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ABSTRACT: Radiotherapy plays a critical role in the treatment of non-small cell lung cancer (NSCLC). However, radioresistance is a major barrier against increasing the efficiency of radiotherapy for NSCLC. To understand the mechanisms underlying NSCLC radioresistance, we previously focused on the potential involvement of PIM1, PRAS40, FOXO3a, 14-3-3, and protein phosphatases. Among these proteins, PIM1 functioned as an oncogene and was found to act as a crucial mediator in radioresistant NSCLC cells. Therefore, we investigated the use of PIM1-specific inhibitors as novel therapeutic drugs to regulate radiosensitivity in NSCLC. After structure-based drug selection, SGI-1776, ETP-45299, and tryptanthrin were selected as candidates of PIM1 inhibitors that act as radiosensitizers. With irradiation, these drugs inhibited only PIM1 kinase activity without affecting PIM1 mRNA/protein levels or cellular localization. When PIM1 kinase activity was suppressed by these inhibitors, PRAS40 was not phosphorylated. Consequently, unphosphorylated PRAS40 did not form trimeric complexes with 14-3-3 and FOXO3a, leading to increased nuclear localization of FOXO3a. Nuclear FOXO3a promoted the expression of pro-apoptotic proteins such as Bim and FasL, resulting in a radiosensitizing effect on radioresistant NSCLC cells. Moreover, an in vivo xenograft mouse model confirmed this radiosensitizing effect induced by PIM1 inhibitors. In these model systems, tumor volume was significantly reduced by a combinational treatment with irradiation and PIM1 inhibitors compared to irradiation alone. Taken together, our findings provided evidence that PIM1-specific inhibitors, SGI-1776, ETP-45299, and tryptanthrin, can act as novel radiosensitizers to enhance the efficacy of radiotherapy by inhibiting irradiation-induced signaling pathway associated with radioresistance.
Pharmacological Research 01/2013; · 4.44 Impact Factor
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ABSTRACT: Radioresistance is a main impediment to effective radiotherapy for non-small cell lung cancer (NSCLC). Despite several experimental and clinical studies of resistance to radiation, the precise mechanism of radioresistance in NSCLC cells and tissues still remains unclear. This result could be explained by limitation of previous researches such as a partial understanding of the cellular radioresistance mechanism at a single molecule level. In this study, we aimed to investigate extensive radiation responses in radioresistant NSCLC cells and to identify radioresistance-associating factors. For the first time, using RNA-seq, a massive sequencing-based approach, we examined whole-transcriptome alteration in radioresistant NSCLC A549 cells under irradiation, and verified significant radiation-altered genes and their chromosome distribution patterns. Also, bioinformatic approaches (GO analysis and IPA) were performed to characterize the radiation responses in radioresistant A549 cells. We found that epithelial-mesenchymal transition (EMT), migration and inflammatory processes could be meaningfully related to regulation of radiation responses in radioresistant A549 cells. Based on the results of bioinformatic analysis for the radiation-induced transcriptome alteration, we selected seven significant radiation-altered genes (SESN2, FN1, TRAF4, CDKN1A, COX-2, DDB2 and FDXR) and then compared radiation effects in two types of NSCLC cells with different radiosensitivity (radioresistant A549 cells and radiosensitive NCI-H460 cells). Interestingly, under irradiation, COX-2 showed the most significant difference in mRNA and protein expression between A549 and NCI-H460 cells. IR-induced increase of COX-2 expression was appeared only in radioresistant A549 cells. Collectively, we suggest that COX-2 (also known as prostaglandin-endoperoxide synthase 2 (PTGS2)) could have possibility as a putative biomarker for radioresistance in NSCLC cells.
