Elimination of TDP-43 inclusions linked to amyotrophic lateral sclerosis by a misfolding-specific intrabody with dual proteolytic signals.
Elimination of TDP-43 inclusions linked to amyotrophic lateral sclerosis by a misfolding-specific intrabody with dual proteolytic signals.
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DOI：
10.1038/s41598-018-24463-3
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发表时间：
2018-04-16
影响因子：
4.6
通讯作者：
Urushitani M
中科院分区：
文献类型：
--
作者：
Tamaki Y;Shodai A;Morimura T;Hikiami R;Minamiyama S;Ayaki T;Tooyama I;Furukawa Y;Takahashi R;Urushitani M
TAR DNA结合蛋白43 kDa（TDP-43）的聚集与肌萎缩侧索硬化症（ALS）的散发性和某些家族性形式的发病机制有关，这表明消除TDP-43聚集体是一种可能的治疗策略。在这里，我们生成并研究了源自3B 12 A单克隆抗体（MAb）的单链可变片段（scFv），该抗体识别TDP-43核输出信号的D247，这是一种在生理状态下被掩蔽的表位。在转染的HEK 293 A细胞中，3B 12 A scFv重现了全长MAb对具有缺陷性核定位信号的错误定位的TDP-43和在RRM 1处具有半胱氨酸至丝氨酸取代的TDP-43包含模拟物的亲和力。此外，3B 12 A scFv加速了蛋白酶体介导的聚集TDP-43的降解，这可能是由于VH结构域CDR 2区中的内源性PEST样蛋白水解信号序列。将分子伴侣介导的自噬（CMA）相关信号添加至3B 12 A scFv诱导HSP 70转录，进一步增强TDP-43聚集体清除和细胞活力。3B 12 A scFv还减少子宫内电穿孔后胚胎小鼠脑中的TDP-43聚集体，同时不引起明显的出生后脑病理学或发育异常。这些结果表明，易于通过蛋白酶体和自噬途径协同蛋白水解的错误折叠特异性胞内抗体是减轻ALS中TDP-43蛋白质病的有希望的策略。
Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is implicated in the pathogenesis of sporadic and certain familial forms of amyotrophic lateral sclerosis (ALS), suggesting elimination of TDP-43 aggregates as a possible therapeutic strategy. Here we generated and investigated a single-chain variable fragment (scFv) derived from the 3B12A monoclonal antibody (MAb) that recognises D247 of the TDP-43 nuclear export signal, an epitope masked in the physiological state. In transfected HEK293A cells, 3B12A scFv recapitulated the affinity of the full-length MAb to mislocalised TDP-43 with a defective nuclear localising signal and to a TDP-43 inclusion mimic with cysteine-to-serine substitution at RRM1. Moreover, 3B12A scFv accelerated proteasome-mediated degradation of aggregated TDP-43, likely due to an endogenous PEST-like proteolytic signal sequence in the VH domain CDR2 region. Addition of the chaperone-mediated autophagy (CMA)-related signal to 3B12A scFv induced HSP70 transcription, further enhancing TDP-43 aggregate clearance and cell viability. The 3B12A scFv also reduced TDP-43 aggregates in embryonic mouse brain following in utero electroporation while causing no overt postnatal brain pathology or developmental anomalies. These results suggest that a misfolding-specific intrabody prone to synergistic proteolysis by proteasomal and autophagic pathways is a promising strategy for mitigation of TDP-43 proteinopathy in ALS.
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影响因子：
2.4
作者：
Kvam, Erik;Sierks, Michael R.;Messer, Anne
通讯作者：
Messer, Anne
DOI：
10.1083/jcb.201302044
发表时间：
2013-04-29
期刊：
The Journal of cell biology
影响因子：
--
作者：
Li YR;King OD;Shorter J;Gitler AD
通讯作者：
Gitler AD
影响因子：
4.8
作者：
Bao, YP;Cook, LJ;Rubinsztein, DC
通讯作者：
Rubinsztein, DC
影响因子：
6
作者：
Cairns, Nigel J.;Neumann, Manuela;Mackenzie, Ian R. A.
通讯作者：
Mackenzie, Ian R. A.
影响因子：
14.9
作者：
Kuo PH;Doudeva LG;Wang YT;Shen CK;Yuan HS
通讯作者：
Yuan HS