PROTEOLYSIS AND MAMMALIAN GL/S CELL CYCLE TRANSITION

蛋白质水解和哺乳动物 GL/S 细胞周期转变

• 批准号: 2871922
• 负责人: HUI ZHANG
• 金额: $ 27.26万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2871922
• 关键词: HeLa cells; SDS polyacrylamide gel electrophoresis; autoradiography; carcinogenesis; cell cycle; cell differentiation; cell growth regulation; cyclin dependent kinase; cyclins; enzyme inhibitors; immunoprecipitation; laboratory rabbit; phosphorylation; polymerase chain reaction; protein purification; protein structure function; proteolysis; synchronous cell division; ubiquitin; western blottings

The cell cycle control is a fundamental regulatory process that ensures the faithful duplication and passage of genetic information during cell division. To prevent the irreversible incorporation of genetic lesions into the genome, the transitions between the G1/S and G2/M phases of cell cycle are highly regulated as part of the checkpoint control in respond to environmental cues. Aberrant cell cycle control abolishes coordination between different phases of the cell cycle and is a hallmark of neoplastic transformation. The broad, long-term objective of this proposal is to understand the mechanism of cell cycle control in cell growth and differentiation, and the consequences of its alteration during tumorigenesis. This proposal focuses on the mechanism of the G1/S cell cycle transition in mammalian cells. In particular, we describe the approaches to investigate the mechanism by which the levels of the mammalian G1 cyclins and CDK inhibitors are controlled through the selective ubiquitin-dependent degradation during the G1/S transition. The accumulation of the G1 cyclins are the rate limiting step during the G1/S transition and the CDK inhibitors usually serve as the G1/S checkpoint control proteins in response to negative growth signals. Over-expression of the G1 cyclins and the loss of CDK inhibitors are often associated with human cancer. We have previously isolated a novel p19SKP1 and p45SKP2 cell cycle complex based on its highly elevated level in many neoplastic transformed cells. We have identified additional components of this complex. Our preliminary data indicate that this complex is involved in the control of the G1/S transition by regulating the levels of the G1 cell cycle regulators through the ubiquitin-dependent proteolysis. To further investigate this important cell cycle control mechanism, we propose the following specific aims: (1) To determine and characterize the components that are involved in the control of the G/S transition through the p19SKP1 and p45SKP2-mediated ubiquitin-dependent proteolysis. (2) To identify the critical targets of this regulation during the G1/S transition. (3) To determine the cell cycle regulatory factors that control this process. (4) To establish in vitro assays to characterize the ubiquitin conjugation reaction and its regulatory mechanism. Our investigation should provide new insights into the mechanism of the mammalian G1/S transition and help to assess how the alteration of these cell cycle regulatory processes may contribute to tumorigenesis. These studies will also provide a molecular basis for designing novel strategies for the diagnostic and therapeutic treatment of human cancer.

细胞周期控制是一个基本的调控过程，可确保 细胞内遗传信息的忠实复制和传递 分配。 防止基因损伤的不可逆转的掺入 进入基因组，G1/S 和 G2/M 阶段之间的转变 细胞周期作为检查点控制的一部分受到高度调控 对环境线索做出反应。 异常的细胞周期控制被废除 细胞周期不同阶段之间的协调 肿瘤转化的标志。 广泛、长期的目标 该提案的目的是了解细胞周期控制的机制 细胞生长和分化及其后果 肿瘤发生过程中的改变。 本提案重点关注机制 哺乳动物细胞的 G1/S 细胞周期转变。 尤其， 我们描述了研究机制的方法 哺乳动物 G1 细胞周期蛋白和 CDK 抑制剂的水平受到控制 通过 G1/S 期间的选择性泛素依赖性降解 过渡。 G1 细胞周期蛋白的积累是速率限制 G1/S 转变过程中的步骤，CDK 抑制剂通常充当 G1/S 检查点控制蛋白响应负生长 信号。 G1 细胞周期蛋白的过度表达和 CDK 的丢失 抑制剂通常与人类癌症有关。 我们之前有过 基于其分离出新型 p19SKP1 和 p45SKP2 细胞周期复合物 许多肿瘤转化细胞中的水平高度升高。 我们有 确定了该复合物的其他成分。 我们的初步数据 表明该复合物参与 G1/S 的控制 通过调节 G1 细胞周期调节因子的水平来实现转变 通过泛素依赖性蛋白水解。 进一步调查 对于这一重要的细胞周期控制机制，我们提出以下建议 具体目标： (1) 确定和表征组件 通过 p19SKP1 参与 G/S 转变的控制 p45SKP2 介导的泛素依赖性蛋白水解。 (2) 识别 G1/S 过渡期间该法规的关键目标。 (3) 至 确定控制该过程的细胞周期调节因子。 (4) 建立体外检测方法来表征泛素 共轭反应及其调控机制。 我们的调查 应该为哺乳动物 G1/S 的机制提供新的见解 转变并帮助评估这些细胞周期的改变 调节过程可能有助于肿瘤发生。 这些研究 还将为设计新策略提供分子基础 人类癌症的诊断和治疗。