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1.
Gen Physiol Biophys ; 43(4): 321-333, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38953574

RESUMO

Vitiligo is featured by manifestation of white maculae and primarily results from oxidative stress. Sphingosine kinase-1 (SPHK1) participates in oxidative stress. This paper was devised to explore the role of SPHK1 in vitiligo and to disclose the mechanism. PIG1 cell viability was appraised utilizing cell counting kit-8 assay while Western blot detected SPHK1 and four and a half LIM domains 2 (FHL2). The transduction efficacy of small interfering RNA (siRNA)-SPHK1, siRNA-FHL2 and pcDNA3.1 plasmid overexpressing FHL2 (Ov-FHL2) was checked using Western blot. Flow cytometry detected cell apoptotisis. Western blot detected mitochondrial cytochrome c (Mit-Cyt-c) and cytosolic cytochrome c (Cyto-Cyt-c). Dichloro-dihydro-fluorescein diacetate (DCFH-DA) detected reactive oxygen species (ROS) activity while oxidative stress markers were evaluated using corresponding assay kits. SPHK1 expression was discovered to be increased in hydrogen peroxide (H2O2)-challenged PIG1 cells and SPHK1 interference alleviated H2O2-challenged viability damage, apoptosis, oxidative stress and FHL2 expression in PIG1 cells. FHL2 depletion could suppress viability damage, apoptosis and oxidative stress in H2O2-challenged PIG1 cells. Rescue experiments demonstrated that the suppressive impacts of SPHK1 deficiency on PIG1 cell viability, apoptosis and oxidative stress induced by H2O2 were offset by FHL2 overexpression. Collectively, SPHK1 knockdown protected against vitiligo via the regulation of FHL2.


Assuntos
Sobrevivência Celular , Peróxido de Hidrogênio , Proteínas com Homeodomínio LIM , Melanócitos , Estresse Oxidativo , Fosfotransferases (Aceptor do Grupo Álcool) , Estresse Oxidativo/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Peróxido de Hidrogênio/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Proteínas com Homeodomínio LIM/genética , Humanos , Melanócitos/metabolismo , Melanócitos/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Linhagem Celular
2.
Plant Physiol Biochem ; 213: 108764, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38879983

RESUMO

The phosphoenolpyruvate carboxylase kinase of Medicago sativa L. (MsPPCK1) modulates the phosphorylation status and activity of the C4 pathway phosphoenolpyruvate carboxylase enzyme, which is pivotal for photosynthetic carbon assimilation in plants. This study investigated the role of MsPPCK1 in alfalfa by creating transgenic plants overexpressing MsPPCK1 under the control of the CaMV35S promoter. The enhanced alkali tolerance of transgenic plants indicated an important role of MsPPCK1 gene in regulating plant alkali tolerance. Transgenic plants exhibited heightened antioxidant activity (SOD, POD, and CAT), reduced MDA, H2O2, OFR and REC% content, increased activity of key photosynthetic enzymes (PEPC, PPDK, NADP-ME, and NADP-MDH), and enhanced photosynthetic parameters (Pn, E, Gs, and Ci). Moreover, MsPPCK1 overexpression increased the content of organic acids (oxaloacetic, malic, citric, and succinic acids) in the plants. The upregulation of MsPPCK1 under rhizobial inoculation showcased its other role in nodule development. In transgenic plants, MsDMI2, MsEnod12, and MsNODL4 expression increased, facilitating root nodule development and augmenting plant nodulation. Accelerated root nodule growth positively influences plant growth and yield and enhances alfalfa resistance to alkali stress. This study highlights the pivotal role of MsPPCK1 in fortifying plant alkali stress tolerance and improving yield, underscoring its potential as a key genetic target for developing alkali-tolerant and high-yielding alfalfa varieties.


Assuntos
Medicago sativa , Fotossíntese , Proteínas de Plantas , Plantas Geneticamente Modificadas , Medicago sativa/genética , Medicago sativa/enzimologia , Medicago sativa/crescimento & desenvolvimento , Fotossíntese/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Álcalis , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Serina-Treonina Quinases
3.
Chem Biol Interact ; 398: 111085, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38823539

