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1.
Nucleic Acids Res ; 49(15): 8535-8555, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34358317

RESUMO

Gene deletion and gene expression alteration can lead to growth defects that are amplified or reduced when a second mutation is present in the same cells. We performed 154 genetic interaction mapping (GIM) screens with query mutants related with RNA metabolism and estimated the growth rates of about 700 000 double mutant Saccharomyces cerevisiae strains. The tested targets included the gene deletion collection and 900 strains in which essential genes were affected by mRNA destabilization (DAmP). To analyze the results, we developed RECAP, a strategy that validates genetic interaction profiles by comparison with gene co-citation frequency, and identified links between 1471 genes and 117 biological processes. In addition to these large-scale results, we validated both enhancement and suppression of slow growth measured for specific RNA-related pathways. Thus, negative genetic interactions identified a role for the OCA inositol polyphosphate hydrolase complex in mRNA translation initiation. By analysis of suppressors, we found that Puf4, a Pumilio family RNA binding protein, inhibits ribosomal protein Rpl9 function, by acting on a conserved UGUAcauUA motif located downstream the stop codon of the RPL9B mRNA. Altogether, the results and their analysis should represent a useful resource for discovery of gene function in yeast.


Assuntos
Genes Fúngicos , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/genética , Alelos , Deleção de Genes , Pleiotropia Genética , Fosfatos de Inositol/metabolismo , Iniciação Traducional da Cadeia Peptídica , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia
2.
Nat Commun ; 12(1): 4847, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381031

RESUMO

Circulating phosphate levels are tightly controlled within a narrow range in mammals. By using a novel small-molecule inhibitor, we show that the enzymatic activity of inositol hexakisphosphate kinases (IP6K) is essential for phosphate regulation in vivo. IP6K inhibition suppressed XPR1, a phosphate exporter, thereby decreasing cellular phosphate export, which resulted in increased intracellular ATP levels. The in vivo inhibition of IP6K decreased plasma phosphate levels without inhibiting gut intake or kidney reuptake of phosphate, demonstrating a pivotal role of IP6K-regulated cellular phosphate export on circulating phosphate levels. IP6K inhibition-induced decrease in intracellular inositol pyrophosphate, an enzymatic product of IP6K, was correlated with phosphate changes. Chronic IP6K inhibition alleviated hyperphosphataemia, increased kidney ATP, and improved kidney functions in chronic kidney disease rats. Our results demonstrate that the enzymatic activity of IP6K regulates circulating phosphate and intracellular ATP and suggest that IP6K inhibition is a potential novel treatment strategy against hyperphosphataemia.


Assuntos
Fosfatos/sangue , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Homeostase/efeitos dos fármacos , Humanos , Hiperfosfatemia/tratamento farmacológico , Fosfatos de Inositol/metabolismo , Mamíferos , Fosfatos/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/antagonistas & inibidores , Ratos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico
3.
Molecules ; 26(16)2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34443630

RESUMO

Inositol phosphates (IPs) are a huge and complex family of biomolecules, important in regulating vital cellular functions, signal transduction, energy transmission, and ion channels physiology and serving as structural components of cell membranes [...].


Assuntos
Fosfatos de Inositol/metabolismo , Animais , Humanos , Inositol/metabolismo , Transdução de Sinais/fisiologia
4.
Methods Mol Biol ; 2295: 365-378, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34047987

