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都起着至关重要的作用 - 在核转运，纺锤体形成，核包膜形成和原发性纤毛形成中起着至关重要的作用。它也与衰老，癌症和病毒感染有关。 RCC1与染色质相关，并在有丝分裂染色体周围产生RANGTP梯度。我们确定了RCC1的新的翻译后修饰，其中切除了启动MET残留物，并且暴露的1-氨基甲基是甲基化的。 RCC1的甲基化突变体引起有丝分裂缺陷。我们已经确定了负责此修饰的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标包括肿瘤抑制蛋白RB。 NRMT沉默会导致有丝分裂缺陷。鉴于RCC1函数的关键重要性，其对1-N-甲基化的调节以及这种异常修改的高生物学意义，我们计划关注以下目标：1。当前的RCC1调节模型是否正确？已经提出，RCC1作为其催化作用的重要组成部分动态循环，而N末端尾巴的磷酸化稳定了有丝分裂期间的染色质缔合。我们将通过用束缚融合蛋白和磷酸化突变体代替内源性RCC1来严格测试该模型。我们将询问RCC1动力学是否在有丝分裂和凋亡中很重要。使用动物模型，我们还将测试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，并询问小鼠是否显示出染色体误差的水平增加。为了确定RCC1与染色质弱的关联是否是主要缺陷，我们将在KO MEF中表达染色质连续的RCC1，并询问我们是否挽救了正常有丝分裂。我们提出了这样的假设，即这种修饰的一般函数是促进染色质结合。 RB的损失会导致基因组不稳定性，我们将测试不可用的RB是否会导致相似的缺陷，以及是否将此类缺陷连接到其将CAP-3D募集到CencerRomeres。 3。确定1-N-甲基化的控制机制。 NRMT的核定位可能会限制对某些靶蛋白的访问，从而阻止它们被甲基化。为了测试这个想法，我们将通过表达具有核出口信号的酶的版本来“侧面” NRMT。通过质谱法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脱甲基酶。我们将使用细胞质提取物净化这种酶活性。最后，我们发现在表达E7癌蛋白的HeLa细胞中，RB未被发现甲基化。我们将解决阻止这些细胞中RB甲基化的潜在机制。

项目成果

期刊论文数量（29）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

The adapter importin-alpha provides flexible control of nuclear import at the expense of efficiency.

适配器 importin-alpha 提供了核导入的灵活控制，但以效率为代价。

DOI：
10.1038/msb4100160
发表时间：
2007
期刊：
Molecular systems biology
影响因子：
9.9
作者：
Riddick,Greg;Macara,IanG
通讯作者：
Macara,IanG

A systems analysis of importin-{alpha}-{beta} mediated nuclear protein import.

DOI：
10.1083/jcb.200409024
发表时间：
2005-03-28
期刊：
The Journal of cell biology
影响因子：
0
作者：
Riddick G;Macara IG
通讯作者：
Macara IG

Exportin-5, a novel karyopherin, mediates nuclear export of double-stranded RNA binding proteins.

DOI：
10.1083/jcb.200110082
发表时间：
2002-01-07
期刊：
The Journal of cell biology
影响因子：
0
作者：
Brownawell AM;Macara IG
通讯作者：
Macara IG

NRMT2 is an N-terminal monomethylase that primes for its homologue NRMT1.

DOI：
10.1042/bj20131163
发表时间：
2013-12-15
期刊：
The Biochemical journal
影响因子：
0
作者：
Petkowski JJ;Bonsignore LA;Tooley JG;Wilkey DW;Merchant ML;Macara IG;Schaner Tooley CE
通讯作者：
Schaner Tooley CE

Depletion of the adaptor protein NCK increases UV-induced p53 phosphorylation and promotes apoptosis.

接头蛋白 NCK 的消耗会增加紫外线诱导的 p53 磷酸化并促进细胞凋亡。