RESUMO
Lateral roots (LRs) increase the contact area of the root with the rhizosphere and thereby improve water and nutrient uptake from the soil. LRs are generated either via a developmentally controlled mechanism or through induction by external stimuli, such as water and nutrient availability. Auxin regulates LR organogenesis via transcriptional activation by an auxin complex receptor. Endocytic trafficking to the vacuole positively regulates LR organogenesis independently of the auxin complex receptor in Arabidopsis (Arabidopsis thaliana). Here, we demonstrate that phosphatidylinositol 4-phosphate (PI4P) biosynthesis regulated by the phosphatidylinositol 4-kinases PI4KIIIß1 and PI4KIIIß2 is essential for the LR organogenesis driven by endocytic trafficking to the vacuole. Stimulation with Sortin2, a biomodulator that promotes protein targeting to the vacuole, altered PI4P abundance at both the plasma membrane and endosomal compartments, a process dependent on PI4K activity. These findings suggest that endocytic trafficking to the vacuole regulated by the enzymatic activities of PI4KIIIß1 and PI4KIIIß2 participates in a mechanism independent of the auxin complex receptor that regulates LR organogenesis in Arabidopsis. Surprisingly, loss-of-function of PI4KIIIß1 and PI4KIIIß2 induced both LR primordium formation and endocytic trafficking toward the vacuole. This LR primordium induction was alleviated by exogenous PI4P, suggesting that PI4KIIIß1 and PI4KIIIß2 activity constitutively negatively regulates LR primordium formation. Overall, this research demonstrates a dual role of PI4KIIIß1 and PI4KIIIß2 in LR primordium formation in Arabidopsis.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Ácidos Indolacéticos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , 1-Fosfatidilinositol 4-Quinase/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Endossomos/metabolismo , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/fisiologia , Transporte Proteico , Transdução de Sinais , Vacúolos/metabolismoRESUMO
BACKGROUND: Undernutrition during childhood leads to chronic diseases in adult life including hypertension, diabetes and chronic kidney disease. Here we explore the hypothesis that physiological alterations in the bioactive lipids pattern within kidney tissue might be involved in the progression of chronic kidney disease. METHODS: Membrane fractions from kidney homogenates of undernourished rats (RBD) were submitted to lipid extraction and analysis by thin layer chromatography and cholesterol determination. RESULTS: Kidneys from RBD rats had 25% lower cholesterol content, which disturb membrane microdomains, affecting Ca2+ homeostasis and the enzymes responsible for important lipid mediators such as phosphatidylinositol-4 kinase, sphingosine kinase, diacylglicerol kinase and phospholipase A2. We observed a decrease in phosphatidylinositol(4)-phosphate (8.8 ± 0.9 vs. 3.6 ± 0.7 pmol.mg-1.mim-1), and an increase in phosphatidic acid (2.2 ± 0.8 vs. 3.8 ± 1.3 pmol.mg-1.mim-1), being these lipid mediators involved in the regulation of key renal functions. Ceramide levels are augmented in kidney tissue from RBD rats (18.7 ± 1.4 vs. 21.7 ± 1.5 fmol.mg-1.min-1) indicating an ongoing renal lesion. CONCLUSION: Results point to an imbalance in the bioactive lipid generation with further consequences to key events related to kidney function, thus contributing to the establishment of chronic kidney disease.
