Ribonucleotide Reductase Research Articles

Dissecting the Cell-Killing Mechanisms of Hydroxyurea Using Spot Assays.

Hydroxyurea is an inhibitor of ribonucleotide reductase and is commonly used in laboratories to induce replication stress or arrest cells in the S phase for cell cycle or checkpoint studies. However, hydroxyurea also causes side effects such as oxidative stress, particularly under chronic exposure conditions. This complicates the interpretation of the cell-killing mechanisms, particularly in a previously uncharacterized mutant, and thus hampers the analyses. Here, we describe a few easy and simple spot assays in fission yeast that allow dissecting the cell-killing mechanisms of hydroxyurea.

The Role of Ribonucleotide Reductase M2 in Lung Cancer Progression and Chemotherapy Resistance: A Bioinformatics Analysis and Review.

The Role of Ribonucleotide Reductase M2 in Lung Cancer Progression and Chemotherapy Resistance: A Bioinformatics Analysis and Review.

Genome editing of WSSV CRISPR/Cas9 and immune activation extends the survival of infected Penaeus vannamei

White spot syndrome virus (WSSV) is an exceptionally harmful virus that generally causes high levels of mortality in cultured shrimp. Attempts at viral suppression have been made to control the disease and have achieved limited efficiency. Recent advances in genome editing technology using CRISPR/Cas9 have led to potential innovations to prevent or treat many viral diseases. In this study, a CRISPR/Cas9 system was applied to WSSV genome cleavage to suppress WSSV infection in shrimp. The U6 promoter sequence was identified. A chimeric DNA vector consisting of the shrimp U6 promoter with gRNA expression sequences specific to two sites of the WSSV genome and the WSSV ribonucleotide reductase promoter with the Cas9 DNA sequence in pAC-sgRNA-Cas9 was constructed. The expression of gRNAs specific to the WSSV genome and Cas9 was determined in primary cultured hemocyte cells and in shrimp tissue via RT‒PCR. The efficacy of CRISPR/Cas9-WSSV for WSSV genome cleavage was determined in vitro and against WSSV-infected Penaeus vannamei. The reaction of synthetic gRNAs and recombinant Cas9 was able to cleave WSSV DNA amplicons, and shrimp that received CRISPR/Cas9-WSSV presented significantly lower WSSV DNA. In addition to interfering with viral DNA propagation, CRISPR/Cas9-WSSV encapsulated with IHHNV-VLP also stimulated an immune-related gene response. Treatment with CRISPR/Cas9-WSSV against WSSV challenge resulted in a significantly longer survival period. This finding has led to the development and application of a CRISPR/Cas9 system for WSSV infectious disease control, which could be used for managing shrimp aquaculture in the future.

2.6-Å resolution cryo-EM structure of a class Ia ribonucleotide reductase trapped with mechanism-based inhibitor N3CDP

Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides using radical-based chemistry. For class Ia RNRs, the radical species is stored in a separate subunit (β2) from the subunit housing the active site (α2), requiring the formation of a short-lived α2β2 complex and long-range radical transfer (RT). RT occurs via proton-coupled electron transfer (PCET) over a long distance (~32-Å) and involves the formation and decay of multiple amino acid radical species. Here, we use cryogenic electron microscopy and a mechanism-based inhibitor 2'-azido-2'-deoxycytidine-5'-diphosphate (N3CDP) to trap a wild-type α2β2 complex of Escherichia coli class Ia RNR. We find that one α subunit has turned over and that the other is trapped, bound to β in a midturnover state. Instead of N3CDP in the active site, forward RT has resulted in N2 loss, migration of the third nitrogen from the ribose C2' to C3' positions, and attachment of this nitrogen to the sulfur of cysteine-225. In this study, an inhibitor has been visualized as an adduct to an RNR. Additionally, this structure reveals the positions of PCET residues following forward RT, complementing the previous structure that depicted a preturnover PCET pathway and suggesting how PCET is gated at the α-β interface. This N3CDP-trapped structure is also of sufficient resolution (2.6 Å) to visualize water molecules, allowing us to evaluate the proposal that water molecules are proton acceptors and donors as part of the PCET process.

