RESUMO
Epilepsy is a multifactorial neurologic disease that often leads to many devastating disabilities and an enormous burden on the healthcare system. Until now, drug-resistant epilepsy has presented a major challenge for approximately 30% of the epileptic population. The present article summarizes the validated rodent models of seizures employed in pharmacological researches and comprehensively reviews updated advances of novel antiseizure candidates in the preclinical phase. Newly discovered compounds that demonstrate antiseizure efficacy in preclinical trials will be discussed in the review. It is inspiring that several candidates exert promising antiseizure activities in drug-resistant seizure models. The representative compounds consist of derivatives of hybrid compounds that integrate multiple approved antiseizure medications, novel positive allosteric modulators targeting subtype-selective γ-Aminobutyric acid type A receptors, and a derivative of cinnamamide. Although the precise molecular mechanism, pharmacokinetic properties, and safety are not yet fully clear in every novel antiseizure candidate, the adapted approaches to design novel antiseizure medications provide new insights to overcome drug-resistant epilepsy.
Assuntos
Epilepsia Resistente a Medicamentos , Convulsões , Animais , Convulsões/tratamento farmacológicoRESUMO
The transcriptional regulators TAZ and YAP (TAZ/YAP) have emerged as pro-tumorigenic factors that drive many oncogenic traits, including induction of cell growth, resistance to cell death, and activation of processes that promote migration and invasion. Here, we report that TAZ/YAP reprogram cellular energetics to promote the dependence of breast cancer cell growth on exogenous glutamine. Rescue experiments with glutamine-derived metabolites suggest an essential role for glutamate and α-ketoglutarate (AKG) in TAZ/YAP-driven cell growth in the absence of glutamine. Analysis of enzymes that mediate the conversion of glutamate to AKG shows that TAZ/YAP induce glutamic-oxaloacetic transaminase (GOT1) and phosphoserine aminotransferase (PSAT1) expression and that TAZ/YAP activity positively correlates with transaminase expression in breast cancer patients. Notably, we find that the transaminase inhibitor aminooxyacetate (AOA) represses cell growth in a TAZ/YAP-dependent manner, identifying transamination as a potential vulnerable metabolic requirement for TAZ/YAP-driven breast cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Aspartato Aminotransferase Citoplasmática/metabolismo , Neoplasias da Mama/metabolismo , Glutamina/metabolismo , Fosfoproteínas/fisiologia , Transaminases/metabolismo , Fatores de Transcrição/fisiologia , Aciltransferases , Carcinogênese , Proliferação de Células , Metabolismo Energético , Feminino , Humanos , Células Tumorais Cultivadas , Proteínas de Sinalização YAPRESUMO
While lysine acetylation in the nucleus is well characterized, comparatively little is known about its significance in cytoplasmic signaling. Here we show that inhibition of the Sirt1 deacetylase, which is primarily cytoplasmic in cancer cell lines, sensitizes these cells to caspase-2-dependent death. To identify relevant Sirt1 substrates, we developed a proteomics strategy, enabling the identification of a range of putative substrates, including 14-3-3ζ, a known direct regulator of caspase-2. We show here that inhibition of Sirtuin activity accelerates caspase activation and overrides caspase-2 suppression by nutrient abundance. Furthermore, 14-3-3ζ is acetylated prior to caspase activation, and supplementation of Xenopus egg extract with glucose-6-phosphate, which promotes caspase-2/14-3-3ζ binding, enhances 14-3-3ζ-directed Sirtuin activity. Conversely, inhibiting Sirtuin activity promotes14-3-3ζ dissociation from caspase-2 in both egg extract and human cultured cells. These data reveal a role for Sirt1 in modulating apoptotic sensitivity, in response to metabolic changes, by antagonizing 14-3-3ζ acetylation.
