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1.
BMC Genomics ; 25(1): 350, 2024 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-38589807

RESUMO

BACKGROUND: In Eukaryotes, inositol polyphosphates (InsPs) represent a large family of secondary messengers and play crucial roes in various cellular processes. InsPs are synthesized through a series of pohophorylation reactions catalyzed by various InsP kinases in a sequential manner. Inositol 1,4,5-trisphosphate 3-kinase (IP3 3-kinase/IP3K), one member of InsP kinase, plays important regulation roles in InsPs metabolism by specifically phosphorylating inositol 1,4,5-trisphosphate (IP3) to inositol 1,3,4,5-tetrakisphosphate (IP4) in animal cells. IP3Ks were widespread in fungi, plants and animals. However, its evolutionary history and patterns have not been examined systematically. RESULTS: A total of 104 and 31 IP3K orthologues were identified across 57 plant genomes and 13 animal genomes, respectively. Phylogenetic analyses indicate that IP3K originated in the common ancestor before the divergence of fungi, plants and animals. In most plants and animals, IP3K maintained low-copy numbers suggesting functional conservation during plant and animal evolution. In Brassicaceae and vertebrate, IP3K underwent one and two duplication events, respectively, resulting in multiple gene copies. Whole-genome duplication (WGD) was the main mechanism for IP3K duplications, and the IP3K duplicates have experienced functional divergence. Finally, a hypothetical evolutionary model for the IP3K proteins is proposed based on phylogenetic theory. CONCLUSION: Our study reveals the evolutionary history of IP3K proteins and guides the future functions of animal, plant, and fungal IP3K proteins.


Assuntos
Inositol 1,4,5-Trifosfato , Fosfotransferases (Aceptor do Grupo Álcool) , Animais , Inositol 1,4,5-Trifosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Filogenia , Plantas/genética , Plantas/metabolismo , Evolução Molecular
2.
Int J Mol Sci ; 25(5)2024 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-38473803

RESUMO

Mevalonate kinase (MevK) is an important enzyme in the mevalonate pathway that catalyzes the phosphorylation of mevalonate into phosphomevalonate and is involved in juvenile hormone biosynthesis. Herein, we present a structure model of MevK from the red flour beetle Tribolium castaneum (TcMevK), which adopts a compact α/ß conformation that can be divided into two parts: an N-terminal domain and a C-terminal domain. A narrow, deep cavity accommodating the substrate and cofactor was observed at the junction between the two domains of TcMevK. Computational simulation combined with site-directed mutagenesis and biochemical analyses allowed us to define the binding mode of TcMevK to cofactors and substrates. Moreover, TcMevK showed optimal enzyme activity at pH 8.0 and an optimal temperature of 40 °C for mevalonate as the substrate. The expression profiles and RNA interference of TcMevK indicated its critical role in controlling juvenile hormone biosynthesis, as well as its participation in the production of other terpenoids in T. castaneum. These findings improve our understanding of the structural and biochemical features of insect Mevk and provide a structural basis for the design of MevK inhibitors.


Assuntos
Besouros , Fosfotransferases (Aceptor do Grupo Álcool) , Tribolium , Animais , Tribolium/genética , Besouros/metabolismo , Ácido Mevalônico/metabolismo , Hormônios Juvenis/metabolismo
3.
Int J Mol Sci ; 25(6)2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38542231

RESUMO

Plant glycerate kinase (GK) was previously considered an exclusively chloroplastic enzyme of the glycolate pathway (photorespiration), and its sole predicted role was to return most of the glycolate-derived carbon (as glycerate) to the Calvin cycle. However, recent discovery of cytosolic GK revealed metabolic links for glycerate to other processes. Although GK was initially proposed as being solely regulated by substrate availability, subsequent discoveries of its redox regulation and the light involvement in the production of chloroplastic and cytosolic GK isoforms have indicated a more refined regulation of the pathways of glycerate conversion. Here, we re-evaluate the importance of GK and emphasize its multifaceted role in plants. Thus, GK can be a major player in several branches of primary metabolism, including the glycolate pathway, gluconeogenesis, glycolysis, and C4 metabolism. In addition, recently, the chloroplastic (but not cytosolic) GK isoform was implicated as part of a light-dependent plant immune response to pathogen attack. The origins of glycerate are also discussed here; it is produced in several cell compartments and undergoes huge fluctuations depending on light/dark conditions. The recent discovery of the vacuolar glycerate transporter adds yet another layer to our understanding of glycerate transport/metabolism and that of other two- and three-carbon metabolites.


