RÉSUMÉ
Bcr-Abl transformation leads to chronic myeloid leukemia (CML). The acquirement of T315I mutation causes tyrosine kinase inhibitors (TKI) resistance. This study develops a compound, JMF4073, inhibiting thymidylate (TMP) and cytidylate (CMP) kinases, aiming for a new therapy against TKI-resistant CML. In vitro and in vivo treatment of JMF4073 eliminates WT-Bcr-Abl-32D CML cells. However, T315I-Bcr-Abl-32D cells are less vulnerable to JMF4073. Evidence is presented that ATF4-mediated upregulation of GSH causes T315I-Bcr-Abl-32D cells to be less sensitive to JMF4073. Reducing GSH biosynthesis generates replication stress in T315I-Bcr-Abl-32D cells that require dTTP/dCTP synthesis for survival, thus enabling JMF4073 susceptibility. It further shows that the levels of ATF4 and GSH in several human CML blast-crisis cell lines are inversely correlated with JMF4073 sensitivity, and the combinatory treatment of JMF4073 with GSH reducing agent leads to synthetic lethality in these CML blast-crisis lines. Altogether, the investigation indicates an alternative option in CML therapy.
Sujet(s)
Glutathion , Leucémie myéloïde chronique BCR-ABL positive , Leucémie myéloïde chronique BCR-ABL positive/traitement médicamenteux , Leucémie myéloïde chronique BCR-ABL positive/métabolisme , Leucémie myéloïde chronique BCR-ABL positive/génétique , Glutathion/métabolisme , Humains , Animaux , Souris , Inhibiteurs de protéines kinases/pharmacologie , Résistance aux médicaments antinéoplasiques/effets des médicaments et des substances chimiques , Lignée cellulaire tumorale , Protéines de fusion bcr-abl/métabolisme , Protéines de fusion bcr-abl/génétique , Protéines de fusion bcr-abl/antagonistes et inhibiteursRÉSUMÉ
NME3 is a member of the nucleoside diphosphate kinase (NDPK) family localized on the mitochondrial outer membrane (MOM). Here, we report a role of NME3 in hypoxia-induced mitophagy dependent on its active site phosphohistidine but not the NDPK function. Mice carrying a knock-in mutation in the Nme3 gene disrupting NME3 active site histidine phosphorylation are vulnerable to ischemia/reperfusion-induced infarction and develop abnormalities in cerebellar function. Our mechanistic analysis reveals that hypoxia-induced phosphatidic acid (PA) on mitochondria is essential for mitophagy and the interaction of DRP1 with NME3. The PA binding function of MOM-localized NME3 is required for hypoxia-induced mitophagy. Further investigation demonstrates that the interaction with active NME3 prevents DRP1 susceptibility to MUL1-mediated ubiquitination, thereby allowing a sufficient amount of active DRP1 to mediate mitophagy. Furthermore, MUL1 overexpression suppresses hypoxia-induced mitophagy, which is reversed by co-expression of ubiquitin-resistant DRP1 mutant or histidine phosphorylatable NME3. Thus, the site-specific interaction with active NME3 provides DRP1 a microenvironment for stabilization to proceed the segregation process in mitophagy.
Sujet(s)
Dynamines , Mitophagie , Animaux , Souris , Dynamines/génétique , Dynamines/métabolisme , Histidine/métabolisme , Hypoxie , Mitophagie/génétique , UbiquitinationRÉSUMÉ
The Akt-Rheb-mTORC1 pathway plays a crucial role in regulating cell growth, but the mechanisms underlying the activation of Rheb-mTORC1 by Akt remain unclear. In our previous study, we found that CBAP was highly expressed in human T-ALL cells and primary tumors, and its deficiency led to reduced phosphorylation of TSC2/S6K1 signaling proteins as well as impaired cell proliferation and leukemogenicity. We also demonstrated that CBAP was required for Akt-mediated TSC2 phosphorylation in vitro. In response to insulin, CBAP was also necessary for the phosphorylation of TSC2/S6K1 and the dissociation of TSC2 from the lysosomal membrane. Here we report that CBAP interacts with AKT and TSC2, and knockout of CBAP or serum starvation leads to an increase in TSC1 in the Akt/TSC2 immunoprecipitation complexes. Lysosomal-anchored CBAP was found to override serum starvation and promote S6K1 and 4EBP1 phosphorylation and c-Myc expression in a TSC2-dependent manner. Additionally, recombinant CBAP inhibited the GAP activity of TSC2 complexes in vitro, leading to increased Rheb-GTP loading, likely due to the competition between TSC1 and CBAP for binding to the HBD domain of TSC2. Overexpression of the N26 region of CBAP, which is crucial for binding to TSC2, resulted in a decrease in mTORC1 signaling and an increase in TSC1 association with the TSC2/AKT complex, ultimately leading to increased GAP activity toward Rheb and impaired cell proliferation. Thus, we propose that CBAP can modulate the stability of TSC1-TSC2 as well as promote the translocation of TSC1/TSC2 complexes away from lysosomes to regulate Rheb-mTORC1 signaling.