RESUMO

The phosphate esters of myo-inositol (Ins) occur ubiquitously in biology. These molecules exist as soluble or membrane-resident derivatives and regulate a plethora of cellular functions including phosphate homeostasis, DNA repair, vesicle trafficking, metabolism, cell polarity, tip-directed growth, and membrane morphogenesis. Phosphorylation of all inositol hydroxyl groups generates phytic acid (InsP6), the most abundant inositol phosphate present in eukaryotic cells. However, phytic acid is not the most highly phosphorylated naturally occurring inositol phosphate. Specialized small molecule kinases catalyze the formation of the so-called myo-inositol pyrophosphates (PP-InsPs), such as InsP7 and InsP8. These molecules are characterized by one or several "high-energy" diphosphate moieties and are ubiquitous in eukaryotic cells. In plants, PP-InsPs play critical roles in immune responses and nutrient sensing. The detection of inositol derivatives in plants is challenging. This is particularly the case for inositol pyrophosphates because diphospho bonds are labile in plant cell extracts due to high amounts of acid phosphatase activity. We present two steady-state inositol labeling-based techniques coupled with strong anion exchange (SAX)-HPLC analyses that allow robust detection and quantification of soluble and membrane-resident inositol polyphosphates in plant extracts. These techniques will be instrumental to uncover the cellular and physiological processes controlled by these intriguing regulatory molecules in plants.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Fosfatos de Inositol/química , Resinas de Troca Aniônica/química , Ânions/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Arabidopsis/metabolismo , Inositol/química , Fosfatos de Inositol/metabolismo , Fosfatidilinositóis/química , Fosforilação , Plantas/química , Plantas/metabolismo , Polifosfatos/química , Sementes/química , Transdução de Sinais/fisiologia
5.
Nat Commun ; 12(1): 2673, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976123

RESUMO

Vesicular traffic and membrane contact sites between organelles enable the exchange of proteins, lipids, and metabolites. Recruitment of tethers to contact sites between the endoplasmic reticulum (ER) and the plasma membrane is often triggered by calcium. Here we reveal a function for calcium in the repression of cholesterol export at membrane contact sites between the ER and the Golgi complex. We show that calcium efflux from ER stores induced by inositol-triphosphate [IP3] accumulation upon loss of the inositol 5-phosphatase INPP5A or receptor signaling triggers depletion of cholesterol and associated Gb3 from the cell surface, resulting in a blockade of clathrin-independent endocytosis (CIE) of Shiga toxin. This phenotype is caused by the calcium-induced dissociation of oxysterol binding protein (OSBP) from the Golgi complex and from VAP-containing membrane contact sites. Our findings reveal a crucial function for INPP5A-mediated IP3 hydrolysis in the control of lipid exchange at membrane contact sites.


Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Fosfatos de Inositol/metabolismo , Lipídeos de Membrana/metabolismo , Animais , Transporte Biológico , Células COS , Chlorocebus aethiops , Colesterol/metabolismo , Endocitose , Células HEK293 , Células HeLa , Humanos , Inositol Polifosfato 5-Fosfatases/genética , Inositol Polifosfato 5-Fosfatases/metabolismo , Microscopia Confocal , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Triexosilceramidas/metabolismo
6.
J Bacteriol ; 203(13): e0014121, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-33875544

RESUMO

ICP2 is a virulent bacteriophage (phage) that preys on Vibrio cholerae. ICP2 was first isolated from cholera patient stool samples. Some of these stools also contained ICP2-resistant isogenic V. cholerae strains harboring missense mutations in the trimeric outer membrane porin protein OmpU, identifying it as the ICP2 receptor. In this study, we identify the ICP2 proteins that mediate interactions with OmpU by selecting for ICP2 host range mutants within infant rabbits infected with a mixture of wild-type and OmpU mutant strains. ICP2 host range mutants that can now infect OmpU mutant strains have missense mutations in the putative tail fiber gene gp25 and the putative adhesin gene gp23. Using site-specific mutagenesis, we show that single or double mutations in gp25 are sufficient to generate the host range mutant phenotype. However, at least one additional mutation in gp23 is required for robust plaque formation on specific OmpU mutants. Mutations in gp23 alone were insufficient to produce a host range mutant phenotype. All ICP2 host range mutants retained the ability to form plaques on wild-type V. cholerae cells. The strength of binding of host range mutants to V. cholerae correlated with plaque morphology, indicating that the selected mutations in gp25 and gp23 restore molecular interactions with the receptor. We propose that ICP2 host range mutants evolve by a two-step process. First, gp25 mutations are selected for their broad host range, albeit accompanied by low-level phage adsorption. Subsequent selection occurs for gp23 mutations that further increase productive binding to specific OmpU alleles, allowing for near-wild-type efficiencies of adsorption and subsequent phage multiplication. IMPORTANCE Concern over multidrug-resistant bacterial pathogens, including Vibrio cholerae, has led to renewed interest in phage biology and the potential for phage therapy. ICP2 is a genetically unique virulent phage isolated from cholera patient stool samples. It is also one of three phages in a prophylactic cocktail that have been shown to be effective in animal models of infection and the only one of the three that requires a protein receptor (OmpU). This study identifies an ICP2 tail fiber and a receptor binding protein and examines how ICP2 responds to the selective pressures of phage-resistant OmpU mutants. We found that this particular coevolutionary arms race presents fitness costs to both ICP2 and V. cholerae.


