的原纤维形成的复杂动力学和热力学。这些疾病的缓慢进展表明，毒物的形成、结合和/或作用可能是速率限制的。候选毒性物质包括前体（有些浓度很低），也称为低聚物和原纤维，以及原纤维。在这里，我们通过两种不同类型的实验-冷却和播种-探索胰岛素原纤维形成的反应途径，研究了人胰岛素低聚物（通过冷冻电镜成像）在原纤维形成条件（pH 1.6和65°C）下的动力学和热力学行为，并证实了成核模型的有效性及其对原纤维生长的影响。冷却和种子研究的结果证实，在原纤维形成之前，存在一个随时间变化的低聚物反应过程，可能包括一个限速成核过程，然后是中间产物的结构重排（变成富含β片的实体），形成低聚物，然后形成原纤维。后一种结构重排步骤即使在没有细胞核的情况下也会发生（即添加了异源种子）。核在纤颤条件下（pH 1.6和65°C）形成，但在pH 1.6和25°C的冷却过程中也连续形成。在实验的时间尺度内，只有当温度升高到65°C时，被捕获的胰岛素核和生成的结构才能诱导结构重排步骤并克服能量屏障形成原纤维。在低温（25°C）下，这种纤颤的延迟和核的积累导致在65°C时纤维的平均长度减少。经验模型对数据的拟合提供了成核过程中滞后时间的延迟和冷却导致的纤维生长速率的随后降低的定量测量。此外，在测量的时间尺度内，播种实验表明，纤维可以与溶解的胰岛素（新鲜的或在滞后期间摄取的）一起引发快速纤颤，而与其他纤维一起则不能。定性地用一个猜想的自由能量空间图来解释这一点。
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 is 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°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 β-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°C) but are also continuously formed during cooling at pH 1.6 and 25°C. Within the time-scale of the experiments, only after increasing the temperature to 65°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°C), result in a decrease in the mean length of the fibers when placed at 65°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.
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DOI：
10.1073/pnas.0308754101
发表时间：
2004-02-24
影响因子：
11.1
作者：
Scheibel, T;Bloom, J;Lindquist, SL
通讯作者：
Lindquist, SL
DOI：
10.1073/pnas.0701250104
发表时间：
2007-05-22
影响因子：
11.1
作者：
Linse, Sara;Cabaleiro-Lago, Celia;Dawson, Kenneth A.
通讯作者：
Dawson, Kenneth A.
DOI：
10.1073/pnas.142459399
发表时间：
2002-07-09
影响因子：
11.1
作者：
Jimenez, JL;Nettleton, EJ;Saibil, HR
通讯作者：
Saibil, HR
影响因子：
168.9
作者：
Holmes, Clive;Boche, Delphine;Nicoll, James A. R.
通讯作者：
Nicoll, James A. R.
影响因子：
16.6
作者：
Gilead, Sharon;Wolfenson, Haguy;Gazit, Ehud
通讯作者：
Gazit, Ehud