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: 10467888
• 负责人: Weidong Wang
• 金额: $ 16.66万
• 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/10467888
• 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.

-当前进展的更新——重点关注项目的最初目标 目标 1，进展：确定 SAP1 和庇护组件 TPP1 之间的交互域 我们创建了多个携带 SAP1 不同缺失突变体的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 中第 800 至 830 个氨基酸的区域是其与 TPP1（庇护蛋白的组成部分）相互作用所必需的。为了研究 SAP1 的这种相互作用结构域是否需要在体内与庇护蛋白结合，我们建立了邻近连接酶测定 (PLA)，这是一种用于原位检测细胞中蛋白质-蛋白质相互作用的强大工具。我们观察到TPP1与细胞中标记的SAP1野生型和SAP1突变体852之间的关联，但与SAP1突变体696和770没有关联。这些数据与我们的IP-Western印迹结果一致（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 中相互作用域的识别为研究 SAP1-SAP2 复合物如何在目标 2 和目标 3 中描述的端粒保护中发挥作用的机制提供了基础。 目标 2，进展：SAP1-SAP2 复合物控制端粒长度稳态。 我们采用了多种方案来检查 SAP1 或 SAP2 敲除 MEF 细胞中的任何端粒缺陷。到目前为止，我们已经可重复地确定这些敲除细胞的主要表型异常是端粒缩短。值得注意的是，我们还检测到 SAP1 消除的小鼠原代 B 细胞的端粒缩短，为得出 SAP1-SAP2 复合物是维持端粒长度所必需的结论提供了额外的证据。为了探索潜在的机制，我们生成了多个 SAP1 敲除 MEF 细胞，稳定表达从 Aim 1 中鉴定出的 SAP1 的各种缺失突变体。我们目前正在确定 SAP1 的庇护蛋白相互作用域对于端粒延长的重要性。 目标 3，进展：SAP1-SAP2 复合物刺激端粒酶活性以延长端粒长度。 我们之前在我们的提案中描述过 SAP1 和 SAP2 敲除 MEF 细胞均表现出端粒长度缩短。为了探索潜在的机制，我们利用端粒重复扩增方案（TRAP）测定来检测 SAP1 或 SAP2 敲除 MEF 细胞中的端粒酶活性，与野生型细胞或通过 SAP1 或 SAP2 重新表达补充的 KO 细胞相比。结果表明，与野生型细胞相比，SAP1 和 SAP2 敲除细胞中的端粒酶活性均降低；并且这种降低可以通过重新引入 SAP1 或 SAP2 来很大程度上逆转，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水平高于 SAP1 突变体 696。这与 SAP1 突变体 830 和 852 均与 TPP1 相互作用，而突变体 696 不相互作用的发现相关，表明 SAP1 与 TPP1 相互作用。 TPP1 可能促进端粒酶功能。已知TPP1是shelterin中唯一直接与shelterin相互作用并将端粒酶募集到shelterin以促进端粒长度延长的成分（Nandakumar等人，Nature 2012；Schmidt等人，Nature 2014；Zhong等人，Cell 2012）。携带杂合单氨基酸缺失 (del K170) 的个体表现出端粒缩短（Kocak 等人，Genes Dev 2014；Guo 等人，Blood 2014），这与 SAP1 和 SAP2 敲除细胞的表型相似。综上所述，我们的数据表明 SAP1-SAP2 复合物可能与 TTP1 相互作用并刺激 TTP1 的活性，从而将端粒酶募集到端粒以维持其正常长度。