RESUMO
Proper subcellular localization is crucial for the functioning of biomacromolecules, including proteins and RNAs. Nuclear transport is a fundamental cellular process that regulates the localization of many macromolecules within the nuclear or cytoplasmic compartments. In humans, approximately 60 proteins are involved in nuclear transport, including nucleoporins that form membrane-embedded nuclear pore complexes, karyopherins that transport cargoes through these complexes, and Ran system proteins that ensure directed and rapid transport. Many of these nuclear transport proteins play additional and essential roles in mitosis, biomolecular condensation, and gene transcription. Dysregulation of nuclear transport is linked to major human diseases such as cancer, neurodegenerative diseases, and viral infections. Selinexor (KPT-330), an inhibitor targeting the nuclear export factor XPO1 (also known as CRM1), was approved in 2019 to treat two types of blood cancers, and dozens of clinical trials of are ongoing. This review summarizes approximately three decades of research data in this field but focuses on the structure and function of individual nuclear transport proteins from recent studies, providing a cutting-edge and holistic view on the role of nuclear transport proteins in health and disease. In-depth knowledge of this rapidly evolving field has the potential to bring new insights into fundamental biology, pathogenic mechanisms, and therapeutic approaches.
Assuntos
Neoplasias , Receptores Citoplasmáticos e Nucleares , Humanos , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/uso terapêutico , Transporte Ativo do Núcleo Celular/genética , Carioferinas/genética , Carioferinas/metabolismo , Carioferinas/uso terapêutico , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Neoplasias/metabolismo , Proteína ran de Ligação ao GTPRESUMO
INTRODUCTION: Triple A (Allgrove) syndrome is a rare autosomal recessive disorder characterized by cardinal features of primary adrenal insufficiency (AI) due to adrenocorticotropic hormone (ACTH) resistance, achalasia, and alacrima. It is frequently associated with neurological manifestations such as autonomic dysfunction, cognitive dysfunction, cranial nerve, or motor involvement. Amyotrophy/motor neuron disease is a rare association. CASE PRESENTATION: We herein report a 19-year-old boy diagnosed with triple A syndrome (TAS), with the classic triad of ACTH-resistant adrenal insufficiency, achalasia, and alacrima. Additionally, he had distal spinal muscle amyotrophy. Alacrima was the earliest feature evident in early childhood, followed by achalasia at 12 years of age. He was diagnosed with AI at the age of 19 years, with involvement of the mineralocorticoid axis. Further evaluation showed a neurogenic pattern on electromyography, consistent with a diagnosis of motor neuron disease. A nerve conduction study revealed no significant neuropathy. Genetic analysis confirmed a pathogenic homozygous mutation in the AAAS gene c.43C>A, p.Gln15Lys. He improved with glucocorticoid and mineralocorticoid supplements for AI, and nifedipine for achalasia and artificial tears. He is planned for esophagomyotomy. CONCLUSION: In any young patient with AI not due to congenital adrenal hyperplasia, Allgrove syndrome should be ruled out. Though mineralocorticoid sparing pattern is classical, it can rarely be involved, as seen in the index case. Various components of the syndrome, as well as amyotrophy and other neurologic features, may present in a metachronous fashion. Hence, a high index of clinical suspicion can aid in early diagnosis and management.
