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1.
Mol Cell ; 70(3): 531-544.e9, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29727621

RESUMO

While the majority of phosphatidylinositol-4, 5-bisphosphate (PI-4, 5-P2) in mammalian cells is generated by the conversion of phosphatidylinositol-4-phosphate (PI-4-P) to PI-4, 5-P2, a small fraction can be made by phosphorylating phosphatidylinositol-5-phosphate (PI-5-P). The physiological relevance of this second pathway is not clear. Here, we show that deletion of the genes encoding the two most active enzymes in this pathway, Pip4k2a and Pip4k2b, in the liver of mice causes a large enrichment in lipid droplets and in autophagic vesicles during fasting. These changes are due to a defect in the clearance of autophagosomes that halts autophagy and reduces the supply of nutrients salvaged through this pathway. Similar defects in autophagy are seen in nutrient-starved Pip4k2a-/-Pip4k2b-/- mouse embryonic fibroblasts and in C. elegans lacking the PI5P4K ortholog. These results suggest that this alternative pathway for PI-4, 5-P2 synthesis evolved, in part, to enhance the ability of multicellular organisms to survive starvation.


Assuntos
Autofagia/fisiologia , Jejum/metabolismo , Metabolismo dos Lipídeos/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Autofagossomos/metabolismo , Caenorhabditis elegans/metabolismo , Linhagem Celular , Fibroblastos/metabolismo , Células HEK293 , Humanos , Fígado/metabolismo , Camundongos , Fosfatos de Fosfatidilinositol/metabolismo , Transdução de Sinais/fisiologia
2.
Proc Natl Acad Sci U S A ; 118(31)2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34312224

RESUMO

Regulatory T cells (Tregs) play fundamental roles in maintaining peripheral tolerance to prevent autoimmunity and limit legitimate immune responses, a feature hijacked in tumor microenvironments in which the recruitment of Tregs often extinguishes immune surveillance through suppression of T-effector cell signaling and tumor cell killing. The pharmacological tuning of Treg activity without impacting on T conventional (Tconv) cell activity would likely be beneficial in the treatment of various human pathologies. PIP4K2A, 2B, and 2C constitute a family of lipid kinases that phosphorylate PtdIns5P to PtdIns(4,5)P2 They are involved in stress signaling, act as synthetic lethal targets in p53-null tumors, and in mice, the loss of PIP4K2C leads to late onset hyperinflammation. Accordingly, a human single nucleotide polymorphism (SNP) near the PIP4K2C gene is linked with susceptibility to autoimmune diseases. How PIP4Ks impact on human T cell signaling is not known. Using ex vivo human primary T cells, we found that PIP4K activity is required for Treg cell signaling and immunosuppressive activity. Genetic and pharmacological inhibition of PIP4K in Tregs reduces signaling through the PI3K, mTORC1/S6, and MAPK pathways, impairs cell proliferation, and increases activation-induced cell death while sparing Tconv. PIP4K and PI3K signaling regulate the expression of the Treg master transcriptional activator FOXP3 and the epigenetic signaling protein Ubiquitin-like containing PHD and RING finger domains 1 (UHRF1). Our studies suggest that the pharmacological inhibition of PIP4K can reprogram human Treg identity while leaving Tconv cell signaling and T-helper differentiation to largely intact potentially enhancing overall immunological activity.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Linfócitos T Reguladores/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proliferação de Células , Sobrevivência Celular , Clonagem Molecular , Fatores de Transcrição Forkhead/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/imunologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Terapia de Imunossupressão , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Quinazolinas/farmacologia , Transdução de Sinais , Tiofenos/farmacologia , Ubiquitina-Proteína Ligases/genética
3.
Mol Cell ; 58(3): 453-67, 2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25866244

