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的缺失废除了磷酸盐饥饿后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，磷酸盐饥饿通过小分子ppGpp水平的增加来感知，并且iraP启动子受到应激报警蛋白ppGpp的正调控。另外两种小蛋白质也在纯化的体外系统中稳定RpoS，它们是CNOM和CNOD。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。这导致了对CNOP的详细分析，包括与X合作的晶体结构。纪与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。我们正在进一步定义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。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，RpoS的另一个调节器，Crl，在从饥饿中恢复起着关键的和意想不到的作用，和C-末端的RpoS是必要的，这种快速恢复。这些结果支持了一个复杂而精确平衡的系统，该系统允许细胞在某些条件下使用RpoS一般应激反应，但在不需要时将其丢弃。研究RpoS是如何被调控的继续产生对转录后调控机制的新见解。例如，H.塔博尔的实验室（NIDDK）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，我们已经证实了这项工作，并证明了RpoS在不存在多胺的情况下可能会非常迅速地降解。然而，这一过程可能同时发生，整合不寻常的密码子使用，导致对蛋白水解的超敏感性;我们正在测试这个模型。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。

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.