Assuntos
Proteínas 14-3-3/genética , Biotina/genética , Caspase 2/genética , Sirtuína 1/metabolismo , Proteínas 14-3-3/metabolismo , Acetilação , Animais , Apoptose , Biotina/metabolismo , Caspase 2/metabolismo , Morte Celular , Linhagem Celular Tumoral , Citoplasma/metabolismo , Humanos , Proteômica , Sirtuína 1/genéticaRESUMO
Gastrodigenin (HBA) and gastrodin (GAS) are phenolic ingredients found in Gastrodia elata Blume (GEB), a traditional Chinese herbal medicine. These compounds have been previously used to treat cognitive dysfunction, convulsion, and dizziness. However, at present, there is no available information regarding their potential ionic effects in electrically excitable cells. In the current study, the possible effects of HBA and GAS on different ionic currents in pituitary GH3 cells and hippocampal mHippoE-14 neurons were investigated using the patch-clamp technique. The addition of HBA or GAS resulted in the differential inhibition of the M-type K+ current (IK(M)) density in a concentration-dependent manner in GH3 cells. HBA resulted in a slowing of the activation time course of IK(M), while GAS elevated it. HBA also mildly suppressed the density of erg-mediated or the delayed-rectifier K+ current in GH3 cells. Neither GAS nor HBA (10 µM) modified the voltage-gated Na+ current density, although they suppressed the L-type Ca2+ current density at the same concentration. In hippocampal mHippoE-14 neurons, HBA was effective at inhibiting IK(M) density as well as slowing the activation time course. Taken together, the present study provided the first evidence that HBA or GAS could act on cellular mechanisms, and could therefore potentially have a functional influence in various neurologic disorders.
Assuntos
Álcoois Benzílicos/farmacologia , Glucosídeos/farmacologia , Hipocampo/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Neurônios/metabolismo , Hipófise/metabolismo , Potássio/metabolismo , Linhagem Celular Tumoral , Hipocampo/citologia , Humanos , Neurônios/citologia , Hipófise/citologiaRESUMO
BACKGROUND: Fahr's disease is a rare neurodegenerative disorder characterized by diffuse intracranial calcium deposition and associated cell loss mainly in bilateral basal ganglia and dentate nuclei of the cerebellum. Subarachnoid hemorrhage and epileptic syncope had been reported as acute presentation of Fahr's disease. We here report a 36-year-old male Indonesian diagnosed as Fahr's disease presenting with young-onset ischemic stroke. CASE PRESENTATION: A 36-year-old male Indonesian without prior systemic disease or neurologic disorder presented with young-onset ischemic stroke involving the right posterior limb of internal capsule. Brain computed tomography and magnetic resonance imaging demonstrated symmetric calcifications in bilateral basal ganglia, internal capsules, cerebellar dentate nuclei, thalami, cerebral white matter, which were all consistent with Fahr's disease. The laboratory studies excluded the presence of other pathologic processes leading to secondary intracranial calcification. Other young stroke surveys were unremarkable. After medical treatment and sustained physical rehabilitation for 3 months, he recovered to carry out daily activities independently. CONCLUSION: We present ischemic stroke in a young patient with sporadic Fahr's disease. The differentiation between Fahr's disease and Fahr's syndrome is specially highlighted when brain CT exhibits diffuse, symmetric calcifications in bilateral basal ganglia, thalami, cerebellar dentate nuclei and cerebral white matter. The association between nonarteriosclerotic vascular calcification and cerebrovascular disease is worth special attention and further investigation.
Assuntos
Doenças dos Gânglios da Base/complicações , Calcinose/complicações , Doenças Neurodegenerativas/complicações , Acidente Vascular Cerebral/etiologia , Adulto , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Síncope/etiologiaRESUMO
Increased understanding of the precise molecular mechanisms involved in cell survival and cell death signaling pathways offers the promise of harnessing these molecules to eliminate cancer cells without damaging normal cells. Tyrosine kinase oncoproteins promote the genesis of leukemias through both increased cell proliferation and inhibition of apoptotic cell death. Although tyrosine kinase inhibitors, such as the BCR-ABL inhibitor imatinib, have demonstrated remarkable efficacy in the clinic, drug-resistant leukemias emerge in some patients because of either the acquisition of point mutations or amplification of the tyrosine kinase, resulting in a poor long-term prognosis. Here, we exploit the molecular mechanisms of caspase activation and tyrosine kinase/adaptor protein signaling to forge a unique approach for selectively killing leukemic cells through the forcible induction of apoptosis. We have engineered caspase variants that can directly be activated in response to BCR-ABL. Because we harness, rather than inhibit, the activity of leukemogenic kinases to kill transformed cells, this approach selectively eliminates leukemic cells regardless of drug-resistant mutations.