RESUMO

Sepsis-induced acute lung injury (SALI) is the common complication of sepsis, resulting in high incidence and mortality rates. The primary pathogenesis of SALI is the interplay between acute inflammation and endothelial barrier damage. Studies have shown that kaempferol (KPF) has anti-sepsis properties. Sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P) signaling pathway's significance in acute lung damage and S1P receptor 1 (S1PR1) agonists potential in myosin light chain 2 (MLC2) phosphorylation are documented. Whether KPF can regulate the SphK1/S1P/S1PR1/MLC2 signaling pathway to protect the lung endothelial barrier remains unclear. This study investigates the KPF's therapeutic effects and molecular mechanisms in repairing endothelial cell barrier damage in both LPS-induced sepsis mice and human umbilical vein endothelial cells (HUVECs). KPF significantly reduced lung tissue damage and showed anti-inflammatory effects by decreasing IL-6 and TNF-α synthesis in the sepsis mice model. Further, KPF administration can reduce the high permeability of the LPS-induced endothelial cell barrier and alleviate lung endothelial cell barrier injury. Mechanistic studies showed that KPF pretreatment can suppress MLC2 hyperphosphorylation and decrease SphK1, S1P, and S1PR1 levels. The SphK1/S1P/S1PR1/MLC2 signaling pathway controls the downstream proteins linked to endothelial barrier damage, and the Western blot (WB) showed that KPF raised the protein levels. These proteins include zonula occludens (ZO)-1, vascular endothelial (VE)-cadherin and Occludin. The present work revealed that in mice exhibiting sepsis triggered by LPS, KPF strengthened the endothelial barrier and reduced the inflammatory response. The SphK1/S1P/S1PR1/MLC2 pathway's modulation is the mechanism underlying this impact.


Assuntos
Lesão Pulmonar Aguda , Miosinas Cardíacas , Células Endoteliais da Veia Umbilical Humana , Quempferóis , Pulmão , Lisofosfolipídeos , Camundongos Endogâmicos C57BL , Cadeias Leves de Miosina , Sepse , Transdução de Sinais , Esfingosina , Animais , Sepse/tratamento farmacológico , Sepse/complicações , Sepse/metabolismo , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/patologia , Humanos , Cadeias Leves de Miosina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Camundongos , Lisofosfolipídeos/metabolismo , Quempferóis/farmacologia , Quempferóis/uso terapêutico , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Esfingosina/farmacologia , Masculino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Miosinas Cardíacas/metabolismo , Pulmão/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Lipopolissacarídeos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Interleucina-6/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo
4.
Eur J Pharmacol ; 977: 176723, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-38851560

RESUMO

Acute lymphoblastic leukemia (ALL), a complex malignancy, displays varying expression profiles of PIP4K2-related genes in adult patients. While PIP4K2A expression is elevated in ALL bone marrow cells compared to healthy bone marrow cells, PIP4K2B is downregulated, and PIP4K2C remains relatively unchanged. Despite the correlation between increased PIP4K2A expression and increased percentage of peripheral blood blasts, clinical outcomes do not strongly correlate with the expression of these genes. Here we investigated the therapeutic potential of three PIP4K2 inhibitors (THZ-P1-2, a131, and CC260) in ALL cell models. THZ-P1-2 emerges as the most effective inhibitor, inducing cell death and mitochondrial damage while reducing cell viability and metabolism significantly. Comparative analyses highlight the superior efficacy of THZ-P1-2 over a131 and CC260. Notably, THZ-P1-2 uniquely disrupts autophagic flux and inhibits the PI3K/AKT/mTOR pathway, indicating a distinct molecular mechanism. In summary, our findings elucidate the differential expression of PIP4K2-related genes in ALL and underscore the potential role of PIP4K2A in disease pathogenesis. The therapeutic promise of THZ-P1-2 in ALL treatment, along with its distinct effects on cell death mechanisms and signaling pathways, enriches our understanding of PIP4K2's involvement in ALL development and offers targeted therapy prospects.


Assuntos
Fosfotransferases (Aceptor do Grupo Álcool) , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Linhagem Celular Tumoral , Transdução de Sinais/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Autofagia/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Apoptose/efeitos dos fármacos
5.
Int J Mol Sci ; 25(12)2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38928345

RESUMO

Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM) are two common diseases that affect the elderly population worldwide. The identification of common genes associated with AD and T2DM holds promise for potential biomarkers and intriguing pathogenesis of these two complicated diseases. This study utilized a comprehensive approach by integrating transcriptome data from multiple cohorts, encompassing both AD and T2DM. The analysis incorporated various data types, including blood and tissue samples as well as single-cell datasets, allowing for a detailed assessment of gene expression patterns. From the brain region-specific single-cell analysis, PIP4K2A, which encodes phosphatidylinositol-5-phosphate 4-kinase type 2 alpha, was found to be expressed mainly in oligodendrocytes compared to other cell types. Elevated levels of PIP4K2A in AD and T2DM patients' blood were found to be associated with key cellular processes such as vesicle-mediated transport, negative regulation of autophagosome assembly, and cytosolic transport. The identification of PIP4K2A's potential roles in the cellular processes of AD and T2DM offers valuable insights into the development of biomarkers for diagnosis and therapy, especially in the complication of these two diseases.


