The role fo the TPP1 peotein in telomerase function and cancer cell survival

TPP1蛋白在端粒酶功能和癌细胞存活中的作用

• 批准号: 8731837
• 负责人: Jayakrishnan Nandakumar
• 金额: $ 22.67万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-03 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8731837
• 关键词: Address; Affect; Affinity; American Association of Cancer Research; Amino Acids; Antineoplastic Agents; Area; Award; Binding; Biochemical; Biochemistry; Biological; Biology; Cancer Biology; Cancer Center; Cancer cell line; Cell Line; Cell Survival; Cells; Cellular biology; Chimeric Proteins; Chromosomes; Chromosomes, Human, Pair 3; Collaborations; Complex; Crystallization; Crystallography; DNA; DNA Binding; DNA Damage; DNA Repair; Defect; Doctor of Philosophy; Drug Design; Drug Targeting; Educational workshop; Engineering; Environment; Enzymatic Biochemistry; Enzymes; Event; Faculty; Fluorescent in Situ Hybridization; Gene Fusion; Generations; Glycine; Goals; Hela Cells; Heterodimerization; Human; Immunofluorescence Immunologic; In Vitro; Indium; Individual; Institution; Knowledge; Lead; Learning; Length; Malignant Neoplasms; Malignant neoplasm of lung; Mammalian Cell; Mammalian Chromosomes; Mentors; Mentorship; Microscopy; Mutation; Oligonucleotides; Phase; Physiological; Positioning Attribute; Postdoctoral Fellow; Proteins; RNA; RNA-Directed DNA Polymerase; Regulation; Research; Research Personnel; Research Training; Resolution; Resources; Role; Running; Serine; Site-Directed Mutagenesis; Somatic Cell; Specificity; Structure; Students; Surface; Tail; Telomerase; Telomerase RNA Component; Testing; Time; Training; Validation; Work; base; cancer cell; career; complement C2a; design; experience; expression vector; fighting; graduate student; in vivo; insight; knock-down; meetings; mutant; overexpression; prevent; protein complex; protein folding; research study; response; responsible research conduct; skills; small molecule; structural biology; telomere

Summary: Mammalian telomerase is a specialized reverse transcriptase that extends the 3' ends of chromosomes with telomeric DNA. Because telomerase is weakly expressed in somatic cells, but is overexpressed in 90% of cancer cells, it serves as an attractive target for anti-cancer drug design. A multi- protein complex known as shelterin associates specifically with telomeric DNA to repress illicit DNA fusions at mammalian chromosome ends. If the normal function of shelterin is to 'protect' chromosome ends, how does telomerase gain access to these ends to extend them? POT1-TPP1 is a shelterin sub-complex that binds single-stranded telomeric DNA with high specificity and affinity. A major function of POT1-TPP1 in vivo is to repress DNA damage recognition events at telomeres. Given its role in chromosome end-protection, POT1- TPP1 might be expected to inhibit telomerase by preventing its access to chromosome ends. Surprisingly, POT1-TPP1 increases telomerase processivity in vitro. Additionally, the OB domain of TPP1 is involved in telomerase recruitment to telomeres. The stimulation of telomerase by POT1-TPP1 has critical physiological significance insofar as the telomerase activity associated with cancer cells might require POT1-TPP1-based stimulation. Here, it is hypothesized that a surface on the OB domain of TPP1 interacts directly with telomerase to give rise to telomerase recruitment and stimulation. Enzymology in combination with mammalian cell biology and structural biology will be used to test this hypothesis and determine the consequence of telomerase stimulation by TPP1 in cancer cells. This will be the first study to assess directly the biological importance of telomerase stimulation by TPP1 or any mammalian shelterin subcomplex. The specific aims of the project are to: 1. Identify structural elements in human TPP1 that lead to telomerase processivity stimulation using a site-directed mutagenesis screen, looking for separation-of-function mutants defective specifically in telomerase stimulation in vitro but not in DNA end-protection. 2. Determine the physiological importance and mechanism of telomerase stimulation by TPP1 in HeLa-based and lung cancer cell-lines that knock down endogenous TPP1 and express wild-type or telomerase stimulation-defective mutants of TPP1 in a stable manner. These cell lines will be tested for telomere length defects and telomerase recruitment defects. 3a. Obtain a high-resolution view of chromosome-end protection by POT1-TPP1 by crystallizing a POT1-TPP1 fusion protein in complex with telomeric DNA. 3b. Obtain insights into telomerase stimulation by TPP1 by crystallizing a biochemically competent, minimal, TPP1-telomerase complex defined through truncation analyses of the individual components. The K99 phase of the proposed aims will be conducted under the mentorship of Dr. Tom Cech, whose mentoring skills have helped more than 30 of his mentees to attain faculty positions in research institutions in the US and worldwide. The Cech lab is a leader in the biochemistry of telomerase and telomeres, and is equipped with the resources required to address the biochemical/structural aims of the proposed study. For the HeLa-based experiments, we have an ongoing collaboration with Dr. Leslie Leinwand of the Mol. Cell and Dev. Biology Department of UC Boulder. For the experiments in the lung cancer cell lines, I will be co- mentored by Dr. James DeGregori of the UC Cancer Center Denver and will have full access to the facilities of his lab and the Cancer Center. Hence, I strongly believe that the facilities at CU Boulder and at the Cancer Center will provide me with the ideal environment to execute the proposed goals of the K99/R00 application. My goal in the K99 phase of research is to complete, in 2 years, Aim 1 and Aim 2A&C of the proposal, and apply for an independent faculty position in the US. Aims 2B&D and Aim 3 will be completed in the R00 phase. In the long-term, I wish to become an independent investigator, running a lab consisting of people from various backgrounds (biochemistry, structural biology, and cell biology) working together to answer critical questions in telomere biology and its implications in cancer. In addition to allowing me to hire staff and buy lab supplies, the K99/R00 award will greatly facilitate my postdoc-to-PI transition by allowing me to attend a cancer biology course, a telomerase-cancer AACR meeting, and a microscopy workshop conducted by Cold Spring Harbor labs. I have obtained formal training in the responsible conduct of research (RCR) during my Ph.D. and will continue to take steps to acquire RCR training during and after my postdoc. I began my research career as an M.S. student synthesizing small molecules, but have since shifted my focus to more to bio-oriented problems. As a technician I studied protein folding, then, as a graduate student I employed biochemistry and x-ray crystallography to study RNA/DNA repair, and now, working as a post-doc with Dr. Tom Cech I am beginning to study telomerase regulation in human cancer cells. During the course of my scientific training, I have learnt theoretical concepts and developed experimental skills in diverse areas of research. I believe that the knowledge and experience I have gained thus far will greatly assist in the successful completion of the aims of the K99/R00 proposal in a timely fashion.