Assuntos
Caspases/genética , Caspases/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzamidas , Caspase 8/genética , Caspase 8/metabolismo , Caspase 8/uso terapêutico , Caspases/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática , Variação Genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Mesilato de Imatinib , Células K562 , Leucemia/patologia , Camundongos , Piperazinas/farmacologia , Engenharia de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapêutico , Transdução GenéticaRESUMO
High levels of metabolic activity confer resistance to apoptosis. Caspase-2, an apoptotic initiator, can be suppressed by high levels of nutrient flux through the pentose phosphate pathway. This metabolic control is exerted via inhibitory phosphorylation of the caspase-2 prodomain by activated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). We show here that this activation of CaMKII depends, in part, on dephosphorylation of CaMKII at novel sites (Thr(393)/Ser(395)) and that this is mediated by metabolic activation of protein phosphatase 2A in complex with the B55ß targeting subunit. This represents a novel locus of CaMKII control and also provides a mechanism contributing to metabolic control of apoptosis. These findings may have implications for metabolic control of the many CaMKII-controlled and protein phosphatase 2A-regulated physiological processes, because both enzymes appear to be responsive to alterations in glucose metabolized via the pentose phosphate pathway.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Caspase 2/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Ativação Enzimática , Glucose-6-Fosfato/fisiologia , Células HEK293 , Humanos , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Xenopus laevisRESUMO
Apoptosis ensures tissue homeostasis in response to developmental cues or cellular damage. Recently reported genome-wide RNAi screens have suggested that several metabolic regulators can modulate caspase activation in Drosophila. Here, we establish a previously unrecognized link between metabolism and Drosophila apoptosis by showing that cellular NADPH levels modulate the initiator caspase Dronc through its phosphorylation at S130. Depletion of NADPH removed this inhibitory phosphorylation, resulting in the activation of Dronc and subsequent cell death. Conversely, upregulation of NADPH prevented Dronc-mediated apoptosis upon DIAP1 RNAi or cycloheximide treatment. Furthermore, this CaMKII-mediated phosphorylation of Dronc hindered Dronc activation, but not its catalytic activity. Blockade of NADPH production aggravated the death-inducing activity of Dronc in specific neurons, but not in the photoreceptor cells of the eyes of transgenic flies; similarly, non-phosphorylatable Dronc was more potent than wild type in triggering specific neuronal apoptosis. Our observations reveal a novel regulatory circuitry in Drosophila apoptosis, and, as NADPH levels are elevated in cancer cells, also provide a genetic model to understand aberrations in cancer cell apoptosis resulting from metabolic alterations.
Assuntos
Apoptose , Caspases/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Neurônios/metabolismo , Animais , Animais Geneticamente Modificados , Western Blotting , Sobrevivência Celular , Células Cultivadas , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Ativação Enzimática , Imunoprecipitação , Malatos/metabolismo , NADP/metabolismo , Neurônios/citologia , RNA Interferente Pequeno/farmacologiaRESUMO
Hyperventilation is a traditional seizure-provoking procedure used mainly in idiopathic generalized epilepsy and with a relatively limited role in partial epilepsy. Ictal fear is a rare seizure semiology seen in temporal lobe epilepsy. It has been suggested that the amygdala and anterior hippocampus are involved in generating ictal fear. We describe a rare patient with nonlesional temporal epilepsy who, while hyperventilating during an electroencephalography recording, developed complex partial seizures presenting as ictal fear. The particular sensitivity of the anterior hippocampus (probably the amygdala) to hypocapnia might be an important factor contributing to seizures. To avoid misdiagnosing this unusual condition as a pseudo-seizure, a detailed history and seizure semiology, as well as a concurrent electroencephalography recording, are mandatory.
