Función de las Metalotioneínas en el metabolismo celular de cobre

Copper is an essential micronutrient for life, it is required by a wide range of species, from bacteria to yeast, plant and humans. Copper essentiality is based in the capacity of the metal to be an intermediary in the transfer of electrons; thus, redox active enzymes that participate in physiological process like cellular respiration, iron transport and metabolism, and neurotransmitter synthesis utilize it. On the other hand, as a consequence of its redox properties, copper is potentially toxic to membranes, DNA and proteins through a Fenton´s type reaction. To prevent excess or deficit, living organisms have developed mechanisms that regulate copper in plasma and tissue stores, through specialized tissues such as intestinal mucosa and hepatic tissue. A number of molecules at a cellular level regulate the uptake, efflux, storage and utilization of the metal. The evidence indicates that copper handling is tightly regulated, thus, under normal conditions it is almost impossible to find a free copper ion inside the cell. Metallothioneins (MTs) are proteins involved in the regulation of intracellular copper metabolism. MTs are found in all eukaryots as well as some prokaryots; the structure is highly conserved across species. Physical-chemical properties allow MTs to coordinate the binding of multiple atoms of copper with a high affinity, and at the same time since these are kinetically labile they can be transferred to other molecules. Copper induces MT expression, thus intracellular MT content is related to an increase in cell resistance to copper toxicity. These properties suggest that MTs have an important role in cellular copper metabolism, storage and/or delivery of copper ions to specific cupro-enzymes. Since the role of MT has not been fully characterized, the goal of this doctoral thesis was to evaluate MT function in copper metabolism of cells maintained in sub, iso- and supra- physiological concentrations of copper. The results of this thesis indicate that in the absence of MT, cultured cell grown in low and normal concentration of copper have a decreased capacity to store copper; moreover they are more sensitive to an increment in intracellular copper, decreasing their viability. The increment in intracellular copper relative to wild cells is not explained by an increase in copper uptake, suggesting a difference in the efflux rate. The analysis of efflux of newly incorporated copper showed that in absence of MT, the retention of this copper is slightly lower; this may explain in part, the lower copper content observed in these cells. Finally, I have demonstrated that in the absence of MTs, the capacity of copper to induce gene expression of MT, SOD1 and Ccs1 its chaperone is lost. This observation could partially explain the increased vulnerability of MT null cells to lower intracellular copper concentrations. I propose as conclusions of this thesis that the presence of MT is important to maintain an appropriate regulation of intracellular copper homeostasis in cells grown at physiological copper concentrations and especially in response to extracellular copper excess. This response is dependent on the capacity of MT to store copper under physiological conditions and in response to excess, sequester intracellular copper in a safe form.