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1.
Sci Adv ; 8(31): eabo5633, 2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-35921423

RESUMO

Trigeminal neuralgia, historically dubbed the "suicide disease," is an exceedingly painful neurologic condition characterized by sudden episodes of intense facial pain. Unfortunately, the only U.S. Food and Drug Administration (FDA)-approved medication for trigeminal neuralgia carries substantial side effects, with many patients requiring surgery. Here, we identify the NRF2 transcriptional network as a potential therapeutic target. We report that cerebrospinal fluid from patients with trigeminal neuralgia accumulates reactive oxygen species, several of which directly activate the pain-transducing channel TRPA1. Similar to our patient cohort, a mouse model of trigeminal neuropathic pain also exhibits notable oxidative stress. We discover that stimulating the NRF2 antioxidant transcriptional network is as analgesic as inhibiting TRPA1, in part by reversing the underlying oxidative stress. Using a transcriptome-guided drug discovery strategy, we identify two NRF2 network modulators as potential treatments. One of these candidates, exemestane, is already FDA-approved and may thus be a promising alternative treatment for trigeminal neuropathic pain.

2.
Sci Signal ; 15(733): eabh3066, 2022 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-35536885

RESUMO

Synapses connect discrete neurons into vast networks that send, receive, and encode diverse forms of information. Synaptic function and plasticity, the neuronal process of adapting to diverse and variable inputs, depend on the dynamic nature of synaptic molecular components, which is mediated in part by cell adhesion signaling pathways. Here, we found that the enzyme biliverdin reductase (BVR) physically links together key focal adhesion signaling molecules at the synapse. BVR-null (BVR-/-) mice exhibited substantial deficits in learning and memory on neurocognitive tests, and hippocampal slices in which BVR was postsynaptically depleted showed deficits in electrophysiological responses to stimuli. RNA sequencing, biochemistry, and pathway analyses suggested that these deficits were mediated through the loss of focal adhesion signaling at both the transcriptional and biochemical level in the hippocampus. Independently of its catalytic function, BVR acted as a bridge between the primary focal adhesion signaling kinases FAK and Pyk2 and the effector kinase Src. Without BVR, FAK and Pyk2 did not bind to and stimulate Src, which then did not phosphorylate the N-methyl-d-aspartate (NMDA) receptor, a critical posttranslational modification for synaptic plasticity. Src itself is a molecular hub on which many signaling pathways converge to stimulate NMDAR-mediated neurotransmission, thus positioning BVR at a prominent intersection of synaptic signaling.


Assuntos
Quinase 2 de Adesão Focal , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Animais , Quinase 2 de Adesão Focal/genética , Quinase 2 de Adesão Focal/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Camundongos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Fosforilação/genética , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Quinases da Família src/metabolismo
3.
Proc Natl Acad Sci U S A ; 119(16): e2200545119, 2022 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-35412917

RESUMO

Cocaine exerts its stimulant effect by inhibiting dopamine (DA) reuptake, leading to increased dopamine signaling. This action is thought to reflect the binding of cocaine to the dopamine transporter (DAT) to inhibit its function. However, cocaine is a relatively weak inhibitor of DAT, and many DAT inhibitors do not share cocaine's behavioral actions. Further, recent reports show more potent actions of the drug, implying the existence of a high-affinity receptor for cocaine. We now report high-affinity binding of cocaine associated with the brain acid soluble protein 1 (BASP1) with a dissociation constant (Kd) of 7 nM. Knocking down BASP1 in the striatum inhibits [3H]cocaine binding to striatal synaptosomes. Depleting BASP1 in the nucleus accumbens but not the dorsal striatum diminishes locomotor stimulation in mice. Our findings imply that BASP1 is a pharmacologically relevant receptor for cocaine.


