RESUMO
We report an improved total synthesis of 4,5-dibromo-9,10-dihydrophenanthrene-2,3,6,7-tetraol, (±)-polysiphenol, via intermolecular McMurray dimerization of 5-bromovanillin and subsequent intramolecular oxidative coupling as the key steps. The synthetic route is applicable to 4,5-dichloro- and 4,5-difluoro-halologues (as well as a 4,5-dialkyl-analogue). Distinctive AA'BB' multiplets in their 1H NMR spectra for the dimethylene bridges of the dibromo and dichloro compounds reveal them to be room-temperature stable atropisomers, while for the difluoro compound they present as a singlet. X-ray crystal structure determinations of their tetramethylated synthetic precursors show atropisomeric twist angles of 48°, 46°, and 32°, respectively, with the former representing the largest yet observed in any 4,5-disubstituted-9,10-dihydrophenanthrene. DFT computational studies reveal an unprecedented two-stage atropisomeric interconversion process involving time-independent asynchronous rotations of the dimethylene bridge and the biaryl axis for halologues containing chlorine or bromine, but a more synchronous rotation for the difluoro analogue.
Assuntos
Fenantrenos , Bromo/química , Dimerização , Espectroscopia de Ressonância Magnética , Fenantrenos/químicaRESUMO
RATIONALE: Glycosyl-inositol-phospho-ceramides (GIPCs) or glycosylphosphatidylinositol-anchored fungal polysaccharides are known to be major lipids in plant and fungal plasma membranes and to play an important role in stress adaption. However, their analysis remains challenging due to the several steps involved for their extractions and purifications prior to mass spectrometric analysis. To address this challenge, we developed a rapid and sensitive method to identify GIPCs from the four common fungal plant pathogens Botrytis cinerea, Fusarium graminearium, Neurospora crassa and Ustilago maydis. METHODS: Fungal plant pathogens were cultured, harvested, heat-inactivated and washed three times with double-distilled water. Intact fungi were deposited on a matrix-assisted laser desorption ionization (MALDI) target plate, mixed with the matrix consisting of a 9:1 mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid solubilized at 10 mg/mL in chloroform-methanol (9:1 v/v) and analyzed using a Bruker MALDI Biotyper Sirius system in the linear negative ion mode. Mass spectra were acquired from m/z 700 to 2000. RESULTS: MALDI time-of-flight (TOF) mass spectrometric analysis of cultured fungi showed clear signature of GIPCs in B. cinerea, F. graminearium, N. crassa and U. maydis. CONCLUSIONS: We have demonstrated that routine MALDI-TOF in the linear negative ion mode combined with an apolar solvent system to solubilize the matrix is applicable to the detection of filamentous fungal GIPCs.
Assuntos
Ceramidas/análise , Fungos/química , Glicosilfosfatidilinositóis/análise , Plantas/microbiologia , Técnicas de Tipagem Micológica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
PTEN (phosphatase and tensin homologue deleted on chromosome 10) is well known as a tumour suppressor. It's PI(3,4,5)P3 lipid phosphatase activity is an important counteracting mechanism in PI 3-kinase (phosphoinositide 3-kinase) signalling. Furthermore, PTEN lies upstream of Akt kinase, a key enzyme in insulin signalling regulating glucose uptake and cell growth. Therefore, PTEN has recently gained attention as a valuable drug target for the treatment of diabetes, stroke, cardiac infarct and fertility. This review summarizes the use of small molecules as PTEN inhibitors. Currently available methodologies and techniques for accessing PTEN inhibition in vitro and in cellulo will be discussed.
