Defining the telomerase holoenzyme in progenitor cells with aging

描述(申请人提供)：端粒是覆盖染色体末端的核蛋白结构，由端粒酶维持，端粒酶是一种多亚单位酶复合体。在端粒酶不足的情况下，包括在衰老的人体组织中，端粒会随着细胞分裂而缩短。在某些人类遗传性疾病中，端粒酶亚基发生突变，如先天性角化不良，端粒缩短加速，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前体细胞功能的严重损害有力地支持了这样的假设，即人类衰老的端粒缩短有助于衰老表型的某些方面。然而，端粒缩短随年龄增长的确切机制以及钝化或逆转端粒缩短作为一种治疗策略的具体方法仍然知之甚少。弄清端粒为什么会随着年龄的增长而缩短，并开发出预防端粒缩短的治疗方法，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是一个非常大的复合体，但直到最近才发现端粒酶全酶的三个组分：端粒酶逆转录酶TERT，端粒酶RNA组分TERC和端粒酶结合蛋白Dyskerin。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体，并通过质谱仪鉴定了新的端粒酶相关蛋白。通过这种方法，我们发现了端粒酶Cajal Body Protein 1的一个新的端粒酶全酶成分，称为TCAB1。TCAB1与所有活性的端粒酶结合，并与人细胞提取物中的所有TERC结合。重要的是，TCAB1专门在Cajal小体中发现，Cajal小体是作为核糖核蛋白复合体修饰或组装的亚核焦点。最近发现Cajal小体是端粒酶的定位部位，而TCAB1是端粒酶的第一个Cajal小体特异性蛋白质组分。使用RNA干扰耗尽人类细胞中的TCAB1可以阻止端粒酶定位于Cajal小体，并导致严重的端粒缩短。因此，TCAB1是端粒酶运输和端粒合成所必需的独特的和必需的端粒酶成分。我们的计划如下：(1)研究TCAB1在端粒酶复合体和Scarna复合体中的生物化学；(2)通过培养细胞的遗传学方法和TCAB1蛋白复合体的分析，研究TCAB1在端粒酶功能中的需求；(3)了解TCAB1在体内端粒酶功能和干细胞调节中的作用。

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.