Assuntos
Epilepsia do Lobo Temporal/psicologia , Medo , Adulto , Eletroencefalografia , Humanos , MasculinoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Temporal lobe epilepsy remains one of the most drug-resistant focal epilepsy, leading to enormous healthcare burden. Among traditional herb medicine, some ingredients have the potential to treat seizure and alleviate the neuronal excitoxicity. The dried rhizome of Gastrodia elata Blume has been used to treat convulsive disorder, dizziness, dementia and migraine in eastern Asia. AIM OF THE STUDY: To determine whether gastrodin, an active ingredient of Gastrodia elata Blume, can reduce lithium-pilocarpine induced seizure severity and neuronal excitotoxicity and explore the underlying mechanism. MATERIALS AND METHODS: We divided the Sprague-Dawley rats into an experimental group (gastrodin group) and a control group (Dimethyl sulfoxide, vehicle group) and performed the behavioral analysis and electroencephalography to determine the effect of gastrodin on the seizure severity induced by lithium-pilocarpine injection. Nissl-stained histopathology elucidated the degree of rat hippocampal neuronal damage as markers of acute and subacute neuronal excitotoxicity. Besides, the Western blotting of dissected hippocampus was carried out to demonstrate the protein expression involving GABAergic transmission and metabolic pathway. RESULTS: Gastrodin reduced the acute seizure severity in lithium-pilocarpine-induced seizure model. In electroencephalography recording, gastrodin exerted inhibitory action on epileptiform discharge. Compared with control group, gastrodin exhibited neuroprotective effect against seizure related hippocampal neuronal damage at acute and subacute stages. The Western blotting showed that gastrodin reversed the degradation of GABAA receptor after pilocarpine-induced seizures. CONCLUSIONS: In the experimental seizure model, gastrodin showed anti-seizure and neuroprotective abilities. Enhancing the expression of GABAA receptor plays an important role in its antiepileptic mechanism. The results offer a new insight of developing new antiepileptic drugs from traditional Chinese medicine.
Assuntos
Anticonvulsivantes/farmacologia , Álcoois Benzílicos/farmacologia , Epilepsia do Lobo Temporal/tratamento farmacológico , Glucosídeos/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Convulsões/tratamento farmacológico , Animais , Anticonvulsivantes/uso terapêutico , Álcoois Benzílicos/uso terapêutico , Modelos Animais de Doenças , Eletroencefalografia/efeitos dos fármacos , Epilepsia do Lobo Temporal/induzido quimicamente , Gastrodia/química , Glucosídeos/uso terapêutico , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Lítio/toxicidade , Masculino , Medicina Tradicional Chinesa , Fármacos Neuroprotetores/uso terapêutico , Pilocarpina/toxicidade , Ratos Sprague-Dawley , Receptores de GABA-A/metabolismo , Rizoma/química , Convulsões/induzido quimicamente , Estado Epiléptico/prevenção & controleRESUMO
How sodium metabisulfite (SMB; Na2S2O5), a popular food preservative and antioxidant, interacts with excitable membrane and induces excitotoxicity is incompletely understood. In this study, the patch-clamp technique was used to investigate and record the electrophysiological effect of SMB on electrically excitable HL-1 cardiomyocytes and NSC-34 neurons, as well as its relationship to pilocarpine-induced seizures and neuronal excitotoxicity in rats. We used Western blotting, to analyze sodium channel expression on hippocampi after chronic SMB treatment. It was found that voltage-gated Na+ current (I Na) was stimulated, and current inactivation and deactivation were slowed in SMB-treated (30 µM) HL-1 cardiomyocytes. SMB-induced increases of I Na were attenuated in cells treated with ranolazine (10 µM) or eugenol (30 µM). The current-voltage relationship of I Na shifted to slightly more negative potentials in SMB-treated cells, the peak I Na with an EC50 value of 18 µM increased, and the steady-state inactivation curve of I Na shifted to a more positive potential. However, the tail component of the rapidly activating delayed-rectifier K+ current (I Kr) was dose-dependently inhibited. Cell-attached voltage-clamp recordings in SMB-treated cells showed that the frequency of action currents and prolonged action potential were higher. In SMB-treated NSC-34 neurons, the peak I Na was higher; however, neither the time to peak nor the inactivation time constant (I Na) changed. Pilocarpine-induced seizures were exacerbated, and acute neuronal damage and chronic mossy fiber sprouting increased in SMB-treated rats. Western blotting showed higher expression of the sodium channel in cells after chronic SMB treatment. We conclude that SMB contributes to the sodium channel-activating mechanism through which it alters cellular excitability and excitotoxicity in wide-spectrum excitable cells.
