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.

摘要 端粒是位于染色体末端的重复DNA和相关蛋白质，对 基因组的稳定性。遮盖素复合体是一组六元蛋白质，对端粒长度很重要。 维持，这是通过调节端粒酶的招募和作用而发生的。此外, 保护素还保护染色体末端不被DNA损伤修复错误识别 机制。端粒生物学的紊乱是许多人类疾病的潜在原因。基因突变 端粒酶或保护素成分，导致端粒维持丧失，导致先天性角化不良， 肺纤维化和再生障碍性贫血。它在医学上的重要性进一步体现在90%的癌症 依赖于端粒酶的过度激活才能持续增殖。尽管庇护所在 基因组稳定性，一直没有一个一致的框架来理解其在端粒维持中的机制 已经成立了。保护素如何保护染色体末端并限制端粒酶访问的领先模型 端粒的尾巴是端粒环模型。其他更简单的模型包括DNA末端封顶或通过 形成隐蔽的结构。虽然这些模型是从DNA重塑属性派生出来的 单独的庇护素蛋白，庇护素蛋白作为一个功能性多亚单位复合体发挥的集体作用是 仍然模棱两可。避难所复合体如何将端粒DNA组织成不同的结构，以实现其调控 端粒酶延长中的功能及其在抑制和允许作用之间的转换 是下一个关键问题。答案在于这些过程的分子机制，因此， 本研究的目的是阐明保护素复合体调节端粒酶功能的结构基础。 端粒的募集和延长。将使用多尺度和跨学科的方法，包括 生物化学、生物物理学、细胞生物学和低温电磁技术。具体地说，本提案旨在确定：(1) 保护素复合体如何将端粒DNA组织成调节端粒酶的各种结构 可及性，(2)与端粒DNA组装的保护素复合体的结构，以及(3)保护如何 复合体从端粒末端封端切换到端粒酶刺激。在K99阶段，在 在Tom Cech博士的指导下，将使用AFM成像和生化分析来表征高阶 不同防护素复合体形成的DNA-蛋白质结构及其对端粒酶募集的影响 并将对活动进行衡量。在于志恒博士的额外支持下，将获得一套冷冻EM技能 同时测定与端粒DNA相关的保护素的冷冻-EM结构。这将便于使用 冷冻-电子显微镜研究在独立的R00期，护色素调节端粒酶的结构基础。 这一建议的结果将为理解防护林的机制提供一个多尺度的框架 在端粒维持中的作用，也潜在地提供了开发治疗和 对抗人类端粒功能障碍疾病的诊断策略。

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.