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

描述（由申请人提供）：哺乳动物端粒酶是一种特殊的逆转录酶，可以用端粒DNA延伸染色体的3'端。由于端粒酶在体细胞中表达较弱，但在90%的癌细胞中过度表达，因此它是抗癌药物设计的一个有吸引力的靶点。一种称为庇护蛋白的多蛋白复合物与端粒DNA特异性结合，抑制哺乳动物染色体末端的非法DNA融合。如果庇护蛋白的正常功能是“保护”染色体末端，那么端粒酶是如何进入这些末端并延长它们的呢？POT1-TPP1是一种具有高特异性和亲和力的结合单链端粒DNA的庇护蛋白亚复合物。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，在体外，POT1-TPP1增加了端粒酶的活性。此外，TPP1的OB结构域参与端粒酶向端粒的募集。POT1-TPP1对端粒酶的刺激具有重要的生理意义，因为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的OB结构域表面直接与端粒酶相互作用，引起端粒酶的募集和刺激。酶学将结合哺乳动物细胞生物学和结构生物学来验证这一假设，并确定TPP1在癌细胞中刺激端粒酶的后果。这将是第一个直接评估TPP1或任何哺乳动物庇护蛋白亚复合物对端粒酶刺激的生物学重要性的研究。

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.