Assuntos
Gluconeogênese , Fosfotransferases (Aceptor do Grupo Álcool) , Fotossíntese , Fotossíntese/fisiologia , Plantas/metabolismo , Imunidade Vegetal , Glicolatos , Carbono/metabolismo
4.
Front Immunol ; 15: 1379220, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38550596

RESUMO

Objective: Bi-allelic pathogenic variants in the MVK gene, which encodes mevalonate kinase (MK), an essential enzyme in isoprenoid biosynthesis, cause the autoinflammatory metabolic disorder mevalonate kinase deficiency (MKD). We generated and characterized MK-deficient monocytic THP-1 cells to identify molecular and cellular mechanisms that contribute to the pro-inflammatory phenotype of MKD. Methods: Using CRISPR/Cas9 genome editing, we generated THP-1 cells with different MK deficiencies mimicking the severe (MKD-MA) and mild end (MKD-HIDS) of the MKD disease spectrum. Following confirmation of previously established disease-specific biochemical hallmarks, we studied the consequences of the different MK deficiencies on LPS-stimulated cytokine release, glycolysis versus oxidative phosphorylation rates, cellular chemotaxis and protein kinase activity. Results: Similar to MKD patients' cells, MK deficiency in the THP-1 cells caused a pro-inflammatory phenotype with a severity correlating with the residual MK protein levels. In the MKD-MA THP-1 cells, MK protein levels were barely detectable, which affected protein prenylation and was accompanied by a profound pro-inflammatory phenotype. This included a markedly increased LPS-stimulated release of pro-inflammatory cytokines and a metabolic switch from oxidative phosphorylation towards glycolysis. We also observed increased activity of protein kinases that are involved in cell migration and proliferation, and in innate and adaptive immune responses. The MKD-HIDS THP-1 cells had approximately 20% residual MK activity and showed a milder phenotype, which manifested mainly upon LPS stimulation or exposure to elevated temperatures. Conclusion: MK-deficient THP-1 cells show the biochemical and pro-inflammatory phenotype of MKD and are a good model to study underlying disease mechanisms and therapeutic options of this autoinflammatory disorder.


Assuntos
Lipopolissacarídeos , Deficiência de Mevalonato Quinase , Fosfotransferases (Aceptor do Grupo Álcool) , Humanos , Lipopolissacarídeos/metabolismo , Células THP-1 , Fenótipo , Deficiência de Mevalonato Quinase/metabolismo , Fosforilação Oxidativa
5.
Hum Exp Toxicol ; 43: 9603271231218707, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38487884

RESUMO

BACKGROUND: Bupivacaine (BUP), a long-acting local anesthetic, has been widely used in analgesia and anesthesia. However, evidence strongly suggests that excessive application of BUP may lead to neurotoxicity in neurons. Sphingosine kinase 2 (SPHK2) has been reported to exert neuroprotective effects. In this study, we intended to investigate the potential role and mechanism of SPHK2 in BUP-induced neurotoxicity in dorsal root ganglion (DRG) neurons. METHODS: DRG neurons were cultured with BUP to simulate BUP-induced neurotoxicity in vitro. CCK-8, LDH, and flow cytometry assays were performed to detect the viability, LDH activity, and apoptosis of DRG neurons. RT-qPCR and western blotting was applied to measure gene and protein expression. Levels. MeRIP-qPCR was applied for quantification of m6A modification. RIP-qPCR was used to analyze the interaction between SPHK2 and YTHDF1. RESULTS: SPHK2 expression significantly declined in DRG neurons upon exposure to BUP. BUP challenge substantially reduced the cell viability and increased the apoptosis rate in DRG neurons, which was partly abolished by SPHK2 upregulation. YTHDF1, an N6-methyladenosine (m6A) reader, promoted SPHK2 expression in BUP-treated DRG neurons in an m6A-dependent manner. YTHDF1 knockdown partly eliminated the increase in SPHK2 protein level and the protection against BUP-triggered neurotoxicity in DRG neurons mediated by SPHK2 overexpression. Moreover, SPHK2 activated the PI3K/AKT signaling to protect against BUP-induced cytotoxic effects on DRG neurons. CONCLUSIONS: In sum, YTHDF1-mediated SPHK2 upregulation ameliorated BUP-induced neurotoxicity in DRG neurons via promoting activation of the PI3K/AKT signaling pathway.


