Molecular Evolution of Unicellular Eukaryote MetallothioneinsTandem Repetition of Coordinating Domains

Resumen

Metallothioneins (MTs) are a superfamily of ubiquitous and small cysteine rich metalloproteins present in all eukaryotes and some prokaryotes organisms, that exhibit preferences to coordinate divalent or monovalent heavy metal ions such as Zn(II), Cd(II) or Cu(I). This ability is used by MTs to participate in toxic metal detoxification, metal ion homeostasis and protection against oxidative stress. Features of MT sequences, as well as their structural arrangements, are crucial to determine their metal-abilities and the related functions. In this PhD thesis, we aimed at expanding the knowledge of some peculiar MTs, which are characterized by their unusual long sequence, their Cys-distribution and their modular folding. Thus, initially, the five Tetrahymena thermophila MTs (MTT1 to MTT5), which represent one of the longest MTs reported so far, were characterized to decipher their divalent or monovalent metal preferences. The modular structure of MTT1, MTT3 and MTT5 isoforms, which contain high occurrence of doublets and triplets confer a clear Zn-character to these MTs. Contrarily, MTT2 and MTT4 in which no modular structures, nor Cys doublets and triplets, are present, exhibit a specific Cu-thionein character. The gradation of metal-binding preferences from Zn-thionein to Cu-thionein shown by each MT, as well as the sequence features, constitute an important information source for MT evolutionary studies. Later, two other MTs from the human pathogenic opportunistic fungus Cryptococcus neoformans (CnMT1 and CnMT2) were characterized. Their unusual long sequence, not known in fungal MTs so far, together with their high Cys content, explained their extraordinary Cu detoxification capacity. The modular structure of CnMT1 and CnMT2, with three and five 7-Cys containing regions, respectively, separated by cysteine-free spacers, leads to their folding into Cu5 clusters upon Cu coordination, which enhances their detoxification capacity. This extraordinary ability becomes decisive for C. neoformans virulence in order to avoid the copper toxicity induced by macrophages during mammalian host infection. The sequence analysis of these Cys-rich regions in CnMTs revealed their homology to other well characterized fungal MT models, as Neurospora crassa and Agaricus bisporus MTs, whose Cys pattern almost exactly coincides with that found in CnMTs, this supporting the emergence of the long C. neoformans MTs by ancient tandem repetitions of a primeval fungal MT unit. Finally, in this work other hypothetical fungal MTs were identified in silico, among which those of other human opportunistic pathogenic fungi such as Histoplasma capsulatum, Paracoccidioides brasiliensis, Coccidioides immitis or Fusarium genus fungi. The strategy used in our group to characterize MTs, allowed us to identify a new fungal MT from F. verticillioides which presents the same Cu-thionein features (Cu5 clusters) than each Cys-building blocks from C. neoformans MTs. Evenly, the characteristics presented in F. verticillioides MT is extensible to F. graminearum and F. oxysporum MTs, given that the MT sequences are identical. Overall, this PhD thesis work contributes to enlarge the current limited knowledge about molecular evolution of unicellular eukaryote MTs and at the same time, it opens the door to characterize new pathogenic fungal MTs, whose results may provide interesting conclusions to decipher the role of MTs as virulent determinants and its function as Cu detoxifying.