Assuntos
Doença de Alzheimer , Diabetes Mellitus Tipo 2 , Oligodendroglia , Fosfotransferases (Aceptor do Grupo Álcool) , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Humanos , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/genética , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Biomarcadores , Transcriptoma , Análise de Célula Única , Perfilação da Expressão Gênica , Multiômica
6.
Int J Mol Sci ; 25(12)2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38928268

RESUMO

Human corneal fibrosis can lead to opacity and ultimately partial or complete vision loss. Currently, corneal transplantation is the only treatment for severe corneal fibrosis and comes with the risk of rejection and donor shortages. Sphingolipids (SPLs) are known to modulate fibrosis in various tissues and organs, including the cornea. We previously reported that SPLs are tightly related to both, transforming growth factor beta (TGF-ß) signaling and corneal fibrogenesis. The aim of this study was to investigate the effects of sphingosine-1-phosphate (S1P) and S1P inhibition on specific TGF-ß and SPL family members in corneal fibrosis. Healthy human corneal fibroblasts (HCFs) were isolated and cultured in EMEM + FBS + VitC (construct medium) on 3D transwells for 4 weeks. The following treatments were prepared in a construct medium: 0.1 ng/mL TGF-ß1 (ß1), 1 µM sphingosine-1-phosphate (S1P), and 5 µM Sphingosine kinase inhibitor 2 (I2). Five groups were tested: (1) control (no treatment); rescue groups; (2) ß1/S1P; (3) ß1/I2; prevention groups; (4) S1P/ß1; and (5) I2/ß1. Each treatment was administered for 2 weeks with one treatment and switched to another for 2 weeks. Using Western blot analysis, the 3D constructs were examined for the expression of fibrotic markers, SPL, and TGF-ß signaling pathway members. Scratch assays from 2D cultures were also utilized to evaluate cell migration We observed reduced fibrotic expression and inactivation of latent TGF-ß binding proteins (LTBPs), TGF-ß receptors, Suppressor of Mothers Against Decapentaplegic homologs (SMADs), and SPL signaling following treatment with I2 prevention and rescue compared to S1P prevention and rescue, respectively. Furthermore, we observed increased cell migration following stimulation with I2 prevention and rescue groups, with decreased cell migration following stimulation with S1P prevention and rescue groups after 12 h and 18 h post-scratch. We have demonstrated that I2 treatment reduced fibrosis and modulated the inactivation of LTBPs, TGF-ß receptors, SPLs, and the canonical downstream SMAD pathway. Further investigations are warranted in order to fully uncover the potential of utilizing SphK I2 as a novel therapy for corneal fibrosis.


Assuntos
Córnea , Fibrose , Lisofosfolipídeos , Transdução de Sinais , Esfingosina , Fator de Crescimento Transformador beta , Humanos , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Esfingosina/farmacologia , Lisofosfolipídeos/metabolismo , Lisofosfolipídeos/farmacologia , Córnea/metabolismo , Córnea/patologia , Córnea/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Células Cultivadas , Esfingolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Doenças da Córnea/metabolismo , Doenças da Córnea/patologia , Doenças da Córnea/tratamento farmacológico
7.
Eur J Med Chem ; 275: 116577, 2024 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-38875809

RESUMO

Sphingosine kinase 2 (SphK2) has emerged as a promising target for cancer therapy due to its critical role in tumor growth. However, the lack of potent and selective inhibitors has hindered its clinical application. Herein, we report the design and synthesis of a series of novel SphK2 inhibitors, culminating in the identification of compound 12q as a highly selective and potent inhibitor of SphK2. Molecular dynamics simulations suggest that the incorporation of larger substitution groups facilitates a more effective occupation of the binding site, thereby stabilizing the complex. Compared to the widely used inhibitor ABC294640, compound 12q exhibits superior anti-proliferative activity against various cancer cells, inducing G2 phase arrest and apoptosis in liver cancer cells HepG2. Notably, 12q inhibited migration and colony formation in HepG2 and altered intracellular sphingolipid content. Moreover, intraperitoneal administration of 12q in mice resulted in decreased levels of S1P. 12q provides a valuable tool compound for exploring the therapeutic potential of targeting SphK2 in cancer.


