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1.
Structure ; 29(4): 345-356.e8, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33333006

RESUMO

TEAD transcription factors regulate gene expression through interactions with DNA and other proteins. They are crucial for the development of eukaryotic organisms and to control the expression of genes involved mostly in cell proliferation and differentiation; however, their deregulation can lead to tumorigenesis. To study the interactions of TEAD1 with M-CAT motifs and their inverted versions, the KD of each complex was determined, and H/D exchange, quantitative chemical cross-linking, molecular docking, and smFRET were utilized for structural characterization. ChIP-qPCR was employed to correlate the results with a cell line model. The results obtained showed that although the inverted motif has 10× higher KD, the same residues were affected by the presence of M-CAT in both orientations. Molecular docking and smFRET revealed that TEAD1 binds the inverted motif rotated 180°. In addition, the inverted motif was proven to be occupied by TEAD1 in Jurkat cells, suggesting that the low-affinity binding sites present in the human genome may possess biological relevance.


Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Proteínas Nucleares/química , Fatores de Transcrição/química , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Células Jurkat , Simulação de Acoplamento Molecular , Proteínas Nucleares/metabolismo , Motivos de Nucleotídeos , Ligação Proteica , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/metabolismo
2.
Int J Mol Sci ; 21(9)2020 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-32375238

RESUMO

Extracellular signal-regulated kinase (ERK) is a part of the mitogen-activated protein kinase (MAPK) signaling pathway which allows the transduction of various cellular signals to final effectors and regulation of elementary cellular processes. Deregulation of the MAPK signaling occurs under many pathological conditions including neurodegenerative disorders, metabolic syndromes and cancers. Targeted inhibition of individual kinases of the MAPK signaling pathway using synthetic compounds represents a promising way to effective anti-cancer therapy. Cross-talk of the MAPK signaling pathway with other proteins and signaling pathways have a crucial impact on clinical outcomes of targeted therapies and plays important role during development of drug resistance in cancers. We discuss cross-talk of the MAPK/ERK signaling pathway with other signaling pathways, in particular interplay with the Hippo/MST pathway. We demonstrate the mechanism of cell death induction shared between MAPK/ERK and Hippo/MST signaling pathways and discuss the potential of combination targeting of these pathways in the development of more effective anti-cancer therapies.


Assuntos
Antineoplásicos/uso terapêutico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/metabolismo , Animais , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Via de Sinalização Hippo , Humanos , Neoplasias/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Transdução de Sinais
3.
Biomolecules ; 9(10)2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31561554

RESUMO

The limited information available on the structure of complexes involving transcription factors and cognate DNA response elements represents a major obstacle in the quest to understand their mechanism of action at the molecular level. We implemented a concerted structural proteomics approach, which combined hydrogen-deuterium exchange (HDX), quantitative protein-protein and protein-nucleic acid cross-linking (XL), and homology analysis, to model the structure of the complex between the full-length DNA binding domain (DBD) of Forkhead box protein O4 (FOXO4) and its DNA binding element (DBE). The results confirmed that FOXO4-DBD assumes the characteristic forkhead topology shared by these types of transcription factors, but its binding mode differs significantly from those of other members of the family. The results showed that the binding interaction stabilized regions that were rather flexible and disordered in the unbound form. Surprisingly, the conformational effects were not limited only to the interface between bound components, but extended also to distal regions that may be essential to recruiting additional factors to the transcription machinery. In addition to providing valuable new insights into the binding mechanism, this project provided an excellent evaluation of the merits of structural proteomics approaches in the investigation of systems that are not directly amenable to traditional high-resolution techniques.


