The role of the first mammalian N-term. methyltransferase, NRMT, in tumorigenesis

第一个哺乳动物 N 项的作用。

• 批准号: 8189662
• 负责人: Christine E Schaner-Tooley
• 金额: $ 8.79万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-05 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8189662
• 关键词: A Mouse; Acetylation; Advisory Committees; Affect; Aneuploidy; Antibodies; Apoptotic; Binding; Biological; Biological Assay; Biological Process; Biology; Cell Cycle; Cell Line; Cell Proliferation; Cell Separation; Cell physiology; Chromatin; Committee Members; Consensus Sequence; Critiques; Cytoplasm; DNA; DNA Binding; DNA repair protein; DNA-Binding Proteins; Data; Defect; Development; Educational workshop; Elements; Embryo; Environment; Epithelial Cells; Exhibits; Fatty acid glycerol esters; Fellowship; Fibroblasts; Flow Cytometry; Fluorescence Resonance Energy Transfer; Fourier Transform; Future; Gene Combinations; Gene Targeting; Genes; Genetic; Gland; Glioblastoma; Goals; Growth; Harvest; Histology; Human; Image; Immunoprecipitation; In Vitro; Individual; Institution; Knockout Mice; Lead; Learning; Malignant Neoplasms; Mammary gland; Mass Spectrum Analysis; Measures; Medicine; Mentors; Methods; Methylation; Methyltransferase; Microarray Analysis; Microscopy; Mitotic; Modeling; Modification; Monitor; Mono-S; Mus; Mutation; N-terminal; Neoplasm Metastasis; Nuclear; Oncogene Proteins; Operative Surgical Procedures; Paraffin Embedding; Patients; Pattern; Pharmaceutical Preparations; Phase; Phenotype; Positioning Attribute; Post-Translational Protein Processing; Process; Proteins; Reagent; Research; Retinoblastoma Protein; Role; Running; Series; Signal Pathway; Site; Slide; System; Techniques; Testing; Therapeutic Agents; Time; Tissue Sample; Training; Transgenic Organisms; Transplantation; Tumor Suppressor Genes; Tumor Suppressor Proteins; Tumor Tissue; Universities; Virginia; Work; Writing; career development; cellular imaging; chromatin immunoprecipitation; design; experience; in vitro Assay; in vivo; interest; leukemia; malignant breast neoplasm; mass spectrometer; meetings; mouse model; mutant; myosin light chain 2; programs; protein function; research study; skills; stoichiometry; tool; tumor; tumor progression; tumorigenesis

DESCRIPTION (provided by applicant): During my postdoctoral fellowship, I successfully purified and identified the first mammalian N-terminal methyltransferase, NRMT. Shown by microarray analysis to have reduced expression/copy number in human breast cancers, I have also illustrated NRMT knockdown results in increased cell proliferation and a multi- spindle phenotype. Given that NRMT knockdown results in these two cancer prone phenotypes, I intend to further study how it is regulated, how N-terminal methylation affects substrate function, and how NRMT misregulation leads to tumorigenesis. My goal after completion of my postdoctoral work is to obtain a tenure- track position at an academic research institution and establish an independent research program. Successful completion of my research goals includes training in a mouse mammary transplant model. As I have no previous mouse research experience, the additional training time provided by a K99 will be imperative for learning these skills. Obtaining a K99, will also allow for further training in mass spectrometry, fluorescent- activating cell sorting, and FRET analysis. The long-term goal of my research is to use the NRMT mouse model to dissect the signaling pathways that lead to breast cancer. As personalized medicine treatments become increasingly necessary, I want to create a model where combinations of genes can be easily manipulated simultaneously. These models can be used to test which current drug treatments are most effective for patients with specific combinations of mutations and to screen for new and more effective therapeutic agents. Research The list of genes involved in tumorigenesis is quite extensive, however, many of their biological