Assuntos
Broncoconstritores/farmacologia , Canais Iônicos/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Convulsões/tratamento farmacológico , Sulfitos/farmacologia , Alopecia/induzido quimicamente , Animais , Biofísica , Peso Corporal/efeitos dos fármacos , Broncoconstritores/uso terapêutico , Linhagem Celular Transformada , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Estimulação Elétrica , Expressão Gênica/efeitos dos fármacos , Canais Iônicos/fisiologia , Masculino , Camundongos , Agonistas Muscarínicos/toxicidade , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Pilocarpina/toxicidade , Ratos , Ratos Sprague-Dawley , Convulsões/induzido quimicamente , Convulsões/patologia , Pele/efeitos dos fármacos , Pele/patologia , Sulfitos/uso terapêuticoRESUMO
RNA editing is a molecular event that alters specific nucleotides in RNA post-transcriptionally. RNA editing has the potential to impact a variety of cellular processes and is implicated in diseases such as cancer. Yet, the precise mechanisms by which RNA editing controls cellular processes are poorly understood. Here, we characterize sequences altered by RNA editing in patient samples from lymphoma, neuroblastoma and head and neck cancers. We show that A-to-I RNA editing sites are highly conserved across samples of the same tissue type and that most editing sites identified in tumors are also detectable in normal tissues. Next, we identify the significant changes in editing levels of known sites between tumor and paired "normal" tissues across 14 cancer types (627 pairs) from The Cancer Genome Atlas project and show that the complexity of RNA editing regulation cannot be captured by the activity of ADAR family genes alone. Our pan-cancer analysis confirms previous results on individual tumor types and suggests that changes of RNA editing levels in coding and 3'UTR regions could be a general mechanism to promote tumor growth. We also propose a model explaining how altered RNA editing levels affect microRNA-mediated post-transcriptional regulation of oncogenes and tumor-suppressors.
Assuntos
Regiões 3' não Traduzidas , Regulação Neoplásica da Expressão Gênica , MicroRNAs/fisiologia , Edição de RNA , Proteínas Supressoras de Tumor/genética , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Oncogenes , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Interferência de RNA , Análise de Sequência de RNA , Proteínas Supressoras de Tumor/metabolismoRESUMO
Proapoptotic Bcl-2 family members, such as Bax, promote release of cytochrome c from mitochondria, leading to caspase activation and cell death. It was previously reported that modulator of apoptosis protein 1 (MOAP-1), an enhancer of Bax activation induced by DNA damage, is stabilized by Trim39, a protein of unknown function. In this paper, we show that MOAP-1 is a novel substrate of the anaphase-promoting complex (APC/C(Cdh1)) ubiquitin ligase. The influence of Trim39 on MOAP-1 levels stems from the ability of Trim39 (a RING domain E3 ligase) to directly inhibit APC/C(Cdh1)-mediated protein ubiquitylation. Accordingly, small interfering ribonucleic acid-mediated knockdown of Cdh1 stabilized MOAP-1, thereby enhancing etoposide-induced Bax activation and apoptosis. These data identify Trim39 as a novel APC/C regulator and provide an unexpected link between the APC/C and apoptotic regulation via MOAP-1.