Assuntos
Insuficiência Adrenal/complicações , Insuficiência Adrenal/genética , Acalasia Esofágica/complicações , Acalasia Esofágica/genética , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Corticosteroides/uso terapêutico , Insuficiência Adrenal/tratamento farmacológico , Bloqueadores dos Canais de Cálcio/uso terapêutico , Acalasia Esofágica/tratamento farmacológico , Humanos , Lubrificantes Oftálmicos , Masculino , Mutação , Nifedipino/uso terapêutico , Adulto JovemRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: As one of the most common female malignant tumors mainly infected by human papillomavirus (HPV) worldwide, cervical cancer is widely distributed in about 90% developing countries. An in-hospital preparation derived from a traditional Chinese herbal formula, Youdujing (YDJ), has been developed and clinically used for more than 20 years in our hospital for treating multiple diseases caused by HPV infection, such as cervical precancerous lesions, recurrent condyloma acuminata, fla t warts, etc. However, few investigations on the effect and mechanism of YDJ extract on treating and preventing HPV infection induced cervical cancer have been reported. AIM OF THE STUDY: Previous reports showed that YDJ extract is effective in triggering human cervical cancer cells (ectocervical Ect1/E6E7) death in a necrotic manner. Herein, we aim to investigate the anti-proliferation effects and potential mechanisms of YDJ extract in inducing necroptosis in ectocervical Ect1/E6E7 cells. MATERIALS AND METHODS: The high-performance liquid chromatography (HPLC) fingerprint method was firstly used for better quality control of the chemical components in YDJ extract. MTT assay and flow cytometer were applied for evaluating cytotoxicity and necroptosis induced by YDJ extract in ectocervical Ect1/E6E7 cells. Besides, Western blotting, receptor-interacting protein serine-threonine kinase 1 (RIP1) inhibitor (necrostatin-1), and RIP1 shRNA and pCDNA transfection assays were employed for investigation on the underlying mechanisms and validation the role of RIP1 in YDJ extract induced necroptosis. RESULTS: YDJ extract induced necroptosis in ectocervical Ect1/E6E7 cells both in time- and concentration-dependent manners, without affecting activation of caspases and elevation of intracellular reactive oxygen species (ROS) level. Moreover, a selective increasing in RIP1expression was observed in YDJ extract treated ectocervical Ect1/E6E7 cells. The induction effect of necroptosis by YDJ extract was partially blocked by the addition of RIP1 inhibitor (necrostatin-1). Co-immunoprecipitation assay demonstrated that the treatment of YDJ extract in ectocervical Ect1/E6E7 cells promoted the combination of RIP1 with RIP3 and MLKL to form necrosome, which facilitates the process of necroptosis. CONCLUSIONS: Taken together, YDJ preparation displays an effective ability of inducing necroptosis in cervical cancer cells through activation of RIP1 kinase. However, although the treatment efficacy and potential mechanisms of YDJ extract in vivo remain unclear and need further investigation, it is believed that YDJ extract has the great potential to be used as a starting point to develop more potent agent for treating or preventing cervical cancer and other proliferative diseases.
Assuntos
Apoptose/efeitos dos fármacos , Colo do Útero/citologia , Medicamentos de Ervas Chinesas/farmacologia , Necrose/induzido quimicamente , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
KEY MESSAGE: Loss-of-function of nucleoporin NUP1 in Arabidopsis causes defect in both male and female gametogenesis. Its ovules are arrested during meiosis, and its pollen grains are aborted at mitosis I. Nuclear pore complex (NPC) plays crucial roles in nucleocytoplasmic trafficking of proteins and RNAs. The NPC contains approximately 30 different proteins termed nucleoporins (NUPs). So far, only a few of plant NUPs have been characterized. The Arabidopsis NUP1 was identified as an ortholog of the yeast NUP1 and animal NUP153. Loss-of-function of NUP1 in Arabidopsis caused fertility defect; however, the molecular mechanism of this defect remains unknown. Here, we found that both male and female gametogenesis of the nup1 mutants were defective. nup1 ovules were arrested from the meiosis stage onward; only approximately 6.7% and 3% ovules of the nup1-1 and nup1-4 mutants developed up to the FG7 stage, respectively. Pollen development of the nup1 mutants was arrested during the first mitotic division. In addition, enlarged pollen grains with increased DNA content were observed in the nup1 mutant. RNA-sequencing showed that expression levels of genes involved in pollen development or regulation of cell size were reduced dramatically in nup1 compared with wild type. These results suggest that NUP1 plays an important role in gametogenesis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Pólen/metabolismo , Pólen/fisiologia , Proteínas de Arabidopsis/genética , Gametogênese/genética , Gametogênese/fisiologia , Poro Nuclear/genética , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismoRESUMO
RIP1 and RIP3 are necroptosis initiators, but their roles in regulation of glycolysis remain elusive. In this study, we found shikonin activated RIP1 and RIP3 in glioma cells in vitro and in vivo, which was accompanied with glycolysis suppression. Further investigation revealed that shikonin-induced decreases of glucose-6-phosphate and pyruvate and downregulation of HK II and PKM2 were significantly prevented when RIP1 or RIP3 was pharmacologically inhibited or genetically knocked down with SiRNA. Moreover, shikonin also triggered accumulation of intracellular H2O2 and depletion of GSH and cysteine. Mitigation of intracellular H2O2 via supplement of GSH reversed shikonin-induced glycolysis suppression. The role of intracellular H2O2 in regulation of glycolysis suppression was further confirmed in the cells treated with exogenous H2O2. Notably, inhibition of RIP1 or RIP3 prevented intracellular H2O2 accumulation, which was correlated with preventing shikonin-induced downregulation of x-CT and depletion of GSH and cysteine. In addition, supplement of pyruvate effectively inhibited shikonin- or exogenous H2O2-induced accumulation of intracellular H2O2 and glioma cell death. Taken together, we demonstrated in this study that RIP1 and RIP3 contributed to shikonin-induced glycolysis suppression via increasing intracellular H2O2.