Assuntos
Acetamidas , Antineoplásicos , Proliferação de Células , Ensaios de Seleção de Medicamentos Antitumorais , Fosfotransferases (Aceptor do Grupo Álcool) , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Humanos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Camundongos , Proliferação de Células/efeitos dos fármacos , Relação Estrutura-Atividade , Acetamidas/farmacologia , Acetamidas/síntese química , Acetamidas/química , Estrutura Molecular , Apoptose/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Descoberta de Drogas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química
8.
Nat Commun ; 15(1): 5107, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38877001

RESUMO

Inositol hexaphosphate (InsP6) is the major storage form of phosphorus in seeds. Reducing seed InsP6 content is a breeding objective in agriculture, as InsP6 negatively impacts animal nutrition and the environment. Nevertheless, how InsP6 accumulation is regulated remains largely unknown. Here, we identify a clade of receptor-like cytoplasmic kinases (RLCKs), named Inositol Polyphosphate-related Cytoplasmic Kinases 1-6 (IPCK1-IPCK6), deeply involved in InsP6 accumulation. The InsP6 concentration is dramatically reduced in seeds of ipck quadruple (T-4m/C-4m) and quintuple (C-5m) mutants, accompanied with the obviously increase of phosphate (Pi) concentration. The plasma membrane-localized IPCKs recruit IPK1 involved in InsP6 synthesis, and facilitate its binding and activity via phosphorylation of GRF 14-3-3 proteins. IPCKs also recruit IPK2s and PI-PLCs required for InsP4/InsP5 and InsP3 biosynthesis respectively, to form a potential IPCK-GRF-PLC-IPK2-IPK1 complex. Our findings therefore uncover a regulatory mechanism of InsP6 accumulation governed by IPCKs, shedding light on the mechanisms of InsP biosynthesis in eukaryotes.


Assuntos
Proteínas 14-3-3 , Proteínas de Arabidopsis , Arabidopsis , Ácido Fítico , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/genética , Ácido Fítico/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Mutação , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Fosfatos de Inositol/metabolismo
9.
NPJ Syst Biol Appl ; 10(1): 64, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38830903

RESUMO

Fructosamine-3-kinases (FN3Ks) are a conserved family of repair enzymes that phosphorylate reactive sugars attached to lysine residues in peptides and proteins. Although FN3Ks are present across the Tree of Life and share detectable sequence similarity to eukaryotic protein kinases, the biological processes regulated by these kinases are largely unknown. To address this knowledge gap, we leveraged the FN3K CRISPR Knock-Out (KO) HepG2 cell line alongside an integrative multi-omics study combining transcriptomics, metabolomics, and interactomics to place these enzymes in a pathway context. The integrative analyses revealed the enrichment of pathways related to oxidative stress response, lipid biosynthesis (cholesterol and fatty acids), and carbon and co-factor metabolism. Moreover, enrichment of nicotinamide adenine dinucleotide (NAD) binding proteins and localization of human FN3K (HsFN3K) to mitochondria suggests potential links between FN3K and NAD-mediated energy metabolism and redox balance. We report specific binding of HsFN3K to NAD compounds in a metal and concentration-dependent manner and provide insight into their binding mode using modeling and experimental site-directed mutagenesis. Our studies provide a framework for targeting these understudied kinases in diabetic complications and metabolic disorders where redox balance and NAD-dependent metabolic processes are altered.


Assuntos
Redes e Vias Metabólicas , Fosfotransferases (Aceptor do Grupo Álcool) , Humanos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Células Hep G2 , Redes e Vias Metabólicas/genética , Metabolômica/métodos , NAD/metabolismo , Estresse Oxidativo/fisiologia , Estresse Oxidativo/genética , Multiômica
10.
Skin Res Technol ; 30(7): e13800, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38925555

RESUMO

BACKGROUND: MicroRNAs (miRNAs) are small RNA molecules that play a regulatory role in various biological processes by acting as intracellular mediators. They hold great potential as therapeutic agents for targeting human disease pathways; however, there is still much to be uncovered about their mechanism of gene regulation. Alopecia areata (AA) is a commonly occurring inflammatory condition characterized by the infiltration of T cells that specifically target the anagen-stage hair follicle. The limited understanding of its precise cellular mechanism may be the reason behind the scarcity of effective treatments for AA. AIM: The significance and function of hsa-miR-193a-5p as a genetic marker for AA and its potential influence on the advancement of the disease. SUBJECTS AND METHODS: A case-control study comprised 77 individuals diagnosed with AA who were matched with 75 healthy controls. In order to measure the expression of miR-200c-3p in both groups, the real-time PCR technique was utilized. The prediction of suitable genes for hsa-miR-193a-5p, as well as the identification of pathways and gene-gene interactions, were carried out using bioinformatic tools. RESULTS: The levels of hsa-miR-193a-5p expression were notably elevated in AA patients in comparison to healthy controls. Our prediction suggests that the involvement of hsa-miR-193a-5p in the development of AA is significant due to its influence on the inositol phosphorylation pathway and the Phosphatidylinositol signaling system, achieved through its direct impact on the IPPK gene. CONCLUSION: For the first time, our study demonstrates the significant over-expression of a new miRNA, hsa-miR-193a-5p, in the blood of AA patients compared to controls, and highlights its impact on the IPPK gene and the inositol phosphorylation and Phosphatidylinositol signaling pathways, suggesting a potential therapeutic role for hsa-miR-193a-5p in AA.


