Sorci Mirco, Grassucci Robert A, Hahn Ingrid, Frank Joachim, Belfort Georges
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.
Proteins. 2009 Oct;77(1):62-73. doi: 10.1002/prot.22417.
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.
在所有淀粉样蛋白相关疾病中识别毒性因子的困难，可能归因于成核过程以及随后的原纤维形成过程复杂的动力学和热力学。这些疾病进展缓慢表明，毒性因子的形成、掺入和/或作用可能是限速步骤。候选毒性因子包括前体（一些浓度极低），也称为寡聚体和原纤维，以及原纤维。在此，我们通过两种不同类型的实验——降温实验和接种实验，探究在原纤维形成条件（pH 1.6和65℃）下人类胰岛素寡聚体（通过冷冻电镜成像）的动力学和热力学行为，以探寻胰岛素原纤维形成的反应途径，并证实成核模型的有效性及其对原纤维生长的影响。降温实验和接种实验的结果均证实，在原纤维形成之前存在一个随时间变化的寡聚体反应过程，这可能涉及一个限速成核过程，随后中间体发生结构重排（形成富含β折叠的实体）以形成寡聚体，进而形成原纤维。即使在没有核（即添加异源种子）的情况下，也会发生后者的结构重排步骤。在原纤维形成条件（pH 1.6和65℃）下会形成核，但在pH 1.6和25℃降温过程中也会持续形成核。在实验的时间尺度内，只有将温度升至65℃后，捕获的胰岛素核及产生的结构才能诱导结构重排步骤并克服能量障碍形成原纤维。这种在低温（25℃）下原纤维形成的延迟以及核的积累，导致在65℃时纤维的平均长度缩短。将一个经验模型拟合到数据中，可对成核过程中滞后时间的延迟以及降温导致的原纤维生长速率随后降低进行定量测量。此外，接种实验在测量的时间尺度内表明，纤维可以引发溶解的胰岛素（新鲜的或在滞后阶段获取的）快速形成原纤维，但不能引发其他纤维快速形成原纤维。定性地说，这可以用一个推测的自由能空间图来解释。
