| Abstract |
Cellular copper homeostasis is a fundamental and continuous process in all living organisms
including S. cerevisiae. However, when uptake exceeds the buffering capacity of the cell,
copper becomes toxic. Thus, yeast cell has developed several mechanisms to control copper
homeostasis. A major regulator is the transcription factor Mac1 (“Metal-binding activator”),
which has a crucial role in sensing changes in copper ion concentrations and activating the
transcription of genes involved in copper uptake in ion-depleted cells. Initial experiments of
our group revealed an unexpected connection between DNA damage and copper
homeostasis. Particularly, using the drug Zeocin to induce DNA damage proved that the
function and specifically the DNA binding ability of the Mac1 transcription factor is hindered.
Zeocin is a radiomimetic drug and apart from DNA damaging agent, is used as antitumor drug
and as antibiotic, due to its wide range of toxicity. The rationale was to investigate which are
the prerequisites of Mac1 DNA binding and identify which of them are perturbed in the
presence of Zeocin. Three hypotheses had arisen which are going to be investigated and
discussed in the following chapters.
In Chapter 2 of this thesis, we have identified a connection between copper homeostasis and
the TORC1 protein complex in Saccharomyces cerevisiae. The connecting ring of this
association is the Ksp1 kinase. Particularly, Ksp1 is a downstream regulator of TORC1 and
phosphorylates the transcription factor Mac1. We found that Zeocin perturbs the Ksp1-
regulated phosphorylation of Mac1 and leads to the deregulation of copper homeostasis.
Analysis of the S. cerevisiae transcriptome in the presence of Zeocin, compared to other DNA
damaging conditions, proved that the deregulation of copper homeostasis does not correlate
with the DNA damage response, but it is a Zeocin-specific effect. According to the Zeocinspecific
transcriptome, Mac1 target genes are the most down-regulated. Additionally, a
negative effect on the conserved signaling pathway TORC1 was uncovered, while the TORC1
downstream pathways such as ribosome biogenesis and autophagy were found affected.
Furthermore, an up-regulation of mitochondrial function was revealed. We verified the
transcriptomic results by a series of functional experiments and assumed that Zeocin induces
metabolic reprogramming in S. cerevisiae, through the TORC1 protein complex function.
In conclusion, our results establish a functional link between the copper-dependent Mac1-
regulated transcription and the TORC1 signaling pathway. Moreover, they have indicated two
new effects of the drug Zeocin apart from its role in the DNA damage induction. First, Zeocin
disturbs copper/iron-regulated homeostasis by inhibiting the DNA binding of the Mac1
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transcription factor. Second, Zeocin induces metabolic reprogramming in the S. cerevisiae,
through the TORC1 protein complex function, an observation with potentially promising
biomedical applications.
In Chapter 3 of the thesis, we investigated the hypothesis that Mac1’s malfunction in Zeocin
conditions could be due to a Mac1-Sod1 dissociation. Our results indicate that the Zeocin
effect on Mac1 function is not via Sod1 protein, although, we provide evidence that the
copper- and zinc-dependent enzyme Sod1 plays an important role in Mac1 transcriptional
activity, in nutrient-rich conditions. Cu/Zn superoxide dismutase Sod1 is a multipotent
cytoplasmic and mitochondrial enzyme, whose main known function is to detoxify the cell
from superoxide ions. Furthermore, it has been reported that Sod1 enters the nucleus and
affects the transcription of several genes, some of which are involved in copper homeostasis
under Cu-depleted conditions and only under specific oxidative stress conditions. We found
that Sod1 physically interacts with Mac1 in nutrient-rich conditions. Our results indicate that
Sod1 is important for both transactivation and binding activity of Mac1 on its target promoter
regions. On the other hand, a constitutively active mutant of Mac1 is not affected functionally
by the ablation of Sod1. Combining our findings, we suggest that Sod1 interaction with Mac1
is vital for Mac1 functionality, due to a conformational alteration of the transcription factor.
Sod1 enzymatic activity may contribute to the maintenance of a reduced cellular environment
and prevents the oxidation and subsequent inactivation of the transcription factor Mac1.
In Chapter 4 of this thesis, we aimed to explore the hypothesis of direct interaction between
the Mac1 transcription factor and the drug Zeocin. Having evidence that Zeocin may inhibit
the function of other copper-fist containing transcription factors, we present our progress in
purifying peptide derivatives of the Mac1 protein, in order to investigate the potential direct
protein-drug interaction using an acoustic biosensor.
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Μεταλλορυθμιζόμενη μεταγραφή - η Zeocin επηρεάζει την χαλκο/σιδηρο-ρυθμιζόμενη μεταγραφή και προκαλεί μεταβολικό αναπρογραμματισμό στον σακχαρομύκητα
