RESUMO
In the Suf Fe-S cluster assembly pathway, the activity of the cysteine desulfurase, SufS, is regulated by interactions with the accessory sulfotransferase protein, SufE. SufE has been shown to stimulate SufS activity, likely by inducing conformational changes in the SufS active site that promote the desulfurase step and by acting as an efficient persulfide acceptor in the transpersulfuration step. Previous results point toward an additional level of regulation through a "half-sites" mechanism that affects the stoichiometry and affinity for SufE as the dimeric SufS shifts between desulfurase and transpersulfuration activities. Investigation of the covalent persulfide intermediate of SufS by backbone amide hydrogen-deuterium exchange mass spectrometry identified two active site peptides (residues 225-236 and 356-366) and two peptides at the dimer interface of SufS (residues 88-100 and 243-255) that exhibit changes in deuterium uptake upon formation of the intermediate. Residues in these peptides are organized to form a conduit between the two active sites upon persulfide formation and include key cross-monomer interactions, suggesting they may play a role in the half-sites regulation. Three evolutionarily conserved residues at the dimer interface (R92, E96, and E250) were investigated by alanine scanning mutagenesis. Two of the substituted enzymes (E96A and E250A SufS) resulted in 6-fold increases in the value of KSufE, confirming a functional role. Re-examination of the dimer interface in reported crystal structures of SufS and the SufS homologue CsdA identified previously unnoticed residue mobility at the dimer interface. The identification of conformational changes at the dimer interface by hydrogen-deuterium exchange confirmed by mutagenesis and structural reports provides a physical mechanism for active site communication in the half-sites regulation of SufS activity. Given the conservation of the interface interactions, this mechanism may be broadly applicable to type II cysteine desulfurase systems.
Assuntos
Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/metabolismo , Escherichia coli/enzimologia , Liases/química , Liases/metabolismo , Enxofre/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Liases de Carbono-Enxofre/genética , Domínio Catalítico , Liases/genética , Mutagênese Sítio-Dirigida , Conformação ProteicaRESUMO
Yes-associated protein (YAP) is a main mediator of the Hippo pathway and promotes cancer development and progression in human lung cancer. We sought to determine whether inhibition of YAP suppresses metastasis of human lung adenocarcinoma in a murine model. We found that metastatic NSCLC cell lines H2030-BrM3(K-rasG12C mutation) and PC9-BrM3 (EGFRΔexon19 mutation) had a significantly decreased p-YAP(S127)/YAP ratio compared to parental H2030 (K-rasG12C mutation) and PC9 (EGFRΔexon19 mutation) cells (P < .05). H2030-BrM3 cells had significantly increased YAP mRNA and expression of Hippo downstream genes CTGF and CYR61 compared to parental H2030 cells (P < .05). Inhibition of YAP by short hairpin RNA (shRNA) and small interfering RNA (siRNA) significantly decreased mRNA expression in downstream genes CTGF and CYR61 in H2030-BrM3 cells (P < .05). In addition, inhibiting YAP by YAP shRNA significantly decreased migration and invasion abilities of H2030-BrM3 cells (P < .05). We are first to show that mice inoculated with YAP shRNA-transfected H2030-BrM3 cells had significantly decreased metastatic tumour burden and survived longer than control mice (P < .05). Collectively, our results suggest that YAP plays an important role in promoting lung adenocarcinoma brain metastasis and that direct inhibition of YAP by shRNA suppresses H2030-BrM3 cell brain metastasis in a murine model.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Adenocarcinoma de Pulmão/genética , Neoplasias Encefálicas/genética , Carcinogênese/genética , Fosfoproteínas/genética , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/terapia , Animais , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/secundário , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Fator de Crescimento do Tecido Conjuntivo/genética , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Mutação , Fosfoproteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Interferente Pequeno/administração & dosagem , Transdução de Sinais , Fatores de Transcrição , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas de Sinalização YAPRESUMO