RESUMO

Phosphoinositides (PI) are important signaling molecules in the nucleus that influence gene expression. However, if and how nuclear PI directly affects the transcriptional machinery is not known. We report that the lipid kinase PIP4K2B regulates nuclear PI5P and the expression of myogenic genes during myoblast differentiation. A targeted screen for PI interactors identified the PHD finger of TAF3, a TATA box binding protein-associated factor with important roles in transcription regulation, pluripotency, and differentiation. We show that the PI interaction site is distinct from the known H3K4me3 binding region of TAF3 and that PI binding modulates association of TAF3 with H3K4me3 in vitro and with chromatin in vivo. Analysis of TAF3 mutants indicates that TAF3 transduces PIP4K2B-mediated alterations in PI into changes in specific gene transcription. Our study reveals TAF3 as a direct target of nuclear PI and further illustrates the importance of basal transcription components as signal transducers.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Homeodomínio/metabolismo , Fosfatidilinositóis/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Animais , Diferenciação Celular/genética , Linhagem Celular , Núcleo Celular/genética , Eletroforese em Gel de Poliacrilamida , Perfilação da Expressão Gênica , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Lisina/metabolismo , Metilação , Camundongos , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Mutação , Mioblastos/citologia , Mioblastos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Interferência de RNA , Homologia de Sequência de Aminoácidos , Fatores Associados à Proteína de Ligação a TATA , Fator de Transcrição TFIID/genética , Fator de Transcrição TFIID/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
4.
Bioorg Med Chem ; 54: 116557, 2022 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-34922306

RESUMO

Phosphatidyl inositol (4,5)-bisphosphate (PI(4,5)P2) plays several key roles in human biology and the lipid kinase that produces PI(4,5)P2, PIP5K, has been hypothesized to provide a potential therapeutic target of interest in the treatment of cancers. To better understand and explore the role of PIP5K in human cancers there remains an urgent need for potent and specific PIP5K inhibitor molecules. Following a high throughput screen of the AstraZeneca collection, a novel, moderately potent and selective inhibitor of PIP5K, 1, was discovered. Detailed exploration of the SAR for this novel scaffold resulted in the considerable optimization of both potency for PIP5K, and selectivity over the closely related kinase PI3Kα, as well as identifying several opportunities for the continued optimization of drug-like properties. As a result, several high quality in vitro tool compounds were identified (8, 20 and 25) that demonstrate the desired biochemical and cellular profiles required to aid better understanding of this complex area of biology.


Assuntos
Amidas/farmacologia , Inibidores Enzimáticos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Amidas/química , Amidas/metabolismo , Animais , Células CACO-2 , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ratos , Relação Estrutura-Atividade
5.
Mol Cell ; 54(6): 905-919, 2014 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-24813945

RESUMO

UHRF1 is a multidomain protein crucially linking histone H3 modification states and DNA methylation. While the interaction properties of its specific domains are well characterized, little is known about the regulation of these functionalities. We show that UHRF1 exists in distinct active states, binding either unmodified H3 or the H3 lysine 9 trimethylation (H3K9me3) modification. A polybasic region (PBR) in the C terminus blocks interaction of a tandem tudor domain (TTD) with H3K9me3 by occupying an essential peptide-binding groove. In this state the plant homeodomain (PHD) mediates interaction with the extreme N terminus of the unmodified H3 tail. Binding of the phosphatidylinositol phosphate PI5P to the PBR of UHRF1 results in a conformational rearrangement of the domains, allowing the TTD to bind H3K9me3. Our results define an allosteric mechanism controlling heterochromatin association of an essential regulatory protein of epigenetic states and identify a functional role for enigmatic nuclear phosphatidylinositol phosphates.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/química , Histonas/química , Fosfatos de Fosfatidilinositol/química , Regulação Alostérica , Sítios de Ligação/fisiologia , Linhagem Celular Tumoral , Metilação de DNA , Células HeLa , Heterocromatina/fisiologia , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Ubiquitina-Proteína Ligases
7.
J Proteome Res ; 18(3): 1032-1042, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30672294

