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Analysis of supramolecular assemblies of NE81, the first lamin-like protein in a unicellular organism

单细胞生物中第一个核纤层蛋白样蛋白 NE81 的超分子组装体分析

基本信息

批准号：
180581162
负责人：
Professor Dr. Ralph Gräf
金额：
--
依托单位：
Arbeitsgruppe Zellbiologie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2010
资助国家：
德国
起止时间：
2009-12-31 至 2018-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/180581162?language=en
关键词：
Analysis supramolecular assemblies NE81 first

项目摘要

Lamins build the nuclear lamina and are required for mechanical stabilization of cells, chromatin organization, gene expression, cell cycle progression and cell migration. This became evident by the pathogenesis of laminopathies, which are caused by mutations in genes encoding lamin A/C. Despite these functions of universal importance, lamins have so far only been found in metazoans. With Dictyostelium NE81 we have now identified a protein associated with the inner nuclear envelope, whose properties justify denomination as a lamin-like protein in a lower eukaryote. This is based both on its primary sequence and an initial functional characterization. Here we propose a work schedule that aims at the verification of NE81 as a primitive lamin and the analysis of its putative lamin-like functions. First, we will generate and analyze NE81 depletion mutants, mutants in CDK1 kinase target sequences and isoprenylation mutants. Second, we will examine the expected assembly properties of NE81 on the biochemical and microscopic level. Third, we will characterize the role of the enzymatic machinery for prelamin processing, which is completely conserved in Dictyostelium and is expected to be required for NE81 function. Fourth we intend to verify putative binding partners of NE81 at the nuclear envelope and its binding to chromatin. The discovery of a lamin-like protein in a unicellular organism is not only very interesting in the light of evolution, it could also provide a simple experimental platform for studies of the molecular basis of laminopathies.

核纤层蛋白构建核纤层，并且是细胞的机械稳定、染色质组织、基因表达、细胞周期进程和细胞迁移所需的。这在核纤层蛋白病的发病机制中变得明显，核纤层蛋白病是由核纤层蛋白A/C编码基因突变引起的。尽管这些功能具有普遍的重要性，核纤层蛋白到目前为止只在后生动物中发现。利用Dictyosteoprotein NE 81，我们现在已经鉴定出与内核被膜相关的蛋白质，其性质证明命名为低等真核生物中的层粘连蛋白样蛋白。这是基于其主要序列和初始功能表征。在这里，我们提出了一个工作计划，旨在验证NE 81作为原始层粘连蛋白并分析其推定的层粘连蛋白样功能。首先，我们将产生并分析NE 81缺失突变体、CDK 1激酶靶序列突变体和异戊二烯化突变体。其次，我们将在生物化学和微观水平上研究NE 81的预期组装特性。第三，我们将表征前层蛋白加工的酶机制的作用，这是完全保守的Dictyosteoprotein，预计需要NE 81功能。第四，我们打算验证NE 81在核膜上的假定结合伴侣及其与染色质的结合。在单细胞生物体中发现层粘连蛋白样蛋白不仅在进化方面非常有趣，而且还可以为研究层粘连蛋白病的分子基础提供一个简单的实验平台。