Malignant mesothelioma is an aggressive cancer that is resistant to current therapy. The poor prognosis of mesothelioma has been associated with elevated Yes-associated protein (YAP) activity. In this study, we evaluated the effect of targeting YAP in mesothelioma. First, we comprehensively studied YAP activity in five mesothelioma cell lines (211H, H2052, H290, MS-1 and H2452) and one normal mesothelial cell line (LP9). We found decreased phospho-YAP to YAP protein ratio and consistently increased GTIIC reporter activity in 211H, H2052 and H290 compared to LP9. The same three cell lines (IC50 s < 1 µM) were more sensitive than LP9 (IC50 = 3.5 µM) to the YAP/TEAD inhibitor verteporfin. We also found that verteporfin significantly reduced YAP protein level, mRNA levels of YAP downstream genes and GTIIC reporter activity in the same three cell lines, indicating inhibition of YAP signaling by verteporfin. Verteporfin also impaired invasion and tumoursphere formation ability of H2052 and H290. To validate the effect of specific targeting YAP in mesothelioma cells, we down-regulated YAP by siRNA. We found siYAP significantly decreased YAP transcriptional activity and impaired invasion and tumoursphere formation ability of H2052 and H290. Furthermore, forced overexpression of YAP rescued GTIIC reporter activity and cell viability after siYAP targeting 3'UTR of YAP. Finally, we found concurrent immunohistochemistry staining of ROCK2 and YAP (P < 0.05). Inhibition of ROCK2 decreased GTIIC reporter activity in H2052 and 211H suggesting that Rho/ROCK signaling also contributed to YAP activation in mesothelioma cells. Our results indicate that YAP may be a potential therapeutic target in mesothelioma.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ligação a DNA/genética , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/genética , Fosfoproteínas/genética , RNA Mensageiro/genética , Fatores de Transcrição/genética , Quinases Associadas a rho/genética , Regiões 3' não Traduzidas , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Genes Reporter , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Mesotelioma/genética , Mesotelioma/metabolismo , Mesotelioma/patologia , Mesotelioma Maligno , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/metabolismo , Fosforilação , Porfirinas/farmacologia , Prognóstico , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Verteporfina , Proteínas de Sinalização YAP , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Glioblastoma multiforme (GBM) is the most aggressive and common brain tumor in adults. Sorafenib, a multi-kinase inhibitor, has been shown to inhibit cell proliferation and induce apoptosis through inhibition of STAT3 signaling in glioblastoma cells and in intracranial gliomas. However, sorafenib also induces cell autophagy. Due to the dual roles of autophagy in tumor cell survival and death, the therapeutic effect of sorafenib on glioblastoma is uncertain. Here, we combined sorafenib treatment in GBM cells (U373 and LN229) and tumors with the autophagy inhibitor chloroquine. We found that blockage of autophagy further inhibited cell proliferation and migration and induced cell apoptosis in vitro and in vivo. These findings suggest the possibility of combination treatment with sorafenib and autophagy inhibitors for GBM.
Assuntos
Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Proliferação de Células/efeitos dos fármacos , Cloroquina/farmacologia , Glioblastoma/patologia , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Animais , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Masculino , Camundongos , Niacinamida/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , SorafenibeRESUMO
Many essential metalloproteins require iron-sulfur (Fe-S) cluster cofactors for their function. In vivo persulfide formation from l-cysteine is a key step in the biogenesis of Fe-S clusters in most organisms. In Escherichia coli, the SufS cysteine desulfurase mobilizes persulfide from l-cysteine via a PLP-dependent ping-pong reaction. SufS requires the SufE partner protein to transfer the persulfide to the SufB Fe-S cluster scaffold. Without SufE, the SufS enzyme fails to efficiently turn over and remains locked in the persulfide-bound state. Coordinated protein-protein interactions mediate sulfur transfer from SufS to SufE. Multiple studies have suggested that SufE must undergo a conformational change to extend its active site Cys loop during sulfur transfer from SufS. To test this putative model, we mutated SufE Asp74 to Arg (D74R) to increase the dynamics of the SufE Cys51 loop. Amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis of SufE D74R revealed an increase in solvent accessibility and dynamics in the loop containing the active site Cys51 used to accept persulfide from SufS. Our results indicate that the mutant protein has a stronger binding affinity for SufS than that of wild-type SufE. In addition, SufE D74R can still enhance SufS desulfurase activity and did not show saturation at higher SufE D74R concentrations, unlike wild-type SufE. These results show that dynamic changes may shift SufE to a sulfur-acceptor state that interacts more strongly with SufS.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Liases/química , Mutação de Sentido Incorreto , Enxofre/química , Substituição de Aminoácidos , Domínio Catalítico , Medição da Troca de Deutério , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Liases/genética , Liases/metabolismo , Espectrometria de Massas , Estrutura Secundária de Proteína , Enxofre/metabolismoRESUMO