RESUMO

DNA methylation is a critical epigenetic modification that is established and maintained across the genome by DNA methyltransferase enzymes (Dnmts). Altered patterns of DNA methylation are a frequent occurrence in many tumor genomes, and inhibitors of Dnmts have become important epigenetic drugs. Azacitidine is a cytidine analog that is incorporated into DNA and induces the specific inhibition and proteasomal-mediated degradation of Dnmts. The downstream effects of azacitidine on CpG methylation and on gene transcription have been widely studied in many systems, but how azacitidine impacts the proteome is not well-understood. In addition, with its specific ability to induce the rapid degradation of Dnmts (in particular, the primary maintenance DNA methyltransferase, Dnmt1), it may be employed as a specific chemical knockdown for investigating the Dnmt1-associated functional or physical interactome. In this study, we use quantitative proteomics to analyze the degradation profile of proteins in the nuclear proteome of cells treated with azacitidine. We identify specific proteins as well as multiple pathways and processes that are impacted by azacitidine. The Dnmt1 interaction partner, Uhrf1, exhibits significant azacitidine-induced degradation, and this azacitidine-induced degradation is independent of the levels of Dnmt1 protein. We identify multiple other chromatin- and epigenetic-associated factors, including the bromodomain-containing transcriptional regulator, Brd2. We show that azacitidine induces highly specific perturbations of the Dnmt1-associated proteome, and while interaction partners such as Uhrf1 are sensitive to azacitidine, others such as the Dnmt1 interaction partner and stability regulator, Usp7, are not. In summary, we have conducted the first comprehensive proteomic analysis of the azacitidine-sensitive nuclear proteome, and we show how 5-azacitidine can be used as a specific probe to explore Dnmt- and chromatin-related protein networks.


Assuntos
Azacitidina/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Proteômica/métodos , Ubiquitina-Proteína Ligases/metabolismo , Núcleo Celular/química , Núcleo Celular/metabolismo , Cromatina/efeitos dos fármacos , Metilação de DNA , Epigênese Genética , Células HCT116 , Humanos , Peptidase 7 Específica de Ubiquitina
8.
Int J Mol Sci ; 20(9)2019 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-31035587

RESUMO

Phosphatidylinositol (PI)-related signaling plays a pivotal role in many cellular aspects, including survival, cell proliferation, differentiation, DNA damage, and trafficking. PI is the core of a network of proteins represented by kinases, phosphatases, and lipases which are able to add, remove or hydrolyze PI, leading to different phosphoinositide products. Among the seven known phosphoinositides, phosphatidylinositol 5 phosphate (PI5P) was the last to be discovered. PI5P presence in cells is very low compared to other PIs. However, much evidence collected throughout the years has described the role of this mono-phosphoinositide in cell cycles, stress response, T-cell activation, and chromatin remodeling. Interestingly, PI5P has been found in different cellular compartments, including the nucleus. Here, we will review the nuclear role of PI5P, describing how it is synthesized and regulated, and how changes in the levels of this rare phosphoinositide can lead to different nuclear outputs.


Assuntos
Núcleo Celular/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Animais , Humanos , Metabolismo dos Lipídeos , Proteínas Nucleares/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais , Estresse Fisiológico
9.
Nature ; 492(7428): 276-9, 2012 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-23235882

RESUMO

At the end of cell division, cytokinesis splits the cytoplasm of nascent daughter cells and partitions segregated sister genomes. To coordinate cell division with chromosome segregation, the mitotic spindle controls cytokinetic events at the cell envelope. The spindle midzone stimulates the actomyosin-driven contraction of the cleavage furrow, which proceeds until the formation of a microtubule-rich intercellular bridge with the midbody at its centre. The midbody directs the final membrane abscission reaction and has been proposed to attach the cleavage furrow to the intercellular bridge. How the mitotic spindle is connected to the plasma membrane during cytokinesis is not understood. Here we identify a plasma membrane tethering activity in the centralspindlin protein complex, a conserved component of the spindle midzone and midbody. We demonstrate that the C1 domain of the centralspindlin subunit MgcRacGAP associates with the plasma membrane by interacting with polyanionic phosphoinositide lipids. Using X-ray crystallography we determine the structure of this atypical C1 domain. Mutations in the hydrophobic cap and in basic residues of the C1 domain of MgcRacGAP prevent association of the protein with the plasma membrane, and abrogate cytokinesis in human and chicken cells. Artificial membrane tethering of centralspindlin restores cell division in the absence of the C1 domain of MgcRacGAP. Although C1 domain function is dispensable for the formation of the midzone and midbody, it promotes contractility and is required for the attachment of the plasma membrane to the midbody, a long-postulated function of this organelle. Our analysis suggests that centralspindlin links the mitotic spindle to the plasma membrane to secure the final cut during cytokinesis in animal cells.


