Defining the telomerase holoenzyme in progenitor cells with aging

描述（由申请人提供）：端粒是一种覆盖染色体末端的核蛋白结构，由端粒酶（一种多亚基酶复合物）维持。在端粒酶不足的情况下，包括在老化的人体组织中，端粒随着细胞分裂而缩短。端粒酶亚基在某些人类遗传性疾病中发生突变，例如先天性角化不良，其中端粒缩短加速并且组织祖细胞功能障碍。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害为以下假设提供了强有力的支持：衰老人类中的端粒缩短有助于衰老表型的各个方面。然而，端粒随着衰老而缩短的确切机制以及作为治疗策略的钝化或逆转端粒缩短的具体方法仍然知之甚少。确定为什么端粒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组分：端粒酶逆转录酶（TERT）、端粒酶RNA组分TERC和一种TERC结合蛋白dyskerin。为了解决这一关键领域的老化研究，我们已经从人类细胞中纯化端粒酶复合物，并确定新的端粒酶相关蛋白质的质谱。通过这种方法，我们确定了端粒酶全酶的一个新的组成部分，称为TCAB 1，端粒酶卡哈尔体蛋白1。TCAB 1与所有活性端粒酶相关，并与人细胞提取物中的所有TERC相关。重要的是，TCAB 1特异性地存在于卡哈尔体中，卡哈尔体是作为核糖核蛋白复合物修饰或组装的位点的亚核病灶。Cajal小体最近被证明是端粒酶定位的位点，TCAB 1是全酶的第一个Cajal小体特异性蛋白组分。使用RNA干扰在人类细胞中耗尽TCAB 1阻止端粒酶定位在Cajal小体中并导致端粒显著缩短。因此，TCAB 1是端粒酶运输和端粒合成所需的独特且必需的端粒酶组分。我们计划如下：（1）研究TCAB 1在端粒酶复合物和scaRNA复合物中的生物化学作用;（2）通过体外培养细胞的遗传学方法和TCAB 1蛋白复合物的分析，研究TCAB 1在端粒酶功能中的必要性; 公共卫生相关性：衰老是一个过程，在此过程中，健康随着时间的推移而减少，导致组织功能受损和对压力的反应减少。老龄化和老年人所患的疾病是一个巨大的公共卫生问题，特别是随着美国和其他工业化国家人口的老龄化。表征和引起衰老和衰老相关疾病的分子变化刚刚被揭开。其中一个变化是端粒的缩短，端粒是保护我们染色体末端的帽子。这项提议将研究维持端粒所需的端粒酶的一种新成分。对端粒酶的进一步了解对于设计延缓或逆转衰老某些方面的疗法至关重要。

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.