Assuntos
Bupivacaína , Síndromes Neurotóxicas , Fosfotransferases (Aceptor do Grupo Álcool) , Humanos , Bupivacaína/toxicidade , Regulação para Cima , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Síndromes Neurotóxicas/prevenção & controle , Apoptose , Proteínas de Ligação a RNA
6.
Asian Pac J Cancer Prev ; 25(3): 725-733, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38546054

RESUMO

OBJECTIVE: Availability of multimodal treatment strategies, including targeted therapies and immunotherapies, have improved the survival of non-small cell lung carcinoma (NSCLC). However, some patients still progress or respond poorly due to inherent resistance, acquired resistance, or lack of druggable driver mutations. Sphingosine-1-phosphate (S1P) and receptor tyrosine kinase-like orphan receptor (ROR1/2) signaling pathways are activated during lung carcinogenesis. METHODS: In this study, we have evaluated the crosstalk of S1P and ROR1/2 signaling pathways in lung cancer cells. RESULTS: S1P treatment of lung cancer cells decreases ROR1 and ROR2 transcript levels. While treatment with PF-543, a pharmacological SphK1 inhibitor or genetic knockdown of SPHK1 by shRNA, raises ROR1 and ROR2. Furthermore, simultaneous inhibition of SphK1 along with ROR1 reduced the migration of lung cancer cells. CONCLUSION: These findings demonstrate the reciprocal regulation of both pathways, suggesting that both pathways have an inverse relation i.e, in the absence of one pathway, another pathway may take charge of the other pathway. Therefore, simultaneously targeting both pathways could serve as a potential therapeutic target for lung cancer treatment.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Lisofosfolipídeos , Esfingosina/análogos & derivados , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Transdução de Sinais , Pulmão/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo
7.
Artigo em Inglês | MEDLINE | ID: mdl-38408517

RESUMO

Euryhaline organisms can accumulate organic osmolytes to maintain osmotic balance between their internal and external environments. Proline is a pivotal organic small molecule and plays an important role in osmoregulation that enables marine shellfish to tolerate high-salinity conditions. During high-salinity challenge, NAD kinase (NADK) is involved in de novo synthesis of NADP(H) in living organisms, which serves as a reducing agent for the biosynthetic reactions. However, the role of shellfish NADK in proline biosynthesis remains elusive. In this study, we show the modulation of NADK on proline synthesis in the razor clam (Sinonovacula constricta) in response to osmotic stress. Under acute hypersaline conditions, gill tissues exhibited a significant increase in the expression of ScNADK. To elucidate the role of ScNADK in proline biosynthesis, we performed dsRNA interference in the expression of ScNADK in gill tissues to assess proline content and the expression levels of key enzyme genes involved in proline biosynthesis. The results indicate that the knock-down of ScNADK led to a significant decrease in proline content (P<0.01), as well as the expression levels of two proline synthetase genes P5CS and P5CR involved in the glutamate pathway. Razor clams preferred to use ornithine as substrate for proline synthesis when the glutamate pathway is blocked. Exogenous administration of proline greatly improved cell viability and mitigated cell apoptosis in gills. In conclusion, our results demonstrate the important role of ScNADK in augmenting proline production under high-salinity stress, by which the razor clam is able to accommodate salinity variations in the ecological niche.