PLoS ONE 01/2013; 8(3):e59319. · 4.09 Impact Factor
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ABSTRACT: Radioresistance is considered as a main factor restricting efficacy of radiotherapy. However, the exact molecular mechanism of radioresistance has not been explained yet. In this study, to elucidate radioresistance mechanism in lung cancer, we compared radiation responses in two types of non-small cell lung cancer (NSCLC) cells with different radiosensitivity, and identified key molecules conferring radioresistance. In radioresistant NSCLC cells, ionizing radiation (IR) led to CK2α- and PKC-mediated phosphorylation of rpS3 and TRAF2, respectively, which induced dissociation of rpS3-TRAF2 complex and NF-κB activation, resulting in significant up-regulation of pro-survival genes (cIAP1, cIAP2 and survivin). Also, dissociated phospho-rpS3 translocated into nucleus and bound with NF-κB complex (p65 and p50) contributing to p65 DNA binding property and specificity. However, in radiosensitive NSCLC cells, IR-mediated rpS3 phosphorylation was not detected, due to absence of CK2α overexpression. Consequently, IR-induced rpS3-TRAF2 complex dissociation, NF-κB activation and pro-survival gene expression were not presented. Taken together, our findings revealed a novel radioresistance mechanism through functional orchestration of rpS3, TRAF2 and NF-κB in NSCLC cells. Moreover, we provided the first evidence for function of rpS3 as a new TRAF2-binding protein, and demonstrated that phosphorylation of both rpS3 and TRAF2 is a key control point of radioresistance in NSCLC cells. These results suggest that regulation of rpS3 and TRAF2 in combination with radiotherapy could have high pharmacological therapeutic potency for radioresistance of NSCLC.
Journal of Biological Chemistry 11/2012; · 4.77 Impact Factor
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ABSTRACT: Beta 1,4-galactosyltransferase 1 (B4GALT1) synthesizes galactose β-1,4-N-acetylglucosamine (Galβ1-4GlcNAc) groups on N-linked sugar chains of glycoproteins, which play important roles in many biological events, including the proliferation and migration of cancer cells. A previous microarray study reported that this gene is expressed by estrogen treatment in breast cancer. In this study, we examined the regulatory mechanisms and biological functions of estrogen-induced B4GALT1 expression. Our data showed that estrogen-induced expression of B4GALT1 is localized in intracellular compartments and in the plasma membrane. In addition, B4GALT1 has an enzyme activity involved in the production of the Galβ1-4GlcNAc structure. The result from a promoter assay and chromatin immunoprecipitation revealed that 3 different estrogen response elements (EREs) in the B4GALT1 promoter are critical for responsiveness to estrogen. In addition, the estrogen antagonists ICI 182,780 and ER-α-ERE binding blocker TPBM inhibit the expression of estrogen-induced B4GALT1. However, the inhibition of signal molecules relating to the extra-nuclear pathway, including the G-protein coupled receptors, Ras, and mitogen-activated protein kinases, had no inhibitory effects on B4GALT1 expression. The knock-down of the B4GALT1 gene and the inhibition of membrane B4GALT1 function resulted in the significant inhibition of estrogen-induced proliferation of MCF-7 cells. Considering these results, we propose that estrogen regulates the expression of B4GALT1 through the direct binding of ER-α to ERE and that the expressed B4GALT1 plays a crucial role in the proliferation of MCF-7 cells through its activity as a membrane receptor.
Biochemical and Biophysical Research Communications 09/2012; 426(4):620-5. · 2.48 Impact Factor
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ABSTRACT: Several recent biological science studies have been focused on nanotechnology and nanomaterials due to their potential use in biomedicine. Drug delivery systems are an example of biomedical applications utilizing nanoparticles. Silver nanoparticles (AgNPs) can be used for these drug delivery systems. However, the effects of cytotoxicity caused by AgNPs are not fully understood. Determining the optimal characteristics to facilitate the biocompatibility of AgNPs is an important subject for application. In the present study, human erythrocytes were used as an in vitro model to examine the size, dose, and coating surfactant-dependent cytotoxicity of AgNPs. Our results demonstrated that polyvinylpyrrolidone (PVP) was a more suitable surfactant than polyethylene glycol (PEG) for AgNPs capping. In addition, we determined the appropriate particular size and dosage of AgNPs to reduce human erythrocytes hemolysis. Membrane damages including hemolysis, potassium efflux, protein leakage, and alterations in cell shape and membrane fragility were minimized with 100-nm AgNP particles. This study provides novel insights into AgNPs cytotoxicity and a basis for utilizing AgNPs for diagnostic and therapeutic applications.
