Bacterial Protein Tagging, Degradation and Ribosome Rescue

描述（由申请人提供）：蛋白酶在被数千种潜在蛋白质底物占据的拥挤细胞内环境中工作。因此，了解这些破坏性的酶如何选择“正确”的蛋白质进行降解，对于理解它们的生物学功能和特定底物的生物学功能至关重要。细胞内蛋白酶在消除受损或有害蛋白质、在基因表达变化后重塑蛋白质组、在细胞周期控制中以及作为应激反应途径中的传感器和调节组分中发挥重要作用。我们的主要目标是确定允许底物，衔接子和其他调节分子中的肽信号与蛋白酶相互作用以控制细菌中蛋白质降解的分子机制。第二个实际目标是开发靶向降解的合成系统，测试基本原理并为研究蛋白质功能提供社区工具。AAA+蛋白酶对细胞质蛋白的ATP依赖性降解发生在所有生物体中。生物化学，遗传和结构研究将阐明三种大肠杆菌AAA+蛋白酶（ClpXP，HslUV和Lon）底物识别的基本分子机制，并为理解这些酶的直系同源物如何识别其他细菌和真核生物中正确的细胞内底物提供范例。调节性膜内蛋白水解（RIP）是一种信号转导途径。我们将确定详细的分子机制，允许PDZ-蛋白酶（DegS）的感觉包膜压力的周质中的E。大肠杆菌中，并启动蛋白水解级联反应，将信息跨内膜传递给第二个PDZ蛋白酶（RseP）。该信号传导系统的分子逻辑及其由输入信号和PDZ蛋白酶与两种调节蛋白（RseA和RseB）的相互作用的控制将被阐明。我们也将剖析一个相关的RIP系统（AlgW-MucA-MucB），控制藻酸盐的生物合成在铜绿假单胞菌。了解细胞内降解是基础研究的一个关键目标，在生物技术和医学中有应用。例如，了解底物是如何鉴定的将能够改善重组蛋白的细菌表达，并且受控的降解系统将允许验证新的抗生素靶标。AAA+蛋白酶通常在细菌病原体的毒力中起作用，因此可以是抗生素靶标。此外，铜绿假单胞菌RIP系统的MucA和MucB蛋白的突变增加了囊性纤维化患者的死亡率和发病率。最后，了解DegS功能将与其人类同源物HtrA 2/Omi的研究相关，HtrA 2/Omi有助于caspase非依赖性细胞凋亡和癌症预防。 公共卫生相关性：了解细胞内降解是基础研究的一个关键目标，在生物技术和医学中有应用。例如，知道底物是如何鉴定的将允许重组蛋白的改进的细菌表达，并且受控降解系统将允许验证新的抗生素靶标。AAA+蛋白酶通常在细菌病原体的毒力中起作用，因此可以是抗生素靶标。此外，铜绿假单胞菌RIP系统的MucA和MucB蛋白的突变增加了囊性纤维化患者的死亡率和发病率。最后，了解DegS功能将与其人类同源物HtrA 2/Omi的研究相关，HtrA 2/Omi有助于非半胱天冬酶依赖性细胞凋亡和癌症预防。

DESCRIPTION (provided by applicant): Proteases work in crowded intracellular environments occupied by thousands of potential protein substrates. Thus, knowing how these destructive enzymes choose the "right" proteins for degradation is critical for understanding both their biological functions and those of specific substrates. Intracellular proteases play important roles in eliminating damaged or harmful proteins, in resculpting the proteome following changes in gene expression, in cell-cycle control, and as sensor and regulatory components in stress-response pathways. Our primary goal is to determine the molecular mechanisms that allow peptide signals in substrates, adaptors, and other regulatory molecules to interact with proteases to control protein degradation in bacteria. A second practical goal is to develop synthetic systems of targeted degradation that test basic principles and provide community tools for studying protein function. ATP-dependent degradation of cytoplasmic proteins by AAA+ proteases occurs in all organisms. Biochemical, genetic, and structural studies will elucidate fundamental molecular mechanisms of substrate recognition for three Escherichia coli AAA+ proteases (ClpXP, HslUV, and Lon), and provide paradigms for understanding how orthologs of these enzymes identify the correct intracellular substrates in other bacteria and eukaryotes. Regulated intramembrane proteolysis (RIP) is a method of signal transduction. We will determine the detailed molecular mechanisms that allow a PDZ-protease (DegS) to sense envelope stress in the periplasm of E. coli and initiate a proteolytic cascade that relays information across the inner membrane to a second PDZ-protease (RseP). The molecular logic of this signaling system and its control by input signals and interactions of the PDZproteases with two regulatory proteins (RseA and RseB) will be elucidated. We will also dissect a related RIP system (AlgW-MucA-MucB) that controls alginate biosynthesis in Pseudomonas aeruginosa. Understanding intracellular degradation is a key goal of basic research, with applications in biotechnology and medicine. For example, knowing how substrates are identified will enable improved bacterial expression of recombinant proteins, and controlled degradation systems will permit validation of new antibiotic targets. AAA+ proteases often play roles in the virulence of bacterial pathogens and thus can be antibiotic targets. Moreover, mutations in the MucA and MucB proteins of the P. aeruginosa RIP system increase mortality and morbidity in patients with cystic fibrosis. Finally, understanding DegS function will be relevant to studies of its human homolog, HtrA2/Omi, which contributes to caspase independent apoptosis and cancer prevention. PUBLIC HEALTH RELEVANCE: Understanding intracellular degradation is a key goal of basic research, with applications in biotechnology and medicine. For example, knowing how substrates are identified would allow improved bacterial expression of recombinant proteins, and controlled-degradation systems would permit validation of novel antibiotic targets. AAA+ proteases often play roles in the virulence of bacterial pathogens and thus can be antibiotic targets. Moreover, mutations in the MucA and MucB proteins of the P. aeruginosa RIP system increase mortality and morbidity in patients with cystic fibrosis. Finally, understanding DegS function will be relevant to studies of its human homolog, HtrA2/Omi, which contributes to caspase-independent apoptosis and cancer prevention.