Investigating a novel shelterin-associated protein complex in telomere protection

研究端粒保护中的新型庇护蛋白相关蛋白复合物

• 批准号: 10688810
• 负责人: Weidong Wang
• 金额: $ 16.84万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10688810
• 关键词: Aging; Amino Acids; B-Lymphocytes; Biological Assay; Biology; Blood; Cell Aging; Cell division; Cells; Chromosomes; Chronic; Chronic Lymphocytic Leukemia; Complement; Complex; Coupled; Cutaneous Melanoma; DNA Damage; Data; Defect; Detection; Disease; Dyskeratosis Congenita; Ensure; Exhibits; Functional disorder; Genes; Genetic Recombination; Genome; Goals; Homeostasis; In Situ; Individual; Inflammatory; Knock-out; Length; Ligase; Link; Malignant Neoplasms; Mammals; Mass Spectrum Analysis; Mediating; Mitochondria; Modeling; Mus; Mutation; Nature; Nerve Degeneration; Nucleoproteins; Pathway interactions; Phenotype; Play; Proteins; Protocols documentation; Role; Signal Transduction; Skeletal Muscle; Structure; TERF1 gene; TINF2 gene; Telomerase; Telomere Length Maintenance; Telomere Shortening; Telomere-Binding Proteins; Update; Western Blotting; age related; base; genome integrity; in vivo; mutant; novel; overexpression; protein complex; protein protein interaction; recruit; repaired; response; telomere; tool

-Updates on current progress-focusing on the original aims of the project Aim 1, Progress: Determined the interacting domains between SAP1 and shelterin component TPP1 We have created multiple retroviral expression constructs carrying different deletion mutants of SAP1, and generated MEF cells stably expressing those mutants. We then determined that the region located from 800 to 830 amino acids in SAP1 is required for its interaction with TPP1, an integral component of shelterin. To study if this interaction domain of SAP1 is needed for its association with shelterin in vivo, we established proximity ligase assay (PLA), a powerful tool for in situ detection of the protein-protein interaction in a cell. We have observed the association between TPP1 and flag-tagged SAP1 wildtype and SAP1 mutant 852 in cells, but not with SAP1 mutants 696 and 770. These data are consistent with our IP-Western blotting results (Fig. B). Using similar approaches, we determined the region of TPP1 required for the interaction of SAP1 with TPP1. The identification of the interacting domains in both SAP1 and TPP1 provides a basis for studying the mechanism of how SAP1-SAP2 complex acts in telomere protection described in Aim 2 and Aim 3. Aim 2, Progress: SAP1-SAP2 complex controls telomere length homeostasis. We have employed several protocols to examine any telomere defects in SAP1 or SAP2 knockout MEF cells. Thus far, we have reproducibly identified that the major phenotypic abnormalities of those knockout cells are shortened telomeres. Notably, we have also detected telomere shortening of SAP1-ablated mouse primary B cells, providing additional evidence to conclude that SAP1-SAP2 complex is required for the maintenance of telomere length. To explore the underlying mechanism, we have generated multiple SAP1 knockout MEF cells stably expressing various deletion mutants of SAP1 identified from Aim 1. We are currently determining the importance of shelterin-interacting domain of SAP1 for telomere lengthening. Aim 3, Progress: SAP1-SAP2 complex stimulates telomerase activity to elongate telomere length. We have previously described in our proposal that both SAP1 and SAP2 knockout MEF cells exhibit shortened telomere length. To explore the underlying mechanism, we have utilized telomeric repeat amplification protocol (TRAP) assay to detect the telomerase activity in SAP1 or SAP2 knockout MEF cells, versus the wildtype cells, or KO cells complemented by re-expression of SAP1 or SAP2. The results showed that telomerase activity were decreased in both SAP1 and SAP2 knockout cells, when compared to those in wildtype cells; and this decrease can be largely recused by reintroduction of SAP1 or SAP2, indicating that SAP1-SAP2 complex regulates the telomerase activity in MEF cells. Notably, overexpression of either SAP1 mutant 830 or mutant 852 resulted in higher levels of telomerase activity than SAP1 mutant 696. This correlates with the findings that both SAP1 mutant 830 and 852 interact with TPP1, whereas mutant 696 does not, suggesting that SAP1 interaction with TPP1 may promote telomerase function. TPP1 is known to be the only component of shelterin that directly interacts with and recruits telomerase to shelterin to facilitate the elongation of telomere length (Nandakumar et al, Nature 2012; Schmidt et al, Nature 2014; Zhong et al, Cell 2012). Individuals carrying a heterozygous single amino acid deletion (del K170) exhibit shortened telomeres (Kocak et al., Genes Dev 2014; Guo et al, Blood 2014), which is the similar phenotype of both SAP1 and SAP2 knockout cells. Taken together, our data suggest a model that SAP1-SAP2 complex may interact with and stimulate the activity of TTP1 in recruiting telomerase to telomeres to maintain its normal length.

