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1.
FEBS Lett ; 2020 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-32324254

RESUMO

Pyridoxal 5'-phosphate (PLP) is an essential cofactor for neurotransmitter metabolism. Pyridoxal phosphatase (PDXP) deficiency in mice increases PLP and γ-aminobutyric acid levels in the brain, yet how PDXP is regulated is unclear. Here, we identify the Ca2+ - and integrin-binding protein 1 (CIB1) as a PDXP interactor by yeast two-hybrid screening and find a calmodulin (CaM)-binding motif that overlaps with the PDXP-CIB1 interaction site. Pulldown and crosslinking assays with purified proteins demonstrate that PDXP directly binds to CIB1 or CaM. CIB1 or CaM does not alter PDXP phosphatase activity. However, elevated Ca2+ concentrations promote CaM binding and, thereby, diminish CIB1 binding to PDXP, as both interactors bind in a mutually exclusive way. Hence, the PDXP-CIB1 complex may functionally differ from the PDXP-Ca2+ -CaM complex.

2.
Mol Pharm ; 17(6): 1835-1847, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32315193

RESUMO

Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of the family of statins have been suggested as therapeutic options in various tumors. Atorvastatin is a statin with the potential to cross the blood-brain barrier; however, the concentrations necessary for a cytotoxic effect against cancer cells exceed the concentrations achievable via oral administration, which made the development of a novel atorvastatin formulation necessary. We characterized the drug loading and basic physicochemical characteristics of micellar atorvastatin formulations and tested their cytotoxicity against a panel of different glioblastoma cell lines. In addition, activity against tumor spheroids formed from mouse glioma and mouse cancer stem cells, respectively, was evaluated. Our results show good activity of atorvastatin against all tested cell lines. Interestingly, in the three-dimensional (3D) models, growth inhibition was more pronounced for the micellar formulation compared to free atorvastatin. Finally, atorvastatin penetration across a blood-brain barrier model obtained from human induced-pluripotent stem cells was evaluated. Our results suggest that the presented micelles may enable much higher serum concentrations than possible by oral administration; however, if transport across the blood-brain barrier is sufficient to reach the therapeutic atorvastatin concentration for the treatment of glioblastoma via intravenous administration remains unclear.

3.
Biochim Biophys Acta Mol Cell Res ; 1866(1): 153-166, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30030002

RESUMO

Mammalian haloacid dehalogenase (HAD)-type phosphatases have evolved to dephosphorylate a wide range of small metabolites, but can also target macromolecules such as serine/threonine, tyrosine-, and histidine-phosphorylated proteins. To accomplish these tasks, HAD phosphatases are equipped with cap domains that control access to the active site and provide substrate specificity determinants. A number of capped HAD phosphatases impact protein phosphorylation, although structural data are consistent with small metabolite substrates rather than protein substrates. This review discusses the structures, functions and disease implications of the three closely related, capped HAD phosphatases pyridoxal phosphatase (PDXP or chronophin), phosphoglycolate phosphatase (PGP, also termed AUM or glycerol phosphatase) and phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP or HDHD2B). Evidence in support of small metabolite and protein phosphatase activity is discussed in the context of the diversity of their biological functions.


Assuntos
Pirofosfatase Inorgânica/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Citoesqueleto de Actina/fisiologia , Animais , Humanos , Hidrolases , Pirofosfatase Inorgânica/química , Pirofosfatase Inorgânica/fisiologia , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/fisiologia , Fosfoproteínas Fosfatases/ultraestrutura , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/fisiologia , Fosforilação , Proteínas Tirosina Fosfatases/metabolismo
4.
Biochim Biophys Acta Mol Basis Dis ; 1865(1): 193-205, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30327125

