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Functional Analysis of LEM-domain Nuclear Proteins

LEM 结构域核蛋白的功能分析

基本信息

批准号：
6891432
负责人：
Katherine L Wilson
金额：
$ 26.75万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-05-01 至 2007-04-30
项目状态：
已结题

项目摘要

Emerin is a nuclear membrane protein that bines lamina filaments. Emerin mutations cause Emery-Dreifuss muscular dystrophy, affecting heart, skeletal muscle, and tendons. Emerin belongs to the conserved LEM-domain family of nuclear proteins. The LEM-domain of emerin binds BAF (barrier to autointegration factor), a conserved chromatin protein of unknown function. We propose that LEM proteins structurally link chromatin (via BAF) to the lamina, and also bind partners that mediate DNA replication or transcriptional repression. We propose to determine the essential function of three conserved LEM proteins in C.elegans: emerin, MAN1 and lem-3. In C. elelgans, all or most cells express emerin and MAN1, but the RNAi-induced loss of emerin or MAN1 has no phenotype. However, loss of both proteins is lethal in embryos. Thus, emerin and MAN1 have essential function(s). We will test the hypothesis that all three LEM proteins bind directly to BAF and lamin, and use site-directed mutagenesis to map their binding regions. We will use RNAi to deplete each LEM protein, and pairs of LEM proteins to test for overlapping and cell-type-specific functions in vivo. By identifying and characterizing new binding partners for emerin, MAN1 and lem- 3, we will test our hypothesis that LEM proteins are directly involved in replication, transcription, or lamin dynamics. We will determine if mutant LEM proteins, whose binding activities are defined in vitro, can rescue lethality of emerin: MAN1 (RNAi) embryos. WE will screen for mutations that are synthetically lethal in an emerin null or MAN1 null background, to identify proteins that mediate the essential functions of emerin and MAN1. This work will be the first genetic analysis of the nuclear lamina. Our work will yield basic knowledge about the functions of LEM proteins, and their interactions with lamins and BAF. This work can be extended to humans to predict the molecular mechanisms of heart disease and muscular dystrophy caused by loss of emerin.

Emerin 是一种结合核纤丝的核膜蛋白。 Emeryin 突变会导致 Emery-Dreifuss 肌营养不良症，影响心脏、骨骼肌和肌腱。 Emerin 属于保守的核蛋白 LEM 结构域家族。 emerin 的 LEM 结构域结合 BAF（自整合因子屏障），这是一种功能未知的保守染色质蛋白。我们提出 LEM 蛋白在结构上将染色质（通过 BAF）连接到核纤层，并且还结合介导 DNA 复制或转录抑制的伙伴。我们建议确定线虫中三种保守的 LEM 蛋白的基本功能：emerin、MAN1 和 lem-3。在线虫中，所有或大多数细胞表达 emerin 和 MAN1，但 RNAi 诱导的 emerin 或 MAN1 丢失没有表型。然而，这两种蛋白质的丢失对于胚胎来说是致命的。因此，emerin 和 MAN1 具有重要的功能。我们将测试所有三种 LEM 蛋白直接与 BAF 和核纤层蛋白结合的假设，并使用定点诱变来绘制它们的结合区域。我们将使用 RNAi 来消耗每个 LEM 蛋白，并使用成对的 LEM 蛋白来测试体内重叠和细胞类型特异性功能。通过鉴定和表征 emerin、MAN1 和 lem-3 的新结合伴侣，我们将检验我们的假设，即 LEM 蛋白直接参与复制、转录或核纤层蛋白动力学。我们将确定突变的 LEM 蛋白（其结合活性在体外确定）是否可以挽救 emerin: MAN1 (RNAi) 胚胎的致死率。我们将筛选在 emerin 缺失或 MAN1 缺失背景下合成致死的突变，以鉴定介导 emerin 和 MAN1 基本功能的蛋白质。这项工作将是首次对核层进行遗传分析。我们的工作将产生有关 LEM 蛋白功能及其与核纤层蛋白和 BAF 相互作用的基本知识。这项工作可以扩展到人类，以预测由艾默林缺失引起的心脏病和肌肉萎缩症的分子机制。