Assuntos
Proteínas de Ligação a Calmodulina , Proteínas de Transporte , Cocaína , Proteínas do Citoesqueleto , Proteínas do Tecido Nervoso , Receptores de Droga , Animais , Sítios de Ligação , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cocaína/metabolismo , Cocaína/farmacologia , Corpo Estriado/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Técnicas de Introdução de Genes , Humanos , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ratos , Receptores de Droga/genética , Receptores de Droga/metabolismo
4.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33431651

RESUMO

Alzheimer's disease (AD), the most common cause of dementia and neurodegeneration in the elderly, is characterized by deterioration of memory and executive and motor functions. Neuropathologic hallmarks of AD include neurofibrillary tangles (NFTs), paired helical filaments, and amyloid plaques. Mutations in the microtubule-associated protein Tau, a major component of the NFTs, cause its hyperphosphorylation in AD. We have shown that signaling by the gaseous molecule hydrogen sulfide (H2S) is dysregulated during aging. H2S signals via a posttranslational modification termed sulfhydration/persulfidation, which participates in diverse cellular processes. Here we show that cystathionine γ-lyase (CSE), the biosynthetic enzyme for H2S, binds wild type Tau, which enhances its catalytic activity. By contrast, CSE fails to bind Tau P301L, a mutant that is present in the 3xTg-AD mouse model of AD. We further show that CSE is depleted in 3xTg-AD mice as well as in human AD brains, and that H2S prevents hyperphosphorylation of Tau by sulfhydrating its kinase, glycogen synthase kinase 3ß (GSK3ß). Finally, we demonstrate that sulfhydration is diminished in AD, while administering the H2S donor sodium GYY4137 (NaGYY) to 3xTg-AD mice ameliorates motor and cognitive deficits in AD.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Cistationina gama-Liase/genética , Glicogênio Sintase Quinase 3 beta/genética , Sulfeto de Hidrogênio/farmacologia , Morfolinas/farmacologia , Fármacos Neuroprotetores/farmacologia , Compostos Organotiofosforados/farmacologia , Proteínas tau/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Cistationina gama-Liase/metabolismo , Modelos Animais de Doenças , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HEK293 , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Emaranhados Neurofibrilares/efeitos dos fármacos , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologia , Fosforilação , Placa Amiloide/genética , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Placa Amiloide/prevenção & controle , Ligação Proteica , Processamento de Proteína Pós-Traducional , Sulfatos/metabolismo , Proteínas tau/metabolismo
5.
Cell Chem Biol ; 26(10): 1450-1460.e7, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31353321

RESUMO

Bilirubin is one of the most frequently measured metabolites in medicine, yet its physiologic roles remain unclear. Bilirubin can act as an antioxidant in vitro, but whether its redox activity is physiologically relevant is unclear because many other antioxidants are far more abundant in vivo. Here, we report that depleting endogenous bilirubin renders mice hypersensitive to oxidative stress. We find that mice lacking bilirubin are particularly vulnerable to superoxide (O2⋅-) over other tested reactive oxidants and electrophiles. Whereas major antioxidants such as glutathione and cysteine exhibit little to no reactivity toward O2⋅-, bilirubin readily scavenges O2⋅-. We find that bilirubin's redox activity is particularly important in the brain, where it prevents excitotoxicity and neuronal death by scavenging O2⋅- during NMDA neurotransmission. Bilirubin's unique redox activity toward O2⋅- may underlie a prominent physiologic role despite being significantly less abundant than other endogenous and exogenous antioxidants.


Assuntos
Antioxidantes/metabolismo , Bilirrubina/metabolismo , Heme/metabolismo , Superóxidos/metabolismo , Animais , Antioxidantes/química , Bilirrubina/química , Bilirrubina/deficiência , Células Cultivadas , Heme/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuroproteção , Oxirredução , Estresse Oxidativo
6.
Proc Natl Acad Sci U S A ; 116(7): 2701-2706, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30692251

RESUMO

Glutamate is the most abundant excitatory neurotransmitter, present at the bulk of cortical synapses, and participating in many physiologic and pathologic processes ranging from learning and memory to stroke. The tripeptide, glutathione, is one-third glutamate and present at up to low millimolar intracellular concentrations in brain, mediating antioxidant defenses and drug detoxification. Because of the substantial amounts of brain glutathione and its rapid turnover under homeostatic control, we hypothesized that glutathione is a relevant reservoir of glutamate and could influence synaptic excitability. We find that drugs that inhibit generation of glutamate by the glutathione cycle elicit decreases in cytosolic glutamate and decreased miniature excitatory postsynaptic potential (mEPSC) frequency. In contrast, pharmacologically decreasing the biosynthesis of glutathione leads to increases in cytosolic glutamate and enhanced mEPSC frequency. The glutathione cycle can compensate for decreased excitatory neurotransmission when the glutamate-glutamine shuttle is inhibited. Glutathione may be a physiologic reservoir of glutamate neurotransmitter.