Assuntos
Glioma/tratamento farmacológico , Glicólise/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Naftoquinonas/administração & dosagem , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Linhagem Celular Tumoral , Cisteína/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/metabolismo , Glutationa/metabolismo , Humanos , Camundongos , Naftoquinonas/farmacologia , Ratos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Treatments targeting hormone receptors typically fail to provide a positive clinical outcome against triple-negative breast cancers (TNBC), which lack expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2/neu). Towards identifying viable treatments for aggressive breast cancer, we have tested an extract of the tropical plant Lippia origanoides (LOE) on TNBC and normal cells lines to uncover its potential anticancer effects. Treatment with LOE reduced TNBC cell viability in a dose-dependent manner to a greater extent than in normal mammary epithelial MCF10A cells. In MDA-MB231 cells, LOE was found to halt the cell cycle in the G0/G1 phase via cyclin D1 and cIAP2 regulation, and induce apoptosis without promoting necrosis via caspase-8/-3 and PARP cleavage. Constitutive nuclear factor-κB (NF-κB) signaling has been shown to contribute to the heightened inflammatory state and survival in TNBC cells. Herein, we also provide evidence that LOE inhibits NF-κB signaling by reducing RIP1 protein levels in MDA-MB-231 cells. These studies reveal that LOE suppresses key features of the progression of aggressive breast cancer cells and provides a basis for further definition of its underlying mechanisms of action and anticancer potential.
Assuntos
Lippia/química , NF-kappa B/metabolismo , Extratos Vegetais/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , NF-kappa B/antagonistas & inibidores , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
BACKGROUND/AIMS: Traumatic brain injury (TBI) is a major public health problem in the world and causes high rates of mortality and disability. Recent evidence suggests that vitamin D (VD) has neuroprotective actions and can promote function recovery after TBI. In vitro and in vivo studies have demonstrated that autophagy could be enhanced following supplementation with an active metabolite of VD (calcitriol). However, it is unclear whether autophagy participates in the protective effects of calcitriol after TBI. To test this hypothesis, we examined the protective effects of calcitriol on TBI-induced neurological impairment and further investigated whether calcitriol could modulate autophagy dysfunction-mediated cell death in the cortex region of rat brain. METHODS: Eighty-five male rats (250-280 g) were randomly assigned to sham (n=15), TBI model (TBI, n=35) and calcitriol treatment (calcitriol, n=35) groups. Rats were injected intraperitoneally with calcitriol (1 µg/kg) at 30 min, 24 h and 48 h post-TBI in the calcitriol group. The lysosomal inhibitor, chloroquine (CQ), was used to evaluate autophagic flux in the TBI and calcitriol groups. Neurological functions were evaluated via the modified neurological severity score test at 1-7 days after TBI or sham operation, and the terminal deoxynucleotidyl transferase-mediated FITC-dUTP nick-end labeling method was used to evaluate the ability of calcitriol to inhibit apoptosis. The expression of VDR, LC3 and p62 proteins was measured by western blot analysis at 1, 3 and 7 days post-injury Results: Calcitriol treatment attenuated mNSS at 2-7 days post-TBI (P < 0.05 versus TBI group). Calcitriol dramatically increased VDR protein expression compared with the untreated counterparts at 1, 3 and 7 days post-TBI (P < 0.05). The rate of apoptotic cells in calcitriol-treated rats was significantly reduced compared to that observed in the TBI group (P < 0.05). The LC3II/LC3I ratio was decreased in the cortex region at 1, 3 and 7 days post-TBI in rats treated with calcitriol (p < 0.05 versus TBI group), and the p62 expression was also attenuated (p < 0.05 versus TBI group). The LC3II/LC3I ratio in the calcitriol group was significantly increased when pretreated with CQ (P < 0.05). CONCLUSION: Calcitriol treatment activated VDR protein expression and attenuated neurological deficits in this rat TBI model. The protective effects might be associated with the restoration of autophagy flux and the decrease in apoptosis in the cortex region of rat brain.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Lesões Encefálicas Traumáticas/patologia , Calcitriol/farmacologia , Receptores de Calcitriol/metabolismo , Animais , Lesões Encefálicas Traumáticas/metabolismo , Cloroquina/toxicidade , Modelos Animais de Doenças , Injeções Intraperitoneais , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fármacos Neuroprotetores/farmacologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Ratos , Receptores de Calcitriol/agonistasRESUMO
PP2A-B55 is one of the major phosphatases regulating cell division. Despite its importance for temporal control during mitotic exit, how B55 substrates are recognized and differentially dephosphorylated is unclear. Using phosphoproteomics combined with kinetic modeling to extract B55-dependent rate constants, we have systematically identified B55 substrates and assigned their temporal order in mitotic exit. These substrates share a bipartite polybasic recognition determinant (BPR) flanking a Cdk1 phosphorylation site. Experiments and modeling show that dephosphorylation rate is encoded into B55 substrates, including its inhibitor ENSA, by cooperative action of basic residues within the BPR. A complementary acidic surface on B55 decodes this signal, supporting a cooperative electrostatic mechanism for substrate selection. A further level of specificity is encoded into B55 substrates because B55 displays selectivity for phosphothreonine. These simple biochemical properties, combined with feedback control of B55 activity by the phosphoserine-containing substrate/inhibitor ENSA, can help explain the temporal sequence of events during exit from mitosis.