Assuntos
Alopecia em Áreas , Inositol , MicroRNAs , Humanos , Alopecia em Áreas/genética , Alopecia em Áreas/metabolismo , MicroRNAs/metabolismo , MicroRNAs/genética , Masculino , Estudos de Casos e Controles , Feminino , Adulto , Inositol/metabolismo , Pessoa de Meia-Idade , Adulto Jovem , Marcadores Genéticos/genética , Fosfotransferases (Aceptor do Grupo Álcool)
11.
Biomed Pharmacother ; 176: 116826, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38838507

RESUMO

BACKGROUND: Phosphatidylinositol-4-phosphate 5-kinase type 1 alpha (PIP5K1A) acts upstream of the Akt regulatory pathway and is abnormally expressed in many types of malignancies. However, the role and mechanism of PIP5K1A in colorectal cancer (CRC) have not yet been reported. In this study, we aimed to determine the association between PIP5K1A and progression of CRC and assess the efficacy and mechanism by which rupatadine targets PIP5K1A. METHODS: Firstly, expression and function of PIP5K1A in CRC were investigated by human colon cancer tissue chip analysis and cell proliferation assay. Next, rupatadine was screened by computational screening and cytotoxicity assay and interactions between PIP5K1A and rupatadine assessed by kinase activity detection assay and bio-layer interferometry analysis. Next, rupatadine's anti-tumor effect was evaluated by in vivo and in vitro pharmacodynamic assays. Finally, rupatadine's anti-tumor mechanism was explored by quantitative real-time reverse-transcription polymerase chain reaction, western blot, and immunofluorescence. RESULTS: We found that PIP5K1A exerts tumor-promoting effects as a proto-oncogene in CRC and aberrant PIP5K1A expression correlates with CRC malignancy. We also found that rupatadine down-regulates cyclin-dependent kinase 2 and cyclin D1 protein expression by inhibiting the PIP5K1A/Akt/GSK-3ß pathway, induces cell cycle arrest, and inhibits CRC cell proliferation in vitro and in vivo. CONCLUSIONS: PIP5K1A is a potential drug target for treating CRC. Rupatadine, which targets PIP5K1A, could serve as a new option for treating CRC, its therapeutic mechanism being related to regulation of the Akt/GSK-3ß signaling pathway.


Assuntos
Proliferação de Células , Neoplasias Colorretais , Ciproeptadina , Fosfotransferases (Aceptor do Grupo Álcool) , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Humanos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Transdução de Sinais/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ciproeptadina/farmacologia , Ciproeptadina/análogos & derivados , Camundongos Nus , Linhagem Celular Tumoral , Camundongos Endogâmicos BALB C , Masculino , Proto-Oncogene Mas , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos , Antineoplásicos/farmacologia
12.
Molecules ; 29(11)2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38893478

RESUMO

Transient receptor potential melastatin-8 (TRPM8) is a cation channel that is activated by cold and "cooling agents" such as menthol and icilin, which induce a cold sensation. The stimulation of TRPM8 activates an intracellular signaling cascade that ultimately leads to a change in the gene expression pattern of the cells. Here, we investigate the TRPM8-induced signaling pathway that links TRPM8 channel activation to gene transcription. Using a pharmacological approach, we show that the inhibition of phosphatidylinositol 4-phosphate 5 kinase α (PIP5K), an enzyme essential for the biosynthesis of phosphatidylinositol 4,5-bisphosphate, attenuates TRPM8-induced gene transcription. Analyzing the link between TRPM8 and Gq proteins, we show that the pharmacological inhibition of the ßγ subunits impairs TRPM8 signaling. In addition, genetic studies show that TRPM8 requires an activated Gα subunit for signaling. In the nucleus, the TRPM8-induced signaling cascade triggers the activation of the transcription factor AP-1, a complex consisting of a dimer of basic region leucine zipper (bZIP) transcription factors. Here, we identify the bZIP protein c-Jun as an essential component of AP-1 within the TRPM8-induced signaling cascade. In summary, with PIP5K, Gq subunits, and c-Jun, we identified key molecules in TRPM8-induced signaling from the plasma membrane to the nucleus.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Fosfotransferases (Aceptor do Grupo Álcool) , Transdução de Sinais , Canais de Cátion TRPM , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPM/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fator de Transcrição AP-1/metabolismo , Células HEK293 , Proteínas Proto-Oncogênicas c-jun/metabolismo , Animais
13.
Mol Cell Biol ; 44(5): 178-193, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38767243