Iron-sulfur (Fe-S) cluster metalloproteins conduct essential functions in nearly all contemporary forms of life. The nearly ubiquitous presence of Fe-S clusters and the fundamental requirement for Fe-S clusters in both aerobic and anaerobic Archaea, Bacteria, and Eukarya suggest that these clusters were likely integrated into central metabolic pathways early in the evolution of life prior to the widespread oxidation of Earth's atmosphere. Intriguingly, Fe-S cluster-dependent metabolism is sensitive to disruption by oxygen because of the decreased bioavailability of ferric iron as well as direct oxidation of sulfur trafficking intermediates and Fe-S clusters by reactive oxygen species. This fact, coupled with the ubiquity of Fe-S clusters in aerobic organisms, suggests that organisms evolved with mechanisms that facilitate the biogenesis and use of these essential cofactors in the presence of oxygen, which gradually began to accumulate around 2.5 billion years ago as oxygenic photosynthesis proliferated and reduced minerals that buffered against oxidation were depleted. This review highlights the most ancient of the Fe-S cluster biogenesis pathways, the Suf system, which likely was present in early anaerobic forms of life. Herein, we use the evolution of the Suf pathway to assess the relationships between the biochemical functions and physiological roles of Suf proteins, with an emphasis on the selective pressure of oxygen toxicity. Our analysis suggests that diversification into oxygen-containing environments disrupted iron and sulfur metabolism and was a main driving force in the acquisition of accessory Suf proteins (such as SufD, SufE, and SufS) by the core SufB-SufC scaffold complex. This analysis provides a new framework for the study of Fe-S cluster biogenesis pathways and Fe-S cluster-containing metalloenzymes and their complicated patterns of divergence in response to oxygen.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Oxigênio/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Redes e Vias Metabólicas , Methanosarcina/química , Modelos Moleculares , Óperon , Filogenia , Conformação ProteicaRESUMO
Fe-S clusters are critical metallocofactors required for cell function. Fe-S cluster biogenesis is carried out by assembly machinery consisting of multiple proteins. Fe-S cluster biogenesis proteins work together to mobilize sulfide and iron, form the nascent cluster, traffic the cluster to target metalloproteins, and regulate the assembly machinery in response to cellular Fe-S cluster demand. A complex series of protein-protein interactions is required for the assembly machinery to function properly. Despite considerable progress in obtaining static three-dimensional structures of the assembly proteins, little is known about transient protein-protein interactions during cluster assembly or the role of protein dynamics in the cluster assembly process. The Escherichia coli cysteine desulfurase SufS (EC 2.8.1.7) and its accessory protein SufE work together to mobilize persulfide from L-cysteine, which is then donated to the SufB Fe-S cluster scaffold. Here we use amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize SufS-SufE interactions and protein dynamics in solution. HDX-MS analysis shows that SufE binds near the SufS active site to accept persulfide from Cys-364. Furthermore, SufE binding initiates allosteric changes in other parts of the SufS structure that likely affect SufS catalysis and alter SufS monomer-monomer interactions. SufE enhances the initial l-cysteine substrate binding to SufS and formation of the external aldimine with pyridoxal phosphate required for early steps in SufS catalysis. Together, these results provide a new picture of the SufS-SufE sulfur transferase pathway and suggest a more active role for SufE in promoting the SufS cysteine desulfurase reaction for Fe-S cluster assembly.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Liases/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Proteínas de Transporte/química , Cisteína/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Hidrogênio/metabolismo , Liases/química , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Sulfetos/química , Sulfetos/metabolismoRESUMO
Orchestrated regulation of neuronal migration and morphogenesis is critical for neuronal development and establishment of functional circuits, but its regulatory mechanism is incompletely defined. We established and analyzed mice with neural-specific knock-out of Trio, a guanine nucleotide exchange factor with multiple guanine nucleotide exchange factor domains. Knock-out mice showed defective cerebella and severe signs of ataxia. Mutant cerebella had no granule cells in the internal granule cell layer due to aberrant granule cell migration as well as abnormal neurite growth. Trio-deficient granule cells showed reduced extension of neurites and highly branched and misguided processes with perturbed stabilization of actin and microtubules. Trio deletion caused down-regulation of the activation of Rac1, RhoA, and Cdc42, and mutant granule cells appeared to be unresponsive to neurite growth-promoting molecules such as Netrin-1 and Semaphorin 6A. These results suggest that Trio may be a key signal module for the orchestrated regulation of neuronal migration and morphogenesis during cerebellar development. Trio may serve as a signal integrator decoding extrinsic signals to Rho GTPases for cytoskeleton organization.