Assuntos
Bivalves , Fosfotransferases (Aceptor do Grupo Álcool) , Tolerância ao Sal , Animais , Bivalves/metabolismo , Prolina/metabolismo , Glutamatos/metabolismo
8.
Arterioscler Thromb Vasc Biol ; 44(4): 883-897, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38328936

RESUMO

BACKGROUND: Myeloid cells (MCs) reside in the aortic intima at regions predisposed to atherosclerosis. Systemic inflammation triggers reverse transendothelial migration (RTM) of intimal MCs into the arterial blood, which orchestrates a protective immune response that clears intracellular pathogens from the arterial intima. Molecular pathways that regulate RTM remain poorly understood. S1P (sphingosine-1-phosphate) is a lipid mediator that regulates immune cell trafficking by signaling via 5 G-protein-coupled receptors (S1PRs [S1P receptors]). We investigated the role of S1P in the RTM of aortic intimal MCs. METHODS: Intravenous injection of lipopolysaccharide was used to model a systemic inflammatory stimulus that triggers RTM. CD11c+ intimal MCs in the lesser curvature of the ascending aortic arch were enumerated by en face confocal microscopy. Local gene expression was evaluated by transcriptomic analysis of microdissected intimal cells. RESULTS: In wild-type C57BL/6 mice, lipopolysaccharide induced intimal cell expression of S1pr1, S1pr3, and Sphk1 (a kinase responsible for S1P production). Pharmacological modulation of multiple S1PRs blocked lipopolysaccharide-induced RTM and modulation of S1PR1 and S1PR3 reduced RTM in an additive manner. Cre-mediated deletion of S1pr1 in MCs blocked lipopolysaccharide-induced RTM, confirming a role for myeloid-specific S1PR1 signaling. Global or hematopoietic deficiency of Sphk1 reduced plasma S1P levels, the abundance of CD11c+ MCs in the aortic intima, and blunted lipopolysaccharide-induced RTM. In contrast, plasma S1P levels, the abundance of intimal MCs, and lipopolysaccharide-induced RTM were rescued in Sphk1-/- mice transplanted with Sphk1+/+ or mixed Sphk1+/+ and Sphk1-/- bone marrow. Stimulation with lipopolysaccharide increased endothelial permeability and intimal MC exposure to circulating factors such as S1P. CONCLUSIONS: Functional and expression studies support a novel role for S1P signaling in the regulation of lipopolysaccharide-induced RTM and the homeostatic maintenance of aortic intimal MCs. Our data provide insight into how circulating plasma mediators help orchestrate intimal MC dynamics.


Assuntos
Receptores de Lisoesfingolipídeo , Migração Transendotelial e Transepitelial , Camundongos , Animais , Receptores de Lisoesfingolipídeo/genética , Receptores de Lisoesfingolipídeo/metabolismo , Lipopolissacarídeos/toxicidade , Camundongos Endogâmicos C57BL , Esfingosina/metabolismo , Células Mieloides/metabolismo , Lisofosfolipídeos/metabolismo , Túnica Íntima/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo
9.
Nat Commun ; 15(1): 1502, 2024 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-38374076

RESUMO

D-myo-inositol 1,4,5-trisphosphate (InsP3) is a fundamental second messenger in cellular Ca2+ mobilization. InsP3 3-kinase, a highly specific enzyme binding InsP3 in just one mode, phosphorylates InsP3 specifically at its secondary 3-hydroxyl group to generate a tetrakisphosphate. Using a chemical biology approach with both synthetised and established ligands, combining synthesis, crystallography, computational docking, HPLC and fluorescence polarization binding assays using fluorescently-tagged InsP3, we have surveyed the limits of InsP3 3-kinase ligand specificity and uncovered surprisingly unforeseen biosynthetic capacity. Structurally-modified ligands exploit active site plasticity generating a helix-tilt. These facilitated uncovering of unexpected substrates phosphorylated at a surrogate extended primary hydroxyl at the inositol pseudo 3-position, applicable even to carbohydrate-based substrates. Crystallization experiments designed to allow reactions to proceed in situ facilitated unequivocal characterization of the atypical tetrakisphosphate products. In summary, we define features of InsP3 3-kinase plasticity and substrate tolerance that may be more widely exploitable.