Assuntos
Glutationa/metabolismo , Sinapses/metabolismo , Animais , Células Cultivadas , Potenciais Pós-Sinápticos Excitadores/fisiologia , Ácido Glutâmico/metabolismo , Homeostase , Neurônios/fisiologia , Ratos Sprague-Dawley , Transmissão Sináptica/fisiologia
7.
Nat Commun ; 9(1): 1526, 2018 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-29670103

RESUMO

ATM drives DNA repair by phosphorylating the histone variant H2AX. While ATM mutations elicit prominent neurobehavioral phenotypes, neural roles for H2AX have been elusive. We report impaired motor learning and balance in H2AX-deficient mice. Mitigation of reactive oxygen species (ROS) with N-acetylcysteine (NAC) reverses the behavioral deficits. Mouse embryonic fibroblasts deficient for H2AX exhibit increased ROS production and failure to activate the antioxidant response pathway controlled by the transcription factor NRF2. The NRF2 targets GCLC and NQO1 are depleted in the striatum of H2AX knockouts, one of the regions most vulnerable to ROS-mediated damage. These findings establish a role for ROS in the behavioral deficits of H2AX knockout mice and reveal a physiologic function of H2AX in mediating influences of oxidative stress on NRF2-transcriptional targets and behavior.


Assuntos
Comportamento Animal , Histonas/deficiência , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Acetilcisteína/química , Animais , Antioxidantes/química , Corpo Estriado/metabolismo , Dano ao DNA , Fibroblastos/metabolismo , Células HEK293 , Heterozigoto , Histonas/fisiologia , Humanos , Camundongos , Camundongos Knockout , Microscopia Confocal , Modelos Neurológicos , Destreza Motora , Oxirredução , Fenótipo , Fosforilação , Espécies Reativas de Oxigênio/metabolismo
8.
Science ; 360(6387): 449-453, 2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29599194

RESUMO

Activated immune cells undergo a metabolic switch to aerobic glycolysis akin to the Warburg effect, thereby presenting a potential therapeutic target in autoimmune disease. Dimethyl fumarate (DMF), a derivative of the Krebs cycle intermediate fumarate, is an immunomodulatory drug used to treat multiple sclerosis and psoriasis. Although its therapeutic mechanism remains uncertain, DMF covalently modifies cysteine residues in a process termed succination. We found that DMF succinates and inactivates the catalytic cysteine of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in mice and humans, both in vitro and in vivo. It thereby down-regulates aerobic glycolysis in activated myeloid and lymphoid cells, which mediates its anti-inflammatory effects. Our results provide mechanistic insight into immune modulation by DMF and represent a proof of concept that aerobic glycolysis is a therapeutic target in autoimmunity.


Assuntos
Autoimunidade/efeitos dos fármacos , Fumarato de Dimetilo/farmacologia , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Glicólise/efeitos dos fármacos , Imunossupressores/farmacologia , Animais , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/enzimologia , Ciclo do Ácido Cítrico , Cisteína/metabolismo , Fumarato de Dimetilo/uso terapêutico , Humanos , Imunossupressores/uso terapêutico , Linfócitos/efeitos dos fármacos , Linfócitos/enzimologia , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/efeitos dos fármacos , Células Mieloides/enzimologia , Células Mieloides/imunologia , Succinatos/química
9.
Circ Res ; 122(3): 457-472, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29279301