RESUMO

Transcription factor 12 (TCF12) is a known oncogene in many cancers. However, whether TCF12 can regulate malignant phenotypes and angiogenesis in osteosarcoma is not elucidated. In this study, we demonstrated increased expression of TCF12 in osteosarcoma tissues and cell lines. High TCF12 expression was associated with metastasis and poor survival rate of osteosarcoma patients. Knockdown of TCF12 reduced the proliferation, migration, and invasion of osteosarcoma cells. TCF12 was found to bind to the promoter region of sphingosine kinase 1 (SPHK1) to induce transcriptional activation of SPHK1 expression and enhance the secretion of sphingosine-1-phosphate (S1P), which eventually resulted in the malignant phenotypes of osteosarcoma cells. In addition, S1P secreted by osteosarcoma cells promoted the angiogenesis of HUVECs by targeting S1PR4 on the cell membrane to activate the STAT3 signaling pathway. These findings suggest that TCF12 may induce transcriptional activation of SPHK1 to promote the synthesis and secretion of S1P. This process likely enhances the malignant phenotypes of osteosarcoma cells and induces angiogenesis via the S1PR4/STAT3 signaling pathway.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Lisofosfolipídeos , Neovascularização Patológica , Osteossarcoma , Fosfotransferases (Aceptor do Grupo Álcool) , Fator de Transcrição STAT3 , Transdução de Sinais , Esfingosina , Humanos , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Lisofosfolipídeos/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/genética , Linhagem Celular Tumoral , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação Neoplásica da Expressão Gênica , Proliferação de Células/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Ativação Transcricional/genética , Receptores de Esfingosina-1-Fosfato/metabolismo , Receptores de Esfingosina-1-Fosfato/genética , Receptores de Lisoesfingolipídeo/metabolismo , Receptores de Lisoesfingolipídeo/genética , Movimento Celular/genética , Masculino , Animais , Feminino , Angiogênese
14.
PLoS One ; 19(5): e0303296, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38753743

RESUMO

AIM: Metabolic dysfunction-associated steatohepatitis (MASH) is one of the most prevalent liver diseases and is characterized by steatosis and the accumulation of bioactive lipids. This study aims to understand the specific lipid species responsible for the progression of liver fibrosis in MASH. METHODS: Changes in bioactive lipid levels were examined in the livers of MASH mice fed a choline-deficient diet (CDD). Additionally, sphingosine kinase (SphK)1 mRNA, which generates sphingosine 1 phosphate (S1P), was examined in the livers of patients with MASH. RESULTS: CDD induced MASH and liver fibrosis were accompanied by elevated levels of S1P and increased expression of SphK1 in capillarized liver sinusoidal endothelial cells (LSECs) in mice. SphK1 mRNA also increased in the livers of patients with MASH. Treatment of primary cultured mouse hepatic stellate cells (HSCs) with S1P stimulated their activation, which was mitigated by the S1P receptor (S1PR)2 inhibitor, JTE013. The inhibition of S1PR2 or its knockout in mice suppressed liver fibrosis without reducing steatosis or hepatocellular damage. CONCLUSION: S1P level is increased in MASH livers and contributes to liver fibrosis via S1PR2.


Assuntos
Fígado Gorduroso , Células Estreladas do Fígado , Cirrose Hepática , Lisofosfolipídeos , Fosfotransferases (Aceptor do Grupo Álcool) , Receptores de Esfingosina-1-Fosfato , Esfingosina , Animais , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Lisofosfolipídeos/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/genética , Cirrose Hepática/etiologia , Camundongos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Humanos , Receptores de Esfingosina-1-Fosfato/metabolismo , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Masculino , Camundongos Knockout , Camundongos Endogâmicos C57BL , Fígado/metabolismo , Fígado/patologia , Deficiência de Colina/complicações , Deficiência de Colina/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Receptores de Lisoesfingolipídeo/metabolismo , Receptores de Lisoesfingolipídeo/genética , Pirazóis , Piridinas
15.
Mol Genet Genomic Med ; 12(5): e2445, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38722107

