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: 8724761
• 负责人: Jayakrishnan Nandakumar
• 金额: $ 23.39万
• 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/8724761
• 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，可延长端粒的 3' 端。 带有端粒DNA的染色体。因为端粒酶在体细胞中表达较弱，但 它在 90% 的癌细胞中过度表达，是抗癌药物设计的一个有吸引力的靶点。一个多 称为庇护蛋白的蛋白质复合物与端粒 DNA 特异性结合，以抑制非法 DNA 融合 哺乳动物染色体末端。如果庇护蛋白的正常功能是“保护”染色体末端，那么如何 端粒酶是否能够接近这些末端并延长它们？ POT1-TPP1 是一个庇护子复合体 以高特异性和亲和力结合单链端粒 DNA。 POT1-TPP1 在体内的主要功能是 抑制端粒 DNA 损伤识别事件。鉴于其在染色体末端保护中的作用，POT1- TPP1 可能会通过阻止端粒酶接近染色体末端来抑制端粒酶。出奇， POT1-TPP1 增加体外端粒酶的持续合成能力。此外，TPP1 的 OB 结构域参与 端粒酶招募到端粒。 POT1-TPP1对端粒酶的刺激具有关键的生理作用 意义在于与癌细胞相关的端粒酶活性可能需要基于 POT1-TPP1 的 刺激。这里，假设 TPP1 OB 结构域上的表面直接与 端粒酶引起端粒酶募集和刺激。酶学结合 哺乳动物细胞生物学和结构生物学将用于检验这一假设并确定 癌细胞中 TPP1 刺激端粒酶的结果。这将是第一项直接评估 TPP1 或任何哺乳动物庇护蛋白亚复合物刺激端粒酶的生物学重要性。 该项目的具体目标是： 1. 鉴定人 TPP1 中导致端粒酶的结构元件 使用定点诱变筛选进行持续刺激，寻找功能分离突变体 特别是在体外端粒酶刺激方面有缺陷，但在 DNA 末端保护方面没有缺陷。 2. 确定 TPP1 刺激端粒酶在 HeLa 细胞和肺癌中的生理重要性和机制 敲低内源性 TPP1 并表达野生型或端粒酶刺激缺陷的细胞系 TPP1 突变体以稳定的方式。将测试这些细胞系的端粒长度缺陷和端粒酶 招聘缺陷。 3a.通过 POT1-TPP1 获得染色体末端保护的高分辨率视图 结晶 POT1-TPP1 融合蛋白与端粒 DNA 的复合物。 3b.深入了解端粒酶 通过结晶具有生化能力的最小 TPP1-端粒酶复合物来刺激 TPP1 通过对各个成分的截断分析。 拟议目标的 K99 阶段将在 Tom Cech 博士的指导下进行，他的 指导技能帮助他的 30 多名学员获得了美国研究机构的教职 美国和全世界。切赫实验室是端粒酶和端粒生物化学领域的领导者， 配备解决拟议研究的生化/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。细胞和 开发。加州大学博尔德分校生物学系。对于肺癌细胞系的实验，我将共同- 由加州大学丹佛分校癌症中心的 James DeGregori 博士指导，并将可以完全使用以下机构的设施： 他的实验室和癌症中心。因此，我坚信 CU Boulder 和 Cancer 的设施 中心将为我提供理想的环境来执行 K99/R00 应用程序的拟议目标。 我在 K99 阶段研究的目标是在 2 年内完成提案的目标 1 和目标 2A&C，并且 申请美国独立教职职位。目标 2B&D 和目标 3 将在 R00 阶段完成。 从长远来看，我希望成为一名独立调查员，经营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）共同努力回答关键问题 端粒生物学及其对癌症的影响。除了允许我雇用员工和购买实验室用品之外， K99/R00 奖允许我参加癌症生物学课程，极大地促进我从博士后到 PI 的过渡 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。我在攻读博士学位期间获得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之后继续采取措施获得 RCR 培训。 我以硕士学位开始了我的研究生涯。学生合成小分子，但此后我的注意力转移了 更多以生物为导向的问题。作为一名技术人员，我研究了蛋白质折叠，然后，作为一名研究生，我 利用生物化学和 X 射线晶体学研究 RNA/DNA 修复，现在担任博士后 我和 Tom Cech 博士一起开始研究人类癌细胞中的端粒酶调节。期间 在我的科学训练中，我学习了理论概念并培养了各个领域的实验技能 研究。我相信我迄今为止所获得的知识和经验将极大地帮助我 及时成功地完成了 K99/R00 提案的目标。