RESUMO
BACKGROUND: Oolonghomobisflavans are unique polyphenols found in oolong teas. Oolonghomobisflavan B (OHBFB), a dimer of (-)-epigallocatechin-3-O-gallate (EGCG), is an active compound found in green tea. PURPOSE: OHBFB has been reported to exert an inhibitory effect on lipase enzyme activity. However, little is known regarding its intercellular signaling induction effect. Further, there are no reports describing the anti-cancer effects of OHBFB. METHODS: The effect of OFBFB on B16 melanoma cells was evaluated by cell counting, and its mechanisms were determined by western blot analysis with or without protein phosphatase 2A (PP2A) inhibitor treatment. Intracellular cyclic adenosine monophosphate (cAMP) levels were evaluated by time-resolved fluorescence resonance energy transfer analysis. Quartz crystal microbalance (QCM) analysis was performed to assess the binding of OHBFB to 67LR. RESULTS: Cell growth assay and western blot analyses showed that OHBFB inhibited melanoma cell growth, followed by myosin phosphatase target subunit 1 (MYPT1) and myosin regulatory light chain (MRLC) dephosphorylation via protein phosphatase 2A (PP2A)-dependent mechanisms. These effects are mediated by intracellular cAMP- and protein kinase A (PKA) A-dependent mechanisms. QCM analysis identified the 67-kDa laminin receptor (67LR) as an OHBFB receptor with a Kd of 3.7 µM. We also demonstrated for the first time that OHBFB intake suppresses tumor growth in vivo. CONCLUSIONS: Taken together, these results indicate that the cAMP/PKA/PP2A/MYPT1/MRLC pathway is a key mediator of melanoma cell growth inhibition following OHBFB binding to 67LR and that OHBFB suppresses tumor growth in vivo.
Assuntos
Catequina , Melanoma Experimental , Animais , Humanos , Proteína Fosfatase 2/metabolismo , Polifenóis/farmacologia , Catequina/farmacologia , Ciclo Celular , Melanoma Experimental/tratamento farmacológico , Receptores de Laminina/química , Receptores de Laminina/metabolismoRESUMO
BACKGROUND/AIMS: Pigment epithelium-derived factor (PEDF) is a potent endogenous inhibitor of angiogenesis, and a promising anticancer agent. We have previously shown that PEDF can be phosphorylated, and that distinct phosphorylations differentially regulate its physiological functions. We also demonstrated that triple phosphomimetic mutant (EEE-PEDF), has significantly increased antiangiogenic activity, and is much more efficient than WT-PEDF in inhibiting neovascularization and tumor growth. The enhanced antiangiogenic effect was associated with a direct ability to facilitate apoptosis of tumor-residing endothelial cells (EC), and subsequently, disruption of intratumoral vascularization. In the present report, we elucidated the molecular mechanism by which EEE-PEDF exerts more profound effects at the cellular level. METHODS: Here we used Western blotting, as well as in vitro binding, proliferation, apoptosis and migration assays to follow the signaling components responsible for the PEDF and EEE-PEDF effects. RESULTS: We found that EEE-PEDF suppresses EC proliferation due to caspase-3-dependent apoptosis, and also inhibits migration of the EC much better than WT-PEDF. Although WT-PEDF and EEE-PEDF did not affect proliferation and did not induce apoptosis of cancer cells, these agents efficiently inhibited cancer cell motility, with EEE-PEDF showing stronger effect. The stronger activity of EEE-PEDF was correlated to a better binding to laminin receptors. Furthermore, the proapoptotic and antimigratory activities of WT-PEDF and EEE-PEDF were found respectively regulated by differential activation of two distinct MAPK pathways, namely JNK and p38. We show that JNK and p38 phosphorylation is much higher in cells treated with EEE-PEDF. JNK leads to apoptosis of ECs, while p38 leads to antimigratory effect in both EC and cancer cells. CONCLUSION: These results reveal the molecular signaling mechanism by which the phosphorylated PEDF exerts its stronger antiangiogenic, antitumor activities.