RESUMO

Pyridoxal 5'-phosphate (PLP) is an essential cofactor in the catalysis of ~140 different enzymatic reactions. A pharmacological elevation of cellular PLP concentrations is of interest in neuropsychiatric diseases, but whole-body consequences of higher intracellular PLP levels are unknown. To address this question, we have generated mice allowing a conditional ablation of the PLP phosphatase PDXP. Ubiquitous PDXP deletion increased PLP levels in brain, skeletal muscle and red blood cells up to 3-fold compared to control mice, demonstrating that PDXP acts as a major regulator of cellular PLP concentrations in vivo. Neurotransmitter analysis revealed that the concentrations of dopamine, serotonin, epinephrine and glutamate were unchanged in the brains of PDXP knockout mice. However, the levels of γ-aminobutyric acid (GABA) increased by ~20%, demonstrating that elevated PLP levels can drive additional GABA production. Behavioral phenotyping of PDXP knockout mice revealed improved spatial learning and memory, and a mild anxiety-like behavior. Consistent with elevated GABA levels in the brain, PDXP loss in neural cells decreased performance in motor tests, whereas PDXP-deficiency in skeletal muscle increased grip strength. Our findings suggest that PDXP is involved in the fine-tuning of GABA biosynthesis. Pharmacological inhibition of PDXP might correct the excitatory/inhibitory imbalance in some neuropsychiatric diseases.


Assuntos
Ansiedade/metabolismo , Encéfalo/metabolismo , Cognição/fisiologia , Músculo Esquelético/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosfato de Piridoxal/metabolismo , Animais , Comportamento Animal , Dopamina/metabolismo , Epinefrina/metabolismo , Eritrócitos/metabolismo , Ácido Glutâmico/metabolismo , Masculino , Memória , Camundongos , Camundongos Knockout , Modelos Animais , Neurotransmissores , Desempenho Psicomotor , Serotonina/metabolismo , Aprendizagem Espacial , Vitamina B 6/metabolismo , Ácido gama-Aminobutírico/metabolismo
5.
Cardiovasc Res ; 115(1): 71-82, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29931050

RESUMO

Aims: Chronic heart failure is becoming increasingly prevalent and is still associated with a high mortality rate. Myocardial hypertrophy and fibrosis drive cardiac remodelling and heart failure, but they are not sufficiently inhibited by current treatment strategies. Furthermore, despite increasing knowledge on cardiomyocyte intracellular signalling proteins inducing pathological hypertrophy, therapeutic approaches to target these molecules are currently unavailable. In this study, we aimed to establish and test a therapeutic tool to counteract the 22 kDa calcium and integrin binding protein (CIB) 1, which we have previously identified as nodal regulator of pathological cardiac hypertrophy and as activator of the maladaptive calcineurin/NFAT axis. Methods and results: Among three different sequences, we selected a shRNA construct (shCIB1) to specifically down-regulate CIB1 by 50% upon adenoviral overexpression in neonatal rat cardiomyocytes (NRCM), and upon overexpression by an adeno-associated-virus (AAV) 9 vector in mouse hearts. Overexpression of shCIB1 in NRCM markedly reduced cellular growth, improved contractility of bioartificial cardiac tissue and reduced calcineurin/NFAT activation in response to hypertrophic stimulation. In mice, administration of AAV-shCIB1 strongly ameliorated eccentric cardiac hypertrophy and cardiac dysfunction during 2 weeks of pressure overload by transverse aortic constriction (TAC). Ultrastructural and molecular analyses revealed markedly reduced myocardial fibrosis, inhibition of hypertrophy associated gene expression and calcineurin/NFAT as well as ERK MAP kinase activation after TAC in AAV-shCIB1 vs. AAV-shControl treated mice. During long-term exposure to pressure overload for 10 weeks, AAV-shCIB1 treatment maintained its anti-hypertrophic and anti-fibrotic effects, but cardiac function was no longer improved vs. AAV-shControl treatment, most likely resulting from a reduction in myocardial angiogenesis upon downregulation of CIB1. Conclusions: Inhibition of CIB1 by a shRNA-mediated gene therapy potently inhibits pathological cardiac hypertrophy and fibrosis during pressure overload. While cardiac function is initially improved by shCIB1, this cannot be kept up during persisting overload.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Insuficiência Cardíaca/terapia , Hipertrofia Ventricular Esquerda/terapia , Miócitos Cardíacos/metabolismo , RNA Interferente Pequeno/metabolismo , Terapêutica com RNAi , Disfunção Ventricular Esquerda/terapia , Função Ventricular Esquerda , Remodelação Ventricular , Animais , Calcineurina/metabolismo , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Modelos Animais de Doenças , Fibrose , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/patologia , Fatores de Transcrição NFATC/metabolismo , Neovascularização Fisiológica , RNA Interferente Pequeno/genética , Ratos Sprague-Dawley , Transdução de Sinais , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/fisiopatologia
6.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1863(6): 584-594, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29524543