Assuntos
DNA/química , Fatores de Transcrição/química , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Medição da Troca de Deutério , Espectrometria de Massas , Estrutura Molecular , Elementos de Resposta , Fatores de Transcrição/metabolismo
4.
Cells ; 8(2)2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30795621

RESUMO

The discrete activation of individual caspases is essential during T-cell development, activation, and apoptosis. Humans carrying nonfunctional caspase-8 and caspase-8 conditional knockout mice exhibit several defects in the progression of naive CD4⁺ T cells to the effector stage. MST1, a key kinase of the Hippo signaling pathway, is often presented as a substrate of caspases, and its cleavage by caspases potentiates its activity. Several studies have focused on the involvement of MST1 in caspase activation and also reported several defects in the immune system function caused by MST1 deficiency. Here, we show the rapid activation of the MEK-ERK-MST1 axis together with the cleavage and activation of caspase-3, -6, -7, -8, and -9 after PI3K signaling blockade by the selective inhibitor GDC-0941 in Jurkat T cells. We determined the phosphorylation pattern of MST1 using a phosphoproteomic approach and identified two amino acid residues phosphorylated in an ERK-dependent manner after GDC-0941 treatment together with a novel phosphorylation site at S21 residue, which was extensively phosphorylated in an ERK-independent manner during PI3K signaling blockade. Using caspase inhibitors and the inhibition of MST1 expression using siRNA, we identified an exclusive role of the MEK-ERK-MST1 axis in the activation of initiator caspase-8, which in turn activates executive caspase-3/-7 that finally potentiate MST1 proteolytic cleavage. This mechanism forms a positive feed-back loop that amplifies the activation of MST1 together with apoptotic response in Jurkat T cells during PI3K inhibition. Altogether, we propose a novel MEK-ERK-MST1-CASP8-CASP3/7 apoptotic pathway in Jurkat T cells and believe that the regulation of this pathway can open novel possibilities in systemic and cancer therapies.


Assuntos
Apoptose/efeitos dos fármacos , Fator de Crescimento de Hepatócito/metabolismo , Indazóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sulfonamidas/farmacologia , Sequência de Aminoácidos , Inibidores de Caspase/farmacologia , Caspases/metabolismo , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Fator de Crescimento de Hepatócito/química , Humanos , Células Jurkat , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismo , Piperazinas/farmacologia , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas/química
5.
Oncotarget ; 8(61): 103137-103153, 2017 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-29262552

RESUMO

Abnormalities in cancer metabolism represent potential targets for cancer therapy. We have recently identified a natural compound Quambalarine B (QB), which inhibits proliferation of several leukemic cell lines followed by cell death. We have predicted ubiquinone binding sites of mitochondrial respiratory complexes as potential molecular targets of QB in leukemia cells. Hence, we tracked the effect of QB on leukemia metabolism by applying several omics and biochemical techniques. We have confirmed the inhibition of respiratory complexes by QB and found an increase in the intracellular AMP levels together with respiratory substrates. Inhibition of mitochondrial respiration by QB triggered reprogramming of leukemic cell metabolism involving disproportions in glycolytic flux, inhibition of proteins O-glycosylation, stimulation of glycine synthesis pathway, and pyruvate kinase activity, followed by an increase in pyruvate and a decrease in lactate levels. Inhibition of mitochondrial complex I by QB suppressed folate metabolism as determined by a decrease in formate production. We have also observed an increase in cellular levels of several amino acids except for aspartate, indicating the dependence of Jurkat (T-ALL) cells on aspartate synthesis. These results indicate blockade of mitochondrial complex I and II activity by QB and reduction in aspartate and folate metabolism as therapeutic targets in T-ALL cells. Anti-cancer activity of QB was also confirmed during in vivo studies, suggesting the therapeutic potential of this natural compound.