Assuntos
Proteínas do Olho/farmacologia , Fatores de Crescimento Neural/farmacologia , Serpinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Bovinos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipase/química , Lipase/genética , Lipase/metabolismo , Mutagênese , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Receptores de Laminina/química , Receptores de Laminina/genética , Receptores de Laminina/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Serpinas/genética , Serpinas/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) has been attributed to the activation of its cell surface sensing receptor 67 kDa laminin receptor (67LR). However, the action of EGCG to activate 67LR remains unknown. Here we show that EGCG undergoes oligomer formation on its surface receptor 67LR.
Assuntos
Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Catequina/análogos & derivados , Receptores de Laminina/metabolismo , Chá/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Antioxidantes/química , Antioxidantes/metabolismo , Catequina/antagonistas & inibidores , Catequina/química , Catequina/farmacologia , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células Hep G2 , Humanos , Estrutura Molecular , Peptídeos/química , Peptídeos/farmacologia , Receptores de Laminina/química , Ressonância de Plasmônio de Superfície , TermodinâmicaRESUMO
Lysyl-tRNA synthetase (KRS) is an enzyme that conjugates lysine to its cognate tRNAs in the process of protein synthesis. In addition to its catalytic function, KRS binds to the 67-kDa laminin receptor (LR) on the cell membrane and facilitates cell migration and metastasis. Modulation of this interaction by small-molecule inhibitors can be exploited to suppress cancer metastasis. In this study, we present fragment-based methods for the identification of inhibitors and monitoring protein-protein interactions between KRS and LR. First, we identified the amino acid residues, located on the KRS anticodon-binding domain, which interact with the C-terminal extension of the LR. One-dimensional (1D) relaxation-edited nuclear magnetic resonance spectroscopy (NMR) and competition experiments were designed and optimized to screen the fragment library. For screening using two-dimensional (2D) NMR, we identified the indicative signals in the KRS anticodon-binding domain and selected inhibitors that bind to KRS and compete with LR at the KRS-LR binding interface. These methods may offer an efficient approach for the discovery of anti-metastatic drugs.
Assuntos
Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Lisina-tRNA Ligase/antagonistas & inibidores , Lisina/metabolismo , Receptores de Laminina/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Motivos de Aminoácidos , Anticódon/química , Anticódon/metabolismo , Antineoplásicos/farmacologia , Sítios de Ligação , Descoberta de Drogas/métodos , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Humanos , Lisina-tRNA Ligase/química , Lisina-tRNA Ligase/genética , Lisina-tRNA Ligase/metabolismo , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA de Transferência de Lisina/genética , RNA de Transferência de Lisina/metabolismo , Receptores de Laminina/química , Receptores de Laminina/genética , Receptores de Laminina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Aminoacilação de RNA de TransferênciaRESUMO
Aminoacyl-tRNA synthetases (ARSs) are essential enzymes that conjugate specific amino acids to their cognate tRNAs for protein synthesis. Besides their catalytic activity, recent studies have uncovered many additional functions of these enzymes through their interactions with diverse cellular factors. Among human ARSs, cytosolic lysyl-tRNA synthetase (KRS) is often highly expressed in cancer cells and tissues, and facilitates cancer cell migration and invasion through the interaction with the 67kDa laminin receptor on the plasma membrane. Specific modulation of this interaction by small molecule inhibitors has revealed a new way to control metastasis. Here, we summarize the pro-metastatic functions of KRS and their patho-physiological implications.
Assuntos
Carcinogênese/metabolismo , Lisina-tRNA Ligase/química , Lisina-tRNA Ligase/metabolismo , Transporte Ativo do Núcleo Celular , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Biocatálise , Membrana Celular/enzimologia , Movimento Celular , Transição Epitelial-Mesenquimal , Humanos , Modelos Biológicos , Modelos Moleculares , Invasividade Neoplásica , Metástase Neoplásica , Domínios e Motivos de Interação entre Proteínas , Receptores de Laminina/química , Receptores de Laminina/metabolismoRESUMO
The 37-kDa laminin receptor (37LRP or RPSA) is a remarkable, multifaceted protein that functions in processes ranging from matrix adhesion to ribosome biogenesis. Its ability to engage extracellular laminin is further thought to contribute to cellular migration and invasion. Most commonly associated with metastatic cancer, RPSA is also increasingly found to be important in other pathologies, including microbial infection, neurodegenerative disease and developmental malformations. Importantly, it is thought to have higher molecular weight forms, including a 67-kDa species (67LR), the expression of which is linked to strong laminin binding and metastatic behavior. The composition of these larger forms has remained elusive and controversial. Homo- and heterodimerization have been proposed as events capable of building the larger species from the monomeric 37-kDa precursor, but solid evidence is lacking. Here, we present data suggesting that higher molecular weight species require SUMOylation to form. We also comment on the difficulty of isolating larger RPSA species for unambiguous identification and demonstrate that cell lines stably expressing tagged RPSA for long periods of time fail to produce tagged higher molecular weight RPSA. It is possible that higher molecular weight species like 67LR are not derived from RPSA.