Assuntos
Bacteriófagos/fisiologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Fosfatos de Inositol/metabolismo , Vibrio cholerae/virologia , Proteínas da Cauda Viral/metabolismo , Adesinas Bacterianas , Alelos , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Bacteriófagos/genética , Proteínas do Capsídeo/genética , Cólera , Interações entre Hospedeiro e Microrganismos/genética , Especificidade de Hospedeiro , Humanos , Fosfatos de Inositol/química , Fosfatos de Inositol/genética , Modelos Animais , Mutação , Mutação de Sentido Incorreto , Fenótipo , Porinas/química , Porinas/genética , Porinas/metabolismo , Coelhos , Vibrio cholerae/genética , Proteínas da Cauda Viral/química , Proteínas da Cauda Viral/genética
7.
J Med Chem ; 64(7): 3813-3826, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33724834

RESUMO

Src homology 2 domain-containing inositol phosphate phosphatase 2 (SHIP2) is one of the 10 human inositol phosphate 5-phosphatases. One of its physiological functions is dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate, PtdIns(3,4,5)P3. It is therefore a therapeutic target for pathophysiologies dependent on PtdIns(3,4,5)P3 and PtdIns(3,4)P2. Therapeutic interventions are limited by the dearth of crystallographic data describing ligand/inhibitor binding. An active site-directed fluorescent probe facilitated screening of compound libraries for SHIP2 ligands. With two additional orthogonal assays, several ligands including galloflavin were identified as low micromolar Ki inhibitors. One ligand, an oxo-linked ethylene-bridged dimer of benzene 1,2,4-trisphosphate, was shown to be an uncompetitive inhibitor that binds to a regulatory site on the catalytic domain. We posit that binding of ligands to this site restrains L4 loop motions that are key to interdomain communications that accompany high catalytic activity with phosphoinositide substrate. This site may, therefore, be a future druggable target for medicinal chemistry.


Assuntos
Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Fosfatos de Inositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/antagonistas & inibidores , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Sítio Alostérico , Sequência de Aminoácidos , Animais , Domínio Catalítico , Linhagem Celular Tumoral , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Ligantes , Camundongos , Simulação de Acoplamento Molecular , Células NIH 3T3 , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/química , Ligação Proteica
8.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33443153

RESUMO

The differentiation of cells depends on a precise control of their internal organization, which is the result of a complex dynamic interplay between the cytoskeleton, molecular motors, signaling molecules, and membranes. For example, in the developing neuron, the protein ADAP1 (ADP-ribosylation factor GTPase-activating protein [ArfGAP] with dual pleckstrin homology [PH] domains 1) has been suggested to control dendrite branching by regulating the small GTPase ARF6. Together with the motor protein KIF13B, ADAP1 is also thought to mediate delivery of the second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to the axon tip, thus contributing to PIP3 polarity. However, what defines the function of ADAP1 and how its different roles are coordinated are still not clear. Here, we studied ADAP1's functions using in vitro reconstitutions. We found that KIF13B transports ADAP1 along microtubules, but that PIP3 as well as PI(3,4)P2 act as stop signals for this transport instead of being transported. We also demonstrate that these phosphoinositides activate ADAP1's enzymatic activity to catalyze GTP hydrolysis by ARF6. Together, our results support a model for the cellular function of ADAP1, where KIF13B transports ADAP1 until it encounters high PIP3/PI(3,4)P2 concentrations in the plasma membrane. Here, ADAP1 disassociates from the motor to inactivate ARF6, promoting dendrite branching.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfatidilinositóis/metabolismo , Fatores de Ribosilação do ADP/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Axônios/metabolismo , Transporte Biológico/fisiologia , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Fosfatos de Inositol/metabolismo , Cinesina/metabolismo , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Transdução de Sinais
9.
FASEB J ; 35(2): e21275, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33475202