6.
Exp Cell Res ; 349(2): 273-281, 2016 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-27793648

RESUMO

The general mechanism underlying the tumor suppressor activity of the Hippo signaling pathway remains unclear. In this study, we explore the molecular mechanisms connecting the Hippo signaling pathway with glucose metabolism. We have found that two key regulators of glycolysis, C-MYC and GLUT1, are targets of the Hippo signaling pathway in human leukemia cells. Our results revealed that activation of MST1 by the natural compound shikonin inhibited the expression of GLUT1 and C-MYC. Furthermore, RNAi experiments confirmed the regulation of GLUT1 and C-MYC expression via the MST1-YAP1-TEAD1 axis. Surprisingly, YAP1 was found to positively regulate C-MYC mRNA levels in complex with TEAD1, while it negatively regulates C-MYC levels in cooperation with MST1. Hence, YAP1 serves as a rheostat for C-MYC, which is regulated by MST1. In addition, depletion of MST1 stimulates lactate production, whereas the specific depletion of TEAD1 has an opposite effect. The inhibition of lactate production and cellular proliferation induced by shikonin also depends on the Hippo pathway activity. Finally, a bioinformatic analysis revealed conserved TEAD-binding motifs in the C-MYC and GLUT1 promoters providing another molecular data supporting our observations. In summary, regulation of glucose metabolism could serve as a new tumor suppressor mechanism orchestrated by the Hippo signaling pathway.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Genes myc/efeitos dos fármacos , Naftoquinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Apoptose/genética , Proliferação de Células/genética , Proteínas de Ligação a DNA/efeitos dos fármacos , Transportador de Glucose Tipo 1/metabolismo , Fator de Crescimento de Hepatócito , Humanos , Proteínas Nucleares/efeitos dos fármacos , Fosfoproteínas/efeitos dos fármacos , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/efeitos dos fármacos , Proteínas de Sinalização YAP
7.
J Nat Prod ; 79(9): 2304-14, 2016 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-27571379

RESUMO

Quambalarine B (QB) is a secondary metabolite produced by the basidiomycete Quambalaria cyanescens with potential anticancer activity. Here we report that QB at low micromolar concentration inhibits proliferation of several model leukemic cell lines (Jurkat, NALM6, and REH), whereas higher concentrations induce cell death. By contrast, the effect of QB on primary leukocytes (peripheral blood mononuclear cells) is significantly milder with lower toxicity and cytostatic activity. Moreover, QB inhibited expression of the C-MYC oncoprotein and mRNA expression of its target genes, LDHA, PKM2, and GLS. Finally, QB blocked the phosphorylation of P70S6K, a downstream effector kinase in mTOR signaling that regulates translation of C-MYC. This observation could explain the molecular mechanism behind the antiproliferative and cytotoxic effects of QB on leukemic cells. Altogether, our results establish QB as a promising molecule in anticancer treatment.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Basidiomycota/química , Naftoquinonas/química , Naftoquinonas/farmacologia , Antineoplásicos/sangue , Antineoplásicos/isolamento & purificação , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células Jurkat/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Estrutura Molecular , Naftoquinonas/sangue , Naftoquinonas/síntese química , Naftoquinonas/isolamento & purificação , Fosforilação , Proteínas Quinases S6 Ribossômicas 70-kDa , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR
8.
J Mass Spectrom ; 50(6): 802-11, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26169134

RESUMO

We report an MS-based workflow for identification of phosphorylated peptides from trypsinized protein mixtures and cell lysates that is suitable for high-throughput sample analysis. The workflow is based on an in situ enrichment on matrix-assisted laser desorption/ionization (MALDI) plates that were functionalized by TiO2 using automated ion landing apparatus that can operate unsupervised. The MALDI plate can be functionalized by TiO2 into any array of predefined geometry (here, 96 positions for samples and 24 for mass calibration standards) made compatible with a standard MALDI spotter and coupled with high-performance liquid chromatography. The in situ MALDI plate enrichment was compared with a standard precolumn-based separation and achieved comparable or better results than the standard method. The performance of this new workflow was demonstrated on a model mixture of proteins as well as on Jurkat cells lysates. The method showed improved signal-to-noise ratio in a single MS spectrum, which resulted in better identification by MS/MS and a subsequent database search. Using the workflow, we also found specific phosphorylations in Jurkat cells that were nonspecifically activated by phorbol 12-myristate 13-acetate. These phosphorylations concerned the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and its targets and were in agreement with the current knowledge of this signaling cascade. Control sample of non-activated cells was devoid of these phosphorylations. Overall, the presented analytical workflow is able to detect dynamic phosphorylation events in minimally processed mammalian cells while using only a short high-performance liquid chromatography gradient.


