Growth of β-amyloid(1-40) protofibrils by monomer elongation and lateral association.: Characterization of distinct products by light scattering and atomic force microscopy
Growth of β-amyloid(1-40) protofibrils by monomer elongation and lateral association.: Characterization of distinct products by light scattering and atomic force microscopy
复制标题
DOI：
10.1021/bi015985r
复制
发表时间：
2002-05-14
期刊：
影响因子：
2.9
通讯作者：
Rosenberry, TL
中科院分区：
文献类型：
--
作者：
Nichols, MR;Moss, MA;Rosenberry, TL
脑组织中的淀粉样斑块是阿尔茨海默病的标志。这些斑块的主要成分是40-残基肽和42-残基肽，分别表示为Abeta（1-40）和Abeta(1-42)，它们是通过细胞淀粉样前体蛋白的蛋白水解而产生的。合成的β(1-40)和β(1-42)在体外形成淀粉样蛋白原纤维，与斑块中的淀粉样蛋白有许多共同的特征。Abeta纤维形成的可溶性中间体，称为原原纤维，以前已经确定，在这里，我们描述了Abeta（1-40）原原纤维的体外形成和分离的大小排斥色谱。在一些实验中，Abeta（140）被放射性甲基化，以更好地量化各种Abeta物种。机理研究明确了原原纤维生长的两种不同模式，即单体沉积延伸和原原纤维-原原纤维结合，这两种模式可以通过改变NaCl浓度来解决。在稀释的Tris-HCl缓冲液中，分离的小原原纤维通过添加Abeta（1-40）单体或NaCl引导其沿缔合途径生长。多角度光散射分析表明，在这两种生长过程中，初始分子质量M-w为（7-30）× 10(3) kDa的原纤维的M-w值增长到高达250 × 10(3) kDa。然而，单位长度的原原纤维的质量约为延长原原纤维的2-3倍。当原始原原纤维的浓度相等时，单体沉积在伸长、结合和初始原原纤维池中进一步伸长的速率常数是相同的，这表明原始原原纤维末端的原始数量没有被伸长或结合过程改变。原子力显微镜显示非均匀的原始原纤维在伸长反应后变得更像棒状。我们的数据表明，在没有NaCl的情况下，原原纤维的伸长仅仅是由于单体沉积在原原纤维的末端，并且原原纤维的直径没有增加。相反，在没有单体的情况下，当NaCl加入时，原原纤维发生缔合，但这种缔合涉及横向相互作用，导致原纤维结构相对无序。
Amyloid plaques in brain tissue are a hallmark of Alzheimer's disease. Primary components of these plaques are 40- and 42-residue peptides, denoted Abeta(1-40) and Abeta(1-42), that are derived by proteolysis of cellular amyloid precursor protein. Synthetic Abeta(1-40) and Abeta(1-42) form amyloid fibrils in vitro that share many features with the amyloid in plaques. Soluble intermediates in Abeta fibrillogenesis, termed protofibrils, have been identified previously, and here we describe the in vitro formation and isolation of Abeta(1-40) protofibrils by size exclusion chromatography. In some experiments, the Abeta(140) was radiomethylated to better quantify various Abeta species. Mechanistic studies clarified two separate modes of protofibril growth, elongation by monomer deposition and protofibril-protofibril association, that could be resolved by varying the NaCl concentration. Small isolated protofibrils in dilute Tris-HCl buffers were directed along the elongation pathway by addition of Abeta(1-40) monomer or along the association pathway by addition of NaCl. Multi-angle light scattering analysis revealed that protofibrils with initial molecular masses M-w of (7-30) x 10(3) kDa grew to M-w values of up to 250 x 10(3) kDa by these two growth processes. However, the mass per unit length of the associated protofibrils was about 2-3 times that of the elongated protofibrils. Rate constants for further elongation by monomer deposition with the elongated, associated, and initial protofibril pools were identical when equal number concentrations of original protofibrils were compared, indicating that the original number of protofibril ends had not been altered by the elongation or association processes. Atomic force microscopy revealed heterogeneous initial protofibrils that became more rodlike following the elongation reaction. Our data indicate that protofibril elongation in the absence of NaCl results from monomer deposition only at the ends of protofibrils and proceeds without an increase in protofibril diameter. In contrast, protofibril association occurs in the absence of monomer when NaCl is introduced, but this association involves lateral interactions that result in a relatively disordered fibril structure.