Assuntos
Mitose , Proteína Fosfatase 2/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Aminoácidos/metabolismo , Anáfase/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Cinética , Mitose/efeitos dos fármacos , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2/química , Subunidades Proteicas/metabolismo , Eletricidade Estática , Especificidade por Substrato/efeitos dos fármacos , Fatores de TempoRESUMO
Honokiol (HNK) is a pharmacologically active small molecule that is isolated from the traditional Chinese medicinal herb, houpu. It may induce diversified types of regulated cell death, which are dependent on different cell types and varying concentrations of therapeutic agent. We previously reported that HNK triggers a cyclophilin D (CypD)-mediated regulated necrosis in various cell lines at certain concentrations (twofold higher than its half maximal inhibitory concentration). Subsequent study revealed that HNK induced cell death transition from early apoptosis to regulated necrosis in parallel with the increase of HNK dose. In the current study, a lower concentration of HNK (30 µg/ml) than previously reported also induced simplex CypDmediated mitochondrial permeability transition (MPT)associated regulated necrosis in the HEK293 human embryonic kidney cell line. HNK, at concentration of 30 µg/ml, induced necrotic cell death in HEK293 cells, which was demonstrated by positive staining for propidium iodide. No DNA ladder patterns or apoptotic bodies were detected in cells that underwent this type of necrotic cell death. Caspase8 and 3 were not activated during the process of HNKinduced necrosis. In addition, pancaspase inhibitor, zVADfmk and receptorinteracting protein 1 inhibitor, necrostatin1 did not inhibit HNKinduced necrosis. However, CypD inhibitor, cyclosporin A (CsA), blocked HNKinduced necrosis. These findings indicate that 30 µg/ml HNK induced simplex CypD-mediated MPTassociated regulated necrosis in HEK293 cells. Furthermore, the findings demonstrated that during HNK-triggered regulated necrosis the mammalian target of rapamycin (mTOR) signaling pathway is also inhibited. Pretreatment with CsA, therefore, inhibits HNKtriggered regulated necrosis and reverses dephosphorylation of Akt, eIF4Ebinding protein 1 and S6 kinase. This indicated that the mTOR signaling pathway is effective downstream of the CypDmediated MPT and before the onset of plasma membrane breakdown during the regulated necrosis process. Therefore, it has been demonstrated for the first time, to the best of our knowledge, that the mTOR signaling pathway was inhibited downstream of the CypD-mediated MPT in the process of HNK-induced regulated necrosis.
Assuntos
Apoptose/efeitos dos fármacos , Compostos de Bifenilo/toxicidade , Ciclofilinas/farmacologia , Lignanas/toxicidade , Mitocôndrias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Western Blotting , Caspase 3/metabolismo , Caspase 8/metabolismo , Peptidil-Prolil Isomerase F , Ciclosporina/farmacologia , Células HEK293 , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Permeabilidade/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismoRESUMO
The BioID method uses a promiscuous biotin ligase to detect protein-protein associations as well as proximate proteins in living cells. Here we report improvements to the BioID method centered on BioID2, a substantially smaller promiscuous biotin ligase. BioID2 enables more-selective targeting of fusion proteins, requires less biotin supplementation, and exhibits enhanced labeling of proximate proteins. Thus BioID2 improves the efficiency of screening for protein-protein associations. We also demonstrate that the biotinylation range of BioID2 can be considerably modulated using flexible linkers, thus enabling application-specific adjustment of the biotin-labeling radius.