Assuntos
Cromatografia Líquida/métodos , Ensaios de Triagem em Larga Escala/métodos , Fosfopeptídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Desenho de Equipamento , Humanos , Células Jurkat , Fosfopeptídeos/química
10.
Free Radic Biol Med ; 50(11): 1546-55, 2011 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21402148

RESUMO

Mitochondria are emerging as intriguing targets for anti-cancer agents. We tested here a novel approach, whereby the mitochondrially targeted delivery of anti-cancer drugs is enhanced by the addition of a triphenylphosphonium group (TPP(+)). A mitochondrially targeted analog of vitamin E succinate (MitoVES), modified by tagging the parental compound with TPP(+), induced considerably more robust apoptosis in cancer cells with a 1-2 log gain in anti-cancer activity compared to the unmodified counterpart, while maintaining selectivity for malignant cells. This is because MitoVES associates with mitochondria and causes fast generation of reactive oxygen species that then trigger mitochondria-dependent apoptosis, involving transcriptional modulation of the Bcl-2 family proteins. MitoVES proved superior in suppression of experimental tumors compared to the untargeted analog. We propose that mitochondrially targeted delivery of anti-cancer agents offers a new paradigm for increasing the efficacy of compounds with anti-cancer activity.


Assuntos
Sistemas de Liberação de Medicamentos , Mitocôndrias/metabolismo , Compostos Organofosforados , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Tocoferóis , Animais , Apoptose/efeitos dos fármacos , Tratamento Farmacológico/tendências , Humanos , Células Jurkat , Modelos Animais , Terapia de Alvo Molecular , Compostos Organofosforados/química , Compostos Organofosforados/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Espécies Reativas de Oxigênio/metabolismo , Tocoferóis/química , Tocoferóis/farmacologia , Transcrição Gênica/efeitos dos fármacos
11.
Cancer Res ; 71(3): 946-54, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21245099

RESUMO

The proapoptotic protein Noxa, a member of the BH3-only Bcl-2 protein family, can effectively induce apoptosis in cancer cells, although the relevant regulatory pathways have been obscure. Previous studies of the cytotoxic effects of α-tocopheryl succinate (α-TOS) on cancer cells identified a mechanism whereby α-TOS caused apoptosis requiring the Noxa-Bak axis. In the present study, ab initio analysis revealed a conserved FoxO-binding site (DBE; DAF-16 binding element) in the NOXA promoter, and specific affinity of FoxO proteins to this DBE was confirmed by fluorescence anisotropy. FoxO1 and FoxO3a proteins accumulated in the nucleus of α-TOS-treated cells, and the drug-induced specific FoxO1 association with the NOXA promoter and its activation were validated by chromatin immunoprecipitation. Using siRNA knockdown, a specific role for the FoxO1 protein in activating NOXA transcription in cancer cells was identified. Furthermore, the proapoptotic kinase Hippo/Mst1 was found to be strongly activated by α-TOS, and inhibiting Hippo/Mst1 by specific siRNA prevented phosphorylation of FoxO1 and its nuclear translocation, thereby reducing levels of NOXA transcription and apoptosis in cancer cells exposed to α-TOS. Thus, we have demonstrated that anticancer drugs, exemplified by α-TOS, induce apoptosis by a mechanism involving the Hippo/Mst1-FoxO1-Noxa pathway. We propose that activation of this pathway provides a new paradigm for developing targeted cancer treatments.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , alfa-Tocoferol/farmacologia , Apoptose/fisiologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Linhagem Celular Tumoral , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células Jurkat , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , Linfoma de Células T/patologia , Linfoma de Células T/terapia , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transcrição Gênica
12.
Apoptosis ; 15(7): 782-94, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20217235

