FUNCTION AND REGULATION OF THE PROTEASOME

描述：本研究计划的长期目标是 提供对生物化学机制的全面了解， 细胞内蛋白质降解的生理调节。这 这一目标对于理解许多细胞的 利用蛋白质降解作为其 调节，并用于减轻以组织萎缩为特征的疾病。 本项目的总体目标是建立 PA 28，蛋白酶体的蛋白活化。蛋白酶体是一种 细胞内蛋白酶所需的所有泛素依赖的-和一个未知的 真核细胞中泛素非依赖性蛋白质降解的量。 蛋白酶体在体外和体内的功能都是由多种 相对细胞作用不明确的调节蛋白。PA28 是这些蛋白质中了解最少的一种。细胞的作用 PA 28将通过在哺乳动物细胞中过表达蛋白质来定义。 这些研究将检验PA 28增加总体发病率的假设。 细胞内蛋白质的降解。PA 28的细胞功能将 通过密切相关的生物化学实验进一步研究。这些 实验将确定PA 28可逆磷酸化的作用 作为一种控制其活动的机制， PA 28的磷酸化抑制其蛋白酶体刺激活性。 PA 28对模型蛋白降解的影响将在 通过分析PA 28-蛋白酶体降解蛋白质的动力学， 单独的复合物和与其他蛋白酶体调节复合物的组合 蛋白质复合物这些实验将验证PA 28的假设， 细胞功能是促进大肽的降解 直接和由其他蛋白酶体作用产生的大肽 最后，PA 28蛋白酶体复合物的细胞底物 将通过使用表达克隆的筛选策略进行鉴定。 这些高度整合的研究将确定PA 28的细胞作用， 可能的生化基础。因此，该项目将大大 提高对机制和调节的理解， 细胞内蛋白质降解

DESCRIPTION: The long-term objective of this research program is to provide a comprehensive understanding of the biochemical mechanisms and physiological regulation of intracellular protein degradation. This objective has broad significance for understanding numerous cellular processes that utilize protein degradation as a mechanism for their regulation, and for alleviating diseases characterize by tissue atrophy. The general goal of this project is to establish the physiological role of PA28, a protein activation of the proteasome. The proteasome is an intracellular protease required for all ubiquitin-dependent-and an unknown amount of ubiquitin-independent protein degradation in eukaryotic cells. Proteasome function both in vitro and in vivo is mediated by multiple regulatory proteins whose relative cellular roles are poorly defined. PA28 is one of the least understood of these proteins. The cellular role of PA28 will be defined by over-expressing the protein in mammalian cells. These studies will test the hypothesis that PA28 increases overall rates of intracellular protein degradation. The cellular function of PA28 will be examined further by closely related biochemical experiments. These experiments will establish the role of reversible phosphorylation of PA28 as a mechanism for control of its activity by testing the hypothesis that phosphorylation of the PA28 inhibits its proteasome-stimulatory activity. The effect of PA28 on the degradation of model proteins will be studied in vitro by analyzing the kinetics of protein degradation by PA28-proteasome complexes alone and in combination with other proteasome-regulatory protein complexes. These experiments will test the hypothesis that PA28's cellular function is to promote degradation both of large peptides directly and of large peptides generated by the action of other proteasome complexes Finally, cellular substrates for the PA28 proteasome complex will be identified with a screening strategy using expression cloning. These highly integrated studies will define cellular role of PA28 and the possible biochemical bases for them. Therefore, this project will greatly increase the understanding of the mechanisms and regulation of intracellular protein degradation.