权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Analysis of the Molecular Mechanisms of Telomerase Recruitment to Telomeres and Telomerase Catalysis

端粒酶招募端粒及端粒酶催化的分子机制分析

基本信息

批准号：
9331708
负责人：
Jens Christopher Schmidt
金额：
$ 3.61万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-15 至 2017-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9331708
关键词：
Address Affinity Affinity Chromatography Alpha Cell Aplastic Anemia Apoptosis Binding Biochemical Biological Biological Assay Biology Biophysics Catalysis Cell Cycle Cell Cycle Stage Cell Line Cell Nucleus Cells Cellular Structures Cellular biology Chromosomes Complex Core Facility DNA Damage DNA Sequence DNA biosynthesis Defect Dyskeratosis Congenita Ensure Environment Enzymes Event Faculty Failure Fluorescent in Situ Hybridization Foundations Future Genetic Germ Cells Goals Grant Human Human Chromosomes Immunofluorescence Immunologic Institutes Institution Lead Learning Length Malignant Neoplasms Mass Spectrum Analysis Measures Mentorship Methods Microscopy Molecular Molecular Analysis Monitor Nucleotides Phase Phosphotransferases Play Positioning Attribute Post-Translational Modification Alteration Post-Translational Protein Processing Process Property Proteins Proteomics Pulmonary Fibrosis RNA RNA Processing RNA-Directed DNA Polymerase Recruitment Activity Regulation Research Research Personnel Resolution Role Running S Phase Secure Single-Stranded DNA Site Stem cells TERF1 gene TINF2 gene Telomerase Telomerase RNA Component Telomere Maintenance Therapeutic Time Training Visit base biophysical properties cancer cell career experience experimental study genome analysis genome editing human disease interdisciplinary approach kinase inhibitor live cell imaging novel strategies overexpression post-doctoral training prevent programs protein complex protein protein interaction senescence single molecule spatiotemporal telomere tool trafficking

项目摘要

Summary/Abstract Human chromosomes end in telomeres, repetitive DNA sequences that are bound by the Shelterin protein complex (1). During semi-conservative DNA replication the extreme ends of a chromosome are unable to be duplicated, leading to successive chromosome shortening. Once telomeres reach a critical length, cells enter senescence or undergo apoptosis (2). To counteract chromosome shortening, continuously dividing cells, such as germ cells, stem cells, and most cancer cells, express telomerase, an RNA-containing reverse transcriptase (3). Telomerase is a unique enzyme that processively adds telomeric repeats, copied from its RNA component, to the single-stranded DNA overhang of chromosome ends (4). The molecular mechanisms that govern telomerase processivity are poorly defined, but are critical to understand telomere maintenance. The Shelterin complex carries out two key functions at telomeres; it prevents telomeres from being recognized as sites of DNA damage, and it recruits telomerase to telomeres (5,6). Telomerase recruitment to telomeres is a tightly regulated process. Telomerase resides in Cajal bodies, specialized RNA-processing compartments in the nucleus, throughout most of the cell cycle. During S-phase, telomerase is recruited to telomeres to maintain telomere length (7). Although the protein-protein interactions required for telomerase to associate with telomeres are well understood, the spatio- temporal control of telomerase recruitment is poorly defined (7). Potential mechanisms for regulating telomerase recruitment include alterations in composition of telomerase and the shelterin complex or post-translational modification of its components. Telomere maintenance plays an important role in multiple human diseases. Deficiencies in telomerase assembly, activity, or recruitment to telomeres cause dyskeratosis congenita, pulmonary fibrosis, and aplastic anemia, severe human conditions characterized by stem cell failure (8). In addition, 90% of cancers rely on telomerase activity to allow them to divide indefinitely (9). Therefore, understanding the basic biology of telomerase recruitment to telomeres and telomerase catalysis could lead to novel approaches to modulate this process as a therapeutic approach for several human diseases. I propose to analyze the molecular mechanisms underlying telomerase recruitment to telomeres and telomerase catalysis using genome editing and a combination of cell biological, proteomic, biochemical, and single-molecule approaches. In particular I will: 1. Determine the molecular mechanisms that drive telomerase recruitment to telomeres in S- Phase. Using genome-edited cell lines expressing tagged telomerase and shelterin components, I will conduct live cell imaging of telomerase trafficking to telomeres, analyze the assembly state of telomerase and the shelterin complex throughout the cell cycle using cell biological and proteomic approaches, and identify kinases that modulate telomerase trafficking. 2. Define the biochemical and biophysical properties of telomerase. Using single molecule approaches, I will assess the oligomeric state of telomerase, the biophysical properties that control its intrinsic processivity, and the impact of the interaction of TPP1 with telomerase on its catalytic cycle. The K99 phase of the proposed aims will be conducted under the mentorship of Dr. Tom Cech, who has an extraordinary track record in training post-doctoral fellows, with over 30 former mentees in faculty positions at prestigious research institutions worldwide. The Cech lab is an established leader in the biochemical and structural analysis of telomerase. In combination with my strong expertise in cell biological and microscopy-based approaches, the Cech lab provides an ideal environment to carry out the majority of the proposed research. For the proteomic analysis of shelterin assembly I will collaborate with the lab of Dr. Natalie Ahn, a leading researcher in using mass spectrometry to study protein post-translational modifications. Dr. Ahn's expertise and the proteomics core facility at the BioFrontiers Institute will allow me develop a strong foundation in using mass-spectrometry as a core discovery tool, a critical learning experience that will facilitate my short term goals and my future independent career. My goal for the K99 phase is to initiate Aims 1 and 2 of the proposal and build a strong foundation for the transition to becoming an independent investigator at a US research institution. My long term goal is to run a research program focused on the molecular mechanisms that ensure chromosomal integrity, a process defective in a large number of human diseases, using multi- disciplinary approaches including cell biological, biochemical, biophysical, proteomic, and genetic methods. A K99 grant would greatly aid me by providing critical training, helping me secure a faculty position, and allowing me to jumpstart my career as independent researcher.

