Proteolysis and Regulation of Bacterial Cell Growth Control

细菌细胞生长控制的蛋白水解和调节

• 批准号: 10486787
• 负责人: SUSAN GOTTESMAN
• 金额: $ 88.76万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/10486787
• 关键词: ATP phosphohydrolase; ATP-Dependent Proteases; ATPase Domain; Adaptor Signaling Protein; Affect; Bacteria; Bacteria sigma factor KatF protein; Biochemical; Biological; Cell division; Cells; Collaborations; Complex; Cues; DNA Damage; DNA-Directed RNA Polymerase; Dissection; Ensure; Escherichia coli; Feedback; Gene Expression; Gene Expression Regulation; Genes; Genetic; Growth; In Vitro; Libraries; Magnesium; Metabolism; Mutation Analysis; N-terminal; Names; National Institute of Diabetes and Digestive and Kidney Diseases; Organism; Peptide Hydrolases; Plasmids; Play; Polyamines; Polymerase; Polysaccharides; Process; Protease Domain; Proteins; Proteolysis; Quality Control; Recovery; Regulation; Ribose; Role; Sigma Factor; Signal Transduction; Small RNA; Starvation; Stress; Structure; System; Translational Activation; Translations; Universities; Work; base; biological adaptation to stress; cell growth; endopeptidase Clp; environmental change; falls; feeding; flexibility; in vivo; inhibitor/antagonist; inorganic phosphate; insight; misfolded protein; multicatalytic endopeptidase complex; mutant; novel; programs; promoter; protein degradation; reconstitution; response; screening; small molecule; transcription factor

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 regulation is carried out. In the past, 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 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, now named IraP (inhibitor of RssB activity after phosphate starvation). Mutants of iraP 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 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. Other anti-adaptors are likely to exist, based on a variety of results. 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 this 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 may increase the synthesis of RssB; we have found that another regulator of RpoS, Crl, plays a critical and unexpected role in the recovery from starvation. 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; this is being examined in vitro. Other regulatory inputs to the degradation of RpoS are being investigated as well. 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. Our work suggests that the lack of polyamines may allow rapid co-translational degradation of RpoS. In another project, screening a plasmid library for negative regulation of RpoS has led to the characterization of a role for ribose metabolism in the negative regulation of RpoS, as well as identification of a transcription factor that acts to counteract translational activation of RpoS. 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控中的作用。ATP依赖性细胞质蛋白酶，类似于真核生物蛋白酶体，含有ATP酶结构域或亚基，其识别底物并将其展开，将其供给蛋白水解结构域。细菌含有多种ATP依赖性蛋白酶，其中五种已在E.杆菌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外，蛋白酶还降解天然不稳定的蛋白质;对于这些蛋白质，降解可能发挥重要的生物学作用。这种蛋白酶底物分为两大类：总是降解的蛋白质，因此其丰度的调节主要取决于合成的变化，以及显示调节蛋白水解的蛋白质。在所有情况下，确定底物如何被蛋白酶识别以及识别如何受到生长条件的影响对于理解如何进行调节是重要的。在过去，我们的实验室表明，依赖于Lon ATP的蛋白酶通过降解RcsA和苏拉蛋白来调节荚膜多糖的合成和细胞分裂，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。近年来，我们的重点一直是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对于细胞切换到稳定或应激反应基因表达程序是重要的。细胞以多种方式调节RpoS积累，包括通过翻译的小RNA激活剂在翻译水平上，以及通过调节蛋白水解。我们一直在研究这种蛋白水解，这是大肠杆菌中调节蛋白质周转的最好例子之一。杆菌RpoS在活跃生长期间迅速降解，在需要能量依赖性ClpXP蛋白酶和衔接蛋白RssB（将RpoS呈递给蛋白酶的可磷酸化蛋白）的过程中。RpoS在各种应激或饥饿处理后变得稳定;稳定模式是一个谜，直到我们实验室的工作导致发现了一种以前未表征的小蛋白质，现在命名为RssB（磷酸盐饥饿后RssB活性抑制剂）。iraP突变体在磷酸盐饥饿后破坏了RpoS的稳定性。在纯化的体外系统中，RssP阻断RpoS周转，并直接与RssB相互作用。在大肠在大肠杆菌中，由于小分子ppGpp水平的增加，磷酸盐饥饿导致IraP诱导; IraP启动子已成为ppGpp如何正向调节启动子的最佳实例。另外两种小蛋白质也在纯化的体外系统中稳定RpoS，它们分别是Rp 3 M和Rp 3D。这些蛋白质在预测的结构上不类似于CNOMP。镁缺乏时，依赖于PhoP和PhoQ调节剂，而镁缺乏则会产生镁缺乏;在DNA损伤后，镁缺乏是很重要的。抗衔接子定义了一个新的调控水平，与RssB衔接子蛋白相互作用并阻断其作用能力;环境信号通过调节不同抗衔接子的表达来调节RpoS周转。在继续与Sue Wickner（NCI）合作研究RssB及其抗适配器的结构和功能时，我们使用体内遗传学和体外重建来了解抗适配器和适配器蛋白如何工作。与A的合作。Deaconescu（布朗大学）已经导致了一种BHD/RssB复合物的结构，为BHD如何使RssB失活提供了有价值的新见解，并完全支持了我们早期的遗传和生物化学研究。我们正在进一步确定RssB如何与ClpX相互作用，ClpXP蛋白酶的ATP酶亚基。已知ClpX的N-末端结构域与一些其他衔接子和底物相互作用，与RssB C-末端相互作用。对这一系统的继续解剖将使我们深入了解这一过程在细胞中是如何平衡的。基于各种结果，其他抗适应者可能存在。一个长期存在的问题是细胞如何从压力中恢复，特别是从抗适应因子中恢复。我们已经研究了从磷酸盐饥饿中恢复的过程。在这种饥饿过程中，诱导了RPOP并使RPOS稳定。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，意味着主动失活的RpoS，是依赖于一个反馈回路，其中RpoS可能会增加RssB的合成;我们发现，另一个调节RpoS，Crl，在恢复饥饿中起着关键的和意想不到的作用。突变分析表明，这种抗适配器的C-末端是至关重要的快速恢复，这表明它调节的相互作用的CIPTP与RssB，这是在体外检查。RpoS降解的其他监管输入也正在调查中。H.塔博尔的实验室（NIDDK; 2020年去世）观察到缺乏多胺的细胞具有非常低的RpoS水平;在与他们的合作中，我们已经证实并扩展了这项工作。我们的工作表明，多胺的缺乏可能允许快速共翻译降解RpoS。在另一个项目中，筛选RpoS负调控的质粒文库导致了核糖代谢在RpoS负调控中的作用的表征，以及鉴定了用于抵消RpoS的翻译激活的转录因子。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。