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，并且在个体寿命之后，其可以在几分钟、几小时甚至几天之间变化，在大多数情况下，由于功能缺陷，它们被蛋白水解机器破坏。Clp蛋白酶代表最重要的细胞内蛋白水解系统，其中蛋白酶ClpP是枯草芽孢杆菌中大量蛋白质周转的主要决定因素。在B中，ATP依赖的ClpCP蛋白酶主要负责降解热损伤蛋白、热敏转录抑制因子CtsR以及葡萄糖饥饿期间的生物合成酶。枯草杆菌。ClpXP对于在标准生长条件下通过ssrA标记的蛋白质的蛋白水解来拯救停滞的核糖体以及对于“二次诱导的”氧化应激调节剂如B来说似乎是重要的。subtilis MgsR和Spx.最近，我们和其他人描述了蛋白质精氨酸磷酸化施加的精氨酸激酶Mcs B在B。枯草芽孢杆菌，其充当ClpCP蛋白酶的衔接蛋白。精氨酸磷酸化似乎在革兰氏阳性菌中起着至关重要但迄今被低估的作用。精氨酸磷酸化，蛋白质的寿命和蛋白酶活性之间的联系的启示是一个新的和令人信服的策略内的ATP依赖性蛋白水解研究在低GC革兰氏阳性菌。本研究旨在探讨：1）。蛋白质质量控制调节剂CtsR的体内识别和降解的McsB依赖性机制，2.）在不同的压力和饥饿条件下使用特定的陷阱突变体，3.）在缺乏mcsB的细菌如乳球菌或链球菌中存在磷酸精氨酸蛋白，以及与L.乳酸菌ClpC ATP酶将揭示B中CtsR的ClpCL依赖性降解对Mcs B的严格要求。subtilis，4.）选择的“无用的”和“失业的”蛋白质，如固有失活的谷氨酸脱氢酶GudB和非遗传性生长条件下的遗传性GapB的Clp依赖性降解，5.）CtsR和generell应激调节因子SigmaB之间的联系（RsbW-SigmaB复合物的降解谱，应激期间MgsR和Spx氧化调节因子的命运的详细分析以及McsB精氨酸激酶的影响，MazEF毒素-抗毒素系统、SigmaB和Clp蛋白水解之间的推定关系）。

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).