RESUMO

RATIONALE: Inositol polyphosphate multikinase (IPMK) and its major product inositol pentakisphosphate (IP5) regulate a variety of cellular functions, but their role in vascular biology remains unexplored. OBJECTIVE: We have investigated the role of IPMK in regulating angiogenesis. METHODS AND RESULTS: Deletion of IPMK in fibroblasts induces angiogenesis in both in vitro and in vivo models. IPMK deletion elicits a substantial increase of VEGF (vascular endothelial growth factor), which mediates the regulation of angiogenesis by IPMK. The regulation of VEGF by IPMK requires its catalytic activity. IPMK is predominantly nuclear and regulates gene transcription. However, IPMK does not apparently serve as a transcription factor for VEGF. HIF (hypoxia-inducible factor)-1α is a major determinant of angiogenesis and induces VEGF transcription. IPMK deletion elicits a major enrichment of HIF-1α protein and thus VEGF. HIF-1α is constitutively ubiquitinated by pVHL (von Hippel-Lindau protein) followed by proteasomal degradation under normal conditions. However, HIF-1α is not recognized and ubiquitinated by pVHL in IPMK KO (knockout) cells. IP5 reinstates the interaction of HIF-1α and pVHL. HIF-1α prolyl hydroxylation, which is prerequisite for pVHL recognition, is interrupted in IPMK-deleted cells. IP5 promotes HIF-1α prolyl hydroxylation and thus pVHL-dependent degradation of HIF-1α. Deletion of IPMK in mouse brain increases HIF-1α/VEGF levels and vascularization. The increased VEGF in IPMK KO disrupts blood-brain barrier and enhances brain blood vessel permeability. CONCLUSIONS: IPMK, via its product IP5, negatively regulates angiogenesis by inhibiting VEGF expression. IP5 acts by enhancing HIF-1α hydroxylation and thus pVHL-dependent degradation of HIF-1α.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fosfatos de Inositol/metabolismo , Neovascularização Fisiológica/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Animais , Barreira Hematoencefálica , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteólise , RNA Interferente Pequeno/genética , Organismos Livres de Patógenos Específicos , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
10.
Proc Natl Acad Sci U S A ; 114(8): 2036-2041, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28154132

RESUMO

Inositol hexakisphosphate kinase 1 (IP6K1), which generates 5-diphosphoinositol pentakisphosphate (5-IP7), physiologically mediates numerous functions. We report that IP6K1 deletion leads to brain malformation and abnormalities of neuronal migration. IP6K1 physiologically associates with α-actinin and localizes to focal adhesions. IP6K1 deletion disrupts α-actinin's intracellular localization and function. The IP6K1 deleted cells display substantial decreases of stress fiber formation and impaired cell migration and spreading. Regulation of α-actinin by IP6K1 requires its kinase activity. Deletion of IP6K1 abolishes α-actinin tyrosine phosphorylation, which is known to be regulated by focal adhesion kinase (FAK). FAK phosphorylation is substantially decreased in IP6K1 deleted cells. 5-IP7, a product of IP6K1, promotes FAK autophosphorylation. Pharmacologic inhibition of IP6K by TNP [N2-(m-Trifluorobenzyl), N6-(p-nitrobenzyl)purine] recapitulates the phenotype of IP6K1 deletion. These findings establish that IP6K1 physiologically regulates neuronal migration by binding to α-actinin and influencing phosphorylation of both FAK and α-actinin through its product 5-IP7.


Assuntos
Actinina/metabolismo , Movimento Celular/fisiologia , Quinase 1 de Adesão Focal/metabolismo , Neurônios/fisiologia , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Animais , Encéfalo/anormalidades , Encéfalo/enzimologia , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Fosfatos de Inositol/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , Fosfotransferases (Aceptor do Grupo Fosfato)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo
11.
J Neurosci ; 35(31): 11056-67, 2015 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-26245967

RESUMO

The inositol hexakisphosphate kinases (IP6Ks) are the principal enzymes that generate inositol pyrophosphates. There are three IP6Ks (IP6K1, 2, and 3). Functions of IP6K1 and IP6K2 have been substantially delineated, but little is known of IP6K3's role in normal physiology, especially in the brain. To elucidate functions of IP6K3, we generated mice with targeted deletion of IP6K3. We demonstrate that IP6K3 is highly concentrated in the brain in cerebellar Purkinje cells. IP6K3 physiologically binds to the cytoskeletal proteins adducin and spectrin, whose mutual interactions are perturbed in IP6K3-null mutants. Consequently, IP6K3 knock-out cerebella manifest abnormalities in Purkinje cell structure and synapse number, and the mutant mice display deficits in motor learning and coordination. Thus, IP6K3 is a major determinant of cytoskeletal disposition and function of cerebellar Purkinje cells. SIGNIFICANCE STATEMENT: We identified and cloned a family of three inositol hexakisphosphate kinases (IP6Ks) that generate the inositol pyrophosphates, most notably 5-diphosphoinositol pentakisphosphate (IP7). Of these, IP6K3 has been least characterized. In the present study we generated IP6K3 knock-out mice and show that IP6K3 is highly expressed in cerebellar Purkinje cells. IP6K3-deleted mice display defects of motor learning and coordination. IP6K3-null mice manifest aberrations of Purkinje cells with a diminished number of synapses. IP6K3 interacts with the cytoskeletal proteins spectrin and adducin whose altered disposition in IP6K3 knock-out mice may mediate phenotypic features of the mutant mice. These findings afford molecular/cytoskeletal mechanisms by which the inositol polyphosphate system impacts brain function.


Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Células de Purkinje/metabolismo , Espectrina/metabolismo , Sinapses/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Forma Celular/fisiologia , Camundongos , Camundongos Knockout , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Células de Purkinje/citologia
12.
Nature ; 509(7498): 96-100, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24670645

RESUMO

Huntington's disease is an autosomal dominant disease associated with a mutation in the gene encoding huntingtin (Htt) leading to expanded polyglutamine repeats of mutant Htt (mHtt) that elicit oxidative stress, neurotoxicity, and motor and behavioural changes. Huntington's disease is characterized by highly selective and profound damage to the corpus striatum, which regulates motor function. Striatal selectivity of Huntington's disease may reflect the striatally selective small G protein Rhes binding to mHtt and enhancing its neurotoxicity. Specific molecular mechanisms by which mHtt elicits neurodegeneration have been hard to determine. Here we show a major depletion of cystathionine γ-lyase (CSE), the biosynthetic enzyme for cysteine, in Huntington's disease tissues, which may mediate Huntington's disease pathophysiology. The defect occurs at the transcriptional level and seems to reflect influences of mHtt on specificity protein 1, a transcriptional activator for CSE. Consistent with the notion of loss of CSE as a pathogenic mechanism, supplementation with cysteine reverses abnormalities in cultures of Huntington's disease tissues and in intact mouse models of Huntington's disease, suggesting therapeutic potential.


Assuntos
Cistationina gama-Liase/deficiência , Doença de Huntington/enzimologia , Doença de Huntington/patologia , Animais , Encéfalo/enzimologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/enzimologia , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Cistationina gama-Liase/genética , Cisteína/administração & dosagem , Cisteína/biossíntese , Cisteína/farmacologia , Cisteína/uso terapêutico , Suplementos Nutricionais , Modelos Animais de Doenças , Água Potável/química , Deleção de Genes , Regulação Enzimológica da Expressão Gênica/genética , Proteína Huntingtina , Doença de Huntington/tratamento farmacológico , Doença de Huntington/genética , Masculino , Camundongos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Fator de Transcrição Sp1/antagonistas & inibidores , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica/genética
13.
Sci Signal ; 6(269): ra22, 2013 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-23550211

RESUMO

The tumor suppressor protein p53 is a critical stress response transcription factor that induces the expression of genes leading to cell cycle arrest, apoptosis, and tumor suppression. We found that mammalian inositol polyphosphate multikinase (IPMK) stimulated p53-mediated transcription by binding to p53 and enhancing its acetylation by the acetyltransferase p300 independently of its inositol phosphate and lipid kinase activities. Genetic or RNA interference (RNAi)-mediated knockdown of IPMK resulted in decreased activation of p53, decreased recruitment of p53 and p300 to target gene promoters, abrogated transcription of p53 target genes, and enhanced cell viability. Additionally, blocking the IPMK-p53 interaction decreased the extent of p53-mediated transcription. These results suggest that IPMK acts as a transcriptional coactivator for p53 and that it is an integral part of the p53 transcriptional complex facilitating cell death.


Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/genética , Transativadores/genética , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Acetilação , Animais , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Embrião de Mamíferos/citologia , Etoposídeo/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HCT116 , Humanos , Camundongos , Camundongos Knockout , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo
14.
Nat Commun ; 4: 1626, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23535647

RESUMO

Increases in S-nitrosylation and inactivation of the neuroprotective ubiquitin E3 ligase, parkin, in the brains of patients with Parkinson's disease are thought to be pathogenic and suggest a possible mechanism linking parkin to sporadic Parkinson's disease. Here we demonstrate that physiologic modification of parkin by hydrogen sulfide, termed sulfhydration, enhances its catalytic activity. Sulfhydration sites are identified by mass spectrometry analysis and are investigated by site-directed mutagenesis. Parkin sulfhydration is markedly depleted in the brains of patients with Parkinson's disease, suggesting that this loss may be pathologic. This implies that hydrogen sulfide donors may be therapeutic.


