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The RAN GTPase

RAN GTP 酶

基本信息

批准号：
8688255
负责人：
IAN G MACARA
金额：
$ 34.7万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-08-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8688255
关键词：
Address Affinity Aging Animal Model Apoptosis Area Binding Biological Biological Preservation Biological Process Cell Line Cell division Cells Centromere Chimeric Proteins Chromatin Chromosomes Cilia DNA Defect Embryo Enzymes Eukaryotic Cell Excision Fibroblasts Funding Genetic Genomic Instability Guanine Nucleotide Exchange Factors Guanosine Triphosphate Phosphohydrolases Hela Cells Kinetochores Knockout Mice Laboratories Life Malignant Neoplasms Mass Spectrum Analysis Methylation Methyltransferase Mitosis Mitotic Mitotic Chromosome Modeling Modification Mus Mutation N-terminal Names Nuclear Nuclear Envelope Nuclear Export Oncogene Proteins Other Genetics Phosphorylation Play Post-Translational Protein Processing Premature aging syndrome Proteins Reagent Regulation Role Running Signal Transduction Tail Testing Tumor Suppressor Proteins Virus Diseases amino group centromere autoantigen 80K centromere protein A enzyme activity genetic manipulation insight interest meetings mutant novel nucleocytoplasmic transport prevent segregation tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): RCC1 is a guanine nucleotide exchange factor for the Ran GTPase. It plays vital roles in all eukaryotic cells - in nuclear transport, spindle formation, nuclear envelope formation, and primary cilium formation. It has also been implicated in aging, cancer, and viral infection. RCC1 associates with chromatin, and generates a RanGTP gradient around mitotic chromosomes. We identified a new post-translational modification of RCC1, in which the initiating Met residue is excised, and the exposed 1-amino group is methylated. Mon- methylatable mutants of RCC1 cause mitotic defects. We have identified the 1-N-methyltransferase responsible for this modification, which we named NRMT. Other interesting targets for methylation by NRMT include the tumor suppressor protein RB. Silencing of NRMT causes mitotic defects. Given the pivotal importance of RCC1 function, its regulation by 1-N-methylation, and the high biological significance of this unusual modification, we plan to focus on the following aims: 1. Are current models for RCC1 regulation correct? It has been proposed that RCC1 cycles dynamically on and off chromatin, as an essential part of its catalytic action, and that phosphorylation of the N-terminal tail stabilizes chromatin association during mitosis. We will rigorously test this model, by replacing endogenous RCC1 with tethered fusion proteins and phosphorylation mutants. We will ask if RCC1 dynamics are important in mitosis and apoptosis. Using an animal model, we will also test whether Ran and RCC1 are involved in tumorigenesis. 2. Identification of the biological functions of NRMT, using a knockout mouse and MEFs. We will generate KO mice lacking NRMT, and ask if the mice display increased levels of chromosome mis- segregation. To determine if weakened association of RCC1 with chromatin is the primary defect we will express a chromatin-tethered RCC1 in KO MEFs and ask if we rescue normal mitosis. We propose the hypothesis that a general function for this modification is to facilitate chromatin binding. Loss of RB causes genomic instability, and we will test if a non-methylatable RB causes similar defects, and if such defects are connected to its recruitment of CAP-3D to centromeres. 3. Determination of control mechanisms for 1-N-methylation. The nuclear localization of NRMT might limit access to certain target proteins, thereby preventing them from being methylated. To test this idea, we will "knock sideways" the NRMT by expressing a version of the enzyme that possesses a nuclear export signal. Methylated proteins will be compared by mass spectrometry to cells expressing wild type NRMT. A second hypothesis is that there exists a cytoplasmic demethylase. We will use cytoplasmic extracts to purify such an enzyme activity. Finally, we discovered that in HeLa cells, which express the E7 oncoprotein, RB is not detectably methylated. We will address the underlying mechanism that blocks RB methylation in these cells.

描述（由申请人提供）：RCC1 是 Ran GTPase 的鸟嘌呤核苷酸交换因子。它在所有真核细胞中发挥着至关重要的作用——核运输、纺锤体形成、核膜形成和初级纤毛形成。它还与衰老、癌症和病毒感染有关。 RCC1 与染色质结合，并在有丝分裂染色体周围产生 RanGTP 梯度。我们发现了 RCC1 的一种新的翻译后修饰，其中起始 Met 残基被切除，暴露的 1-氨基被甲基化。 RCC1 的单甲基化突变体会导致有丝分裂缺陷。我们已经确定了负责这种修饰的 1-N-甲基转移酶，我们将其命名为 NRMT。 NRMT 甲基化的其他有趣靶点包括肿瘤抑制蛋白 RB。 NRMT 沉默会导致有丝分裂缺陷。鉴于 RCC1 功能的关键重要性、其通过 1-N-甲基化的调节以及这种不寻常修饰的高度生物学意义，我们计划重点关注以下目标： 1. 目前的 RCC1 调节模型是否正确？有人提出，RCC1 动态地循环打开和关闭染色质，这是其催化作用的重要组成部分，并且 N 末端尾部的磷酸化可稳定有丝分裂期间的染色质结合。我们将通过用束缚融合蛋白和磷酸化突变体替换内源性 RCC1 来严格测试该模型。我们将询问 RCC1 动态在有丝分裂和细胞凋亡中是否重要。我们还将使用动物模型来测试 Ran 和 RCC1 是否参与肿瘤发生。 2. 使用基因敲除小鼠和 MEF 鉴定 NRMT 的生物学功能。我们将产生缺乏 NRMT 的 KO 小鼠，并询问小鼠是否表现出染色体错误分离水平增加。为了确定 RCC1 与染色质关联减弱是否是主要缺陷，我们将在 KO MEF 中表达染色质束缚的 RCC1，并询问我们是否可以挽救正常的有丝分裂。我们提出这样的假设：这种修饰的一般功能是促进染色质结合。 RB 的丢失会导致基因组不稳定，我们将测试不可甲基化的 RB 是否会导致类似的缺陷，以及这些缺陷是否与其将 CAP-3D 招募到着丝粒有关。 3.确定1-N-甲基化的控制机制。 NRMT 的核定位可能会限制某些靶蛋白的进入，从而防止它们被甲基化。为了测试这个想法，我们将通过表达具有核输出信号的酶版本来“敲击”NRMT。将通过质谱分析将甲基化蛋白与表达野生型 NRMT 的细胞进行比较。第二个假设是存在细胞质去甲基酶。我们将使用细胞质提取物来纯化这种酶活性。最后，我们发现在表达 E7 癌蛋白的 HeLa 细胞中，未检测到 RB 甲基化。我们将探讨阻止这些细胞中 RB 甲基化的潜在机制。