RESUMO

Mitocans are drugs selectively killing cancer cells by destabilizing mitochondria and many induce apoptosis via generation of reactive oxygen species (ROS). However, the molecular events by which ROS production leads to apoptosis has not been clearly defined. In this study with the mitocan alpha-tocopheryl succinate (alpha-TOS) the role of the Bcl-2 family proteins in the mechanism of malignant cell apoptosis has been determined. Exposure of several different cancer cell lines to alpha-TOS increased expression of the Noxa protein, but none of the other proteins of the Bcl-2 family, an event that was independent of the cellular p53 status. alpha-TOS caused a profound conformational change in the pro-apoptotic protein, Bak, involving oligomerization in all cell types, and this also applied to the Bax protein, but only in non-small cell lung cancer cells. Immunoprecipitation studies indicated that alpha-TOS activates the two BH1-3 proteins, Bak or Bax, to form high molecular weight complexes in the mitochondria. RNAi knockdown revealed that Noxa and Bak are required for alpha-TOS-induced apoptosis, and the role of Bak was confirmed using Bak- and/or Bax-deficient cells. We conclude that the major events induced by alpha-TOS in cancer cells downstream of ROS production leading to mitochondrial apoptosis involve the Noxa-Bak axis. It is proposed that this represents a common mechanism for mitochondrial destabilization activated by a variety of mitocans that induce accumulation of ROS in the early phases of apoptosis.


Assuntos
Antineoplásicos/toxicidade , Apoptose , Mitocôndrias/efeitos dos fármacos , alfa-Tocoferol/toxicidade , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Antineoplásicos/química , Humanos , Células Jurkat , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/genética , alfa-Tocoferol/química , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/genética
13.
Clin Cancer Res ; 15(5): 1593-600, 2009 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19223492

RESUMO

PURPOSE: Vitamin E analogues are potent novel anticancer drugs. The purpose of this study was to elucidate the cellular target by which these agents, represented by alpha-tocopoheryl succinate (alpha-TOS), suppress tumors in vivo, with the focus on the mitochondrial complex II (CII). EXPERIMENTAL DESIGN: Chinese hamster lung fibroblasts with functional, dysfunctional, and reconstituted CII were transformed using H-Ras. The cells were then used to form xenografts in immunocompromized mice, and response of the cells and the tumors to alpha-TOS was studied. RESULTS: The CII-functional and CII-reconstituted cells, unlike their CII-dysfunctional counterparts, responded to alpha-TOS by reactive oxygen species generation and apoptosis execution. Tumors derived from these cell lines reciprocated their responses to alpha-TOS. Thus, growth of CII-functional and CII-reconstituted tumors was strongly suppressed by the agent, and this was accompanied by high level of apoptosis induction in the tumor cells. On the other hand, alpha-TOS did not inhibit the CII-dysfunctional tumors. CONCLUSIONS: We document in this report a novel paradigm, according to which the mitochondrial CII, which rarely mutates in human neoplasias, is a plausible target for anticancer drugs from the group of vitamin E analogues, providing support for their testing in clinical trials.


Assuntos
Antioxidantes/uso terapêutico , Complexo II de Transporte de Elétrons/metabolismo , Neoplasias Pulmonares/prevenção & controle , Mitocôndrias/metabolismo , alfa-Tocoferol/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Transformação Celular Neoplásica , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Cricetinae , Cricetulus , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/genética , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Consumo de Oxigênio , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Mol Cell Biochem ; 311(1-2): 225-31, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18278581

RESUMO

We have shown previously that iron deprivation significantly stimulates the uptake of non-transferrin ferric iron from ferric citrate by erythroleukemia K562 cells and that this stimulation depends on protein synthesis. However, we have not detected increased expression of any known iron transport protein (Kovar J. et al. (2006) Blood Cells Mol Dis 37:95-99). Therefore, in order to identify membrane proteins of K562 cells with increased expression under iron deprivation, we employed the isolation of membrane proteins by two-phase partitioning system, protein separation by high-resolution 2D electrophoresis, computer differential analysis, and tandem mass spectrometry. Employing these techniques we identified two proteins with statistically significant upregulation, i.e., aldolase A (ALDA) and voltage-dependent anion channel 2 (VDAC2). The upregulation of aldolase A and VDAC2 in K562 cells under iron deprivation was also confirmed by western blot analysis. This is the first time when the control of aldolase A and VDAC2 levels by iron status of the cell is demonstrated.


Assuntos
Frutose-Bifosfato Aldolase/metabolismo , Regulação da Expressão Gênica , Deficiências de Ferro , Células K562 , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Frutose-Bifosfato Aldolase/genética , Humanos , Regulação para Cima , Canal de Ânion 2 Dependente de Voltagem/genética
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