Assuntos
Marcação de Genes/métodos , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Supressoras de Tumor/genética , Animais , Humanos , PTEN Fosfo-Hidrolase/análise , Proteínas Supressoras de Tumor/análiseRESUMO
In this paper evidence is presented that the fatty acid component of an inositide substrate affects the kinetic parameters of the lipid phosphatase Salmonella Outer Protein B (SopB). A succinct route was used to prepare the naturally occurring enantiomer of phosphatidylinositol 4-phosphate (PI-4-P) with saturated, as well as singly, triply and quadruply unsaturated, fatty acid esters, in four stages: (1) The enantiomers of 2,3:5,6-O-dicyclohexylidene-myo-inositol were resolved by crystallisation of their di(acetylmandelate) diastereoisomers. (2) The resulting diol was phosphorylated regio-selectively exclusively on the 1-O using the new reagent tri(2-cyanoethyl)phosphite. (3) With the 4-OH still unprotected, the glyceride was coupled using phosphate tri-ester methodology. (4) A final phosphorylation of the 4-O, followed by global deprotection under basic then acidic conditions, provided PI-4-P bearing a range of sn-1-stearoyl, sn-2-stearoyl, -oleoyl, -γ-linolenoyl and arachidonoyl, glycerides. Enzymological studies showed that the introduction of cis-unsaturated bonds has a measurable influence on the activity (relative Vmax) of SopB. Mono-unsaturated PI-4-P exhibited a five-fold higher activity, with a two-fold higher KM, over the saturated substrate, when presented in DOPC vesicles. Poly-unsaturated PI-4-P showed little further change with respect to the singly unsaturated species. This result, coupled with our previous report that saturated PI-4-P has much higher stored curvature elastic stress than PI, supports the hypothesis that the activity of inositide phosphatase SopB has a physical role in vivo.
Assuntos
Proteínas de Bactérias/metabolismo , Fosfatos de Fosfatidilinositol/biossíntese , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Bactérias/química , Ativação Enzimática , Conformação Molecular , Fosfatos de Fosfatidilinositol/química , Monoéster Fosfórico Hidrolases/química , Especificidade por SubstratoRESUMO
Chemical intervention tools have been beneficial to many investigations elucidating signalling networks and interactions. The present review summarizes the current status of chemical tools to probe phosphoinositide metabolism and signalling. In particular, phosphoinositide-targeting tools are compared with protein-targeting tools with respect to their unique advantages and possible applications.
Assuntos
Modelos Biológicos , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Animais , Inibidores Enzimáticos/farmacologia , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/química , Fosfatidilinositol 3-Quinase/química , Inibidores de Fosfoinositídeo-3 Quinase , Estrutura Terciária de ProteínaRESUMO
Cellulose is the most abundant biopolymer in the world, the main load-bearing element in plant cell walls, and represents a major sink for carbon fixed during photosynthesis. Previous work has shown that photosynthetic activity is partially regulated by carbohydrate sinks. However, the coordination of cellulose biosynthesis with carbohydrate metabolism and photosynthesis is not well understood. Here, we demonstrate that cellulose biosynthesis inhibition (CBI) leads to reductions in transcript levels of genes involved in photosynthesis, the Calvin cycle, and starch degradation in Arabidopsis (Arabidopsis thaliana) seedlings. In parallel, we show that CBI induces changes in carbohydrate distribution and influences Rubisco activase levels. We find that the effects of CBI on gene expression and carbohydrate metabolism can be neutralized by osmotic support in a concentration-dependent manner. However, osmotic support does not suppress CBI-induced metabolic changes in seedlings impaired in mechanoperception (mid1 complementing activity1 [mca1]) and osmoperception (cytokinin receptor1 [cre1]) or reactive oxygen species production (respiratory burst oxidase homolog DF [rbohDF]). These results show that carbohydrate metabolism is responsive to changes in cellulose biosynthesis activity and turgor pressure. The data suggest that MCA1, CRE1, and RBOHDF-derived reactive oxygen species are involved in the regulation of osmosensitive metabolic changes. The evidence presented here supports the notion that cellulose and carbohydrate metabolism may be coordinated via an osmosensitive mechanism.
