CAREER: Protein Unfolding by Energy Dependent Proteases

9875857本项目的目标是确定底物蛋白在ATP依赖的蛋白酶降解过程中的解折叠机制。在液泡或溶酶体以外的细胞中，大多数蛋白质的降解是由依赖于ATP的蛋白酶催化的。底物蛋白质的去折叠是降解过程的关键早期步骤。短寿命蛋白质的降解对于细胞周期控制、信号转导和抗原递呈等广泛的细胞功能是必不可少的。蛋白质错误折叠的分解是细胞对压力反应的一部分。Barnase具有良好的体外折叠特性，被选为ATP依赖蛋白水解酶、离子蛋白水解酶、CLP水解酶、线粒体AAA水解酶和蛋白酶体的人工底物蛋白。将降解过程中的Barnase去折叠与自发去折叠进行比较，以确定蛋白质去折叠是否是由蛋白酶催化的。不稳定的Barnase突变体将被用作错误折叠蛋白质的模型，带有C末端降解标签的Barnase突变体将被用作短期调节蛋白的模型。如果去折叠被催化，那么催化的机理就会被确定。该奖项的教育目的是向本科生传授了解细胞生物学过程的生物化学和生物物理学的先决条件。为此，将开发一门关于定量生物物理技术的实验室课程。概述蛋白质是由称为氨基酸的亚单位组成的链。为了执行其生物学功能，蛋白质折叠成特定的三维形状。然而，在细胞中有一些必要的过程，蛋白质必须重新展开，例如当蛋白质受损或调节蛋白质被蛋白酶降解时。不能降解调节蛋白和错误折叠的蛋白会导致细胞功能异常。这项提议研究了蛋白水解酶是如何展开蛋白质的。生物学已经到了可以确定细胞内过程的详细机制的阶段。为了让学生为这个实验生物学的新时代做好准备，将开设一门关于细胞生物学定量方法的实验室课程。

9875857The goal of this project is to determine the mechanism of unfolding of substrate proteins during degradation by ATP-dependent proteases. Most protein degradation in cells outside the vacuole or the lysosome is catalyzed by ATP-dependent proteases. Unfolding of the substrate protein is a critical early step in the degradation process. Degradation of short-lived proteins is essential for a wide range of cellular functions including cell cycle control, signal transduction and antigen presentation. Proteolysis of misfolded proteins is part of the cellular response to stress. Barnase, whose folding is well characterized in vitro, was chosen as an artificial substrate protein for the ATP-dependent proteases lon-protease, Clp-protease, mitochondrial AAA-protease and the proteasome. Barnase unfolding during degradation will be compared to spontaneous unfolding to determine whether protein unfolding is catalyzed by the proteases. Destabilized barnase mutants will be used as models for misfolded proteins and barnase mutants with C-terminal degradation tags will be used as models for short-lived regulatory proteins. If unfolding is catalyzed, the mechanism of catalysis will be determined. The educational aim of this award is to teach undergraduate students the prerequisites for a biochemical and biophysical understanding of cell biological processes. For this purpose a laboratory course on quantitative biophysical techniques will be developed.Lay summaryProteins are chains made from subunits called amino acids. To perform their biological function, proteins fold into a specific three-dimensional shape. However, there are essential processes in the cell for which proteins must unfold again, such as when damaged or regulatory proteins are degraded by proteases. Failure to degrade regulatory proteins and misfolded proteins leads to abnormal cellular function. This proposal investigates how proteases unfold proteins.Biology has reached a stage where it is possible to determine thedetailed mechanisms of processes in the cell. To prepare students for this new era in experimental biology, a laboratory course on quantitative methods in cell biology will be developed.