| Abstract |
The presence of copper within living organisms is extremely important as a cofactor proteins and enzymes necessary for carrying out biological functions. In the yeast S. cerevisiae, a large number of genes of the proteins responsible for the reduction, introduction, distribution and storage of copper within the cell are transcribed by the protein Mac1 (Metal binding Activator). In cultures grown under DNA damage conditions using the antibiotic, radiomimetic Zeocin, it has been observed that the CTR1 gene, encoding a plasma membrane high-affinity copper transporter, exhibits significantly reduced levels of transcription. In parallel, iron-regulated genes are upregulated. In the present study, we confirmed that the Zeocin-dependent transcriptional reduction of CTR1 is due to the reduced functionality of the transcription factor, Mac1. Mac1 protein was detected quantitatively at similar levels in the presence or absence of Zeocin in the cells.
Understanding the mechanism of the Zeocin effect in the transcription of specific genes is important due to the wide use of this drug as anticancer, antibiotic in cell cultures and radiomimetic for the study of DNA damage response.
To clarify the reason Mac1 dysfunctionality in the presence of Zeocin, different approaches were used. We first examined RNA polymerase II recruitment on CTR1 gene and found reduced amounts of the protein on CTR1 promoter and coding region in cells cultured in the presence of Zeocin. This reduction is due to reduced levels of recruited Mac1 protein under the same conditions as seen by chromatin immunoprecipitation experiments.
We then examined a potentially direct Zeocin-Mac1 interaction and found that the transactivation potential of the hybrid LexA-Mac1 was unaffected in the presence of Zeocin. Therefore, the drug does not interfere with the carboxy-terminal part of Mac1 that includes the transactivation domain. Moreover, Zeocin does not induce the intermolecular conformation known to inactivate the transcription function of Mac1.
However, by using a strain expressing the constitutively active mutant Mac1up, we concluded that Zeocin affects the amino-terminal part of Mac1 protein. This region includes a Cu-fist involves in the binding of the protein to the target promoter DNA. We propose that copper ion in the Cu-fist region, that facilitates Mac1 binding on DNA under physiological conditions, reacts with the copper-chelating region of the Zeocin glycopeptide. The Zeocin –Mac1 complex has reduced capacity for binding to DNA and therefore transactivation of the Mac1-gene targets.
We have also examined the possible involvement of proteins collaborating with Mac1 with roles in cell stress response. Rad9 protein, mediator of the DNA damage response signal, physically interacts with Mac1 and reduces its activity under physiological conditions (Gkouskou 2007). We found no additional effect in its absence on Mac1 reduced function in the presence of Zeocin. Therefore it has no role in the Zeocin-Mac1 interaction.
The Cu / Zn superoxide dismutase Sod1 was recently found to localize also in the nucleus and affect the transcription of several genes including those regulating Cu/Fe homeostasis (Tsang et al. 2014). In the absence of Sod1 we found further reduction of CTR1 gene in rich medium cultures in the presence of Zeocin.
Therefore, we propose that Zeocin affects Mac1 function by interacting directly with Cu-fist interfering with its DNA binding. The Zeocin-Mac1 complex is further modulated by Sod1 which also binds copper ions. Verification of the proposed model is quite important since many proteins use copper or iron ions as cofactors imperative for their function in all organisms.
|
Το αντικαρκινικό φάρμακο Zeocin διαταράσσει την ομοιόσταση χαλκού-σιδήρου στον Saccharomyces cerevisiae