Assuntos
Arabidopsis/metabolismo , Metabolismo dos Carboidratos , Celulose/biossíntese , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Benzamidas/farmacologia , Sobrevivência Celular , Celulose/antagonistas & inibidores , Celulose/genética , Ativação Enzimática , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Pressão Osmótica , Fotossíntese , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polietilenoglicóis/farmacologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
Mechanical properties of biological membranes are known to regulate membrane protein function. Despite this, current models of protein communication typically feature only direct protein-protein or protein-small molecule interactions. Here we show for the first time that, by harnessing nanoscale mechanical energy within biological membranes, it is possible to promote controlled communication between proteins. By coupling lipid-protein modules and matching their response to the mechanical properties of the membrane, we have shown that the action of phospholipase A(2) on acyl-based phospholipids triggers the opening of the mechanosensitive channel, MscL, by generating membrane asymmetry. Our findings confirm that the global physical properties of biological membranes can act as information pathways between proteins, a novel mechanism of membrane-mediated protein-protein communication that has important implications for (i) the underlying structure of signaling pathways, (ii) our understanding of in vivo communication networks, and (iii) the generation of building blocks for artificial protein networks.
Assuntos
Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Engenharia Genética/métodos , Canais Iônicos/metabolismo , Fosfolipases A2 Secretórias/metabolismo , Fenômenos Biomecânicos , Proteínas de Escherichia coli/genética , Canais Iônicos/genética , Bicamadas Lipídicas/metabolismo , Fosfolipídeos/metabolismo , Ligação ProteicaRESUMO
Arylstibonates structurally resemble phosphotyrosine side chains in proteins and here we addressed the ability of such compounds to act as inhibitors of a panel of mammalian tyrosine and dual-specificity phosphatases. Two arylstibonates both possessing a carboxylate side chain were identified as potent inhibitors of the protein tyrosine phosphatase PTP-ß. In addition, they inhibited the dual-specificity, cell cycle regulatory phosphatases Cdc25a and Cdc25b with sub-micromolar potency. However, the Cdc25c phosphatase was not affected demonstrating that arylstibonates may be viable leads from which to develop isoform specific Cdc25 inhibitors.
Assuntos
Antimônio/química , Inibidores Enzimáticos/química , Compostos Organometálicos/química , Propionatos/química , Fosfatases cdc25/antagonistas & inibidores , Cinética , Relação Estrutura-Atividade , Fosfatases cdc25/metabolismoRESUMO
Phospholipase D reacts with alcohols or water, transphosphatidylating or hydrolysing lipids such as phosphatidylcholine, generating phosphatidylalcohols or phosphatidic acid, respectively. The enzyme has been employed in many applications making use of the transphosphatidylation reaction and the enzyme's tolerance for organic solvents in order to synthesize natural and artificial phospholipids. Yet, its catalytic properties with respect to the transphosphatidylation reaction are not well understood. Here, we introduce a novel high-throughput assay, making use of 96-well plates, that employs Fluorescamine for the detection of transphosphatidylated amino alcohols. This assay allowed to monitor the KM and VMax at different temperatures, revealing that the former will be elevated by the temperature, while the latter is increased by a combination of both temperature and alcohol acceptor concentration being elevated, suggesting that increase in temperature may open up a new binding site for the alcohol acceptor.
Assuntos
Fosfolipase D , Etanol , Fosfatidilcolinas , Fosfolipase D/metabolismo , Solventes , TemperaturaRESUMO
The discovery of small-molecule modulators of signaling pathways is currently a particularly active area of research. We aimed at developing unprecedented metal-based activators of Akt signaling which can potentially find applications as tools for regulating glucose metabolism downstream of Akt or serve as lead structures for developing antidiabetic drugs. In this context, a highly diverse library of 11 new zinc(II) complexes with phenolic, picolinic, pyridino, and hydroxamic ligands, all containing features beneficial for medicinal purposes, was prepared and screened in an assay that detected levels of phospho-Akt in lysates from NIH3T3 cells after treatment with the compounds. The complexes featuring hydroxamic ligands were found to be the most prominent activators of Akt among the molecules prepared, with the most efficient compound acting at submicromolar concentrations. Further characterization revealed that this compound induces phosphorylation of the Akt downstream effector glycogen synthase kinase 3ß, but does not act as an inhibitor of tyrosine phosphatases or PTEN.
