-
Cell Reports Jul 2023The protein kinase ATR is essential for replication stress responses in all eukaryotes. Ribonucleotide reductase (RNR) catalyzes the formation of deoxyribonucleotide...
The protein kinase ATR is essential for replication stress responses in all eukaryotes. Ribonucleotide reductase (RNR) catalyzes the formation of deoxyribonucleotide (dNTP), the universal building block for DNA replication and repair. However, the relationship between ATR and RNR is not well understood. Here, we show that ATR promotes the protein stability of RNR in Arabidopsis. Through an activation tagging-based genetic screen, we found that overexpression of TSO2, a small subunit of RNR, partially suppresses the hypersensitivity of the atr mutant to replication stress. Biochemically, TSO2 interacts with PRL1, a central subunit of the Cullin4-based E3 ubiquitin ligase CRL4, which polyubiquitinates TSO2 and promotes its degradation. ATR inhibits CRL4 to attenuate TSO2 degradation. Our work provides an important insight into the replication stress responses and a post-translational regulatory mechanism for RNR. Given the evolutionary conservation of the proteins involved, the ATR-PRL1-RNR module may act across eukaryotes.
Topics: Arabidopsis; Arabidopsis Proteins; Ataxia Telangiectasia Mutated Proteins; DNA Damage; DNA Replication; Ribonucleotide Reductases; Ubiquitin-Protein Ligases
PubMed: 37354461
DOI: 10.1016/j.celrep.2023.112685 -
Molecular Medicine Reports Aug 2023Polydeoxyribonucleotide (PDRN) is a mixture of deoxyribonucleotides. It serves as an anti‑inflammatory and tissue‑regenerating agent. The mitogen‑activated protein...
Polydeoxyribonucleotide (PDRN) is a mixture of deoxyribonucleotides. It serves as an anti‑inflammatory and tissue‑regenerating agent. The mitogen‑activated protein kinase pathway modulates cell growth and collagen accumulation. It also regulates inflammation by suppressing the expression of proinflammatory cytokines. In the present study, it was attempted to elucidate the molecular mechanism of PDRN in skin healing by confirming the effects of PDRN treatment on skin keratinocytes and fibroblasts, and by assessing the levels of collagen and inflammatory cytokines regulated by the extracellular signal‑regulated kinase (ERK) pathway. The potential effects of PDRN on skin regeneration were investigated. Fibroblast and keratinocyte proliferation and migration were analyzed using the water‑soluble tetrazolium‑8 and wound healing assays. The upregulation of collagen synthesis by PDRN‑induced ERK activation was analyzed in fibroblasts with or without an ERK inhibitor. Inflammatory cytokine expression levels in keratinocytes were determined using reverse transcription‑quantitative polymerase chain reaction. PDRN promoted the proliferation and migration of keratinocytes and fibroblasts. However, PDRN‑induced ERK phosphorylation differed between keratinocytes and fibroblasts; PDRN increased ERK phosphorylation and collagen accumulation in fibroblasts, while it inhibited matrix metalloproteinase expression. By contrast, PDRN inhibited ERK phosphorylation in keratinocytes, and it decreased inflammatory cytokine expression levels. PDRN affects skin cell proliferation and migration, and collagen and inflammatory cytokine expression levels via ERK signaling. Overall, PDRN exerts a positive effect on skin regeneration, but the mechanism by which it promotes skin regeneration varies among different skin cell types.
Topics: Humans; Phosphorylation; Polydeoxyribonucleotides; Skin; Keratinocytes; Collagen; Extracellular Signal-Regulated MAP Kinases; Cytokines; Fibroblasts
PubMed: 37350391
DOI: 10.3892/mmr.2023.13035 -
Molecular Carcinogenesis Oct 2023RRM2 is the catalytic subunit of ribonucleotide reductase (RNR), which catalyzes de novo synthesis of deoxyribonucleotide triphosphates (dNTPs) and plays critical roles...
