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

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重复序列重新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要的。新的证据表明，在不朽的人类细胞中，组成型TERT的表达也通过多种机制促进应激生长条件下的细胞存活。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，但负责这些活动的确切机制和TERT的结构形式在很大程度上仍然未知。我的实验室报告了端粒酶的重组表达赋予细胞生长优势，并以与端粒长度维持无关的方式促进非锚定生长。TERT表达还可以防止dna损伤剂引起的遗传毒性。我们发现TERT变体和选择性剪接（截断蛋白）形式，缺乏端粒合成活性，也对多种基因毒性刺激表现出相同的保护作用，这表明端粒酶参与了其规范（端粒合成相关）和非规范（端粒合成独立）活性的结构和功能不同的复合物。假设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体来促进细胞生长和增殖。我们将绘制负责其DNA损伤修复/保护活性的最小TERT结构域，生成跨越已识别结构域序列的短肽，并测试这些肽作为破坏该TERT活性的策略。目的2：不同TERT复合物的亲和纯化和质谱鉴定。我们将从表达FL-TERT或截断TERT变体的ALT细胞中进行串联亲和力标签（TAP）纯化和LC-MS-MS分析纯化的TERT复合物。选定的非规范TERT蛋白伴侣的生物学功能将通过遗传和/或化学手段进行评估。我们对不同TERT结构形式及其独特生物复合物成员的功能和生化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，其中包括2名高级研究生和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化，蛋白质科学和分子遗传学培训，建立一个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.