RESUMO

Nudix hydrolases attract considerable attention for their wide range of specialized activities in all domains of life. One particular group of Nudix phosphohydrolases (DIPPs), through their metabolism of diphosphoinositol polyphosphates (PP-InsPs), regulates the actions of these polyphosphates upon bioenergetic homeostasis. In the current study, we describe, at an atomic level, hitherto unknown properties of human DIPP1.We provide X-ray analysis of the catalytic core of DIPP1 in crystals complexed with either natural PP-InsPs, alternative PP-InsP stereoisomers, or non-hydrolysable methylene bisphosphonate analogs ("PCP-InsPs"). The conclusions that we draw from these data are interrogated by studying the impact upon catalytic activity upon mutagenesis of certain key residues. We present a picture of a V-shaped catalytic furrow with overhanging ridges constructed from flexible positively charged side chains; within this cavity, the labile phosphoanhydride bond is appropriately positioned at the catalytic site by an extensive series of interlocking polar contacts which we analogize as "suspension cables." We demonstrate functionality for a triglycine peptide within a ß-strand which represents a non-canonical addition to the standard Nudix catalytic core structure. We describe pre-reaction enzyme/substrate states which we posit to reflect a role for electrostatic steering in substrate capture. Finally, through time-resolved analysis, we uncover a chronological sequence of DIPP1/product post-reaction states, one of which may rationalize a role for InsP6 as an inhibitor of catalytic activity.


Assuntos
Hidrolases Anidrido Ácido/química , Fosfatos de Inositol/metabolismo , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Humanos , Hidrólise , Fosfatos de Inositol/química , Cinética , Simulação de Acoplamento Molecular , Ligação Proteica
10.
Gastroenterology ; 160(5): 1709-1724, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33421512

RESUMO

BACKGROUND & AIMS: Recent literature has implicated a key role for mast cells in murine models of colonic inflammation, but their role in human ulcerative colitis (UC) is not well established. A major advance has been the identification of mrgprb2 (human orthologue, MRGPX2) as mediating IgE-independent mast cell activation. We sought to define mechanisms of mast cell activation and MRGPRX2 in human UC. METHODS: Colon tissues were collected from patients with UC for bulk RNA sequencing and lamina propria cells were isolated for MRGPRX2 activation studies and single-cell RNA sequencing. Genetic association of all protein-altering G-protein coupled receptor single-nucleotide polymorphism was performed in an Ashkenazi Jewish UC case-control cohort. Variants of MRGPRX2 were transfected into Chinese hamster ovary (CHO) and human mast cell (HMC) 1.1 cells to detect genotype-dependent effects on ß-arrestin recruitment, IP-1 accumulation, and phosphorylated extracellular signal-regulated kinase. RESULTS: Mast cell-specific mediators and adrenomedullin (proteolytic precursor of PAMP-12, an MRGPRX2 agonist) are up-regulated in inflamed compared to uninflamed UC. MRGPRX2 stimulation induces carboxypeptidase secretion from inflamed UC. Of all protein-altering GPCR alleles, a unique variant of MRGPRX2, Asn62Ser, was most associated with and was bioinformatically predicted to alter arrestin recruitment. We validated that the UC protective serine allele enhances ß-arrestin recruitment, decreases IP-1, and increases phosphorylated extracellular signal-regulated kinase with MRGPRX2 agonists. Single-cell RNA sequencing defines that adrenomedullin is expressed by activated fibroblasts and epithelial cells and that interferon gamma is a key upstream regulator of mast cell gene expression. CONCLUSION: Inflamed UC regions are distinguished by MRGPRX2-mediated activation of mast cells, with decreased activation observed with a UC-protective genetic variant. These results define cell modules of UC activation and a new therapeutic target.