RESUMO

Mammalian phosphoglycolate phosphatase (PGP, also known as AUM or glycerol-3-phosphate phosphatase) is a small molecule-directed phosphatase important for metabolite repair and lipid metabolism. Although PGP was first characterized as an enzyme involved in epidermal growth factor (EGF) signaling, PGP protein substrates have remained elusive. Here we show that PGP depletion facilitates fatty acid flux through the intracellular triacylglycerol/fatty acid cycle, and that phosphatidylinositol-4,5-bisphosphate (PIP2), produced in a side branch of this cycle, is critical for the impact of PGP activity on EGF-induced signaling. Loss of endogenous PGP expression amplified both EGF-induced EGF receptor autophosphorylation and Src-dependent tyrosine phosphorylation of phospholipase C-γ1 (PLCγ1). Furthermore, EGF enhanced the formation of circular dorsal ruffles in PGP-depleted cells via Src/PLCγ1/protein kinase C (PKC)-dependent signaling to the cytoskeleton. Inhibition of adipose triglyceride lipase normalized the increased PIP2 content, reduced EGF-dependent PLCγ1 hyperphosphorylation, and decreased the elevated dorsal ruffle formation of PGP-depleted cells. Our data explain how PGP exerts control over EGF-induced cellular protein tyrosine phosphorylation, and reveal an unexpected influence of triacylglycerol turnover on growth factor signaling.


Assuntos
Fator de Crescimento Epidérmico/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transdução de Sinais , Triglicerídeos/metabolismo , Linhagem Celular , Fator de Crescimento Epidérmico/genética , Humanos , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Monoéster Fosfórico Hidrolases/genética , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Triglicerídeos/genética
7.
J Vis Exp ; (131)2018 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-29364244

RESUMO

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental insults that disturb placenta development or function can cause early pregnancy loss in mice and humans. Nevertheless, simple in vitro assays to screen for potential effects on placenta formation are lacking. Here, we focus on modeling the first and critical step in placenta formation, which consists of the attachment of the allantois to the chorion. We describe a method to rapidly assess the attachment of allantoic explants on immobilized α4ß1 integrin, which serves as a chorio-mimetic substrate.This in vitro approach enables a qualitative evaluation of the attachment and spreading behavior of multiple allantois explants at different consecutive time points. The protocol may be used to investigate the effect of targeted mouse mutations, drugs, or various environmental factors that have been linked to pregnancy complications or fetal loss on allantois attachment ex vivo.


Assuntos
Alantoide/citologia , Alantoide/cirurgia , Animais , Técnicas de Cultura de Células/métodos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/cirurgia , Feminino , Camundongos , Gravidez
8.
Sci Rep ; 6: 35160, 2016 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-27731369

RESUMO

Mammalian phosphoglycolate phosphatase (PGP) is thought to target phosphoglycolate, a 2-deoxyribose fragment derived from the repair of oxidative DNA lesions. However, the physiological role of this activity and the biological function of the DNA damage product phosphoglycolate is unknown. We now show that knockin replacement of murine Pgp with its phosphatase-inactive PgpD34N mutant is embryonically lethal due to intrauterine growth arrest and developmental delay in midgestation. PGP inactivation attenuated triosephosphate isomerase activity, increased triglyceride levels at the expense of the cellular phosphatidylcholine content, and inhibited cell proliferation. These effects were prevented under hypoxic conditions or by blocking phosphoglycolate release from damaged DNA. Thus, PGP is essential to sustain cell proliferation in the presence of oxygen. Collectively, our findings reveal a previously unknown mechanism coupling a DNA damage repair product to the control of intermediary metabolism and cell proliferation.