Assuntos
Ácidos Hidroxâmicos/química , Compostos Organometálicos/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Zinco/química , Animais , Camundongos , Modelos Biológicos , Células NIH 3T3 , Compostos Organometálicos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacosRESUMO
Analogues of the novel inhibitor of the PI3-K/PKB pathway, 2-[5-(2-chloroethyl)-2-acetoxy-benzyl]-4-(2-chloroethyl)-phenyl acetate (E1), have been prepared and preliminary SAR performed. This established that at least one of the chloroethyl para-substituents could be removed or modified and the ability to inhibit PKB/Akt activation retained. Synthetic methodologies were then developed to methylene-linked aryl acetates for use as molecular probes to identify the target of compound E1.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fenilacetatos/química , Fenilacetatos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Inibidores Enzimáticos/síntese química , Feminino , Humanos , Fenilacetatos/síntese química , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Relação Estrutura-AtividadeRESUMO
Phosphatidylinositol phosphates (PIPs) are membrane phospholipids that play crucial roles in a wide range of cellular processes. Their function is dictated by the number and positions of the phosphate groups in the inositol ring (with seven different PIPs being active in the cell). Therefore, there is significant interest in developing small-molecule receptors that can bind selectively to these species and in doing so affect their cellular function or be the basis for molecular probes. However, to date there are very few examples of such molecular receptors. Towards this aim, herein we report a novel tripodal molecule that acts as receptor for mono- and bis-phosphorylated PIPs in a cell free environment. To assess their affinity to PIPs we have developed a new cell free assay based on the ability of the receptor to prevent alkaline phosphatase from hydrolysing these substrates. The new receptor displays selectivity towards two out of the seven PIPs, namely PI(3)P and PI(3,4)P2. To rationalise these results, a DFT computational study was performed which corroborated the experimental results and provided insight into the host-guest binding mode.
Assuntos
Fosfatos de Fosfatidilinositol/químicaRESUMO
This chapter describes an indirect approach to measure PTEN's lipid phosphatase activity in vivo. PTEN counteracts phosphatidylinositol 3-kinase action in dephosphorylating 3-phosphorylated phosphoinositides. Therefore, PtdIns(3,4,5)P3-dependent activation and phosphorylation of the survival kinase Akt can be used as readout for cellular PTEN activity. Here we have outlined a detailed procedure employing a phosphoserine-specific anti-Akt antibody to examine the content of phosphorylated Akt by immunofluorescence and its dependence on PTEN activity.
Assuntos
PTEN Fosfo-Hidrolase/análise , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/análise , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células 3T3 , Animais , Anticorpos/imunologia , Especificidade de Anticorpos , Camundongos , Microscopia de Fluorescência , PTEN Fosfo-Hidrolase/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Proto-Oncogênicas c-akt/imunologia , Coloração e RotulagemRESUMO
This chapter describes a method for the preparation of giant unilamellar vesicles containing phosphatidylinositol 4,5-bisphosphate that are larger than 20 microm in size. The phospholipids composition of the vesicular membrane is such that fluid lamellar and liquid-ordered or gel phases are formed and separate within the confines of one vesicle. It outlines the preparation of a protein fluorescent label, pleckstrin homology domain from phospholipase C-delta 1, that binds specifically to phosphatidylinositol 4,5-bisphosphate. Using fluorescence microscopy, the presence and spatial position of this phosphorylated phosphatidylinositol lipid on the lipid membrane have been located with the pleckstrin homology domain. We show that phosphatidylinositol 4,5-bisphosphate and the phospholipase C-delta 1 pleckstrin homology domain are located to the fluid phase of the vesicle membrane. This approach can therefore show how membrane physical properties can affect enzyme binding to phosphatidylinositol 4,5-bisphosphate and thus further the understanding of important membrane processes such as endocytosis.