Assuntos
Degranulação Celular , Colite Ulcerativa/metabolismo , Colo/metabolismo , Mastócitos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Adrenomedulina/genética , Adrenomedulina/metabolismo , Animais , Células CHO , Estudos de Casos e Controles , Colite Ulcerativa/genética , Colite Ulcerativa/imunologia , Colo/imunologia , Cricetulus , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Variação Genética , Humanos , Fosfatos de Inositol/metabolismo , Ligantes , Mastócitos/imunologia , Proteínas do Tecido Nervoso/genética , Fosforilação , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropeptídeos/genética , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismo
11.
Nat Commun ; 12(1): 384, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33452263

RESUMO

Phosphorus is an essential nutrient taken up by organisms in the form of inorganic phosphate (Pi). Eukaryotes have evolved sophisticated Pi sensing and signaling cascades, enabling them to stably maintain cellular Pi concentrations. Pi homeostasis is regulated by inositol pyrophosphate signaling molecules (PP-InsPs), which are sensed by SPX domain-containing proteins. In plants, PP-InsP-bound SPX receptors inactivate Myb coiled-coil (MYB-CC) Pi starvation response transcription factors (PHRs) by an unknown mechanism. Here we report that a InsP8-SPX complex targets the plant-unique CC domain of PHRs. Crystal structures of the CC domain reveal an unusual four-stranded anti-parallel arrangement. Interface mutations in the CC domain yield monomeric PHR1, which is no longer able to bind DNA with high affinity. Mutation of conserved basic residues located at the surface of the CC domain disrupt interaction with the SPX receptor in vitro and in planta, resulting in constitutive Pi starvation responses. Together, our findings suggest that InsP8 regulates plant Pi homeostasis by controlling the oligomeric state and hence the promoter binding capability of PHRs via their SPX receptors.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Difosfatos/metabolismo , Regulação da Expressão Gênica de Plantas , Fosfatos de Inositol/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Arabidopsis/ultraestrutura , Cristalografia por Raios X , Mutação , Proteínas Nucleares/genética , Ligação Proteica/genética , Domínios Proteicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/ultraestrutura
12.
PLoS Pathog ; 17(1): e1009190, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33476323

RESUMO

Gag polymerization with viral RNA at the plasma membrane initiates HIV-1 assembly. Assembly processes are inefficient in vitro but are stimulated by inositol (1,3,4,5,6) pentakisphosphate (IP5) and inositol hexakisphosphate (IP6) metabolites. Previous studies have shown that depletion of these inositol phosphate species from HEK293T cells reduced HIV-1 particle production but did not alter the infectivity of the resulting progeny virions. Moreover, HIV-1 substitutions bearing Gag/CA mutations ablating IP6 binding are noninfectious with destabilized viral cores. In this study, we analyzed the effects of cellular depletion of IP5 and IP6 on HIV-1 replication in T cells in which we disrupted the genes encoding the kinases required for IP6 generation, IP5 2-kinase (IPPK) and Inositol Polyphosphate Multikinase (IPMK). Knockout (KO) of IPPK from CEM and MT-4 cells depleted cellular IP6 in both T cell lines, and IPMK disruption reduced the levels of both IP5 and IP6. In the KO lines, HIV-1 spread was delayed relative to parental wild-type (WT) cells and was rescued by complementation. Virus release was decreased in all IPPK or IPMK KO lines relative to WT cells. Infected IPMK KO cells exhibited elevated levels of intracellular Gag protein, indicative of impaired particle assembly. IPMK KO compromised virus production to a greater extent than IPPK KO suggesting that IP5 promotes HIV-1 particle assembly in IPPK KO cells. HIV-1 particles released from infected IPPK or IPMK KO cells were less infectious than those from WT cells. These viruses exhibited partially cleaved Gag proteins, decreased virion-associated p24, and higher frequencies of aberrant particles, indicative of a maturation defect. Our data demonstrate that IP6 enhances the quantity and quality of virions produced from T cells, thereby preventing defects in HIV-1 replication.


Assuntos
Linfócitos T CD4-Positivos/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Fosfatos de Inositol/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/virologia , Montagem de Vírus , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Produtos do Gene gag/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Células Tumorais Cultivadas , Vírion/fisiologia
13.
Molecules ; 25(24)2020 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-33302391

