CAREER: Protein Unfolding by Energy Dependent Proteases

9875857本项目的目的是确定atp依赖性蛋白酶降解过程中底物蛋白展开的机制。在液泡或溶酶体外的细胞中，大多数蛋白质降解是由atp依赖性蛋白酶催化的。底物蛋白的展开是降解过程中关键的早期步骤。短寿命蛋白的降解对于细胞周期控制、信号转导和抗原呈递等多种细胞功能至关重要。错误折叠蛋白质的蛋白质水解是细胞对压力反应的一部分。选择Barnase作为atp依赖性蛋白酶long -蛋白酶、clp蛋白酶、线粒体aaa蛋白酶和蛋白酶体的人工底物蛋白，Barnase在体外折叠特性良好。降解过程中的巴纳酶展开将与自发展开进行比较，以确定蛋白质展开是否由蛋白酶催化。不稳定的藤壶酶突变体将被用作错误折叠蛋白的模型，而带有c端降解标签的藤壶酶突变体将被用作短寿命调节蛋白的模型。如果展开是催化的，则确定了催化的机理。该奖项的教育目的是教授本科生对细胞生物过程的生化和生物物理理解的先决条件。为此目的，将开设一门定量生物物理技术的实验课程。蛋白质是由称为氨基酸的亚基组成的链。为了发挥其生物学功能，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，蛋白质必须再次展开，例如当受损或调节蛋白被蛋白酶降解时。不能降解调节蛋白和错误折叠蛋白导致细胞功能异常。这项提议研究了蛋白酶如何展开蛋白质。生物学已经发展到可以确定细胞过程的详细机制的阶段。为了让学生为实验生物学的新时代做好准备，我们将开设一门细胞生物学定量方法的实验课程。

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.