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

文献报道端粒酶逆转录酶(TERT)是一种核糖核蛋白逆转录酶，负责将端粒DNA重复序列从头添加到人类染色体上。在一些需要延长增殖寿命的特殊细胞中，如干细胞和生殖细胞，端粒酶活性是端粒修复所必需的，以延长这些细胞的增殖能力。新的证据表明，在永生的人类细胞中，结构性TERT的表达也通过多种机制促进细胞在应激生长条件下的存活。TERT提供的这些生存优势是通过与端粒修复无关的细胞功能介导的，但导致这些活动的确切机制和TERT的结构形式在很大程度上仍不清楚。最近进展我的实验室报道，端粒酶的重组表达赋予细胞生长优势，并以一种与端粒长度维持无关的方式促进非锚定生长。TERT的表达还可以防止DNA损伤剂造成的遗传毒性。我们发现，缺乏端粒合成活性的TERT变异体和选择性剪接(截断蛋白)形式也对多种遗传毒性刺激表现出相同的保护作用，这表明端粒酶参与了结构上和功能上不同的复合体的典型(端粒合成相关)和非典型(端粒合成非依赖性)活性。假设端粒酶在细胞毒性和代谢应激后促进细胞存活，并通过参与多种结构复合体而促进生长和增殖。目的和方法目标1：DNA损伤修复活动中最小TERT结构域的生化特征我们将定位负责其DNA损伤修复/保护活性的最小TERT结构域，并产生跨越已鉴定结构域序列的短肽，并测试这些多肽作为干扰这种TERT活性的策略。目的2：亲和纯化和质谱鉴定不同的TERT络合物。我们将对表达FL-TERT或截短TERT变体的ALT细胞进行串联亲和标签(TAP)纯化和LC-MS-MS分析纯化的TERT-复合体。将用遗传和/或化学手段评估选定的非典型TERT蛋白伙伴的生物学功能。影响我们的功能和生化特性不同的TERT结构形式及其在独特的生物复合体中的成员将进一步加深对人类生物学中过多的端粒酶活性的理解。这项工作还将支持7个总部基地，包括2个高级研究生和5个UGS。我们的建议与NSERC的2020年研究战略计划一致，即通过为下一代研究人员提供尖端的生化、蛋白质科学和分子遗传学培训，建立一个多元化和具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.