The Current Role of Hydroxyurea in the Treatment of Sickle Cell Anemia.

Despite advancements in treatment of sickle cell disease (SCD), hydroxyurea, a ribonucleotide reductase inhibitor, remains the cornerstone of therapy. While its primary effect is the elevation of fetal hemoglobin (HbF), hydroxyurea's mechanisms of action are multifaceted. Hydroxyurea (HU) reduces leukocyte and platelet counts, decreases the expression of endothelial adhesion molecules CD36 and CD49d, and increases nitric oxide and cyclic nucleotide levels, which may facilitate vascular dilation and further HbF induction. Numerous studies have demonstrated that hydroxyurea therapy reduces the frequency of painful episodes, acute chest syndrome, and the need for erythrocyte transfusions and hospitalizations. Long-term use of hydroxyurea leads to reduced morbidity and mortality. Hydroxyurea should be initiated in children from 9 months of age, including asymptomatic individuals, and is recommended for adults experiencing pain crises that significantly interfere with daily activities or quality of life, as well as those with severe or recurrent vaso-occlusive crises, ACS, or severe symptomatic anemia. Hydroxyurea is not recommended during pregnancy or lactation due to potential teratogenic effects and transfer into breast milk. However, its use may be considered in high-risk patients, particularly during the second and third trimesters. Concerns about secondary tumor development have not been substantiated in long-term follow-up studies. Alternative therapies, including L-glutamine, crizanlizumab, and voxelotor, are not presently approved or available for clinical use in Europe.

Hydroxyurea Pharmacokinetic Evaluation in Patients with Sickle Cell Disease

Background: Hydroxyurea (HU), also known as hydroxycarbamide, is an oral ribonucleotide reductase inhibitor. In 1999, the United States Food and Drug Administration (FDA) approved HU for the treatment of sickle cell disease (SCD). Since then, it has become the cornerstone in the management of SCD patients, helping to reduce vaso-occlusive crises, acute chest syndrome, the need for blood transfusions, hospitalizations and mortality. There is considerable variability among individuals in HU pharmacokinetic (Pk) parameters that can influence treatment efficacy and toxicity. The objective of this work is part of a clinical study aimed at investigating HU Pk and determining the optimal sampling time to estimate the Area Under the Curve (AUC) in SCD patients. Methods: HU plasma concentration in 80 patients at various time points (2, 4, 6, 24 h) following a 48-h drug washout was quantified using High-Pressure Liquid Chromatography (HPLC) coupled with an ultraviolet (UV) detection method previously described in the literature and adapted to new conditions with partial modifications. Results: The mean HU administered dose was 19.5 ± 5.1 mg/kg (range: 7.7–37.5 mg/kg). The median AUC quantified in plasma patients was 101.3 mg/L/h (Interquartile Range (IQR): 72.5–355.9) and it was not influenced by the weight-based dose. However, there was a strong positive correlation between AUC and Body Mass Index (BMI) as well as dose per Body Surface Area (BSA). Along with a three-point approach for AUC determination present in the literature, we show results obtained from a four-point sampling strategy, which is more useful and effective for better optimizing dose escalation to the maximum tolerated dose (MTD). Moreover, we observed that most patients achieved the maximum HU plasma concentration two hours after drug administration, regardless of age differences. Conclusions: HU treatment, which represents a milestone in the treatment of SCD due to its ability to reduce disease complications and improve patients’ quality of life, requires careful monitoring to optimize the individual dose for saving potential side effects and/or adverse events.

MARC1 Is the Main Contributor to Metabolic Reduction of N-Hydroxyurea.

N-Hydroxyurea has been known since the 1960s as an antiproliferative drug and is used both in oncology and for treatment of hematological disorders such as sickle cell anemia where very high daily doses are administered. It is assumed that the cellular effect of N-hydroxyurea is caused by inhibition of ribonucleotide reductase, while alternative mechanisms, e.g., generation of nitric oxide, have also been proposed. Despite its many therapeutic applications, the metabolism of hydroxyurea is largely unexplored. The major elimination pathway of N-hydroxyurea is the reduction to urea. Since the mitochondrial amidoxime reducing component (mARC) is known for its N-reductive activity, we investigated the reduction of NHU by this enzyme system. This study presents in vitro and in vivo evidence that this reductive biotransformation is specifically mediated by the mARC1. Inactivation by mARC1 is a possible explanation for the high doses of NHU required for treatment.

