Arsenite inhibition of mitotic progression

亚砷酸盐抑制有丝分裂进展

基本信息

批准号：
6834668
负责人：
BEN F TAYLOR
金额：
$ 2.98万
依托单位：
UNIVERSITY OF LOUISVILLE
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-06-15 至 2008-06-14
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6834668
关键词：
SDS polyacrylamide gel electrophoresis arsenic autoradiography cell cycle cytotoxicity enzyme activity immunoprecipitation ligase mass spectrometry molecular genetics p53 gene /protein predoctoral investigator toxicology ubiquitin western blottings

项目摘要

DESCRIPTION (provided by applicant): This project will determine whether the mechanism by which arsenate induces mitotic effects on the Anaphase Promoting Complex (APC) mediate arrest. We hypothesize that arsenite inhibits the APC and subsequently mitotic progression. The APC orchestrates progress through the cell cycle and mitosis by targeting specific mitotic substrates for proteasomal degradation via ubiquitination. Specific aim 1 will determine whether arsenite treatment inhibits APC substrate degradation necessary for mitotic exit. In Specific aim 2 we will immunoprecipitate (IP) the APC and identify its subunits in order to investigate arsenite binding to APC subunits using radiolabeled Na[73As]O2. Determination of arsenite binding requires resolution of APC subunits using SDS-PAGE, examination of protein banding pattern using Sipro-Red and fluorescence imaging, and subsequent autoradiography to elucidate bands incorporating radiolabeled arsenite. Western blot analysis of SDS-PAGE resolved APC subunits will be used to identify specific APC subunits. Once arsenite association is determined, the proteins will be excised from the gel and identity confirmed by mass spectrometry. Specific aim 3 will determine whether arsenite inhibits APC ubiquitin ligase activity. Isolated APC will be used in an ubiquitin conjugating assay with radiolabeled APC substrates treated with or without arsenite. Reaction products will be resolved on SDS-PAGE and autoradiography will be used to determine the degree of substrate laddering due to ubiquitin conjugation. Identification of the molecular targets of arsenite is important for chemotherapeutic drug development.

描述（由申请人提供）：该项目将确定砷酸盐诱导后期促进复合物（APC）有丝分裂效应的机制是否介导停滞。我们假设亚砷酸盐抑制 APC 和随后的有丝分裂进展。 APC 通过泛素化靶向特定有丝分裂底物进行蛋白酶体降解，从而协调细胞周期和有丝分裂的进展。具体目标 1 将确定亚砷酸盐处理是否抑制有丝分裂退出所需的 APC 底物降解。在具体目标 2 中，我们将对 APC 进行免疫沉淀 (IP) 并鉴定其亚基，以便使用放射性标记的 Na[73As]O2 研究亚砷酸盐与 APC 亚基的结合。亚砷酸盐结合的测定需要使用 SDS-PAGE 解析 APC 亚基，使用 Sipro-Red 和荧光成像检查蛋白质条带模式，以及随后的放射自显影以阐明掺有放射性标记亚砷酸盐的条带。 SDS-PAGE 解析的 APC 亚基的蛋白质印迹分析将用于鉴定特定的 APC 亚基。一旦亚砷酸盐结合被确定，蛋白质将从凝胶中切除并通过质谱法确认其身份。具体目标 3 将确定亚砷酸盐是否抑制 APC 泛素连接酶活性。分离的 APC 将用于泛素缀合测定，其中放射性标记的 APC 底物经过或不经过亚砷酸盐处理。反应产物将在 SDS-PAGE 上解析，并且放射自显影将用于确定由于泛素缀合而导致的底物梯状化程度。亚砷酸盐分子靶点的鉴定对于化疗药物的开发非常重要。