Examining the Turnover of the Catalytic Subunit of RNA Polymerase I to Elucidate the Enzyme Stability and Regulation

检查 RNA 聚合酶 I 催化亚基的周转率以阐明酶的稳定性和调节

• 批准号: 10065264
• 负责人: Stephanie Pitts
• 金额: $ 4.55万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10065264
• 关键词: Affect; Biochemical; Biogenesis; Biological Assay; Cancer Biology; Cancer cell line; Catalytic Domain; Cell Death; Cells; Complex; Core Facility; Cullin Proteins; Data; Development; Educational workshop; Environment; Enzyme Stability; Enzymes; Exhibits; F Box Domain; F-Box Proteins; FRAP1 gene; Faculty; Fellowship; Foundations; Genes; Genetic; Genetic Transcription; Glean; Goals; Grant; Immunologics; Institution; Interview; Knock-out; Knowledge; Link; Malignant Neoplasms; Manuscripts; Mediating; Modeling; Molecular; Nature; Normal Cell; Occupations; Oncogenic; Pathway interactions; Polymerase; Polyubiquitin; Predisposition; Process; RNA Polymerase I; RNA interference screen; Regulation; Research; Research Training; Ribosomal DNA; Ribosomes; Scientist; Signaling Protein; Site; Site-Directed Mutagenesis; System; Testing; Therapeutic; Therapeutic Agents; Training; Ubiquitin; Ubiquitination; Universities; Work; Writing; anti-cancer therapeutic; base; biomarker development; cancer cell; career; cell type; chromatin immunoprecipitation; experimental study; fitness; improved; insight; knock-down; medical schools; member; multicatalytic endopeptidase complex; mutant; novel; novel therapeutics; overexpression; protein complex; small molecule; symposium; tool; ubiquitin-protein ligase

Project Summary While RNA polymerase I (Pol I) activity is upregulated in many cancers, it remains unclear how to target the enzyme. Little is known about the stability of Pol I and the regulation of its activity. It is also unclear how to selectively target Pol I transcription in cancer cells without adversely affecting wild-type cells. We have recently discovered a first-in-class small molecule, BMH-21, that inhibits Pol I transcription and induces the degradation of RPA194, the catalytic subunit of Pol I. Treatment with this molecule results in cancer cell-specific cell death. Yet the mechanisms of this inducible degradation of RPA194 remain unknown. This project will investigate the mechanisms and regulation of RPA194 degradation and use the small molecule as a tool. Our prior data suggests that BMH-21 induces the ubiquitination of RPA194 and targets it for degradation by the proteasome. To test this premise and elucidate the mechanisms involved, this research training plan will pursue three primary goals. (1) To determine the site(s) of ubiquitination on RPA194; (2) To identify the E3 ligase(s) responsible for the ubiquitination of RPA194; (3) To elucidate the regulation of RPA194 degradation. These goals will be accomplished through the use of site-directed mutagenesis, ubiquitination analyses, and the identification of the ubiquitin chain linkages. E3 ligase candidates, identified in a previous screen, will be validated by knockdown and expression analyses, ubiquitination assays, and interaction analyses. Transcription assays, chromatin immunoprecipitation, and assays for Pol I complex assembly will be used to determine the regulation of RPA194 degradation. By studying the mechanisms and regulation of the turnover of RPA194, the study will provide fundamental new knowledge about the stability and regulation of Pol I. This knowledge will improve understanding of how to target Pol I as an anti-cancer therapeutic. The study will be conducted at the Johns Hopkins University School of Medicine, an institution which takes pride in the broad expertise of its faculty, numerous core facilities, and outstanding training environment. In addition to undertaking the proposed research, the trainee will take elective courses and participate in seminars to establish a strong foundation in molecular cancer biology. She will also collaborate with experts in ubiquitination and the regulation of Pol I transcription to enhance her training. She will present her research at internal retreats and external conferences, and she will write abstracts and manuscripts. Lastly, she will participate in workshops provided by the professional development office that cover topics such as interviewing, grant writing, and preparing a job talk. The fellowship will therefore have considerable impact in preparing the trainee for her career goal as an independent research scientist.

项目摘要 虽然RNA聚合酶I（Pol I）活性在许多癌症中上调，但仍不清楚如何靶向 酶关于Pol I的稳定性及其活性的调节知之甚少。目前还不清楚如何 选择性靶向癌细胞中的Pol I转录，而不会对野生型细胞产生不利影响。我们最近 发现了一种一流的小分子BMH-21，它抑制Pol I的转录并诱导其降解。 RPA 194是Pol I的催化亚基。用这种分子治疗导致癌细胞特异性细胞死亡。 然而，RPA 194的这种诱导性降解的机制仍然未知。该项目将调查 RPA 194降解的机制和调控，并使用小分子作为工具。 我们先前的数据表明，BMH-21诱导RPA 194的泛素化，并将其靶向于 通过蛋白酶体降解。为了验证这一前提并阐明相关机制，本研究 培训计划将追求三个主要目标。(1)确定RPA 194上的泛素化位点;（2） 鉴定负责RPA 194遍在化的E3连接酶;（3）阐明 RPA 194降解。这些目标将通过使用定点诱变来实现， 泛素化分析和泛素链连接的鉴定。E3连接酶候选物，在 将通过敲低和表达分析、泛素化测定和 互动分析转录测定、染色质免疫沉淀和Pol I复合物测定 组装将用于确定RPA 194降解的调节。通过研究这些机制， 调节RPA 194的营业额，该研究将提供有关稳定性和 Pol I的调节。这些知识将提高对如何靶向Pol I作为抗癌药物的理解。 有治疗作用的 这项研究将在约翰霍普金斯大学医学院进行，该机构 以其教师的广泛专业知识，众多的核心设施和出色的培训环境而自豪。 除了进行拟议的研究外，受训人员还将选修课程，并参加 研讨会，以建立在分子癌症生物学的坚实基础。她还将与专家合作， 泛素化和Pol I转录的调节来增强她的训练。她将介绍她的研究在 内部务虚会和外部会议，她将写摘要和手稿。最后，她将 参加专业发展办公室提供的讲习班，内容包括： 面试、撰写论文、准备求职演讲。因此，研究金将在以下方面产生重大影响： 使受训者为成为独立研究科学家的职业目标做好准备。