Intracellular distribution of Cu(I): De-regulation & exploitation in pathogen-control.

铜作为杀菌剂广泛应用于农化工业，先正达正在研究新的铜基配方，以提高效率和/或减少对环境的铜投入。幸运的是，先正达合成化学家生产的一种Cu(I)螯合剂化合物似乎可以解除对酿酒酵母中铜稳态的调节，并抑制几种致病真菌的生长。通过这种去调控，这种Cu(I)螯合剂有可能深入了解将这种金属运送到活细胞内目的地的途径。具体来说，初步数据表明，每种铜输送途径的铜来源并不相同。这是一个难得的机会，可以探索金属细胞生物学核心的基本问题，同时解决与农用化学品公司直接相关的问题。先正达公司想要了解Cu(I)螯合剂的生物化学基础，如果它要被商业化，纽卡斯尔大学的“细胞中的金属小组”渴望使用Cu(I)螯合剂来探索铜是如何在细胞内正确定位的。铜是细胞色素氧化酶、超氧化物歧化酶1和（在植物中）质体青素等酶的必需物质。有些金属，尤其是铜，倾向于与蛋白质形成比其他金属紧密得多的复合物。细胞必须保持极低的缓冲细胞质铜浓度，以尽量减少蛋白质的错误种群，这些蛋白质需要竞争力较弱的金属。铜也必须严格控制，因为它倾向于参与氧化还原化学，如芬顿反应，产生致命的羟基自由基。为了避免铜在细胞质中释放，它需要在动力学控制下的蛋白质来供应铜，这意味着铜通过特定的“铜金属伴侣”被运送到正确的目的地。金属通过一系列的配体交换反应从铜金属伴侣体传递到它们的伴侣体。在大多数真核细胞中，包括真菌，它们包括线粒体中细胞色素氧化酶的铜金属伴侣蛋白，细胞质中超氧化物歧化酶1的铜金属伴侣蛋白，以及反式高尔基网络的铜金属伴侣蛋白。然而，目前尚不清楚铜金属伴侣本身从哪里获得铜，也不清楚铜如何优先到达这些不同的细胞目的地，特别是在铜供应短缺的情况下。这些都是关于所有生物体内铜稳态的基本未知数；植物，真菌，细菌和动物，包括人类。一个有趣的假设是，细胞色素氧化酶的铜伴侣可以获得铜蛋白周转时释放的铜，而SOD1和反式高尔基网络的铜伴侣主要可以获得新进口的铜。这将确保随着铜水平的下降，金属离子主要被输送到最重要的细胞内目的地，即细胞色素氧化酶。用先正达化学家生产的Cu(I)螯合剂处理过的真菌细胞似乎可以通过打开金属硫蛋白Cup1-1和Cup1-2基因的表达来检测细胞内的高铜浓度，这些基因的产物可以清除多余的铜。然而，处理后的细胞同时表现出与到达细胞色素氧化酶的铜不足一致的表型。该方案的一个目标是测量各自的铜酶活性，并量化到达不同目的地的铜的数量。这将确定不同的铜输送途径是否有不同的铜来源。

Copper is widely used in the agrochemical industry as a fungicide and Syngenta are investigating new copper based formulations with greater efficacy and/or requiring lower environmental copper input. Fortuitously, one of the Cu(I)-chelator compounds produced by synthetic chemists at Syngenta appears to de-regulate copper homeostasis in S. cereviasiae, as well as inhibiting growth of several pathogenic fungi. Via such de-regulation, this Cu(I)-chelator has the potential to provide insight into the pathways that deliver this metal to its destinations inside living cells. Specifically, preliminary data suggest that the sources of copper for each copper delivery pathway are not identical. This is a rare opportunity to explore fundamental questions at the heart of the cell biology of metals, while simultaneously tackling issues of direct relevance to an agrochemical company. Syngenta would like to understand the biochemical basis via which the Cu(I)-chelator acts if it is to be pursued commercially, and the 'Metals in Cells Group' at Newcastle University are eager to use the Cu(I)-chelator to explore how copper is correctly targeted inside cells. Copper is essential for enzymes such as cytochrome oxidase, superoxide dismutase 1, and (in plants) plastocyanin. Some metals, especially copper, have a tendency to form much tighter complexes with proteins than do others. Cells must maintain exceptionally low buffered cytosolic concentrations of copper in order to minimise the mis-population of proteins that require the less competitive metals. Copper must also be tightly controlled due to its propensity to engage in redox chemistry such as the Fenton reaction which generates deadly hydroxyl radicals. To avoid copper-release in the cytosol it is supplied to copper requiring proteins under kinetic control, meaning that copper is delivered to its correct destinations by specific 'copper metallochaperones'. The metal is passed from the copper metallochaperones to their partners by sequences of ligand-exchange reactions. In most eukaryotic cells, including fungi, these include copper metallochaperones for cytochrome oxidase in mitochondria, one for superoxide dismutase 1 in the cytosol and finally one for the trans-Golgi network. However, it is unclear where the copper metallochaperones themselves obtain copper and it is also unclear how the routing of copper to these different cellular destinations is prioritised, especially when copper is in short supply. These are fundamental unknowns in regard to copper homeostasis in all organisms; plants, fungi, bacteria and animals including humans. An intriguing hypothesis is that the copper chaperones for cytochrome oxidase have access to copper released at cuproprotein turnover, while those for SOD1 and for the trans-Golgi network predominantly have access to newly imported copper. This would ensure that as copper levels decline the metal ions become predominantly routed to a most vital intracellular destination, namely cytochrome oxidase. Fungal cells treated with the Cu(I)-chelator generated by Syngenta chemists appear to detect high intracellular copper concentrations by switching on expression of (metallothionein Cup1-1 and Cup1-2) genes whose products mop up surplus copper. However, the treated cells concurrently exhibit phenotypes consistent with insufficient copper reaching cytochrome oxidase. A goal of this programme is to measure the respective cupro-enzyme activities and quantify the amounts of copper reaching the different destinations. This will establish if there are distinct sources of copper for the different copper delivery pathways.