Assuntos
Membrana Celular/metabolismo , Citocinese/efeitos da radiação , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Animais , Citocinese/genética , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/química , Microtúbulos/metabolismo , Modelos Moleculares , Ligação Proteica , Proteína Quinase C-alfa/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologia
10.
Biochim Biophys Acta ; 1851(6): 898-910, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25728392

RESUMO

Phosphatidylinositol-5-phosphate (PtdIns5P)-4-kinases (PIP4Ks) are stress-regulated lipid kinases that phosphorylate PtdIns5P to generate PtdIns(4,5)P2. There are three isoforms of PIP4Ks: PIP4K2A, 2B and 2C, which localise to different subcellular compartments with the PIP4K2B isoform being localised predominantly in the nucleus. Suppression of PIP4K expression selectively prevents tumour cell growth in vitro and prevents tumour development in mice that have lost the tumour suppressor p53. p53 is lost or mutated in over 70% of all human tumours. These studies suggest that inhibition of PIP4K signalling constitutes a novel anti-cancer therapeutic target. In this review we will discuss the role of PIP4K in tumour suppression and speculate on how PIP4K modulates nuclear phosphoinositides (PPIns) and how this might impact on nuclear functions to regulate cell growth. This article is part of a Special Issue entitled Phosphoinositides.


Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Núcleo Celular/enzimologia , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/enzimologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , 1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , 1-Fosfatidilinositol 4-Quinase/genética , Animais , Antineoplásicos/farmacologia , Citoplasma/enzimologia , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética
11.
Biochem Soc Trans ; 44(1): 299-306, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26862219

RESUMO

Phosphoinositides are a family of phospholipid messenger molecules that control various aspects of cell biology in part by interacting with and regulating downstream protein partners. Importantly, phosphoinositides are present in the nucleus. They form part of the nuclear envelope and are present within the nucleus in nuclear speckles, intra nuclear chromatin domains, the nuclear matrix and in chromatin. What their exact role is within these compartments is not completely clear, but the identification of nuclear specific proteins that contain phosphoinositide interaction domains suggest that they are important regulators of DNA topology, chromatin conformation and RNA maturation and export. The plant homeo domain (PHD) finger is a phosphoinositide binding motif that is largely present in nuclear proteins that regulate chromatin conformation. In the present study I outline how changes in the levels of the nuclear phosphoinositide PtdIns5P impact on muscle cell differentiation through the PHD finger of TAF3 (TAF, TATA box binding protein (TBP)-associated factor), which is a core component of a number of different basal transcription complexes.


Assuntos
Diferenciação Celular , Núcleo Celular/metabolismo , Desenvolvimento Muscular , Músculos/metabolismo , Fosfatidilinositóis/metabolismo , Animais , Humanos , Músculos/citologia , Transdução de Sinais
12.
Proc Natl Acad Sci U S A ; 110(15): 5963-8, 2013 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-23530222

RESUMO

During development, Drosophila larvae undergo a dramatic increase in body mass wherein nutritional and developmental cues are transduced into growth through the activity of complex signaling pathways. Class I phosphoinositide 3-kinases have an established role in this process. In this study we identify Drosophila phosphatidylinositol 5-phosphate 4-kinase (dPIP4K) as a phosphoinositide kinase that regulates growth during larval development. Loss-of-function mutants in dPIP4K show reduced body weight and prolonged larval development, whereas overexpression of dPIP4K results both in an increase in body weight and shortening of larval development. The growth defect associated with dPIP4K loss of function is accompanied by a reduction in the average cell size of larval endoreplicative tissues. Our findings reveal that these phenotypes are underpinned by changes in the signaling input into the target of rapamycin (TOR) signaling complex and changes in the activity of its direct downstream target p70 S6 kinase. Together, these results define dPIP4K activity as a regulator of cell growth and TOR signaling during larval development.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Sequência de Aminoácidos , Animais , Proliferação de Células , Drosophila melanogaster/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Microscopia Confocal , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos
13.
Hum Mol Genet ; 21(8): 1744-59, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22210625