Assuntos
Proliferação de Células/fisiologia , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Dano ao DNA , Reparo do DNA , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Feminino , Técnicas de Introdução de Genes , Glicolatos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oxirredução , Fosfatidilcolinas/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/genética , Gravidez , Triglicerídeos/metabolismo , Triose-Fosfato Isomerase/metabolismo
9.
Free Radic Biol Med ; 97: 75-84, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27179418

RESUMO

Redox-dependent switches of enzyme activity are emerging as important fine-tuning mechanisms in cell signaling. For example, protein tyrosine phosphatases employ a conserved cysteine residue for catalysis, which also renders them highly susceptible to reversible inactivation by oxidation. In contrast, haloacid dehalogenase (HAD)-type phosphatases perform catalysis via a phosphoaspartyltransferase reaction. The potential regulation of HAD phosphatases by reversible oxidation has not yet been explored. Here, we investigate the redox-sensitivity of the HAD-type phosphoglycolate phosphatase PGP, also known as AUM or glycerol-3-phosphate phosphatase. We show that recombinant, purified murine PGP is inhibited by oxidation and re-activated by reduction. We identify three reactive cysteine residues in the catalytic core domain of PGP (Cys35, Cys104 and Cys243) that mediate the reversible inhibition of PGP activity and the associated, redox-dependent conformational changes. Structural analysis suggests that Cys35 oxidation weakens van-der-Waals interactions with Thr67, a conserved catalytic residue required for substrate coordination. Cys104 and Cys243 form a redox-dependent disulfide bridge between the PGP catalytic core and cap domains, which may impair the open/close-dynamics of the catalytic cycle. In addition, we demonstrate that Cys297 in the PGP cap domain is essential for redox-dependent PGP oligomerization, and that PGP oxidation/oligomerization occurs in response to stimulation of cells with EGF. Finally, employing a modified cysteinyl-labeling assay, we show that cysteines of cellular PGP are transiently oxidized to sulfenic acids. Taken together, our findings establish that PGP, an aspartate-dependent HAD phosphatase, is transiently inactivated by reversible oxidation in cells.


Assuntos
Hidrolases/metabolismo , Estresse Oxidativo/genética , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Ácido Aspártico/metabolismo , Catálise , Domínio Catalítico/genética , Cisteína/metabolismo , Dissulfetos/química , Peróxido de Hidrogênio/metabolismo , Hidrolases/química , Hidrolases/genética , Camundongos , Oxirredução , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
10.
Proc Natl Acad Sci U S A ; 113(4): E430-9, 2016 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-26755581

RESUMO

Obesity, and the associated disturbed glycerolipid/fatty acid (GL/FA) cycle, contribute to insulin resistance, islet ß-cell failure, and type 2 diabetes. Flux through the GL/FA cycle is regulated by the availability of glycerol-3-phosphate (Gro3P) and fatty acyl-CoA. We describe here a mammalian Gro3P phosphatase (G3PP), which was not known to exist in mammalian cells, that can directly hydrolyze Gro3P to glycerol. We identified that mammalian phosphoglycolate phosphatase, with an uncertain function, acts in fact as a G3PP. We found that G3PP, by controlling Gro3P levels, regulates glycolysis and glucose oxidation, cellular redox and ATP production, gluconeogenesis, glycerolipid synthesis, and fatty acid oxidation in pancreatic islet ß-cells and hepatocytes, and that glucose stimulated insulin secretion and the response to metabolic stress, e.g., glucolipotoxicity, in ß-cells. In vivo overexpression of G3PP in rat liver lowers body weight gain and hepatic glucose production from glycerol and elevates plasma HDL levels. G3PP is expressed at various levels in different tissues, and its expression varies according to the nutritional state in some tissues. As Gro3P lies at the crossroads of glucose, lipid, and energy metabolism, control of its availability by G3PP adds a key level of metabolic regulation in mammalian cells, and G3PP offers a potential target for type 2 diabetes and cardiometabolic disorders.