Assuntos
Fármacos Neuroprotetores/metabolismo , Compostos de Sulfidrila/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Sequência de Aminoácidos , Catálise , Sulfeto de Hidrogênio/farmacologia , Espectrometria de Massas , Dados de Sequência Molecular , Compostos Nitrosos/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo
15.
Cell Metab ; 13(2): 215-21, 2011 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-21284988

RESUMO

mTOR complex 1 (mTORC1; mammalian target of rapamycin [mTOR] in complex with raptor) is a key regulator of protein synthesis and cell growth in response to nutrient amino acids. Here we report that inositol polyphosphate multikinase (IPMK), which possesses both inositol phosphate kinase and lipid kinase activities, regulates amino acid signaling to mTORC1. This regulation is independent of IPMK's catalytic function, instead reflecting its binding with mTOR and raptor, which maintains the mTOR-raptor association. Thus, IPMK appears to be a physiologic mTOR cofactor, serving as a determinant of mTORC1 stability and amino acid-induced mTOR signaling. Substances that block IPMK-mTORC1 binding may afford therapeutic benefit in nutrient amino acid-regulated conditions such as obesity and diabetes.


Assuntos
Aminoácidos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Substituição de Aminoácidos , Animais , Biocatálise , Linhagem Celular , Fibroblastos/metabolismo , Humanos , Camundongos , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ligação Proteica , Transdução de Sinais
16.
Proc Natl Acad Sci U S A ; 108(6): 2205-9, 2011 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-21262846

RESUMO

The inositol pyrophosphate, diphosphoinositol pentakisphosphate, regulates p53 and protein kinase Akt signaling, and its aberrant increase in cells has been implicated in apoptosis and insulin resistance. Inositol hexakisphosphate kinase-2 (IP6K2), one of the major inositol pyrophosphate synthesizing enzymes, mediates p53-linked apoptotic cell death. Casein kinase-2 (CK2) promotes cell survival and is upregulated in tumors. We show that CK2 mediated cell survival involves IP6K2 destabilization. CK2 physiologically phosphorylates IP6K2 at amino acid residues S347 and S356 contained within a PEST sequence, a consensus site for ubiquitination. HCT116 cells depleted of IP6K2 are resistant to cell death elicited by CK2 inhibitors. CK2 phosphorylation at the degradation motif of IP6K2 enhances its ubiquitination and subsequent degradation. IP6K2 mutants at the CK2 sites that are resistant to CK2 phosphorylation are metabolically stable.


Assuntos
Apoptose , Caseína Quinase II/metabolismo , Regulação Enzimológica da Expressão Gênica , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Transdução de Sinais , Regulação para Cima , Motivos de Aminoácidos , Sobrevivência Celular , Estabilidade Enzimática , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Resistência à Insulina , Neoplasias/enzimologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação
17.
Cell ; 143(6): 897-910, 2010 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-21145457

RESUMO

The inositol pyrophosphate IP7 (5-diphosphoinositolpentakisphosphate), formed by a family of three inositol hexakisphosphate kinases (IP6Ks), modulates diverse cellular activities. We now report that IP7 is a physiologic inhibitor of Akt, a serine/threonine kinase that regulates glucose homeostasis and protein translation, respectively, via the GSK3ß and mTOR pathways. Thus, Akt and mTOR signaling are dramatically augmented and GSK3ß signaling reduced in skeletal muscle, white adipose tissue, and liver of mice with targeted deletion of IP6K1. IP7 affects this pathway by potently inhibiting the PDK1 phosphorylation of Akt, preventing its activation and thereby affecting insulin signaling. IP6K1 knockout mice manifest insulin sensitivity and are resistant to obesity elicited by high-fat diet or aging. Inhibition of IP6K1 may afford a therapeutic approach to obesity and diabetes.