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína da Polipose Adenomatosa do Colo/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Caderinas/metabolismo , Proteínas de Transporte/metabolismo , Ubiquitina/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína da Polipose Adenomatosa do Colo/genética , Antígenos CD , Apoptose , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Western Blotting , Caderinas/antagonistas & inibidores , Caderinas/genética , Proteínas de Transporte/genética , Dano ao DNA , Citometria de Fluxo , Fase G1/fisiologia , Células HeLa , Humanos , Imunoprecipitação , RNA Interferente Pequeno/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitina-Proteína Ligases , UbiquitinaçãoRESUMO
Bostrycin, a red antibacterial agent with tetrahydroanthraquinone structure, has been isolated from Nigrospora sp. No. 407. This study investigated the potential antibacterial and multifunctional properties of matrixes through immobilization of bostrycin on their surface for immobilization of protein and prevention of bacterial growth. Bostrycin was immobilized on nonwoven polypropylene (PP) fabric by a technique using glutaraldehyde and polyethyleneimine for the activation of the surface. Glucose oxidase immobilized on bostrycin-treated nonwoven PP fabric showed high activity. The immobilization process improved thermal stability of the enzymes. During repeated assay for 30 cycles, the enzyme activity dropped to only 70% of the initial activity. Both bostrycin-treated nonwoven PP fabric sample and subsequently immobilized glucose oxidase sample on the surface also still exhibited a bacteriostatic effect. This is the first study to show that bostrycin is a promising coupling agent for surface modification on matrix and its potential applications in protein immobilization and biomaterial-centered infection.
Assuntos
Antraquinonas/química , Antibacterianos/farmacologia , Ascomicetos/enzimologia , Enzimas Imobilizadas/química , Glucose Oxidase/química , Têxteis , Antraquinonas/metabolismo , Antraquinonas/farmacologia , Antibacterianos/biossíntese , Antibacterianos/química , Biotecnologia/métodos , Clostridium botulinum/efeitos dos fármacos , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Glucose Oxidase/metabolismo , Testes de Sensibilidade Microbiana , Polipropilenos/química , Staphylococcus aureus/efeitos dos fármacos , Propriedades de SuperfícieRESUMO
Homeostatic maintenance of cellular mitochondria requires a dynamic balance between fission and fusion, and controlled changes in morphology are important for processes such as apoptosis and cellular division. Interphase mitochondria have been described as an interconnected network that fragments as cells enter mitosis, and this mitotic mitochondrial fragmentation is known to be regulated by the dynamin-related GTPase Drp1 (dynamin-related protein 1), a key component of the mitochondrial division machinery. Loss of Drp1 function and the subsequent failure of mitochondrial division during mitosis lead to incomplete cytokinesis and the unequal distribution of mitochondria into daughter cells. During mitotic exit and interphase, the mitochondrial network reforms. Here we demonstrate that changes in mitochondrial dynamics as cells exit mitosis are driven in part through ubiquitylation of Drp1, catalyzed by the APC/C(Cdh1) (anaphase-promoting complex/cyclosome and its coactivator Cdh1) E3 ubiquitin ligase complex. Importantly, inhibition of Cdh1-mediated Drp1 ubiquitylation and proteasomal degradation during interphase prevents the normal G1 phase regrowth of mitochondrial networks following cell division.