RESUMO

Lowe syndrome, which is characterized by defects in the central nervous system, eyes and kidneys, is caused by mutation of the phosphoinositide 5-phosphatase OCRL1. The mechanisms by which loss of OCRL1 leads to the phenotypic manifestations of Lowe syndrome are currently unclear, in part, owing to the lack of an animal model that recapitulates the disease phenotype. Here, we describe a zebrafish model for Lowe syndrome using stable and transient suppression of OCRL1 expression. Deficiency of OCRL1, which is enriched in the brain, leads to neurological defects similar to those reported in Lowe syndrome patients, namely increased susceptibility to heat-induced seizures and cystic brain lesions. In OCRL1-deficient embryos, Akt signalling is reduced and there is both increased apoptosis and reduced proliferation, most strikingly in the neural tissue. Rescue experiments indicate that catalytic activity and binding to the vesicle coat protein clathrin are essential for OCRL1 function in these processes. Our results indicate a novel role for OCRL1 in neural development, and support a model whereby dysregulation of phosphoinositide metabolism and clathrin-mediated membrane traffic leads to the neurological symptoms of Lowe syndrome.


Assuntos
Encéfalo/embriologia , Modelos Animais de Doenças , Síndrome Oculocerebrorrenal , Monoéster Fosfórico Hidrolases/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra , Animais , Encéfalo/patologia , Sobrevivência Celular , Clatrina/metabolismo , Embrião não Mamífero , Desenvolvimento Embrionário , Endossomos/metabolismo , Perfilação da Expressão Gênica , Complexo de Golgi/metabolismo , Temperatura Alta , Fosfatidilinositol 4,5-Difosfato/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Processamento de Proteína , Proteínas Proto-Oncogênicas c-akt/metabolismo , Convulsões/fisiopatologia , Transdução de Sinais , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/metabolismo
14.
FASEB J ; 27(4): 1644-56, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23241309

RESUMO

Oxidative stress initiates signaling pathways, which protect from stress-induced cellular damage, initiate apoptosis, or drive cells into senescence or into tumorigenesis. Oxidative stress regulates the activity of the cell survival factor PKB, through the regulation of PtdIns(3,4,5)P3 synthesis. Whether oxidative stress regulates other phosphoinositides to control PKB activation is not clear. Here we show that PtdIns5P is a redox-regulated second messenger. In response to hydrogen peroxide (H2O2), we measured an increase in PtdIns5P in cells derived from human osteosarcoma, U2OS (5-fold); breast tumors, MDA-MB-468 (2-fold); and fibrosarcoma, HT1080 (3-fold); and in p53-null murine embryonic fibroblasts (8-fold). In U2OS cells, the increase in H2O2-dependent PtdIns5P did not require mTOR, PDK1, PKB, ERK, and p38 signaling or PIKfyve, a lipid kinase that increases PtdIns5P in response to osmotic and oncogenic signaling. A reduction in H2O2-induced PtdIns5P levels by the overexpression of PIP4K revealed its role in PKB activation. Suppression of H2O2-induced PtdIns5P generation reduced PKB activation and, surprisingly, reduced cell sensitivity to growth inhibition by H2O2. These data suggest that inhibition of PIP4K signaling might be useful as a novel strategy to increase the susceptibility of tumor cells to therapeutics that function through increased oxidative stress.


Assuntos
Estresse Oxidativo/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática , Humanos , Peróxido de Hidrogênio/farmacologia , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
Biochem J ; 455(3): 347-58, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23909401

