A "Two-hit" Hypothesis for Inclusion Formation by Carboxyl-terminal Fragments of TDP-43 Protein Linked to RNA Depletion and Impaired Microtubule-dependent Transport
A "Two-hit" Hypothesis for Inclusion Formation by Carboxyl-terminal Fragments of TDP-43 Protein Linked to RNA Depletion and Impaired Microtubule-dependent Transport
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DOI：
10.1074/jbc.m111.231118
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发表时间：
2011-05-27
影响因子：
4.8
通讯作者：
Lee, Virginia M. -Y.
中科院分区：
文献类型：
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作者：
Pesiridis, G. Scott;Tripathy, Kalyan;Lee, Virginia M. -Y.
TDP-43的羧基末端片段（CTFs）聚集形成额颞叶变性和肌萎缩侧索硬化症的诊断标志性内含物，但这些CTFs的生物学意义以及它们是如何产生的仍然是个谜。为了解决这些问题，我们用诱导型烟草蚀纹病毒（TEV）蛋白酶改造哺乳动物细胞，所述蛋白酶切割含有TEV切割位点的TDP-43。TDP-43侧翼的第二RNA识别基序（RRM 2）的区域被TEV有效地切割，而该结构域内的位点对切割更具抗性。含有从细胞核TDP-43的从头切割产生的RRM 2的CTF被转运到细胞质并被有效清除，表明单独的切割不足以引发CTF聚集。然而，当动力蛋白介导的微管转运被破坏、RNA被耗尽或天然错误折叠的CTF被引入这些细胞中时，CTF在从头切割后迅速聚集成稳定的细胞质内含物。我们的数据支持依赖于TDP-43切割的CTF聚集的“两次打击”机制。
Carboxyl-terminal fragments (CTFs) of TDP-43 aggregate to form the diagnostic signature inclusions of frontotemporal lobar degeneration and amyotrophic lateral sclerosis, but the biological significance of these CTFs and how they are generated remain enigmatic. To address these issues, we engineered mammalian cells with an inducible tobacco etch virus (TEV) protease that cleaves TDP-43 containing a TEV cleavage site. Regions of TDP-43 flanking the second RNA recognition motif (RRM2) are efficiently cleaved by TEV, whereas sites within this domain are more resistant to cleavage. CTFs containing RRM2 generated from de novo cleavage of nuclear TDP-43 are transported to the cytoplasm and efficiently cleared, indicating that cleavage alone is not sufficient to initiate CTF aggregation. However, CTFs rapidly aggregated into stable cytoplasmic inclusions following de novo cleavage when dynein-mediated microtubule transport was disrupted, RNA was depleted, or natively misfolded CTFs were introduced into these cells. Our data support a "two-hit" mechanism of CTF aggregation dependent on TDP-43 cleavage.