Axotrophin/MARCH7 acts as an E3 ubiquitin ligase and ubiquitinates tau protein in vitro impairing microtubule binding.
Axotrophin/MARCH7 acts as an E3 ubiquitin ligase and ubiquitinates tau protein in vitro impairing microtubule binding.
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DOI：
10.1016/j.bbadis.2014.05.029
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发表时间：
2014-09
期刊：
影响因子：
--
通讯作者：
Holzer M
中科院分区：
文献类型：
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作者：
Flach K;Ramminger E;Hilbrich I;Arsalan-Werner A;Albrecht F;Herrmann L;Goedert M;Arendt T;Holzer M
Tau是神经元中参与轴突隔室中微管稳定的主要微管相关蛋白。tau基因表达、选择性剪接和翻译后修饰的变化调节tau功能，并且在tau蛋白病中可导致tau错误定位和功能障碍，引起tau聚集和细胞死亡。为了揭示参与tau蛋白病发展的蛋白质，使用酵母双杂交系统来筛选tau相互作用蛋白。我们发现，轴突生长因子/MARCH 7，一个环变异域含有蛋白质与E3泛素连接酶的相似性与tau相互作用。我们将tau结合结构域定义为包含RING-变体结构域的促轴素的氨基酸552-682。免疫共沉淀和共定位证实了相互作用的特异性。轴突生长因子的细胞内定位由N-末端核靶向信号和C-末端核输出信号决定。在AD中，脑核定位丢失，并且促轴素与神经元缠结相当相关。我们在这里发现，tau蛋白被重组tau蛋白相互作用的RING变体结构域单泛素化，这减少了其微管结合。四重复tau的体外泛素化导致最多四个泛素分子的掺入，而三重复tau中只有两个分子。总之，我们提出了一种新的tau修饰优先发生在4-重复tau蛋白上，其修饰微管结合并可能影响tau蛋白病的发病机制。我们使用cytotrap酵母双杂交试验寻找tau相互作用蛋白。MARCH 7被鉴定为tau结合蛋白，并通过多种方法证实。重组MARCH 7环变体结构域使用Ubc 5进行E3自身泛素化活性。MARCH 7环变体结构域在包括微管结合结构域在内的多个位点单泛素化tau蛋白。tau蛋白的单泛素化减少了其微管结合。
Tau is the major microtubule-associated protein in neurons involved in microtubule stabilization in the axonal compartment. Changes in tau gene expression, alternative splicing and posttranslational modification regulate tau function and in tauopathies can result in tau mislocalization and dysfunction, causing tau aggregation and cell death. To uncover proteins involved in the development of tauopathies, a yeast two-hybrid system was used to screen for tau-interacting proteins. We show that axotrophin/MARCH7, a RING-variant domain containing protein with similarity to E3 ubiquitin ligases interacts with tau. We defined the tau binding domain to amino acids 552–682 of axotrophin comprising the RING-variant domain. Co-immunoprecipitation and co-localization confirmed the specificity of the interaction. Intracellular localization of axotrophin is determined by an N-terminal nuclear targeting signal and a C-terminal nuclear export signal. In AD brain nuclear localization is lost and axotrophin is rather associated with neurofibrillary tangles. We find here that tau becomes mono-ubiquitinated by recombinant tau-interacting RING-variant domain, which diminishes its microtubule-binding. In vitro ubiquitination of four-repeat tau results in incorporation of up to four ubiquitin molecules compared to two molecules in three-repeat tau. In summary, we present a novel tau modification occurring preferentially on 4-repeat tau protein which modifies microtubule-binding and may impact on the pathogenesis of tauopathies. We search for tau-interacting proteins using a cytotrap yeast two-hybrid assay. MARCH7 was identified as a tau-binding protein and confirmed by several methods. Recombinant MARCH7 Ring-variant domain uses Ubc5 for E3 self-ubiquitinating activity. MARCH7 Ring-variant domain mono-ubiquitinates tau protein at multiple sites including the microtubule-binding domain. Mono-ubiquitination of tau protein diminishes its microtubule-binding.