Molecular mechanisms of heavy metal homeostasis in flies and humans

Abstract

Heavy metal ions, like Zn2+ and Cu+/2+, are important for the structure and function of many cellular proteins. If an organism is supplied with inadequate amounts of these heavy metals, it is suffering from severe deficiency syndromes, including growth retardation and a compromised immune function. On the other hand, accumulation of these ions is toxic for the organism. For these reasons every organism has to find a way to keep the concentration of essential heavy metals at an optimal level. To achieve this, the expression of metal ion importers, exporters and metallothioneins is tightly regulated. Metallothioneins are small, cysteine-rich proteins that bind metal ions and thereby detoxify the cell. In most higher organisms the metal-responsive transcription factor-1 (MTF-1) plays a central role in protecting the cell from the adverse effects generated by heavy metal ions as it activates the expression of metallothioneins and other cytoprotective proteins. In this work we characterize the function of a cluster of cysteines, which is conserved in all vertebrate homologs of MTF-1. If these cysteines are mutated, MTF-1 is severely impaired in activating its target genes under normal and especially under metal-induced conditions, but is still able to bind DNA. We can show that these cysteines mediate homodimerization of MTF-1. The dimerization of MTF-1 cannot be boosted by treating cells with zinc and rather seems to be constitutive. We suggest that only the MTF-1 dimer is able to recruit proteins of the general transcriptional machinery to the promoter region of MTF-1 target genes. The interaction of MTF-1 and p300 will be discussed in the second part of this work. p300 is a histone acetyltransferase and is part of the general transcriptional machinery. Our data demonstrate that MTF-1 is interacting with p300 and is acetylated by p300 in a cell free test system. The third part deals with the characterization of the functional domains of the Drosophila MTF-1 homolog (dMTF-1) and preliminary results will be shown. The aim is to identify the protein segments that mediate transcriptional activity, regulate subcellular localization of the protein, and sense heavy metal concentrations. The experiments and results presented here will elucidate regulatory mechanisms of dMTF-1 and allow a comparison with mammalian MTF-1, whose domain structure has been studied intensively over the past years. In the last part we study a Drosophila melanogaster mutant, which lacks the Atox1 gene. Atox1 is a so called chaperone which transports copper ions from the plasma membrane to ATP7, a copper exporter. ATP7 delivers copper to proteins located in the trans-Golgi network. From there, copper is exported out of the cell via exocytosis. This route of transport is particularly important in enterocytes, where copper is taken up from the food and is provided to the organism. If Atox1 is missing, copper accumulates in the gut cells, with copper deficiency in the rest of the body as a consequence. Larvae that do not express Atox1 cannot develop under copper starvation conditions that pose no problem for wild type flies. On the other hand, these flies are resistant to the anticancer drug cisplatin, a compound whose uptake and distribution in the body is mediated by copper transport mechanisms.