Sujet(s)
Complexe-1 cible mécanistique de la rapamycine , Protéines membranaires , Protéines proto-oncogènes c-akt , Protéine-1 du complexe de la sclérose tubéreuse , Protéine-2 du complexe de la sclérose tubéreuse , Humains , Prolifération cellulaire , Guanosine triphosphate/métabolisme , Immunoprécipitation , Lysosomes/métabolisme , Complexe-1 cible mécanistique de la rapamycine/métabolisme , Protéines membranaires/déficit , Protéines membranaires/génétique , Protéines membranaires/métabolisme , Phosphorylation , Protéines proto-oncogènes c-akt/métabolisme , Protéine homologue de Ras enrichie dans le cerveau/métabolisme , Sérine-thréonine kinases TOR/métabolisme , Protéine-1 du complexe de la sclérose tubéreuse/métabolisme , Protéine-2 du complexe de la sclérose tubéreuse/métabolismeRÉSUMÉ
Mitochondria are dynamic organelles regulated by fission and fusion processes. The fusion of membranes requires elaborative coordination of proteins and lipids and is particularly crucial for the function and quality control of mitochondria. Phosphatidic acid (PA) on the mitochondrial outer membrane generated by PLD6 facilitates the fusion of mitochondria. However, how PA promotes mitochondrial fusion remains unclear. Here, we show that a mitochondrial outer membrane protein, NME3, is required for PLD6-induced mitochondrial tethering or clustering. NME3 is enriched at the contact interface of two closely positioned mitochondria depending on PLD6, and NME3 binds directly to PA-exposed lipid packing defects via its N-terminal amphipathic helix. The PA binding function and hexamerization confer NME3 mitochondrial tethering activity. Importantly, nutrient starvation enhances the enrichment efficiency of NME3 at the mitochondrial contact interface, and the tethering ability of NME3 contributes to fusion efficiency. Together, our findings demonstrate NME3 as a tethering protein promoting selective fusion between PLD6-remodeled mitochondria for quality control.
Sujet(s)
Mitochondries , NM23 Nucleoside Diphosphate kinases , Acides phosphatidiques , Phospholipase D , Humains , Mitochondries/métabolisme , Dynamique mitochondriale , Membranes mitochondriales/métabolisme , Protéines mitochondriales/génétique , Protéines mitochondriales/métabolisme , NM23 Nucleoside Diphosphate kinases/métabolisme , Acides phosphatidiques/métabolisme , Phospholipase D/métabolismeRÉSUMÉ
This study used DNA methyltransferase 3b (DNMT3b) knockout cells and the functional loss of DNMT3b mutation in immunodeficiency-centromeric instability-facial anomalies syndrome (ICF) cells to understand how DNMT3b dysfunction causes genome instability. We demonstrated that R-loops contribute to DNA damages in DNMT3b knockout and ICF cells. More prominent DNA damage signal in DNMT3b knockout cells was due to the loss of DNMT3b expression and the acquirement of p53 mutation. Genome-wide ChIP-sequencing mapped DNA damage sites at satellite repetitive DNA sequences including (peri-)centromere regions. However, the steady-state levels of (peri-)centromeric R-loops were reduced in DNMT3b knockout and ICF cells. Our analysis indicates that XPG and XPF endonucleases-mediated cleavages remove (peri-)centromeric R-loops to generate DNA beaks, causing chromosome instability. DNMT3b dysfunctions clearly increase R-loops susceptibility to the cleavage process. Finally, we showed that DNA double-strand breaks (DSBs) in centromere are probably repaired by error-prone end-joining pathway in ICF cells. Thus, DNMT3 dysfunctions undermine the integrity of centromere by R-loop-mediated DNA damages and repair.
