Structural understanding of human shelterin complex assembly at telomere and its regulation mechanism of telomerase activity

端粒端粒蛋白复合物组装结构及其端粒酶活性调控机制

• 批准号: 10470875
• 负责人: Ci Ji Lim
• 金额: $ 23.74万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-10 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10470875
• 关键词: 3-Dimensional; Address; Affect; Aging; Aplastic Anemia; Architecture; Atomic Force Microscopy; Automobile Driving; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Biology; Biophysics; Cells; Cellular biology; Chromosomes; Complex; Cryoelectron Microscopy; Cultured Cells; DNA; DNA Repair; Disease; Dyskeratosis Congenita; Elongation by Telomerase; Enzymes; Failure; Functional disorder; Gel; Genome Stability; Genomic Instability; Goals; Human; Image Analysis; Individual; Knowledge; Lead; Length; Malignant Neoplasms; Measures; Mediating; Medical; Mentorship; Modeling; Molecular; Molecular Conformation; Mutation; Negative Staining; Outcome Study; Pathway interactions; Phase; Play; Process; Property; Proteins; Pulmonary Fibrosis; Recombinants; Recruitment Activity; Regulation; Resolution; Role; Structure; Switching Complex; Tail; Techniques; Telomerase; Telomere Length Maintenance; Telomere Maintenance; Therapeutic; base; combat; diagnostic strategy; human disease; interdisciplinary approach; member; microscopic imaging; particle; protein complex; protein protein interaction; reconstruction; recruit; single molecule; skills; telomere; telomere loss; tumor progression

Summary Telomeres are repetitive DNA and associated proteins at the ends of the chromosomes that are important for genome stability. The shelterin complex, a six-member group of proteins, is important for telomere length maintenance, which occurs through regulating the recruitment and action of the telomerase enzyme. In addition, shelterin also protects the chromosome ends from being incorrectly recognized by DNA damage repair mechanisms. Disorders in telomere biology are an underlying cause of many human diseases. Mutation of telomerase or shelterin components, resulting in loss of telomere maintenance, leads to dyskeratosis congenita, pulmonary fibrosis and aplastic anemia. Its medical importance is further shown by the fact that 90% of cancers depend on hyper-activation of telomerase for persistent proliferation. Despite the critical roles shelterin plays in genome stability, a consistent framework of understanding its mechanism in telomere maintenance has not been established. A leading model of how shelterin can protect chromosome ends and restrict telomerase access to the telomeric tail is the telomere-loop model. Other simpler models include DNA end-capping or compaction by shelterin to form reclusive structures. While these models are derived from the DNA remodeling properties of individual shelterin proteins, the collective roles shelterin proteins play as a functional multisubunit complex are still ambiguous. How a shelterin complex organizes telomeric DNA into various architectures for their regulatory functions and how they switch between inhibitory and permissive roles in telomerase elongation of telomeres are the next key questions. The answers lie in the molecular mechanisms of these processes and hence, the goal of this proposal is to elucidate the structural basis of shelterin complex functions in regulating telomerase recruitment and elongation of telomeres. A multiscale and interdisciplinary approach will be used, which includes biochemistry, biophysics, cell biology and cryo-EM techniques. Specifically, this proposal aims to determine: (1) How the shelterin complex organizes telomeric DNA into various architectures that regulate telomerase accessibility, (2) The structure of the shelterin complex assembled with telomeric DNA, and (3) How a shelterin complex switches from telomere end-capping to telomerase stimulation. During the K99 phase, under the mentorship of Dr. Tom Cech, AFM imaging with biochemical assays will be used to characterize higher-order DNA-protein architectures formed by various shelterin complexes, and their effects on telomerase recruitment and activity will be measured. With additional support from Dr. Zhiheng Yu, a cryo-EM skill set will be acquired while determining the cryo-EM structure of shelterin in association with telomeric DNA. This will facilitate using cryo-EM to study the structural basis of telomerase regulation by shelterin during the independent R00 phase. The results of this proposal would provide a multiscale framework for understanding the mechanisms of shelterin functions in telomere maintenance, and also potentially provide new avenues in developing therapeutic and diagnostic strategies to combat human diseases of telomere dysfunction.

