The Ape1-NPM1 Axis and Telomere Maintenance

 描述(申请人提供)：端粒需要特殊的机制来维持，这些机制或端粒结构本身的破坏，几乎在所有癌症中都会发生。端粒DNA的损伤会破坏端粒的维持，并可能导致染色体重排。我们最近发现，APE1蛋白是碱基切除DNA修复的中心角色，在正常哺乳动物细胞和肿瘤细胞中都需要适当的端粒维持。APE1是保护蛋白TRF2正常结合所必需的，APE1缺乏会导致POT1端粒结合增加。APE1的AP内切酶活性(DNA修复功能)是端粒维持所必需的。虽然APE1在细胞核中广泛分布，但它通过与核磷蛋白(NPM1)的相互作用优先在核仁中蓄积，在DNA损伤的细胞中通过APE1N-末端附近的赖氨酸乙酰化而释放。虽然APE1对DNA损伤和端粒破坏有机制上的联系，但NPM1对APE1的调控在这一过程中发挥的作用尚不清楚。此外，约1/3的急性髓系白血病(AML)中发生的NPM1c突变导致NPM1重新定位到细胞质，携带APE1。在这个修订的应用中，我们将检查NPM1-APE1相互作用对端粒维持的影响，以及它在AML和其他癌症的病因学中的可能作用。虽然APE1不能被消除，但我们已经建立了RNAi协议来有效地下调。与NPM1的相互作用可以通过用丙氨酸(模拟不带电的乙酰化状态)或精氨酸(防止乙酰化和保留电荷)取代APE1的关键N-末端赖氨酸(27，31，32，35)来控制。这些工具和表达NPM1c蛋白的细胞将被用来确定APE1-NPM1相互作用在两种正常条件下对端粒维持是否重要，以及在面临氧化或烷基化试剂造成的DNA损伤时，后者会产生通过碱基切除修复处理的损伤。端粒长度和TRF2蛋白的占有率将通过我们已经建立的方法进行监测。

 DESCRIPTION (provided by applicant): Telomeres require special mechanisms for their maintenance, and disruption of these mechanisms, or of the telomeric structures themselves, occur in nearly all cancers. Damage to telomeric DNA disrupts telomere maintenance and can result in chromosome rearrangements. We found recently that Ape1 protein, the central player of base excision DNA repair, is required for proper telomere maintenance in both normal mammalian cells and in tumor lines. Ape1 is required for normal binding of the protective protein TRF2, and Ape1 deficiency leads to increased telomeric binding of POT1. The AP endonuclease activity (DNA repair function) of Ape1 is required for telomere maintenance. Although Ape1 is found throughout the nucleus, it accumulates preferentially in the nucleolus via interaction with nucleophosmin (NPM1), from which it is released in DNA-damaged cells through acetylation of lysines near the Ape1 N- terminus. While DNA damage and telomere disruption are mechanistically related by Ape1, it is unknown how the modulation of Ape1 by NPM1 plays a role in this process. Moreover, the NPM1c+ mutations that occur in about 1/3 of acute myelogenous leukemias (AML) lead to relocation of NPM1 to the cytoplasm, carrying Ape1 with it. In this revised application, we will examine the effects of the NPM1-Ape1 interaction on telomere maintenance, and its possible role in the etiology of AML and other cancers. Although Ape1 cannot be eliminated, we have established RNAi protocols for its efficient down-regulation. The interaction with NPM1 can be controlled by replacement of the key N-terminal lysines (27, 31, 32, 35) of Ape1 with alanines (to mimic the uncharged acetylated state) or arginines (to prevent actylation and retain charge). These tools, and cells expressing the NPM1c+ protein, will be used to determine whether the Ape1-NPM1 interaction is important for telomere maintenance under both normal conditions, and in the face of DNA damage by oxidative or alkylating agents, which generate lesions processed by base excision repair. Telomere length, and the occupation by TRF2 protein, will be monitored by methods we have already established.