Assuntos
Cerebelo/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Troca do Nucleotídeo Guanina/química , Fosfoproteínas/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Movimento Celular , Cromossomos Artificiais Bacterianos/metabolismo , Citoesqueleto/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Proteínas de Filamentos Intermediários/metabolismo , Camundongos , Camundongos Knockout , Morfogênese , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neurônios/metabolismo , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Homeodomain-interacting protein kinase-2 (HIPK2) can either promote or inhibit transcription depending on cellular context. In this study, we show that a new HIPK2 isoform increases TEAD reporter activity in NSCLC cells. We detected HIPK2 copy number gain in 5/6 (83.3%) NSCLC cell lines. In NSCLC patients with high HIPK2 mRNA expression in the Human Protein Atlas, the five-year survival rate is significantly lower than in patients with low expression (38% vs 47%; p = 0.047). We also found that 70/78 (89.7%) of NSCLC tissues have moderate to strong expression of the N-terminal HIPK2 protein. We detected and cloned a novel HIPK2 isoform 3 and found that its forced overexpression promotes TEAD reporter activity in NSCLC cells. Expressing HIPK2 isoform 3_K228A kinase-dead plasmid failed to increase TEAD reporter activity in NSCLC cells. Next, we showed that two siRNAs targeting HIPK2 decreased HIPK2 isoform 3 and YAP protein levels in NSCLC cells. Degradation of the YAP protein was accelerated after HIPK2 knockdown in NSCLC cells. Inhibition of HIPK2 isoform 3 decreased the mRNA expression of YAP downstream gene CTGF. The specific HIPK2 kinase inhibitor TBID decreased TEAD reporter activity, reduced cancer side populations, and inhibited tumorsphere formation of NSCLC cells. In summary, this study indicates that HIPK2 isoform 3, the main HIPK2 isoform expressed in NSCLC, promotes YAP/TEAD transcriptional activity in NSCLC cells. Our results suggest that HIPK2 isoform 3 may be a potential therapeutic target for NSCLC.
RESUMO
Glioma is the most common primary malignant brain tumor with poor survival and limited therapeutic options. The non-psychoactive phytocannabinoid cannabidiol (CBD) has been shown to be effective against glioma; however, the molecular target and mechanism of action of CBD in glioma are poorly understood. Here we investigated the molecular mechanisms underlying the antitumor effect of CBD in preclinical models of human glioma. Our results showed that CBD induced autophagic rather than apoptotic cell death in glioma cells. We also showed that CBD induced mitochondrial dysfunction and lethal mitophagy arrest, leading to autophagic cell death. Mechanistically, calcium flux induced by CBD through TRPV4 (transient receptor potential cation channel subfamily V member 4) activation played a key role in mitophagy initiation. We further confirmed TRPV4 levels correlated with both tumor grade and poor survival in glioma patients. Transcriptome analysis and other results demonstrated that ER stress and the ATF4-DDIT3-TRIB3-AKT-MTOR axis downstream of TRPV4 were involved in CBD-induced mitophagy in glioma cells. Lastly, CBD and temozolomide combination therapy in patient-derived neurosphere cultures and mouse orthotopic models showed significant synergistic effect in both controlling tumor size and improving survival. Altogether, these findings showed for the first time that the antitumor effect of CBD in glioma is caused by lethal mitophagy and identified TRPV4 as a molecular target and potential biomarker of CBD in glioma. Given the low toxicity and high tolerability of CBD, we therefore propose CBD should be tested clinically for glioma, both alone and in combination with temozolomide.Abbreviations: 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATF4: activating transcription factor 4; Baf-A1: bafilomycin A1; CANX: calnexin; CASP3: caspase 3; CAT: catalase; CBD: cannabidiol; CQ: chloroquine; DDIT3: DNA damage inducible transcript 3; ER: endoplasmic reticulum; GBM: glioblastoma multiforme; GFP: green fluorescent protein; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PARP1: poly(ADP-ribose) polymerase; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; SLC8A1: solute carrier family 8 member A1; SQSTM1: sequestosome 1; TCGA: The cancer genome atlas; TEM: transmission electron microscopy; TMZ: temozolomide; TRIB3: tribbles pseudokinase 3; TRPC: transient receptor potential cation channel subfamily C; TRPV4: transient receptor potential cation channel subfamily V member 4.
