Proteomics in Drosophila to Identify Autism Candidate Substrates of UBE3A

描述（由申请人提供）：自闭症谱系障碍（ASD）影响约60 / 10000的儿童，但在这些个体中只有约10%是与已知原因相关的自闭症。Angelman综合征（AS）是一种罕见且严重的与自闭症相关的发育障碍，了解其分子通路失调可能为识别自闭症易感基因和通路提供关键见解。大约3%的自闭症病例是由于母亲复制了含有AS基因UBE3A的区域。因此，编码泛素连接酶UBE3A蛋白靶点的基因变异可能赋予自闭症的遗传易感性，甚至导致自闭症表型。在这里，我们描述了一种蛋白质组学策略，利用强大的遗传模式生物黑腹果蝇来鉴定人类UBE3A和苍蝇Dube3a的蛋白质靶点。使用GAL4/UAS系统，UBE3A将在果蝇的大脑中过度表达，以增加或降低UBE3A/Dube3a蛋白靶标的水平。然后，我们将通过rotofor辅助蛋白质组学分析和质谱法鉴定这些目标。潜在靶点将通过眼睛和神经肌肉连接处的遗传相互作用、293T细胞的结合试验和ube3a缺陷和过表达小鼠大脑的免疫组织化学来验证。我们预计这些研究将为我们提供新的自闭症候选基因，用于未来在具有遗传性自闭症风险的家庭中进行验证。为此，我们提出以下具体目标：具体目标1：利用果蝇头部提取物的蛋白质组学分析鉴定潜在的UBE3A和Dube3a调节蛋白。这一目标的完成将导致收集潜在的Dube3a和UBE3A调节蛋白，用于后续验证和自闭症遗传研究。具体目标2：验证潜在靶点与Dube3a之间的物理、生化和遗传相互作用。这一目的将验证aim #1中检测到的蛋白质直接或间接受Dube3a调节的假设。特异性目的3：确定UBE3A调节蛋白表达模式是否在UBE3A缺陷和过表达小鼠的大脑中发生改变。这一目标将证明Ube3a靶蛋白与天使综合征和近端15q重复自闭症小鼠模型中脑功能改变之间的联系。公共卫生相关性：本研究的主要目标是确定由UBE3A调节的蛋白质，并调查这些蛋白质在某些自闭症病例中也失调或突变的可能性。在分子水平上了解UBE3A水平的升高是如何导致自闭症表型的，将有助于我们更好地识别和治疗特发性自闭症的潜在风险。

DESCRIPTION (provided by applicant): Autism spectrum disorders (ASD) affect ~60 in 10,000 children but in only ~10% of these individuals is autism associated with a recognized cause. Understanding the molecular pathways dysregulated in Angelman syndrome (AS), a rare and severe developmental disorder related to autism, may provide key insights leading to identification of autism susceptibility genes and pathways. Approximately 3% of all autism cases result from maternal duplications of the region containing the AS gene UBE3A. Variants in genes encoding protein targets of the ubiquitin ligase UBE3A may, therefore, confer a genetic susceptibility to autism or even cause an ASD phenotype. Here we describe a proteomics strategy utilizing the powerful genetic model organism Drosophila melanogaster to identify protein targets of human UBE3A and fly Dube3a. UBE3A will be over-expressed in the brains of flies using the GAL4/UAS system in order to increase or decrease the levels of UBE3A/Dube3a protein targets. We will then identify these targets by Rotofor-assisted proteomic profiling and mass spectrometry. Potential targets will be validated though genetic interactions in the eye and neuromuscular junction, binding assays in 293T cells and immunohistochemistry in the brains of both Ube3a-deficient and over-expressing mice. We anticipate that these studies will provide us with new autism candidate genes for future validation in families that demonstrate heritable autism risk. To this end, we propose the following specific aims: Specific Aim 1: To identify potential UBE3A and Dube3a regulated proteins using proteomic profiling in Drosophila head extracts. Completion of this aim will result in a collection of potential Dube3a and UBE3A regulated proteins for subsequent validation and autism genetic studies. Specific Aim 2: To validate physical, biochemical and genetic interactions between potential targets and Dube3a. This aim will test the hypothesis that the proteins detected in Aim #1 are regulated directly or indirectly by Dube3a. Specific Aim 3: To determine if UBE3A regulated protein expression patterns are altered in the brains of Ube3a deficient and over-expression mice. This aim will demonstrate a link between Ube3a target proteins and altered brain function in the mouse models of Angelman syndrome and proximal 15q duplication autism. PUBLIC HEALTH RELEVANCE: The primary goal of this research is to identify proteins regulated by UBE3A and to investigate the possibility that these proteins are also dysregulated or mutated in some cases of autism. Understanding how increased levels of UBE3A result in an autism phenotype at the molecular level will help us better identify and treat the underlying liability in idopathic autism.