RESUMO

The spatial and temporal regulation of the second messenger PtdIns(4,5)P2 has been shown to be crucial for regulating numerous processes in the cytoplasm and in the nucleus. Three isoforms of PIP5K1 (phosphatidylinositol 4-phosphate 5-kinase), A, B and C, are responsible for the regulation of the major pools of cellular PtdIns(4,5)P2. PIP5K1B is negatively regulated in response to oxidative stress although it remains unclear which pathways regulate its activity. In the present study, we have investigated the regulation of PIP5K1B by protein phosphorylation. Using MS analysis, we identified 12 phosphorylation sites on PIP5K1B. We developed a phospho-specific antibody against Ser413 and showed that its phosphorylation was increased in response to treatment of cells with phorbol ester, H2O2 or energy restriction. Using inhibitors, we define a stress-dependent pathway that requires the activity of the cellular energy sensor AMPK (AMP-activated protein kinase) and PKC (protein kinase C) to regulate Ser413 phosphorylation. Furthermore, we demonstrate that PKC can directly phosphorylate Ser413 in vitro. Mutation of Ser413 to aspartate to mimic serine phosphorylation decreased both PIP5K1B activity in vitro and PtdIns(4,5)P2 synthesis in vivo. Our studies show that collaboration between AMPK and PKC dictates the extent of Ser413 phosphorylation on PIP5K1B and regulates PtdIns(4,5)P2 synthesis.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Metabolismo Energético , Estresse Oxidativo , Serina/genética , Células HEK293 , Células HeLa , Humanos , Peróxido de Hidrogênio/metabolismo , Mutação , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Serina/metabolismo
16.
ACS Photonics ; 11(1): 42-52, 2024 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-38249683

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) has had a tremendous impact on humanity. Prevention of transmission by disinfection of surfaces and aerosols through a chemical-free method is highly desirable. Ultraviolet C (UVC) light is uniquely positioned to achieve inactivation of pathogens. We report the inactivation of SARS-CoV-2 virus by UVC radiation and explore its mechanisms. A dose of 50 mJ/cm2 using a UVC laser at 266 nm achieved an inactivation efficiency of 99.89%, while infectious virions were undetectable at 75 mJ/cm2 indicating >99.99% inactivation. Infection by SARS-CoV-2 involves viral entry mediated by the spike glycoprotein (S), and viral reproduction, reliant on translation of its genome. We demonstrate that UVC radiation damages ribonucleic acid (RNA) and provide in-depth characterization of UVC-induced damage of the S protein. We find that UVC severely impacts SARS-CoV- 2 spike protein's ability to bind human angiotensin-converting enzyme 2 (hACE2) and this correlates with loss of native protein conformation and aromatic amino acid integrity. This report has important implications for the design and development of rapid and effective disinfection systems against the SARS-CoV-2 virus and other pathogens.

17.
Plant J ; 70(5): 866-78, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22324391

RESUMO

Myotubularin and myotubularin-related proteins are evolutionarily conserved in eukaryotes. Defects in their function result in muscular dystrophy, neuronal diseases and leukemia in humans. In contrast to the animal lineage, where genes encoding both active and inactive myotubularins (phosphoinositide 3-phosphatases) have appeared and proliferated in the basal metazoan group, myotubularin genes are not found in the unicellular relatives of green plants. However, they are present in land plants encoding proteins highly similar to the active metazoan enzymes. Despite their remarkable structural conservation, plant and animal myotubularins have significantly diverged in their functions. While loss of myotubularin function causes severe disease phenotypes in humans it is not essential for the cellular homeostasis under normal conditions in Arabidopsis thaliana. Instead, myotubularin deficiency is associated with altered tolerance to dehydration stress. The two Arabidopsis genes AtMTM1 and AtMTM2 have originated from a segmental chromosomal duplication and encode catalytically active enzymes. However, only AtMTM1 is involved in elevating the cellular level of phosphatidylinositol 5-phosphate in response to dehydration stress, and the two myotubularins differentially affect the Arabidopsis dehydration stress-responding transcriptome. AtMTM1 and AtMTM2 display different localization patterns in the cell, consistent with the idea that they associate with different membranes to perform specific functions. A single amino acid mutation in AtMTM2 (L250W) results in a dramatic loss of subcellular localization. Mutations in this region are linked to disease conditions in humans.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Evolução Molecular , Proteínas Mitocondriais/metabolismo , Substituição de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Duplicação Cromossômica , Cromossomos de Plantas/genética , Cromossomos de Plantas/metabolismo , Desidratação/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Proteínas Mitocondriais/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Células Vegetais/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Solo , Estresse Fisiológico , Transcriptoma
18.
Mol Cell Proteomics ; 10(2): M110.003376, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21048195