Journal of Nanoscience and Nanotechnology 08/2012; 12(8):6168-75. · 1.56 Impact Factor
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12/2011; , ISBN: 978-953-307-746-8
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ABSTRACT: Resistance of cancer cells to ionizing radiation plays an important role in the clinical setting of lung cancer treatment. To date, however, the exact molecular mechanism of radiosensitivity has not been well explained. In this study, we compared radioresistance in two types of non-small cell lung cancer (NSCLC) cells, NCI-H460 and A549, and investigated the signaling pathways that confer radioresistance. In radioresistant cells, exposure to radiation led to overexpression of PIM1 and reduction of protein phosphatases (PP2A and PP5), which induced translocation of PIM1 into the nucleus. Increased nuclear PIM1 phosphorylated PRAS40. Consequently, pPRAS40 made a trimeric complex with 14-3-3 and AKT-activated pFOXO3a, which then moved rapidly to the cytoplasm. Cytoplasmic retention of FOXO3a was associated with downregulation of proapoptotic genes and possibly radioresistance. On the other hand, no suppressive effect of radiation on protein phosphatases was detected and, concomitantly, protein phosphatases downregulated PIM1 in radiosensitive cells. In this setting, PIM1-activated pPRAS40, AKT-activated pFOXO3a, and their complex formation with 14-3-3 could be key regulators of the radiation-induced radioresistance in NSCLC cells.
Radiation Research 09/2011; 176(5):539-52. · 2.68 Impact Factor
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ABSTRACT: Radiotherapy is the most significant non-surgical cure for the elimination of tumor, however it is restricted by two major problems: radioresistance and normal tissue damage. Efficiency improvement on radiotherapy is demanded to achieve cancer treatment. We focused on radiation-induced normal cell damage, and are concerned about inflammation reported to act as a main limiting factor in the radiotherapy. Psoralidin, a coumestan derivative isolated from the seed of Psoralea corylifolia, has been studied for anti-cancer and anti-bacterial properties. However, little is known regarding its effects on IR-induced pulmonary inflammation. The aim of this study is to investigate mechanisms of IR-induced inflammation and to examine therapeutic mechanisms of psoralidin in human normal lung fibroblasts and mice. Here, we demonstrated that IR-induced ROS activated cyclooxygenases-2 (COX-2) and 5-lipoxygenase (5-LOX) pathway in HFL-1 and MRC-5 cells. Psoralidin inhibited the IR-induced COX-2 expression and PGE(2) production through regulation of PI3K/Akt and NF-κB pathway. Also, psoralidin blocked IR-induced LTB(4) production, and it was due to direct interaction of psoralidin and 5-lipoxygenase activating protein (FLAP) in 5-LOX pathway. IR-induced fibroblast migration was notably attenuated in the presence of psoralidin. Moreover, in vivo results from mouse lung indicate that psoralidin suppresses IR-induced expression of pro-inflammatory cytokines (TNF-α, TGF-β, IL-6 and IL-1 α/β) and ICAM-1. Taken together, our findings reveal a regulatory mechanism of IR-induced pulmonary inflammation in human normal lung fibroblast and mice, and suggest that psoralidin may be useful as a potential lead compound for development of a better radiopreventive agent against radiation-induced normal tissue injury.
Biochemical pharmacology 06/2011; 82(5):524-34. · 4.25 Impact Factor
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ABSTRACT: Invasive infections of Candida albicans are life-threatening clinical conditions affecting immunosuppressed patients. To maintain genome integrity and diversity, C. albicans utilizes DNA repair systems, such as nucleotide excision repair (NER), to escape from attack by macrophages. Rad3 helicase is a component of the TFIIH complex, which plays a role in transcription and the NER pathway. Accumulated evidence of studies from Archaea to humans has revealed that the conserved structure, including an iron-containing domain, is essential in the function of Rad3 helicase activity. However, no study of the Rad3 protein of C. albicans has yet been reported. In the present study, putative C. albicans Rad3 (CaRad3) has been cloned with orf19.7119 of the Candida genome. CaRad3 proteins were over-expressed and purified from E. coli and S. cerevisiae using a Ni-NTA column and a size exclusion column for physicochemical and functional characterization. Through EMR and spectrometric analysis, we have proven that the purified CaRad3 protein has a Fe-S cluster. We also revealed that CaRad3 protein has a helicase activity on a duplex DNA substrate. Furthermore, we showed that the CaRad3 protein purified from yeasts was N-glycosylated, and that this protein complemented the defects in both the NER pathway and transcription. These data suggest that the Rad3 helicase in C. albicans is the product of the orf19.7119 gene.