Assuntos
Canabidiol/uso terapêutico , Glioma/tratamento farmacológico , Mitofagia/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Animais , Antineoplásicos Alquilantes/farmacologia , Morte Celular Autofágica/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Canabidiol/farmacologia , Linhagem Celular Tumoral , Glioma/metabolismo , Glioma/patologia , Humanos , Masculino , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Transplante de Neoplasias , Canais de Cátion TRPV/fisiologia , Temozolomida/farmacologiaRESUMO
Overcoming temozolomide (TMZ) resistance in glioma cancer cells remains a major challenge to the effective treatment of the disease. Increasing TMZ efficacy for patients with glioblastoma (GBM) is urgently needed because TMZ treatment is the standard chemotherapy protocol for adult patients with glioblastoma. O6-methylguanine-DNA-methyltransferase (MGMT) overexpression is associated with TMZ resistance, and low MGMT is a positive response marker for TMZ therapy. Here, we used 3 glioma cell lines (SF767, U373, and LN229), which had different levels of TMZ sensitivity. We found TMZ sensitivity is positively correlated with MGMT expression and multidrug-resistance protein ABC subfamily G member 2 (ABCG2) in these cells. CK2-STAT3 signaling and Hippo-YAP signaling are reported to regulate MGMT expression and ABCG2 expression, respectively. We combined CK2 inhibitor CX-4945 and YAP inhibitor verteporfin with TMZ treatment. We found that CX-4945 but not verteporfin can sensitize TMZ-resistant cells SF767 to TMZ and that CX-4945 and TMZ combinational treatment was effective for glioma treatment in mouse models compared with TMZ alone. IMPLICATIONS: A combination of CK2 inhibitor with TMZ may improve the therapeutic efficiency of TMZ toward GBM with acquired resistance.
RESUMO
Iron-sulfur clusters are key iron cofactors in biological pathways ranging from nitrogen fixation to respiration. Because of the toxicity of ferrous iron and sulfide to the cell, in vivo Fe-S cluster assembly transpires via multiprotein biosynthetic pathways. Fe-S cluster assembly proteins traffic iron and sulfide, assemble nascent Fe-S clusters, and correctly transfer Fe-S clusters to the appropriate target metalloproteins in vivo. The Gram-negative bacterium Escherichia coli contains a stress-responsive Fe-S cluster assembly system, the SufABCDSE pathway, that functions under iron starvation and oxidative stress conditions that compromise Fe-S homeostasis. Using a combination of protein-protein interaction and in vitro Fe-S cluster assembly assays, we have characterized the relative roles of the SufBCD complex and the SufA protein during Suf Fe-S cluster biosynthesis. These studies reveal that SufA interacts with SufBCD to accept Fe-S clusters formed de novo on the SufBCD complex. Our results represent the first biochemical evidence that the SufBCD complex within the Suf pathway functions as a novel Fe-S scaffold system to assemble nascent clusters and transfer them to the SufA Fe-S shuttle.
Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Sequência de Bases , Dicroísmo Circular , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Homeostase , Proteínas Ferro-Enxofre/química , Espectrofotometria UltravioletaRESUMO
Programmed death-ligand 1 (PD-L1) is a membrane protein on tumor cells that binds to the PD-1 receptor expressed on immune cells, leading to the immune escape of tumor cells. Yes-associated protein (YAP) is a main effector of the Hippo/YAP signaling pathway, which plays important roles in cancer development. Here we show that YAP regulates PD-L1 expression in human non-small cell lung cancer (NSCLC) cells. First, we investigated YAP and PD-L1 expression at the protein level in 142 NSCLC samples and 15 normal lung samples. In tumor tissue, immunohistochemistry showed positive staining for YAP and PD-L1, which correlated significantly (n = 142, r = 0.514, P < 0.001). Second, in cell lines that express high levels of PD-L1 (H460, SKLU-1, and H1299), the ratio of p-YAP/YAP was lower and GTIIC reporter activity of the Hippo pathway was higher than those in three cell lines expressing low levels of PD-L1 (A549, H2030, and PC9) (P < 0.05). Third, in the same three cell lines, inhibition of YAP by two small interfering RNAs (siRNAs) decreased the mRNA and protein level of PD-L1 (P < 0.05). Fourth, forced overexpression of the YAP gene rescued the PD-L1 mRNA and protein level after siRNA knockdown targeting 3'UTR of the endogenous YAP gene. Finally, chromatin immunoprecipitation (ChIP) assays using a YAP-specific monoclonal antibody resulted in the precipitation of PD-L1 enhancer region encompassing two putative TEAD binding sites. Our results indicate that YAP regulates the transcription of PD-L1 in NSCLC.
RESUMO
A small population of cancer cells called cancer-initiating cells or cancer stem cells (CSCs) are involved in drug resistance, metastasis, and cancer relapse. Finding pathways that regulate CSC is very important for clinical therapy. ATP-binding cassette sub-family G member 2 (ABCG2) plays a role in side population (SP) cell formation and contributes to chemotherapy resistance in common forms of cancer. Yes-associated protein 1 (YAP1) is a major transcriptional effector of the Hippo pathway, which plays important roles in organ size control and tumorigenesis. In this study, we found ABCG2 and YAP1 were both overexpressed in lung cancer SP cells. Disruption of YAP1 expression by siRNA attenuated the expression of ABCG2 transcript and significantly reduced the percentage of SP cells and sphere formation in lung cancer cells. Overexpression of YAP1 in lung cancers led to an increase in ABCG2 expression and increased the percentage of SP cells. However, overexpression of YAP1 in purified non-SP cells did not increase ABCG2 expression and the percentage of SP cells, which may be due to the inhibition of YAP activity through phosphorylation. YAP1 directly transcriptionally regulated ABCG2 by binding to the promoter of ABCG2. Moreover, the YAP1 inhibitor verteporfin and YAP1 siRNA downregulated ABCG2 level through inhibition of YAP1 in lung cancer cells and sensitized them to the chemotherapy drug doxorubicin. Our study adds a new function for YAP1 that may be relevant to drug resistance and cancer therapy through regulation of ABCG2 and side population cell formation in lung cancer.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Células da Side Population/metabolismo , Células A549 , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Fosforilação , Porfirinas/farmacologia , Regiões Promotoras Genéticas , Fatores de Transcrição , Regulação para Cima , Verteporfina , Proteínas de Sinalização YAPRESUMO
Malignant pleural mesothelioma (MPM) is an aggressive cancer for which more effective treatments are needed. In this study, strong to moderate staining of MET and ERK5 was detected in 67.1 and 48% of the analyzed 73 human mesothelioma tumors, and significant correlation of MET and ERK5 expression was identified (P<0.05). We evaluated the doublecortin-like kinase 1 (DCLK1) expression in human mesothelioma tumors. Our results showed that 50.7% of the immunohistochemistry analyzed human mesothelioma tumors have strong to moderate staining of DCLK1, and its expression is significantly correlated with MET or ERK5 expression (P<0.05). Also, the upregulation of DCLK1 is correlated with poor prognosis in MPM patients (P=0.0235). To investigate whether DCLK1 is downstream of MET/ERK5 signaling in human mesothelioma, the effect of DCLK1 expression was analyzed after treatments with either the MET inhibitor XL184 or the ERK5 inhibitor XMD8-92 in human mesothelioma cell lines. Our results showed that the MET inhibitor XL184 reduced the expression of phosphoERK5 and DCLK1 expression in human mesothelioma cell lines. In addition, the ERK5 inhibitor XMD8-92 reduced the expression of phospho-ERK5 and DCLK1 expression in human mesothelioma cell lines. Furthermore, XML184 and XMD8-92 treatment impaired invasion and tumor sphere formation ability of H290 mesothelioma cells. These results suggest that DCLK1 is regulated by MET/ERK5 signaling in human mesothelioma, and the MET/ERK5/DCLK1 signaling cascade could be further developed into a promising therapeutic target against mesothelioma.
