Yeast Ribonucleotide Reductase Is a Direct Target of the Proteasome and Provides Hyper Resistance to the Carcinogen 4-NQO

Abstract

Various external and internal factors damaging DNA constantly disrupt the stability of the genome. Cells use numerous dedicated DNA repair systems to detect damage and restore genomic integrity in a timely manner. Ribonucleotide reductase (RNR) is a key enzyme providing dNTPs for DNA repair. Molecular mechanisms of indirect regulation of yeast RNR activity are well understood, whereas little is known about its direct regulation. The study was aimed at elucidation of the proteasome-dependent mechanism of direct regulation of RNR subunits in Saccharomyces cerevisiae. Proteome analysis followed by Western blot, RT-PCR, and yeast plating analysis showed that upregulation of RNR by proteasome deregulation is associated with yeast hyper resistance to 4-nitroquinoline-1-oxide (4-NQO), a UV-mimetic DNA-damaging drug used in animal models to study oncogenesis. Inhibition of RNR or deletion of RNR regulatory proteins reverses the phenotype of yeast hyper resistance to 4-NQO. We have shown for the first time that the yeast Rnr1 subunit is a substrate of the proteasome, which suggests a common mechanism of RNR regulation in yeast and mammals.

Synergistic Effect of a Combination of Proteasome and Ribonucleotide Reductase Inhibitors in a Biochemical Model of the Yeast Saccharomyces cerevisiae and a Glioblastoma Cell Line

“…Our data provide new evidence of the similarity between the mechanisms of RNR activity regulation in yeast and mammals. So, we have previously shown that the yeast subunit Rnr1 [23], as well as its orthologue in mammalian cells RRM1 [35], serves as a substrate of the proteasome. Since RRM2, the ortholog of the yeast Rnr2 subunit, is controlled in a cell cycle phase-dependent manner by the ubiquitin-proteasome system [34,57], we can assume that the yeast Rnr2 subunit also serves as a proteasome substrate under DNA-damaging stress.…”

“…We have previously shown that Rnr1p is a substrate of the proteasome [23], which is evidenced by an increase in the content of Rnr1p under the action of the proteasome inhibitor bortezomib or in a mutant strain with reduced proteasome activity due to decreased expression of the PRE1 gene encoding an essential structural subunit of the proteasome (called pre1-8). In the present study, Western blot analysis showed that in the mutant strain rpn4-Δ with the deletion of the RPN4 gene, which encodes a key transcriptional regulator of proteasomal genes [45][46][47], an increase in the level of the large catalytic subunit of ribonucleotide reductase Rnr1p was also observed under normal conditions (Figure 1A,B), while the level of RNR1 mRNA did not change significantly (Figure 1C).…”

“…Wild-type yeast strain BY4742 (MAT α; his3∆1; leu2∆0; lys2∆0; ura3∆0, Euroscarf, Oberursel, Germany), its derivative strain with reduced proteasome activity pre1-8 (BY4742 MAT α; his3∆1; leu2∆0; lys2∆0; ura3∆0; pre1-8) [21], as well as derivatives of strain BY4741 (MAT a; his3∆1; leu2∆0; lys2∆0; ura3∆0, Euroscarf, Oberursel, Germany) and pre1-8 with deletions of genes encoding RNR regulators, sml1-∆ (BY4741 YML058W::LEU2) [23], ydj1-∆ (BY4741 YNL064C::LEU2) [23], pre1-8 sml1-∆ (pre1-8 YML058W::LEU2) [23] and pre1-8 ydj1-∆ (pre1-8 YNL064C::LEU2) [23], were used in the experiments.…”

“…Using yeast as a biochemical model of tumors, we have previously obtained results indicating that the use of proteasome inhibitors in combination with DNA-damaging compounds may not be effective in tumor therapy [21,22]. In the same model, we have shown that the use of DNA repair inhibitors [22] or ribonucleotide reductase (RNR) inhibitors [23] under conditions of proteasome inhibition can be an effective way to control tumors. RNR is a highly conserved enzyme that catalyzes the limiting step of the de novo pathway for the synthesis of deoxyribonucleoside triphosphates (dNTPs), which are used for DNA replication and repair [24].…”

“…The enzyme is a heterotetramer in which two large subunits perform the function of catalysis, and two small subunits are responsible for the regulation of complex activity. In yeast and mammalian cells, RNR activity is controlled at several levels by the regulation of gene expression [25][26][27][28], oligomerization of subunits, post-translational modifications of subunits [29,30], subcellular localization [31,32], allosteric inhibition by dNTPs [33] as well as through the degradation of subunits [23,34,35] and regulatory proteins [36,37] in the proteasome or by autophagy [38]. RNR activity has been found to increase in tumors, which is associated with both active tumor cell division and more active DNA repair processes [39,40].…”