Separation-of-function Mutants to Study the Biological Significance of Telomerase

功能分离突变体研究端粒酶的生物学意义

• 批准号: 8298843
• 负责人: Jayakrishnan Nandakumar
• 金额: $ 9万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-15 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8298843
• 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; 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 Delivery Systems; Drug Design; 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

DESCRIPTION (provided by applicant): 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 bu 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. PUBLIC HEALTH RELEVANCE: Telomerase is an enzyme that is overproduced in 90% of human cancers and is therefore considered a major drug target for fighting cancer. Biochemically, this enzyme is stimulated by a protein called TPP1, and the importance of this stimulation in cancer cells will be tested in the proposed study. From this study, it will be possible to understand how TPP1 stimulates telomerase to full potency in cancer cells, and the knowledge gained may pave the path to the design of a new generation of anti-cancer drugs.

描述（由申请人提供）：哺乳动物端粒酶是一种专门的逆转录酶，它用端粒DNA扩展了染色体的3'末端。由于端粒酶在体细胞中弱表达，但在90％的癌细胞中过表达，因此它是抗癌药物设计的有吸引力的靶标。一种被称为庇护素的多蛋白复合物与端粒DNA相关联，以抑制哺乳动物染色体末端的非法DNA融合。如果庇护所的正常功能是“保护”染色体末端，端粒酶如何访问这些末端以扩展它们？ POT1-TPP1是一种庇护素子复合物，它结合具有高特异性和亲和力的单链端粒DNA。 POT1-TPP1在体内的主要功能是抑制端粒的DNA损伤识别事件。鉴于其在染色体末端保护中的作用，POT1-TPP1可能会通过防止染色体末端的获取来抑制端粒酶。出乎意料的是，POT1-TPP1在体外提高了端粒酶加工性。此外，TPP1的OB结构域参与端粒募集到端粒。 POT1-TPP1刺激端粒酶具有至关重要的生理意义，而与癌细胞相关的端粒酶活性可能需要基于POT1-TPP1的刺激。在这里，假设TPP1 OB结构域的表面与端粒酶直接相互作用，从而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结合，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的研究。 该项目的具体目的是：1。确定人类TPP1中的结构元素，这些元素使用位置定向的诱变筛选导致端粒酶加工性刺激，寻找功能分离突变体在体外特有在端粒酶刺激中有缺陷，但在体外却不有缺陷 DNA末端保护。 2。确定TPP1在基于HELA的和肺癌细胞线中TPP1刺激端粒酶刺激的生理重要性和机制，这些细胞会以稳定的方式击倒TPP1的内源性TPP1和表达TPP1的野生型或端粒酶刺激性突变体。这些细胞系将测试端粒长度缺陷和端粒酶募集缺陷。 3a。通过与端粒DNA复合物中的POT1-TPP1融合蛋白结晶，通过POT1-TPP1来获得染色体末端保护的高分辨率视图。 3b。通过结晶的生物化学能力，最小，TPP1-纤维素酶复合物通过单个成分的截断分析来定义，通过TPP1刺激端粒酶刺激。 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，他的指导技巧帮助他的30多名受训者在美国和全球的研究机构中获得了教师职位。 Cech Lab是端粒酶和端粒生物化学的领导者，并配备了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，我将由丹佛分校癌症中心的James Degregori博士共同指导，并将完全访问其实验室和癌症中心的设施。因此，我坚信Cu Boulder和癌症中心的设施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，并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研究者，运营一个由来自各种背景的人（生物化学，结构生物学和细胞生物学）组成的实验室，以回答端粒生物学及其对癌症的影响的关键问题。除了允许我雇用员工和BU实验室用品外，K99/R00奖还将通过允许我参加癌症生物学课程，端粒酶CACRACR AACR会议以及Cold Springshore Harbour Labs进行的显微镜工作室，从而极大地促进了我的博士后过渡。我在博士学位期间接受了负责任的研究（RCR）的正式培训。并将继续采取措施在我的博士后中和之后获得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，但此后将我的重点转移到了面向生物的问题上。作为技术人员，我研究了蛋白质折叠，因此，作为一名研究生，我采用了生物化学和X射线晶体学来研究RNA/DNA修复，现在，与Tom Cech博士一起工作后，我开始研究人类癌细胞中的端粒酶调节。在我的科学培训过程中，我学习了理论概念并在不同的研究领域发展了实验技能。我相信，到目前为止，我获得的知识和经验将极大地帮助及时完成K99/R00提案的目标。 公共卫生相关性：端粒酶是一种酶，在90％的人类癌症中过量生产，因此被认为是与癌症作斗争的主要药物靶标。从生化上讲，该酶是由称为TPP1的蛋白刺激的，并且在拟议的研究中将测试这种刺激在癌细胞中的重要性。从这项研究中，可以了解TPP1如何刺激端粒酶到癌细胞的全部效力，并且获得的知识可能铺平了新一代抗癌药物的设计。