Department of Biotechnology, Pondicherry Central University, R. V. Nagar, Kalapet, Pondicherry, 605014, India.
Translational Gerontology Branch, National Institute ON Aging, National Institutes of Health, Baltimore, MD, 21224, USA.
Mol Cell Biochem. 2018 Feb;439(1-2):75-85. doi: 10.1007/s11010-017-3137-3. Epub 2017 Aug 4.
In the cell, misfolded proteins are processed by molecular chaperone-mediated refolding or through ubiquitin-mediated proteosome system. Dysregulation of these mechanisms facilitates the aggregation of misfolded proteins and forms aggresomes in the juxta nuclear position of the cell which are removed by lysosome-mediated autophagy pathway in the subsequent cell division. Accumulation of misfolded proteins in the cell is hallmark of several neurological disorders and other diseases including cancer. However, the exact mechanism of aggresome formation and clearance is not thoroughly understood. Reports have shown that several proteins including p300, p53, TAU, α-synuclein, SOD, etc. contain intrinsically disordered region (IDR) which has the tendency to form aggresome. To study the nature of aggresome formation and stability of the aggresome, we have chosen Twist1 as a model protein since it has IDR regions. Twist1 is a bHLH transcription factor which plays a major role in epithelial mesenchymal transition (EMT) and shown to interact with HAT domain of p300 and p53. In the present study, we generated several deletion mutants of human Twist1 with different fluorescent tags and delineated the regions responsible for aggresome formation. The Twist1 protein contains two NLS motifs at the N-terminal region. We showed that the deletions of regions spanning the amino acids 30-46 (Twist1Δ30-46) which lacks the first NLS motif form larger and intense aggregates while the deletion of residues from 47 to 100 (Twist1Δ47-100) which lacks the second NLS motif generates smaller and less intense aggregates in the juxta nuclear position. This suggests that both the NLS motifs are needed for the proper nuclear localization of Twist1. The aggresome formation of the Twist1 deletion mutants was confirmed by counterstaining with known aggresome markers: Vimentin, HDAC6, and gamma tubulin and further validated by MG-132 treatment. In addition, it was found that the aggresomes generated by the Twist1Δ30-46 construct are more stable than the aggresome produced by the Twist1Δ47-100 construct as well as the wild-type Twist1 protein. Taken together, our data provide an important understanding on the role of IDR regions on the formation and stability of aggresomes.
在细胞中，错误折叠的蛋白质通过分子伴侣介导的重折叠或通过泛素介导的蛋白酶体系统进行处理。这些机制的失调促进了错误折叠蛋白质的聚集，并在细胞的核周位置形成聚集体，随后在细胞分裂过程中通过溶酶体介导的自噬途径被清除。错误折叠蛋白质在细胞中的积累是几种神经退行性疾病和其他疾病（包括癌症）的标志。然而，聚集体形成和清除的确切机制还没有被完全理解。有报道表明，包括 p300、p53、TAU、α-突触核蛋白、SOD 等在内的几种蛋白质都含有内在无序区域（IDR），这些区域有形成聚集体的趋势。为了研究聚集体形成的本质和聚集体的稳定性，我们选择 Twist1 作为模型蛋白，因为它含有 IDR 区域。Twist1 是一种 bHLH 转录因子，在上皮-间充质转化（EMT）中起主要作用，并显示与 p300 和 p53 的 HAT 结构域相互作用。在本研究中，我们生成了具有不同荧光标签的人类 Twist1 的几种缺失突变体，并描绘了负责聚集体形成的区域。Twist1 蛋白在 N 端区域含有两个核定位信号（NLS）基序。我们表明，缺失跨越氨基酸 30-46 的区域（Twist1Δ30-46）会导致更大、更强的聚集，而缺失从 47 到 100 的残基（Twist1Δ47-100）会导致更小、更弱的聚集在核周位置。这表明两个 NLS 基序都需要 Twist1 的正确核定位。通过用已知的聚集体标记物（波形蛋白、HDAC6 和γ微管蛋白）进行复染，证实了 Twist1 缺失突变体的聚集体形成，并用 MG-132 处理进一步验证。此外，还发现由 Twist1Δ30-46 构建体产生的聚集体比由 Twist1Δ47-100 构建体以及野生型 Twist1 蛋白产生的聚集体更稳定。总之，我们的数据提供了关于 IDR 区域在聚集体形成和稳定性中的作用的重要理解。
