Biogenesis and Regulation of Human Telomerase

人类端粒酶的生物发生和调控

• 批准号: 8257065
• 负责人: Kathleen Collins
• 金额: $ 37.99万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 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/8257065
• 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

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延长染色体结束。这个新的DNA 需要综合才能平衡不完整复制中固有的DNA损失 染色体以常规DNA聚合酶结束。单细胞真核生物组成性激活 端粒酶并保持端粒长度的体内平衡。令人惊讶的是，人体细胞没有： 它们显示出随着增殖的端粒重复阵列的进行性缩短。一些人类细胞 胚胎，种系，上皮组织和造血系统具有可检测的水平 细胞裂解物中的端粒酶催化活性，但是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。累积损失最终会产生 重复阵列太短，无法保护染色体末端，从而导致牢房的强迫退出 循环。癌细胞急剧上调端粒酶以允许无限期的生长。为此原因， 作为广泛有效的抗癌治疗剂，端粒酶抑制剂具有巨大的希望。端粒酶 激活剂可能同样具有显着的应用来扩大正常的更新能力 由遗传学，疾病，年龄或环境引起的端粒非常短的体细胞。 端粒酶RNA亚基（TER）表示为必须处理，折叠， 并作为稳定的核糖核蛋白（RNP）复合物组装，以积累至可检测 体内水平。然后，此RNP募集端粒酶逆转录酶（TERT）生成活动 酶。柯林斯实验室的努力在以前的资助期间为关于 人类前体加工和RNP组装的内源性途径并发现 基于X连锁和常染色体显性的成熟端粒酶RNP积累的缺陷 骨髓衰竭综合征的形式性障碍性脑膜炎。 下一个资金期的具体目的解决了有关人类知识的差距 端粒酶RNP积累和体内催化活化。 AIM 1利用瞬态和 人类细胞中稳定的TER表达，发现和表征其他RNA基序和蛋白质 成熟和生物稳定性所必需的。 AIM 2应用Collins实验室专业知识 互动测定和亲和力净化，以定义遗传的生化缺陷 端粒酶缺乏的人类疾病。 AIM 3研究端粒酶的组装和活动 带有TERT的RNP。体内重建方法将与体外和体内活性相结合 在催化循环中定义TER基序函数的测定。 RNA和 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 研究是为了了解端粒酶RNP组装，催化活化和细胞调节 正常细胞和疾病，并利用这种理解来改善人类健康。