Functional analysis of the Human Copper Transporters using yeast as a host

In this study we present the investigation of pore formation of human copper transporter (hCtr) proteins in live cancer cells using the number and brightness (N&B) analysis. Copper is one of the most important metals for cell metabolism, considered a crucial cofactor for several processes in eukaryotic cells such as respiration, detoxification and secretion1. Any imbalance on copper homeostasis can lead to severe diseases. In this homeostasis process, the hCtrs are the proteins responsible for copper uptake by the cell. It is known that hCtrs can exists as monomers, dimers or trimers and that the trimers seem to be responsible for the functional pore formation2. The N&B analysis is a potent statistical method based on fluorescent intensity fluctuations of single pixels recorded using a photon-counting confocal microscopy3. Our results indicate that after stimulation with a cooper chelator the hCtrs aggregate. We were able to map the aggregation of hCtr monomers by using N&B. In order to understand the pore formation in cancer cells, we will treat the cells with different concentrations of copper. Copper (Cu) is a crucial ion for both eukaryotic and prokaryotic organisms. It serves as a co-factor of metalloenzymes that participate in important cellular processes involved in growth, development and physiology of the organisms. Although its importance in maintaining cell health, high level of this ion is extremely toxic (Wang et al., 2011). Therefore, cells possess tight regulated systems to preserve copper homeostasis. Indeed, at high Cu levels, the Copper Transporter 1 (Ctr1) is endocytosed, a process already verified in yeast and human cells (Liu et al., 2007; Maryon et al., 2013). Besides these new advances, the molecular mechanisms that are behind the intracellular trafficking of the hCtr1 protein are still poor understood. So, to better understand this process, an heterologous expression system was created using the yeast Saccharomyces cerevisiae as host (Pereira et al., 2016). Human CTR1and CTR2optimized genes tagged with GFP were cloned into pYPKpw plasmid and transformed into a S. cerevisiaestrain disrupted for copper transporters. Importantly, phenotypic assays demonstrated that human Ctr1 complemented the yeast ctr-mutant strain for the ability to grow in a medium containing non-fermentable carbon sources. Moreover, hCtr1 and hCtr2 were localized at the plasma membrane and intracellularly. Data will be presented regarding the expression of hCTRs in different conditionsFCT (PD/BD/135208/2017); Centro de Biologia Molecular e Ambiental (CBMA