Assuntos
Receptores de Laminina/química , Receptores de Laminina/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Animais , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Imunoprecipitação , Camundongos , Peso Molecular , Células NIH 3T3 , Receptores de Laminina/genética , Proteínas Ribossômicas/genética , Sumoilação , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
The 67 kDa laminin receptor (67LR) is a non-integrin receptor for laminin (LM) that derives from a 37 kDa precursor (37LRP). 67LR expression is increased in neoplastic cells and correlates with an enhanced invasive and metastatic potential. We used structure-based virtual screening (SB-VS) to search for 67LR inhibitory small molecules, by focusing on a 37LRP sequence, the peptide G, able to specifically bind LM. Forty-six compounds were identified and tested on HEK-293 cells transfected with 37LRP/67LR (LR-293 cells). One compound, NSC47924, selectively inhibited LR-293 cell adhesion to LM with IC50 and Ki values of 19.35 and 2.45 µmol/L. NSC47924 engaged residues W176 and L173 of peptide G, critical for specific LM binding. Indeed, NSC47924 inhibited in vitro binding of recombinant 37LRP to both LM and its YIGSR fragment. NSC47924 also impaired LR-293 cell migration to LM and cell invasion. A subsequent hierarchical similarity search with NSC47924 led to the identification of additional four compounds inhibiting LR-293 cell binding to LM: NSC47923, NSC48478, NSC48861, and NSC48869, with IC50 values of 1.99, 1.76, 3.4, and 4.0 µmol/L, respectively, and able to block in vitro cancer cell invasion. These compounds are promising scaffolds for future drug design and discovery efforts in cancer progression.
Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Movimento Celular/efeitos dos fármacos , Descoberta de Drogas/métodos , Laminina/antagonistas & inibidores , Naftóis/farmacologia , Neoplasias/tratamento farmacológico , Receptores de Laminina/antagonistas & inibidores , Proteínas Ribossômicas/antagonistas & inibidores , Compostos de Anilina/química , Antineoplásicos/química , Adesão Celular/efeitos dos fármacos , Simulação por Computador , Desenho Assistido por Computador , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Laminina/metabolismo , Modelos Moleculares , Naftóis/química , Invasividade Neoplásica , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Conformação Proteica , Receptores de Laminina/química , Receptores de Laminina/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , TransfecçãoRESUMO
Insufficient trophoblast invasion often occurs in patients experiencing preeclampsia. The 67-kDa laminin receptor (LR1) is a multifunctional protein that binds to laminin and interacts with the extracellular matrix. We recently demonstrated that LR1 is implicated in trophoblast migration and invasion. However, whether LR1 is involved in hypoxia-mediated trophoblastic invasion remains unclear and requires further investigation. This study demonstrates that two trophoblast-like cell lines (JEG3 and BeWo cells) cultured at 3% oxygen exerted enhanced migratory and invasive capabilities as compared with their counterparts exposed to 20% oxygen. LR1 expression was increased in hypoxic JEG3 cells but decreased after transfection with hypoxia-inducible factor 1 alpha (HIF-1α) specific siRNA. Moreover, shRNA targeting LR1 mRNA significantly inhibited hypoxia-induced increase in matrix metalloproteinase (MMP)-9 activity in JEG3 cells. Forced overexpression of LR1 augmented JEG3 cell migration and invasion, and enhanced MMP-9 expression and activity. Additionally, the blockade of the MMP-9 effect with its neutralizing antibody reduced LR1 elevation-promoted trophoblastic invasion. In summary, this study demonstrates that LR1 contributes to hypoxia-induced migration and invasion of trophoblast cells at least partly by mediating MMP-9 in vitro.
