Clp dependent proteolysis and the role of the McsB arginine kinase in the protein quality control network of low GC Gram+ bacteria

蛋白质通常在核糖体中产生，在个体寿命（可能在几分钟、几小时甚至几天之间）之后，它们在大多数情况下由于功能缺陷而被蛋白质水解机器破坏。Clp蛋白酶是枯草芽孢杆菌中最重要的细胞内蛋白水解系统，ClpP蛋白酶是大量蛋白周转的主要决定因素。atp依赖性ClpCP蛋白酶主要负责枯草芽孢杆菌在葡萄糖饥饿期间降解热损伤蛋白、热敏转录抑制因子CtsR以及生物合成酶。ClpXP似乎对在标准生长条件下通过水解ssra标记的蛋白质来挽救停滞的核糖体以及“二次诱导”的氧化应激调节因子（如枯草芽孢杆菌MgsR和Spx）很重要。最近，我们和其他人描述了枯草芽孢杆菌中精氨酸激酶McsB对蛋白质精氨酸的磷酸化作用，该蛋白作为ClpCP蛋白酶的适配蛋白。精氨酸磷酸化似乎在革兰氏+细菌中起着至关重要的作用，但迄今为止被低估了。揭示精氨酸磷酸化、蛋白质寿命和蛋白酶活性之间的联系是低GC Gram+细菌中atp依赖性蛋白水解研究的一个新的和引人注目的策略。本拨款提案探讨：1)；mcsB在体内识别和降解蛋白质质量控制调节因子CtsR的依赖机制，2.)在不同应激和饥饿条件下使用特定诱变突变体介导的mcsB介导的精氨酸磷酸化的作用，3.缺乏mcsB的细菌（如乳酸菌或链球菌）中可能存在磷酸甘氨酸蛋白，以及与L. lactis ClpC atp酶的互补研究将有助于揭示ClpCL对mcsB的严格要求。枯草芽孢杆菌中CtsR的乳酸依赖性降解，4。在非糖异生生长条件下，特定的“无用”和“失业”蛋白质（如本质上不活性的谷氨酸脱氢酶GudB和糖异生GapB）的clp依赖性降解，5. CtsR和一般应激调节因子SigmaB之间的联系（RsbW-SigmaB复合物的降解概况，对应激期间MgsR和Spx氧化调节因子的命运和McsB精氨酸激酶的影响的详细分析）。MazEF毒素-抗毒素系统、SigmaB和Clp蛋白水解之间的推测关系)。

Proteins are usually born at the ribosome and after an individual lifespan, which can vary between minutes, hours or even days they are destroyed by proteolytic machines in most cases due to functional deficiency. Clp proteases represent the most important intracellular proteolytic systems with the protease ClpP as the major determinant of bulk protein turnover in Bacillus subtilis. The ATP-dependent ClpCP protease is mainly responsible for the degradation of heat-damaged proteins, the heat-sensitive transcriptional repressor CtsR as well as biosynthetic enzymes during glucose starvation in B. subtilis. ClpXP appears to be important for the rescue of stalled ribosomes under standard growth conditions by proteolysis of ssrA-tagged proteins as well as for "secondarily-induced" oxidative stress regulators like B. subtilis MgsR and Spx. Recently, we and others described a protein arginine phosphorylation exerted by the arginine kinase McsB in B. subtilis, which acts as an adaptor protein for the ClpCP protease. Arginine phosphorylation seems to play a crucial and so far underestimated role in Gram+ bacteria. The revelation of a link between arginine phosphorylation, lifespan of proteins and protease activity is a new and compelling strategy within the ATP-dependent proteolysis research in low GC Gram+ bacteria. This grant proposal explores the: 1.) McsB-dependent mechanism of in vivo recognition and degradation of the protein quality control regulator CtsR, 2.) role of McsB-mediated arginine phosphorylation during different stress and starvation conditions using specific trap mutants, 3.) putative existence of phosphoarginine proteins in mcsB lacking bacteria such as Lacto- or Streptococci as well as complementation studies with the L. lactis ClpC ATPase will shed some light on a strict requirement of McsB for a ClpCL.lactis-dependent degradation of CtsR in B. subtilis, 4.) Clp-dependent degradation of selected "useless" and "unemployed" proteins such as the intrinsically inactive glutamate dehydrogenase GudB and gluconeogenetic GapB under non-gluconeogenetic growth conditions, 5.) connections between CtsR and the generell stress regulator SigmaB (degradation profile of the RsbW-SigmaB complex, detailed analyses to the fate of the MgsR and Spx oxidative regulators during stress and the influence of McsB arginine kinase, a putative relation between the MazEF toxin-antitoxin system, SigmaB and Clp proteolysis).