Assuntos
Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Fosfatidilinositol 4,5-Difosfato/análise , Fosfolipase C delta/química , Fosfolipase C delta/metabolismo , Lipossomas Unilamelares/química , Lipossomas Unilamelares/isolamento & purificação , Animais , Fluorescência , Micromanipulação , Microscopia de Fluorescência , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Estrutura Terciária de ProteínaRESUMO
The secretagogue compound 48/80 (c48/80) is a well known activator of calcium mediated processes and PKCs, and is a potent inducer of mast cell degranulation. As the latter process is a phosphoinositide 3-kinase (PI 3-kinase) mediated event, we wished to address whether or not c48/80 was an activator of PI 3-kinases. The data presented here reveal that c48/80 is an effective activator of PI 3-kinases as judged by the increased phosphorylation of PKB and p70(S6K) in fibroblasts in a PI 3-kinase dependent fashion. Compound 48/80 effectively translocates PKB to the plasma membrane and induces phosphorylation at serine 473 (S473), detected by fluorescence imaging of fixed cells. At higher concentrations the secretagogue is inhibitory towards PKB phosphorylation on S473. Conversely, p70(S6K) phosphorylation on T389 is unaffected at high doses. We provide evidence that the differential effect on the two PI 3-kinase effectors is due to activation of PKCalpha by c48/80, itself a PI 3-kinase dependent process. We conclude that compound 48/80 is an effective activator of PI 3-kinase dependent pathways, leading to the activation of effectors including PKB/Akt, p70(S6K) and PKCalpha. The latter is only activated by higher doses of c48/80 resulting in an inhibition of the c48/80 induced PKB phosphorylation, thus explaining the observed biphasic activation profile for PKB in response to this secretagogue.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C-alfa/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , p-Metoxi-N-metilfenetilamina/farmacologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Cálcio/metabolismo , Catálise , Linhagem Celular , Membrana Celular/metabolismo , Ativação Enzimática , Humanos , Camundongos , Modelos Biológicos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Transdução de SinaisRESUMO
We report an effective strategy for generating N-terminal cysteinyl proteins by proteolytic cleavage using the enzyme 3C pro, suitable for a wide range of applications via native chemical ligation.
Assuntos
Cisteína Endopeptidases/metabolismo , Cisteína/química , Proteínas/metabolismo , Proteínas Virais/metabolismo , Proteases Virais 3C , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Vírus da Febre Aftosa/enzimologia , Estrutura Terciária de Proteína , Proteínas Recombinantes de FusãoRESUMO
The development of effective molecular probes to detect and image the levels of oxidative stress in cells remains a challenge. Herein we report the design, synthesis and preliminary biological evaluation of a novel optical probe to monitor oxidation of thiol groups in cysteine-based phosphatases (CBPs). Following orthogonal protecting approaches we synthesised a new vanadyl complex designed to bind to CBPs. This complex is functionalised with a well-known dimedone derivative (to covalently trap sulfenic acids, SOHs) and a coumarin-based fluorophore for optical visualization. We show that this new probe efficiently binds to a range of phosphatases in vitro with nanomolar affinity. Moreover, preliminary flow cytometry and microscopy studies in live HCT116 cells show that this probe can successfully image cellular levels of sulfenic acids - one of the species resulting from protein oxidative damage.