Assuntos
Caderinas/metabolismo , Citocinese , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Mitose , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Antígenos CD , Caderinas/antagonistas & inibidores , Caderinas/genética , Dinaminas , Estabilidade Enzimática , Fase G1/genética , GTP Fosfo-Hidrolases/deficiência , GTP Fosfo-Hidrolases/genética , Expressão Gênica , Inativação Gênica , Células HEK293 , Células HeLa , Humanos , Interfase/genética , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Mitocôndrias/genética , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Interferente Pequeno/metabolismo , Transfecção , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidores , Complexos Ubiquitina-Proteína Ligase/genética , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Vertebrate eggs are arrested at Metaphase II by Emi2, the meiotic anaphase-promoting complex/cyclosome (APC/C) inhibitor. Although the importance of Emi2 during oocyte maturation has been widely recognized and its regulation extensively studied, its mechanism of action remained elusive. Many APC/C inhibitors have been reported to act as pseudosubstrates, inhibiting the APC/C by preventing substrate binding. Here we show that a previously identified zinc-binding region is critical for the function of Emi2, whereas the D-box is largely dispensable. We further demonstrate that instead of acting through a "pseudosubstrate" mechanism as previously hypothesized, Emi2 can inhibit Cdc20-dependent activation of the APC/C substoichiometrically, blocking ubiquitin transfer from the ubiquitin-charged E2 to the substrate. These findings provide a novel mechanism of APC/C inhibition wherein the final step of ubiquitin transfer is targeted and raise the interesting possibility that APC/C is inhibited by Emi2 in a catalytic manner.
Assuntos
Proteínas F-Box/química , Proteínas F-Box/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Motivos de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Animais , Biocatálise , Ativação Enzimática , Humanos , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidoresRESUMO
Loss of cell division cycle 2 (Cdc2, also known as Cdk1) activity after cyclin B degradation is necessary, but not sufficient, for mitotic exit. Proteins phosphorylated by Cdc2 and downstream mitotic kinases must be dephosphorylated. We report here that protein phosphatase-1 (PP1) is the main catalyst of mitotic phosphoprotein dephosphorylation. Suppression of PP1 during early mitosis is maintained through dual inhibition by Cdc2 phosphorylation and the binding of inhibitor-1. Protein kinase A (PKA) phosphorylates inhibitor-1, mediating binding to PP1. As Cdc2 levels drop after cyclin B degradation, auto-dephosphorylation of PP1 at its Cdc2 phosphorylation site (Thr 320) allows partial PP1 activation. This promotes PP1-regulated dephosphorylation at the activating site of inhibitor-1 (Thr 35) followed by dissociation of the inhibitor-1-PP1 complex and then full PP1 activation to promote mitotic exit. Thus, Cdc2 both phosphorylates multiple mitotic substrates and inhibits their PP1-mediated dephosphorylation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Mitose/fisiologia , Fosfoproteínas/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Proteína Quinase CDC2 , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ciclina B/metabolismo , Ciclina B/farmacologia , Quinases Ciclina-Dependentes , Células HeLa , Humanos , Modelos Biológicos , Ácido Okadáico/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Fosforilação , Ligação Proteica/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Proteína Fosfatase 1/antagonistas & inibidores , Proteína Fosfatase 1/farmacologia , Proteínas/farmacologia , Purinas/farmacologia , Roscovitina , Treonina/metabolismo , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
The GCM proteins GCMa/1 and GCMb/2 are novel zinc-containing transcription factors critical for glial cell differentiation in fly and for placental as well as parathyroid gland development in mouse. Previous pulse-chase experiments have demonstrated differential protein stabilities of GCM proteins with half-lives from approximately 30 min to 2 h (Tuerk, E. E., Schreiber, J., and Wegner, M. (2000) J. Biol. Chem. 275, 4774-4782). However, little is known about the machinery that controls GCM protein degradation. Here, we report the identification of an SCF complex as the GCM ubiquitin-protein isopeptide ligase (E3) that regulates human GCMa (hGCMa) degradation. We found that SKP1 and CUL1, two key components of the SCF complex, associate with hGCMa in vivo. We further identify the human F-box protein FBW2 (hFBW2) as the substrate recognition subunit in the SCF E3 complex for hGCMa. We show that hFBW2 interacts with hGCMa in a phosphorylation-dependent manner and promotes hGCMa ubiquitination. Supporting a critical role for hFBW2 in hGCMa degradation, knockdown of hFBW2 expression by RNA interference leads to a reduction in hGCMa ubiquitination and a concomitant increase in hGCMa protein stability. Our study identifies the SCF(hFBW2) E3 complex as the key machinery that targets hGCMa to the ubiquitin-proteasome degradation system.