2.6-Å resolution cryo-EM structure of a class Ia ribonucleotide reductase trapped with mechanism-based inhibitor N3CDP.

Several FDA-approved cancer drugs target human ribonucleotide reductase (RNR), a radical enzyme that produces the requisite deoxyribonucleotides for DNA biosynthesis and repair. Human RNR is a class Ia enzyme that requires radical transfer (RT) from a β2 subunit to an α2 subunit on every round of turnover. Long-range RT is both a remarkable feature and an Achilles heel, given that inhibitors can intercept the radical species. Here we present a cryogenic electron microscopy (cryo-EM) structure of the best studied class Ia RNR, the enzyme from E. coli , in which α2 and β2 subunits have been trapped together using a mechanism-based inhibitor. This structure provides insight into both the mechanism of RNR inhibition and the mechanism of long-range RT.

Revised mechanism of hydroxyurea-induced cell cycle arrest and an improved alternative

Replication stress describes endogenous and exogenous challenges to DNA replication in the S-phase. Stress during this critical process causes helicase-polymerase decoupling at replication forks, triggering the S-phase checkpoint, which orchestrates global replication fork stalling and delayed entry into G2. The replication stressor most often used to induce the checkpoint response in yeast is hydroxyurea (HU), a clinically used chemotherapeutic. The primary mechanism of S-phase checkpoint activation by HU has thus far been considered to be a reduction of deoxynucleotide triphosphate synthesis by inhibition of ribonucleotide reductase (RNR), leading to helicase-polymerase decoupling and subsequent activation of the checkpoint, facilitated by the replisome-associated mediator Mrc1. In contrast, we observe that HU causes cell cycle arrest in budding yeast independent of both the Mrc1-mediated replication checkpoint response and the Psk1-Mrc1 oxidative signaling pathway. We demonstrate a direct relationship between HU incubation and reactive oxygen species (ROS) production in yeast and human cells and show that antioxidants restore growth of yeast in HU. We further observe that ROS strongly inhibits the in vitro polymerase activity of replicative polymerases (Pols), Pol α, Pol δ, and Pol ε, causing polymerase complex dissociation and subsequent loss of DNA substrate binding, likely through oxidation of their integral iron-sulfur (Fe-S) clusters. Finally, we present "RNR-deg," a genetically engineered alternative to HU in yeast with greatly increased specificity of RNR inhibition, allowing researchers to achieve fast, nontoxic, and more readily reversible checkpoint activation compared to HU, avoiding harmful ROS generation and associated downstream cellular effects that may confound interpretation of results.

Deferasirox's Anti-Chemoresistance and Anti-Metastatic Effect on Non-Small Cell Lung Carcinoma.

Clinically approved iron chelators, originally designed to address iron overload disorders, have emerged as potential anticancer agents. Deferasirox (Def), a tridentate iron chelator, has demonstrated antiproliferative effects in cancer. Background/Objectives: This study aims to elucidate the mechanism of action of Def and its impact on non-small cell lung carcinoma (NSCLC). Methods: NSCLC A549 cells were treated with Def to assess cytotoxicity, the effect on nuclear and mitochondrial pathways, and iron-containing proteins and genes to evaluate anti-metastasis and chemoresistance. A lung carcinoma mouse model was used for in vivo studies. Results: Our findings revealed that Def induced cytotoxicity, effectively chelated intracellular iron, and triggered apoptosis through the increase in phosphatidylserine externalization and caspase 3 activity. Additionally, Def caused G0/G1 cell cycle arrest by downregulating the ribonucleotide reductase catalytic subunit. Furthermore, Def perturbed mitochondrial function by promoting the production of reactive oxygen species and the inhibition of glutathione as a measurement of ferroptosis activation. Def demonstrated inhibitory effects on cell migration in scratch assays, which was supported by the upregulation of n-myc downstream-regulated gene 1 and downregulation of the epidermal growth factor receptor protein. Also, Def downregulated one of the main markers of chemoresistance, the ABCB1 gene. In vivo experiments using a lung carcinoma mouse model showed that Def treatment did not affect the animal's body weight and showed a significant decrease in tumor growth. Conclusions: This investigation lays the groundwork for unraveling Def action's molecular targets and mechanisms in lung carcinoma, particularly within iron-related pathways, pointing out its anti-metastasis and anti-chemoresistance effect.