Assuntos
Carbono-Nitrogênio Ligases/metabolismo , Proteínas de Escherichia coli/metabolismo , Biologia Molecular/métodos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Animais , Biotina/metabolismo , Biotinilação , Carbono-Nitrogênio Ligases/genética , Proteínas de Escherichia coli/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Células NIH 3T3 , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Engenharia de Proteínas/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas Repressoras/genéticaRESUMO
The nuclear factor-κB (NF-κB) transcription factors control many physiological processes including inflammation, apoptosis, and angiogenesis. In our search for NF-κB inhibitors from natural resources, we identified 4',6-dihydroxy-4-methoxyisoaurone (ISOA) as an inhibitor of NF-κB activation from the seeds of Trichosanthes kirilowii. However, the mechanism by which ISOA inhibits NF-κB activation is not fully understood. In the present study, we demonstrated the effect of ISOA on NF-κB activation in TNF-α-stimulated HeLa cells. This compound suppressed NF-κB activation through the inhibition of IκB kinase (IKK) activation. ISOA also has an influence on upstream signaling of IKK through the inhibition of expression of adaptor proteins, TNF receptor-associated factor 2 (TRAF2) and receptor interacting protein 1 (RIP1). Consequently, ISOA blocked the phosphorylation and degradation of the inhibitor of NF-κB alpha (IκBα), and subsequent phosphorylation and nuclear translocation of p65. The suppression of NF-κB activation by ISOA led to the down-regulation of target genes involved in inflammation, proliferation, as well as potentiation of TNF-α-induced apoptosis. Taken together, this study extends our understanding on the mechanisms underlying the anti-inflammatory and anti-cancer activities of ISOA. Our findings provide new insight into the molecular mechanisms and a potential application of ISOA for inflammatory diseases as well as certain cancers.
Assuntos
Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , NF-kappa B/metabolismo , Sesquiterpenos/farmacologia , Fator de Necrose Tumoral alfa , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células HeLa , Humanos , Quinase I-kappa B/antagonistas & inibidores , Inflamação/tratamento farmacológico , Inflamação/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Fitoterapia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Sementes/química , Sesquiterpenos/isolamento & purificação , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Fator de Transcrição RelA/metabolismo , Trichosanthes/químicaRESUMO
BACKGROUND: Osteosarcoma is the most frequent primary malignant bone tumor, notorious for its lung metastasis. Shikonin, an effective constituent extracted from Chinese medicinal herb, was demonstrated to induce necroptosis in some cancers. METHODS: MTT assay was performed to detect cell survival rate in vitro. Flow cytometry was used to analyze cell cycle and cell death. Western blot was performed to determine the expression levels of RIP1, RIP3, caspase-3, caspase-6 and PARP. The tibial primary and lung metastatic osteosarcoma models were used to evaluate the anti-tumor effect of shikonin in vivo. RESULTS: The cell survival rate was decreased in a dose and time dependent manner when treated with shikonin. No major change in cell cycle was observed after shikonin treatment. The cell death induced by shikonin could be mostly rescued by specific necroptosis inhibitor necrostatin-1, but not by general caspase inhibitor Z-VAD-FMK. The number of necrotic cells caused by shikonin was decreased after being pretreated with Nec-1 detected by flow cytometry in K7 cells. After 8-hour treatment of shikonin, the expression levels of RIP1 and RIP3 were increased while caspase-3, caspase-6 and PARP were not activated in K7 and U2OS cells determined by Western blot. Size of primary tumor and lung metastasis in shikonin treated group were significantly reduced. The protein levels of RIP1 and RIP3 in primary tumor tissues were increased by shikonin. The overall survival of lung metastatic models was longer compared with control group (p < 0.001). CONCLUSIONS: Shikonin had prompt but profound anti-tumor effect on both primary and metastatic osteosarcoma, probably by inducing RIP1 and RIP3 dependent necroptosis. Shikonin would be a potential anti-tumor agent on the treatment of primary and metastatic osteosarcoma.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Naftoquinonas/farmacologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Osteossarcoma/tratamento farmacológico , Proteínas de Ligação a RNA/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Antineoplásicos/uso terapêutico , Apoptose , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Naftoquinonas/uso terapêutico , Necrose , Transplante de Neoplasias , Osteossarcoma/metabolismo , Osteossarcoma/secundário , Regulação para CimaRESUMO
