RNA inteference and "in cellulo" reconstitution assays as tools to uncover the cellular functions of the ubiquitin-protein ligase E6-AP

UBE3A基因的基因改变与Angelman综合征(AS)的发展有关，AS是一种神经疾病。UBE3A基因编码泛素蛋白连接酶E6-AP，AS患者中发现的所有基因改变都使其泛素蛋白连接酶功能失活。然而，对于E6-AP的细胞功能(S)以及E6-AP活性的丧失如何影响神经元的功能，人们通常知之甚少。我们最近发现，RNA干扰(RNAi)对E6-AP表达水平的调节会影响细胞的活力，并建立了蛋白质组学方法来确定E6-AP的相互作用伙伴和底物。在这项建议中，将使用基于RNAi的筛选系统来识别E6-AP下游工作和/或E6-AP发挥其细胞功能所需的蛋白质和过程。此外，E6-AP与本筛选确定的假定结合伙伴的相互作用将通过生化和细胞生物学手段进行表征。最后，分别来自Ube3a缺陷小鼠和野生型小鼠的神经细胞系和原代神经元将被用来深入了解E6-AP在神经元中的功能。综上所述，拟议的研究将有助于识别由E6-AP控制的细胞过程，并作为E6-AP活性丧失的结果，在AS患者中解除调控。

Genetic alterations of the UBE3A gene have been causally involved in the development of the Angelman syndrome (AS), a neurological disorder. The UBE3A gene encodes the ubiquitin-protein ligase E6-AP and all of the genetic alterations found in AS patients inactivate its ubiquitin-protein ligase function. However, only little is known about the cellular function(s) of E6-AP in general and how loss of E6-AP activity affects the functionality of neurons in particular. We have recently shown that modulation of E6-AP expression levels by RNA interference (RNAi) affects the viability of cells and have set up proteomic approaches to identify interaction partners and substrates of E6-AP. In this proposal, an RNAi-based screening system will be employed to identify proteins and processes that operate downstream of E6-AP and/or are required for E6-AP to exert its cellular functions. Furthermore, the interaction of E6-AP with putative binding partners identified by this screen will be characterized by biochemical and cell biological means. Finally, neuronal cell lines and primary neurons derived from Ube3a deficient mice and wild-type mice, respectively, will be used to obtain insight into the function of E6-AP in neurons. Taken together, the proposed studies will contribute to the identification of cellular processes that are controlled by E6-AP and, as a consequence of loss of E6-AP activity, are deregulated in AS patients.