Section of Biochemistry, Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy.
European Laboratory for Non-linear Spectroscopy, Sesto Fiorentino, Italy.
Amyloid. 2021 Mar;28(1):56-65. doi: 10.1080/13506129.2020.1826425. Epub 2020 Oct 7.
Accumulation of ubiquitin-positive, tau- and α-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Such inclusions have variably been described as amorphous aggregates or more structured deposits having amyloid properties. Here we have purified full-length TDP-43 (FL TDP-43) and its C-terminal domain (Ct TDP-43) to investigate the morphological, structural and tinctorial features of aggregates formed by them at pH 7.4 and 37 °C. AFM images indicate that both protein variants show a tendency to form filaments. Moreover, we show that both FL TDP-43 and Ct TDP-43 filaments possess a largely disordered secondary structure, as ascertained by far-UV circular dichroism and Fourier transform infra-red spectroscopy, do not bind Congo red and induce a very weak increase of thioflavin T fluorescence, indicating the absence of a clear amyloid-like signature.
在中枢神经系统中，TDP-43 阳性、tau 和 α-突触核蛋白阴性的泛素阳性细胞内包涵体的积累是与肌萎缩侧索硬化症（ALS）和伴有泛素阳性包涵体的额颞叶变性（FTLD-U）相关的主要特征。这些包涵体曾被描述为具有不同形态的无定形聚集物或具有淀粉样特性的更具结构的沉积物。在这里，我们纯化了全长 TDP-43（FL TDP-43）及其 C 末端结构域（Ct TDP-43），以研究它们在 pH 7.4 和 37°C 下形成的聚集体的形态、结构和染色特征。原子力显微镜图像表明，这两种蛋白质变体都有形成纤维的趋势。此外，我们表明，FL TDP-43 和 Ct TDP-43 纤维都具有很大的无序二级结构，这可以通过远紫外圆二色性和傅里叶变换红外光谱来确定，它们不结合刚果红并诱导较弱的硫代黄素 T 荧光增强，表明不存在明确的淀粉样特征。
