Proteolysis and Regulation of Bacterial Cell Growth Control

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依赖的细胞质蛋白酶，类似于真核蛋白酶体，含有atp酶结构域或亚基，识别底物并展开它们，将它们送入蛋白水解结构域。细菌含有多种atp依赖性蛋白酶；其中五种在大肠杆菌中被鉴定出来。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，蛋白酶还能降解天然不稳定的蛋白质；对于这些蛋白质，降解可能起着重要的生物学作用。这类蛋白酶底物分为两大类：一类是总是被降解的蛋白质，因此对其丰度的调节主要取决于合成的变化；另一类是蛋白质水解受到调节的蛋白质。在所有情况下，确定蛋白酶如何识别底物以及识别如何受到生长条件的影响对于理解调控如何进行非常重要。我们实验室在过去的研究中发现，Lon atp依赖的蛋白酶通过降解RcsA和SulA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，我们的重点一直放在RpoS sigma因子的调控降解上，RpoS sigma因子是RNA聚合酶的一个亚基，指导聚合酶到特定的启动子。RpoS对于细胞切换到静止或应激反应基因表达程序是重要的。细胞通过多种方式调节RpoS的积累，包括在翻译水平上通过翻译的小RNA激活剂和受调节的蛋白质水解。我们一直在研究这种蛋白质水解，这是大肠杆菌中调节蛋白质周转的最好例子之一。在活性生长过程中，RpoS会迅速降解，这一过程需要能量依赖性ClpXP蛋白酶和适配器蛋白RssB（一种可磷酸化的蛋白，可将RpoS呈现给蛋白酶）。RpoS经过各种应激或饥饿处理后趋于稳定；这种稳定模式一直是个谜，直到我们实验室的工作发现了一种小的、以前未被表征的蛋白质，现在被命名为IraP（磷酸盐饥饿后RssB活性抑制剂）。iraP突变体破坏了磷酸盐饥饿后RpoS的稳定性。在纯化的体外系统中，IraP阻断RpoS的转换，并直接与RssB相互作用。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启动子已经成为ppGpp如何积极调节启动子的最佳例子。在纯化的体外系统中，另外两种小蛋白IraM和IraD也能稳定RpoS。这些蛋白质在预测结构上与IraP不相似。IraM是对镁缺乏的反应，依赖于PhoP和PhoQ调节因子；IraD在DNA损伤后很重要。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抗适配器的表达来调节RpoS的周转。在与Sue Wickner （NCI）继续合作研究RssB及其抗接子的结构和功能时，我们使用体内遗传学和体外重构来了解抗接子和接子蛋白是如何工作的。与A. Deaconescu （Brown University）的合作已经获得了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，并充分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atp酶亚基。ClpX的n端结构域，已知与其他一些接头和底物相互作用，与RssB的c端相互作用。对这一系统的进一步剖析有助于我们深入了解这一过程是如何在细胞内实现平衡的。根据各种结果，可能存在其他反适配器。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这个过程。在饥饿期间，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的主动失活，依赖于RpoS可能增加RssB合成的反馈回路；我们发现RpoS的另一个调节因子，Crl，在饥饿后的恢复中起着关键和意想不到的作用。IraP的突变分析表明，该抗接头的c端对于快速恢复至关重要，这表明它调节了IraP与RssB的相互作用；这正在进行体外试验。目前也正在调查对rpo降解的其他管制投入。H. Tabor的实验室（NIDDK，于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，我们确认并扩展了这项工作。我们的工作表明，缺乏多胺可能会导致RpoS的快速共翻译降解。在另一个项目中，筛选RpoS负调控的质粒文库，确定了核糖代谢在RpoS负调控中的作用，并鉴定了一种可抵消RpoS翻译激活的转录因子。总的来说，我们的蛋白质水解研究继续为细菌使用的调节机制提供新的见解。

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.