The role of WWP-family E3 ubiquitin ligases in neuronal development and function

泛素化是一种翻译后修饰，由E1、E2和E3Ub连接酶三种酶的顺序作用催化。泛素化的底物特异性主要由E3连接酶决定，其中约600种不同的亚型在人类基因组中编码。底物蛋白质可以与单体形式的Ub(单泛素化)或串联的多Ub链(多泛素化)结合。单素化作用影响多种蛋白质特性和控制，例如，受体的内吞作用、DNA修复或病毒萌发。多泛素化的结果取决于多Ub链的类型。为了形成多Ub链，一个Ub的C-末端甘氨酸残基与供体Ub(K6、K11、K27、K29、K33、K48或K63)表面的赖氨酸残基连接。K48连接的多Ub链被蛋白酶体识别，目的蛋白被降解，而K63连接的多Ub链参与蛋白质功能的非蛋白酶体调节。有趣的是，在哺乳动物脑中，K63连接的PolyUb链几乎和K48连接的链一样丰富，这表明K-63连接的链对细胞过程的调节可能与Ub介导的蛋白酶体降解一样重要。WWP1和WWP2(WWP1/2)是E3连接酶Nedd4超家族的成员。Nedd4家族E3连接酶主要以非蛋白酶体的方式调节底物的表达、定位或功能。WWP1/2和其他Nedd4超家族E3连接酶一样，优先结合单Ub或K63连接的多Ub链，而不是K48连接的多Ub链。尽管在培养的非神经细胞中参与调节单泛素化或K63连接的多泛素化的分子机制已经有了很大程度的探索，但我们对这种泛素化在体内神经细胞发育和功能中的非蛋白酶体作用的理解是非常初级的。我们最近报道了Nedd4-1和Nedd4-2在轴突分支中发挥关键作用(Kawabe等人，Neuron 65,358,2010；Hsia等人，Proc.娜塔莉。阿卡德。SCI。美国第111、13205、2014年)。另一个Nedd4超家族成员S-1对轴突的获得至关重要。相比之下，WWP1/2在发育和成熟神经元中的作用基本上是未知的。我们已经开始系统地解决这个问题，对WWP1/2双KO小鼠进行了初步鉴定，发现双KO神经元在轴突形成、迁移和突触形成方面有几个显著的表型变化。在这里提出的项目中，我们将通过鉴定和表征WWP1/2的底物和确定WWP E3连接酶是如何通过上游信号通路调节WWP E3连接酶来研究WWP1/2调控轴突形成、神经元迁移和突触形成的机制。

Ubiquitination is a posttranslational modification catalyzed by the sequential action of three classes of enzymes, E1, E2 enzymes, and E3 Ub ligases. Substrate specificity of ubiquitination is mainly determined by the E3 ligases, of which some 600 different isoforms are encoded in the human genome. Substrate proteins can be conjugated either with the monomeric form of Ub (monoubiquitination) or with a tandem poly-Ub chain (polyubiquitination). Monoubiquitination affects multiple protein characteristics and controls, for instance, endocytosis of receptors, DNA repair, or virus budding. The consequence of polyubiquitination depends on the type of the poly-Ub chain. To form a poly-Ub chain, the C-terminal glycine residue of one Ub is conjugated to a lysine residue on the surface of the donor Ub (K6, K11, K27, K29, K33, K48, or K63). K48-linked poly-Ub chains are recognized by the proteasome, where the target protein is degraded, while K63-linked poly-Ub chains are involved in the proteasome-independent regulation of protein functions. Interestingly, K63-linked poly-Ub chains are almost as abundant as K48-linked chains in the mammalian brain, indicating that K-63 linked chain-dependent regulation of cellular processes may be as important as Ub mediated proteasomal degradation. WWP1 and WWP2 (WWP1/2) are members of the Nedd4 superfamily of E3 ligases. Nedd4 family E3 ligases regulate the expression, localization, or function of substrates mainly in a proteasome-independent manner. Consistently, WWP1/2 as well as other Nedd4 superfamily E3 ligases preferentially conjugate mono-Ub or K63-linked poly-Ub chains rather than K48-linked poly-Ub chains. Although the molecular machinery involved in the regulation of monoubiquitination or K63-linked polyubiquitination in cultured non-neuronal cells has been explored to a substantial degree, our understanding of such 'non-proteasomal' roles of ubiquitination in nerve cell development and function in vivo is very rudimentary. We reported recently that Nedd4-1 and Nedd4-2 play key roles in neurite branching (Kawabe et al., Neuron 65, 358, 2010; Hsia et al., Proc. Natl. Acad. Sci. U.S.A. 111, 13205, 2014). Another Nedd4 superfamily member, Smurf1, is crucial for axon acquisition. In contrast, the roles of WWP1/2 in developing and mature neurons are essentially unknown. We have started to address this issue systematically, performed a preliminary characterization of WWP1/2 double KO mice, and discovered several prominent phenotypic changes in axon formation, migration, and synapse formation in double KO neurons. In the project proposed here, we will study the mechanisms through which WWP1/2 regulate axon formation, neuronal migration, and synapse formation, by identifying and characterizing the substrates of WWP1/2 and by determining how WWP E3 ligases are regulated via upstream signaling pathways.