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: 10262261
• 负责人: SUSAN GOTTESMAN
• 金额: $ 78.6万
• 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/10262261
• 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; Codon Nucleotides; Collaborations; Complex; Crystallization; Cues; DNA Damage; DNA-Directed RNA Polymerase; Dissection; Ensure; Escherichia coli; Feedback; Gene Expression; Gene Expression Regulation; Genes; Genetic; Growth; Hypersensitivity; In Vitro; Investigation; Lead; Magnesium; Modeling; N-terminal; Names; National Institute of Diabetes and Digestive and Kidney Diseases; Organism; Peptide Hydrolases; Play; Polyamines; Polymerase; Polysaccharides; Post-Transcriptional Regulation; Process; Protease Domain; Proteins; Proteolysis; Quality Control; Recovery; Regulation; Role; Sigma Factor; Signal Transduction; Small RNA; Starvation; Stress; Structure; System; Testing; Translations; Universities; Work; 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; novel; programs; promoter; protein degradation; reconstitution; response; small molecule

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). Deletion of iraP abolishes 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 is sensed by an increase in the levels of the small molecule ppGpp, and the iraP promoter is positively regulated by the stress alarmone ppGpp. 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. This has led to a detailed analysis of IraP, including a crystal structure in collaboration with X. Ji. 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 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 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, and that the C-terminus of IraP is necessary for this rapid recovery. These results support a complex and precisely balanced system that allows the cell to use the RpoS general stress response under some conditions but dispose of it when it is not needed. Investigations of how RpoS is regulated continue to yield new insights into mechanisms of post-transcriptional regulation. For instance, H. Tabor's lab (NIDDK) had observed that cells devoid of polyamines have very low levels of RpoS. In a collaboration with them, we have confirmed this work and demonstrated that RpoS is likely subject to very rapid degradation in the absence of polyamines. However, this process may occur co-translationally, integrating unusual codon usage to lead to hyper-sensitivity to proteolysis; we are testing this model. 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依赖能量的蛋白分解在细菌基因表达调控中的作用。依赖于ATP的细胞质蛋白类似于真核蛋白酶体，含有识别底物并将其解开的ATPase结构域或亚单位，将它们提供给蛋白水解域。细菌含有多种依赖于三磷酸腺苷的蛋白水解酶；其中五种已在大肠杆菌中被鉴定。异常或错误折叠的蛋白质会被这些酶降解。除了这种质量控制作用外，蛋白酶还会降解天然不稳定的蛋白质；对于这些蛋白质来说，降解很可能起到重要的生物学作用。这类酶的底物一般分为两类：一类是总是被降解的蛋白质，因此它们的丰度主要取决于合成的变化；另一类是表现出受调控的蛋白分解作用的蛋白质。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调控是如何进行的非常重要。过去，我们的实验室发现Lon-ATP依赖的蛋白酶通过降解RCSA和Sula蛋白来调节囊膜多糖的合成和细胞分裂，发现并鉴定了两种CLP蛋白酶ClpAP和ClpXP，并研究了这些酶在体内和体外的作用。近年来，我们的重点一直放在rpos sigma因子的受控降解上，rpos sigma因子是RNA聚合酶的一个亚单位，它将聚合酶导向特定的启动子。RPOS对于细胞切换到稳定的或应激反应的基因表达程序很重要。该细胞以多种方式调节rpos的积累，包括通过翻译的小RNA激活剂在翻译水平上，以及通过调节的蛋白分解。我们一直在研究这种蛋白质分解，这是大肠杆菌中调节蛋白质周转的最好例子之一。在活跃的生长过程中，RPOS被迅速降解，这一过程需要依赖能量的ClpXP蛋白酶和适配器蛋白RSSB，RSSB是一种可磷酸化的蛋白质，将RPOS呈现给蛋白酶。RPOS在各种应激或饥饿处理后变得稳定；稳定的模式一直是一个谜，直到我们实验室的工作导致发现了一种以前没有特征的小蛋白，现在被称为IRAP(磷酸盐饥饿后RSSB活性的抑制物)。IRAP的缺失取消了磷酸饥饿后rpos的稳定性。IRAP在体外纯化的系统中阻断rpos的转换，并直接与rSSB相互作用。在大肠杆菌中，磷饥饿是通过小分子ppGpp水平的增加来感受的，IRAP启动子受到应激警报蛋白ppGpp的正调控。另外两个小蛋白也稳定了体外纯化系统中的rpos，IRAM和IRAD。这些蛋白质在预测的结构上与IRAP不同。IRAM是对镁饥饿的反应，依赖于Phop和PhoQ调节器；IRAD在DNA损伤后是重要的。抗接头定义了一个新的调控水平，与RSSB接头蛋白相互作用并阻断其作用能力；环境信号通过调节不同的抗接头的表达来调节RPO的周转。在与Sue Wickner(NCI)继续合作研究RSSB及其抗接头的结构和功能时，我们使用体内遗传学和体外重组来了解抗接头和接头蛋白是如何工作的。这导致了对IRAP的详细分析，包括与X.Ji合作的晶体结构。与A.Deaconescu(布朗大学)的合作导致了IRAD/RSSB复合体的结构，为IRAD如何使RSSB失活提供了宝贵的新见解，并完全支持了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相互作用，ClpX是ClpXP蛋白酶的ATPase亚单位。ClpX的N-末端结构域与其他一些适配器和底物相互作用，与RSSB的C-末端相互作用。对这一系统的持续剖析提供了对这一过程是如何在细胞内平衡的洞察。一个长期存在的问题是，细胞如何从压力中恢复，特别是从抗适配器恢复。我们研究了从磷酸盐饥饿中恢复的这一过程。在饥饿过程中，IRAP被诱导并稳定RPOS。我们发现，在磷酸盐返回细胞后，RPOS的降解迅速恢复，并且这种快速恢复意味着IRAP的失活，依赖于RPOS可能增加RSSB合成的反馈环；我们发现RPOS的另一种调节因子CRL在饥饿恢复过程中发挥了关键和意想不到的作用，并且IRAP的C末端是这种快速恢复所必需的。这些结果支持一个复杂且精确平衡的系统，该系统允许细胞在某些条件下使用RPOS一般应激反应，但在不需要时处理它。对rpos如何调控的研究继续对转录后调控机制产生新的见解。例如，H.Tabor的实验室(NIDDK)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，我们确认了这项工作，并证明了在没有多胺的情况下，RPO可能会非常迅速地降解。然而，这一过程可能会发生共翻译，整合不寻常的密码子使用导致对蛋白质降解高度敏感；我们正在测试这个模型。总体而言，我们的蛋白质分解研究继续为细菌使用的调节机制提供新的见解。