Assuntos
Metabolismo dos Carboidratos/fisiologia , Glicerofosfatos/metabolismo , Hepatócitos/enzimologia , Células Secretoras de Insulina/enzimologia , Metabolismo dos Lipídeos/fisiologia , Monoéster Fosfórico Hidrolases/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Ácidos Graxos/metabolismo , Glicerol/metabolismo , Hidrólise , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Lactonas/farmacologia , Masculino , Camundongos , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Estado Nutricional , Orlistate , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/genética , Interferência de RNA , Ratos , Homologia de Sequência de Aminoácidos , Estresse Fisiológico/fisiologia
11.
Proc Natl Acad Sci U S A ; 112(37): E5150-9, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26324884

RESUMO

Cofilin, a critical player of actin dynamics, is spatially and temporally regulated to control the direction and force of membrane extension required for cell locomotion. In carcinoma cells, although the signaling pathways regulating cofilin activity to control cell direction have been established, the molecular machinery required to generate the force of the protrusion remains unclear. We show that the cofilin phosphatase chronophin (CIN) spatiotemporally regulates cofilin activity at the cell edge to generate persistent membrane extension. We show that CIN translocates to the leading edge in a PI3-kinase-, Rac1-, and cofilin-dependent manner after EGF stimulation to activate cofilin, promotes actin free barbed end formation, accelerates actin turnover, and enhances membrane protrusion. In addition, we establish that CIN is crucial for the balance of protrusion/retraction events during cell migration. Thus, CIN coordinates the leading edge dynamics by controlling active cofilin levels to promote MTLn3 cell protrusion.


Assuntos
Cofilina 1/fisiologia , Regulação da Expressão Gênica , Fosfoproteínas Fosfatases/fisiologia , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Animais , Neoplasias da Mama/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Receptores ErbB/metabolismo , Humanos , Proteínas dos Microfilamentos/fisiologia , Metástase Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Ratos , Transdução de Sinais
12.
Bioorg Med Chem ; 23(12): 2819-27, 2015 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-25783190

RESUMO

A set of phosphonic acid derivatives (1-4) of pyridoxal 5'-phosphate (PLP) was synthesized and characterized biochemically using purified murine pyridoxal phosphatase (PDXP), also known as chronophin. The most promising compound 1 displayed primarily competitive PDXP inhibitory activity with an IC50 value of 79µM, which was in the range of the Km of the physiological substrate PLP. We also report the X-ray crystal structure of PDXP bound to compound 3, which we solved to 2.75Å resolution (PDB code 5AES). The co-crystal structure proves that compound 3 binds in the same orientation as PLP, and confirms the mode of inhibition to be competitive. Thus, we identify compound 1 as a PDXP phosphatase inhibitor. Our results suggest a strategy to design new, potent and selective PDXP inhibitors, which may be useful to increase the sensitivity of tumor cells to treatment with cytotoxic agents.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Animais , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Hidrólise , Camundongos , Simulação de Acoplamento Molecular , Monoéster Fosfórico Hidrolases/química , Fosfato de Piridoxal/síntese química
13.
PLoS One ; 9(3): e92605, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24663062

RESUMO

BACKGROUND: RGS9-deficient mice show drug-induced dyskinesia but normal locomotor activity under unchallenged conditions. RESULTS: Genes related to Ca2+ signaling and their functions were regulated in RGS9-deficient mice. CONCLUSION: Changes in Ca2+ signaling that compensate for RGS9 loss-of-function can explain the normal locomotor activity in RGS9-deficient mice under unchallenged conditions. SIGNIFICANCE: Identified signaling components may represent novel targets in antidyskinetic therapy. The long splice variant of the regulator of G-protein signaling 9 (RGS9-2) is enriched in striatal medium spiny neurons and dampens dopamine D2 receptor signaling. Lack of RGS9-2 can promote while its overexpression prevents drug-induced dyskinesia. Other animal models of drug-induced dyskinesia rather pointed towards overactivity of dopamine receptor-mediated signaling. To evaluate changes in signaling pathways mRNA expression levels were determined and compared in wild-type and RGS9-deficient mice. Unexpectedly, expression levels of dopamine receptors were unchanged in RGS9-deficient mice, while several genes related to Ca2+ signaling and long-term depression were differentially expressed when compared to wild type animals. Detailed investigations at the protein level revealed hyperphosphorylation of DARPP32 at Thr34 and of ERK1/2 in striata of RGS9-deficient mice. Whole cell patch clamp recordings showed that spontaneous synaptic events are increased (frequency and size) in RGS9-deficient mice while long-term depression is reduced in acute brain slices. These changes are compatible with a Ca2+-induced potentiation of dopamine receptor signaling which may contribute to the drug-induced dyskinesia in RGS9-deficient mice.