Sujet(s)
Déficits immunitaires , Structures en boucle R , Animaux , Centromère/génétique , Centromère/métabolisme , ADN/métabolisme , DNA (cytosine-5-)-methyltransferase/génétique , DNA (cytosine-5-)-methyltransferase/métabolisme , Altération de l'ADN/génétique , Méthylation de l'ADN , Déficits immunitaires/génétique , Déficits immunitaires/métabolisme , Mutation ,RÉSUMÉ
Measurement of four dNTP pools is important for investigating metabolism, genome stability, and drug action. In this report, we developed a two-step method for quantitating dNTPs by the combination of rolling circle amplification (RCA) and quantitative polymerase chain reaction (qPCR). We used CircLigase to generate a single-strand DNA in circular monomeric configuration, which was then used for the first step of RCA reaction that contained three dNTPs in excess for quantification of one dNTP at limiting levels. The second step is the amplification of RCA products by qPCR, in which one primer was designed to be completely annealed with the polymeric ssDNA product but not the monomeric template DNA. Using 1 amol of the template in the assay, each dNTP from 0.02 to 2.5 pmol gave a linearity with r2 > 0.99, and the quantification was not affected by the presence of rNTPs. We further found that the preparation of biological samples for the RCA reaction required methanol and chloroform extraction. The method was so sensitive that 1 × 104 cells were sufficient for dNTP quantification with the results similar to those determined by a radio-isotope method using 2 × 105 cells. Thus, the RCA/qPCR method is convenient, cost-effective, and highly sensitive for dNTP quantification.
Sujet(s)
ADN , Polyphosphates , Dosage biologique , ADN/génétique , Techniques d'amplification d'acides nucléiques , Réaction de polymérisation en chaîneRÉSUMÉ
Mitochondria regulate the immune response after dengue virus (DENV) infection. Microarray analysis of genes identified the upregulation of mitochondrial cytidine/uridine monophosphate kinase 2 (CMPK2) by DENV infection. We used small interfering RNA-mediated knockdown (KD) and CRISPR-Cas9 knockout (KO) approaches, to investigate the role of CMPK2 in mouse and human cells. The results showed that CMPK2 was critical in DENV-induced antiviral cytokine release and mitochondrial oxidative stress and mitochondrial DNA release to the cytosol. The DENV-induced activation of Toll-like receptor (TLR)-9, inflammasome pathway, and cell migration was suppressed by CMPK2 depletion; however, viral production increased under CMPK2 deficiency. Examining mouse bone marrow-derived dendritic cells from interferon-alpha (IFN-α) receptor-KO mice and signal transducer and activator of transcription 1 (STAT1)-KO mice, we confirmed that CMPK2-mediated antiviral activity occurred in IFN-dependent and IFN-independent manners. In sum, CMPK2 is a critical factor in DENV-induced immune responses to determine innate immunity.