Assuntos
Movimento Celular , Metaloproteinase 9 da Matriz/metabolismo , Trofoblastos/citologia , Hipóxia Celular , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Metaloproteinase 9 da Matriz/genética , Peso Molecular , Receptores de Laminina/química , Receptores de Laminina/deficiência , Receptores de Laminina/genética , Receptores de Laminina/metabolismo , Proteínas RibossômicasRESUMO
Lysyl-tRNA synthetase (KRS) interacts with the laminin receptor (LR/RPSA) and enhances laminin-induced cell migration in cancer metastasis. In this nuclear magnetic resonance (NMR)-based study, we show that the anticodon-binding domain of KRS binds directly to the C-terminal region of 37LRP, and the previously found inhibitors BC-K-01 and BC-K-YH16899 interfere with KRS-37LRP binding. In addition, the anticodon-binding domain of KRS binds to laminin, observed by NMR and SPR. These results provide crucial insights into the structural characteristics of the KRS-LR interaction on the cell surface.
Assuntos
Lisina-tRNA Ligase/metabolismo , Ressonância Magnética Nuclear Biomolecular , Receptores de Laminina/metabolismo , Anticódon/metabolismo , Membrana Celular/metabolismo , Humanos , Lisina-tRNA Ligase/química , Modelos Moleculares , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Laminina/químicaRESUMO
Delivery of optimal amounts of brain-derived neurotrophic factor (BDNF) to regions of the brain affected by neurodegenerative diseases is a daunting task. Using natural products with neuroprotective properties, such as green tea polyphenols, would be a highly useful complementary approach for inexpensive long-term treatment of these diseases. In this study, we used PC12(TrkB) cells which ectopically express TrkB, a high affinity receptor for BDNF. They differentiate and induce neurite outgrowth in response to BDNF. Using this model, we show for the first time that treatment with extremely low concentrations (<0.1 µg/ml) of unfractionated green tea polyphenols (GTPP) and low concentrations (<0.5 µM) of their active ingredient, epigallocatechin-3-gallate (EGCG), potentiated the neuritogenic ability of a low concentration (2 ng/ml) of BDNF. A synergistic interaction was observed between GTPP constituents, where epigallocatechin and epicatechin, both individually lacking this activity, promoted the action of EGCG. GTPP-induced potentiation of BDNF action required the cell-surface associated 67 kDa laminin receptor (67LR) to which EGCG binds with high affinity. A cell-permeable catalase abolished GTPP/EGCG-induced potentiation of BDNF action, suggesting the possible involvement of H2O2 in the potentiation. Consistently, exogenous sublethal concentrations of H2O2, added as a bolus dose (5 µM) or more effectively through a steady-state generation (1 µM), potentiated BDNF action. Collectively, these results suggest that EGCG, dependent on 67 LR and H2O2, potentiates the neuritogenic action of BDNF. Intriguingly, this effect requires only submicromolar concentrations of EGCG. This is significant as extremely low concentrations of polyphenols are believed to reach the brain after drinking green tea.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Catequina/farmacologia , Neuritos/efeitos dos fármacos , Chá/química , Animais , Antioxidantes/farmacologia , Catequina/análogos & derivados , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Peso Molecular , Neuritos/fisiologia , Oxidantes/metabolismo , Oxidantes/farmacologia , Células PC12 , Polifenóis/farmacologia , Ratos , Receptor trkB/genética , Receptor trkB/metabolismo , Receptores de Laminina/química , Receptores de Laminina/metabolismo , Receptores de Laminina/fisiologiaRESUMO
Parkin was originally identified as a protein associated with Parkinson's disease. Recently, numerous research studies have suggested that parkin acts as a tumor suppressor. In accordance with these studies, we previously reported that overexpression of parkin in HeLa cells induced growth inhibition. To elucidate possible mechanisms by which parkin may inhibit cell growth, HeLa cells were infected with adenoviruses expressing either the parkin gene or adenovirus alone for 72 h and a total proteomic analysis was performed using 2-D gel electrophoresis followed by LC-MS/MS. We identified three proteins whose expression changed between the two groups: the 40S ribosomal protein SA (RPSA) was downregulated in parkin virus-infected cells, and cytokeratins 8 and 18 exhibited an acid shift in pI value without a change in molecular weight, suggesting that these proteins became phosphorylated in parkin virus-infected cells. The changes in these three proteins were first observed at 60 h postinfection and were most dramatic at 72 h postinfection. Because upregulation of RPSA and dephosphorylation of cytokeratins 8/18 have been linked with tumor progression, these data suggest that parkin may inhibit cell growth, at least in part, by decreasing RPSA expression and inducing phosphorylation of cytokeratin 8/18.
