Defining the telomerase holoenzyme in progenitor cells with aging

描述（由申请人提供）：端粒，覆盖染色体末端的核蛋白结构，由端粒酶维持，端粒酶是一种多亚基酶复合物。在端粒酶不足的情况下，包括在衰老的人体组织中，端粒随着细胞分裂而缩短。端粒酶亚基在某些人类遗传疾病中发生突变，如先天性角化不良，其中端粒缩短加速，组织祖细胞功能失调。在人类遗传综合征和基因敲除小鼠模型中，端粒缩短引起的组织祖细胞功能的严重损伤为衰老人类端粒缩短有助于衰老表型的假设提供了强有力的支持。然而，随着年龄的增长，端粒缩短的确切机制以及作为一种治疗策略来减弱或逆转端粒缩短的具体方法仍然知之甚少。确定端粒为什么随着年龄的增长而缩短，并开发治疗方法来防止端粒缩短，将需要对端粒酶有更全面的了解。虽然从人类癌细胞中提取的端粒酶表现为一个非常大的复合物，但直到最近才确定了端粒酶全酶的三种成分：TERT，端粒酶逆转录酶，TERC，端粒酶RNA成分和dyskerin，一种TERC结合蛋白。为了解决衰老研究中的这一关键领域，我们从人类细胞中纯化了端粒酶复合物，并通过质谱法鉴定了新的端粒酶相关蛋白。通过这种方法，我们确定了端粒酶全酶的新成分，称为TCAB1，端粒酶Cajal体蛋白1。TCAB1与人类细胞提取物中所有活性端粒酶和所有TERC相关。重要的是，TCAB1特异存在于Cajal小体中，这是作为核糖核蛋白复合物修饰或组装位点的亚核病灶。Cajal体最近被证明是端粒酶定位的位点，TCAB1是该全酶的第一个Cajal体特异性蛋白质成分。利用RNA干扰减少人类细胞中的TCAB1可阻止端粒酶在Cajal体中的定位，并导致端粒深度缩短。因此，TCAB1是端粒酶运输和端粒合成所需的独特而必需的端粒酶成分。我们计划：(1)研究TCAB1在端粒酶复合体和scaRNA复合体中的生物化学作用(2)通过培养细胞的遗传方法和TCAB1蛋白复合体的分析，研究端粒酶功能对TCAB1的要求(3)了解TCAB1在体内端粒酶功能和干细胞调控中的作用。

DESCRIPTION (provided by applicant): Telomeres, the nucleoprotein structures that cap chromosome ends, are maintained by telomerase, a multi-subunit enzyme complex. In settings of insufficient telomerase, including in aging human tissues, telomeres shorten with cell division. Telomerase subunits are mutated in certain human genetic diseases, such as dyskeratosis congenita, in which telomere shortening is accelerated and tissue progenitor cells are dysfunctional. The profound impairment of tissue progenitor cell function caused by telomere shortening in both human genetic syndromes and in knockout mouse models provides strong support for the hypothesis that telomere shortening in aging humans contributes to aspects of the aging phenotype. However, the precise mechanisms that underlie telomere shortening with aging and the specific approaches for blunting or reversing telomere shortening as a therapeutic strategy remain very poorly understood. Defining why telomeres shorten with advancing age and developing therapeutics to prevent such telomere shortening will require a much more complete understanding of telomerase. Although telomerase enzyme extracted from human cancer cells behaves as a very large complex, only three components of the telomerase holoenzyme were identified until recently: TERT, the telomerase reverse transcriptase, TERC, the telomerase RNA component, and dyskerin, a TERC-binding protein. To address this critical area in aging research, we have purified telomerase complexes from human cells and identified new telomerase-associated proteins by mass spectrometry. Through this approach, we identified a new component of the telomerase holoenzyme, termed TCAB1, for Telomerase Cajal Body Protein 1. TCAB1 associates with all active telomerase enzyme and associates with all TERC in human cell extracts. Importantly, TCAB1 is specifically found in Cajal bodies, subnuclear foci that serve as sites of ribonucleoprotein complex modification or assembly. Cajal Bodies were recently shown to be sites of telomerase localization and TCAB1 is the first Cajal body-specific protein component of the holoenzyme. Depletion of TCAB1 in human cells using RNA interference prevents telomerase from localizing in Cajal bodies and leads to profound telomere shortening. Thus, TCAB1 is a unique and essential telomerase component required for telomerase trafficking and telomere synthesis. We plan the following: (1) To study the biochemistry of TCAB1 in the telomerase complex and in scaRNA complexes (2) To study the requirement for TCAB1 in telomerase function through genetic approaches in cultured cells and through analysis of the TCAB1 protein complex (3) To understand the role of TCAB1 in telomerase function and stem cell regulation in vivo. PUBLIC HEALTH RELEVANCE: Aging is a process during which fitness diminishes over time, resulting in impaired tissue function and reduced responses to stress. Aging, and the diseases to which aged individuals succumb, represent an enormous public health problem, particularly with the aging of the population in the United States and in other industrialized nations. The molecular changes that characterize and cause aging and aging-related disease are just being unraveled. One such change is the shortening of telomeres, the caps that protect the ends of our chromosomes. This proposal will study a new component of the enzyme telomerase that is required for maintaining telomeres. An improved understanding of telomerase is crucial for designing therapies that will delay or reverse certain aspects of aging.