RÉSUMÉ
Genotoxic insult causes nuclear and mitochondrial DNA damages with macroautophagy/autophagy induction. The role of mitochondrial DNA (mtDNA) damage in the requirement of autophagy for nuclear DNA (nDNA) stability is unclear. Using site-specific DNA damage approaches, we show that specific nDNA damage alone does not require autophagy for repair unless in the presence of mtDNA damage. We provide evidence that after IR exposure-induced mtDNA and nDNA damages, autophagy suppression causes non-apoptotic mitochondrial permeability, by which mitochondrial ENDOG (endonuclease G) is released and translocated to nuclei to sustain nDNA damage in a TET (tet methylcytosine dioxygenase)-dependent manner. Furthermore, blocking lysosome function is sufficient to increase the amount of mtDNA leakage to the cytosol, accompanied by ENDOG-free mitochondrial puncta formation with concurrent ENDOG nuclear accumulation. We proposed that autophagy eliminates the mitochondria specified by mtDNA damage-driven mitochondrial permeability to prevent ENDOG-mediated genome instability. Finally, we showed that HBx, a hepatitis B viral protein capable of suppressing autophagy, also causes mitochondrial permeability-dependent ENDOG mis-localization in nuclei and is linked to hepatitis B virus (HBV)-mediated hepatocellular carcinoma development.Abbreviations: 3-MA: 3-methyladenine; 5-hmC: 5-hydroxymethylcytosine; ACTB: actin beta; ATG5: autophagy related 5; ATM: ATM serine/threonine kinase; DFFB/CAD: DNA fragmentation factor subunit beta; cmtDNA: cytosolic mitochondrial DNA; ConA: concanamycin A; CQ: chloroquine; CsA: cyclosporin A; Dox: doxycycline; DSB: double-strand break; ENDOG: endonuclease G; GFP: green fluorescent protein; Gy: gray; H2AX: H2A.X variant histone; HBV: hepatitis B virus; HBx: hepatitis B virus X protein; HCC: hepatocellular carcinoma; I-PpoI: intron-encoded endonuclease; IR: ionizing radiation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOMP: mitochondrial outer membrane permeability; mPTP: mitochondrial permeability transition pore; mtDNA: mitochondrial DNA; nDNA: nuclear DNA; 4-OHT: 4-hydroxytamoxifen; rDNA: ribosomal DNA; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TET: tet methylcytosine dioxygenase; TFAM: transcription factor A, mitochondrial; TOMM20: translocase of outer mitochondrial membrane 20; VDAC: voltage dependent anion channel.
Sujet(s)
Autophagie/génétique , Altération de l'ADN , ADN mitochondrial/génétique , ADN mitochondrial/métabolisme , Endodeoxyribonucleases/métabolisme , Instabilité du génome , Transport nucléaire actif , Carcinome hépatocellulaire/métabolisme , Lignée cellulaire tumorale , Protéines de liaison à l'ADN/antagonistes et inhibiteurs , Protéines de liaison à l'ADN/génétique , Protéines de liaison à l'ADN/métabolisme , Dioxygenases/antagonistes et inhibiteurs , Dioxygenases/génétique , Dioxygenases/métabolisme , Endodeoxyribonucleases/antagonistes et inhibiteurs , Endodeoxyribonucleases/génétique , Techniques de knock-down de gènes , Cellules HeLa , Humains , Tumeurs du foie/métabolisme , Mitochondries/enzymologie , Mitochondries/génétique , PerméabilitéRÉSUMÉ
Peroxisomes proliferate by sequential processes comprising elongation, constriction, and scission of peroxisomal membrane. It is known that the constriction step is mediated by a GTPase named dynamin-like protein 1 (DLP1) upon efficient loading of GTP. However, mechanism of fuelling GTP to DLP1 remains unknown in mammals. We earlier show that nucleoside diphosphate (NDP) kinase-like protein, termed dynamin-based ring motive-force organizer 1 (DYNAMO1), generates GTP for DLP1 in a red alga, Cyanidioschyzon merolae. In the present study, we identified that nucleoside diphosphate kinase 3 (NME3), a mammalian homologue of DYNAMO1, localizes to peroxisomes. Elongated peroxisomes were observed in cells with suppressed expression of NME3 and fibroblasts from a patient lacking NME3 due to the homozygous mutation at the initiation codon of NME3. Peroxisomes proliferated by elevation of NME3 upon silencing the expression of ATPase family AAA domain containing 1, ATAD1. In the wild-type cells expressing catalytically-inactive NME3, peroxisomes were elongated. These results suggest that NME3 plays an important role in peroxisome division in a manner dependent on its NDP kinase activity. Moreover, the impairment of peroxisome division reduces the level of ether-linked glycerophospholipids, ethanolamine plasmalogens, implying the physiological importance of regulation of peroxisome morphology.