BACKGROUND AND PURPOSE: Shikonin was reported to induce necroptosis in leukemia cells, but apoptosis in glioma cell lines. Thus, it is needed to clarify whether shikonin could cause necroptosis in glioma cells and investigate its underlying mechanisms. METHODS: Shikonin and rat C6 glioma cell line and Human U87 glioma cell line were used in this study. The cellular viability was assayed by MTT. Flow cytometry with annexin V-FITC and PI double staining was used to analyze cellular death modes. Morphological alterations in C6 glioma cells treated with shikoinin were evaluated by electronic transmission microscopy and fluorescence microscopy with Hoechst 33342 and PI double staining. The level of reactive oxygen species was assessed by using redox-sensitive dye DCFH-DA. The expressional level of necroptosis associated protein RIP-1 was analyzed by western blotting. RESULTS: Shikonin induced cell death in C6 and U87 glioma cells in a dose and time dependent manner. The cell death in C6 and U87 glioma cells could be inhibited by necroptosis inhibitor necrotatin-1, not by pan-caspase inhibitor z-VAD-fmk. Shikonin treated C6 glioma cells presented electron-lucent cytoplasm, loss of plasma membrane integrity and intact nuclear membrane in morphology. The increased ROS level caused by shikonin was attenuated by necrostatin-1 and blocking ROS by anti-oxidant NAC rescued shikonin-induced cell death in both C6 and U87 glioma cells. Moreover, the expressional level of RIP-1 was up-regulated by shikonin in a dose and time dependent manner as well, but NAC suppressed RIP-1 expression. CONCLUSIONS: We demonstrated that the cell death caused by shikonin in C6 and U87 glioma cells was mainly via necroptosis. Moreover, not only RIP-1 pathway, but also oxidative stress participated in the activation of shikonin induced necroptosis.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Naftoquinonas/farmacologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Glioma , Humanos , Medicina Tradicional Chinesa , Necrose , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
DEAD-box protein (Dbp) family members are essential for gene expression; however, their precise roles and regulation are not fully defined. During messenger (m)RNA export, Gle1 bound to inositol hexakisphosphate (IP(6)) acts via Dbp5 to facilitate remodeling of mRNA-protein complexes. In contrast, here we define a novel Gle1 role in translation initiation through regulation of a different DEAD-box protein, the initiation factor Ded1. We find that Gle1 physically and genetically interacts with Ded1. Surprisingly, whereas Gle1 stimulates Dbp5, it inhibits Ded1 ATPase activity in vitro, and IP(6) does not affect this inhibition. Functionally, a gle1-4 mutant specifically suppresses initiation defects in a ded1-120 mutant, and ded1 and gle1 mutants have complementary perturbations in AUG start site recognition. Consistent with this role in initiation, Gle1 inhibits translation in vitro in competent extracts. These results indicate that Gle1 has a direct role in initiation and negatively regulates Ded1. Together, the differential regulation of two distinct DEAD-box proteins by a common factor (Gle1) establishes a new paradigm for controlling gene expression and coupling translation with mRNA export.
Assuntos
RNA Helicases DEAD-box/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Iniciação Traducional da Cadeia Peptídica/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico Ativo/fisiologia , RNA Helicases DEAD-box/genética , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Ultrastructural studies of neurofibrillary tangles in Alzheimer disease (AD) have demonstrated a close relationship between nuclear pores and the cytoplasmic paired helical filaments comprising the tangles, as well as nuclear irregularity in many tangle-bearing neurons; nuclear pore aggregation has been observed in nearby neurons. These observations prompted examination of the nuclear pore complex (NPC) and proteins critical to nucleocytoplasmic transport in neurons with and without tangles in AD and control cases. Light microscopic study of hippocampus and neocortex in AD and controls revealed that all nuclei were labeled by antibodies to NPC proteins, including the central transporter nucleoporin Nup62. Nucleoporin and tau label revealed significantly more nuclear irregularity in AD, often associated with neurofibrillary tangles. Double label of Nup62 with apoptotic markers (TUNEL and active caspase-3) and a cell-cycle protein (cyclin B1) revealed no clear relationship of nuclear irregularity to apoptosis or cell-cycle protein expression. However, cytoplasmic accumulation of nuclear transport factor 2 (NTF2), a protein that transports cargo from the cytoplasm into the nucleus, was observed in a subset of hippocampal neurons with and without tangles in AD but not control cases. Further investigation of the NPC and nucleocytoplasmic transport in AD is warranted.