R2 Subunit Of Ribonucleotide Reductase Research Articles

Analysis of glutathione mediated S-(de)nitrosylation in complex biological matrices by immuno-spin trapping and identification of two novel substrates

The intracellular concentration of reduced glutathione (GSH) lies in the range of 1–10 mM, thereby indisputably making it the most abundant intracellular thiol. Such a copious amount of GSH makes it the most potent and robust cellular antioxidant that plays a crucial role in cellular defence against redox stress. The role of GSH as a denitrosylating agent is well established; in this study, we demonstrate GSH mediated denitrosylation of HepG2 cell-derived protein nitrosothiols (PSNOs), by a unique spin-trapping mechanism, using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as the spin trapping agent, followed by a western blot analysis. We also report our findings of two, hitherto unidentified substrates of GSH mediated S-denitrosylation, namely S-nitrosoglutaredoxin 1 (Grx1-SNO) and S-nitrosylated R1 subunit of ribonucleotide reductase (R1-SNO).

Unsymmetrical Trifluoromethyl Methoxyphenyl β-Diketones: Effect of the Position of Methoxy Group and Coordination at Cu(II) on Biological Activity.

Copper(II) complexes with 1,1,1-trifluoro-4-(4-methoxyphenyl)butan-2,4-dione (HL1) were synthesized and characterized by elemental analysis, FT-IR spectroscopy, and single crystal X-ray diffraction. The biological properties of HL1 and cis-[Cu(L1)2(DMSO)] (3) were examined against Gram-positive and Gram-negative bacteria and opportunistic unicellular fungi. The cytotoxicity was estimated towards the HeLa and Vero cell lines. Complex 3 demonstrated antibacterial activity towards S. aureus comparable to that of streptomycin, lower antifungal activity than the ligand HL1 and moderate cytotoxicity. The bioactivity was compared with the activity of compounds of similar structures. The effect of changing the position of the methoxy group at the aromatic ring in the ligand moiety of the complexes on their antimicrobial and cytotoxic activity was explored. We propose that complex 3 has lower bioavailability and reduced bioactivity than expected due to strong intermolecular contacts. In addition, molecular docking studies provided theoretical information on the interactions of tested compounds with ribonucleotide reductase subunit R2, as well as the chaperones Hsp70 and Hsp90, which are important biomolecular targets for antitumor and antimicrobial drug search and design. The obtained results revealed that the complexes displayed enhanced affinity over organic ligands. Taken together, the copper(II) complexes with the trifluoromethyl methoxyphenyl-substituted β-diketones could be considered as promising anticancer agents with antibacterial properties.

In silico screening identifies a novel small molecule inhibitor that counteracts PARP inhibitor resistance in ovarian cancer

Poly ADP-ribose polymerase (PARP) inhibitors are promising targeted therapy for epithelial ovarian cancer (EOC) with BRCA mutations or defective homologous recombination (HR) repair. However, reversion of BRCA mutation and restoration of HR repair in EOC lead to PARP inhibitor resistance and reduced clinical efficacy of PARP inhibitors. We have previously shown that triapine, a small molecule inhibitor of ribonucleotide reductase (RNR), impaired HR repair and sensitized HR repair-proficient EOC to PARP inhibitors. In this study, we performed in silico screening of small molecule libraries to identify novel compounds that bind to the triapine-binding pocket on the R2 subunit of RNR and inhibit RNR in EOC cells. Following experimental validation of selected top-ranking in silico hits for inhibition of dNTP and DNA synthesis, we identified, DB4, a putative RNR pocket-binding inhibitor markedly abrogated HR repair and sensitized BRCA-wild-type EOC cells to the PARP inhibitor olaparib. Furthermore, we demonstrated that the combination of DB4 and olaparib deterred the progression of BRCA-wild type EOC xenografts and significantly prolonged the survival time of tumor-bearing mice. Herein we report the discovery of a putative small molecule inhibitor of RNR and HR repair for combination with PARP inhibitors to treat PARP inhibitor-resistant and HR repair-proficient EOC.