Biochemistry (Moscow) 06/2011; 76(6):666-76. · 1.06 Impact Factor
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ABSTRACT: Cytochrome P450s (P450s) are the most versatile biological catalysts in plants; however, because the structure of the P450s has not been fully established, their broad substrate specificity has been limitedly discussed. p-coumarate-3-hydroxylase (C3H) is an essential enzyme for the biosynthesis of phenolic natural products in plants, but all attempts to express and purify C3H, have failed. In this research, we developed a bacterial expression of Arabidopsis C3H by combinational mutagenesis and purified C3H as a catalytically active form. The modified C3H could be purified in the absence of detergent, and crystallized in two forms (orthorhombic and trigonal space group) under different conditions. X-ray diffraction was processed to a 4.0 Å resolution (first type crystal) and a 3.8 Å resolution (second type crystal). Although the diffraction results of C3H(mod) crystals are not enough to determine crystallographic structure due to low resolution, the simplicity and rapidity of this technology are competitive advantages in comparison with other methods, and may contribute to structural analyses of other membrane proteins including P450s family.
Protein Expression and Purification 04/2011; 79(1):149-55. · 1.59 Impact Factor
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ABSTRACT: Radiotherapy, frequently used for treatment of solid tumors, carries two main obstacles including acquired radioresistance in cancer cells during radiotherapy and normal tissue injury. Phenylpropanoids, which are naturally occurring phytochemicals found in plants, have been identified as potential radiotherapeutic agents due to their anti-cancer activity and relatively safe levels of cytotoxicity. Various studies have proposed that these compounds could not only sensitize cancer cells to radiation resulting in inhibition of growth and cell death but also protect normal cells against radiation-induced damage. This review is intended to provide an overview of recent investigations on the usage of phenylpropanoids in combination with radiotherapy in cancer treatment.
Experimental and Molecular Medicine 04/2011; 43(6):323-33. · 2.48 Impact Factor
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ABSTRACT: Plants in general have multiple lipoxygenase isoforms that exhibit distinct biochemical attributes and discrete cellular and sub-cellular localization patterns. This suggests that plant lipoxygenases are multifunctional and contribute to several physiological processes during growth and development. Soybeans have at least eight isoforms. Three that are localized to the seed (L-1; L-2; L-3) have been characterized extensively. The vegetative organs have five additional isoforms that are not well characterized. To better understand the potential cellular function of these vegetative lipoxygenases (VLXs), kinetic parameters and pH profiles with multiple substrates coupled with structural studies reveal differences within this family of enzymes. In addition, clear differences exist between VLX-A, -B & -E and VLX-C & -D in the exposed loops of the -barrel, O 2 cavity and entry site of the subcavity IIa. Therefore, based on distinct patterns of activity and structure, the existence of functional subgroups among the five VLX isoforms was confirmed.
International Journal of Biology. 02/2011; 3:1916-968.
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ABSTRACT: Deregulation of cell survival pathways and resistance to apoptosis are generally accepted as crucial aspects of tumorigenesis. As in many tumors, increasing occurrence of human skin cancer and other conflicting effects of solar ultraviolet (UV) radiation enhance the demand for novel chemoprevention agents. Myricetin, a naturally occurring phytochemical, is potent in anti-cancer promoting activity and affords to the chemopreventive potential of several healthy-foods, including fruits and vegetables. We demonstrate here that myricetin inhibits Akt activity to induce apoptosis in a low dose ('repairable dose') UVB-irradiated keratinocytes. Treatment of UVB-irradiated HaCaT cells with an apoptosis-inducing concentration of myricetin (20 microM) resulted in a decrease in phosphorylation of Akt leading to inhibition of its kinase activity. Myricetin treatment also caused a decrease in phosphorylation of Bad (a pro-apoptotic protein), a direct target of Akt in signaling pathway. Interaction between Bad and 14-3-3beta was reduced markedly in UVB-irradiated cells upon a treatment with myricetin. Comparable to these results, myricetin treatment promoted mitochondrial translocation of Bad, loss of the mitochondrial membrane potential, and release of the mitochondrial apoptotic proteins including cytochrome c, Smac, and AIF. Ectopic expression of constitutively active Akt granted statistically significant protection against myricetin-induced apoptosis. In addition, myricetin-induced apoptosis in UVB-irradiated cells was notably attenuated in the presence of caspase inhibitors. Together, these results indicate that myricetin might take on potent chemopreventive activity by inhibiting the Akt-mediated survival signaling axis in UVB-induced skin carcinogenesis.