Assuntos
Queratina-18/metabolismo , Queratina-8/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Receptores de Laminina/metabolismo , Proteínas Ribossômicas/metabolismo , Ubiquitina-Proteína Ligases/farmacologia , Regulação para Cima/efeitos dos fármacos , Neoplasias do Colo do Útero/patologia , Sequência de Aminoácidos , Proliferação de Células/efeitos dos fármacos , Feminino , Células HeLa , Humanos , Queratina-18/química , Queratina-8/química , Dados de Sequência Molecular , Receptores de Laminina/química , Proteínas Ribossômicas/químicaRESUMO
The 67-kDa laminin receptor (67LR) is a laminin-binding protein overexpressed in various types of cancer, including bile duct carcinoma, colorectal carcinoma, cervical cancer, and breast carcinoma. 67LR plays a vital role in growth and metastasis of tumor cells and resistance to chemotherapy. Here, we show that 67LR functions as a cancer-specific death receptor. In this cell death receptor pathway, cGMP initiated cancer-specific cell death by activating the PKCδ/acid sphingomyelinase (PKCδ/ASM) pathway. Furthermore, upregulation of cGMP was a rate-determining process of 67LR-dependent cell death induced by the green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG), a natural ligand of 67LR. We found that phosphodiesterase 5 (PDE5), a negative regulator of cGMP, was abnormally expressed in multiple cancers and attenuated 67LR-mediated cell death. Vardenafil, a PDE5 inhibitor that is used to treat erectile dysfunction, significantly potentiated the EGCG-activated 67LR-dependent apoptosis without affecting normal cells and prolonged the survival time in a mouse xenograft model. These results suggest that PDE5 inhibitors could be used to elevate cGMP levels to induce 67LR-mediated, cancer-specific cell death.
Assuntos
Apoptose/fisiologia , GMP Cíclico/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Receptores de Laminina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Catequina/análogos & derivados , Catequina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Feminino , Humanos , Imidazóis/farmacologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Neoplasias/tratamento farmacológico , Inibidores da Fosfodiesterase 5/farmacologia , Piperazinas/farmacologia , Receptores de Laminina/química , Transdução de Sinais , Sulfonas/farmacologia , Triazinas/farmacologia , Dicloridrato de Vardenafila , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The 67 kDa high affinity laminin receptor (67LR) is a non integrin cell surface receptor for the extracellular matrix whose expression is increased in neoplastic cells and directly correlates with an enhanced invasive and metastatic potential. 67LR derives from homo- or hetero-dimerization of a 37 kDa cytosolic precursor (37LRP), by fatty acid acylation. Interestingly, 37LRP is a multifunctional protein involved in the translational machinery and has also been found in the nucleus, where it is tightly associated with nuclear structures. Acting as a receptor for laminin is not the only function of this protein; indeed, 67LR also acts as a receptor for viruses, such as Sindbis virus and Dengue virus, and is involved in the internalization of the prion protein. Here, we review the current understanding of the structure and function of this molecule, highlighting its role in cancer and infection diseases.