functions remain unknown. One such gene, Mettl11a (now renamed NRMT), has been shown to be under-expressed in breast cancers, but has only recently been identified as the first mammalian N-terminal methyltransferase. Reduction of NRMT also results in a multi-spindle phenotype, and the associated aneuploidy is often considered to contribute to cancer progression. As NRMT is a newly discovered protein, the goals of this proposal are to understand the role of N-terminal methylation on protein and cellular function, in order to study how its misregulation leads to tumorigenesis. The first two aims, determining whether N-terminal methylation is constitutive and determining whether N-terminal methylation universally alters the DNA binding of its substrates, are designed to better understand the basic biology of N-terminal methylation. These aims will involve mass spectrometry, fluorescent-activating cell sorting, FRET analysis, and construction of a knockout mouse. The objective of the third aim is to determine the role of NRMT in development and tumorigenesis. A mouse mammary transplant model will be established for assaying if the multi-spindle NRMT knockdown phenotype leads to developmental defects and/or tumorigenesis. The key element of the career development aspect of this proposal will be learning the mouse system needed for successful completion of the third aim. The experiments of aims one and two have been designed for the downtime between surgeries and gland or tumor harvesting. Environment The University of Virginia is well equipped for completion of all three aims of this proposal. The on-campus mass spectrometry facility, of advisory committee member Dr. Don Hunt, has a Fourier transform mass spectrometer necessary for distinguishing N-terminal methylation from acetylation. The UVA Flow Cytometry Core will provide the training necessary for all fluorescent-activating cell sorting experiments. The UVA Gene Targeting and Transgenic Facility will create the NRMT knockout mouse. The Research Histology Core will paraffin embed and make slides of all normal mouse tissue and tumors. My advisory committee member Dr. Amy Bouton will aid in interpretation of the histology. The expertise and reagents of advisory committee member Dr. Todd Stukenberg will aid in characterization of the NRMT multi-spindle phenotype. The UVA W.M. Keck Center for Cellular Imaging supplies state-of-the art imaging facilities for immunofluorescent imaging of mouse tissue samples and training in FRET analysis. The Macara lab currently has three expert mouse biologists available for training in mouse handling and surgical techniques. In addition, UVA offers numerous courses aimed at career development/training, including the UVA transgenic methods and applications workshop, the Flow Cytometry Training Workshop, the Workshop on FRET Microscopy, and a monthly career development seminar series offered by the Office of Postdoctoral Professional Development. PUBLIC HEALTH RELEVANCE: The biological roles of many genes involved in tumorigenesis remain unknown. We need to study their functions in order to achieve a complete understanding of cancer progression and obtain an inclusive list of potential druggable targets. One such protein, NRMT, the newly identified mammalian N-terminal methyltransferase, is under-expressed in breast cancers, and its knockdown results in mitotic defects often considered to contribute to cancer progression. The goal of this proposal is to understand how NRMT is regulated and how its misregulation contributes to tumorigenesis. The written critiques and criteria scores of individual reviewers are provided in essentially unedited form in the "Critique" section below. Please note that these critiques and criteria scores were prepared prior to the meeting and may not have been revised subsequent to any discussions at the review meeting. The "Resume and Summary of Discussion" above summarizes the final opinions of the committee.