摘要：哺乳动物端粒酶是一种特殊的逆转录酶，它延长了端粒酶的3‘端。 带有端粒DNA的染色体。因为端粒酶在体细胞中表达很弱，但 它在90%的癌细胞中过表达，是抗癌药物设计的一个有吸引力的靶点。一个多- 一种名为Shelterin的蛋白质复合体与端粒DNA特异性结合，以抑制非法DNA融合 哺乳动物的染色体末端。如果保护素的正常功能是‘保护’染色体末端，那么如何 端粒酶能接触到这些末端来延长它们吗？POT1-TPP1是一种保护性亚复合体， 结合单链端粒DNA具有高度的特异性和亲和力。POT1-TPP1在体内的一个主要功能是 以抑制端粒上的DNA损伤识别事件。考虑到它在染色体末端保护中的作用，POT1- TPP1可能通过阻止端粒酶进入染色体末端来抑制端粒酶。令人惊讶的是， POT1-TPP1在体外可增加端粒酶活性。此外，TPP1的OB结构域还涉及 端粒酶重新聚集到端粒。POT1-TPP1对端粒酶的刺激具有重要的生理意义 与癌细胞相关的端粒酶活性可能需要基于POT1-TPP1的意义 刺激。这里，假设TPP1的OB结构域上的表面直接与 端粒酶引起端粒酶的募集和刺激。与酶学相结合 将使用哺乳动物细胞生物学和结构生物学来检验这一假说，并确定 TPP1刺激端粒酶对癌细胞的影响。这将是第一个直接评估的研究 TPP1或任何哺乳动物保护素亚复合体刺激端粒酶的生物学意义。 该项目的具体目标是：1.确定人类TPP1中导致端粒酶的结构元件 使用定点突变筛选的加工性刺激，寻找功能分离突变体 在体外刺激端粒酶方面存在缺陷，但在DNA末端保护方面没有缺陷。2.确定 TPP1激活端粒酶在HeLa和肺癌中的生理学意义及机制 敲除内源性TPP1并表达野生型或端粒酶刺激的细胞系-缺陷 突变型TPP1的表达方式稳定。这些细胞系将接受端粒长度缺陷和端粒酶的测试 招聘缺陷。3A.通过以下方式获得POT1-TPP1对染色体末端保护的高分辨率视图 与端粒DNA形成复合体结晶POT1-TPP1融合蛋白。3B.深入了解端粒酶 TPP1通过结晶具有生物化学活性的最小端粒酶复合体来刺激 通过对各个组件的截断分析。 拟议AIMS的K99阶段将在Tom Cech博士的指导下进行，他 指导技能帮助他的30多名学员在年获得研究机构的教职 美国和世界各地。切赫实验室在端粒酶和端粒的生物化学方面处于领先地位， 具备实现拟议研究的生化/结构目标所需的资源。对于 基于HeLa的实验，我们与Mol的Leslie Leinwand博士正在进行合作。单元格和 戴夫。加州大学博尔德分校生物系。对于肺癌细胞系的实验，我将与 由加州大学丹佛分校癌症中心的詹姆斯·德格雷戈里博士指导，将完全访问 他的实验室和癌症中心。因此，我坚信科罗拉多大学博尔德分校和癌症中心的设施 中心将为我提供执行K99/R00应用程序建议目标的理想环境。 我在K99研究阶段的目标是在两年内完成提案的目标1和目标2A和C，以及 申请美国的独立教职。AIMS 2B&D和AIMS 3将在R00阶段完成。 从长远来看，我希望成为一名独立的调查员，管理一个由来自不同领域的人组成的实验室 背景(生物化学、结构生物学和细胞生物学)共同回答 端粒生物学及其在癌症中的意义。除了允许我雇佣员工和购买实验室用品外， K99/R00奖将极大地促进我从博士后到PI的过渡，因为我可以参加一个癌症生物学课程 课程，端粒酶-癌症AACR会议，以及冷泉港举办的显微镜研讨会 实验室。我在博士期间接受了负责任的研究行为(RCR)的正式培训，并将 在我的博士后期间和毕业后，继续采取措施获得RCR培训。 我作为一名合成小分子的理科学生开始了我的研究生涯，但后来转移了我的重点 更多地涉及到面向生物的问题。作为一名技术人员，我研究蛋白质折叠，然后，作为研究生，我 利用生物化学和x射线结晶学来研究RNA/DNA修复，现在是一名博士后 我和汤姆·切赫博士一起开始研究人类癌细胞中的端粒酶调节。在…的过程中 在我的科学训练中，我学到了理论概念，并在不同的领域发展了实验技能 研究。我相信我到目前为止所获得的知识和经验将对 及时成功完成K99/R00提案的目标。