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

端粒上人庇护蛋白复合物组装的结构及其端粒酶活性调控机制

• 批准号: 10226388
• 负责人: Ci Ji Lim
• 金额: $ 24.9万
• 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/10226388
• 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; Diagnostic; 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; 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 端粒是位于染色体末端的重复 DNA 和相关蛋白质，对于生命活动非常重要 基因组稳定性。庇护蛋白复合物是一组六元蛋白质，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来实现的。此外， 保护蛋白还可以保护染色体末端不被 DNA 损伤修复错误识别 机制。端粒生物学紊乱是许多人类疾病的根本原因。突变 端粒酶或庇护蛋白成分，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。 90% 的癌症 依赖于端粒酶的过度激活来实现持续增殖。尽管庇护林在其中发挥着关键作用 基因组稳定性，理解其端粒维持机制的一致框架尚未建立 已确立的。保护蛋白如何保护染色体末端并限制端粒酶接触的领先模型 端粒尾部是端粒环模型。其他更简单的模型包括 DNA 封端或压缩 庇护形成隐居结构。虽然这些模型源自 DNA 重塑特性 单个庇护蛋白，庇护蛋白作为功能性多亚基复合物发挥的集体作用是 还是暧昧。庇护蛋白复合体如何将端粒 DNA 组织成各种结构以进行调节 端粒酶延长的功能以及它们如何在抑制和许可作用之间切换 是下一个关键问题。答案在于这些过程的分子机制，因此 该提案的目标是阐明庇护蛋白复合物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，其中包括 生物化学、生物物理学、细胞生物学和冷冻电镜技术。具体来说，该提案旨在确定：(1) 庇护蛋白复合物如何将端粒 DNA 组织成调节端粒酶的各种结构 可及性，(2) 与端粒 DNA 组装的庇护蛋白复合物的结构，以及 (3) 庇护蛋白如何 从端粒封端到端粒酶刺激的复杂转换。在K99阶段，在 在 Tom Cech 博士的指导下，AFM 成像与生化分析将用于表征高阶 由各种庇护蛋白复合物形成的DNA-蛋白质结构及其对端粒酶招募的影响 并将测量活动。在余志恒博士的额外支持下，我们将获得冷冻电镜技能 同时确定与端粒 DNA 相关的庇护蛋白的冷冻电镜结构。这将有助于使用 冷冻电镜研究独立 R00 相期间庇护蛋白调节端粒酶的结构基础。 该提案的结果将为理解庇护所的机制提供一个多尺度框架 端粒维持的功能，也可能为开发治疗和治疗提供新途径 对抗人类端粒功能障碍疾病的诊断策略。