描述（由申请人提供）：在博士后研究期间，我成功纯化并鉴定了第一个哺乳动物 N 末端甲基转移酶 NRMT。微阵列分析显示人类乳腺癌中的表达/拷贝数减少，我还说明了 NRMT 敲低会导致细胞增殖增加和多纺锤体表型。鉴于 NRMT 敲低会导致这两种易患癌症的表型，我打算进一步研究它是如何调节的、N 端甲基化如何影响底物功能，以及 NRMT 失调如何导致肿瘤发生。完成博士后工作后，我的目标是在学术研究机构获得终身职位并建立独立的研究项目。成功完成我的研究目标包括对小鼠乳腺移植模型进行培训。由于我之前没有鼠标研究经验，因此 K99 提供的额外培训时间对于学习这些技能至关重要。获得 K99 还可以接受质谱分析、荧光激活细胞分选和 FRET 分析方面的进一步培训。我研究的长期目标是利用 NRMT 小鼠模型来剖析导致乳腺癌的信号通路。随着个性化医学治疗变得越来越必要，我想创建一个可以轻松同时操纵基因组合的模型。这些模型可用于测试当前哪些药物治疗对于具有特定突变组合的患者最有效，并筛选新的更有效的治疗药物。研究 与肿瘤发生有关的基因列表相当广泛，然而，它们的许多生物学功能仍然未知。其中一个基因 Mettl11a（现已更名为 NRMT）已被证明在乳腺癌中表达不足，但最近才被确定为第一个哺乳动物 N 末端甲基转移酶。 NRMT 的减少还会导致多纺锤体表型，并且相关的非整倍性通常被认为有助于癌症进展。由于NRMT是一种新发现的蛋白质，该提案的目标是了解N端甲基化对蛋白质和细胞功能的作用，以研究其错误调节如何导致肿瘤发生。前两个目标是确定 N 末端甲基化是否是组成型的，以及确定 N 末端甲基化是否普遍改变其底物的 DNA 结合，旨在更好地了解 N 末端甲基化的基本生物学。这些目标将涉及质谱分析、荧光激活细胞分选、FRET 分析和基因敲除小鼠的构建。第三个目标是确定 NRMT 在发育和肿瘤发生中的作用。将建立小鼠乳腺移植模型来检测多轴NRMT敲低表型是否导致发育缺陷和/或肿瘤发生。该提案职业发展方面的关键要素将是学习成功完成第三个目标所需的鼠标系统。目标一和目标二的实验是针对手术和腺体或肿瘤采集之间的停机时间而设计的。环境 弗吉尼亚大学有能力完成该提案的所有三个目标。顾问委员会成员 Don Hunt 博士的校内质谱设施拥有一台区分 N 末端甲基化和乙酰化所必需的傅里叶变换质谱仪。 UVA 流式细胞术核心将为所有荧光激活细胞分选实验提供必要的培训。 UVA 基因靶向和转基因设施将创建 NRMT 敲除小鼠。研究组织学核心将对所有正常小鼠组织和肿瘤进行石蜡包埋并制作玻片。我的顾问委员会成员 Amy Bouton 博士将帮助解释组织学。咨询委员会成员 Todd Stukenberg 博士的专业知识和试剂将有助于表征 NRMT 多轴表型。 UVA W.M.凯克细胞成像中心提供最先进的成像设施，用于小鼠组织样本的免疫荧光成像和 FRET 分析培训。马卡拉实验室目前拥有三名小鼠生物学专家，可以接受小鼠处理和手术技术方面的培训。此外，UVA还提供众多旨在职业发展/培训的课程，包括UVA转基因方法和应用研讨会、流式细胞术培训研讨会、FRET显微镜研讨会以及博士后专业发展办公室每月提供的职业发展研讨会系列。 公共卫生相关性：许多与肿瘤发生有关的基因的生物学作用仍然未知。我们需要研究它们的功能，以便全面了解癌症进展并获得潜在可药物靶点的包容性列表。其中一种蛋白质 NRMT 是新发现的哺乳动物 N 末端甲基转移酶，在乳腺癌中表达不足，其敲低会导致有丝分裂缺陷，通常被认为是导致癌症进展的原因。该提案的目标是了解 NRMT 是如何调节的以及它的错误调节如何导致肿瘤发生。 下面的“评论”部分以基本上未经编辑的形式提供了个人评论者的书面评论和标准分数。请注意，这些批评和标准分数是在会议之前准备的，在审查会议上进行任何讨论后可能不会进行修改。上述“讨论简历和摘要”总结了委员会的最终意见。