Assuntos
Regulação da Expressão Gênica , Neostriado/metabolismo , Proteínas RGS/deficiência , Receptores de Dopamina D2/metabolismo , Animais , Sinalização do Cálcio , AMP Cíclico/metabolismo , Discinesia Induzida por Medicamentos/genética , Discinesia Induzida por Medicamentos/metabolismo , Discinesia Induzida por Medicamentos/patologia , Discinesia Induzida por Medicamentos/fisiopatologia , Depressão Sináptica de Longo Prazo , Masculino , Camundongos , Plasticidade Neuronal , Fosforilação , Sinapses/metabolismo
14.
Nanomedicine (Lond) ; 9(1): 21-33, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23731458

RESUMO

AIM: To characterize molecular mechanisms underlying photocatalytic cell death of head and neck squamous cell carcinoma (HNSCC) by zinc oxide nanoparticles (ZnO-NPs). MATERIALS & METHODS: Human HNSCC-derived FaDu cells were incubated with ZnO-NPs followed by UVA-1 irradiation. Cytotoxicity was assessed by MTT assay and annexin-V propidium iodide test. Autophagy was detected by autophagosome accumulation, conversion of light chain 3 I to II, and lysosomal activity. The generation of reactive oxygen species was measured using the 2',7'-dichlorofluorescein-diacetate test. RESULTS: Apoptosis-independent cytotoxic effects were induced by 0.2- and 2-µg/ml ZnO-NPs and UVA-1. FaDu cells promoted autophagosome formation. Significantly elevated light chain 3 II and reactive oxygen species were seen after the combined application of both ZnO-NPs and UVA-1 as photocatalytic treatment. Autophagy probably mediates cell survival under UVA-1 or ZnO-NP exposure alone but induces self-digestive cell death after combined treatment. CONCLUSION: The effect of autophagy on HNSCC viability after nanoparticle-induced photocatalytic treatment seems to depend on the impact of the physicochemical trigger.


Assuntos
Autofagia/efeitos dos fármacos , Carcinoma de Células Escamosas/genética , Neoplasias de Cabeça e Pescoço/genética , Nanopartículas Metálicas/toxicidade , Óxido de Zinco/toxicidade , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/induzido quimicamente , Carcinoma de Células Escamosas/etiologia , Catálise , Linhagem Celular Tumoral , Neoplasias de Cabeça e Pescoço/induzido quimicamente , Neoplasias de Cabeça e Pescoço/etiologia , Humanos , Nanopartículas Metálicas/química , Fotoquímica , Espécies Reativas de Oxigênio/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço , Óxido de Zinco/farmacologia
15.
J Biol Chem ; 289(6): 3416-31, 2014 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-24338473

RESUMO

Mammalian haloacid dehalogenase (HAD)-type phosphatases are an emerging family of phosphatases with important functions in physiology and disease, yet little is known about the basis of their substrate specificity. Here, we characterize a previously unexplored HAD family member (gene annotation, phosphoglycolate phosphatase), which we termed AUM, for aspartate-based, ubiquitous, Mg(2+)-dependent phosphatase. AUM is a tyrosine-specific paralog of the serine/threonine-specific protein and pyridoxal 5'-phosphate-directed HAD phosphatase chronophin. Comparative evolutionary and biochemical analyses reveal that a single, differently conserved residue in the cap domain of either AUM or chronophin is crucial for phosphatase specificity. We have solved the x-ray crystal structure of the AUM cap fused to the catalytic core of chronophin to 2.65 Å resolution and present a detailed view of the catalytic clefts of AUM and chronophin that explains their substrate preferences. Our findings identify a small number of cap domain residues that encode the different substrate specificities of AUM and chronophin.