RESUMO

BACKGROUND: FCSK-congenital disorder of glycosylation (FCSK-CDG) is a recently discovered rare autosomal recessive genetic disorder with defective fucosylation due to mutations in the fucokinase encoding gene, FCSK. Despite the essential role of fucokinase in the fucose salvage pathway and severe multisystem manifestations of FCSK-CDG patients, it is not elucidated which cells or which types of fucosylation are affected by its deficiency. METHODS: In this study, CRISPR/Cas9 was employed to construct an FCSK-CDG cell model and explore the molecular mechanisms of the disease by lectin flow cytometry and real-time PCR analyses. RESULTS: Comparison of cellular fucosylation by lectin flow cytometry in the created CRISPR/Cas9 FCSK knockout and the same unedited cell lines showed no significant change in the amount of cell surface fucosylated glycans, which is consistent with the only documented previous study on different cell types. It suggests a probable effect of this disease on secretory glycoproteins. Investigating O-fucosylation by analysis of the NOTCH3 gene expression as a potential target revealed a significant decrease in the FCSK knockout cells compared with the same unedited ones, proving the effect of fucokinase deficiency on EGF-like repeats O-fucosylation. CONCLUSION: This study expands insight into the FCSK-CDG molecular mechanism; to the best of our knowledge, it is the first research conducted to reveal a gene whose expression level alters due to this disease.


Assuntos
Sistemas CRISPR-Cas , Defeitos Congênitos da Glicosilação , Defeitos Congênitos da Glicosilação/genética , Defeitos Congênitos da Glicosilação/patologia , Defeitos Congênitos da Glicosilação/metabolismo , Humanos , Fucose/metabolismo , Glicosilação , Receptores Notch/metabolismo , Receptores Notch/genética , Fosfotransferases (Aceptor do Grupo Álcool)
16.
Nat Plants ; 10(6): 880-889, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38740943

RESUMO

In plants, the rapid accumulation of proline is a common response to combat abiotic stress1-7. Delta-1-pyrroline-5-carboxylate synthase (P5CS) is a rate-limiting enzyme in proline synthesis, catalysing the initial two-step conversion from glutamate to proline8. Here we determine the first structure of plant P5CS. Our results show that Arabidopsis thaliana P5CS1 (AtP5CS1) and P5CS2 (AtP5CS2) can form enzymatic filaments in a substrate-sensitive manner. The destruction of AtP5CS filaments by mutagenesis leads to a significant reduction in enzymatic activity. Furthermore, separate activity tests on two domains reveal that filament-based substrate channelling is essential for maintaining the high catalytic efficiency of AtP5CS. Our study demonstrates the unique mechanism for the efficient catalysis of AtP5CS, shedding light on the intricate mechanisms underlying plant proline metabolism and stress response.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Prolina/metabolismo , Complexos Multienzimáticos , Fosfotransferases (Aceptor do Grupo Álcool) , Glutamato-5-Semialdeído Desidrogenase
17.
Int J Mol Sci ; 25(10)2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38791117

RESUMO

Antimicrobial resistance (AMR) is one of the biggest threats in modern times. It was estimated that in 2019, 1.27 million deaths occurred around the globe due to AMR. Methicillin-resistant Staphylococcus aureus (MRSA) strains, a pathogen considered of high priority by the World Health Organization, have proven to be resistant to most of the actual antimicrobial treatments. Therefore, new treatments are required to be able to manage this increasing threat. Under this perspective, an important metabolic pathway for MRSA survival, and absent in mammals, is the shikimate pathway, which is involved in the biosynthesis of chorismate, an intermediate for the synthesis of aromatic amino acids, folates, and ubiquinone. Therefore, the enzymes of this route have been considered good targets to design novel antibiotics. The fifth step of the route is performed by shikimate kinase (SK). In this study, an in-house chemical library of 170 benzimidazole derivatives was screened against MRSA shikimate kinase (SaSK). This effort led to the identification of the first SaSK inhibitors, and the two inhibitors with the greatest inhibition activity (C1 and C2) were characterized. Kinetic studies showed that both compounds were competitive inhibitors with respect to ATP and non-competitive for shikimate. Structural analysis through molecular docking and molecular dynamics simulations indicated that both inhibitors interacted with ARG113, an important residue involved in ATP binding, and formed stable complexes during the simulation period. Biological activity evaluation showed that both compounds were able to inhibit the growth of a MRSA strain. Mitochondrial assays showed that both compounds modify the activity of electron transport chain complexes. Finally, ADMETox predictions suggested that, in general, C1 and C2 can be considered as potential drug candidates. Therefore, the benzimidazole derivatives reported here are the first SaSK inhibitors, representing a promising scaffold and a guide to design new drugs against MRSA.


