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在正常和恶性细胞中的功能，并希望最终改善癌症治疗。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，topors功能作为E3型泛素和SUMO连接酶，并刺激类小泛素化的拓扑异构酶I和p53在体外。此外，topors动态地与早幼粒细胞白血病核小体相关，这与细胞sumoylation过程和致癌有关。蛋白质和mRNA表达研究表明，topors广泛表达于正常人体组织中，但不在癌细胞系中。与正常结肠组织相反，topors蛋白表达在人结肠癌中经常检测不到。这些发现将使用生物化学和遗传学方法进行。第一个目标是详细绘制参与泛素和SUMO结合过程的topors结构域。此外，拓扑结构诱导的SUMO化对拓扑异构酶I的影响将在体外和细胞中进行研究。第二个目标是探索拓扑结构与早幼粒细胞白血病核小体相关的机制，包括分析拓扑结构与早幼粒细胞白血病蛋白的结合，以及确定拓扑结构是否在细胞中被SUMO化。第三个目标集中于研究topors在正常与恶性人体组织中的差异表达。除了进一步表征恶性肿瘤中topors表达的缺失外，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潜在机制。这些研究将通过使用目的4中的转基因小鼠研究topors功能丧失的影响来补充。topors在抑制肿瘤发生和细胞对喜树碱敏感性中的潜在作用将被研究。拟议的实验应显着增加目前的知识有关的功能topors，并可能导致新的战略，在预防和治疗癌症

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