RESUMO

Solid-state fermentation with food-grade fungal strains can be applied to enhance the bioactive parameters of agro-industrial by-products. Tempe-type fermentation can be adapted to various substrates, but the key factor is the appropriate strain selection. The aim of this study was to compare the potential of Rhizopus strains for obtaining products of improved antioxidant activity from pumpkin oil cake. For this purpose, substances reacting with the Folin-Ciocalteu reagent, with free radical scavenging potential, as well as reducing power were assessed. The effect of the fermentation on the phytate level and inositol phosphate profile in the material was also monitored. The fermentation resulted in the significant enhancement of the antioxidant potential of pumpkin oil cake in the case of all the strains tested, but the most efficient one was R. oligosporus ATCC 64063. During the course of fermentation, the level of phytate in the material decreased (the highest reduction rate was observed in the oil cake fermented with R. oryzae CBS 372.63), while peptides and fungal glucosamine were accumulated. Tempe-type fermentation can be considered as an alternative way of improving the bioactive parameters of pumpkin oil cake and, thanks to the various activities of different Rhizopus strains, it is possible to obtain products of desired parameters.


Assuntos
Cucurbita/química , Fermentação , Manipulação de Alimentos , Microbiologia de Alimentos , Óleos Vegetais/metabolismo , Rhizopus/metabolismo , Antioxidantes/química , Antioxidantes/farmacologia , Análise de Alimentos , Glucosamina/análise , Fosfatos de Inositol/metabolismo , Peptídeos/análise , Proteínas/análise , Especificidade da Espécie
14.
Molecules ; 25(21)2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-33139672

RESUMO

Recently, inositols, especially myo-inositol and inositol hexakisphosphate, also known as phytic acid or IP6, with their biological activities received much attention for their role in multiple health beneficial effects. Although their roles in cancer treatment and prevention have been extensively reported, interestingly, they may also have distinctive properties in energy metabolism and metabolic disorders. We review inositols and inositol phosphate metabolism in mammalian cells to establish their biological activities and highlight their potential roles in energy metabolism. These molecules are known to decrease insulin resistance, increase insulin sensitivity, and have diverse properties with importance from cell signaling to metabolism. Evidence showed that inositol phosphates might enhance the browning of white adipocytes and directly improve insulin sensitivity through adipocytes. In addition, inositol pyrophosphates containing high-energy phosphate bonds are considered in increasing cellular energetics. Despite all recent advances, many aspects of the bioactivity of inositol phosphates are still not clear, especially their effects on insulin resistance and alteration of metabolism, so more research is needed.


Assuntos
Adipócitos Brancos/metabolismo , Metabolismo Energético , Fosfatos de Inositol/metabolismo , Resistência à Insulina , Transdução de Sinais , Adipócitos Brancos/patologia , Animais , Humanos
15.
Nat Commun ; 11(1): 6035, 2020 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-33247133

RESUMO

The analysis of myo-inositol phosphates (InsPs) and myo-inositol pyrophosphates (PP-InsPs) is a daunting challenge due to the large number of possible isomers, the absence of a chromophore, the high charge density, the low abundance, and the instability of the esters and anhydrides. Given their importance in biology, an analytical approach to follow and understand this complex signaling hub is desirable. Here, capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESI-MS) is implemented to analyze complex mixtures of InsPs and PP-InsPs with high sensitivity. Stable isotope labeled (SIL) internal standards allow for matrix-independent quantitative assignment. The method is validated in wild-type and knockout mammalian cell lines and in model organisms. SIL-CE-ESI-MS enables the accurate monitoring of InsPs and PP-InsPs arising from compartmentalized cellular synthesis pathways, by feeding cells with either [13C6]-myo-inositol or [13C6]-D-glucose. In doing so, we provide evidence for the existence of unknown inositol synthesis pathways in mammals, highlighting the potential of this method to dissect inositol phosphate metabolism and signalling.


Assuntos
Eletroforese Capilar , Fosfatos de Inositol/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Arabidopsis/metabolismo , Vias Biossintéticas , Dictyostelium/metabolismo , Células HCT116 , Humanos , Fosfatos de Inositol/química , Brotos de Planta/metabolismo , Saccharomyces cerevisiae/metabolismo
16.
Molecules ; 25(22)2020 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-33198256

RESUMO

Several studies have identified specific signalling functions for inositol polyphosphates (IPs) in different cell types and have led to the accumulation of new information regarding their cellular roles as well as new insights into their cellular production. These studies have revealed that interaction of IPs with several proteins is critical for stabilization of protein complexes and for modulation of enzymatic activity. This has not only revealed their importance in regulation of several cellular processes but it has also highlighted the possibility of new pharmacological interventions in multiple diseases, including cancer. In this review, we describe some of the intracellular roles of IPs and we discuss the pharmacological opportunities that modulation of IPs levels can provide.


