Functional studies of the top 1-binding protein topors

描述（由申请人提供）：拓扑异构酶I是一类重要的抗癌药物喜树碱的靶点。该项目的总体目标是更全面地了解拓扑异构酶I在正常和恶性细胞中的功能，并希望最终改善癌症治疗。为此，该建议将重点放在topors上，topors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，topors在体外同时具有e3型泛素和SUMO连接酶的功能，并刺激拓扑异构酶I和p53的聚合化。此外，topors与早幼粒细胞白血病核体动态相关，这与细胞聚合过程和癌变有关。蛋白质和mRNA的表达研究表明，topors在正常人体组织中广泛表达，但在癌细胞系中不表达。与正常的结肠组织相比，topors蛋白的表达在人类结肠癌中经常检测不到。这些发现将采用生化和遗传方法进行研究。第一个目标包括在泛素和SUMO偶联过程中涉及的拓扑域的详细映射。此外，将在体外和细胞中研究topors诱导的拓扑异构化对拓扑异构酶I的影响。第二个目标，topors与早幼粒细胞白血病核体关联的机制将被探索，包括topors与早幼粒细胞白血病蛋白结合的分析，以及topors是否在细胞中被sumoylation的确定。第三个目的是研究topors在正常和恶性人体组织中的差异表达。除了进一步表征topors在恶性肿瘤中的表达缺失外，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机制。这些研究将通过在Aim 4中使用转基因小鼠研究topor功能丧失的影响来补充。topors在抑制肿瘤发生和喜树碱细胞敏感性方面的潜在作用将被研究。所提出的实验将显著增加当前关于topors功能的知识，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