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

铜在农化工业中被广泛用作杀菌剂，先正达正在研究更有效和/或对环境铜投入更低的新铜基配方。幸运的是，先正达合成化学家生产的一种铜(I)-螯合剂化合物似乎破坏了酿酒酵母中铜的动态平衡，并抑制了几种病原真菌的生长。通过这种去调节，这种铜(I)-螯合剂有可能提供洞察的途径，将这种金属运送到活细胞内的目的地。具体地说，初步数据表明，每条铜交割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。如果要进行商业研究，先正达希望了解铜(I)-螯合剂的生化基础，纽卡斯尔大学的“细胞中的金属”渴望使用铜(I)-螯合剂来探索如何在细胞内正确定位铜。铜是细胞色素氧化酶、超氧化物歧化酶1和(在植物中)叶绿体蛋白等酶的必需物质。一些金属，尤其是铜，往往比其他金属与蛋白质形成更紧密的络合物。细胞必须保持极低的缓冲胞浆铜浓度，以最大限度地减少蛋白质的错配，这些蛋白质需要竞争力较低的金属。铜还必须受到严格控制，因为它倾向于参与氧化还原化学，如芬顿反应，它会产生致命的羟基自由基。为了避免铜在胞质中释放，它在动力学控制下被提供给需要铜的蛋白质，这意味着铜通过特定的“铜金属配位络合物”被输送到正确的目的地。通过一系列的配体交换反应，金属从铜金属配位络合物传递到它们的伙伴。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配位体，胞浆中超氧化物歧化酶1的铜金属配位体，最后是反式高尔基体网络的铜金属配位体。然而，目前尚不清楚铜金属配位体本身从哪里获得铜，也不清楚将铜输送到这些不同的蜂窝目的地的优先顺序，特别是在铜供应短缺的情况下。这些都是关于所有生物体中铜的动态平衡的基本未知因素；植物、真菌、细菌和动物包括人类。一个耐人寻味的假设是，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，金属离子主要被传递到细胞内最重要的目的地，即细胞色素氧化酶。用先正达化学家生产的铜(I)螯合剂处理的真菌细胞似乎通过启动(金属硫蛋白Cup1-1和Cup1-2)基因的表达来检测到细胞内高浓度的铜，这些基因的产物可以吸收多余的铜。然而，处理的细胞同时表现出与铜不足的细胞色素氧化酶一致的表型。该方案的一个目标是衡量各自的铜酶活性，并量化到达不同目的地的铜的数量。这将确定不同的铜运输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.