Assuntos
Inositol 1,4,5-Trifosfato , Fosfotransferases (Aceptor do Grupo Álcool) , Inositol 1,4,5-Trifosfato/metabolismo , Domínio Catalítico , Ligantes , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfatos de Inositol/metabolismo , Cálcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo
10.
Cell Metab ; 36(4): 839-856.e8, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38367623

RESUMO

Utilization of lipids as energy substrates after birth causes cardiomyocyte (CM) cell-cycle arrest and loss of regenerative capacity in mammalian hearts. Beyond energy provision, proper management of lipid composition is crucial for cellular and organismal health, but its role in heart regeneration remains unclear. Here, we demonstrate widespread sphingolipid metabolism remodeling in neonatal hearts after injury and find that SphK1 and SphK2, isoenzymes producing the same sphingolipid metabolite sphingosine-1-phosphate (S1P), differently regulate cardiac regeneration. SphK2 is downregulated during heart development and determines CM proliferation via nuclear S1P-dependent modulation of histone acetylation. Reactivation of SphK2 induces adult CM cell-cycle re-entry and cytokinesis, thereby enhancing regeneration. Conversely, SphK1 is upregulated during development and promotes fibrosis through an S1P autocrine mechanism in cardiac fibroblasts. By fine-tuning the activity of each SphK isoform, we develop a therapy that simultaneously promotes myocardial repair and restricts fibrotic scarring to regenerate the infarcted adult hearts.


Assuntos
Coração , Lisofosfolipídeos , Esfingolipídeos , Esfingosina/análogos & derivados , Animais , Esfingolipídeos/metabolismo , Isoenzimas , Mamíferos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo
11.
Acta Biochim Biophys Sin (Shanghai) ; 56(3): 427-439, 2024 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-38327186

RESUMO

Mitochondrial homeostasis plays a pivotal role in oocyte maturation and embryonic development. Deoxyguanosine kinase (DGUOK) is a nucleoside kinase that salvages purine nucleosides in mitochondria and is critical for mitochondrial DNA replication and homeostasis in non-proliferating cells. Dguok loss-of-function mutations and deletions lead to hepatocerebral mitochondrial DNA deletion syndrome. However, its potential role in reproduction remains largely unknown. In this study, we find that Dguok knockout results in female infertility. Mechanistically, DGUOK deficiency hinders ovarian development and oocyte maturation. Moreover, DGUOK deficiency in oocytes causes a significant reduction in mitochondrial DNA copy number and abnormal mitochondrial dynamics and impairs germinal vesicle breakdown. Only few DGUOK-deficient oocytes can extrude their first polar body during in vitro maturation, and these oocytes exhibit irregular chromosome arrangements and different spindle lengths. In addition, DGUOK deficiency elevates reactive oxygen species levels and accelerates oocyte apoptosis. Our findings reveal novel physiological roles for the mitochondrial nucleoside salvage pathway in oocyte maturation and implicate DGUOK as a potential marker for the diagnosis of female infertility.


Assuntos
Infertilidade Feminina , Doenças Mitocondriais , Fosfotransferases (Aceptor do Grupo Álcool) , Humanos , Gravidez , Camundongos , Feminino , Animais , Infertilidade Feminina/genética , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Oócitos/metabolismo , Fertilidade/genética
12.
Acta Crystallogr D Struct Biol ; 80(Pt 3): 203-215, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38411551

RESUMO

Mevalonate kinase is central to the isoprenoid biosynthesis pathway. Here, high-resolution X-ray crystal structures of two mevalonate kinases are presented: a eukaryotic protein from Ramazzottius varieornatus and an archaeal protein from Methanococcoides burtonii. Both enzymes possess the highly conserved motifs of the GHMP enzyme superfamily, with notable differences between the two enzymes in the N-terminal part of the structures. Biochemical characterization of the two enzymes revealed major differences in their sensitivity to geranyl pyrophosphate and farnesyl pyrophosphate, and in their thermal stabilities. This work adds to the understanding of the structural basis of enzyme inhibition and thermostability in mevalonate kinases.


