Proteolysis and Regulation of Bacterial Cell Growth Control

细菌细胞生长控制的蛋白水解和调节

基本信息

批准号：
10926159
负责人：
SUSAN GOTTESMAN
金额：
$ 63.58万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10926159
关键词：
ATP phosphohydrolase ATP-Dependent Proteases ATPase Domain Adaptor Signaling Protein Affect Amino Acids Bacteria Bacteria sigma factor KatF protein Biochemical Biological Cell division Cells Codon Nucleotides Collaborations Complex Cues Cytoplasm DNA Damage DNA-Directed RNA Polymerase Defect Dissection Ensure Escherichia coli Feedback Gammaproteobacteria Gene Expression Gene Expression Regulation Gene Proteins Genes Genetic Growth Hyperactivity In Vitro Investigation Magnesium Mutate Mutation Mutation Analysis N-terminal Names National Institute of Diabetes and Digestive and Kidney Diseases Organism Peptide Hydrolases Play Polyamines Polymerase Polysaccharides Process Protease Domain Proteins Proteolysis Pseudomonas aeruginosa Quality Control Recovery Regulation Resistance Ribosomes Role Sigma Factor Signal Transduction Silent Mutation Small RNA Starvation Stress Structure System Translations Universities Work biological adaptation to stress cell growth endopeptidase Clp environmental change extreme temperature falls feeding flexibility in vivo inhibitor inorganic phosphate insight misfolded protein multicatalytic endopeptidase complex mutant novel programs promoter protein degradation reconstitution response small molecule transmission process

项目摘要

For many years, our lab has investigated the role of energy-dependent proteolysis in regulation of gene expression in bacteria. The ATP-dependent cytoplasmic proteases, akin to the eukaryotic proteasome, contain ATPase domains or subunits that recognize substrates and unfold them, feeding them to the proteolytic domains. Bacteria contain multiple ATP-dependent proteases; five of them have been characterized in E. coli. Abnormal or misfolded proteins are degraded by these proteases. In addition to this quality control role, the proteases degrade proteins that are naturally unstable; for these proteins, degradation is likely to play an important biological role. Such protease substrates fall into two general classes: proteins that are always degraded, so that regulation of their abundance depends primarily on changes in synthesis, and proteins that show regulated proteolysis. In all cases, identifying how the substrate is recognized by the protease and how recognition is affected by growth conditions is important in understanding how and when regulation is carried out. Our lab showed that the Lon ATP-dependent protease regulated capsular polysaccharide synthesis and cell division by degrading the RcsA and SulA proteins, discovered and characterized the two-component Clp proteases, ClpAP and ClpXP, and investigated the roles of these proteases in vivo and in vitro. In recent years, our focus has been on the regulated degradation of the RpoS sigma factor, a subunit of RNA polymerase that directs the polymerase to specific promoters. RpoS is important for cells to switch to a stationary or stress response gene expression program; the expressed genes provide resistance to starvation, temperature extremes, and other stresses. However, RpoS and its expressed genes are detrimental when the bacteria is under optimal growth conditions. The cell regulates RpoS accumulation in a variety of ways, including at the level of translation via small RNA activators of translation, and by regulated proteolysis. We have been studying this proteolysis, one of the best examples of regulated protein turnover in E. coli. RpoS is rapidly degraded during active growth, in a process that requires the energy-dependent ClpXP protease and the adaptor protein RssB, a phosphorylatable protein that presents RpoS to the protease. RpoS becomes stable after various stress or starvation treatments; the mode of stabilization was a mystery until work from our lab led to discovery of a small, previously uncharacterized protein that acts as an anti-adaptor, blocking the ability of RssB to deliver RpoS to the protease. Mutants in the gene for that protein, now named IraP (inhibitor of RssB activity after phosphate starvation) abolish the stabilization