Functional studies of the top 1-binding protein topors

描述(申请人提供)：拓扑异构酶I是一类重要的抗癌药物喜树碱的靶标。该项目的总体目标是更全面地了解拓扑异构酶I在正常细胞和恶性肿瘤细胞中的功能，希望最终能够改善癌症的治疗。为此，该提案将重点放在Topors上，这是一种结合拓扑异构酶I和p53的蛋白质。目前的研究结果表明，在体外，TOPORS同时作为E3型泛素和相扑连接酶发挥作用，并刺激拓扑异构酶I和P53的相扑甲基化。此外，TOPORS与早幼粒细胞白血病核体动态结合，与细胞苏莫化过程和肿瘤的发生有关。蛋白质和mRNA表达研究表明，TOPORS在正常人体组织中广泛表达，但在癌细胞中不表达。与正常结肠组织相比，TOPORS蛋白在人类结肠癌中的表达通常是检测不到的。这些发现将使用生化和遗传方法进行研究。第一个目标包括对泛素和相扑共轭过程中涉及的TOPORS结构域进行详细的映射。此外，还将在体外和细胞内研究TOPORS诱导的SUMO化对拓扑异构酶I的影响。第二个目的是探索TOPORS与早幼粒细胞白血病核体结合的机制，包括分析TOPORS与早幼粒细胞白血病蛋白的结合，以及确定细胞中是否存在TOPORS总甲基化。第三个目标是研究TOPORS在正常和恶性人体组织中的差异表达。除了进一步研究TOPORS在恶性肿瘤中的表达缺失，还将研究TOPORS基因的杂合性缺失和突变，以及TOPORS在细胞系中抗增殖作用的潜在机制。这些研究将通过在AIM 4中使用转基因小鼠研究TOPORS功能丧失的影响来补充。将研究TOPORS在抑制肿瘤形成和细胞对喜树碱的敏感性方面的潜在作用。拟议中的实验将显著增加目前对TOPORS功能的了解，并可能导致预防和治疗癌症的新策略

DESCRIPTION (provided by applicant): Topoisomerase I is the target of an important class of anti-cancer drugs, the camptothecins. The overall goal of this project is to more completely understand the functions of topoisomerase I in normal and malignant cells, with the hope of eventually improving cancer treatment. Towards this end, the proposal focuses on topors, a protein that binds topoisomerase I and p53. Current results indicate that topors functions as both an E3-type ubiquitin and SUMO ligase and stimulates the sumoylation of topoisomerase I and p53 in vitro. In addition, topors dynamically associates with promyelocytic leukemia nuclear bodies, which are implicated in cellular sumoylation processes and in carcinogenesis. Protein and mRNA expression studies indicate that topors is widely expressed in normal human tissues, but not in cancer cell lines. In contrast to normal colon tissue, topors protein expression is frequently undetectable in human colon cancers. These findings will be pursued using biochemical and genetic approaches. The first aim includes detailed mapping of topors domains involved in ubiquitin and SUMO conjugation processes. In addition, the effects of topors-induced sumoylation on topoisomerase I will be studied in vitro and in cells In the second aim, mechanisms underlying the association of topors with promyelocytic leukemia nuclear bodies will be explored, including analysis of binding of the promyelocytic leukemia protein by topors, and determination of whether topors is sumoylated in cells. The third aim focuses on investigation of the differential expression of topors in normal versus malignant human tissues. In addition to further characterization of the loss of topors expression in malignancies, Ioss-of-heterozygosity and mutation of the topors gene will be investigated, as well as mechanisms underlying the anti-proliferative effects of topors in cell lines. These studies will be complemented by investigation of the effects of loss of topors function using transgenic mice in Aim 4. Potential roles for topors in the suppression of tumorigenesis and in cellular sensitivity to camptothecin will be investigated. The proposed experiments should significantly increase current knowledge regarding the function of topors and may lead to new strategies in the prevention and treatment of cancer