The Absence of Tryptase Mcpt6 Causes Elevated Cellular Stress in Response to Modulation of the Histone Acetylation Status in Mast Cells.

Mast cells contain large amounts of proteases stored within their secretory granules. Previously we showed that one of these proteases, tryptase, in addition to its location within granules, can also be found within the mast cell nucleus, where it has the capacity to affect the acetylation profile of nucleosomal core histones in aging cells. Based on this notion, and on the known sensitivity of mast cells to modulation of histone acetylation, we here asked whether tryptase could impact on the responses against cellular stress caused by disturbed histone acetylation status. To address this, wild-type and tryptase-deficient (Mcpt6−/−) mast cells were subjected to cell stress caused by trichostatin A (TSA), a histone deacetylase inhibitor. Wild-type and Mcpt6−/− mast cells were equally sensitive to TSA at an early stage of culture (~8 weeks). However, in aging mast cells (>50 weeks), tryptase-deficiency led to increased sensitivity to cell death. To address the underlying mechanism, we assessed effects of tryptase deficiency on the expression of markers for proliferation and cell stress. These analyses revealed aberrant regulation of thioredoxin, thioredoxin reductase, glutaredoxin, and glutathione reductase, as well as blunted upregulation of ribonucleotide reductase subunit R2 in response to TSA in aging cells. Moreover, the absence of tryptase led to increased expression of Psme4/PA200, a proteasome variant involved in the processing of acetylated core histones. Altogether, this study identifies a novel role for tryptase in regulating the manifestations of cell stress in aging mast cells.

Nuclear inelastic scattering at the diiron center of ribonucleotide reductase from Escherichia coli

The enzyme ribonucleotide reductase R2 catalyzes an important step in the synthesis of the building blocks of DNA, and harbors a dinuclear iron center required for activity. Not only the iron valence states but also the protonation of the iron ligands govern the enzymatic activity of the enzyme. We have performed Nuclear Inelastic Scattering (NIS) experiments on the 57Fe reconstituted ribonucleotide reductase R2 subunit from Escherichia coli (Ec R2a). Accompanying Mossbauer spectroscopic investigations show that the partial density of vibrational states (pDOS) of the 57Fe reconstituted Ec R2a sample contained contributions from both 57Fe-Ec R2a protein as well as unspecifically bound 57Fe. Subtraction of a featureless pDOS as obtained from protein-coated iron oxide particles allowed modeling of the contribution of non-specifically bound iron and thus the pDOS of 57Fe-Ec R2a could be obtained. Quantum-mechanics/molecular-mechanics (QM/MM) calculations of the whole 57Fe-Ec R2a protein with variations of the cofactor protonation were performed in order to assign characteristic bands to their corresponding molecular vibrational modes.

Long-term HIV-1 infection induces an antiviral state in primary macrophages

HIV-1 infection is thought to impair type I interferon (IFN-I) production in macrophages, a cell type that is also relatively resistant to HIV-1 cytotoxic effects. Here, we show that monocyte differentiation into macrophages by M-CSF led to cell proliferation and susceptibility to HIV-1 infection that induced cell cycle arrest and increased cell death. Established HIV-1 infection of monocyte-derived macrophages induced the upregulation of the pattern recognition receptors MDA5 and Rig-I that serve as virus sensors; production of interferon-β, and transcription of interferon-stimulated genes including CXCL10. Infected macrophages showed increased expression of p21 and subsequent inactivation of cyclin-CDK2 activity leading to a hypo-phosphorylated active retinoblastoma protein (pRb) and deactivation of E2F1-dependent transcription and CDK1 downregulation. Additionally, HIV-1 infection limited deoxynucleotide pool by downregulation of the ribonucleotide reductase subunit R2 (RNR2) and reactivation of the HIV-1 restriction factor SAMHD1 together with increased cell death. In conclusion, HIV-1 induced an innate antiviral mechanism associated to IFN-I production, interferon stimulated gene activation, and p21-mediated G2/M arrest leading to elevated levels of cell death in monocyte derived macrophages. Upregulation of MDA5 and Rig-I may serve as targets for the development of antiviral strategies leading to the elimination of HIV-1 infected cells.

