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: 9556490
• 负责人: SUSAN GOTTESMAN
• 金额: $ 42.05万
• 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/9556490
• 关键词: ATP phosphohydrolase; ATP-Dependent Proteases; ATPase Domain; Adaptor Signaling Protein; Affect; Bacteria; Bacteria sigma factor KatF protein; Biochemical; Biological; Biological Assay; Bypass; C-terminal; Cell division; Cells; Complex; Cues; DNA Damage; DNA-Directed RNA Polymerase; Dimerization; Dissection; Ensure; Enzymes; Escherichia coli; Family; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Growth; In Vitro; Magnesium; Metabolism; Mutation; N-terminal; Names; Organism; Peptide Hydrolases; Peptidoglycan; Phosphoric Monoester Hydrolases; Phosphorylation; Play; Polymerase; Polysaccharides; Process; Protease Domain; Proteins; Proteolysis; Quality Control; Recycling; Regulation; Resistance; Role; Sigma Factor; Signal Transduction; Small RNA; Starvation; Stress; Structure; System; Translations; Two-Hybrid System Techniques; Universities; Work; base; biological adaptation to stress; cell growth; dimer; endopeptidase Clp; environmental change; falls; feeding; flexibility; genetic regulatory protein; genetic selection; in vivo; inhibitor/antagonist; inorganic phosphate; insight; interest; member; misfolded protein; multicatalytic endopeptidase complex; mutant; novel; overexpression; programs; promoter; protein degradation; protein phosphatase 2C; response; small molecule; yeast two hybrid system

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, and 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 is sensed by an increase in the levels of the small molecule ppGpp, and the iraP promoter is positively regulated by 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. RssB structure and function have been further investigated using a range of approaches: 1) a genetic selection to identify mutations in RssB resistant to a specific anti-adaptor, 2) a bacterial two-hybrid system to investigate the interaction of wild-type and mutant derivatives of RssB and its domains with the anti-adaptors and other components of the system, and 3) collaborative in vitro studies with the lab of Sue Wickner (NCI) of RpoS degradation with the mutant proteins and the antiadaptors. The results of these studies demonstrate that both IraP and IraD interact with the N-terminal domain of RssB. This domain of RssB is a member of the widespread response regulator family. Although both anti-adaptors interact with this conserved domain, they do not interact in the same fashion. Thus, mutations in RssB that abolish interaction with IraP retain interactions with IraD. Collaborative studies with X. Ji (NCI) on the structure of IraP reveal that it is a unique protein with similarity to B-Zip dimers. Mutational and biochemical analysis of mutants in both IraP and RssB are defining how these interact and providing insight into how RssB works to deliver RpoS to the protease. We are collaborating with A. Deaconescu (Brown University) to investigate the structural basis for the interaction of IraD with RssB; her structure of a complex is consistent with our earlier conclusions suggesting that IraD interacts with the inactive form of RssB, and provides new insight into the structure of the inactive form. IraM interacts with C-terminal domain of RssB; this domain has homology to an inactive PP2C phosphatase domain. One class of anti-adaptor mutations activates RssB, bypassing the stimulatory effect of phosphorylation. These mutants provide new insight into how RssB works and how regulatory proteins can disrupt the function of the conserved domains that make up RssB. 