Journal of Radiation Research 03/2010; 51(3):285-96. · 1.68 Impact Factor
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ABSTRACT: Burkholderia cepacia AC1100 completely degrades 2,4,5-trichlorophenol, in which an FADH2-dependent monooxygenase (TftD) and an NADH:FAD oxidoreductase (TftC) catalyze the initial steps. TftD oxidizes 2,4,5-trichlorophenol
(2,4,5-TCP) to 2,5-dichloro-p-benzoquinone, which is chemically reduced to 2,5-dichloro-p-hydroquinone (2,5-DiCHQ). Then, TftD oxidizes the latter to 5-chloro-2-hydroxy-p-benzoquinone. In those processes, TftC provides all the required FADH2. We have determined the crystal structures of dimeric TftC and tetrameric TftD at 2.0 and 2.5 Å resolution, respectively.
The structure of TftC was similar to those of related flavin reductases. The stacked nicotinamide:isoalloxazine rings in TftC
and sequential reaction kinetics suggest that the reduced FAD leaves TftC after NADH oxidation. The structure of TftD was
also similar to the known structures of FADH2-dependent monooxygenases. Its His-289 residue in the re-side of the isoalloxazine ring is within hydrogen bonding distance with a hydroxyl group of 2,5-DiCHQ. An H289A mutation
resulted in the complete loss of activity toward 2,5-DiCHQ and a significant decrease in catalytic efficiency toward 2,4,5-TCP.
Thus, His-289 plays different roles in the catalysis of 2,4,5-TCP and 2,5-DiCHQ. The results support that free FADH2 is generated by TftC, and TftD uses FADH2 to separately transform 2,4,5-TCP and 2,5-DiCHQ. Additional experimental data also support the diffusion of FADH2 between TftC and TftD without direct physical interaction between the two enzymes.
Journal of Biological Chemistry 01/2010; 285(3):2014-2027. · 4.77 Impact Factor
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ABSTRACT: Burkholderia cepacia AC1100 completely degrades 2,4,5-trichlorophenol, in which an FADH(2)-dependent monooxygenase (TftD) and an NADH:FAD oxidoreductase (TftC) catalyze the initial steps. TftD oxidizes 2,4,5-trichlorophenol (2,4,5-TCP) to 2,5-dichloro-p-benzoquinone, which is chemically reduced to 2,5-dichloro-p-hydroquinone (2,5-DiCHQ). Then, TftD oxidizes the latter to 5-chloro-2-hydroxy-p-benzoquinone. In those processes, TftC provides all the required FADH(2). We have determined the crystal structures of dimeric TftC and tetrameric TftD at 2.0 and 2.5 A resolution, respectively. The structure of TftC was similar to those of related flavin reductases. The stacked nicotinamide:isoalloxazine rings in TftC and sequential reaction kinetics suggest that the reduced FAD leaves TftC after NADH oxidation. The structure of TftD was also similar to the known structures of FADH(2)-dependent monooxygenases. Its His-289 residue in the re-side of the isoalloxazine ring is within hydrogen bonding distance with a hydroxyl group of 2,5-DiCHQ. An H289A mutation resulted in the complete loss of activity toward 2,5-DiCHQ and a significant decrease in catalytic efficiency toward 2,4,5-TCP. Thus, His-289 plays different roles in the catalysis of 2,4,5-TCP and 2,5-DiCHQ. The results support that free FADH(2) is generated by TftC, and TftD uses FADH(2) to separately transform 2,4,5-TCP and 2,5-DiCHQ. Additional experimental data also support the diffusion of FADH(2) between TftC and TftD without direct physical interaction between the two enzymes.