Assuntos
Archaea , Ácido Mevalônico , Ácido Mevalônico/metabolismo , Archaea/metabolismo , Methanosarcinaceae/química , Methanosarcinaceae/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/química
13.
Antiviral Res ; 222: 105792, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38163624

RESUMO

Therapeutic use of maribavir for human cytomegalovirus infection has renewed attention to the extent of cross-resistance with ganciclovir as the existing standard therapy. Each drug selects in vivo for a characteristic set of resistance mutations in the viral UL97 kinase gene. To improve the calibration of relative susceptibilities to each drug, genetic variants at relevant UL97 codons were extensively phenotyped using the same baseline viral clone, cell culture conditions and growth readout. Ganciclovir-selected mutations at codons 460, 520, 592, 594, 595 and 603 conferred 2.8-fold (C603Y) to 12-fold (M460I) increases in ganciclovir 50% inhibitory concentrations (EC50) over wild type baseline, while conferring maribavir EC50 fold changes ranging from 0.21-fold (M460I) to 1.9-fold (A594V). Maribavir-selected mutations at codons 409, 411 and 480 conferred maribavir EC50 fold changes ranging from 17 (H411Y) to 210 (C480F), while conferring ganciclovir EC50 fold changes ranging from 0.7 (H411Y) to 2.3 (C480F). The P-loop substitution F342Y, selected by either drug, is confirmed to confer 4.7-fold and 6-fold increases in maribavir and ganciclovir EC50s respectively, and suggests this part of the ATP-binding domain of UL97 to be involved in moderate resistance to both drugs. The maribavir hypersensitivity of M460I and M460V may be advantageous.


Assuntos
Citomegalovirus , Diclororribofuranosilbenzimidazol/análogos & derivados , Ganciclovir , Humanos , Ganciclovir/farmacologia , Antivirais/farmacologia , Mutação , Códon , Farmacorresistência Viral/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética
14.
Nucleic Acids Res ; 52(5): 2372-2388, 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38214234

RESUMO

Pediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent mechanism, which fosters genome instability in H3.3 mutant pHGG, and opens new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme polynucleotide kinase 3'-phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting.


Assuntos
Neoplasias Encefálicas , Glioma , Histonas , Criança , Humanos , Neoplasias Encefálicas/patologia , Reparo do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Glioma/patologia , Histonas/genética , Histonas/metabolismo , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética
15.
Nat Cancer ; 5(3): 433-447, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38286827

RESUMO

Liver metastasis (LM) confers poor survival and therapy resistance across cancer types, but the mechanisms of liver-metastatic organotropism remain unknown. Here, through in vivo CRISPR-Cas9 screens, we found that Pip4k2c loss conferred LM but had no impact on lung metastasis or primary tumor growth. Pip4k2c-deficient cells were hypersensitized to insulin-mediated PI3K/AKT signaling and exploited the insulin-rich liver milieu for organ-specific metastasis. We observed concordant changes in PIP4K2C expression and distinct metabolic changes in 3,511 patient melanomas, including primary tumors, LMs and lung metastases. We found that systemic PI3K inhibition exacerbated LM burden in mice injected with Pip4k2c-deficient cancer cells through host-mediated increase in hepatic insulin levels; however, this circuit could be broken by concurrent administration of an SGLT2 inhibitor or feeding of a ketogenic diet. Thus, this work demonstrates a rare example of metastatic organotropism through co-optation of physiological metabolic cues and proposes therapeutic avenues to counteract these mechanisms.