Non-covalent complexes of folic acid and oleic acid conjugated polyethylenimine: An efficient vehicle for antisense oligonucleotide delivery

Polyethylenimine (PEI) was conjugated to oleic acid (PEI-OA) and evaluated as a delivery agent for LOR-2501, an antisense oligonucleotide against ribonucleotide reductase R1 subunit. PEI-OA/LOR-2501 complexes were further coated with folic acid (FA/PEI-OA/LOR-2501) and evaluated in tumor cells. The level of cellular uptake of FA/PEI-OA/LOR-2501 was more than double that of PEI/LOR-2501 complexes, and was not affected by the expression level of folate receptor (FR) on the cell surface. Efficient delivery was seen in several cell lines. Furthermore, pathway specific cellular internalization inhibitors and markers were used to reveal the principal mechanism of cellular uptake. FA/PEI-OA/LOR-2501 significantly induced the downregulation of R1 mRNA and R1 protein. This novel formulation of FA/PEI-OA provides a reliable and highly efficient method for delivery of oligonucleotide and warrants further investigation.

CD33-Targeted Lipid Nanoparticles (aCD33LNs) for Therapeutic Delivery of GTI-2040 to Acute Myelogenous Leukemia.

CD33-targeted lipid nanoparticles (aCD33LNs) were synthesized for delivery of GTI-2040, an antisense oligonucleotide (ASO) against the R2 subunit of ribonucleotide reductase, to acute myelogenous leukemia (AML). These LNs incorporated a deoxycholate-polyethylenimine (DOC-PEI) conjugate, which has shown significant activity to facilitate oligonucleotide delivery. Anti-CD33 scFv (aCD33) was added as a targeting ligand. The delivery efficiency of this system was investigated both in vitro and in vivo. When cells were treated with aCD33LN/GTI-2040, significant uptake was observed in CD33 positive Kasumi-1 cells. aCD33LNs loaded with GTI-2040 induced significant down-regulation of R2 mRNA and protein levels in AML cells. Moreover, aCD33LN/GTI-2040 showed a 15-fold reduction in the IC50 of antileukemic drug Ara-C in Kasumi-1 cells. In Kasumi-1 xenograft model, aCD33LN/GTI-2040 showed significant R2 downregulation compared to LN/GTI-2040. Furthermore, aCD33LN/GTI-2040 coadministered with Ara-C was shown to be highly effective in tumor growth inhibition and to greatly increase survival time of mice bearing Kasumi-1 xenograft tumors. The conjugate DOC-PEI has shown an ability to include calcein release from lipid nanoparticles, suggesting a potential mechanism contributing to efficient endosome release by DOC-PEI2K. These results indicate that aCD33LNs are a highly effective vehicle for the therapeutic delivery of antisense agents to AML.

A novel reduction-sensitive modified polyethylenimine oligonucleotide vector

A reduction-sensitive cross-linked polyethylenimine derivative PEI-SS-OA was synthesized and evaluated for oligonucleotide delivery. PEI-SS-OA was shown to condense LOR-2501, an oligonucleotide targeting ribonucleotide reductase R1 subunit (RRM1), into positively charged complexes. The reductive degradation of the PEI-SS-OA induced by dithiothreitol was confirmed by a gel retardation assay. In vitro experiments revealed that the reduction-sensitive PEI-SS-OA was less cytotoxic and more effective in oligonucleotide delivery than the control 25kDa PEI. This study demonstrates that a reducibly degradable cationic polymer PEI-SS-OA possesses both higher oligonucleotide delivery efficiency and lower cytotoxicity than PEI (25kDa), therefore is an attractive candidate for further in vivo evaluation.

Design and virtual screening towards synthesis of novel substituted thiosemicarbozones as ribonuleotide reductase (RNR) inhibitors with improved cellular trafficking and anticancer activity.

