Human Ribonucleotide Reductase Subunit Research Articles

Impact of N221S missense mutation in human ribonucleotide reductase small subunit b on mitochondrial DNA depletion syndrome

The impact of N221S mutation in hRRM2B gene, which encodes the small subunit of human ribonucleotide reductase (RNR), on RNR activity and the pathogenesis of mitochondrial DNA depletion syndrome (MDDS) was investigated. Our results demonstrate that N221 mutations significantly reduce RNR activity, suggesting its role in the development of MDDS. We proposed an allosteric regulation pathway involving a chain of three phenylalanine residues on the αE helix of RNR small subunit β. This pathway connects the C-terminal loop of β2, transfers the activation signal from the large catalytic subunit α to β active site, and controls access of oxygen for radical generation. N221 is near this pathway and likely plays a role in regulating RNR activity. Mutagenesis studies on residues involved in the phenylalanine chain and the regulation pathway were conducted to confirm our proposed mechanism. We also performed molecular dynamic simulation and protein contact network analysis to support our findings. This study sheds new light on RNR small subunit regulation and provides insight on the pathogenesis of MDDS.

Synthesis and in silico evaluation of novel uridyl sulfamoylbenzoate derivatives as potential anticancer agents targeting M1 subunit of human ribonucleotide reductase (hRRM1)

Synthesis and in silico evaluation of novel uridyl sulfamoylbenzoate derivatives as potential anticancer agents targeting M1 subunit of human ribonucleotide reductase (hRRM1)

Activity and electrochemical properties: iron complexes of the anticancer drug triapine and its analogs.

Triapine (3-AP), is an iron-binding ligand and anticancer drug that is an inhibitor of human ribonucleotide reductase (RNR). Inhibition of RNR by 3-AP results in the depletion of dNTP precursors of DNA, thereby selectively starving fast-replicating cancer cells of nucleotides for survival. The redox-active form of 3-AP directly responsible for inhibition of RNR is the Fe(II)(3-AP)2 complex. In this work, we synthesize 12 analogs of 3-AP, test their inhibition of RNR in vitro, and study the electronic properties of their iron complexes. The reduction and oxidation events of 3-AP iron complexes that are crucial for the inhibition of RNR are modeled with solution studies. We monitor the pH necessary to induce reduction in iron complexes of 3-AP analogs in a reducing environment, as well as the kinetics of oxidation in an oxidizing environment. The oxidation state of the complex is monitored using UV-Vis spectroscopy. Isoquinoline analogs of 3-AP favor the maintenance of the biologically active reduced complex and possess oxidation kinetics that allow redox cycling, consistent with their effective inhibition of RNR seen in our in vitro experiments. In contrast, methylation on the thiosemicarbazone secondary amine moiety of 3-AP produces analogs that form iron complexes with much higher redox potentials, that do not redox cycle, and are inactive against RNR in vitro. The catalytic subunit of human Ribonucleotide Reductase (RNR), contains a tyrosyl radical in the enzyme active site. Fe(II) complexes of 3-AP and its analogs can quench the radical and, subsequently, inactivate RNR. The potency of RNR inhibitors is highly dependent on the redox properties of the iron complexes, which can be tuned by ligand modifications. Complexes are found to be active within a narrow redox window imposed by the cellular environment.

Novel regulators and molecular mechanisms of p53R2 and its disease relevance.

p53R2 is a p53-inducible human ribonucleotide reductase subunit involved in critical cellular mechanisms, such as DNA repair, cell cycle arrest, and mitochondrial homeostasis. Molecular investigations and animal studies have revealed functional regulations of p53R2 and its disease relevance. The relationship between p53R2 expression and disease progression in different cancers has been evaluated, and researchers have discovered novel transcription factors and cellular mechanisms that control p53R2 in a p53-independent manner. In addition, p53R2-Mediated mechanisms that affect mitochondria, inflammation, and cancer have been addressed. The role of p53R2 in mitochondria diseases and in cancer is discussed. Finally, p53R2 is taken as a potential target for cancer treatment. This review summarizes the general background, novel regulatory findings, and medical prospect of p53R2.

DNTP Supply Gene Expression Patterns after P53 Loss.

