ATP-dependent intracellular proteolysis is essential for all living organisms. ClpP, the proteolytic subunit of the ATP-dependent Clp proteases, shares 56% protein identity between B. subtilis and man. The aim of this study was to verify, whether human ClpP (HClpP) is able to substitute the bacterial pendant, BClpP, irrespectively of the huge evolutionary distance. For this reason hclpP was expressed from the natural B. subtilis promoters at the original chromosomal site. Growth at 37 °C as well as sporulation in the presence of hclpP depict an intermediate phenotype between wild type and clpP mutant suggesting a partial functional substitution of BClpP by HClpP. Northern as well as Western blot analyses show a similar induction pattern of both, bclpP and hclpP during heat stress on the mRNA as well as on the protein levels. Co-immunoprecipitation experiments imply specific interaction of HClpP with bacterial ClpC, ClpX and ClpE during control as well as heat stress conditions. Radioactive pulse-chase labeling and immunoprecipitation revealed that a ClpXP substrate, the short-living regulatory protein MgsR, is degraded by HClpP, although with an extremely slower rate in comparison to BClpP. The occurrence of an exceptional thickened cell wall of a clpP mutant can be almost fully reversed by the complementation with HClpP. The utilization of the HClpP expressing strain as a test system for new biological or synthetic active substances targeting BClpP is discussed.

ATP依赖的细胞内蛋白水解对于所有活生物都是必不可少的。ClpP是ATP依赖的Clp蛋白酶的蛋白水解亚基，在枯草芽孢杆菌和人之间具有56％的蛋白质同一性。这项研究的目的是验证人类ClpP（HClpP）是否能够替代细菌垂饰BClpP，而不论其进化距离如何。因此，hclpP从天然枯草芽孢杆菌启动子在原始染色体位点表达。在37°C的生长以及hclpP存在下的孢子形成描述了野生型和clpP之间的中间表型突变体提示BClpP被HClpP部分取代。Northern和Western印迹分析显示，在mRNA和蛋白质水平上的热应激期间，bclpP和hclpP的诱导方式相似。免疫共沉淀实验表明，在控制以及热应激条件下，HClpP与细菌ClpC，ClpX和ClpE发生特异性相互作用。放射性脉冲追踪标记和免疫沉淀法显示，HClpP可降解ClpXP底物，即短暂的调节蛋白MgsR，但其降解速度比BClpP慢得多。clpP异常增厚的细胞壁的发生突变体可以通过与HClpP互补而几乎完全逆转。讨论了表达HClpP的菌株作为针对BClpP的新型生物或合成活性物质的测试系统的应用。