Ribonucleotide reductase subunit R2 regulates catalytic action of the enzyme to provide DNA synthesis material via reduction. It has been continuously investigated as anticancer drug target for design and discovery of its inhibitors. Present studies aim to design novel heterocyclic/aryl substituted and adamantyl added thiosemicarbazones out fitted with improved cell permeability and effective RNR inhibition. Design strategy renders significant use of virtual screening and molecular docking studies to converge search of selective molecules for synthesis and further experimental studies. Selected candidates were synthesized and evaluated in vitro for their RNR inhibitory activity (IC₅₀, uM) on MCF-7 cells, breast cancer cell lines. Molecular docking results (docking scores) and experimental results (IC₅₀, uM) were found to be correlated and in agreement. Structure-based and ligandbased studies of results substantiate regulative role of water molecules at catalytic site (H₂O: 2057) as well as at RNR inhibitor binding site (H₂O: 2023, 2047, 2060 and 2070). Admantyl group has testified constant spatial position in docked poses and involved in steric interactions with Cys271, Asp272, Phe237, Gly234 and Val238. Heterocyclic/aryl substitutions equally offered H-bonds with water molecules (H₂O: 2028, 2054, 2061 and 2073) along with amino acids Ser264, Asp272, Tyr324 and Asn346. Present efforts to design new inhibitors incur new characteristics in RNR Inhibition.

Magnolol Affects Cellular Proliferation, Polyamine Biosynthesis and Catabolism-Linked Protein Expression and Associated Cellular Signaling Pathways in Human Prostate Cancer Cells in vitro

Background: Prostate cancer is the most commonly diagnosed form of cancer in men in Canada and the United States. Both genetic and environmental factors contribute to the development and progression of many cancers, including prostate cancer.Context and purpose of this study: This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on cellular proliferation and proliferation-linked activities of PC3 human prostate cancer cells in vitro.Results: PC3 cells exposed to magnolol at a concentration of 80 μM for 6 hours exhibited decreased protein expression of ornithine decarboxylase, a key regulator in polyamine biosynthesis, as well as affecting the expression of other proteins involved in polyamine biosynthesis and catabolism. Furthermore, protein expression of the R2 subunit of ribonucleotide reductase, a key regulatory protein associated with DNA synthesis, was significantly decreased. Finally, the MAPK (mitogen-activated protein kinase), PI3K (phosphatidylinositol 3-kinase), NFκB (nuclear factor of kappa-light-chain-enhancer of activated B cells) and AP-1 (activator protein 1) cellular signaling pathways were assayed to determine which, if any, of these pathways magnolol exposure would alter. Protein expressions of p-JNK-1 and c-jun were significantly increased while p-p38, JNK-1/2, PI3Kp85, p-PI3Kp85, p-Akt, NFκBp65, p-IκBα and IκBα protein expressions were significantly decreased.Conclusions: These alterations further support the anti-proliferative effects of magnolol on PC3 human prostate cancer cells in vitro and suggest that magnolol may have potential as a novel anti-prostate cancer agent.Key Words: prostate cancer cells, magnolol, polyamines, MAPK, PI3K, NFκB

Microfluidic assembly of lipid nanoparticles for delivery of antisense oligonucleotides.

Microfluidically (MF) synthesized lipid nanoparticles (LNPs) for antisense oligonucleotides (ODN) delivery have been shown to be superior to those prepared by bulk mixing (BM). In this study, a 5-inlet MF chip was used to synthesize LNPs loaded with LOR-2501, an antisense ODN targeting the ribonucleotide reductase R1 subunit. The size distribution of ODN- LNPs was measured by dynamic light scattering. The cytotoxicity of ODN- LNPs was determined by MTS assay. Gene silencing activity of ODN- LNPs was investigated by qRT-PCR and by Western blot. Results showed that MF synthesis produced ODN-LNPs that have lower average size and polydispersity values. The highest antisense activity was shown by LNs synthesized by the MF T2 chip, with downregulation of R1 mRNA by 32.5%. In conclusion, given their simplicity, affordability and reproducibility, MF is an attractive method for synthesis of LNs for ODN delivery.

Enhanced antisense oligonucleotide delivery using cationic liposomes incorporating fatty acid-modified polyethylenimine.