Loss of the transcription factor p53 implies mRNA losses of target genes such as the p53R2 subunit of human ribonucleotide reductase (RNR). We hypothesized that other genes in the dNTP supply system would compensate for such p53R2 losses and looked for this in our own data and in data of the Gene Expression Omnibus (GEO). We found that the de novo dNTP supply system compensates for p53R2 losses with increases in RNR subunit R1, R2, or both. We also found compensatory increases in cytosolic deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) and in mitochondrial deoxyguanosine kinase (dGK), all of the salvage dNTP supply system; in contrast, the remaining mitochondrial salvage enzyme thymidine kinase 2 (TK2) decreased with p53 loss. Thus, TK2 may be more dedicated to meeting mitochondrial dNTP demands than dGK which may be more obligated to assist cytosolic dNTP supply in meeting nuclear DNA dNTP demands.

Targeting the Large Subunit of Human Ribonucleotide Reductase for Cancer Chemotherapy

Ribonucleotide reductase (RR) is a crucial enzyme in de novo DNA synthesis, where it catalyses the rate determining step of dNTP synthesis. RRs consist of a large subunit called RR1 (α), that contains two allosteric sites and one catalytic site, and a small subunit called RR2 (β), which houses a tyrosyl free radical essential for initiating catalysis. The active form of mammalian RR is an αnβm hetero oligomer. RR inhibitors are cytotoxic to proliferating cancer cells. In this brief review we will discuss the three classes of RR, the catalytic mechanism of RR, the regulation of the dNTP pool, the substrate selection, the allosteric activation, inactivation by ATP and dATP, and the nucleoside drugs that target RR. We will also discuss possible strategies for developing a new class of drugs that disrupts the RR assembly.

The ribonucleotide reductase large subunit (RRM1) as a predictive factor in patients with cancer

The large subunit of human ribonucleotide reductase, RRM1, is involved in the regulation of cell proliferation, cell migration, tumour and metastasis development, and the synthesis of deoxyribonucleotides for DNA synthesis. It is also a cellular target for the chemotherapeutic agent, gemcitabine. RRM1 has been studied in a large number of patients with different types of cancer, such as non-small-cell lung cancer, pancreatic cancer, breast cancer, and biliary tract cancer, to establish its prognostic or predictive value when patients were treated with gemcitabine, and mRNA expression and genetic variants as determined by genotyping have in some cases been associated with clinical outcome of patients with cancer. Here, we review preclinical and clinical studies of RRM1 assessment and discuss the further steps in the development of this clinically pertinent biomarker.

Regulation of p53R2 and its role as potential target for cancer therapy

p53R2, a recently discovered small subunit of human ribonucleotide reductase, is believed to play essential roles in DNA repair, mtDNA synthesis, and protection against oxidative stress. Because of the positive correlation between the level of this protein and drug sensitivity and tumor invasiveness, it constitutes a potential target for anticancer drugs as well as a diagnostic marker in cancer.

MAb Against the Small Subunit of Human Ribonucleotide Reductase (hRRM2)

MAb Against the Small Subunit of Human Ribonucleotide Reductase (hRRM2)

GTI-2501, an antisense agent targeting R1, the large subunit of human ribonucleotide reductase, shows potent anti-tumor activity against a variety of tumors

GTI-2501 is a 20-mer oligonucleotide that is complementary to a coding region in the mRNA of R1, the large subunit of ribonucleotide reductase (RNR). In vitro studies, have demonstrated that GTI-2501 decreases mRNA and protein levels of R1 in a sequence-specific and dose-dependent manner. Furthermore, GTI-2501 inhibits the growth of human lung, liver, ovary, brain, melanoma, breast and pancreatic tumor cells in colony forming assays. In vivo studies have shown that GTI-2501 significantly inhibits growth of human colon, pancreas, lung, breast, renal, ovarian, melanoma, brain glioblastoma-astrocytoma, and prostatic tumors in CD-1 nude, Balb/c nude and/or SCID mice. GTI-2501 treatment caused total regression of human breast and renal tumor xenografts in mice. These effects are not observed with a scrambled control oligonucleotide containing the same base content but not complementary to R1. GTI-2501 specifically inhibits metastasis of human melanoma cells to the lungs in CD-1 athymic nude mice and prolongs the survival of mice bearing human lymphoma. Taken together these results suggest that an antisense mechanism of action is responsible for growth inhibition in vitro and in vivo and that GTI-2501 can act as a selective and specific anti-tumor agent.