of RpoS after phosphate starvation. IraP blocks RpoS turnover in a purified in vitro system, and directly interacts with RssB. In E. coli, phosphate starvation leads to IraP induction, due to an increase in the levels of the small molecule alarmone ppGpp; the iraP promoter has become the best example of how ppGpp positively regulates promoters. Two other small proteins also stabilize RpoS in a purified in vitro system, IraM, and IraD. These proteins are not similar in predicted structure to IraP. IraM is made in response to magnesium starvation, dependent on the PhoP and PhoQ regulators; IraD is important after DNA damage. The anti-adaptors define a new level of regulatory control, interacting with the RssB adaptor protein and blocking its ability to act; environmental signals regulate RpoS turnover by regulating expression of different anti-adaptors. In continuing collaborative studies with Sue Wickner (NCI) on the structure and function of RssB and its anti-adaptors, we use in vivo genetics and in vitro reconstitution to understand how the antiadaptors and adaptor protein work. A collaboration with A. Deaconescu (Brown University) has led to a structure of an IraD/RssB complex, providing valuable new insight into how IraD inactivates RssB and fully supporting our earlier genetic and biochemical studies. We are further defining how RssB interacts with ClpX, the ATPase subunit of the ClpXP protease. The N-terminal domain of ClpX, known to interact with some other adaptors and substrates, interacts with the RssB C-terminus. Continued dissection of this system is providing insight into how this process is balanced in the cell. A long-standing question has been how the cell recovers from stress, in particular from the antiadaptors. We have investigated this process for recovery from phosphate starvation. During starvation, IraP is induced and stabilizes RpoS. We find that degradation of RpoS is restored rapidly after phosphate is returned to cells, and that this rapid recovery, implying active inactivation of IraP, is dependent on a feedback loop in which RpoS increases the synthesis of RssB. Another regulator of RpoS, Crl, plays a critical and unexpected role in the recovery from starvation. Crl promotes the association of RpoS with the core RNA polymerase, thus favoring expression of RpoS-dependent promoters, including the promoter for the rssB gene, encoding the adaptor. Mutational analysis of IraP demonstrates that the C-terminus of this anti-adaptor is critically necessary for rapid recovery, suggesting that it modulates the interaction of IraP with RssB. In vitro and in vivo, IraP lacking or mutated for critical residues in the C-terminus are, unexpectedly, hyperactive. Therefore, the C-terminus acts as a critical negative regulator of IraP, ensuring that it is active only when required. In another aspect of RpoS regulation, H. Tabor's lab (NIDDK; deceased in 2020) had observed that cells devoid of polyamines have very low levels of RpoS. In a collaboration with them, we have confirmed and extended this work. We find that the lack of polyamines allows rapid co-translational degradation of RpoS. Ribosomal mutations that increase translational proofreading have a similar, if not as drastic, effect. In both cases, changing codon usage within the rpoS gene is sufficient to overcome much of the defect. These results suggest that previously unrecognized aspects of codon usage poise some genes, including rpoS, to be particularly sensitive to translational stress, and reinforce the importance of so-called "silent" mutations that change codon usage within a protein without changing the encoded amino acids. While our work focuses on RpoS and its adaptor in RssB in E. coli, both proteins are found in a range of other gammaproteobacteria. In a recent project, comparisons of RssB from selected other species shows that the role of RssB in regulating RpoS is widespread but that in some organisms, for instance P. aeruginosa, RssB may act as an anti-sigma rather than as an adaptor for proteolysis. The basis of this difference is under investigation. Overall, our proteolysis studies continue to provide novel insights into regulatory mechanisms used by bacteria.

