Lilge Lars, Reder Alexander, Tippmann Frank, Morgenroth Friedrich, Grohmann Janice, Becher Dörte, Riedel Katharina, Völker Uwe, Hecker Michael, Gerth Ulf
Institute of Microbiology, University of Greifswald, Greifswald, Germany.
Interfaculty Institute for Genetics and Functional Genomics, University of Greifswald, Greifswald, Germany.
Front Microbiol. 2020 May 12;11:900. doi: 10.3389/fmicb.2020.00900. eCollection 2020.
Regulated ATP-dependent proteolysis is a common feature of developmental processes and plays also a crucial role during environmental perturbations such as stress and starvation. The MgsR regulator controls a subregulon within the stress- and stationary phase σ regulon. After ethanol exposition and a short time-window of activity, MgsR is ClpXP-dependently degraded with a half-life of approximately 6 min. Surprisingly, a protein interaction analysis with MgsR revealed an association with the McsB arginine kinase and an degradation assay confirmed a strong impact of McsB on MgsR degradation. phosphorylation experiments with arginine (R) by lysine (K) substitutions in McsB and its activator McsA unraveled all R residues, which are essentially needed for the arginine kinase reaction. Subsequently, site directed mutagenesis of the MgsR substrate was used to substitute all arginine residues with glutamate (R-E) to mimic arginine phosphorylation and to test their influence on MgsR degradation . It turned out, that especially the R33E and R94/95E residues (RRPI motif), the latter are adjacently located to the two redox-sensitive cysteines in a 3D model, have the potential to accelerate MgsR degradation. These results imply that selective arginine phosphorylation may have favorable effects for Clp dependent degradation of short-living regulatory proteins. We speculate that in addition to its kinase activity and adaptor function for the ClpC ATPase, McsB might also serve as a proteolytic adaptor for the ClpX ATPase in the degradation mechanism of MgsR.
受调控的ATP依赖性蛋白水解是发育过程的一个共同特征，在诸如应激和饥饿等环境扰动期间也起着关键作用。MgsR调节因子控制应激和稳定期σ调节子内的一个亚调节子。在乙醇暴露和短时间的活性窗口后，MgsR以大约6分钟的半衰期被ClpXP依赖性降解。令人惊讶的是，与MgsR的蛋白质相互作用分析揭示了它与McsB精氨酸激酶的关联，并且降解试验证实了McsB对MgsR降解有强烈影响。通过在McsB及其激活剂McsA中用赖氨酸（K）替代精氨酸（R）进行磷酸化实验，揭示了精氨酸激酶反应基本所需的所有R残基。随后，对MgsR底物进行定点诱变，用谷氨酸（R-E）替代所有精氨酸残基以模拟精氨酸磷酸化，并测试它们对MgsR降解的影响。结果表明，特别是R33E和R94/95E残基（RRPI基序），后者在三维模型中与两个氧化还原敏感的半胱氨酸相邻，有可能加速MgsR降解。这些结果表明，选择性精氨酸磷酸化可能对短命调节蛋白的Clp依赖性降解有有利影响。我们推测，除了其激酶活性和作为ClpC ATP酶的衔接子功能外，McsB在MgsR的降解机制中也可能作为ClpX ATP酶的蛋白水解衔接子。
