From the Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts 01605.
From the Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts 01605
J Biol Chem. 2019 Mar 29;294(13):4934-4945. doi: 10.1074/jbc.RA118.006316. Epub 2019 Feb 4.
Copper homeostasis in pathogenic bacteria is critical for cuproprotein assembly and virulence. However, biochemical analyses of these processes are challenging, which has prevented defining and quantifying the homeostatic interplay between Cu-sensing transcriptional regulators, chaperones, and sequestering molecules. The cytoplasm of contains a Cu-sensing transcriptional regulator, CueR, and two homologous metal chaperones, CopZ1 and CopZ2, forming a unique system for studying Cu homeostasis. We found here that both chaperones exchange Cu, albeit at a slow rate, reaching equilibrium after 3 h, a time much longer than duplication time. Therefore, they appeared as two separate cellular Cu pools. Although both chaperones transferred Cu to CueR , experiments indicated that CopZ1 metallates CueR, eliciting the translation of Cu efflux transporters involved in metal tolerance. Although this observation was consistent with the relative Cu affinities of the three proteins (CopZ1 < CueR < CopZ2), and analyses also indicated a stronger interaction between CopZ1 and CueR that was independent of Cu In contrast, CopZ2 function was defined by its distinctly high abundance during Cu stress. Under resting conditions, CopZ2 remained largely in its form. Metal stress quickly induced CopZ2 expression, and its form predominated, reaching levels commensurate with the cytoplasmic Cu levels. In summary, these results show that CopZ1 acts as chaperone delivering Cu to the CueR sensor, whereas CopZ2 functions as a fast-response Cu-sequestering storage protein. We propose that equivalent proteins likely play similar roles in most bacterial systems.
铜在病原细菌中的稳态对于铜蛋白的组装和毒力至关重要。然而，这些过程的生化分析具有挑战性，这阻止了定义和量化铜感应转录调节因子、伴侣和螯合分子之间的稳态相互作用。细胞质中含有铜感应转录调节因子 CueR 和两种同源金属伴侣 CopZ1 和 CopZ2，形成了研究铜稳态的独特系统。我们在这里发现，两种伴侣都可以交换 Cu，尽管速度较慢，在 3 小时后达到平衡，这一时间远远长于细胞分裂时间。因此，它们似乎是两个独立的细胞 Cu 池。尽管两种伴侣都将 Cu 转移给 CueR，但实验表明 CopZ1 使 CueR 金属化，引发涉及金属耐受性的 Cu 外排转运蛋白的翻译。尽管这一观察结果与三种蛋白质的相对 Cu 亲和力（CopZ1 < CueR < CopZ2）一致，但 和 分析还表明 CopZ1 与 CueR 之间存在更强的相互作用，而这种相互作用与 Cu 无关。相比之下，CopZ2 的功能是由其在 Cu 应激下的高丰度所定义的。在静止条件下，CopZ2 主要以其 形式存在。金属应激会迅速诱导 CopZ2 的表达，其 形式占主导地位，达到与细胞质 Cu 水平相当的水平。总之，这些结果表明 CopZ1 作为伴侣将 Cu 递送到 CueR 传感器，而 CopZ2 作为快速反应的 Cu 螯合储存蛋白发挥作用。我们提出，类似的蛋白可能在大多数细菌系统中发挥类似的作用。
