Biogenesis and Regulation of Human Telomerase

人类端粒酶的生物发生和调控

基本信息

批准号：
8463827
负责人：
Kathleen Collins
金额：
$ 36.17万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-09-30 至 2014-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8463827
关键词：
Address Affinity Chromatography Age Amino Acid Motifs Biochemical Biogenesis Biological Biological Assay Cell Cycle Cells Chromosomes Clinical Collaborations Complex DNA DNA biosynthesis DNA-Directed DNA Polymerase Data Defect Disease Dyskeratosis Congenita Embryo Environment Enzymes Epithelial Equilibrium Eukaryota Event Failure Funding Goals Growth Health Hematopoietic System Hereditary Disease Homeostasis Human In Vitro Inherited Knowledge Length Link Malignant Neoplasms Medical Methods Molecular Normal Cell Pancytopenia Pathway interactions Patients Pharmaceutical Preparations Physiological Process Proteins Publishing RNA RNA-Protein Interaction Recruitment Activity Regulation Ribonucleoproteins Somatic Cell Specificity Syndrome Telomerase Telomerase Inhibitor Telomerase RNA Component Tertiary Protein Structure Tissues Work anti-cancer therapeutic cancer cell cell growth regulation chromosome replication design human TERT protein human disease human tissue in vivo insight prevent reconstitution telomerase reverse transcriptase telomere

项目摘要

Telomerase elongates chromosome ends by addition of tandem telomeric repeats. This new DNA synthesis is required to balance the loss of DNA that is inherent in the incomplete replication of chromosome ends by conventional DNA polymerases. Single-celled eukaryotes constitutively activate telomerase and maintain a homeostasis of telomere length. Surprisingly, human somatic cells do not: they show progressive shortening of the telomeric repeat array with proliferation. Some human cells in the embryo, germline, epithelial tissues, and hematopoietic system have detectable levels of telomerase catalytic activity in cell lysates, but this level of activation is insufficient to prevent an overall loss of telomere length in all human tissues with age. Cumulative loss eventually produces a repeat array that is too short to protect the chromosome end, resulting in a forced exit from the cell cycle. Cancer cells dramatically up-regulate telomerase to permit indefinite growth. For this reason, telomerase inhibitors have great promise as broadly effective anti-cancer therapeutics. Telomerase activators may have equally significant application for expanding the renewal capacity of normal somatic cells with critically short telomeres arising from genetics, disease, age, or environment. The telomerase RNA subunit (TER) is expressed as a precursor that must be processed, folded, and assembled as a stable ribonucleoprotein (RNP) complex in order to accumulate to detectable level in vivo. This RNP then recruits telomerase reverse transcriptase (TERT) to generate the active enzyme. Collins lab efforts in previous funding periods have contributed pioneering insights about the endogenous pathway of human TER precursor processing and RNP assembly and discovered defects in the accumulation of mature telomerase RNP that underlie X-linked and autosomal dominant forms of the bone marrow failure syndrome dyskeratosis congenita. The Specific Aims of the next funding period address remaining gaps in knowledge about human telomerase RNP accumulation and catalytic activation in vivo. Aim 1 exploits methods of transient and stable TER expression in human cells to discover and characterize additional RNA motifs and proteins required for TER maturation and biological stability. Aim 2 applies Collins lab expertise in RNA-protein interaction assays and affinity purification to define the biochemical defects that underlie inherited human diseases of telomerase deficiency. Aim 3 investigates the assembly and activity of telomerase RNP with TERT. In vivo reconstitution methods will be combined with in vitro and in vivo activity assays to define TER motif functions in the catalytic cycle. The physiological specificity of RNA and protein domain interactions within the active RNP will be established. The long-term goal of these studies is to understand telomerase RNP assembly, catalytic activation, and cellular regulation in normal cells and disease and to exploit this understanding for improvement of human health.

端粒酶通过添加串联端粒重复序列来延长染色体末端。这个新的DNA 需要合成来平衡 DNA 的不完全复制所固有的损失染色体末端由常规 DNA 聚合酶完成。单细胞真核生物组成型激活端粒酶并维持端粒长度的稳态。令人惊讶的是，人类体细胞不会：它们显示端粒重复阵列随着增殖逐渐缩短。一些人体细胞在胚胎、种系、上皮组织和造血系统均具有可检测水平端粒酶在细胞裂解物中的催化活性，但这种激活水平不足以防止随着年龄的增长，所有人体组织中端粒长度的总体损失。累积损失最终产生重复阵列太短，无法保护染色体末端，导致强制退出细胞循环。癌细胞显着上调端粒酶以允许无限期生长。为此原因，端粒酶抑制剂作为广泛有效的抗癌疗法具有广阔的前景。端粒酶活化剂在扩大正常细胞的更新能力方面可能具有同样重要的应用由于遗传、疾病、年龄或环境而导致端粒极短的体细胞。端粒酶 RNA 亚基 (TER) 表达为前体，必须经过加工、折叠、并组装成稳定的核糖核蛋白 (RNP) 复合物，以便积累到可检测的水平体内水平。然后，该 RNP 招募端粒酶逆转录酶 (TERT) 来生成活性酶。柯林斯实验室在之前的资助期间的努力贡献了有关该领域的开创性见解人类 TER 前体加工和 RNP 组装的内源性途径并被发现成熟端粒酶 RNP 积累缺陷是 X 连锁和常染色体显性遗传的基础先天性角化不良的骨髓衰竭综合征的形式。下一个资助期的具体目标是解决人类知识方面的剩余差距端粒酶 RNP 积累和体内催化激活。目标 1 利用瞬态和人类细胞中稳定的 TER 表达，以发现和表征其他 RNA 基序和蛋白质 TER 成熟和生物稳定性所需的。目标 2 应用柯林斯实验室在 RNA 蛋白质方面的专业知识相互作用测定和亲和纯化来确定遗传性的生化缺陷人类端粒酶缺乏症。目标 3 研究端粒酶的组装和活性 RNP 与 TERT。体内重构方法将与体外和体内活性相结合测定催化循环中的 TER 基序功能。 RNA的生理特异性将建立活性 RNP 内的蛋白质结构域相互作用。这些的长期目标研究的目的是了解端粒酶 RNP 组装、催化激活和细胞调节正常细胞和疾病，并利用这种理解来改善人类健康。