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, including the observation that the transcriptional regulator AppY stabilizes RpoS in the absence of all three known anti-adaptors. Other mutations and conditions that stabilize RpoS work through the known anti-adaptors. For instance, deletion of the global repressor, H-NS, also stabilize RpoS. Expression of IraD and IraM is repressed by H-NS, and in the absence of these two anti-adaptors, much but not all of the stabilizing effect of H-NS is lost. Mutations in aceE, a component of central metabolism, also leads to stabilization of RpoS, dependent on IraP and IraD. High pH also leads to induction of RpoS by stabilization, via the known anti-adaptors. The bacterial two-hybrid system has also been used to identify other proteins that interact with RssB. Interacting proteins may be additional anti-adaptors or other substrates for RssB. A number of interesting regulatory proteins have been identified and found to affect RpoS turnover, and thus may be acting as anti-adaptors or competing substrates, both of which are of significant interest. One of them, AnmK, has been studied in some detail. AnmK is an enzyme involved in recycling of peptidoglycan. In addition to interacting with RssB in the bacterial two-hybrid assay, it interacts in a pull-down assay. Overexpression of AnmK stabilizes RpoS, consistent with it acting either as a competitive inhibitor or an anti-adaptor; deletion of anmK may have an effect on stability of RpoS under particular stress conditions. Further study of these interacting proteins is in process. 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调控是如何进行的非常重要。过去，我们实验室证明了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的作用体内和体外。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非常重要，并且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；稳定的模式一直是个谜，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，现在命名为 IraP（磷酸盐饥饿后 RssB 活性的抑制剂）。 iraP 突变体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，磷酸盐饥饿是通过小分子 ppGpp 水平的增加来感知的，并且 iraP 启动子受到 ppGpp 的正向调节。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。使用一系列方法进一步研究了 RssB 的结构和功能：1) 遗传选择来识别对特定抗接头有抗性的 RssB 突变，2) 细菌双杂交系统来研究野生型和突变体的相互作用RssB 的衍生物及其结构域与反适配器和系统的其他组件，以及 3) 与 Sue Wickner (NCI) 实验室合作进行突变蛋白和 RpoS 降解的体外研究反适配器。这些研究的结果表明 IraP 和 IraD 均与 RssB 的 N 末端结构域相互作用。 RssB 的这个结构域是广泛的响应调节器家族的成员。尽管两个反适配器都与这个保守域相互作用，但它们的相互作用方式不同。因此，消除与 IraP 相互作用的 RssB 突变保留了与 IraD 的相互作用。与 X. Ji (NCI) 合作对 IraP 结构的研究表明，它是一种与 B-Zip 二聚体相似的独特蛋白质。对 IraP 和 RssB 突变体的突变和生化分析正在定义它们如何相互作用，并深入了解 RssB 如何将 RpoS 传递给蛋白酶。我们正在与 A. Deaconescu（布朗大学）合作，研究 IraD 与 RssB 相互作用的结构基础；她的复合物结构与我们之前的结论一致，表明 IraD 与 RssB 的非活性形式相互作用，并为非活性形式的结构提供了新的见解。 IraM 与 RssB 的 C 端结构域相互作用；该结构域与无活性 PP2C 磷酸酶结构域具有同源性。一类抗接头突变会激活 RssB，绕过磷酸化的刺激作用。这些突变体为了解 RssB 的工作原理以及调节蛋白如何破坏组成 RssB 的保守结构域的功能提供了新的见解。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。根据各种结果，包括转录调节因子 AppY 在所有三种已知反适配器都不存在的情况下稳定 RpoS 的观察结果，可能还存在其他反适配器。其他稳定 RpoS 的突变和条件通过已知的反适配器发挥作用。例如，删除全局阻遏蛋白 H-NS 也可以稳定 RpoS。 IraD 和 IraM 的表达受到 H-NS 的抑制，并且在缺乏这两种抗接头的情况下，H-NS 的大部分（但不是全部）稳定作用会丧失。 aceE（中央代谢的一个组成部分）的突变也会导致 RpoS 的稳定，依赖于 IraP 和 IraD。高 pH 值还可以通过已知的反接头通过稳定化诱导 RpoS。细菌双杂交系统也已被用来鉴定与 RssB 相互作用的其他蛋白质。相互作用的蛋白质可能是额外的抗接头蛋白或 RssB 的其他底物。已经鉴定出许多有趣的调节蛋白，并发现它们会影响 RpoS 周转，因此可能充当抗接头蛋白或竞争底物，这两者都具有重要意义。其中之一，AnmK，已经得到了一些详细的研究。 AnmK 是一种参与肽聚糖回收的酶。除了在细菌双杂交测定中与 RssB 相互作用外，它还在 Pull-down 测定中相互作用。 AnmK 的过度表达可稳定 RpoS，这与它作为竞争性抑制剂或抗适配器的作用一致； anmK 的缺失可能会影响 RpoS 在特定胁迫条件下的稳定性。对这些相互作用蛋白质的进一步研究正在进行中。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。