Bacterial Protein Tagging, Degradation and Ribosome Rescue

描述(由申请人提供)：蛋白酶在拥挤的细胞内环境中工作，被数千种潜在的蛋白质底物占据。因此，了解这些破坏性酶如何选择“正确的”蛋白质进行降解，对于了解它们的生物学功能和特定底物的功能至关重要。细胞内的蛋白水解酶在清除受损或有害的蛋白质、在基因表达改变后蛋白质组的重建、细胞周期控制以及作为应激反应通路的传感器和调节元件方面发挥着重要作用。我们的主要目标是确定允许底物、接头和其他调节分子中的肽信号与蛋白酶相互作用的分子机制，以控制细菌中的蛋白质降解。第二个实际目标是开发靶向降解的合成系统，以测试基本原理，并为研究蛋白质功能提供社区工具。AAA+酶对细胞质蛋白的降解依赖于ATP，存在于所有生物体中。生化、遗传和结构研究将阐明三种大肠杆菌AAA+酶(ClpXP、HslUV和Lon)底物识别的基本分子机制，并为理解这些酶的同源酶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。调节膜内蛋白分解(RIP)是一种信号转导方法。我们将确定详细的分子机制，使PDZ-蛋白酶(DEGS)能够感知大肠杆菌周质中的包膜应力，并启动蛋白水解级联反应，通过内膜将信息传递到第二个PDZ-蛋白酶(RSEP)。这一信号系统的分子逻辑及其输入信号的控制以及PDZ蛋白酶与两种调节蛋白(RseA和RseB)的相互作用将被阐明。我们还将剖析一个控制铜绿假单胞菌藻酸盐生物合成的相关RIP系统(AlgW-MucA-MucB)。了解细胞内降解是基础研究的一个关键目标，并在生物技术和医学中得到应用。例如，了解底物是如何识别的将有助于改善重组蛋白的细菌表达，而受控降解系统将允许验证新的抗生素靶标。AAA+蛋白水解酶通常在细菌病原体的毒力中发挥作用，因此可以成为抗生素的靶标。此外，铜绿假单胞菌RIP系统的粘蛋白A和粘蛋白B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的功能将与其人类同源基因HtrA2/Omi的研究有关，HtrA2/Omi有助于caspase非依赖的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的应用。例如，了解底物是如何识别的将有助于改善重组蛋白的细菌表达，而受控降解系统将允许验证新的抗生素靶标。AAA+蛋白水解酶通常在细菌病原体的毒力中发挥作用，因此可以成为抗生素的靶标。此外，铜绿假单胞菌RIP系统的粘蛋白A和粘蛋白B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的功能将与其人类同源基因HtrA2/Omi的研究相关，HtrA2/Omi有助于caspase非依赖的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.