Altered dNTP pools accelerate tumor formation in mice.

Alterations in deoxyribonucleoside triphosphate (dNTP) pools have been linked to increased mutation rates and genome instability in unicellular organisms and cell cultures. However, the role of dNTP poolchanges in tumor development in mammals remains unclear. In this study, we present a mouse model with a point mutation at the allosteric specificity site of ribonucleotide reductase, RRM1-Y285A. This mutation reduced ribonucleotide reductase activity, impairing the synthesis of deoxyadenosine triphosphate (dATP) and deoxyguanosinetriphosphate (dGTP). Heterozygous Rrm1+/Y285A mice exhibited distinct alterations in dNTP pools across various organs, shorter lifespans and earlier tumor onset compared with wild-type controls. Mutational spectrum analysis of tumors revealed two distinct signatures, one resembling a signature extracted from a human cancer harboring a mutation of the same amino acid residue in ribonucleotide reductase, RRM1Y285C. Our findings suggest that mutations in enzymes involved in dNTP metabolism can serve as drivers of cancer development.

The five homologous CiaR-controlled Ccn sRNAs of Streptococcus pneumoniae modulate Zn-resistance.

Zinc is a vital transition metal for all bacteria; however, elevated intracellular free Zn levels can result in mis-metalation of Mn-dependent enzymes. For Mn-centric bacteria such as Streptococcus pneumoniae that primarily use Mn instead of Fe as an enzyme cofactor, Zn is particularly toxic at high concentrations. Here, we report our identification and characterization of the function of the five homologous, CiaRH-regulated Ccn sRNAs in controlling S. pneumoniae virulence and metal homeostasis. We show that deletion of all five ccn genes (ccnA, ccnB, ccnC, ccnD, and ccnE) from S. pneumoniae strains D39 (serotype 2) and TIGR4 (serotype 4) causes Zn hypersensitivity and an attenuation of virulence in a murine invasive pneumonia model. We provide evidence that bioavailable Zn disproportionately increases in S. pneumoniae strains lacking the five ccn genes. Consistent with a response to Zn intoxication or relatively high intracellular free Zn levels, expression of genes encoding the CzcD Zn exporter and the Mn-independent ribonucleotide reductase, NrdD-NrdG, were increased in the ΔccnABCDE mutant relative to its isogenic ccn+ parent strain. The growth inhibition by Zn that occurs as the result of loss of the ccn genes is rescued by supplementation with Mn or Oxyrase, a reagent that removes dissolved oxygen. Lastly, we found that the Zn-dependent growth inhibition of the ΔccnABCDE strain was not altered by deletion of sodA, whereas the ccn+ ΔsodA strain phenocopied the ΔccnABCDE strain. Overall, our results indicate that the Ccn sRNAs have a crucial role in preventing Zn intoxication in S. pneumoniae.

Insights into the functional properties of thioredoxin domain-containing protein 12 (TXNDC12): Antioxidant activity, immunological expression, and wound-healing effect in yellowtail clownfish (Amphiprion clarkii)