Assuntos
Fosfoproteínas Fosfatases/química , Animais , Cristalografia por Raios X , Humanos , Masculino , Camundongos , Fosfoproteínas Fosfatases/classificação , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Estrutura Terciária de Proteína , Ratos , Especificidade por Substrato
16.
J Biol Chem ; 289(5): 3094-103, 2014 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-24338687

RESUMO

Mammalian phosphatases of the haloacid dehalogenase (HAD) superfamily have emerged as important regulators of physiology and disease. Many of these enzymes are stable homodimers; however, the role of their dimerization is largely unknown. Here, we explore the function of the obligatory homodimerization of chronophin, a mammalian HAD phosphatase known to dephosphorylate pyridoxal 5'-phosphate (PLP) and serine/threonine-phosphorylated proteins. The exchange of two residues in the murine chronophin homodimerization interface (chronophin(A194K,A195K)) yields a constitutive monomer both in vitro and in cells. The catalytic activity of monomeric chronophin toward PLP is strongly impaired. X-ray crystallographic studies of chronophin(A194K,A195K) revealed that dimer formation is essential for an intermolecular arginine-arginine-tryptophan stacking interaction that positions a critical histidine residue in the substrate specificity loop of chronophin for PLP coordination. Analysis of all available crystal structures of HAD hydrolases that are grouped together with chronophin in the C2a-type structural subfamily uncovered a highly conserved mode of dimerization that results in intermolecular contacts involving the substrate specificity loop. Our results explain how the dimerization of HAD hydrolases contributes to their catalytic efficiency and substrate specificity.


Assuntos
Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Fosfato de Piridoxal/metabolismo , Fatores Etários , Regulação Alostérica , Animais , Cristalografia por Raios X , Dimerização , Hidrolases/química , Hidrolases/metabolismo , Camundongos , Fosforilação , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato
17.
Int J Biochem Cell Biol ; 45(8): 1720-9, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23702034

RESUMO

The role of cytoskeleton-associated proteins during TNF-induced apoptosis is not fully understood. A potential candidate kinase that might connect TNF signaling to actin reorganization is the death-associated protein kinase (DAPK). To identify new DAPK interaction partners in TNF-induced apoptosis, we performed a peptide array screen. We show that TNF-treatment enhanced the phosphorylation of LIMK at threonine508 and its downstream target cofilin at serine3 (p-cofilin(Ser3)). Modulation of DAPK activity and expression by DAPK inhibitor treatment, siRNA knockdown, and overexpression affected the phosphorylation of both proteins. We propose a 3D structural model where DAPK functions as a scaffold for the LIMK/cofilin complex and triggers a closer interaction of both proteins under TNF stimulation. Upon TNF a striking redistribution of LIMK, DAPK, and cofilin to the perinuclear compartment was observed. The pro-apoptotic DAPK/LIMK/cofilin multiprotein complex was abrogated in detached cells, indicating that its signaling was no longer needed if cells committed to apoptosis. P-cofilin(Ser3) was strongly accumulated in cells with condensed chromatin, pronounced membrane blebs and Annexin V up-regulation. From studying different cofilin(Ser3) mutants we suggest that p-cofilin(Ser3) is an indicator of TNF-induced apoptosis. Collectively, our findings identify a novel molecular cytoskeleton-associated mechanism in TNF-induced DAPK-dependent apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Cofilina 1/metabolismo , Proteínas Quinases Associadas com Morte Celular/metabolismo , Quinases Lim/metabolismo , Complexos Multiproteicos/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Sequência de Aminoácidos , Adesão Celular/efeitos dos fármacos , Cofilina 1/química , Células HCT116 , Humanos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
18.
FEBS J ; 280(2): 549-71, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22607316

RESUMO

Phosphatases of the haloacid dehalogenase (HAD) superfamily of hydrolases are an ancient and very large class of enzymes that have evolved to dephosphorylate a wide range of low- and high molecular weight substrates with often exquisite specificities. HAD phosphatases constitute approximately one-fifth of all human phosphatase catalytic subunits. While the overall sequence similarity between HAD phosphatases is generally very low, family members can be identified based on the presence of a characteristic Rossmann-like fold and the active site sequence DxDx(V/T). HAD phosphatases employ an aspartate residue as a nucleophile in a magnesium-dependent phosphoaspartyl transferase reaction. Although there is genetic evidence demonstrating a causal involvement of some HAD phosphatases in diseases such as cancer, cardiovascular, metabolic and neurological disorders, the physiological roles of many of these enzymes are still poorly understood. In this review, we discuss the structure and evolution of human HAD phosphatases, and summarize their known functions in health and disease.


