UBE2E Ubiquitin-conjugating Enzymes and Ubiquitin Isopeptidase Y Regulate TDP-43 Protein Ubiquitination
UBE2E Ubiquitin-conjugating Enzymes and Ubiquitin Isopeptidase Y Regulate TDP-43 Protein Ubiquitination
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DOI：
10.1074/jbc.m114.561704
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发表时间：
2014-07-11
影响因子：
4.8
通讯作者：
Kahle, Philipp J.
中科院分区：
文献类型：
--
作者：
Hans, Friederike;Fiesel, Fabienne C.;Kahle, Philipp J.
43 kDa的反式激活元件DNA结合蛋白（TDP-43）表征额颞叶变性和肌萎缩侧索硬化症不同亚型中的不溶性蛋白质聚集体。TDP-43介导不同蛋白质复合物内的许多RNA加工步骤。在这里，我们确定了新的TDP-43蛋白质相互作用发现在酵母双杂交筛选成人人脑cDNA文库。我们通过免疫共沉淀证实了7个命中的TDP-43相互作用，并评估了它们在HEK 293 E细胞中的共定位。由于病理性TDP-43是泛素化的，因此我们集中于泛素缀合酶UBE 2 E3和泛素异肽酶Y（UBPY）。当细胞用蛋白酶体抑制剂处理时，泛素化的和不溶性的TDP-43物质积累。所有三个UBE 2 E家族成员都可以增强TDP-43的泛素化，而无催化活性的UBE 2 E3（C145 S）的效率要低得多。相反，UBE 2 E3的沉默减少了TDP-43泛素化。我们检查了48个已知的疾病相关TDP-43突变体中的15个，发现其中一个过度泛素化。这种强烈的TDP-43（K263 E）泛素化通过蛋白酶体抑制以及UBE 2 E3表达进一步增强。相反，UBE 2 E3沉默和UBPY表达减少TDP-43（K263 E）泛素化。此外，野生型，但不是活性位点突变体UBPY减少泛素化的TDP-43 C-末端片段和核进口受损的突变体。在果蝇中，UBPY沉默增强了神经退行性TDP-43表型和不溶性高分子量TDP-43和泛素种类的积累。因此，UBE 2 E3和UBPY参与TDP-43泛素化、溶解性和神经变性的调节。
Trans-activation element DNA-binding protein of 43 kDa (TDP-43) characterizes insoluble protein aggregates in distinct subtypes of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. TDP-43 mediates many RNA processing steps within distinct protein complexes. Here we identify novel TDP-43 protein interactors found in a yeast two-hybrid screen using an adult human brain cDNA library. We confirmed the TDP-43 interaction of seven hits by co-immunoprecipitation and assessed their co-localization in HEK293E cells. As pathological TDP-43 is ubiquitinated, we focused on the ubiquitin- conjugating enzyme UBE2E3 and the ubiquitin isopeptidase Y (UBPY). When cells were treated with proteasome inhibitor, ubiquitinated and insoluble TDP-43 species accumulated. All three UBE2E family members could enhance the ubiquitination of TDP-43, whereas catalytically inactive UBE2E3(C145S) was much less efficient. Conversely, silencing of UBE2E3 reduced TDP-43 ubiquitination. We examined 15 of the 48 known disease-associated TDP-43 mutants and found that one was excessively ubiquitinated. This strong TDP-43(K263E) ubiquitination was further enhanced by proteasomal inhibition as well as UBE2E3 expression. Conversely, UBE2E3 silencing and expression of UBPY reduced TDP-43(K263E) ubiquitination. Moreover, wild-type but not active site mutant UBPY reduced ubiquitination of TDP-43 C-terminal fragments and of a nuclear import-impaired mutant. In Drosophila melanogaster, UBPY silencing enhanced neurodegenerative TDP-43 phenotypes and the accumulation of insoluble high molecular weight TDP-43 and ubiquitin species. Thus, UBE2E3 and UBPY participate in the regulation of TDP-43 ubiquitination, solubility, and neurodegeneration.