Assuntos
Fosfatos de Inositol/metabolismo , Inositol/química , Ácido Fítico/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/química , Endocitose , Exocitose , Humanos , Fosfatos de Inositol/química , Camundongos , Ácido Fítico/química , Agregação Plaquetária , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Replicação Viral
17.
Biochem J ; 477(20): 4071-4084, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33026061

RESUMO

Hydrolysis of the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) at the cell membrane induces the release of inositol 1,4,5-trisphosphate (IP3) into the cytoplasm and diffusion of diacylglycerol (DAG) through the membrane, respectively. Release of IP3 subsequently increases Ca2+ levels in the cytoplasm, which results in activation of protein kinase C α (PKCα) by Ca2+ and DAG, and finally the translocation of PKCα from the cytoplasm to the membrane. In this study, we developed a metabolic reaction-diffusion framework to simulate PKCα translocation via PIP2 hydrolysis in an endothelial cell. A three-dimensional cell model, divided into membrane and cytoplasm domains, was reconstructed from confocal microscopy images. The associated metabolic reactions were divided into their corresponding domain; PIP2 hydrolysis at the membrane domain resulted in DAG diffusion at the membrane domain and IP3 release into the cytoplasm domain. In the cytoplasm domain, Ca2+ was released from the endoplasmic reticulum, and IP3, Ca2+, and PKCα diffused through the cytoplasm. PKCα bound Ca2+ at, and diffused through, the cytoplasm, and was finally activated by binding with DAG at the membrane. Using our model, we analyzed IP3 and DAG dynamics, Ca2+ waves, and PKCα translocation in response to a microscopic stimulus. We found a qualitative agreement between our simulation results and our experimental results obtained by live-cell imaging. Interestingly, our results suggest that PKCα translocation is dominated by DAG dynamics. This three-dimensional reaction-diffusion mathematical framework could be used to investigate the link between PKCα activation in a cell and cell function.


Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Diglicerídeos/metabolismo , Células Endoteliais/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteína Quinase C-alfa/metabolismo , Transdução de Sinais/fisiologia , Animais , Bovinos , Biologia Computacional , Simulação por Computador , Hidrólise , Fosfatos de Inositol/metabolismo
18.
Nucleic Acids Res ; 48(19): 10739-10752, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33010152

RESUMO

Fission yeast phosphate homeostasis genes are repressed in phosphate-rich medium by transcription of upstream lncRNAs that interferes with activation of the flanking mRNA promoters. lncRNA control of PHO gene expression is influenced by the Thr4 phospho-site in the RNA polymerase II CTD and the 3' processing/termination factors CPF and Rhn1, mutations of which result in hyper-repression of the PHO regulon. Here, we performed a forward genetic screen for mutations that de-repress Pho1 acid phosphatase expression in CTD-T4A cells. Sequencing of 18 independent STF (Suppressor of Threonine Four) isolates revealed, in every case, a mutation in the C-terminal pyrophosphatase domain of Asp1, a bifunctional inositol pyrophosphate (IPP) kinase/pyrophosphatase that interconverts 5-IP7 and 1,5-IP8. Focused characterization of two STF strains identified 51 coding genes coordinately upregulated vis-à-vis the parental T4A strain, including all three PHO regulon genes (pho1, pho84, tgp1). Whereas these STF alleles-asp1-386(Stop) and asp1-493(Stop)-were lethal in a wild-type CTD background, they were viable in combination with mutations in CPF and Rhn1, in which context Pho1 was also de-repressed. Our findings implicate Asp1 pyrophosphatase in constraining 1,5-IP8 or 1-IP7 synthesis by Asp1 kinase, without which 1-IPPs can accumulate to toxic levels that elicit precocious termination by CPF/Rhn1.


