Seilheimer B, Bohrmann B, Bondolfi L, Müller F, Stüber D, Döbeli H
Hoffmann-La Roche Ltd., Basel, Switzerland.
J Struct Biol. 1997 Jun;119(1):59-71. doi: 10.1006/jsbi.1997.3859.
In an attempt to elucidate the relationship among aggregation properties, fiber morphology, and cellular toxicity several beta-amyloid peptides (A beta) were prepared according to a standardized procedure. Peptides either carried mutations inside the membrane anchor segment around amino acid position 35 or their carboxy terminus was shortened from 42 to 41, 40, or 39 amino acids. The time-dependent self-assembly of monomeric A beta into fibers was simultaneously monitored by electron microscopy, circular dichroism spectroscopy, analytical ultracentrifugation, and A beta-mediated cellular toxicity using the reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to measure cell viability. The transition of A beta monomers into fibers was analyzed by more than 600 electron micrographs. Distinct morphological changes from seed-like structures to immature and mature fibers were observed. Seeds were of spherical appearance. Immature fibers were typically elongated structures with a rough surface and with varying thickness depending on the A beta sequence. Mature fibers were characterized by a periodic variation of their thickness along the fiber axis. The proportion of these different structures and the total amount of aggregated A beta was amino acid sequence-dependent. Wild-type A beta 1-42 and its oxidized derivative carrying a methionine sulfoxide residue at position 35 showed the highest rate of fiber formation and exerted toxic activity in the MTT assay at very low nanomolar concentrations. The fibers formed by these two peptides were predominantly of the mature type. In contrast, carboxyl-terminus truncated peptides A beta 1-41, A beta 1-40, and A beta 1-39 or most A beta 1-42 derivatives mutated around amino acid position 35 showed a reduced aggregation rate, the immature fibers predominated, and the toxicity was orders of magnitude lower. Thus, a correlation can be drawn among the chemical structure, aggregation properties, fiber morphology, and cellular toxicity.
为了阐明聚集特性、纤维形态和细胞毒性之间的关系，按照标准化程序制备了几种β-淀粉样肽（Aβ）。肽段要么在氨基酸位置35周围的膜锚定段内携带突变，要么其羧基末端从42个氨基酸缩短至41、40或39个氨基酸。通过电子显微镜、圆二色光谱、分析超速离心法以及使用3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴盐（MTT）还原法测量细胞活力来监测Aβ单体随时间自组装成纤维的过程以及Aβ介导的细胞毒性。通过600多张电子显微照片分析了Aβ单体向纤维的转变。观察到从种子样结构到未成熟和成熟纤维的明显形态变化。种子呈球形。未成熟纤维通常是细长结构，表面粗糙，厚度因Aβ序列而异。成熟纤维的特征是其厚度沿纤维轴呈周期性变化。这些不同结构的比例和聚集的Aβ总量取决于氨基酸序列。野生型Aβ1-42及其在35位携带甲硫氨酸亚砜残基的氧化衍生物显示出最高的纤维形成速率，并且在极低的纳摩尔浓度下在MTT试验中发挥毒性活性。由这两种肽形成的纤维主要是成熟类型。相比之下，羧基末端截短的肽段Aβ1-41、Aβ1-40和Aβ1-39或大多数在氨基酸位置35周围突变的Aβ1-42衍生物显示出降低的聚集速率，未成熟纤维占主导，并且毒性低几个数量级。因此，可以得出化学结构、聚集特性、纤维形态和细胞毒性之间的相关性。
