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

摘要：哺乳动物端粒酶是一种特异性逆转录酶，它延伸了端粒酶的3'端， 染色体端粒DNA。由于端粒酶在体细胞中表达较弱， 在90%的癌细胞中过表达，它是抗癌药物设计的一个有吸引力的靶点。一个多- 一种称为shelterin的蛋白质复合物特异性地与端粒DNA结合， 哺乳动物染色体末端。如果shelterin的正常功能是“保护”染色体末端， 端粒酶是否能进入这些末端并延伸它们呢？POT 1-TPP 1是一种shelterin亚复合物， 以高特异性和亲和力结合单链端粒DNA。POT 1-TPP 1在体内的主要功能是 来抑制端粒的DNA损伤识别。鉴于其在染色体末端保护中的作用，POT 1- TPP 1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， POT 1-TPP 1在体外增加端粒酶的合成能力。此外，TPP 1的OB结构域参与了 端粒酶向端粒的募集。POT 1-TPP 1对端粒酶的刺激具有重要的生理学意义。 与癌细胞相关的端粒酶活性可能需要基于POT 1-TPP 1的 刺激.在此，假设TPP 1的OB结构域上的表面直接与 端粒酶，以引起端粒酶募集和刺激。酶学与 哺乳动物细胞生物学和结构生物学将用于测试这一假设，并确定 这是癌细胞中TPP 1对端粒酶刺激的结果。这将是第一项直接评估 TPP 1或任何哺乳动物shelterin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TPP 1中导致端粒酶的结构元件 使用定点诱变筛选进行持续合成能力刺激，寻找功能分离突变体 在体外特异性地在端粒酶刺激中有缺陷，但在DNA末端保护中没有缺陷。2.确定 TPP 1刺激端粒酶活性在HeLa细胞和肺癌中的生理意义及机制 敲低内源性TPP 1并表达野生型或端粒酶刺激缺陷型的细胞系 TPP 1突变体。这些细胞系将进行端粒长度缺陷和端粒酶检测 招聘缺陷。3a.通过以下方法获得POT 1-TPP 1对染色体末端保护的高分辨率视图： 结晶与端粒DNA复合的POT 1-TPP 1融合蛋白。3b.深入了解端粒酶 TPP 1通过结晶化生物化学活性的、最小的TPP 1-端粒酶复合物的刺激， 通过对单个组件的截断分析。 拟议目标的K99阶段将在汤姆·切赫博士的指导下进行，他 指导技能帮助他的30多名学员获得了研究机构的教职， 美国和全世界。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， 配备所需的资源，以解决拟议研究的生化/结构目标。为 基于HeLa的实验，我们正在与Mol的Leslie Leinwand博士合作。细胞和 Dev.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- 由丹佛加州大学癌症中心的James DeGregori博士指导，并将完全使用 他的实验室和癌症中心因此，我坚信，在CU博尔德和癌症的设施， 中心将为我提供理想的环境来执行K99/R 00应用程序的拟议目标。 我在K99研究阶段的目标是在2年内完成提案的目标1和目标2A & C， 在美国申请一个独立的教师职位。目标2B&D和目标3将在R 00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）一起工作，以回答关键问题， 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/R 00奖将大大促进我的博士后到PI的过渡，让我参加癌症生物学 课程，端粒酶癌症AACR会议，以及由冷泉港举办的显微镜研讨会 labs.在我的博士学位期间，我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之后，我将继续采取措施获得RCR培训。 我开始我的研究生涯作为一个MS。我是一个学生，合成小分子，但后来我把重点转移到了 to more to bio-oriented生物导向problems问题.作为一名技术人员，我研究蛋白质折叠，然后，作为一名研究生，我 我利用生物化学和X射线晶体学来研究RNA/DNA修复，现在，我是一名博士后， 我和汤姆·切赫博士开始研究人类癌细胞中的端粒酶调节。过程中 我的科学训练，我已经学会了理论概念，并在不同领域的发展实验技能， research.我相信，我迄今为止所获得的知识和经验将大大有助于 及时成功完成K99/R 00提案的目标。

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.