Assuntos
Fosfatase Ácida/genética , Proteínas do Citoesqueleto/genética , DNA Polimerase II/genética , Fosfatos de Inositol/metabolismo , Mutação , RNA Longo não Codificante/genética , Proteínas de Schizosaccharomyces pombe/genética , Fosfatase Ácida/metabolismo , Domínio Catalítico , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , DNA Polimerase II/química , DNA Polimerase II/metabolismo , Regulação Fúngica da Expressão Gênica , Regulon , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Mutações Sintéticas Letais , Terminação da Transcrição Genética , Regulação para Cima
19.
Cells ; 9(10)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-33003441

RESUMO

Diverse extracellular signals induce plasma membrane translocation of sphingosine kinase-1 (SphK1), thereby enabling inside-out signaling of sphingosine-1-phosphate. We have shown before that Gq-coupled receptors and constitutively active Gαq/11 specifically induced a rapid and long-lasting SphK1 translocation, independently of canonical Gq/phospholipase C (PLC) signaling. Here, we further characterized Gq/11 regulation of SphK1. SphK1 translocation by the M3 receptor in HEK-293 cells was delayed by expression of catalytically inactive G-protein-coupled receptor kinase-2, p63Rho guanine nucleotide exchange factor (p63RhoGEF), and catalytically inactive PLCß3, but accelerated by wild-type PLCß3 and the PLCδ PH domain. Both wild-type SphK1 and catalytically inactive SphK1-G82D reduced M3 receptor-stimulated inositol phosphate production, suggesting competition at Gαq. Embryonic fibroblasts from Gαq/11 double-deficient mice were used to show that amino acids W263 and T257 of Gαq, which interact directly with PLCß3 and p63RhoGEF, were important for bradykinin B2 receptor-induced SphK1 translocation. Finally, an AIXXPL motif was identified in vertebrate SphK1 (positions 100-105 in human SphK1a), which resembles the Gαq binding motif, ALXXPI, in PLCß and p63RhoGEF. After M3 receptor stimulation, SphK1-A100E-I101E and SphK1-P104A-L105A translocated in only 25% and 56% of cells, respectively, and translocation efficiency was significantly reduced. The data suggest that both the AIXXPL motif and currently unknown consequences of PLCß/PLCδ(PH) expression are important for regulation of SphK1 by Gq/11.


Assuntos
Membrana Celular/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Lisofosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/análogos & derivados , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Membrana Celular/genética , Cromatografia Líquida de Alta Pressão , Fibroblastos , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Fosfatos de Inositol/metabolismo , Camundongos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ligação Proteica , Receptor B2 da Bradicinina/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais/genética , Esfingosina/metabolismo , Espectrometria de Massas em Tandem , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo
20.
Int J Mol Sci ; 21(19)2020 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-32998310

RESUMO

The presence of abnormal ovarian ratios of myo-inositol (MI) to D-chiro-inositol (DCI) is a recurrent feature in PCOS. Available evidence suggests that MI and DCI may modulate steroid biosynthesis, likely in an opposite manner. Specifically, MI seems to induce estrogen production, while DCI has a role in the synthesis of androgens. Elevated insulin levels, generally associated with PCOS, alter the physiological MI/DCI ratio, increasing MI-to-DCI conversion through activation of a specific epimerase enzyme. DCI directly increases testosterone biosynthesis in thecal cells and reduces its conversion to estradiol by downregulating aromatase enzyme in granulosa cells. This manuscript reviews the literature that supports the connection between altered MI/DCI ratios and pathological steroidogenesis observed in PCOS women. Furthermore, it discusses the application of inositol-based treatment protocols in managing PCOS symptoms and improving the quality of patients' life.


Assuntos
Células da Granulosa/metabolismo , Fosfatos de Inositol/metabolismo , Inositol/metabolismo , Síndrome do Ovário Policístico/metabolismo , Células Tecais/metabolismo , Androgênios/metabolismo , Aromatase/genética , Aromatase/metabolismo , Estrogênios/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/patologia , Humanos , Inositol/uso terapêutico , Fosfatos de Inositol/uso terapêutico , Insulina/metabolismo , Síndrome do Ovário Policístico/tratamento farmacológico , Síndrome do Ovário Policístico/genética , Síndrome do Ovário Policístico/patologia , Qualidade de Vida , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Células Tecais/efeitos dos fármacos , Células Tecais/patologia
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