总结/摘要人类染色体以端粒结束，端粒是由庇护蛋白结合的重复DNA序列。蛋白复合物（1）。在半保守的DNA复制过程中，染色体的末端是无法复制，导致染色体连续缩短。一旦端粒达到一个临界点，长度，细胞进入衰老或经历凋亡（2）。为了抵消染色体缩短，连续分裂的细胞如生殖细胞、干细胞和大多数癌细胞，表达端粒酶，含RNA的逆转录酶（3）。端粒酶是一种独特的酶，端粒重复序列，从其RNA组分复制到单链DNA突出端，染色体末端（4）。控制端粒酶持续合成能力的分子机制定义，但对理解端粒的维护至关重要。 Shelterin复合物在端粒上执行两个关键功能：它被认为是DNA损伤的位点，并且它将端粒酶招募到端粒（5，6）。端粒酶端粒的补充是一个严格调控的过程。端粒酶存在于卡哈尔体中，在细胞周期的大部分时间里，细胞核中的RNA加工区室。在S期，端粒酶被募集到端粒以维持端粒长度（7）。虽然蛋白质-蛋白质端粒酶与端粒结合所需的相互作用是很好理解的，端粒酶募集的时间控制定义不明确（7）。潜在的调节机制端粒酶募集包括端粒酶和shelterin复合物组成的改变，其组分的翻译后修饰。端粒的维持在多种人类疾病中起着重要作用。缺陷端粒酶组装、活性或募集至端粒引起先天性肺角化不良纤维化和再生障碍性贫血，严重的人类疾病特征是干细胞衰竭（8）。在此外，90%的癌症依赖于端粒酶活性，使它们能够无限分裂（9）。因此，我们认为，了解端粒酶募集到端粒和端粒酶催化的基础生物学，从而导致调节该过程新方法作为几种人的治疗方法疾病我建议分析端粒酶募集的分子机制，端粒和端粒酶催化使用基因组编辑和细胞生物学，蛋白质组学、生物化学和单分子方法。我尤其将： 1.确定驱动端粒酶募集到S-细胞端粒的分子机制，相位使用基因组编辑的表达标记端粒酶和shelterin成分的细胞系，我将进行端粒酶向端粒运输的活细胞成像，分析端粒的组装状态，端粒酶和shelterin复合物在整个细胞周期中的作用方法，并确定调节端粒酶运输的激酶。 2.定义端粒酶的生物化学和生物物理特性。使用单分子方法，我将评估端粒酶的寡聚状态，控制其生物物理特性，内在的持续合成能力，以及TPP 1与端粒酶的相互作用对其催化循环的影响。拟议目标的K99阶段将在Tom Cech博士的指导下进行，他在培养博士后研究员方面有着非凡的记录，在全球著名研究机构担任教职。切赫实验室是一个公认的领导者，端粒酶的生化和结构分析。结合我在细胞方面的丰富专业知识生物和显微镜为基础的方法，切赫实验室提供了一个理想的环境，大部分的研究建议。对于shelterin组装的蛋白质组学分析，与Natalie Ahn博士的实验室合作，Natalie Ahn博士是使用质谱法研究蛋白质翻译后修饰。安博士的专业知识和蛋白质组学的核心设施，生物前沿研究所将使我在使用质谱作为核心方面打下坚实的基础发现工具，一个关键的学习经验，将促进我的短期目标和我的未来独立的职业生涯。我在K99阶段的目标是启动提案的目标1和2，并建立一个强大的为过渡到成为美国研究机构的独立调查员奠定了基础。我长期目标是开展一项研究计划，重点是确保染色体完整性，在大量的人类疾病中有缺陷的过程，使用多- 包括细胞生物学、生物化学、生物物理学、蛋白质组学和遗传学在内的学科方法方法. K99的资助将通过提供关键性培训来帮助我，帮助我获得一名教师，这让我有机会开始我的独立研究员生涯。