【作者】 王婷婷；

【导师】 廖鲁剑；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2019， 博士

【摘要】 天使综合征(AS)是一种较为罕见的神经发育性疾病,该疾病是由于母源的基因UBE3A突变同时父源基因印记不表达引起。该基因的编码蛋白UBE3A,是一个E3泛素连接酶。尽管有关于UBE3A的底物报道有一些并被证实,但较多都是在细胞系中的验证,这些蛋白在神经系统的功能研究依然不清楚。本研究采用稳定同位素标记小鼠(SILAM)结合大规模蛋白质组学的研究手段探究UBE3A在神经系统中的作用底物。在AS小鼠中表达上调的潜在底物中,我们挑选了Huntington舞蹈症(HD)中致病蛋白Htt的结合蛋白HAP1做进一步的机制及功能探究。我们用蛋白稳定性实验及体内泛素化实验验证了HAP1作为UBE3A的底物特性,接着利用质谱实验找到了HAP1受UBE3A调控的泛素化位点。我们发现AS小鼠及原代神经元中自噬水平增强,但不依赖于m TOR1的调控。进一步,我们探究HAP1在自噬调控中的作用。我们的研究揭示了HAP1通过其Coiled coil结构域与Ptd Ins3K复合体中的ATG14相互作用以促进自噬的发生。进一步,HAP1参与到ATG14-VPS34-Beclin1的复合体的组装过程中以形成更大的复合体,这一结果在HEK293T及小鼠脑组织中均得到验证。在AS神经元中,Ptd Ins3K活性过度增强,敲低HAP1的表达显著降低了自噬水平。在自噬小体的成核阶段,HAP1进一步与VPS34的产物Ptd Ins3P的效应底物DFCP1在内质网的隔离膜上共定位。随着自噬小体的形成,HAP1与自噬小体的重要标志蛋白MAP1LC3(LC3)共定位并促进LC3点状聚集,说明了HAP1参与了自噬小体的形成及延伸过程。自噬的最后阶段为自噬小体与溶酶体的融合并降解包裹的蛋白及细胞器的过程。实验结果显示HAP1与Stx17相互作用并部分共定位,推测HAP1通过结合Stx17参与自噬小体与溶酶体的融合过程。利用RFPGFP-LC3的质粒表达系统实验发现过表达的HAP1显著增强自噬流的发生,而利用葡萄糖饥饿处理则进一步加剧了这一现象。在AS的原代神经元中,利用siRNA敲低HAP1的表达显著降低AS中过度增强的自噬水平。同时,我们发现AS皮质及海马神经元中加入自噬的抑制剂处理显著增多其由于自噬过强而减少的树突上神经嵴(spine)的数量。综上所述,我们的研究发现并验证了HAP1是UBE3A的一个内源底物,AS小鼠的中枢神经系统中由于UBE3A的失活导致HAP1的过度积累,高表达的HAP1加剧了AS小鼠的自噬水平严重影响了突触功能的发挥而导致疾病的发生。更多还原

【Abstract】 Angelman syndrome(AS)is a severe neurodevelopmental disorder caused by maternal mutation and paternal imprinting of the gene encoding UBE3 A,an E3 ubiquitin ligase.Although several potential target proteins of UBE3 A have been reported,how these proteins regulate neuronal development remains unclear.We performed a large-scale quantitative proteomic analysis using stable-isotope labeling of amino acids in mammals(SILAM)in mice with maternal Ube3 a mutation.Among the up-regμlated potential substrates,we identified huntingtin(Htt)-associated protein(HAP1),a protein that is involved in Huntington’s disease(HD),as a new in vivo target of UBE3 A.Stability and ubiquitination experiments for HAP1 were performed for substrate confirmation.Mass spectrometry analysis of HAP1 modifications further identified the ubiquitination sites regulated by UBE3 A.Remarkably,we observed the accelerated autophagy existed both in AS mouse and cortical neuron,while m TOR1 activity was not inhibited.Further,we demonstrate that HAP1 acts as a critical regμlator of the Ptd Ins3 K complex to promote autophagy by interacting with ATG14,and reveal that the coiled coil domain of HAP1 is sufficient for the interaction.What’s more,HAP1 participates in the formation of ATG14-VPS34-Beclin1 complex to a larger one in HEK293 T cells and mouse brain lysate.The increased activity of Ptd Ins3 K was mitigated by knocking down the expression of HAP1 in AS primary cortical neuron.HAP1 is co-localized with DFCP1,and this distribution pattern coincides with Ptd Ins3 P targeting to isolation membranes and promotion the nucleation of autophagosomes.MAP1LC3(LC3),as the mature autophagosome marker,is also co-expressed with HAP1 in the form of puncta,suggesting a role of HAP1 in mediating the autophagosome formation.In the final step,HAP1 targets Stx17 to enhance the fusion between autophagosome and lysosome.As a consequence,the autophagic flux is accelerated upon overexpressed HAP1 by monitoring the tandem RFP-GFP-LC3 expression signal,and glucose starvation treatment further enhances the flux progress.Strikingly,knocking down of HAP1 alleviated aberrant autophagy in primary neurons from AS mice.Concordantly,treatment of AS neurons with an autophagy inhibitor alleviated the reduction in density of dendritic spines.Together,our results identify HAP1 as an in vivo UBE3 A target that contributes to deregulated autophagy and synaptic dysfunction in the central nervous system of AS mouse.更多还原