Assuntos
Benzimidazóis , Staphylococcus aureus Resistente à Meticilina , Simulação de Acoplamento Molecular , Fosfotransferases (Aceptor do Grupo Álcool) , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/química , Benzimidazóis/farmacologia , Benzimidazóis/química , Cinética , Antibacterianos/farmacologia , Antibacterianos/química , Simulação de Dinâmica Molecular , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Humanos , Testes de Sensibilidade Microbiana , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química
18.
Int J Mol Sci ; 25(10)2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38791156

RESUMO

The deterioration of osteoblast-led bone formation and the upregulation of osteoclast-regulated bone resorption are the primary causes of bone diseases, including osteoporosis. Numerous circulating factors play a role in bone homeostasis by regulating osteoblast and osteoclast activity, including the sphingolipid-sphingosine-1-phosphate (S1P). However, to date no comprehensive studies have investigated the impact of S1P activity on human and murine osteoblasts and osteoclasts. We observed species-specific responses to S1P in both osteoblasts and osteoclasts, where S1P stimulated human osteoblast mineralisation and reduced human pre-osteoclast differentiation and mineral resorption, thereby favouring bone formation. The opposite was true for murine osteoblasts and osteoclasts, resulting in more mineral resorption and less mineral deposition. Species-specific differences in osteoblast responses to S1P were potentially explained by differential expression of S1P receptor 1. By contrast, human and murine osteoclasts expressed comparable levels of S1P receptors but showed differential expression patterns of the two sphingosine kinase enzymes responsible for S1P production. Ultimately, we reveal that murine models may not accurately represent how human bone cells will respond to S1P, and thus are not a suitable model for exploring S1P physiology or potential therapeutic agents.


Assuntos
Diferenciação Celular , Lisofosfolipídeos , Osteoblastos , Osteoclastos , Especificidade da Espécie , Esfingosina , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Lisofosfolipídeos/metabolismo , Humanos , Animais , Camundongos , Osteoclastos/metabolismo , Osteoclastos/citologia , Osteoblastos/metabolismo , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Receptores de Esfingosina-1-Fosfato/metabolismo , Osso e Ossos/metabolismo , Reabsorção Óssea/metabolismo , Células Cultivadas
19.
Biotechnol J ; 19(5): e2400014, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38719614

RESUMO

Microbial production of L-malic acid from renewable carbon sources has attracted extensive attention. The reduced cofactor NADPH plays a key role in biotransformation because it participates in both biosynthetic reactions and cellular stress responses. In this study, NADPH or its precursors nicotinamide and nicotinic acid were added to the fermentation medium of Aspergillus niger RG0095, which significantly increased the yield of malic acid by 11%. To further improve the titer and productivity of L-malic acid, we increased the cytoplasmic NADPH levels of A. niger by upregulating the NAD kinases Utr1p and Yef1p. Biochemical analyses demonstrated that overexpression of Utr1p and Yef1p reduced oxidative stress, while also providing more NADPH to catalyze the conversion of glucose into malic acid. Notably, the strain overexpressing Utr1p reached a malate titer of 110.72 ± 1.91 g L-1 after 108 h, corresponding to a productivity of 1.03 ± 0.02 g L-1 h-1. Thus, the titer and productivity of malate were increased by 24.5% and 44.7%, respectively. The strategies developed in this study may also be useful for the metabolic engineering of fungi to produce other industrially relevant bulk chemicals.


Assuntos
Aspergillus niger , Fermentação , Malatos , Engenharia Metabólica , NADP , Aspergillus niger/metabolismo , Aspergillus niger/genética , Malatos/metabolismo , Engenharia Metabólica/métodos , NADP/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucose/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo
20.
Clin Neurol Neurosurg ; 241: 108306, 2024 06.
Artigo em Inglês | MEDLINE | ID: mdl-38713962

RESUMO

BACKGROUND: Pantothenate kinase-associated neurodegeneration (PKAN) is a type of inherited metabolic disorder caused by mutation in the PANK2 gene. The metabolic disorder mainly affects the basal ganglia region and eventually manifests as dystonia. For patients of dystonia, their dystonic symptom may progress to life-threatening emergency--status dystonicus. OBJECTIVE: We described a case of a child with PKAN who had developed status dystonicus and was successfully treated with deep brain stimulation (DBS). Based on this rare condition, we analysed the clinical features of PKAN with status dystonicus and reviewed the reasonable management process of this condition. CONCLUSION: This case confirmed the rationality of choosing DBS for the treatment of status dystonicus. Meanwhile, we found that children with classic PKAN have a cluster of risk factors for developing status dystonicus. Once children diagnosed with similar neurodegenerative diseases are under status dystonicus, DBS can be active considered because it has showed high control rate of this emergent condition.


Assuntos
Estimulação Encefálica Profunda , Neurodegeneração Associada a Pantotenato-Quinase , Humanos , Neurodegeneração Associada a Pantotenato-Quinase/genética , Estimulação Encefálica Profunda/métodos , Masculino , Criança , Distonia/terapia , Feminino , Distúrbios Distônicos/terapia , Distúrbios Distônicos/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética
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