Assuntos
Complexos de Coordenação/química , Cisteína/análise , Vanádio/química , Complexos de Coordenação/síntese química , Complexos de Coordenação/metabolismo , Cumarínicos/química , Cisteína/química , Espectroscopia de Ressonância de Spin Eletrônica , Células HCT116 , Humanos , Microscopia de Fluorescência , Oxirredução , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Ligação ProteicaRESUMO
Phosphatase and tensin homolog (PTEN) is involved in a number of different cellular processes including metabolism, apoptosis, cell proliferation and survival. It is a redox-sensitive dual-specificity protein phosphatase that acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of redox regulation of PTEN downstream signaling has been reported, the effect of PTEN redox status on its protein-protein interactions is poorly understood. PTEN-GST in its reduced and a DTT-reversible H2O2-oxidized form was immobilized on a glutathione-sepharose support and incubated with cell lysate to capture interacting proteins. Captured proteins were analyzed by LC-MSMS and comparatively quantified using label-free methods. 97 Potential protein interactors were identified, including a significant number that are novel. The abundance of fourteen interactors was found to vary significantly with the redox status of PTEN. Altered binding to PTEN was confirmed by affinity pull-down and Western blotting for Prdx1, Trx, and Anxa2, while DDB1 was validated as a novel interactor with unaltered binding. These results suggest that the redox status of PTEN causes a functional variation in the PTEN interactome. The resin capture method developed had distinct advantages in that the redox status of PTEN could be directly controlled and measured.
Assuntos
PTEN Fosfo-Hidrolase/fisiologia , Transdução de Sinais , Proteínas de Ligação a DNA/metabolismo , Dissulfetos/química , Glutationa/metabolismo , Células HCT116 , Humanos , Oxirredução , Peroxirredoxinas/metabolismo , Proteômica , Tiorredoxinas/metabolismoRESUMO
Mutation of the inositol 5-phosphatase OCRL1 causes Lowe syndrome and Dent-2 disease. Loss of OCRL1 function perturbs several cellular processes, including membrane traffic, but the underlying mechanisms remain poorly defined. Here we show that OCRL1 is part of the membrane-trafficking machinery operating at the trans-Golgi network (TGN)/endosome interface. OCRL1 interacts via IPIP27A with the F-BAR protein pacsin 2. OCRL1 and IPIP27A localize to mannose 6-phosphate receptor (MPR)-containing trafficking intermediates, and loss of either protein leads to defective MPR carrier biogenesis at the TGN and endosomes. OCRL1 5-phosphatase activity, which is membrane curvature sensitive, is stimulated by IPIP27A-mediated engagement of OCRL1 with pacsin 2 and promotes scission of MPR-containing carriers. Our data indicate a role for OCRL1, via IPIP27A, in regulating the formation of pacsin 2-dependent trafficking intermediates and reveal a mechanism for coupling PtdIns(4,5)P2 hydrolysis with carrier biogenesis on endomembranes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Células COS , Endocitose , Endossomos/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Células HEK293 , Células HeLa , Humanos , Inositol Polifosfato 5-Fosfatases , Nefrolitíase/genética , Nefrolitíase/metabolismo , Nefrolitíase/patologia , Proteínas do Tecido Nervoso/metabolismo , Síndrome Oculocerebrorrenal/genética , Síndrome Oculocerebrorrenal/metabolismo , Síndrome Oculocerebrorrenal/patologia , Fosfatidilinositóis/biossíntese , Fosfatidilinositóis/metabolismo , Transporte Proteico , Receptor IGF Tipo 2/metabolismo , Rede trans-Golgi/metabolismoRESUMO
A new chemical sensor for cellular imaging of NO is presented. This cell-permeable probe is based on a complex where copper(ii) is coordinated to a tridentate ligand substituted with a fluorophore (NBD) and an octyl group. The fluorescence response of this complex towards a range of reactive species (namely NO, NO2-, NO3-, H2O2, ClO-, O2- and ONOO-) has been studied in vitro showing that the probe is highly selective for NO. The probe is readily taken up by cells and is able to image the cellular concentrations of NO.