解析泛素连接酶Ube3a和蛋白激酶Pk17e如何调控BMP通路并影响神经突触的发育

Angelman syndrome (AS) is a neurodevelopmental disorder characterized by severe mental retardation, motor dysfunction and developmental delay. It is caused by the absence of ubiquitin ligase E3a (Ube3a) which is also associated with autism spectrum disorder. However, the function of ube3a in the nervous system and how ube3a mutations lead to Angelman syndrome are not clear. Drosophila has a unique homolog of Ube3a, encoded by ube3a. We generated multiple mutations of ube3a in Drosophila melanogaster and produced specific monoclonal antibody against Ube3a. We found that the bouton number and large vesicle number in ube3a mutants were significantly increased. ube3a mutants had a endocytosis defect in the synapses of neuromuscular junction (NMJ). Furthermore, electrophysiological analysis under high-frequency stimulation showed compromised neurotransmission in the synapses of ube3a mutants, confirming an insufficient replenishment of synaptic vesicles. But what is the mechanism by which ube3a regulated synapse development? Further study showed Tkv expression level was increased in the ube3a mutants and Co-IP experiments indicated that Tkv and Ube3a were in a complex. Tkv can be ubiquinated by Ube3a in S2 cells. Genetic interaction showed that heterozygous tkv mutation rescued the synapse phenotype of ube3a mutants. Our results suggested that Ube3a negatively regulated Tkv to control synapse development. We also found that pk17e mutants had the similar NMJ morphology as ube3a and pk17e also negatively regulated Tkv expression level. We want to know whether pk17e plays a role in synapse function by dye-loading and electrophysiology experiments and whether Tkv ubiquitination by Ube3a is dependent on its phosphorylation by Pk17e. We will study the relationship among ube3a, pk17e and tkv by genetic and biochemical method. This project will reveal the pathological mechanism of Angelman syndrome.

泛素连接酶3a (Ube3a)的缺失导致认知障碍疾病Angelman综合征，而Ube3a过高表达则导致自闭症，但Ube3a如何调控神经系统的发育和功能目前知之甚少。利用模式生物果蝇，我们发现ube3a突变体中神经突触过多生长；突触囊泡内吞功能减弱，突触传导异常。ube3a突变体突触中BMP（bone morphogenetic protein）信号通路中的Tkv受体升高，且Ube3a可以泛素化Tkv。Pk17e属于核糖体蛋白激酶S6家族,其突变体与ube3a有类似的突触结构，且也负调控Tkv表达量。我们的工作假设是Tkv先被Pk17e磷酸化，再由Ube3a泛素化降解。本项目将利用遗传、生化、电生理等手段解析Pk17e是否与Ube3a协同负调控BMP信号通路受体Tkv，进而影响突触的发育和功能。本项研究将增进对Angelman综合征致病机理的认识，为预防和治疗认知障碍疾病提供新的思路。

Angelman syndrome（AS）是一种神经发育疾病，它的主要临床表现是严重的智力障碍，运动失调，语言缺失，共济失调和发育迟缓。AS是由E3泛素连接酶UBE3A的缺失导致的，UBE3A通过蛋白酶体途径参与蛋白质的降解。然而，UBE3A在神经元中的底物以及它的缺失是如何导致AS的，目前我们了解的并不多。UBE3A在果蝇中唯一的同源蛋白是Ube3a。为了研究在神经发育过程中Ube3a发挥作用的机制，我们构建了一系列ube3a突变体和RNAi转基因果蝇，并制备了特异的针对Ube3a的抗体。我们的研究发现，ube3a突变体的神经肌肉接头突触（NMJs）的形态发育异常，突触扣结和卫星扣结数目都明显增加。透射电镜结果显示，ube3a突变体的突触前囊泡的体积变大。荧光染料FM 1-43摄取实验表明，ube3a突变体中神经肌肉突触内吞能力明显减弱，而电生理结果则表明ube3a突变体在低频刺激下突触传导正常，但在高频刺激下则不能有效维持突触传递。遗传互作分析表明，在ube3a突变体背景下，去掉一个拷贝的tkv（编码BMPI型受体）或一个拷贝的mad（编码BMP信号效应蛋白即转录因子）都能挽救NMJ过度生长的表型；而去掉一个拷贝的wit（编码BMP II型受体）则不能挽救这种表型。免疫染色结果表明，ube3a突变体中磷酸化的Mad水平升高，表明突变体中BMP信号通路上调。生化分析进一步表明，在ube3a突变体中，BMPI型受体Tkv的蛋白水平特异性增加，磷酸化的Mad水平也伴随增加，而BMPII型受体Wit和BMPI型受体Sax蛋白水平则没有明显的变化。体内免疫共沉淀以及体外GST pull-down结果表明，Ube3a与Tkv存在相互作用；我们进一步鉴定了Ube3a与Tkv结合的区段，发现Ube3a 201-640氨基酸内的连续两个肽段能够与Tkv结合；而Tkv蛋白具有丝氨酸/苏氨酸激酶结构域的STYKc则主要介导它与Ube3a的互作。同时，在S2细胞中，Ube3a能调控Tkv蛋白的稳定性。Ube3a泛素化Tkv，并且特异的泛素化Tkv C端的227位点的赖氨酸，最终促进它通过蛋白酶体途径进行降解。Ube3a对Tkv的负调控机制在哺乳动物细胞中是保守的。总之，这些结果表明BMP信号通路的增加可部分的解释AS的发病机制，并且能够为研究AS的病因学及机制提供一个有效模型。