Assuntos
Fosfatos de Inositol/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Aumento de Peso , Adipogenia , Envelhecimento/metabolismo , Animais , Técnicas de Cultura de Células , Dieta , Difosfatos/metabolismo , Inositol/metabolismo , Insulina/metabolismo , Resistência à Insulina , Camundongos , Obesidade/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Fosfato)/genética
18.
Proc Natl Acad Sci U S A ; 107(49): 20947-51, 2010 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-21078964

RESUMO

Inositol pyrophosphates have been implicated in numerous biological processes. Inositol hexakisphosphate kinase-2 (IP6K2), which generates the inositol pyrophosphate, diphosphoinositol pentakisphosphate (IP7), influences apoptotic cell death. The tumor suppressor p53 responds to genotoxic stress by engaging a transcriptional program leading to cell-cycle arrest or apoptosis. We demonstrate that IP6K2 is required for p53-mediated apoptosis and modulates the outcome of the p53 response. Gene disruption of IP6K2 in colorectal cancer cells selectively impairs p53-mediated apoptosis, instead favoring cell-cycle arrest. IP6K2 acts by binding directly to p53 and decreasing expression of proarrest gene targets such as the cyclin-dependent kinase inhibitor p21.


Assuntos
Apoptose/genética , Neoplasias do Colo/patologia , Fosfotransferases (Aceptor do Grupo Fosfato)/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p21/genética , Dano ao DNA , Humanos , Fosfotransferases (Aceptor do Grupo Fosfato)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Ligação Proteica , Proteína Supressora de Tumor p53/metabolismo
19.
Nat Cell Biol ; 12(11): 1094-100, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20972425

RESUMO

S-nitrosylation of proteins by nitric oxide is a major mode of signalling in cells. S-nitrosylation can mediate the regulation of a range of proteins, including prominent nuclear proteins, such as HDAC2 (ref. 2) and PARP1 (ref. 3). The high reactivity of the nitric oxide group with protein thiols, but the selective nature of nitrosylation within the cell, implies the existence of targeting mechanisms. Specificity of nitric oxide signalling is often achieved by the binding of nitric oxide synthase (NOS) to target proteins, either directly or through scaffolding proteins such as PSD-95 (ref. 5) and CAPON. As the three principal isoforms of NOS--neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS)--are primarily non-nuclear, the mechanisms by which nuclear proteins are selectively nitrosylated have been elusive. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is physiologically nitrosylated at its Cys 150 residue. Nitrosylated GAPDH (SNO-GAPDH) binds to Siah1, which possesses a nuclear localization signal, and is transported to the nucleus. Here, we show that SNO-GAPDH physiologically transnitrosylates nuclear proteins, including the deacetylating enzyme sirtuin-1 (SIRT1), histone deacetylase-2 (HDAC2) and DNA-activated protein kinase (DNA-PK). Our findings reveal a novel mechanism for targeted nitrosylation of nuclear proteins and suggest that protein-protein transfer of nitric oxide groups may be a general mechanism in cellular signal transduction.


Assuntos
Proteína Quinase Ativada por DNA/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Histona Desacetilase 2/metabolismo , Proteínas Nucleares/metabolismo , Sirtuína 1/metabolismo , Células Cultivadas , Humanos , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Transdução de Sinais , Sirtuína 1/antagonistas & inibidores
20.
Proc Natl Acad Sci U S A ; 105(4): 1134-9, 2008 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-18195352

RESUMO

Heat-shock proteins (HSPs) are abundant, inducible proteins best known for their ability to maintain the conformation of proteins and to refold damaged proteins. Some HSPs, especially HSP90, can be antiapoptotic and the targets of anticancer drugs. Inositol hexakisphosphate kinase-2 (IP6K2), one of a family of enzymes generating the inositol pyrophosphate IP7 [diphosphoinositol pentakisphosphate (5-PP-IP5)], mediates apoptosis. Increased IP6K2 activity sensitizes cancer cells to stressors, whereas its depletion blocks cell death. We now show that HSP90 physiologically binds IP6K2 and inhibits its catalytic activity. Drugs and selective mutations that abolish HSP90-IP6K2 binding elicit activation of IP6K2, leading to cell death. Thus, the prosurvival actions of HSP90 reflect the inhibition of IP6K2, suggesting that selectively blocking this interaction could provide effective and safer modes of chemotherapy.


Assuntos
Proteínas de Choque Térmico HSP90/fisiologia , Fosfotransferases (Aceptor do Grupo Fosfato)/fisiologia , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Cisplatino/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Novobiocina/farmacologia , Fosfotransferases (Aceptor do Grupo Fosfato)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Fosfato)/deficiência , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Ligação Proteica/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia
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