Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy.
FEBS J. 2012 Feb;279(3):451-63. doi: 10.1111/j.1742-4658.2011.08438.x. Epub 2012 Jan 9.
Aggregation of human ataxin-3 (AT3) into amyloid fibrils is responsible for spinocerebellar ataxia type 3. This protein consists of a folded N-terminal domain (Josephin domain, residues 1-182), a central flexible region (residues 183-291), a poly-glutamine sequence of variable length and a short C-terminal flexible region. Very little is known about the influence of the central flexible region on the conformational and aggregation properties of this protein. The present study aimed to investigate the specific role of this portion of the protein (residues 183-291). Accordingly, protein fragments 1-182 (AT3/182) and 1-291 (AT3/291) were produced and compared by thioflavin-T fluorescence, Fourier transform infrared spectroscopy, CD, intrinsic fluorescence and ESI-MS. It is shown that the central flexible region enhances protein aggregation and can populate conformational states with different degrees of compactness. Both monomeric and dimeric partially-folded forms are identified for both protein fragments under denaturing conditions. Partially-folded monomers and dimers accumulate to a larger extent in AT3/291. These species represent good candidates for early intermediates of the aggregation process under the experimental conditions employed in the present study.
人源共济失调蛋白 3（AT3）聚集形成淀粉样纤维是造成脊髓小脑共济失调 3 型的原因。该蛋白由一个折叠的 N 端结构域（Josephin 结构域，残基 1-182）、一个中间的柔性区域（残基 183-291）、一个可变长度的多聚谷氨酰胺序列和一个短的 C 端柔性区域组成。对于中间柔性区域对该蛋白构象和聚集性质的影响，人们知之甚少。本研究旨在探讨该蛋白这部分（残基 183-291）的特定作用。为此，制备并比较了蛋白片段 1-182（AT3/182）和 1-291（AT3/291），通过硫黄素 T 荧光、傅里叶变换红外光谱、CD、内源荧光和 ESI-MS 进行分析。结果表明，中间柔性区域增强了蛋白的聚集，并能形成不同程度紧凑性的构象状态。在变性条件下，两种蛋白片段都存在单体和二聚体的部分折叠形式。部分折叠的单体和二聚体在 AT3/291 中积累程度更大。这些物质在本研究中所采用的实验条件下，是聚集过程早期中间产物的良好候选物。