RRM2 is the catalytic subunit of ribonucleotide reductase (RNR), which catalyzes de novo synthesis of deoxyribonucleotide triphosphates (dNTPs) and plays critical roles in cancer cell proliferation. RRM2 protein level is controlled by ubiquitination mediated protein degradation system; however, its deubiquitinase has not been identified yet. Here we showed that ubiquitin-specific peptidase 12 (USP12) directly interacts with and deubiquitinates RRM2 in non-small cell lung cancer (NSCLC) cells. Knockdown of USP12 causes DNA replication stress and retards tumor growth in vivo and in vitro. Meanwhile, USP12 protein levels were positively correlated to RRM2 protein levels in human NSCLC tissues. In addition, high expression of USP12 was associated with poor prognosis in NSCLC patients. Therefore, our study reveals that USP12 is a RRM2 regulator and targeting USP12 could be considered as a potential therapeutical strategy for NSCLC treatment.
Topics: Humans; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Lung Neoplasms; Ubiquitin Thiolesterase; Ubiquitination
PubMed: 37341611
DOI: 10.1002/mc.23593 -
Journal of Immunology (Baltimore, Md. :... Aug 2023CMV can elicit adaptive immune features in both mouse and human NK cells. Mouse Ly49H+ NK cells expand 100- to 1000-fold in response to mouse CMV infection and persist...
CMV can elicit adaptive immune features in both mouse and human NK cells. Mouse Ly49H+ NK cells expand 100- to 1000-fold in response to mouse CMV infection and persist for months after exposure. Human NKG2C+ NK cells also expand after human CMV (HCMV) infection and persist for months. The clonal expansion of adaptive NK cells is likely an energy-intensive process, and the metabolic requirements that support adaptive NK cell expansion and persistence remain largely uncharacterized. We previously reported that NK cells from HCMV-seropositive donors had increased maximum capacity for both glycolysis and mitochondrial oxidative phosphorylation relative to NK cells from HCMV-seronegative donors. In this article, we report an extension of this work in which we analyzed the metabolomes of NK cells from HCMV-seropositive donors with NKG2C+ expansions and NK cells from HCMV seronegative donors without such expansions. NK cells from HCMV+ donors exhibited striking elevations in purine and pyrimidine deoxyribonucleotides, along with moderate increases in plasma membrane components. Mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that, as a part of mTOR complex 1 (mTORC1), bridges nutrient signaling to metabolic processes necessary for cell growth. Signaling through mTORC1 induces both nucleotide and lipid synthesis. We observed elevated mTORC1 signaling on activation in both NKG2C- and NKG2C+ NK cells from HCMV+ donors relative to those from HCMV- donors, demonstrating a correlation between higher mTORC1 activity and synthesis of key metabolites for cell growth and division.
Topics: Humans; Animals; Mice; Cytomegalovirus; Cytomegalovirus Infections; Killer Cells, Natural; TOR Serine-Threonine Kinases; Mechanistic Target of Rapamycin Complex 1; Metabolome; NK Cell Lectin-Like Receptor Subfamily C
PubMed: 37341510
DOI: 10.4049/jimmunol.2200851 -
Journal of the American Chemical Society Jul 2023Bacterial glycomes are rich in prokaryote-specific or "rare" sugars that are absent in mammals. Like common sugars found across organisms, rare sugars are typically...
Bacterial glycomes are rich in prokaryote-specific or "rare" sugars that are absent in mammals. Like common sugars found across organisms, rare sugars are typically activated as nucleoside diphosphate sugars (NDP-sugars) by nucleotidyltransferases. In bacteria, the nucleotidyltransferase RmlA initiates the production of several rare NDP-sugars, which in turn regulate downstream glycan assembly through feedback inhibition of RmlA via binding to an allosteric site. , RmlA activates a range of common sugar-1-phosphates to produce NDP-sugars for biochemical and synthetic applications. However, our ability to probe bacterial glycan biosynthesis is hindered by limited chemoenzymatic access to rare NDP-sugars. We postulate that natural feedback mechanisms impact nucleotidyltransferase utility. Here, we use synthetic rare NDP-sugars to identify structural features required for regulation of RmlA from diverse bacterial species. We find that mutation of RmlA to eliminate allosteric binding of an abundant rare NDP-sugar facilitates the activation of noncanonical rare sugar-1-phosphate substrates, as products no longer affect turnover. In addition to promoting an understanding of nucleotidyltransferase regulation by metabolites, this work provides new routes to access rare sugar substrates for the study of important bacteria-specific glycan pathways.