Assuntos
Neoplasias Hepáticas , Proteínas Proto-Oncogênicas c-akt , Humanos , Camundongos , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases , Transdução de Sinais , Insulina , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo
16.
Chem Biol Interact ; 390: 110868, 2024 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-38218310

RESUMO

Acute lung injury (ALI) is a frequent complication of sepsis, with pyroptosis playing a pivotal role. Analysis of Gene Expression Omnibus (GEO) mouse sepsis datasets revealed the upregulation of sphingosine kinase 1 (SphK1) in septic mouse lung tissues, which was validated in lipopolysaccharide (LPS)-treated mice. Therefore, this study aimed to explore the potential role and underlying mechanisms of SphK1, the primary kinase responsible for catalyzing the formation of the bioactive lipid sphingosine-1-phosphat, in sepsis development. Mice received an intraperitoneal injection of SphK1 inhibitor prior to LPS administration. Mouse lung vascular endothelial cells (MLVECs) were exposed to LPS and SphK1 inhibitor. The SphK1 inhibitor mitigated ALI, as evidenced by hematoxylin and eosin (H&E) staining and the wet-to-dry (W/D) weight ratio and reduced Evans blue dye leakage. Furthermore, the SphK1 inhibitor inhibited the activation of the NOD-like receptor protein 3 inflammasome and the subsequent induction of pyroptosis both in vivo and in vitro. Intriguingly, using co-immunoprecipitation (Co-IP) combined with mass spectrometry, our findings revealed that SphK1 associates with pyruvate kinase M2 (PKM2), facilitating PKM2 phosphorylation and its nuclear translocation. TEPP-46, which has the ability to stabilize PKM2 and inhibit the phosphorylation and nuclear translocation of PKM2, markedly reduced the expression of pyroptosis-associated markers and alleviated lung injury. Concludingly, our results suggest that targeting SphK1 is a promising therapeutic strategy for ALI.


Assuntos
Lesão Pulmonar Aguda , Fosfotransferases (Aceptor do Grupo Álcool) , Sepse , Animais , Camundongos , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/tratamento farmacológico , Células Endoteliais/metabolismo , Lipopolissacarídeos/toxicidade , Pulmão/metabolismo , Piroptose , Esfingosina
17.
J Virol ; 98(2): e0174923, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38189249

RESUMO

Enterovirus 71 (EV71) is one of the major pathogens causing hand, foot, and mouth disease in children under 5 years old, which can result in severe neurological complications and even death. Due to limited treatments for EV71 infection, the identification of novel host factors and elucidation of mechanisms involved will help to counter this viral infection. N-terminal acetyltransferase 6 (NAT6) was identified as an essential host factor for EV71 infection with genome-wide CRISPR/Cas9 screening. NAT6 facilitates EV71 viral replication depending on its acetyltransferase activity but has little effect on viral release. In addition, NAT6 is also required for Echovirus 7 and coxsackievirus B5 infection, suggesting it might be a pan-enterovirus host factor. We further demonstrated that NAT6 is required for Golgi integrity and viral replication organelle (RO) biogenesis. NAT6 knockout significantly inhibited phosphatidylinositol 4-kinase IIIß (PI4KB) expression and PI4P production, both of which are key host factors for enterovirus infection and RO biogenesis. Further mechanism studies confirmed that NAT6 formed a complex with its substrate actin and one of the PI4KB recruiters-acyl-coenzyme A binding domain containing 3 (ACBD3). Through modulating actin dynamics, NAT6 maintained the integrity of the Golgi and the stability of ACBD3, thereby enhancing EV71 infection. Collectively, these results uncovered a novel mechanism of N-acetyltransferase supporting EV71 infection.IMPORTANCEEnterovirus 71 (EV71) is an important pathogen for children under the age of five, and currently, no effective treatment is available. Elucidating the mechanism of novel host factors supporting viral infection will reveal potential antiviral targets and aid antiviral development. Here, we demonstrated that a novel N-acetyltransferase, NAT6, is an essential host factor for EV71 replication. NAT6 could promote viral replication organelle (RO) formation to enhance viral replication. The formation of enterovirus ROs requires numerous host factors, including acyl-coenzyme A binding domain containing 3 (ACBD3) and phosphatidylinositol 4-kinase IIIß (PI4KB). NAT6 could stabilize the PI4KB recruiter, ACBD3, by inhibiting the autophagy degradation pathway. This study provides a fresh insight into the relationship between N-acetyltransferase and viral infection.


