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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

基本信息

批准号：
8292310
负责人：
LAWRENCE T REITER
金额：
$ 2.96万
依托单位：
UNIVERSITY OF TENNESSEE HEALTH SCI CTR
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-09-01 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8292310
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 Defect Developmental Delay Disorders Drosophila genus Drosophila melanogaster Etiology Exhibits Eye Family Future Gene Mutation Gene Proteins Genes Genetic Genetic Models Genetic Predisposition to Disease Goals Hand Head Health Heterogeneity Human Immunohistochemistry Individual Inherited Lesion Link Mammals Mass Spectrum Analysis Mediating Minor Modeling Molecular Molecular Cytogenetics Mus Mutate Neurodevelopmental Disorder Neuromuscular Junction Neurons Parents Pathogenesis Pathway interactions Patients Pattern Phenotype Play Predisposition Proteins Proteomics 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

项目摘要

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) 影响约 10,000 名儿童中的 60 名，但其中只有约 10% 的人患有与已知原因相关的自闭症。了解天使综合征（AS）（一种与自闭症相关的罕见且严重的发育障碍）中失调的分子通路，可能会为识别自闭症易感性基因和通路提供关键见解。大约 3% 的自闭症病例是由母体含有 AS 基因 UBE3A 的区域重复引起的。因此，编码泛素连接酶 UBE3A 的蛋白质靶标的基因变异可能会赋予自闭症遗传易感性，甚至导致自闭症谱系障碍 (ASD) 表型。在这里，我们描述了利用强大的遗传模型生物体果蝇的蛋白质组学策略来识别人类 UBE3A 和果蝇 Dube3a 的蛋白质靶标。使用 GAL4/UAS 系统，UBE3A 将在果蝇大脑中过度表达，以增加或减少 UBE3A/Dube3a 蛋白质靶标的水平。然后，我们将通过 Rotofor 辅助的蛋白质组分析和质谱分析来识别这些靶标。潜在靶标将通过眼睛和神经肌肉接头中的遗传相互作用、293T 细胞中的结合测定以及 Ube3a 缺陷和过度表达小鼠大脑中的免疫组织化学进行验证。我们预计这些研究将为我们提供新的自闭症候选基因，以便将来在显示出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中的蛋白质组学分析来鉴定潜在的 UBE3A 和 Dube3a 调节蛋白。这一目标的完成将收集潜在的 Dube3a 和 UBE3A 调节蛋白，用于后续验证和自闭症遗传学研究。具体目标 2：验证潜在靶标与 Dube3a 之间的物理、生化和遗传相互作用。该目标将检验目标 #1 中检测到的蛋白质直接或间接受 Dube3a 调节的假设。具体目标 3：确定 Ube3a 缺陷和过度表达小鼠大脑中 UBE3A 调节的蛋白表达模式是否发生改变。这一目标将证明 Ube3a 靶蛋白与 Angelman 综合征和近端 15q 重复自闭症小鼠模型中大脑功能改变之间的联系。公共健康相关性：这项研究的主要目标是鉴定受 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。了解 UBE3A 水平升高如何在分子水平上导致自闭症表型将有助于我们更好地识别和治疗特发性自闭症的潜在原因。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Variation in Dube3a expression affects neurotransmission at the Drosophila neuromuscular junction.

DOI：
10.1242/bio.20148045
发表时间：
2015-05-06
期刊：
Biology open
影响因子：
2.4
作者：
Valdez C;Scroggs R;Chassen R;Reiter LT
通讯作者：
Reiter LT

Drosophila Ube3a regulates monoamine synthesis by increasing GTP cyclohydrolase I activity via a non-ubiquitin ligase mechanism.

DOI：
10.1016/j.nbd.2010.12.001
发表时间：
2011-03
期刊：
NEUROBIOLOGY OF DISEASE
影响因子：
6.1
作者：
Ferdousy, Faiza;Bodeen, William;Summers, Kyle;Doherty, Olugbenga;Wright, O'Neil;Elsisi, Nahed;Hilliard, George;O'Donnell, Janis M.;Reiter, Lawrence T.
通讯作者：
Reiter, Lawrence T.

Altered serotonin, dopamine and norepinepherine levels in 15q duplication and Angelman syndrome mouse models.