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

RNA 干扰和“细胞内”重构测定作为揭示泛素蛋白连接酶 E6-AP 细胞功能的工具

• 批准号: 70345950
• 负责人: Professor Dr. Martin Scheffner
• 金额: --
• 依托单位: Arbeitsgruppe Zelluläre Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2008
• 资助国家: 德国
• 起止时间: 2007-12-31 至 2013-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/70345950?language=en
• 关键词: RNA; inteference; cellulo; reconstitution; assays

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.

UBE 3A基因的遗传改变与Angelman综合征（AS）的发生有关，AS是一种神经系统疾病。UBE 3A基因编码泛素-蛋白连接酶E6-AP，在AS患者中发现的所有遗传改变都破坏了其泛素-蛋白连接酶功能。然而，关于E6-AP的细胞功能以及E6-AP活性的丧失如何影响神经元的功能，人们知之甚少。我们最近已经表明，通过RNA干扰（RNAi）调节E6-AP表达水平会影响细胞的活力，并建立了蛋白质组学方法来鉴定E6-AP的相互作用伙伴和底物。在该提案中，将采用基于RNAi的筛选系统来鉴定在E6-AP下游操作和/或E6-AP发挥其细胞功能所需的蛋白质和过程。此外，E6-AP与通过该筛选鉴定的推定结合配偶体的相互作用将通过生物化学和细胞生物学手段表征。最后，神经元细胞系和原代神经元来源于Ube 3a缺陷型小鼠和野生型小鼠，分别将用于获得洞察E6-AP在神经元中的功能。两者合计，拟议的研究将有助于确定的细胞过程，由E6-AP控制，并作为E6-AP活性丧失的结果，在AS患者中解除管制。

项目成果

Mutation of HERC2 causes developmental delay with Angelman-like features

• DOI: 10.1136/jmedgenet-2012-101367
• 发表时间: 2013-02-01
• 期刊: JOURNAL OF MEDICAL GENETICS
• 影响因子: 4
• 作者: Harlalka, Gaurav V.;Baple, Emma L.;Crosby, Andrew H.
• 通讯作者: Crosby, Andrew H.

Role of the ubiquitin ligase E6AP/UBE3A in controlling levels of the synaptic protein Arc

• DOI: 10.1073/pnas.1302792110
• 发表时间: 2013-05-28
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Kuehnle, Simone;Mothes, Benedikt;Scheffner, Martin
• 通讯作者: Scheffner, Martin