Antisense oligonucleotides (ASOs) have promising therapeutic potential in oncotherapy. However, low stability and efficacy limit their application in the clinic. Cationic liposomes have been investigated as delivery vehicles for ASOs. Here, we report the synthesis and evaluation of an ASO delivery vehicle comprising cationic liposomes incorporating fatty acid-modified polyethylenimine. An oleic acid derivative of branched polyethylenimine (PEI-OA) and a linoleic acid derivative of branched polyethylenimine (PEI-LA) were synthesized and incorporated into liposomes. The PEI-modified liposomes were synthesized by an ethanol injection method with composition of PEI-modified lipid/Chol/TPGS. The properties of these liposomes, including cytotoxicity, cellular uptake, ASO target silencing activity, based on mRNA and protein downregulation, were investigated. LOR-2501, an ASOs targeting ribonucleotide reductase R1 subunit (R1) was used as the therapeutic cargo. The PEI-modified liposomes showed relatively compact particle size and excellent colloidal stability for at least 25 days. PEI-modified liposomes effectively delivered LOR-2501 into KB cells and efficiently induced down-regulation of R1 mRNA and protein. Compared with regular cationic liposomes, PEI-modified liposomes was more effective, reducing R1 mRNA and protein by ~10%.

A microfluidic method to synthesize transferrin-lipid nanoparticles loaded with siRNA LOR-1284 for therapy of acute myeloid leukemia.

The siRNA LOR-1284 targets the R2 subunit of ribonucleotide reductase (RRM2) and has shown promise in cancer therapy. In this study, transferrin (Tf) conjugated lipid nanoparticles (Tf-NP-LOR-1284) were synthesized by microfluidic hydrodynamic focusing (MHF) and evaluated for the targeted delivery of LOR-1284 siRNA into acute myeloid leukemia (AML) cells. The in vitro study showed that Tf-NP-LOR-1284 can protect LOR-1284 from serum nuclease degradation. Selective uptake of Tf-NP-LOR-1284 was observed in MV4-11 cells. In addition, qRT-PCR and Western blot results revealed that Tf-NP-LOR-1284 was more effective than the free LOR-1284 in reducing the R2 mRNA and protein levels. The Tf-NP-LOR-1284 showed prolonged circulation time and increased AUC after i.v. administration relative to the free LOR-1284. Furthermore, Tf-NP-LOR-1284 facilitated increased accumulation at the tumor site along with the decreased R2 mRNA and protein expression in a murine xenograft model. These results suggest that Tf-conjugated NPs prepared by MHF provide a suitable platform for efficient and specific therapeutic delivery of LOR-1284 into AML cells.

P21 regulates the HIV-1 restriction factor SAMHD1

Allouch et al. (1) have shown that CDKN1A (p21) restricts HIV-1 replication in monocyte-derived macrophages (MDM) by controlling the expression of the ribonucleotide reductase subunit R2 (RNR2) of the ribonucleotide reductase enzyme that, in turn, controls the intracellular deoxynucleotide (dNTP) pool required for HIV-1 reverse transcription. dNTP levels are also tightly controlled by the dNTP triphosphohydrolase SAM domain and HD domain-containing protein 1 (SAMHD1), which is constitutively expressed in myeloid and lymphoid cells and is counteracted by the lentiviral virus protein x (Vpx) (reviewed in ref. 2). SAMHD1 is deactivated in proliferating cells by a mechanism that requires phosphorylation of SAMHD1 (3). Allouch et al. (1) conclude that p21-driven HIV-1 restriction in macrophages is independent of SAMHD1 because ( i ) p21 did not affect SAMHD1 expression and ( ii ) Vpx did not affect p21 expression. Here, we show that M-CSF induces monocyte differentiation into macrophages and cell proliferation (Fig. 1 A ), and RNA interference of p21 leads to an increase in the number of proliferating cells (Fig. 1 B ). Macrophages become susceptible to HIV-1 replication (Fig. 1 C ) because SAMHD1 is inactivated as measured by specific SAMHD1 phosphorylation at residue T592 (Fig. 1 D ). Delivery of simian immunodeficiency virus (SIV) mac Vpx-induced SAMHD1 degradation (Fig. 1 E ) and subsequently increased virus infection (Fig. 1 F ) (2, 4, 5). Of importance, siRNA-induced down-regulation of p21 (Fig. 2 A ) strongly enhanced the phosphorylation of SAMHD1 (Fig. 2 B and C ), followed by an increase in … [↵][1]2To whom correspondence should be addressed. E-mail: jaeste{at}irsicaixa.es. [1]: #xref-corresp-1-1