Enzymatic property analysis of p53R2 subunit of human ribonucleotide reductase

Enzymatic property analysis of p53R2 subunit of human ribonucleotide reductase

Increased expression of the large subunit of ribonucleotide reductase is involved in resistance to gemcitabine in human mammary adenocarcinoma cells

Resistance to cytotoxic nucleoside analogues is a major problem in cancer treatment. The cellular mechanisms involved in this phenomenon have been studied for several years, and some factors have been identified. However, this resistance seems to be multifactorial and more studies are needed to gain better insight into this domain. For this purpose, we developed a gemcitabine-resistant cell line (MCF7 1K) from the human mammary adenocarcinoma MCF7 strain by prolonged exposure to gemcitabine in vitro. MCF7 1K cells are highly resistant to gemcitabine (533-fold) and cross-resistance is observed with araC (47-fold), triapine (14-fold), and hydroxyurea (6.7-fold). Quantitative real-time reverse transcription-PCR and Western blot analysis showed an increase in the gene and protein expression of the large subunit of ribonucleotide reductase, R1. Ribonucleotide reductase activity was also significantly increased in the gemcitabine-resistant cells. Study of genomic DNA showed 12-fold increase in R1 gene dosage in MCF7 1K cells. In contrast, the gene and protein expression of the small subunit of ribonucleotide reductase, R2, were not modified in this cell line. These results show that gemcitabine resistance can be associated with genetic modifications of target genes in malignant cells, and suggest that the large subunit of human ribonucleotide reductase is involved in the cellular response to gemcitabine.

Nuclear factor Y regulation and promoter transactivation of human ribonucleotide reductase subunit M2 gene in a Gemcitabine resistant KB clone

Overexpression of human ribonucleotide reductase subunit M2 ( hRRM2) has been shown as a potential factor causing Gemcitabine (Gem) resistance. We hypothesized the nuclear factor Y (NF-Y) would transcriptionally regulate hRRM2 and contribute to overexpression of hRRM2 in a Gem resistant clone. A luciferase and gel shift assay, and a Southwestern blot were employed to analyze the promoter activity of hRRM2. The data exhibited the hRRM2 promoter was upregulated almost 5-fold in the Gem resistant KB clone (KBGem) via three sequential CCAAT boxes located in the proximal promoter region. Nuclear extracts from KB and KBGem could interact with the CCAAT motif of the hRRM2 proximal promoter region, and could form DNA–protein complexes with different binding patterns. The complexes could be further recognized with antibodies against NF-Y subunits A and B. Histone deacetylases (HDAC) involvement in NF-Y transcription repression in the KBGem clone was examined. A HDAC activity assay revealed a 3-fold decrease of HDAC activity in the KBGem clone compared to KB cells. Parental cells were treated with trichostatin A (TSA), a HDAC inhibitor. NF-Y transactivation was induced, resulting in an increase of hRRM2 expression. This led to an expanded dCTP pool and an abrogated [ 3 H ]Gemcitabine incorporation. In addition, microarray analysis results showed most of the proliferation-related genes were upregulated in KBGem. This finding was consistent with enhanced NF-Y transactivation in KBGem. In summary, upregulation of NF-Y transactivation increased hRRM2 transcription, which played a pivotal role in the Gem resistant KB clone.

In vitro characterization of enzymatic properties and inhibition of the p53R2 subunit of human ribonucleotide reductase.

p53R2 is a newly identified subunit of ribonucleotide reductase (RR) and plays a crucial role in supplying precursors for DNA repair in a p53-dependent manner. In our current work, all three human RR subunit proteins (p53R2, hRRM2, and hRRM1) were prokaryotically expressed and highly purified. Using an in vitro [(3)H]CDP reduction assay, the activity of RR reconstituted with either p53R2 or hRRM2 was found to be time, concentration, and hRRM1 dependent. The kinetic activity of p53R2-containing RR was about 20-50% lower than that of hRRM2-containing RR. Using a synthetic heptapeptide to inhibit RR activity, it was shown that p53R2 bound to hRRM1 through the same COOH-terminal heptapeptide as hRRM2. However, hRRM2 had a 4.76-fold higher binding affinity for hRRM1 than p53R2, which may explain the reduced RR activity of p53R2 relative to hRRM2. Of interest, p53R2 was 158-fold more susceptible to the iron chelator deferoxamine mesylate than hRRM2, although the iron content of the two proteins determined by atomic absorption spectrometer was almost the same. To the contrary, p53R2 was 2.50-fold less sensitive than hRRM2 to the radical scavenger hydroxyurea, whereas EPR showed similar spectra of the tyrosyl radical in two proteins. Triapine, a new RR inhibitor, was equally potent for p53R2 and hRRM2. These inhibition studies showed that the iron center and tyrosyl radical are involved in RR activity for both p53R2 and hRRM2. The susceptibility differences to RR inhibitors between p53R2 and hRRM2 may lead to a new direction in drug design for human cancer treatment.