Assuntos
Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Estrutura Terciária de Proteína , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Monoéster Fosfórico Hidrolases/genética , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Homologia de Sequência de Aminoácidos
19.
Mol Pharmacol ; 82(2): 217-25, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22554804

RESUMO

The parathyroid hormone receptor (PTHR) is a class B G protein-coupled receptor (GPCR) that mediates the endocrine and paracrine effects of parathyroid hormone and related peptides through the activation of phospholipase Cß-, adenylyl cyclase-, mitogen-activated protein kinase-, and ß-arrestin-initiated signaling pathways. It is currently not clear how specificity among these downstream signaling pathways is achieved. A possible mechanism involves adaptor proteins that affect receptor/effector coupling. In a proteomic screen with the PTHR C terminus, we identified vav2, a guanine nucleotide exchange factor (GEF) for Rho GTPases, as a PTHR-interacting protein. The core domains of vav2 bound to the intracellular domains of the PTHR independent of receptor activation. In addition, vav2 specifically interacted with activated Gα(q) but not with Gα(s) subunits, and it competed with PTHR for coupling to Gα(q). Consistent with its specific interaction with Gα(q), vav2 impaired G(q)-mediated inositol phosphate generation but not G(s)-mediated cAMP generation. This inhibition of G(q) signaling was specific for PTHR signaling, compared with other G(q)-coupled GPCRs. Moreover, the benefit for PTHR-mediated inositol phosphate generation in the absence of vav2 required the ezrin binding domain of Na(+)/H(+)-exchanger regulatory factor 1. Our results show that a RhoA GEF can specifically interact with a GPCR and modulate its G protein signaling specificity.


Assuntos
Regulação para Baixo/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Proteínas Proto-Oncogênicas c-vav/fisiologia , Receptor Tipo 1 de Hormônio Paratireóideo/fisiologia , Transdução de Sinais/fisiologia , Animais , Ligação Competitiva/fisiologia , Células COS , Chlorocebus aethiops , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Células HEK293 , Humanos , Inositol/metabolismo , Inositol/farmacologia , Ligação Proteica/fisiologia , Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos
20.
Toxicol Sci ; 122(2): 317-29, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21551354

RESUMO

Ochratoxin A (OTA) is one of the most potent rodent renal carcinogens studied to date. Although controversial results regarding OTA genotoxicity have been published, it is now widely accepted that OTA is not a mutagenic, DNA-reactive carcinogen. Instead, increasing evidence from both in vivo and in vitro studies suggests that OTA may promote genomic instability and tumorigenesis through interference with cell division. The aim of the present study was to provide further support for disruption of mitosis as a key event in OTA toxicity and to understand how OTA mediates these effects. Immortalized human kidney epithelial cells (IHKE) were treated with OTA and monitored by differential interference contrast microscopy for 15 h. Image analysis confirmed that OTA at concentrations ≥ 5 µM, which correlate with plasma concentrations in rats under conditions of carcinogenesis, causes sustained mitotic arrest and exit from mitosis without nuclear or cellular division. Mitotic chromosomes were characterized by aberrant condensation and premature sister chromatid separation associated with altered phosphorylation and acetylation of core histones. To test if OTA directly interferes with histone acetyltransferases (HATs) which regulate lysine acetylation of histones and nonhistone proteins, a cell-free HAT activity assay was conducted using total nuclear extracts of IHKE cells. In this assay, OTA significantly blocked HAT activity in a concentration-dependent manner Overall, results from this study provide further support for a mechanism of OTA carcinogenicity involving interference with the mitotic machinery and suggest HATs as a primary cellular target of OTA.


Assuntos
Carcinógenos/toxicidade , Histona Acetiltransferases/antagonistas & inibidores , Mitose/efeitos dos fármacos , Ocratoxinas/toxicidade , Acetilação/efeitos dos fármacos , Divisão Celular , Linhagem Celular , Dano ao DNA , Células Epiteliais/efeitos dos fármacos , Histonas/metabolismo , Humanos , Rim/citologia , Rim/efeitos dos fármacos , Mutagênicos/toxicidade , Fosforilação/efeitos dos fármacos
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