Assuntos
Biomarcadores Tumorais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/patologia , Mesotelioma/patologia , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Neoplasias Pleurais/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Idoso , Apoptose , Movimento Celular , Proliferação de Células , Quinases Semelhantes a Duplacortina , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Mesotelioma/metabolismo , Mesotelioma Maligno , Neoplasias Pleurais/metabolismo , Prognóstico , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
Glioblastoma multiforme (GBM) is one of the deadliest forms of cancer due to its limited sensitivity to chemotherapy and radiotherapy. Cisplatin (CCDP) is a widely used chemotherapeutic agent for tumors, but the agent often results in the development of chemo-resistance. In several cancers, cisplatin resistance is associated with autophagy induction. Here, we found that in glioma cells cisplatin treatment induced autophagy. Our data indicates that the autophagy induction plays a critical role in cisplatin resistance of glioma cells, knockdown of RB inhibited autophagy induced by cisplatin, and inhibition of autophagy improved cisplatin-induced apoptosis. It suggests that a combination of autophagy inhibitors with cisplatin may improve the therapeutic efficiency of cisplatin towards GBM with acquired resistance.
Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Cisplatino/farmacologia , Glioma/patologia , Proteína do Retinoblastoma/genética , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Técnicas de Silenciamento de Genes , Glioma/genética , Humanos , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Yes-associated protein (YAP) is a main mediator of the Hippo pathway, which promotes cancer development. Here we show that YAP promotes resistance to erlotinib in human non-small cell lung cancer (NSCLC) cells. We found that forced YAP overexpression through YAP plasmid transfection promotes erlotinib resistance in HCC827 (exon 19 deletion) cells. In YAP plasmid-transfected HCC827 cells, GTIIC reporter activity and Hippo downstream gene expression of AREG and CTGF increased significantly (P<0.05), as did ERBB3 mRNA expression (P<0.05). GTIIC reporter activity, ERBB3 protein and mRNA expression all increased in HCC827 erlotinib-resistance (ER) cells compared to parental HCC827 cells. Inhibition of YAP by small interfering RNA (siRNA) increased the cytotoxicity of erlotinib to H1975 (L858R+T790M) cells. In YAP siRNA-transfected H1975 cells, GTIIC reporter activity and downstream gene expression of AREG and CTGF decreased significantly (P<0.05). Verteporfin, YAP inhibitor had an effect similar to that of YAP siRNA; it increased sensitivity of H1975 cells to erlotinib and in combination with erlotinib, synergistically reduced migration, invasion and tumor sphere formation abilities in H1975 cells. Our results indicate that YAP promotes erlotinib resistance in the erlotinib-sensitive NSCLC cell line HCC827. Inhibition of YAP by siRNA increases sensitivity of erlotinib-resistant NSCLC cell line H1975 to erlotinib.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias Pulmonares/patologia , Fosfoproteínas/metabolismo , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Cloridrato de Erlotinib/farmacologia , Humanos , Neoplasias Pulmonares/metabolismo , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
Alterations of the EGFR/ERK and Hippo/YAP pathway have been found in non-small cell lung cancer (NSCLC). Herein, we show that ERK1 and ERK2 have an effect on the Hippo/YAP pathway in human NSCLC cells. Firstly, inhibition of ERK1/2 by siRNA or small-molecular inhibitors decreased the YAP protein level, the reporter activity of the Hippo pathway, and the mRNA levels of the Hippo downstream genes, CTGF, Gli2, and BIRC5. Secondly, degradation of YAP protein was accelerated after ERK1/2 depletion in NSCLC cell lines, in which YAP mRNA level was not decreased. Thirdly, forced over-expression of the ERK2 gene rescued the YAP protein level and Hippo reporter activity after