Assuntos
Enterovirus Humano A , Infecções por Enterovirus , Acetiltransferases N-Terminal , Fosfotransferases (Aceptor do Grupo Álcool) , Criança , Pré-Escolar , Humanos , 1-Fosfatidilinositol 4-Quinase/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antivirais , Coenzima A/metabolismo , Infecções por Coxsackievirus , Enterovirus Humano A/fisiologia , Infecções por Enterovirus/metabolismo , Infecções por Enterovirus/virologia , Proteínas de Membrana/metabolismo , Acetiltransferases N-Terminal/metabolismo , Biogênese de Organelas , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Replicação Viral/fisiologia
18.
Nucleic Acids Res ; 52(5): 2416-2433, 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38224455

RESUMO

Mammalian polynucleotide kinase 3'-phosphatase (PNKP), a DNA end-processing enzyme with 3'-phosphatase and 5'-kinase activities, is involved in multiple DNA repair pathways, including base excision (BER), single-strand break (SSBR), and double-strand break repair (DSBR). However, little is known as to how PNKP functions in such diverse repair processes. Here we report that PNKP is acetylated at K142 (AcK142) by p300 constitutively but at K226 (AcK226) by CBP, only after DSB induction. Co-immunoprecipitation analysis using AcK142 or AcK226 PNKP-specific antibodies showed that AcK142-PNKP associates only with BER/SSBR, and AcK226 PNKP with DSBR proteins. Despite the modest effect of acetylation on PNKP's enzymatic activity in vitro, cells expressing non-acetylable PNKP (K142R or K226R) accumulated DNA damage in transcribed genes. Intriguingly, in striatal neuronal cells of a Huntington's Disease (HD)-based mouse model, K142, but not K226, was acetylated. This is consistent with the reported degradation of CBP, but not p300, in HD cells. Moreover, transcribed genomes of HD cells progressively accumulated DSBs. Chromatin-immunoprecipitation analysis demonstrated the association of Ac-PNKP with the transcribed genes, consistent with PNKP's role in transcription-coupled repair. Thus, our findings demonstrate that acetylation at two lysine residues, located in different domains of PNKP, regulates its distinct role in BER/SSBR versus DSBR.


Assuntos
Enzimas Reparadoras do DNA , Fosfotransferases (Aceptor do Grupo Álcool) , Animais , Humanos , Camundongos , Acetilação , Dano ao DNA , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , Mamíferos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/química , Polinucleotídeo 5'-Hidroxiquinase/genética
19.
Bioorg Chem ; 144: 107112, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38237390

RESUMO

We report here the virtual screening design, synthesis and activity of eight new inhibitors of SphK1. For this study we used a pre-trained Graph Convolutional Network (GCN) combined with docking calculations. This exploratory analysis proposed nine compounds from which eight displayed significant inhibitory effect against sphingosine kinase 1 (SphK1) demonstrating a high level of efficacy for this approach. Four of these compounds also displayed anticancer activity against different tumor cell lines, and three of them (5), (6) and (7) have shown a wide inhibitory action against many of the cancer cell line tested, with GI50 below 5 µM, being (5) the most promising with TGI below 10 µM for the half of cell lines. Our results suggest that the three most promising compounds reported here are the pyrimidine-quinolone hybrids (1) and (6) linked by p-aminophenylsulfanyl and o-aminophenol fragments respectively, and (8) without such aryl linker. We also performed an exhaustive study about the molecular interactions that stabilize the different ligands at the binding site of SphK1. This molecular modeling analysis was carried out by using combined techniques: docking calculations, MD simulations and QTAIM analysis. In this study we also included PF543, as reference compound, in order to better understand the molecular behavior of these ligands at the binding site of SphK1.These results provide useful information for the design of new inhibitors of SphK1 possessing these structural scaffolds.


Assuntos
Antineoplásicos , Fosfotransferases (Aceptor do Grupo Álcool) , Quinolonas , Quinolonas/farmacologia , Inibidores de Proteínas Quinases , Antineoplásicos/química , Modelos Moleculares , Linhagem Celular Tumoral , Simulação de Acoplamento Molecular , Ensaios de Seleção de Medicamentos Antitumorais , Proliferação de Células , Relação Estrutura-Atividade , Estrutura Molecular
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