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

基本信息

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
项目类别：
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

项目摘要

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专门的逆转录酶，扩展了3'的末端带有端粒DNA的染色体。因为端粒酶在体细胞中弱表达，但在90％的癌细胞中过表达，它是抗癌药物设计的有吸引力的靶标。多蛋白质复合物被称为庇护素与端粒DNA相关，以抑制非法DNA融合哺乳动物染色体结束。如果庇护所的正常功能是“保护”染色体结束，那么端粒酶是否可以访问这些目的以扩展它们？ POT1-TPP1是一个庇护所的子复合物结合具有高特异性和亲和力的单链端粒DNA。 pot1-tpp1体内的主要功能是抑制端粒的DNA损伤识别事件。鉴于其在染色体终端保护中的作用，Pot1- TPP1可能会通过防止其进入染色体末端抑制端粒酶。出奇， POT1-TPP1在体外提高了端粒酶加工性。此外，TPP1的OB域参与端粒酶招募到端粒。 POT1-TPP1刺激端粒酶具有关键的生理在与癌细胞相关的端粒酶活性可能需要基于POT1-TPP1的情况下刺激。在这里，假设TPP1 OB域上的表面与产生端粒酶募集和刺激的端粒酶。酶学哺乳动物细胞生物学和结构生物学将用于检验该假设并确定 TPP1在癌细胞中端粒酶刺激的结果。这将是第一个直接评估的研究 TPP1或任何哺乳动物庇护素子复杂性端粒酶刺激的生物学重要性。该项目的具体目的是：1。确定人类TPP1中导致端粒酶的结构元素使用位置定向的诱变屏幕刺激加工性刺激，寻找功能分离突变体在体外刺激方面有缺陷，但在DNA末端保护中没有缺陷。 2。确定 TPP1在HELA基和肺癌中端粒酶刺激的生理重要性和机制击倒内源性TPP1并表达野生型或端粒酶刺激缺陷的细胞线 TPP1的突变体以稳定的方式。这些细胞系将测试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与端粒DNA复合物中的POT1-TPP1融合蛋白结晶。 3b。获得端粒酶的见解通过结晶的生物化学能力，最小，TPP1-纤维素酶复合物来刺激TPP1。通过对各个组件的截断分析。提议的目标的K99阶段将在汤姆·塞奇（Tom Cech）博士的指导下进行。指导技能帮助他的30多个受训者在研究机构中获得教师职务美国和全球。 Cech Lab是端粒酶和端粒生物化学的领导者，是配备了拟议研究的生化/结构目标所需的资源。为了基于HELA的实验，我们与Mol的Leslie Leinwand博士进行了持续的合作。细胞和开发UC Boulder生物学系。对于肺癌细胞系中的实验，我将是共同的由UC癌症中心丹佛的James DeGregori博士指导，将完全访问他的实验室和癌症中心。因此，我坚信Cu Boulder和癌症的设施中心将为我提供理想的环境，以执行K99/R00应用程序的拟议目标。我在K99研究阶段的目标是在2年内完成AIM 1和AIM 2A＆C的提案，并完成该提案的目标2a＆c，以及在美国申请独立的教师职位。 AIMS 2B＆D和AIM 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，经营一个由来自各种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外， K99/R00奖将通过允许我参加癌症生物学，从而极大地促进我的博士后过渡当然，端粒酶癌的AACR会议和Cold Spring Harbor进行的显微镜研讨会实验室。我在博士学位期间接受了负责任的研究（RCR）的正式培训。并将在我的博士后期间和之后，继续采取步骤以获取RCR培训。我从事硕士学位开始了我的研究生涯学生合成小分子，但此后改变了我的注意力更多面向生物的问题。作为技术人员，我研究了蛋白质折叠，然后，作为研究生使用生物化学和X射线晶体学来研究RNA/DNA修复，现在作为DOC工作对于Tom Cech博士，我开始研究人类癌细胞中的端粒酶调节。在我的科学培训，我学到了理论概念，并在不同领域的不同领域发展了实验技能研究。我相信，到目前为止，我获得的知识和经验将极大地帮助成功地完成了K99/R00提案的目标。