Sujet(s)
Dynamines/métabolisme , NM23 Nucleoside Diphosphate kinases/métabolisme , Péroxysomes/métabolisme , Fractions subcellulaires/métabolisme , Séquence d'acides aminés , Animaux , Cellules HeLa , Homozygote , Humains , Rhodophyta , Similitude de séquencesRÉSUMÉ
NME3 is a member of the nucleoside diphosphate kinase (NDPK) family that binds to the mitochondrial outer membrane to stimulate mitochondrial fusion. In this study, we showed that NME3 knockdown delayed DNA repair without reducing the cellular levels of nucleotide triphosphates. Further analyses revealed that NME3 knockdown increased fragmentation of mitochondria, which in turn led to mitochondrial oxidative stress-mediated DNA single-strand breaks (SSBs) in nuclear DNA. Re-expression of wild-type NME3 or inhibition of mitochondrial fission markedly reduced SSBs and facilitated DNA repair in NME3 knockdown cells, while expression of N-terminal deleted mutant defective in mitochondrial binding had no rescue effect. We further showed that disruption of mitochondrial fusion by knockdown of NME4 or MFN1 also caused mitochondrial oxidative stress-mediated genome instability. In conclusion, the contribution of NME3 to redox-regulated genome stability lies in its function in mitochondrial fusion.
Sujet(s)
Altération de l'ADN , Mitochondries/métabolisme , NM23 Nucleoside Diphosphate kinases/métabolisme , Stress oxydatif , Espèces réactives de l'oxygène/métabolisme , Techniques de knock-down de gènes , Instabilité du génome , Cellules HEK293 , Cellules HeLa , Humains , Mitochondries/génétique , NM23 Nucleoside Diphosphate kinases/génétiqueRÉSUMÉ
The levels of the four deoxynucleoside triphosphates (dNTPs) are under strict control in the cell, as improper or imbalanced dNTP pools may lead to growth defects and oncogenesis. Upon treatment of cancer cells with therapeutic agents, changes in the canonical dNTPs levels may provide critical information for evaluating drug response and mode of action. The radioisotope-labeling enzymatic assay has been commonly used for quantitation of cellular dNTP levels. However, the disadvantage of this method is the handling of biohazard materials. Here, we described the use of click chemistry to replace radioisotope-labeling in template-dependent DNA polymerization for quantitation of the four canonical dNTPs. Specific oligomers were designed for dCTP, dTTP, dATP and dGTP measurement, and the incorporation of 5-ethynyl-dUTP or C8-alkyne-dCTP during the polymerization reaction allowed for fluorophore conjugation on immobilized oligonucleotides. The four reactions gave a linear correlation coefficient >0.99 in the range of the concentration of dNTPs present in 106 cells, with little interference of cellular rNTPs. We present evidence indicating that data generated by this methodology is comparable to radioisotope-labeling data. Furthermore, the design and utilization of a robust microplate assay based on this technology evidenced the modulation of dNTPs in response to different chemotherapeutic agents in cancer cells.
Sujet(s)
Chimie click/méthodes , Cuivre/composition chimique , Désoxyribonucléotides/analyse , Nucléotides désoxyuridyliques/composition chimique , Réaction de cycloaddition , Nucléotide désoxyadenylique/analyse , Nucléotide désoxyadenylique/composition chimique , Nucléotides désoxycytidyliques/analyse , Nucléotides désoxycytidyliques/composition chimique , Nucléotide désoxyguanylique/analyse , Nucléotide désoxyguanylique/composition chimique , Désoxyribonucléotides/composition chimique , Cellules HCT116 , Cellules HEK293 , Humains , Cellules K562 , Rhodamines/composition chimique , Coloration et marquage , Nucléotides thymidyliques/analyse , Nucléotides thymidyliques/composition chimiqueRÉSUMÉ
The yeast Candida albicans is the most prevalent opportunistic fungal pathogen in humans. Drug resistance among C. albicans isolates poses a common challenge, and overcoming this resistance represents an unmet need in managing this common pathogen. Here, we investigated CDC8, encoding thymidylate kinase (TMPK), as a potential drug target for the management of C. albicans infections. We found that the region spanning amino acids 106-123, namely the Ca-loop of C. albicans TMPK (CaTMPK), contributes to the hyperactivity of this enzyme compared with the human enzyme (hTMPK) and to the utilization of deoxyuridine monophosphate (dUMP)/deoxy-5-fluorouridine monophosphate (5-FdUMP) as a substrate. Notably, expression of CaTMPK, but not of hTMPK, produced dUTP/5-FdUTP-mediated DNA toxicity in budding yeast (Saccharomyces cerevisiae). CRISPR-mediated deletion of this Ca-loop in C. albicans revealed that the Ca-loop is critical for fungal growth and susceptibility to 5-fluorouridine (5-FUrd). Of note, pathogenic and drug-resistant C. albicans clones were similarly sensitive to 5-FUrd, and we also found that CaTMPK is essential for the growth of C. albicans In conclusion, these findings not only identified a target site for the development of CaTMPK-selective drugs, but also revealed that 5-FUrd may have potential utility as drug for managing C. albicans infections.