- 对当前的进度推进到了项目的原始目标 目标1，进度：确定SAP1和庇护素组件TPP1之间的相互作用域 我们创建了携带SAP1不同缺失突变体的多个逆转录病毒表达构建体，并生成了稳定表达这些突变体的MEF细胞。然后，我们确定SAP1中的800至830个氨基酸的区域与TPP1的相互作用是庇护素不可或缺的组成部分。为了研究SAP1与体内庇护素关联的相互作用结构域，我们建立了接近连接酶测定（PLA），这是一种可以原位检测细胞中蛋白质 - 蛋白质相互作用的强大工具。我们已经观察到细胞中TPP1和标记为标记的SAP1 WildType和SAP1突变体852之间的关联，但与SAP1突变体696和770之间没有相关性。这些数据与我们的IP-Western墨水斑点结果一致（图B）。使用类似的方法，我们确定了SAP1与TPP1相互作用所需的TPP1区域。 SAP1和TPP1中相互作用域的鉴定提供了研究SAP1-SAP2复合物如何在AIM 2和AIM 3中描述的端粒保护中如何作用的机制。 AIM 2，进度：SAP1-SAP2复合物控制端粒长度稳态。 我们已经采用了几种方案来检查SAP1或SAP2基因敲除MEF单元中的任何端粒缺陷。到目前为止，我们已经可重复地识别出那些基因敲除细胞的主要表型异常是缩短的端粒。值得注意的是，我们还检测到SAP1驱动的小鼠原代B细胞的端粒缩短，提供了其他证据来得出结论，即维持端粒长度是必需的。为了探索潜在的机制，我们已经生成了多个SAP1基因敲除MEF细胞，从而稳定地表达了从AIM 1鉴定的SAP1的各种缺失突变体。我们目前正在确定SAP1的庇护素相互作用域对端粒延长的重要性。 AIM 3，进展：SAP1-SAP2复合物刺激端粒酶活性，以拉长端粒长度。 我们先前在提案中描述了SAP1和SAP2基因敲除MEF细胞均显示端粒长度缩短。为了探索潜在的机制，我们利用端粒重复放大方案（TRAP）测定法来检测SAP1或SAP2基因敲除MEF细胞中的端粒酶活性，与Wildtype细胞相比，或通过SAP1或SAP2的重新表达来补充的WildType细胞或KO细胞。结果表明，与野生型细胞相比，SAP1和SAP2基因敲除细胞的端粒酶活性均降低。而且这种减少可以通过重新引入SAP1或SAP2的重新撤回，这表明SAP1-SAP2复合物调节MEF细胞中的端粒酶活性。值得注意的是，SAP1突变体830或突变体852的过表达导致端粒酶活性高于SAP1突变体696。这与SAP1突变体830和852均与TPP1相互作用的发现相关，而SAP1与TPP1相互作用均与TPP1相互作用，这可能会促进Telomasase speal telomasase功能。众所周知，TPP1是庇护素直接相互作用并募集端粒酶以促进端粒长度伸长的唯一组成部分（Nandakumar等人，自然，2012; Schmidt等人，自然，2014; Zhong等，2012）。携带杂合单氨基酸缺失（DEL K170）的个体表现出缩短的端粒（Kocak等，Genes Dev 2014； Guo等，Blood 2014），这是SAP1和SAP2敲除细胞的类似表型。综上所述，我们的数据表明了一个模型，即SAP1-SAP2复合物可以与TTP1相互作用并刺激TTP1在募集端粒酶至端粒以保持其正常长度时的活性。