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

铜作为杀真菌剂广泛用于农业化学工业，先正达正在研究新的铜基配方，其功效更高和/或需要更低的环境铜投入。幸运的是，先正达的合成化学家生产的一种Cu（I）螯合剂化合物似乎可以解除S. cereviasiae，以及抑制几种病原真菌的生长。通过这种去调节，这种Cu（I）螯合剂有可能深入了解将这种金属传递到活细胞内目的地的途径。具体而言，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，同时解决与农业化学公司直接相关的问题。先正达希望了解Cu（I）螯合剂的生物化学基础，如果它要在商业上追求的话，纽卡斯尔大学的“细胞中的金属组"渴望使用Cu（I）螯合剂来探索铜如何在细胞内正确靶向。铜对细胞色素氧化酶、超氧化物歧化酶1和（植物中的）质体蓝素等酶是必需的。有些金属，特别是铜，与蛋白质形成的络合物比其他金属更紧密。细胞必须维持极低的缓冲细胞溶质铜浓度，以最大限度地减少需要竞争性较低的金属的蛋白质的错误群体。铜也必须严格控制，因为它倾向于参与氧化还原化学，如产生致命羟基自由基的芬顿反应。为了避免铜在胞质溶胶中释放，它被提供给动力学控制下的铜需要蛋白质，这意味着铜通过特定的“铜金属伴侣”被递送到其正确的目的地。金属通过一系列配体交换反应从铜金属伴侣传递到它们的伴侣。在大多数真核细胞，包括真菌，这些包括铜金属伴侣细胞色素氧化酶在线粒体中，一个超氧化物歧化酶1在胞质溶胶中，最后一个为trans-Golgi网络。然而，目前尚不清楚铜金属伴侣本身从何处获得铜，也不清楚如何优先考虑将铜路由到这些不同的细胞目的地，特别是当铜供应短缺时。这些是关于所有生物体中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，铜分子伴侣细胞色素氧化酶有机会获得铜蛋白周转释放的铜，而那些SOD 1和trans-Golgi网络主要有机会获得新进口的铜。这将确保随着铜水平下降，金属离子主要被路由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）螯合剂处理的真菌细胞似乎通过开启（金属硫蛋白Cup 1 -1和Cup 1 -2）基因的表达来检测细胞内高浓度的铜，这些基因的产物会清除多余的铜。然而，经处理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相应的铜酶活性，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.