Rescue Analysis of Utrophin & NMJ Support by Syntrophin

Utropin的拯救分析

• 批准号: 6925532
• 负责人: ROBERT W SEALOCK
• 金额: $ 30.39万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6925532
• 关键词: acetylcholine; acetylcholinesterase; agrin; binding proteins; chimeric proteins; genetically modified animals; in situ hybridization; laboratory mouse; laboratory rat; membrane proteins; messenger RNA; muscle proteins; muscular dystrophy; neuromuscular junction; neurotransmitter receptor; phenotype; protein protein interaction; protein sequence; proteomics; receptor binding; site directed mutagenesis; striated muscles

DESCRIPTION (provided by applicant): The syntrophins are a family of peripheral membrane adapter proteins that function in association with dystrophin, utrophin, and the dystrobrevins, all of which are proteins of the surface membrane of skeletal muscle and implicated in the important human disease, Duchenne muscular dystrophy (DMD). Alpha-syntrophin is the major syntrophin in muscle. In the alpha-syntrophin knockout mouse, the postsynaptic membrane at the neuromuscular junction shows major biochemical and morphological defects including low amounts of acetylcholine receptors and acetylcholinesterase, a complete absence of utrophin, immature appearing contacts, junctional folds that are disorganized and few in number, and altered distribution of AChR. Thus, there is a utrophin and NMJ support function of alpha-syntrophin. The first two aims of the project are to intended to extend current understanding that the other syntrophins of muscle (beta1, beta2, and probably gamma2) are not redundant with alpha-syntrophin. They are: 1) Test the hypothesis that transgenically expressed beta 1-syntrophin will restore utrophin to adult alpha-syntrophin 4- junctions but will not rescue other, or all other, aspects of the phenotype. 2) Test the hypothesis that transgenically expressed beta 2-syntrophin will restore no aspects of the alpha-syntrophin -/- phenotype. The results will provide a solid framework for molecular analysis of the mechanisms of the support function. Aim 3) Identify the critical functional domains of the alpha-syntrophin molecule by transgenic expression in alpha-syntrophin -/- mice of a) chimeric proteins containing domains of beta substituted into alpha-syntrophin (or the converse, alpha into beta) and/or b) alpha-syntrophin specifically mutagenized at selected sites. The results will identify domains and implicate syntrophin-dependent pathways. Aim 4) Determine whether low levels of utrophin mRNA, inability of the membrane to accept utrophin incorporation, or both contribute to the lack of utrophin at alpha-syntrophin -/- NMJs. If applicable, use these tools to analyze the transgenic mice. 5) Seek to identify determinants in the AChR accumulation pathway-- AChR mRNA levels, total AChR expression, cell surface AChR expression, AChR clustering response to agrin, stability of agrin-induced clusters--that may contribute to the low AChR content of alpha-syntrophin 4- NMJs. If applicable, use the understanding so generated to analyze the transgenic mice.

描述（由申请人提供）：综合蛋白是与肌营养不良蛋白，乌特罗蛋白和肌肉蛋白蛋白质相关的外周膜衔接蛋白家族，所有这些都与骨骼肌表面膜的蛋白质相关，并且与重要的人类疾病有关，肌肉疾病，二刺肌肉dmdmd（dmdmd）。 α-促链蛋白是肌肉中的主要综合症。在α-促蛋白敲除小鼠中，神经肌肉连接处的突触后膜显示出主要的生化和形态学缺陷，包括低量的乙酰胆碱受体和乙酰胆碱酯酶，完全没有尿蛋白酶，不成熟的出现接触，连接的折叠和几个数字，并且数量不足。因此，α-链蛋白具有乌托蛋白和NMJ支持函数。该项目的前两个目的是旨在扩展当前的理解，即肌肉的其他综合蛋白（Beta1，beta2和可能的γ2）并不是α-链球菌的冗余。它们是：1）检验以下假设：传统表达的β1-链霉素将恢复乌托蛋白为成年α-链链蛋白4-连接，但不会营救表型的其他或所有其他方面。 2）检验以下假设：转基因表达的β2合子将不会恢复α-促链蛋白 - / - 表型的任何方面。结果将为支持函数机制的分子分析提供一个可靠的框架。目标3）通过转基因表达在α-串联蛋白 - / - 小鼠中鉴定α-伴侣蛋白分子的关键功能结构域A）含有β蛋白的嵌合蛋白，这些嵌合蛋白含有β的结构域，被取代为α-串联蛋白（或Converse，或Converse，Alpha，Alpha，Alpha，Alpha）和/或b）alpha-stocepers alphasyntroplins propsyntriphins crothpha crothtrimen。结果将识别域并暗示依赖综合素的途径。目标4）确定低水平的乌托蛋白mRNA是否能够接受乌托蛋白掺入或两者是否有助于在α-链霉素 - / - NMJS上缺乏乌托蛋白。如果适用，请使用这些工具分析转基因小鼠。 5）寻求鉴定ACHR积累途径中的决定因素 - ACHR mRNA水平，总ACHR表达，细胞表面ACHR表达，对Agrin对Agrin的ACHR聚类反应，Agrin诱导的簇的稳定性 - 可能有助于α-串联蛋白4- NMJS的α-syntrophin 4- nmjs的ACHR含量较低。如果适用，请使用如此生成的理解来分析转基因小鼠。