Activation of Dimanganese Class Ib Ribonucleotide Reductase by Hydrogen Peroxide: Mechanistic Insights from Density Functional Theory

Activation of manganese-dependent class Ib ribonucleotide reductase by hydrogen peroxide was modeled using B3LYP* hybrid density functional theory. Class Ib ribonucleotide reductase R2 subunit (R2F) does not react with molecular oxygen. Instead R2F is proposed to react with H2O2 or HO2(-), provided by the unusual flavodoxin protein NrdI, to generate the observed manganese(III) manganese(III) tyrosyl-radical state. On the basis of the calculations, an energetically feasible reaction mechanism is suggested for activation by H2O2, which proceeds through two reductive half-reactions. In the first reductive half-reaction, H2O2 is cleaved with a barrier of 13.1 kcal mol(-1) [Mn(II)Mn(II) → Mn(III)Mn(III)], and in the second reductive half-reaction, H2O2 is cleaved with a barrier of 17.0 kcal mol(-1) [Mn(III)Mn(III) → Mn(IV)Mn(IV)]. Tyrosyl-radical formation from both the Mn(IV)Mn(IV) state and a Mn(III)Mn(IV) state, where an electron and proton have been taken up, is both kinetically and thermodynamically accessible. Hence, chemically, H2O2 is a possible oxidant for the manganese-dependent R2F. The selectivity between the second reductive half-reaction and a competing oxidative reaction, as in manganese catalase, may be the time scale for the availability of H2O2. The role of NrdI may be to provide H2O2 on the correct time scale.

Immunologic Effect of Attenuated Salmonella enteric Serovar Choleraesuis C501 Expressing Recombinant Mycoplasma hyopneumoniae P97R1 Adhesin and NrdF Antigens in Mice

The immunogenicity of the P97 adhesin repeat region R1 (P97R1) and the ribonucleotide reductase R2 subunit (NrdF) antigens of Mycoplasma hyopneumoniae were evaluated as vaccines in mice. Mice were immunized orally or injected intramuscularly (IM) with attenuated Salmonella enterica serovar Choleraesuis strain C501 harboring a prokaryotic expression vector encoding P97R1 and NrdF (pYA97R1N). Local and systemic immune responses were analyzed in vaccinated mice. The results showed that P97R1N-specific serum IgG and IgA antibodies were detected in serum and lung samples. P97R1-specific serum IgG and IgA antibodies were also detected in serum, but in lung sample only P97R1-specific IgG response was induced. ELISA assays demonstrated that IFN-U and IL-4 production was induced in cultural supernatants from splenocytes stimulated with specific antigens in vitro. M. hyopneumoniae whole cell-specific antibody was induced in vaccinated mice and a good immunogenic effect by this vaccine was indicated. Importantly, animals vaccinated by IM had higher levels of P97R1N-specific IgG and IgA than those vaccinated by oral route. Furthermore, mice immunized with the C501 vaccine elicited significantly higher levels of P97R1N-specific IgA and IgG in lungs and serum (P<0.05), and IFN-U and IL-4, than those who received the live M.hyopneumoniae vaccine. Taken together, these results suggest that IM vaccination with the C501 vaccine might provide an effective method of inducing an immune response to M. hyopneumoniae.