Assuntos
Infecções/etiologia , Neoplasias/etiologia , Receptores de Laminina/fisiologia , Proteínas Ribossômicas/fisiologia , Animais , Humanos , Príons/fisiologia , Receptores de Laminina/química , Proteínas Ribossômicas/químicaRESUMO
(-)-Epigallocatechin-3-O-gallate (EGCG) exhibits anti-tumor activity mediated via the 67-kDa laminin receptor (67LR). In this study, we found that 67LR protein levels are reduced by exposure to low O(2) levels (5%), without affecting the expression of HIF-1α. We also found that EGCG-induced anti-cancer activity is abrogated under low O(2) levels (5%) in various cancer cells. Notably, treatment with the proteasome inhibitor, prevented down-regulation of 67LR and restored sensitivity to EGCG under 5% O(2). In summary, 67LR expression is highly sensitive to O(2) partial pressure, and the activity of EGCG can be regulated in cancer cells by O(2) partial pressure.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Catequina/análogos & derivados , Oxigênio/metabolismo , Receptores de Laminina/metabolismo , Animais , Sequência de Bases , Catequina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Primers do DNA/genética , Células HeLa , Células Hep G2 , Humanos , Hipóxia/metabolismo , Hipóxia/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Inibidores de Proteassoma/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Laminina/química , Receptores de Laminina/genética , UbiquitinaçãoRESUMO
Human nonintegrin laminin receptor is a multifunctional protein acting as an integral component of the ribosome and a cell surface receptor for laminin-1. The laminin receptor is overexpressed in several human cancers and is also the cell surface receptor for several viruses and pathogenic prion proteins, making it a pathologically significant protein. This study focused on the proteomic characterization of laminin receptor interacting proteins from Mus musculus. The use of affinity chromatography with immobilized recombinant laminin receptor coupled with mass spectrometry analysis identified 45 proteins with high confidence. Following validation through coimmunoprecipitation, the proteins were classified based on predicted function into ribosomal, RNA processing, signal transduction/metabolism, protein processing, cytoskeleton/cell anchorage, DNA/chromatin, and unknown functions. A significant portion of the identified proteins is related to functions or localizations previously described for laminin receptor. This work represents a comprehensive proteomic approach to studying laminin receptor and provides an essential stepping stone to a better mechanistic understanding of this protein's diverse functions.
Assuntos
Proteoma/química , Animais , Chaperonina com TCP-1/química , Chaperonina com TCP-1/isolamento & purificação , Cromatografia de Afinidade , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/isolamento & purificação , Hexosiltransferases , Histonas/química , Histonas/isolamento & purificação , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Camundongos , Células NIH 3T3 , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/isolamento & purificação , Ligação Proteica , Proteoma/isolamento & purificação , Proteômica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/isolamento & purificação , Receptores de Laminina/química , Proteínas Ribossômicas/química , Proteínas Ribossômicas/isolamento & purificação , Fatores de Transcrição/química , Fatores de Transcrição/isolamento & purificaçãoRESUMO
BACKGROUND: We previously identified the 67-kDa laminin receptor (67LR) as the cell-surface receptor conferring the major green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) responsiveness to cancer cells. However, the underlying mechanism for interaction between EGCG and 67LR remains unclear. In this study, we investigated the possible role of EGCG-67LR interaction responsible for its bioactivities. METHODOLOGY/PRINCIPAL FINDINGS: We synthesized various peptides deduced from the extracellular domain corresponding to the 102-295 region of human 67LR encoding a 295-amino acid. The neutralizing activity of these peptides toward EGCG cell-surface binding and inhibition of cancer cell growth were assayed. Both activities were inhibited by a peptide containing the 10-amino acid residues, IPCNNKGAHS, corresponding to residues 161-170. Furthermore, mass spectrometric analysis revealed the formation of a EGCG-LR161-170 peptide complex. A study of the amino acid deletion/replacement of the peptide LR161-170 indicated that the 10-amino acid length and two basic amino acids, K(166) and H(169), have a critical role in neutralizing EGCG's activities. Moreover, neutralizing activity against the anti-proliferation action of EGCG was observed in a recombinant protein of the extracellular domain of 67LR, and this effect was abrogated by a deletion of residues 161-170. These findings support that the 10 amino-acid sequence, IPCNNKGAHS, might be the functional domain responsible for the anti-cancer activity of EGCG. CONCLUSIONS/SIGNIFICANCE: Overall, our results highlight the nature of the EGCG-67LR interaction and provide novel structural insights into the understanding of 67LR-mediated functions of EGCG, and could aid in the development of potential anti-cancer compounds for chemopreventive or therapeutic uses that can mimic EGCG-67LR interactions.