Thioredoxin domain-containing protein 12 (TXNDC12) is a member of the thioredoxin-like superfamily that contributes to various thiol-dependent metabolic activities in all living organisms. In this research, the TXNDC12 gene from yellowtail clownfish (Amphiprion clarkii) was structurally characterized using in silico tools, assessed for immunological expression, and evaluated for biological activity using recombinant protein and cellular overexpression. The deduced coding sequence of AcTXNDC12 comprised a 522-bp nucleotide, encoding 173 amino acids with a predicted molecular mass of 19.198 kDa. The AcTXNDC12 protein consists of a66WCGAC70 active motif and a170GDEL173 signature. The highest tissue-specific expression of AcTXNDC12 was observed in the brain tissue, with significant modulation observed in the blood and gill tissues following stimulation of polyinosinic: polycytidylic acid, lipopolysaccharides (LPS), and Vibrio harveyi. In functional assays, recombinant AcTXNDC12 protein (rAcTXNDC12) showed insulin disulfide reduction activity, 2,2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) decolorization antioxidant capacity, and ferric (Fe3+) reducing antioxidant potential. Additionally, a significant reduction in nitric oxide production was observed in AcTXNDC12-overexpressed RAW 264.7 cells upon LPS stimulation. Furthermore, genes associated with the regulation of oxidative stress, including nuclear factor erythroid 2-related factor 2 (Nrf2), catalase (Cat), peroxiredoxin 1 (Prx1), and ribonucleotide reductase catalytic subunit M1 (Rrm1) were significantly upregulated in fathead minnow cells overexpressing AcTXNDC12 in response to H2O2 treatment. The scratch wound healing assay demonstrated tissue regeneration and cell proliferation ability upon AcTXNDC12 overexpression. Altogether, the current study elucidated the antioxidant activity, immunological importance, and wound-healing effect of the AcTXNDC12 gene in yellowtail clownfish, providing valuable insights for advancing the aquaculture of A. clarkii fish.

How ATP and dATP Act as Molecular Switches to Regulate Enzymatic Activity in the Prototypical Bacterial Class Ia Ribonucleotide Reductase.

Class Ia ribonucleotide reductases (RNRs) are allosterically regulated by ATP and dATP to maintain the appropriate deoxyribonucleotide levels inside the cell for DNA biosynthesis and repair. RNR activity requires precise positioning of the β2 and α2 subunits for the transfer of a catalytically essential radical species. Excess dATP inhibits RNR through the creation of an α-β interface that restricts the ability of β2 to obtain a position that is capable of radical transfer. ATP breaks the α-β interface, freeing β2 and restoring enzyme activity. Here, we investigate the molecular basis for allosteric activity regulation in the well-studied Escherichia coli class Ia RNR through the determination of six crystal structures and accompanying biochemical and mutagenesis studies. We find that when dATP is bound to the N-terminal regulatory cone domain in α, a helix unwinds, creating a binding surface for β. When ATP displaces dATP, the helix rewinds, dismantling the α-β interface. This reversal of enzyme inhibition requires that two ATP molecules are bound in the cone domain: one in the canonical nucleotide-binding site (site 1) and one in a site (site 2) that is blocked by phenylalanine-87 and tryptophan-28 unless ATP is bound in site 1. When ATP binds to site 1, histidine-59 rearranges, prompting the movement of phenylalanine-87 and trytophan-28, and creating site 2. dATP hydrogen bonds to histidine-59, preventing its movement. The importance of site 2 in the restoration of RNR activity by ATP is confirmed by mutagenesis. These findings have implications for the design of bacterial RNR inhibitors.

Stability of the Tyrosyl Radical in the Ribonucleotide Reductase Beta Subunit of Arcobacter bivalviorum

Arcobacter spp., such as Arcobacter bivalviorum (A. bivalviorum), are free-living organisms found in diverse environments and associated with animals. They are considered emerging enteropathogens and potential zoonotic agents. Ribonucleotide reductase (RNR) is the key enzyme that is used to convert ribonucleotides into deoxyribonucleoside triphosphates (dNTPs). This process utilises radical-based chemistry and is crucial for DNA biosynthesis and repair. There are three RNR classes, with class I RNR the most studied, present in A. bivalviorum, eukaryotes, and many prokaryotes. Class I RNRs are further divided into three subclasses: Ia, Ib, and Ic. Class Ib RNRs use a dimanganese-oxo centre, unlike class Ia RNRs, which use a diiron-oxo centre. A. bivalviorum possesses a class Ia enzyme that requires a diferric tyrosyl radical cofactor located within its beta (β) subunit. Indeed, both the efficiency and fidelity of DNA synthesis are influenced by the stability of the tyrosyl radical (Y•) in the RNR, which is a critical aspect of its enzymatic function. This study investigates the stability of the Y-radical (Y•) site within the RNR β subunit of A. bivalviorum and the nature of the neighbouring amino acid residues. To achieve these goals, we developed a model of the RNR β subunit of A. bivalviorum, using the RNR β subunit of Aquifex aeolicus as a reference template (7aik.1. A PDB). The results provide some important details about the radical site and its surrounding residues, highlighting the influence of the protein structure on the stability of the radical. These findings may guide the development of novel inhibitors targeting this enzyme in A. bivalviorum.