A Polyethylenimine-Linoleic Acid Conjugate for Antisense Oligonucleotide Delivery

A novel antisense oligonucleotide (ASO) carrier, polyethylenimine conjugated to linoleic acid (PEI-LA), was synthesized and evaluated for delivery of LOR-2501 to tumor cells. LOR-2501 is an ASO targeting ribonucleotide reductase R1 subunit (RRM1). In this study, PEI-LA was synthesized by reacting PEI (Mw ~ 800) with linoleoyl chloride. Gel retardation assay showed complete complexation between PEI-LA and LOR-2501 at N/P ratio above 8. No significant cytotoxicity was observed with these complexes at the tested dosage levels. Interestingly, at N/P ratio of >6, levels of cellular uptake of PEI-LA/LOR-2501 were double that of PEI/LOR-2501 complexes of the same N/P ratio. PEI-LA/LOR-2501 induced downregulation of 64% and 70% of RRM1 at mRNA and protein levels, respectively. The highest transfection activity was shown by PEI-LA/LOR-2501 complexes at N/P ratio of 10. Finally, using pathway specific inhibitors, clathrin-mediated endocytosis was shown to be the principle mechanism of cellular internalization of these complexes. In conclusion, PEI-LA is a promising agent for the delivery of ASOs and warrants further investigation.

DFT calculations for intermediate and active states of the diiron center with a tryptophan or tyrosine radical in Escherichia coli ribonucleotide reductase.

Class Ia ribonucleotide reductase subunit R2 contains a diiron active site. In this paper, active-site models for the intermediate X-Trp48(•+) and X-Tyr122(•), the active Fe(III)Fe(III)-Tyr122(•), and the met Fe(III)Fe(III) states of Escherichia coli R2 are studied, using broken-symmetry density functional theory incorporated with the conductor-like screening solvation model. Different structural isomers and different protonation states have been explored. Calculated geometric, energetic, Mössbauer, hyperfine, and redox properties are compared with available experimental data. Feasible detailed structures of these intermediate and active states are proposed. Asp84 and Trp48 are most likely the main contributing residues to the result that the transient Fe(IV)Fe(IV) state is not observed in wild-type class Ia E. coli R2. Asp84 is proposed to serve as a proton-transfer conduit between the diiron cluster and Tyr122 in both the tyrosine radical activation pathway and the first steps of the catalytic proton-coupled electron-transfer pathway. Proton-coupled and simple redox potential calculations show that the kinetic control of proton transfer to Tyr122(•) plays a critical role in preventing reduction from the active Fe(III)Fe(III)-Tyr122(•) state to the met state, which is potentially the reason why Tyr122(•) in the active state can be stable over a very long period.

P53R2 Inhibits the Proliferation of Human Cancer Cells in Association with Cell-Cycle Arrest

Deregulation of the expression of p53R2, a p53-inducible homologue of the R2 subunit of ribonucleotide reductase, has been found in various human cancer tissues; however, the roles p53R2 plays in cancer progression and malignancy remain controversial. In the present study, we examined changes in gene expression profiles associated with p53R2 in cancer cells, using the analysis of cDNA microarray. Gene set enrichment analysis identified that the gene set regulating cell-cycle progression was significantly enriched in p53R2-silencing human oropharyngeal carcinoma KB cells. Attenuation of p53R2 expression significantly reduced p21 expression and moderately increased cyclin D1 expression in both wild-type p53 cancer cells (KB and MCF-7) and mutant p53 cancer cells (PC3 and MDA-MB-231). Conversely, overexpression of p53R2-GFP resulted in an increase in the expression of p21 and decrease in the expression of cyclin D1, which correlated with reduced cell population in S-phase in vitro and suppressed growth in vivo. Furthermore, the MAP/ERK kinase inhibitor PD98059 partially abolished modulation of p21 and cyclin D1 expression by p53R2. Moreover, under the conditions of nonstress and adriamycin-induced genotoxic stress, attenuation of p53R2 in KB cells significantly increased phosphorylated H2AX, which indicates that attenuation of p53R2 may enhance DNA damage induced by adriamycin. Overall, our study shows that p53R2 may suppress cancer cell proliferation partially by upregulation of p21 and downregulation of cyclin D1; p53R2 plays critical roles not only in DNA damage repair but also in proliferation of cancer cells.