Topics: Animals; Nucleotides; Nucleotidyltransferases; Sugars; Feedback; Bacteria; Nucleoside Diphosphate Sugars; Mammals
PubMed: 37283497
DOI: 10.1021/jacs.3c02319 -
Advanced Healthcare Materials Oct 2023DNA origami technology, a unique type of DNA nanotechnology, has attracted much attention from researchers and is applied in various fields. Through exquisite design and... (Review)
Review
DNA origami technology, a unique type of DNA nanotechnology, has attracted much attention from researchers and is applied in various fields. Through exquisite design and precise self-assembly of four kinds of deoxyribonucleotides, DNA origami nanostructures are endowed with excellent programmability and addressability and show outstanding biocompatibility in bio-related applications, especially in cancer treatment. In this review, nanomaterials based on DNA origami for cancer therapy are concluded, whereby chemotherapy and photo-assisted therapy are the main focus. Furthermore, the working mechanisms of the functional materials attached to the rigid DNA structures to enable targeted delivery and circumvent drug resistance are also discussed. DNA origami nanostructures are valuable carriers for delivering multifunctional therapeutic agents and demonstrate great potential in cancer treatment both in vitro and in vivo. It is undoubted that DNA origami technology is a promising strategy for constructing versatile nanodevices in biological fields and will excel in human healthcare.
Topics: Humans; Nanostructures; DNA; Nanotechnology; Drug Carriers; Neoplasms
PubMed: 37252899
DOI: 10.1002/adhm.202301066 -
Journal of Cellular Physiology Jul 2023Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, participates as a cofactor to one carbon (1C) pathway that produces precursors for DNA...
Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, participates as a cofactor to one carbon (1C) pathway that produces precursors for DNA metabolism. The concerted action of PLP-dependent serine hydroxymethyltransferase (SHMT) and thymidylate synthase (TS) leads to the biosynthesis of thymidylate (dTMP), which plays an essential function in DNA synthesis and repair. PLP deficiency causes chromosome aberrations (CABs) in Drosophila and human cells, rising the hypothesis that an altered 1C metabolism may be involved. To test this hypothesis, we used Drosophila as a model system and found, firstly, that in PLP deficient larvae SHMT activity is reduced by 40%. Second, we found that RNAi-induced SHMT depletion causes chromosome damage rescued by PLP supplementation and strongly exacerbated by PLP depletion. RNAi-induced TS depletion causes severe chromosome damage, but this is only slightly enhanced by PLP depletion. dTMP supplementation rescues CABs in both PLP-deficient and PLP-proficient SHMT . Altogether these data suggest that a reduction of SHMT activity caused by PLP deficiency contributes to chromosome damage by reducing dTMP biosynthesis. In addition, our work brings to light a gene-nutrient interaction between SHMT decreased activity and PLP deficiency impacting on genome stability that may be translated to humans.
Topics: Animals; Humans; Chromosome Aberrations; DNA; Drosophila; Glycine Hydroxymethyltransferase; Pyridoxal Phosphate; Thymidine Monophosphate; Vitamin B 6
PubMed: 37183313
DOI: 10.1002/jcp.31033 -
Journal of Experimental Botany Aug 2023The four-celled stomatal complex consists of a pair of guard cells (GCs) and two subsidiary cells (SCs) in grasses, which supports a fast adjustment of stomatal...
The four-celled stomatal complex consists of a pair of guard cells (GCs) and two subsidiary cells (SCs) in grasses, which supports a fast adjustment of stomatal aperture. The formation and development of SCs are thus important for stomatal functionality. Here, we report a maize lost subsidiary cells (lsc) mutant, with many stomata lacking one or two SCs. The loss of SCs is supposed to have resulted from impeded subsidiary mother cell (SMC) polarization and asymmetrical division. Besides the defect in SCs, the lsc mutant also displays a dwarf morphology and pale and striped newly-grown leaves. LSC encodes a large subunit of ribonucleotide reductase (RNR), an enzyme involved in deoxyribonucleotides (dNTPs) synthesis. Consistently, the concentration of dNTPs and expression of genes involved in DNA replication, cell cycle progression, and SC development were significantly reduced in the lsc mutant compared with the wild-type B73 inbred line. Conversely, overexpression of maize LSC increased dNTP synthesis and promoted plant growth in both maize and Arabidopsis. Our data indicate that LSC regulates dNTP production and is required for SMC polarization, SC differentiation, and growth of maize.
