Proteomics in Drosophila to Identify Autism Candidate Substrates of UBE3A

果蝇蛋白质组学鉴定 UBE3A 的自闭症候选底物

• 批准号: 7576681
• 负责人: LAWRENCE T REITER
• 金额: $ 31.33万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7576681
• 关键词: 15q; Adult; Affect; Alleles; Angelman Syndrome; Animal Model; Autistic Disorder; Binding; Biochemical Genetics; Biological Assay; Brain; Brain region; Candidate Disease Gene; Cells; Child; Chromosome abnormality; Chromosomes; Clinical; Collection; Condition; Defect; Developmental Delay Disorders; Drosophila genus; Drosophila melanogaster; Etiology; Exhibits; Eye; Family; Future; Gene Mutation; Gene Proteins; Genes; Genetic; Genetic Predisposition to Disease; Goals; Hand; Head; Heterogeneity; Human; Immunohistochemistry; Individual; Inherited; Lesion; Link; Mammals; Mass Spectrum Analysis; Mediating; Minor; Modeling; Molecular; Molecular Cytogenetics; Mus; Mutate; Neurodevelopmental Disorder; Neuromuscular Junction; Neurons; Numbers; Parents; Pathogenesis; Pathway interactions; Patients; Pattern; Phenotype; Play; Predisposition; Proteins; Proteomics; Public Health; Research; Risk; Role; Seizures; Surgical Flaps; Susceptibility Gene; System; Testing; Tissues; Ubiquitin; Ubiquitin-Protein Ligase Complexes; Ubiquitination; Validation; Variant; autism spectrum disorder; developmental disease; fly; gene function; genetic profiling; imprint; insight; interstitial; mouse model; multicatalytic endopeptidase complex; protein expression; tool; ubiquitin ligase; ubiquitin-protein ligase

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.

描述（由申请人提供）：自闭症谱系障碍（ASD）在10,000名儿童中影响约60个，但其中只有约10％的人是与公认原因相关的自闭症。了解与自闭症相关的一种罕见且严重的发育障碍，在Angelman综合征（AS）中失去失调的分子途径可能会提供关键见解，从而鉴定自闭症易感性基因和途径。所有自闭症病例中约有3％是由于包含AS基因UBE3A的区域的母体重复。因此，编码泛素连接酶UBE3A蛋白质靶标的基因中的变体可能会赋予自闭症的遗传敏感性，甚至引起ASD表型。在这里，我们描述了一种利用强大的遗传模型有机体果蝇的蛋白质组学策略，以鉴定人Ube3a和Fly Dube3a的蛋白质靶标。使用GAL4/UAS系统，UBE3A将在苍蝇的大脑中过表达，以增加或降低UBE3A/DUBE3A蛋白靶标的水平。然后，我们将通过旋转辅助蛋白质组学分析和质谱法识别这些靶标。通过眼睛和神经肌肉连接的遗传相互作用，293T细胞的结合测定以及在UBE3A缺乏和过表达的小鼠的大脑中，将验证潜在靶标。我们预计这些研究将为我们提供新的自闭症候选基因，以供表现出可遗传自闭症风险的家庭的未来验证。为此，我们提出了以下特定目的：特定目标1：使用果蝇头提取物中的蛋白质组学分析鉴定潜在的UBE3A和DUBE3A调节的蛋白质。该目标的完成将导致潜在的Dube3a和UBE3A调节的蛋白质，以进行随后的验证和自闭症遗传研究。特定目的2：验证潜在目标与Dube3a之间的物理，生化和遗传相互作用。该目标将检验以下假设：AIM＃1中检测到的蛋白质由Dube3a直接或间接调节。特定目标3：确定UBE3A不足和过表达小鼠的大脑中UBE3A调节的蛋白质表达模式是否改变。该目标将证明在Angelman综合征和近端15Q重复自闭症的小鼠模型中，UBE3A靶蛋白与大脑功能之间的联系之间存在联系。公共卫生相关性：这项研究的主要目标是鉴定由UBE3A调节的蛋白质，并研究这些蛋白质在某些自闭症情况下也会失调或突变的可能性。了解UBE3A的水平增加如何导致分子水平的自闭症表型将有助于我们更好地识别和治疗拜访的自闭症中的基本责任。