Gallium Uncouples Iron Metabolism to Enhance Glioblastoma Radiosensitivity.

Gallium-based therapy has been considered a potentially effective cancer therapy for decades and has recently re-emerged as a novel therapeutic strategy for the management of glioblastoma tumors. Gallium targets the iron-dependent phenotype associated with aggressive tumors by mimicking iron in circulation and gaining intracellular access through transferrin-receptor-mediated endocytosis. Mechanistically, it is believed that gallium inhibits critical iron-dependent enzymes like ribonucleotide reductase and NADH dehydrogenase (electron transport chain complex I) by replacing iron and removing the ability to transfer electrons through the protein secondary structure. However, information regarding the effects of gallium on cellular iron metabolism is limited. As mitochondrial iron metabolism serves as a central hub of the iron metabolic network, the goal of this study was to investigate the effects of gallium on mitochondrial iron metabolism in glioblastoma cells. Here, it has been discovered that gallium nitrate can induce mitochondrial iron depletion, which is associated with the induction of DNA damage. Moreover, the generation of gallium-resistant cell lines reveals a highly unstable phenotype characterized by impaired colony formation associated with a significant decrease in mitochondrial iron content and loss of the mitochondrial iron uptake transporter, mitoferrin-1. Moreover, gallium-resistant cell lines are significantly more sensitive to radiation and have an impaired ability to repair any sublethal damage and to survive potentially lethal radiation damage when left for 24 h following radiation. These results support the hypothesis that gallium can disrupt mitochondrial iron metabolism and serve as a potential radiosensitizer.

Identification of novel genetic factors that regulate c-di-AMP production in Staphylococcus aureus using a riboswitch-based biosensor.

Nucleotide secondary messengers regulate various processes in bacteria allowing them to rapidly respond to changes in environmental conditions. c-di-AMP is an essential second messenger required for the growth of the human pathogen Staphylococcus aureus, regulating potassium, osmolyte uptake, and beta-lactam resistance. Cellular concentrations of c-di-AMP are regulated by the activities of two enzymes, DacA and GdpP, which synthesize and hydrolyze c-di-AMP, respectively. Besides these, only a limited number of other factors are known to regulate c-di-AMP levels. Using a c-di-AMP biosensor consisting of the Bacillus subtilis c-di-AMP-binding kimA riboswitch and yfp, we were able to efficiently detect differences in cellular c-di-AMP levels in S. aureus. To identify novel factors that regulate c-di-AMP levels, we introduced the biosensor into a library of S. aureus transposon mutants. In this manner, we obtained mutants with increased c-di-AMP levels that contained insertions in gdpP coding for the c-di-AMP hydrolase and ybbR (cdaR) coding for a c-di-AMP cyclase regulator, thus validating our screen. We also identified two high c-di-AMP mutants with insertions upstream of the nrdIEF operon coding for the ribonucleotide reductase enzyme. Further analysis revealed that the insertion down-regulated nrdIEF expression, indicating that the enzyme is a negative regulator of c-di-AMP production. This negative regulation was dependent on rsh, encoding for the synthase of the endogenous GdpP inhibitor (p)ppGpp. The methods established in this work can be readily adapted for use in other bacteria to uncover genetic or environmental factors regulating c-di-AMP levels.IMPORTANCEc-di-AMP is an important secondary messenger, produced by many bacterial species including the opportunistic pathogen Staphylococcus aureus. In this bacterium, c-di-AMP controls cell wall homeostasis, cell size, and osmotic balance. In addition, it has been shown that strains with high c-di-AMP levels exhibit increased resistance to beta-lactam antibiotics. Here, we developed a biosensor-based method for the rapid detection of c-di-AMP levels in S. aureus. We utilized the biosensor in a genetic screen for the identification of novel factors that impact cellular c-di-AMP. In this manner, we identified the ribonucleotide reductase as a novel factor altering cellular c-di-AMP levels and showed that reducing its expression leads to increased cellular c-di-AMP levels. As methicillin-resistant S. aureus strains are considered as a global health threat, it is important to study processes that dictate cellular c-di-AMP levels, which are associated with antibiotic resistance.