RESUMO

Considerable insight into phosphoinositide-regulated cytoplasmic functions has been gained by identifying phosphoinositide-effector proteins. Phosphoinositide-regulated nuclear functions however are fewer and less clear. To address this, we established a proteomic method based on neomycin extraction of intact nuclei to enrich for nuclear phosphoinositide-effector proteins. We identified 168 proteins harboring phosphoinositide-binding domains. Although the vast majority of these contained lysine/arginine-rich patches with the following motif, K/R-(X(n= 3-7)-K-X-K/R-K/R, we also identified a smaller subset of known phosphoinositide-binding proteins containing pleckstrin homology or plant homeodomain modules. Proteins with no prior history of phosphoinositide interaction were identified, some of which have functional roles in RNA splicing and processing and chromatin assembly. The remaining proteins represent potentially other novel nuclear phosphoinositide-effector proteins and as such strengthen our appreciation of phosphoinositide-regulated nuclear functions. DNA topology was exemplar among these: Biochemical assays validated our proteomic data supporting a direct interaction between phosphatidylinositol 4,5-bisphosphate and DNA Topoisomerase IIα. In addition, a subset of neomycin extracted proteins were further validated as phosphatidyl 4,5-bisphosphate-interacting proteins by quantitative lipid pull downs. In summary, data sets such as this serve as a resource for a global view of phosphoinositide-regulated nuclear functions.


Assuntos
Núcleo Celular/metabolismo , Neomicina/farmacologia , Fosfatidilinositol 4,5-Difosfato/química , Proteômica/métodos , Motivos de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Citoplasma/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Glutationa Transferase/metabolismo , Humanos , Células Jurkat , Fosfatidilinositóis/química , Ligação Proteica , Estrutura Terciária de Proteína
19.
Subcell Biochem ; 59: 335-61, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22374096

RESUMO

Lipid signalling in human disease is an important field of investigation and stems from the fact that phosphoinositide signalling has been implicated in the control of nearly all the important cellular pathways including metabolism, cell cycle control, membrane trafficking, apoptosis and neuronal conduction. A distinct nuclear inositide signalling metabolism has been identified, thus defining a new role for inositides in the nucleus, which are now considered essential co-factors for several nuclear processes, including DNA repair, transcription regulation, and RNA dynamics. Deregulation of phoshoinositide metabolism within the nuclear compartment may contribute to disease progression in several disorders, such as chronic inflammation, cancer, metabolic, and degenerative syndromes. In order to utilize these very druggable pathways for human benefit there is a need to identify how nuclear inositides are regulated specifically within this compartment and what downstream nuclear effectors process and integrate inositide signalling cascades in order to specifically control nuclear function. Here we describe some of the facets of nuclear inositide metabolism with a focus on their relationship to cell cycle control and differentiation.


Assuntos
Núcleo Celular/metabolismo , Síndromes Mielodisplásicas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositóis/metabolismo , Fosfolipase C beta/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transporte Biológico , Ciclo Celular/genética , Diferenciação Celular , Regulação da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfolipase C beta/genética , Monoéster Fosfórico Hidrolases/genética , Transdução de Sinais
20.
Front Neurosci ; 17: 1196007, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37342467

RESUMO

In Alzheimer's disease, tau pathology is thought to spread via a prion-like manner along connected neuronal networks. For this to occur, the usually cytosolic tau protein must be secreted via an unconventional mechanism prior to uptake into the connected neuron. While secretion of healthy and pathological tau has been documented, it remains under-investigated whether this occurs via overlapping or distinct processes. Here, we established a sensitive bioluminescence-based assay to assess mechanisms underlying the secretion of pseudohyperphosphorylated and wild-type tau in cultured murine hippocampal neurons. We found that under basal conditions, both wild-type and mutant tau are secreted, with mutant tau being more robustly secreted. Pharmacological stimulation of neuronal activity led to a modest increase of wild-type and mutant tau secretion, whereas inhibition of activity had no effect. Interestingly, inhibition of heparin sulfate proteoglycan (HSPG) biosynthesis drastically decreased secretion of both wild-type and mutant tau without affecting cell viability. This shows that native and pathological tau share release mechanisms; both activity-dependent and non-activity-dependent secretion of tau is facilitated by HSPGs.

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