Topics: Zea mays; Ribonucleotide Reductases; Plant Stomata; Poaceae; Cell Differentiation; Arabidopsis
PubMed: 37103989
DOI: 10.1093/jxb/erad153 -
The ISME Journal Jul 2023Phages are prevalent in diverse environments and play major ecological roles attributed to their tremendous diversity and abundance. Among these viruses, transposable...
Phages are prevalent in diverse environments and play major ecological roles attributed to their tremendous diversity and abundance. Among these viruses, transposable phages (TBPs) are exceptional in terms of their unique lifestyle, especially their replicative transposition. Although several TBPs have been isolated and the life cycle of the representative phage Mu has been extensively studied, the diversity distribution and ecological functions of TBPs on the global scale remain unknown. Here, by mining TBPs from enormous microbial genomes and viromes, we established a TBP genome dataset (TBPGD), that expands the number of accessible TBP genomes 384-fold. TBPs are prevalent in diverse biomes and show great genetic diversity. Based on taxonomic evaluations, we propose the categorization of TBPs into four viral groups, including 11 candidate subfamilies. TBPs infect multiple bacterial phyla, and seem to infect a wider range of hosts than non-TBPs. Diverse auxiliary metabolic genes (AMGs) are identified in the TBP genomes, and genes related to glycoside hydrolases and pyrimidine deoxyribonucleotide biosynthesis are highly enriched. Finally, the influences of TBPs on their hosts are experimentally examined by using the marine bacterium Shewanella psychrophila WP2 and its infecting transposable phage SP2. Collectively, our findings greatly expand the genetic diversity of TBPs, and comprehensively reveal their potential influences in various ecosystems.
Topics: Bacteriophages; Ecosystem; Genome, Viral; Viruses; Bacteria
PubMed: 37069234
DOI: 10.1038/s41396-023-01414-z -
Pain Practice : the Official Journal of... Jul 2023Spinal cord stimulation (SCS) has been proven to be an effective treatment for patients suffering from intractable chronic neuropathic pain. Recent advances in the field...
BACKGROUND
Spinal cord stimulation (SCS) has been proven to be an effective treatment for patients suffering from intractable chronic neuropathic pain. Recent advances in the field include the utilization of programs that multiplex various signals to target different neural structures in the dorsal spinal cord associated with the painful area. Preclinical studies have been fundamental in understanding the mechanism by which this differential target multiplexed programming (DTMP) SCS approach works. Transcriptomic- and proteomic-based studies demonstrated that DTMP can modulate expression levels of genes and proteins involved in pain-related processes that have been affected by a neuropathic pain model. This work studied the effect of the intensity of DTMP signals on mechanical hypersensitivity and cell-specific transcriptomes.
METHODS
The spared nerve injury model (SNI) of neuropathic pain was induced in 20 animals which were 1:1 randomized into two SCS groups in which the intensity of the DTMP was adjusted to either 70% or 40% of the motor threshold (MT). SCS was applied continuously for 48 h via a quadripolar lead implanted in the dorsal epidural space of animals. Controls, which included a group of implanted SNI animals that received no SCS and a group of animals naive to the SNI, were assessed in parallel to the SCS groups. Mechanical hypersensitivity was assessed before SNI, before SCS, and at 48 h of SCS. At the end of SCS, the stimulated segment of the dorsal spinal cord was dissected and subjected to RNA sequencing to quantify expression levels in all experimental groups. Differential effects were assessed via fold-change comparisons of SCS and naive groups versus the no-SCS group for transcriptomes specific to neurons and glial cells. Standard statistical analyses were employed to assess significance of the comparisons (p < 0.05).
RESULTS
SCS treatments provided significant improvement in mechanical sensitivity relative to no SCS treatment. However, the change in the intensity did not provide a significant difference in the improvement of mechanical sensitivity. DTMP regulated expression levels back toward those found in the naive group in the cell-specific transcriptomes analyzed. There were no significant differences related to the intensity of the stimulation in terms of the percentage of genes in each transcriptome in which expression levels were reversed toward the naive state.
CONCLUSIONS
DTMP when applied at either 40% MT or 70% MT provided similar reduction of pain-like behavior in rats and similar effects in neuron- and glia-specific transcriptomes.
Topics: Rats; Animals; Pain Threshold; Pain Measurement; Proteomics; Thymidine Monophosphate; Disease Models, Animal; Neuralgia; Spinal Cord; Spinal Cord Stimulation
PubMed: 37067033
DOI: 10.1111/papr.13235