Sujet(s)
Candida albicans/enzymologie , Protéines fongiques/composition chimique , Nucleoside phosphate kinase/composition chimique , Pyrimidines/pharmacologie , Séquence d'acides aminés , Antifongiques/pharmacologie , Candida albicans/effets des médicaments et des substances chimiques , Candida albicans/croissance et développement , Protéines fongiques/génétique , Protéines fongiques/métabolisme , Édition de gène , Humains , Cinétique , Tests de sensibilité microbienne , Nucleoside phosphate kinase/génétique , Nucleoside phosphate kinase/métabolisme , Protéines recombinantes/génétique , Protéines recombinantes/isolement et purification , Protéines recombinantes/métabolisme , Alignement de séquences , Spécificité du substrat , Uridine/analogues et dérivés , Uridine/pharmacologie , Uridine monophosphate/composition chimique , Uridine monophosphate/métabolismeRÉSUMÉ
We report a patient who presented with congenital hypotonia, hypoventilation, and cerebellar histopathological alterations. Exome analysis revealed a homozygous mutation in the initiation codon of the NME3 gene, which encodes an NDP kinase. The initiation-codon mutation leads to deficiency in NME3 protein expression. NME3 is a mitochondrial outer-membrane protein capable of interacting with MFN1/2, and its depletion causes dysfunction in mitochondrial dynamics. Consistently, the patient's fibroblasts were characterized by a slow rate of mitochondrial dynamics, which was reversed by expression of wild-type or catalytic-dead NME3. Moreover, glucose starvation caused mitochondrial fragmentation and cell death in the patient's cells. The expression of wild-type and catalytic-dead but not oligomerization-attenuated NME3 restored mitochondrial elongation. However, only wild-type NME3 sustained ATP production and viability. Thus, the separate functions of NME3 in mitochondrial fusion and NDP kinase cooperate in metabolic adaptation for cell survival in response to glucose starvation. Given the critical role of mitochondrial dynamics and energy requirements in neuronal development, the homozygous mutation in NME3 is linked to a fatal mitochondrial neurodegenerative disorder.
Sujet(s)
Adénosine triphosphate , Métabolisme énergétique/génétique , Homozygote , Dynamique mitochondriale/génétique , NM23 Nucleoside Diphosphate kinases , Maladies neurodégénératives , Adénosine triphosphate/génétique , Adénosine triphosphate/métabolisme , Lignée cellulaire , Survie cellulaire , Femelle , Humains , Mâle , Mitochondries/enzymologie , Mitochondries/génétique , Mitochondries/anatomopathologie , NM23 Nucleoside Diphosphate kinases/génétique , NM23 Nucleoside Diphosphate kinases/métabolisme , Maladies neurodégénératives/enzymologie , Maladies neurodégénératives/génétique , Maladies neurodégénératives/anatomopathologieRÉSUMÉ
Cellular supply of deoxythymidine triphosphate (dTTP) is crucial for DNA replication and repair. Thymidylate kinase (TMPK) catalyzes the conversion of thymidine monophosphate to thymidine diphosphate, which is an essential step for dTTP synthesis. Despite their major cellular localization in cytosol, TMPK and ribonucleotide reductase (RNR) are detected at DNA damage sites for local dNDP formation. Because deoxyuridine diphosphate is synthesized by RNR, the simultaneous recruitment of TMPK and RNR to DNA damage sites is critical for preventing deoxyuridine triphosphate-mediated toxic repair. This study investigates the mechanism responsible for the recruitment of TMPK to DNA damage sites. Our data demonstrate the requirement of ataxia telangiectasia mutated (ATM) kinase activity for TMPK recruitment to DNA lesion sites. Moreover, we find that TMPK is able to form the complex with histone acetyltransferase Tip60 and RNR. Inhibition of ATM kinase reduces the complex formation and TMPK phosphorylation. Our analysis further shows the presence of TMPK phosphorylation at serine 88, which is an ATM kinase consensus site. A phosphorylation-defective mutation at this site suppresses TMPK recruitment to DNA damage sites and the complex formation with Tip60. Finally, we provide evidence that this site is critical for the function of TMPK in DNA repair but not for catalytic activity. Together, these findings suggest that Tip60-ATM signaling has a functional contribution to the recruitment of TMPK to DNA damage sites, thereby increasing local dTTP synthesis for DNA repair.-Hu, C.-M., Tsao, N., Wang, Y.-T., Chen, Y.-J., Chang, Z.-F. Thymidylate kinase is critical for DNA repair via ATM-dependent Tip60 complex formation.