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Neurônios/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Doença de Alzheimer/fisiopatologia , Biópsia/métodos , Caspase 3 , Caspases/metabolismo , Contagem de Células/métodos , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Humanos , Imuno-Histoquímica/métodos , Marcação In Situ das Extremidades Cortadas/métodos , Microscopia Eletrônica de Transmissão/métodos , Modelos Neurológicos , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologia , Emaranhados Neurofibrilares/ultraestrutura , Neurônios/metabolismo , Neurônios/ultraestrutura , Poro Nuclear/ultraestrutura , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas tau/metabolismoRESUMO
The attachment of the ubiquitin-like protein SUMO to target proteins is involved in a number of important cellular processes. Typically, SUMO modification occurs on lysine residues within the consensus sequence psiKxE/D (psi is a hydrophobic residue and x is any residue), although there are examples of modifications at nonconsensus sites. In most cases, sites of SUMO modification have been inferred from a combination of site-directed mutagenesis and functional analysis; however, these methods have two limitations. They do not directly identify the acceptor lysine, nor are they sufficient to identify acceptor lysine residues in SUMO polymers. Here, we use Fourier transform ion cyclotron resonance (FT-ICR) together with activated-ion electron capture dissociation (AI-ECD) or infrared multiphoton dissociation (IRMPD) mass spectrometry techniques to overcome these restrictions. These approaches were employed to analyze the autoSUMOylation reaction catalyzed by the SUMO E3 ligase RanBP2. Six sites of in vitro SUMOylation in RanBP2 along with four branch-point lysines in SUMO-1 and three in SUMO-2 were identified. In all but one case, SUMOylation occurred within the sequences KxE or KpsiK. These results demonstrate the utility of FT-ICR with AI-ECD or IRMPD mass spectrometry in detecting SUMOylation, and sites of SUMOylation, and their potential roles as complementary tools for proteomic and functional analysis, and provide significant insight into the modification of a SUMO ligase for which conventional techniques have been unsuccessful.
Assuntos
Análise de Fourier , Lisina/análise , Espectrometria de Massas/métodos , Chaperonas Moleculares/análise , Complexo de Proteínas Formadoras de Poros Nucleares/análise , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/análise , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Ciclotrons , Íons/química , Lisina/química , Metilação , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Peso Molecular , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , UbiquitinaRESUMO
AIM: To investigate the anticancer effects and the molecular mechanisms of deguelin on human U937 leukemia cells, and to explore the underlying mechanism regulating nucleoporin 98 (Nup98) and nucleoporin 88 (Nup88) in vitro. METHODS: The effects of deguelin on the growth of U937 cells were studied by MTT assay. The effect of deguelin on the cell cycle of U937 cells was studied by using a propidium iodide method. The localization of the nuclear pore complex proteins Nup98 and Nup88 was investigated by using immunofluorescence and immunoelectron microscopy. The expression of Nup98 and Nup88 in U937 cells was investigated by using flow cytometry and Western blot. RESULTS: The proliferation of U937 cells was inhibited in the deguelin-treated group, with a 24-h IC(50) value of 21.61 nmol/L and a 36-h IC(50) value of 17.07 nmol/L. U937 cells treated with deguelin had reduced percentages of cells in the G(0)/G(1) phase, whereas cells accumulated in the S and G(2)/M phases. Nup88 and Nup98 were found on both the nuclear and cytoplasmic sides of the U937 cells by using immunofluorescence and immunoelectron microscopy. The expression of Nup98 was upregulated and that of the Nup88 protein was downregulated in U937 cells treated with deguelin. CONCLUSION: Deguelin is able to inhibit the proliferation of U937 cells by regulating the cell cycle such that cells are arrested at the S and G(2)/M phases, so that the proportion of cells in the G(0)/G(1) phase decreases. The antitumor effects of deguelin are related to upregulating the expression of Nup98 and downregulating the expression of Nup88 protein in U937 cells.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Rotenona/análogos & derivados , Antineoplásicos Fitogênicos/administração & dosagem , Ciclo Celular , Relação Dose-Resposta a Droga , Fabaceae/química , Humanos , Plantas Medicinais/química , Rotenona/administração & dosagem , Rotenona/isolamento & purificação , Rotenona/farmacologia , Células U937RESUMO
