Structure-Function Relationship Study of Telomerase Reverse Transcriptase's Non-Canonical Activities

端粒酶逆转录酶（Telomerase reverse transcriptase，TERT）是一种核糖核蛋白逆转录酶，负责将端粒DNA重复序列重新添加到人类染色体上。在一些需要延长增殖寿命的特化细胞中，例如干细胞和生殖系细胞，端粒酶活性对于端粒修复以延长这些细胞类型的增殖能力是必需的。新的证据表明，在永生人类细胞中的组成型TERT表达也通过多种机制促进细胞在应激生长条件下的存活。由TERT提供的这些存活优势是通过与端粒修复无关的细胞功能介导的，但是负责这些活动的精确机制和TERT的结构形式在很大程度上仍然未知。最近的进展我的实验室报告说，端粒酶的重组表达赋予细胞生长的优势，并促进锚定非依赖性生长，在一种方式是无关的端粒长度的维护。TERT的表达还可以防止DNA损伤剂引起的遗传毒性。我们发现，端粒酶变体和选择性剪接（截短蛋白）形式，缺乏端粒合成活性，也表现出相同的保护多种遗传毒性刺激，这表明端粒酶从事在结构上，以及功能上不同的复合物，其典型的（端粒合成相关的）和非典型的（端粒合成独立的）活动。端粒酶促进细胞毒性和代谢应激后的细胞存活，并通过参与多种结构复合物促进生长和增殖。目的和方法目的1：最小的TERT结构域在其DNA损伤修复活性中的生物化学表征我们将绘制负责其DNA损伤修复/保护活性的最小的TERT结构域，并产生跨越所鉴定的结构域序列的短肽，并测试这些肽作为破坏这种TERT活性的策略。目的2：亲和纯化和质谱鉴定不同的TERT复合物。我们将对来自表达FL-TERT或截短的TERT变体的ALT细胞的纯化的TERT复合物进行串联亲和标签（TAP）纯化和LC-MS-MS分析。将用遗传和/或化学方法评价所选非典型TERT蛋白配偶体的生物学功能。我们对不同的端粒酶结构形式及其在独特的生物复合物中的成员资格的功能和生物化学表征将进一步了解人类生物学中过多的端粒酶活性。这项工作还将支持7名HQP，包括2名高年级研究生和5名UG。我们的建议与NSERC的2020年研究战略计划一致，通过为下一代研究人员提供尖端的生物化学，蛋白质科学和分子遗传学培训，建立多元化和有竞争力的研究基地。

Literature Telomerase reverse transcriptase (TERT) is a ribonucleoprotein reverse transcriptase responsible for the de novo addition of telomeric DNA repeats to human chromosomes. In some specialized cells that require extended proliferation lifespans, such as stem cells and germ-line cells, telomerase activity is necessary for telomere repair to extend the proliferative capacity of these cell types. New evidence suggests that constitutive TERT expression in immortal human cells also promotes cell survival under stressful growth conditions through multiple mechanisms. These survival advantages provided by TERT are mediated through cellular functions not related to telomere repair, but the precise mechanism and TERT's structural form responsible for these activities remained largely unknown. Recent Progress My laboratory reported that recombinant expression of telomerase confers cellular growth advantages and promotes anchorage-independent growth, in a manner that is unrelated to telomere-length maintenance. TERT expression also protects against genotoxicity caused by DNA-damaging agents. We showed that TERT variants and alternatively spliced (truncated protein) forms, devoid of telomere-synthesis activity, also exhibited the same protection against multiple genotoxic stimuli, suggesting that telomerase engaged in structurally, as well as functionally distinct complexes for its canonical (telomere synthesis-related) and non-canonical (telomere-synthesis-independent) activities. Hypothesis Telomerase promotes cell survival following cytotoxic and metabolic stress, and abets growth and proliferation through participation in multiple structural complexes. Objectives and Methods Aim 1: Biochemical characterization of a minimal TERT domain in its DNA damage repair activities We will map the minimal TERT domain responsible for its DNA damage repair/protection activity, and generate short-peptide spanning the identified domain sequence, and test these peptides as a strategy to disrupt this TERT activity. Aim 2: Affinity purification and mass-spectrometry identification of distinct TERT complexes. We will perform tandem-affinity-tag (TAP) purification and LC-MS-MS analysis of purified TERT-complexes, from ALT cells expressing FL-TERT or truncated TERT variants. The biological functions of selected non-canonical TERT protein partners will be evaluated with genetic and/or chemical means. Impact Our functional and biochemical characterization of the different TERT structural forms and their membership in unique biological complexes will further the understanding on the plethora of telomerase activities in human biology. This work will also support 7 HQP, including 2 senior graduate students and 5 UGs. Our proposal is in agreement with NSERC's 2020 Research Strategic Plan to build a diversify and competitive research base by providing cutting-edge biochemical, protein science and molecular genetics training for the next generation of researchers.