Sujet(s)
Protéines mutées dans l'ataxie-télangiectasie/métabolisme , Altération de l'ADN , Réparation de l'ADN , Lysine acetyltransferase 5/métabolisme , Complexes multienzymatiques/métabolisme , Nucleoside phosphate kinase/métabolisme , Protéines mutées dans l'ataxie-télangiectasie/génétique , Cellules HEK293 , Cellules HeLa , Humains , Lysine acetyltransferase 5/génétique , Complexes multienzymatiques/génétique , Nucleoside phosphate kinase/génétique , Phosphorylation/génétique , Ribonucleotide reductases/génétique , Ribonucleotide reductases/métabolismeRÉSUMÉ
T cells are a versatile immune cell population responding to challenges by differentiation and proliferation followed by contraction and memory formation. Dynamic metabolic reprogramming is essential for T cells to meet the biosynthetic needs and the reutilization of biomolecules, processes that require active participation of metabolite transporters. Here, we show that equilibrative nucleoside transporter 3 (ENT3) is highly expressed in peripheral T cells and has a key role in maintaining T cell homeostasis by supporting the proliferation and survival of T cells. ENT3 deficiency leads to an enlarged and disturbed lysosomal compartment, resulting in accumulation of surplus mitochondria, elevation of intracellular reactive oxygen species, and DNA damage in T cells. Our results identify ENT3 as a vital metabolite transporter that supports T cell homeostasis and activation by regulating lysosomal integrity and the availability of nucleosides. Moreover, we uncovered that T cell lysosomes are an important source of salvaged metabolites for survival and proliferation.
Sujet(s)
Homéostasie , Lysosomes/métabolisme , Transporteurs de nucléosides/métabolisme , Nucléosides/métabolisme , Lymphocytes T/métabolisme , Animaux , Prolifération cellulaire , Taille de la cellule , Survie cellulaire , ADN/biosynthèse , Réparation de l'ADN , Lymphopénie/immunologie , Lymphopénie/anatomopathologie , Lysosomes/ultrastructure , Souris , Mitochondries/métabolisme , Phénotype , Espèces réactives de l'oxygène/métabolisme , Lymphocytes T/cytologie , Lymphocytes T/ultrastructureRÉSUMÉ
The innate immune response is a central process that is activated during pathogenic infection in order to maintain physiological homeostasis. It is well known that dexamethasone (Dex), a synthetic glucocorticoid, is a potent immunosuppressant that inhibits the cytokine production induced by bacterial lipopolysaccharides (LPS). Nevertheless, the extent to which the functional groups of Dex control the excessive activation of inflammatory reactions remains unknown. Furthermore, importantly, the role of Dex in the innate immune response remains unclear. Here we explore the mechanism of LPS-induced TNF-α secretion and reveal p38 MAPK signaling as a target of Dex that is involved in control of tumor necrosis factor-α (TNF-α)-converting enzyme (TACE) activity; that later mediates the shedding of TNF-α that allows its secretion. We further demonstrate that the 11-hydroxyl and 21-hydroxyl groups of Dex are the main groups that are involved in reducing LPS-induced TNF-α secretion by activated macrophages. Blockage of the hydroxyl groups of Dex inhibits immunosuppressant effect of Dex during LPS-induced TNF-α secretion and mouse mortality. Our findings demonstrate Dex signaling is involved in the control of innate immunity.