The CYBB gene encodes gp91Phox; a component of the phagocyte respiratory burst oxidase. CYBB transcription is restricted to myeloid cells differentiated beyond the promyelocyte stage. In undifferentiated myeloid cells, the homeodomain (HD) transcription factor HoxA10 represses CYBB transcription via a cis element in the proximal promoter. During myelopoiesis, phosphorylation of conserved tyrosine residues in the HD decreases HoxA10 binding to this CYBB cis element. In the current studies, we found HoxA9 activates CYBB transcription in differentiated myeloid cells via the same cis element. We find HoxA9-mediated CYBB-transcription requires Pbx1 but is inhibited by Meis1. Additionally, phosphorylation of the conserved HD tyrosines increases HoxA9 binding to the CYBB promoter. The HOXA9 gene is involved in leukemia-associated translocations with the gene encoding Nup98, a nucleopore protein. We find expression of a Nup98-hoxA9 fusion protein blocks HoxA9-induced CYBB transcription in differentiating myeloid cells. In comparison to HoxA9, Nup98-hoxA9 has greater binding affinity for the CYBB cis element, but binding is not altered by HD tyrosine phosphorylation. Therefore, these studies identify CYBB as a common target gene repressed by HoxA10 and activated by HoxA9. These studies also suggest overexpression of Meis1 or Nup98-hoxA9 represses myeloid-specific gene transcription, thereby contributing to differentiation block in leukemogenesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas de Membrana/metabolismo , Células Mieloides/citologia , NADPH Oxidases/metabolismo , Western Blotting , Diferenciação Celular , Linhagem Celular Tumoral , Cloranfenicol O-Acetiltransferase/metabolismo , Cromatina/metabolismo , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Proteínas Homeobox A10 , Proteínas de Homeodomínio/metabolismo , Humanos , Imunoprecipitação , Modelos Genéticos , Mutagênese , Proteína Meis1 , NADPH Oxidase 2 , Proteínas de Neoplasias/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fagocitose , Fosforilação , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Fator de Transcrição 1 de Leucemia de Células Pré-B , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica , Transfecção , Tirosina/química , Células U937RESUMO
Tristetraprolin (TTP) is a widely expressed, zinc finger-containing protein that has been implicated in the regulation of TNFalpha production in mice. Stimulus-dependent cytoplasmic translocation of TTP has been demonstrated in several cells. In this report we used the yeast two-hybrid screen to identify proteins able to interact with full length, human TTP. One of the isolated TTP-interacting clones encoded the FG repeat region of the nuclear pore protein Nup214. Full length Nup214 co-precipitated with TTP from resting and LPS-stimulated THP-1 cells, indicating that this interaction occurred in intact cells. The ability of TTP to associate with Nup214 was dependent on two intact zinc fingers within TTP. In contrast to wild type TTP that localized primarily in the cytosol, a mutant unable to associate with Nup214 localized throughout the cell, suggesting that the interaction with Nup214 regulates TTP localization.
Assuntos
Proteínas de Ligação a DNA , Proteínas Imediatamente Precoces/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Transporte Ativo do Núcleo Celular , Animais , Células COS , Citoplasma/metabolismo , DNA Complementar/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Humanos , Proteínas Imediatamente Precoces/metabolismo , Microscopia Confocal , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Fatores de Tempo , Tristetraprolina , Técnicas do Sistema de Duplo-Híbrido , Dedos de ZincoRESUMO
Mdm2, a ubiquitin ligase that acts on p53, is regulated by sumoylation. In the current study, we identify the enzymes responsible for the sumoylation of Mdm2. When mammalian cells are co-transfected with cDNAs encoding Mdm2 and PIAS1 or PIASxbeta (protein inhibitor of activated STAT) as sumoylation enzymes, Mdm2 is highly sumoylated. Mdm2 is also sumoylated in an in vitro system containing PIASxbeta, PIAS1, and RanBP2. When several lysine residues of Mdm2 were sequentially mutated to arginine, the K182R mutant was not sumoylated in intact cells; however, in the in vitro system this mutant was sumoylated by PIAS1, PIASxbeta, and RanBP2 as efficiently as the wild-type Mdm2 protein. Lysine residues 182 and 185 map within the nuclear localization signal of Mdm2. A K185R mutant of Mdm2 is sumoylated in intact cells, whereas a K182R protein is not. Only a Mdm2 protein bearing the K182R mutation is localized exclusively in the cytoplasm. Because RanBP2 is a nuclear pore protein and PIAS proteins are localized within the nucleus, our data suggest that Mdm2 is sumoylated during nuclear translocation by RanBP2 and then further sumoylated once in the nucleus by PIASxbeta and PIAS1.