siRNA knockdown targeting 3'UTR of the ERK2 gene in NSCLC cells. Fourthly, depletion of ERK1/2 reduced the migration and invasion of NSCLC cells. Combined depletion of ERK1/2 had a greater effect on cell migration than depletion of either one separately. Finally, the MEK1/2 inhibitor Trametinib decreased YAP protein level and transcriptional activity of the Hippo pathway in NSCLC cell lines. Our results suggest that ERK1/2 inhibition participates in reducing YAP protein level, which in turn down-regulates expression of the downstream genes of the Hippo pathway to suppress migration and invasion of NSCLC cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Via de Sinalização Hippo , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/metabolismo , Invasividade Neoplásica/patologia , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Fatores de Transcrição , Transfecção , Proteínas de Sinalização YAPRESUMO
BACKGROUND: The Hedgehog (Hh) signaling pathway has been implicated in stem cell maintenance and its activation is aberrant in several types of cancer including mesothelioma. Protein kinase CK2 affects several cell signaling pathways through the mechanism of phosphorylation. METHODS: Protein and mRNA levels of CK2α and Gli1 were tested by quantitative RT-PCR and immunohistochemistry staining in mesothelioma samples and cell lines. Down-regulated Gli1 expression and transcriptional activity were demonstrated by RT-PCR, Western blot and luciferase reporter assay. RESULTS: In this study, we show that CK2α is over-expressed and a positive regulator of Hegdehog/Gli1 signaling in human malignant pleural mesothelioma. First of all, we found that the mRNA levels of CK2α and Gli1 were broadly elevated and correlated (n = 52, r = 0.401, P < 0.05), compared with LP9 (a normal mesothelial cell line). We then investigated their expression at the protein level, and found that all the 7 mesothelioma cell lines tested showed positive staining in CK2α and Gli1 immunohistochemistry. Correlation analysis by Pearson test for CK2α and Gli1 expression in the 75 mesothelioma tumors and the 7 mesothelioma cell lines showed that the two protein expression was significantly correlated (n = 82, r = 0.554, P < 0.01). Furthermore, we demonstrated that Gli1 expression and transcriptional activity were down-regulated after CK2α was silenced in two mesothelioma cell lines (H28 and H2052). CK2α siRNA also down-regulated the expression of Hh target genes in these cell lines. Moreover, treatment with a small-molecule CK2α inhibitor CX-4945 led to dose-dependent inhibition of Gli1 expression and transcriptional activity. Conversely, forced over-expression of CK2α resulted in an increase in Gli1 transcriptional activity in H28 cells. CONCLUSIONS: Thus, we report for the first time that over-expressed CK2α positively regulate Hh/Gli1 signaling in human mesothelioma.
Assuntos
Proteínas Hedgehog/metabolismo , Neoplasias Pulmonares/enzimologia , Mesotelioma/enzimologia , Neoplasias Pleurais/enzimologia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mesotelioma/genética , Mesotelioma/patologia , Mesotelioma Maligno , Neoplasias Pleurais/genética , Neoplasias Pleurais/patologia , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genética , Transcrição Gênica , Transfecção , Proteína GLI1 em Dedos de ZincoRESUMO
During oxidative stress in Escherichiacoli, the SufABCDSE stress response pathway mediates iron-sulfur (Fe-S) cluster biogenesis rather than the Isc pathway. To determine why the Suf pathway is favored under stress conditions, the stress response SufS-SufE sulfur transfer pathway and the basal housekeeping IscS-IscU pathway were directly compared. We found that SufS-SufE cysteine desulfurase activity is significantly higher than IscS-IscU at physiological cysteine concentrations and after exposure to H(2)O(2). Mass spectrometry analysis demonstrated that IscS-IscU is more susceptible than SufS-SufE to oxidative modification by H(2)O(2). These important results provide biochemical insight into the stress resistance of the Suf pathway.