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。异常蛋白被这些蛋白酶降解。除了这种蛋白质质量控制作用外，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非常重要，细胞通过多种方式调节RpoS的积累，包括通过翻译的小RNA激活剂和受调节的蛋白质水解在翻译水平上。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；稳定模式一直是个谜，直到我们实验室最近的研究。对RpoS降解调节因子的基因筛选发现了一种以前未被表征的小蛋白质，现在被命名为IraP。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，磷酸盐饥饿通过小分子ppGpp水平的增加来感知，并且ppGpp正调控iraP启动子。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。RssB的结构和功能已经通过一系列方法得到了进一步的研究：1)通过遗传选择来鉴定抗特定抗接头的RssB突变；2)通过细菌双杂交系统来研究RssB的野生型和突变型衍生物及其结构域与抗接头和系统其他组分的相互作用；3)在体外合作研究RpoS与突变蛋白和抗接头的降解。这些研究结果表明，IraP和IraD都与RssB的n端结构域相互作用。RssB的这个结构域是广泛反应调控家族的成员。尽管两个反适配器都与这个保守域交互，但它们的交互方式不同。因此，RssB中终止与IraP相互作用的突变保留了与IraD的相互作用。对IraP结构的合作研究表明，它是一种独特的蛋白质，与B-Zip二聚体相似。IraP与RssB的相互作用可能模仿RpoS与RssB的相互作用。另一方面，IraD似乎与RssB的非活性形式相互作用，可能阻断激活。IraM与RssB的c端结构域相互作用；该结构域与失活的PP2C磷酸酶结构域具有同源性。一类抗接头突变激活RssB，绕过磷酸化的刺激作用。这些突变体为RssB如何工作以及调节蛋白如何破坏构成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。其他抗接头可能存在，基于各种结果，包括观察到转录调节剂AppY在缺乏所有三种已知抗接头的情况下稳定RpoS。其他稳定RpoS的突变通过已知的抗适配器起作用。例如，缺失全局抑制因子H-NS也能稳定RpoS。IraD和IraM的表达受到H-NS的抑制，在缺乏这两种抗接子的情况下，H-NS的稳定作用大部分但不是全部丧失。作为中枢代谢的一个组成部分，aceE的突变也会导致RpoS的稳定，这依赖于IraP和IraD。aceE突变体诱导了iraP和iraD的转录水平，显然依赖于信号分子ppGpp。细菌双杂交系统也被用于筛选大肠杆菌蛋白库中与RssB相互作用的其他蛋白。相互作用的蛋白质可能是额外的抗适配器，让我们了解这个调节家族有多广泛；或者，我们可能会发现RssB的其他底物，这些底物以前被认为是RpoS特异性的。许多有趣的调节蛋白已经被确定并发现影响RpoS的周转，因此可能作为抗适配器或竞争底物，这两者都是重要的兴趣。其中的AnmK已经被详细研究过了。AnmK是一种参与肽聚糖循环的酶。除了在细菌双杂交实验中与RssB相互作用外，它还在下拉实验中相互作用。AnmK的过表达可以稳定RpoS，这与它作为竞争性抑制剂或抗适配器的作用一致；在特定的应激条件下，anmK的缺失可能会影响RpoS的稳定性。计划进一步研究这些相互作用的蛋白质。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 proteins are degraded by these proteases. In addition to this protein 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 recent work from our lab. A genetic screen for regulators of RpoS degradation led to discovery of a small, previously uncharacterized protein, now named IraP. 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 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 on the structure of IraP reveal that is a unique protein with similarity to B-Zip dimers. The interaction of IraP with RssB may mimic how RpoS interacts with RssB. IraD, on the other hand, appears to interact with the inactive form of RssB, possibly blocking activation. 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 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. aceE mutants have induced levels of iraP and iraD transcription, apparently dependent on the signaling molecule ppGpp. The bacterial two-hybrid system has also been used to screen a library of E. coli proteins for other proteins that interact with RssB. Interacting proteins may be additional anti-adaptors, giving us a sense of how broad this family of regulators is; alternatively, we may find other substrates for RssB, which has previously been believed to be specific to RpoS. 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 planned. Overall, our proteolysis studies continue to provide novel insights into regulatory mechanisms used by bacteria.