Sujet(s)
Anti-inflammatoires/pharmacologie , Dexaméthasone/pharmacologie , Inflammation/étiologie , Inflammation/métabolisme , Lipopolysaccharides/effets indésirables , Facteur de nécrose tumorale alpha/métabolisme , Protéine ADAM17/métabolisme , Acétylation , Animaux , Anti-inflammatoires/composition chimique , Dexaméthasone/composition chimique , Hydroxydes/composition chimique , Inflammation/traitement médicamenteux , Inflammation/mortalité , Système de signalisation des MAP kinases/effets des médicaments et des substances chimiques , Activation des macrophages , Macrophages/effets des médicaments et des substances chimiques , Macrophages/immunologie , Macrophages/métabolisme , Souris , Modèles biologiques , Structure moléculaire , Cellules RAW 264.7RÉSUMÉ
In this study, we investigated the dosage effect of gemcitabine, an inhibitor of ribonucleotide reductase (RR), on cellular levels of ribonucleotides and deoxyribonucleotides using high performance liquid chromatography-electrospray ionization tandem mass spectrometric method. As anticipated, after 4-h incubation of non-small cell lung cancer (A549) cells with gemcitabine at 0.5 and 2 µM, there were consistent reductions in levels of deoxyribonucleoside diphosphates (dNDP) and their corresponding deoxyribonucleoside triphosphates (dNTP). However, after 24-h exposure to 0.5 µM gemcitabine, the amounts of dNTP were increased by about 3 fold, whereas cells after 24-h 2 µM gemcitabine treatment exhibited deoxycytidine diphosphate (dCDP), deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) levels less than 50% of control values, with deoxycytidine triphosphate (dCTP) and deoxyguanosine triphosphate (dGTP) returning to the control level. Using cell cycle analysis, we found that 24-h incubation at 0.5 µM gemcitabine resulted in a significant increase in S phase arrest, while 2 µM treatment increased G0/G1 population. Our data demonstrated the correlation between the level of RR and the increased levels of dNTPs in the group of 0.5 µM treatment for 24-h with a markedly reduced level of dFdCTP. Accordingly, we proposed that the dosage of dFdC could determine the arrested phase of cell cycle, in turn affecting the recovery of dNTPs pools.
Sujet(s)
Carcinome pulmonaire non à petites cellules/traitement médicamenteux , Désoxycytidine/analogues et dérivés , Désoxyribonucléotides/métabolisme , Tumeurs du poumon/traitement médicamenteux , Ribonucléotides/métabolisme , Cellules A549 , Carcinome pulmonaire non à petites cellules/métabolisme , Carcinome pulmonaire non à petites cellules/anatomopathologie , Désoxycytidine/pharmacologie , Relation dose-effet des médicaments , Phase G1/effets des médicaments et des substances chimiques , Humains , Tumeurs du poumon/métabolisme , Tumeurs du poumon/anatomopathologie , Phase G0/effets des médicaments et des substances chimiques , Points de contrôle de la phase S du cycle cellulaire/effets des médicaments et des substances chimiques ,RÉSUMÉ
Targeting thymidylate kinase (TMPK) that catalyzes the phosphotransfer reaction for formation of dTDP from dTMP is a new strategy for anticancer treatment. This study is to understand the inhibitory mechanism of a previously identified human TMPK (hTMPK) inhibitor YMU1 (1a) by molecular docking, isothermal titration calorimetry, and photoaffinity labeling. The molecular dynamics simulation suggests that 1a prefers binding at the catalytic site of hTMPK, whereas the hTMPK inhibitors that bear pyridino[d]isothiazolone or benzo[d]isothiazolone core structure in lieu of the dimethylpyridine-fused isothiazolone moiety in 1a can have access to both the ATP-binding and catalytic sites. The binding sites of hTMPK inhibitors were validated by photoaffinity labeling and mass spectrometric studies. Taking together, 1a and its analogues stabilize the conformation of ligand-induced degradation (LID) region of hTMPK and block the catalytic site or ATP-binding site, thus attenuating the ATP binding-induced closed conformation that is required for phosphorylation of dTMP.