Assuntos
Catequina/análogos & derivados , Polifenóis/metabolismo , Receptores de Laminina/química , Receptores de Laminina/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Chá/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Catequina/metabolismo , Catequina/farmacologia , Proliferação de Células/efeitos dos fármacos , Espaço Extracelular/metabolismo , Células Hep G2 , Humanos , Dados de Sequência Molecular , Polifenóis/farmacologia , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
The human ribosomal protein SA (RPSA) is a multilocus protein, present in most cellular compartments. It is a multifunctional protein, which belongs to the ribosome but is also a membrane receptor for laminin, growth factors, prion, pathogenic microorganisms, toxins, and the anticarcinogen epigallocatechin gallate. It contributes to the crossing of the blood-brain barrier by neurotropic viruses and bacteria and is used as a biomarker of metastasis. RPSA includes an N-terminal domain, which is homologous to the prokaryotic ribosomal proteins S2, and a C-terminal extension, which is conserved in vertebrates. The structure of its N-domain has been determined from crystals grown at 17 °C. The structure of its C-domain remains unknown. We produced in Escherichia coli and purified the full-length RPSA and its N- and C-domains. We characterized the folding states of these recombinant proteins mainly by methods of fluorescence and circular dichroism spectrometry, in association with quantitative analyses of their unfolding equilibria, induced with heat or urea. The necessary equations were derived from first principles. The results showed that the N-domain unfolded according to a three-state equilibrium. The monomeric intermediate was predominant at the body temperature of 37 °C. It also existed in the full-length RPSA and bound ANS, a small fluorescent molecule. The C-domain was in an intrinsically disordered state. The recombinant N- and C-domains weakly interacted together. These results indicated a high plasticity of RPSA, which could be important for its multiple cellular localizations and functional interactions.
Assuntos
Anticarcinógenos/metabolismo , Laminina/metabolismo , Microbiologia , Dobramento de Proteína , Receptores de Laminina/química , Receptores de Laminina/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Desdobramento de Proteína/efeitos dos fármacos , Receptores de Laminina/isolamento & purificação , Proteínas Ribossômicas/isolamento & purificação , Espectrometria de Fluorescência , Ureia/farmacologiaRESUMO
The 37/67-kDa human laminin receptor(LamR) is a cell surface protein that interacts with molecules located in the extra-cellular matrix. In particular, interactions between LamR and laminins play a major role in mediating changes in the cellular environment that affect cell adhesion, neurite outgrowth, tumor growth and metastasis. The exact interaction mode of laminin-1 and LamR is not fully understood. Laminin-1 is thought to bind to LamR through interaction with the so-called peptide G (residues 161180) and the C-terminal helix (residues 205229). Here we performed 100-ns atomistic force field based molecular dynamics simulations to explore the structure and dynamics of LamR related to laminin-1 interactions. Our main finding is that loop 188197 in the C-terminal region is highly flexible. It undergoes a major change resulting in a conformational switch that partially solvent exposes the R180 residue in the final part of the G peptide. So, R180 could contribute to laminin-1 binding. Projection of the simulations along the first two principal components also confirms the importance of this conformational switch in the LamR. This may be a basic prerequisite to clarify the key structural determinants of the interaction of LamR with laminin-1.
Assuntos
Laminina/metabolismo , Simulação de Dinâmica Molecular , Receptores de Laminina/química , Receptores de Laminina/metabolismo , Sequência de Aminoácidos , Humanos , Laminina/química , Dados de Sequência Molecular , Ligação Proteica , Conformação ProteicaRESUMO
Green tea polyphenols have emerged over the past two decades as an important dietary factor for health promotion. There is considerable evidence that tea polyphenols, in particular (-)-epigallocatechin-3-gallate (EGCG) inhibit carcinogenesis. However, the mechanisms for the cancer-preventive activity of EGCG are not completely characterized and many features remain to be elucidated. Recently we have identified a cell-surface EGCG receptor and the relating molecules that confer EGCG responsiveness to many cancer cells at physiological concentrations. Here, we review some of the reported mechanisms for the cancer chemopreventive action of EGCG and provide an overview of several molecules that sense and manage the physiological functions of EGCG.