多年来，我们的实验室一直在研究能量依赖性蛋白水解在细菌基因表达调节中的作用。 ATP 依赖性细胞质蛋白酶类似于真核蛋白酶体，含有 ATP 酶结构域或亚基，可识别底物并展开它们，将其供给蛋白水解结构域。细菌含有多种ATP依赖性蛋白酶；其中五个已在大肠杆菌中得到表征。异常或错误折叠的蛋白质被这些蛋白酶降解。除了这种质量控制作用之外，蛋白酶还可以降解天然不稳定的蛋白质。对于这些蛋白质来说，降解可能发挥着重要的生物学作用。此类蛋白酶底物分为两大类：总是被降解的蛋白质，因此其丰度的调节主要取决于合成的变化，以及表现出受调节的蛋白水解作用的蛋白质。在所有情况下，确定蛋白酶如何识别底物以及生长条件如何影响识别对于理解如何以及何时进行调节非常重要。我们的实验室表明，Lon ATP 依赖性蛋白酶通过降解 RcsA 和 SulA 蛋白来调节荚膜多糖合成和细胞分裂，发现并表征了双组分 Clp 蛋白酶 ClpAP 和 ClpXP，并研究了这些蛋白酶在体内和体外的作用。体外。近年来，我们的重点是 RpoS sigma 因子的调控降解，RpoS sigma 因子是 RNA 聚合酶的一个亚基，可将聚合酶引导至特定的启动子。 RpoS 对于细胞切换到静止或应激反应基因表达程序很重要；表达的基因提供对饥饿、极端温度和其他压力的抵抗力。然而，当细菌处于最佳生长条件时，RpoS 及其表达的基因是有害的。细胞以多种方式调节 RpoS 积累，包括在翻译水平上通过翻译的小 RNA 激活剂以及通过调节蛋白水解。我们一直在研究这种蛋白水解作用，这是大肠杆菌中调节蛋白质周转的最好例子之一。 RpoS 在活跃生长过程中迅速降解，该过程需要能量依赖性 ClpXP 蛋白酶和接头蛋白 RssB（一种将 RpoS 呈现给蛋白酶的可磷酸化蛋白）。经过各种应激或饥饿处理后，RpoS 变得稳定；稳定模式一直是个谜，直到我们实验室的工作发现了一种以前未表征的小蛋白质，它充当反接头蛋白，阻止 RssB 将 RpoS 传递给蛋白酶的能力。该蛋白质的基因突变体现在被命名为 IraP（磷酸盐饥饿后 RssB 活性抑制剂），在磷酸盐饥饿后消除了 RpoS 的稳定性。 IraP 在纯化的体外系统中阻断 RpoS 周转，并直接与 RssB 相互作用。在大肠杆菌中，磷酸盐饥饿会导致 IraP 诱导，这是由于小分子警报素 ppGpp 水平的增加； iraP启动子已经成为ppGpp如何正向调控启动子的最好例子。另外两种小蛋白 IraM 和 IraD 也在纯化的体外系统中稳定 RpoS。这些蛋白质的预测结构与 IraP 并不相似。 IraM 是为了应对镁缺乏而产生的，依赖于 PhoP 和 PhoQ 调节剂； IraD 在 DNA 损伤后很重要。反适配器定义了新的调控水平，与 RssB 适配器蛋白相互作用并阻断其作用能力；环境信号通过调节不同反接头的表达来调节 RpoS 周转。在与 Sue Wickner (NCI) 继续合作研究 RssB 及其反接头蛋白的结构和功能时，我们利用体内遗传学和体外重构来了解反接头蛋白和接头蛋白的工作原理。与 A. Deaconescu（布朗大学）的合作产生了 IraD/RssB 复合物的结构，为 IraD 如何灭活 RssB 提供了有价值的新见解，并完全支持我们早期的遗传和生化研究。我们正在进一步定义 RssB 如何与 ClpX（ClpXP 蛋白酶的 ATPase 亚基）相互作用。 ClpX 的 N 端结构域已知与一些其他接头和底物相互作用，也与 RssB C 端相互作用。对该系统的继续剖析可以深入了解该过程如何在细胞中保持平衡。一个长期存在的问题是细胞如何从压力中恢复，特别是从抗适应剂中恢复。我们研究了从磷酸盐饥饿中恢复的这一过程。饥饿期间，IraP 被诱导并稳定 RpoS。我们发现，磷酸盐返回细胞后，RpoS 的降解迅速恢复，这种快速恢复意味着 IraP 的主动失活，依赖于 RpoS 增加 RssB 合成的反馈回路。 RpoS 的另一个调节因子 Crl 在饥饿恢复中发挥着关键且意想不到的作用。 Crl 促进 RpoS 与核心 RNA 聚合酶的结合，从而有利于 RpoS 依赖性启动子的表达，包括编码接头的 rssB 基因的启动子。 IraP 的突变分析表明，该反接头的 C 末端对于快速恢复至关重要，表明它调节 IraP 与 RssB 的相互作用。在体外和体内，C 末端关键残基缺失或突变的 IraP 出乎意料地异常活跃。因此，C 末端充当 IraP 的关键负调节因子，确保其仅在需要时才激活。在 RpoS 调节的另一个方面，H. Tabor 实验室（NIDDK；已于 2020 年去世）观察到，缺乏多胺的细胞的 RpoS 水平非常低。在与他们的合作中，我们确认并扩展了这项工作。我们发现多胺的缺乏使得 RpoS 能够快速共翻译降解。增加翻译校对的核糖体突变也有类似的效果，即使不是那么剧烈。在这两种情况下，改变 rpoS 基因内的密码子使用足以克服大部分缺陷。这些结果表明，密码子使用的先前未被认识的方面使包括rpoS在内的一些基因对翻译应激特别敏感，并强化了所谓的“沉默”突变的重要性，这种突变改变蛋白质内的密码子使用而不改变编码的氨基酸。虽然我们的工作重点是大肠杆菌中 RssB 中的 RpoS 及其接头，但这两种蛋白质也在一系列其他伽马变形菌中发现。在最近的一个项目中，对来自选定其他物种的 RssB 的比较表明，RssB 在调节 RpoS 中的作用是广泛的，但在某些生物体中，例如铜绿假单胞菌，RssB 可能充当抗 sigma 而不是蛋白水解的接头。这种差异的基础正在调查中。总的来说，我们的蛋白水解研究继续为细菌使用的调节机制提供新的见解。