Assuntos
Colesterol/metabolismo , Hipertensão/metabolismo , Rim/metabolismo , Desnutrição/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Insuficiência Renal Crônica/metabolismo , 1-Fosfatidilinositol 4-Quinase/genética , 1-Fosfatidilinositol 4-Quinase/metabolismo , Animais , Animais Recém-Nascidos , Ceramidas/metabolismo , Diacilglicerol Quinase/genética , Diacilglicerol Quinase/metabolismo , Regulação da Expressão Gênica , Hipertensão/etiologia , Hipertensão/genética , Hipertensão/patologia , Rim/química , Metabolismo dos Lipídeos , Masculino , Desnutrição/complicações , Desnutrição/genética , Desnutrição/patologia , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosfolipases A2/genética , Fosfolipases A2/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ratos , Ratos Wistar , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologiaRESUMO
Sea Island cotton (Gossypium barbadense) is highly valued for its superior fiber qualities, especially fiber strength. Based on a transcript-derived fragment originated from transcriptome QTL mapping, a fiber strength related candidate gene of phosphatidylinositol 4-kinase cDNA, designated as GbPI4K, was first cloned, and its expression was characterized in the secondary cell wall thickening stage of G. barbadense fibers. The ORF of GbPI4K was found to be 1926 bp in length and encoded a predicted protein of 641 amino acid residues. The putative protein contained a clear PI3/4K kinase catalytic domain and fell into the plant type II PI4K cluster in phylogenetic analysis. In this study, the expression of cotton PI4K protein was also induced in Escherichia coli BL21 (DE3) as a fused protein. Semi-quantitative RT-PCR analysis showed that the gene expressed in the root, hypocotyl and leaf of the cotton plants. Real-time RT-PCR indicated that this gene in Sea Island cotton fibers expressed 10 days longer than that in Upland cotton fibers, and the main expression difference of PI4K between Sea Island cotton and Upland cotton in fibers was located in the secondary cell wall thickening stage of the fiber. Further analysis indicated that PI4K is a crucial factor in the ability of Rac proteins to regulate phospholipid signaling pathways.
Assuntos
1-Fosfatidilinositol 4-Quinase/genética , Mapeamento Cromossômico , Fibra de Algodão , Gossypium/enzimologia , Gossypium/genética , Locos de Características Quantitativas/genética , Transcriptoma/genética , 1-Fosfatidilinositol 4-Quinase/química , 1-Fosfatidilinositol 4-Quinase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Filogenia , Células Procarióticas/metabolismo , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Especificidade da EspécieRESUMO
The oncoprotein c-Fos is a well-recognized AP-1 transcription factor. In addition, this protein associates with the endoplasmic reticulum and activates the synthesis of phospholipids. However, the mechanism by which c-Fos stimulates the synthesis of phospholipids in general and the specific lipid pathways activated are unknown. Here we show that induction of quiescent cells to reenter growth promotes an increase in the labeling of polyphosphoinositides that depends on the expression of c-Fos. We also investigated whether stimulation by c-Fos of the synthesis of phosphatidylinositol and its phosphorylated derivatives depends on the activation of enzymes of the phosphatidylinositolphosphate biosynthetic pathway. We found that c-Fos activates CDP-diacylglycerol synthase and phosphatidylinositol (PtdIns) 4-kinase II α in vitro, whereas no activation of phosphatidylinositol synthase or of PtdIns 4-kinase II ß was observed. Both coimmunoprecipitation and fluorescence resonance energy transfer experiments consistently showed a physical interaction between the N-terminal domain of c-Fos and the enzymes it activates.
Assuntos
Fosfatos de Fosfatidilinositol/biossíntese , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fator de Transcrição AP-1/metabolismo , 1-Fosfatidilinositol 4-Quinase/biossíntese , 1-Fosfatidilinositol 4-Quinase/genética , Animais , Diacilglicerol Colinofosfotransferase/biossíntese , Diacilglicerol Colinofosfotransferase/genética , Ativação Enzimática/fisiologia , Indução Enzimática/fisiologia , Camundongos , Células NIH 3T3 , Fosfatos de Fosfatidilinositol/genética , Fosfolipídeos/biossíntese , Fosfolipídeos/genética , Estrutura Terciária de Proteína/fisiologia , Proteínas Proto-Oncogênicas c-fos/genética , Fator de Transcrição AP-1/genéticaRESUMO
Phosphatidylinositol (PI) kinases are at the heart of one of the major pathways of intracellular signal transduction. Herein, we present the first report on a survey made by similarity searches against the five human pathogenic trypanosomatids Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, Leishmania braziliensis and Leishmania infantum genomes available to date for phosphatidylinositol- and related-kinases (TryPIKs). In addition to generating a panel called "The TryPIKinome", we propose a model of signaling pathways for these TryPIKs. The involvement of TryPIKs in fundamental pathways, such as intracellular signal transduction and host invasion processes, makes the study of TryPIKs an important area for further inquiry. New subtype-specific inhibitors are expected to work on individual members of the PIK family and, therefore, can presumably neutralize trypanosomatid invasion processes.