概括 端粒是重复的DNA和相关蛋白，在染色体的末端对 基因组稳定性。庇护素综合体是一组六人组的蛋白质，对于端粒长度很重要 维护，这是通过调节端粒酶酶的募集和作用而发生的。此外， 庇护素还可以保护染色体末端免于被DNA损伤修复错误地识别 机制。端粒生物学的疾病是许多人类疾病的根本原因。突变 端粒酶或庇护素成分导致端粒维持损失，导致康涅蒂塔病症状， 肺纤维化和性贫血。 90％的癌症的事实进一步表明了它的医学重要性 取决于端粒酶过度激活持续增殖。尽管庇护所扮演着关键的角色 基因组稳定性，是理解其在端粒维护中机制的一致框架 已确立的。庇护所如何保护染色体末端并限制端粒酶的主要模型 端粒尾部是端粒环模型。其他简单的模型包括DNA终端封顶或压实 庇护所形成隐居结构。这些模型是从DNA重塑特性得出的 单个庇护素蛋白，集体角色庇护素蛋白作为功能多育种综合体的发挥作用是 仍然模棱两可。庇护所如何将​​端粒DNA组织成各种体系结构进行监管 功能及其如何在端粒伸长酶伸长率中的抑制性和允许作用之间切换 是下一个关键问题。答案在于这些过程的分子机制，因此 该建议的目标是阐明庇护素复合物在调节端粒酶中的结构基础 端粒的招募和延伸。将使用多尺度和跨学科的方法，其中包括 生物化学，生物物理学，细胞生物学和冷冻EM技术。具体而言，该建议旨在确定：（1） 庇护素综合体如何组织端粒DNA成各种调节端粒酶的体系结构 可访问性，（2）用端粒DNA组装的庇护所综合体的结构，以及（3）庇护所如何 从端粒末端封顶到端粒酶刺激的复杂开关。在K99阶段，在 Tom Cech博士的指导，使用生化测定的AFM成像将用于表征高阶 由各种庇护所络合物形成的DNA-蛋白结构及其对端粒酶募集的影响 和活动将被衡量。在Zhiheng Yu博士的额外支持下，将获得Cryo-EM技能集 同时确定庇护素与端粒DNA相关的冷冻结构。这将有助于使用 冷冻EM研究独立R00阶段中庇护素调节端粒酶调节的结构基础。 该提案的结果将为了解庇护所的机制提供多尺度框架 在端粒维护中的功能，也有可能在发展治疗和发展方面提供新的途径 打击端粒功能障碍的人类疾病的诊断策略。

项目成果

Shaping human telomeres: from shelterin and CST complexes to telomeric chromatin organization.

• DOI: 10.1038/s41580-021-00328-y
• 发表时间: 2021-04
• 期刊: Nature reviews. Molecular cell biology
• 作者: Lim CJ;Cech TR
• 通讯作者: Cech TR

Structures of the human CST-Polα-primase complex bound to telomere templates.

• DOI: 10.1038/s41586-022-05040-1
• 发表时间: 2022-08
• 期刊: NATURE
• 影响因子: 64.8
• 作者: He, Qixiang;Lin, Xiuhua;Chavez, Bianca L.;Agrawal, Sourav;Lusk, Benjamin L.;Lim, Ci Ji
• 通讯作者: Lim, Ci Ji

Structures of human primosome elongation complexes.

• DOI: 10.1038/s41594-023-00971-3
• 发表时间: 2023-05
• 期刊: NATURE STRUCTURAL & MOLECULAR BIOLOGY
• 影响因子: 16.8
• 作者: He, Qixiang;Baranovskiy, Andrey G.;Morstadt, Lucia M.;Lisova, Alisa E.;Babayeva, Nigar D.;Lusk, Benjamin L.;Lim, Ci Ji;Tahirov, Tahir H.
• 通讯作者: Tahirov, Tahir H.