Identification of a new human senescent skin cell marker ribonucleoside-diphosphate reductase subunit M2 B.

In skin aging, it has been hypothesized that aging fibroblasts accumulate within the epidermal basal layer, dermis, and subcutaneous fat, causing abnormal tissue remodeling and extracellular matrix dysfunction, thereby inducing an aging-related secretory phenotype (SASP). A new treatment for skin aging involves the specific elimination of senescent skin cells, especially fibroblasts within the dermis and keratinocytes in the basal layer. This requires the identification of specific protein markers of senescent cells, such as ribonucleoside-diphosphate reductase subunit M2 B (RRM2B), which is upregulated in various malignancies in response to DNA stress damage. However, the behavior and role of RRM2B in skin aging remain unclear. Therefore, we examined whether RRM2B functions as a senescence marker using a human dermal fibroblast model of aging. In a model of cellular senescence induced by replicative aging and exposure to ionizing radiation or UVB, RRM2B was upregulated at the gene and protein levels. This was correlated with decreased uptake of the senescence-associated β-galactosidase activity and proliferation marker bromodeoxyuridine. RRM2B upregulation was concurrent with the increased expression of SASP factor genes. Furthermore, using fluorescence flow cytometry, RRM2B-positive cells were recovered more frequently in the aging cell population. In aging human skin, RRM2B was also found to be more abundant in the dermis and epidermal basal layer than other proteins. Therefore, RRM2B may serve as a clinical marker to identify senescent skin cells.

Unveiling the Connection: Viral Infections and Genes in dNTP Metabolism.

Deoxynucleoside triphosphates (dNTPs) are crucial for the replication and maintenance of genomic information within cells. The balance of the dNTP pool involves several cellular enzymes, including dihydrofolate reductase (DHFR), ribonucleotide reductase (RNR), and SAM and HD domain-containing protein 1 (SAMHD1), among others. DHFR is vital for the de novo synthesis of purines and deoxythymidine monophosphate, which are necessary for DNA synthesis. SAMHD1, a ubiquitously expressed deoxynucleotide triphosphohydrolase, converts dNTPs into deoxynucleosides and inorganic triphosphates. This process counteracts the de novo dNTP synthesis primarily carried out by RNR and cellular deoxynucleoside kinases, which are most active during the S phase of the cell cycle. The intracellular levels of dNTPs can influence various viral infections. This review provides a concise summary of the interactions between different viruses and the genes involved in dNTP metabolism.

Conformational control over proton-coupled electron transfer in metalloenzymes.

From the reduction of dinitrogen to the oxidation of water, thechemical transformations catalysed by metalloenzymes underlie global geochemical and biochemical cycles. These reactions represent some of the most kinetically and thermodynamically challenging processes knownand require the complex choreography of the fundamental building blocks of nature, electrons and protons, to be carried out with utmost precision and accuracy. The rate-determining step of catalysis in many metalloenzymes consists of aprotein structural rearrangement, suggestingthat nature has evolved to leverage macroscopic changes in protein molecularstructure to control subatomicchanges in metallocofactor electronicstructure. The proton-coupled electron transfermechanisms operative in nitrogenase, photosystem II and ribonucleotide reductase exemplify this interplay between molecular and electronic structural control. We present the culmination of decades of study on each of these systems and clarify what is known regarding the interplay between structural changesand functional outcomes in these metalloenzyme linchpins.