Journal of Biological Chemistry 11/2009; 285(3):2014-27. · 4.77 Impact Factor
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ABSTRACT: Nm23-H1 encodes nucleoside diphosphate kinase A (NDPK-A) and is known to have a metastasis suppressive activity in many tumor cells. However, it has many other functions as well. Recent studies have shown that the interacting proteins with Nm23-H1 which mediate the cell proliferation, may act as modulators of the metastasis suppressor activity. The interacting proteins with Nm23-H1 can be classified into 3 groups. The first group of proteins can be classified as upstream kinases of Nm23-H1 such as CKI and Aurora-A/STK15. The second group of proteins acts as downstream effectors for the regulation of specific gene transcriptions, GTP-binding protein functions, and signal transduction in Erk signal cascade. The third group of proteins can be classified as bi-directionally influencing binding partners of Nm23-H1. As a result, the interactions with Nm23-H1 and binding partners have implications in the biochemical characterization involved in metastasis and tumorigenesis.
Molecular and Cellular Biochemistry 05/2009; 329(1-2):167-73. · 2.06 Impact Factor
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ABSTRACT: Potato (Solanum tuberosum) multicystatin (PMC) is a crystalline Cys protease inhibitor present in the subphellogen layer of potato tubers. It consists of eight tandem domains of similar size and sequence. Our in vitro results showed that the pH/PO(4)(-)-dependent oligomeric behavior of PMC was due to its multidomain nature and was not a characteristic of the individual domains. Using a single domain of PMC, which still maintains inhibitor activity, we identified a target protein of PMC, a putative Cys protease. In addition, our crystal structure of a representative repeating unit of PMC, PMC-2, showed structural similarity to both type I and type II cystatins. The N-terminal trunk, alpha-helix, and L2 region of PMC-2 were most similar to those of type I cystatins, while the conformation of L1 more closely resembled that of type II cystatins. The structure of PMC-2 was most similar to the intensely sweet protein monellin from Dioscorephyllum cumminisii (serendipity berry), despite a low level of sequence similarity. We present a model for the possible molecular organization of the eight inhibitory domains in crystalline PMC. The unique molecular properties of the oligomeric PMC crystal are discussed in relation to its potential function in regulating the activity of proteases in potato tubers.
The Plant Cell 04/2009; 21(3):861-75. · 8.99 Impact Factor
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ABSTRACT: Neo-angiogenesis seems to be a critical feature of breast tumor growth, migration and metastasis. Inhibition of angiogenesis may provide information regarding treatment. Since angiogenesis is the result of complex processes, controlled by several angiogenic (pro- and/or -anti) factors and their receptors, multiple ways to prevent or retrogress tumor-induced angiogenesis have been proposed. The clinically significant activity of bevacizumab and other antiangiogenic treatments have attracted a great deal of interest.
We discuss biological aspects of breast cancer angiogenesis and nucleoside diphosphate kinase (NDPK) as a key molecule in this process.
In clinical and experimental trials, it was reported that NDPK is inversely related to breast cancer metastasis and angiogenesis. To inhibit the metastatic potential of cancer cells, Nm23-H1/NDP kinase appears to interact with many proteins involved in cellular signal transduction in angiogenesis and tumorigenesis, and therefore reduces the activation of the extracellular signal-regulated kinase (ERK)/MAPK in response to those signals.
Expert opinion on therapeutic targets 12/2008; 12(11):1419-30. · 3.72 Impact Factor
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ABSTRACT: EDTA has become a major organic pollutant in the environment because of its extreme usage and resistance to biodegradation. Recently, two critical enzymes, EDTA monooxygenase (EmoA) and NADH:FMN oxidoreductase (EmoB), belonging to the newly established two-component flavin-diffusible monooxygenase family, were identified in the EDTA degradation pathway in Mesorhizobium sp. BNC1. EmoA is an FMNH2-dependent enzyme that requires EmoB to provide FMNH2 for the conversion of EDTA to ethylenediaminediacetate. To understand the molecular basis of this FMN-mediated reaction, the crystal structures of the apo-form, FMN.FMN complex, and FMN.NADH complex of EmoB were determined at 2.5 angstroms resolution. The structure of EmoB is a homotetramer consisting of four alpha/beta-single-domain monomers of five parallel beta-strands flanked by five alpha-helices, which is quite different from those of other known two-component flavin-diffusible monooxygenase family members, such as PheA2 and HpaC, in terms of both tertiary and quaternary structures. For the first time, the crystal structures of both the FMN.FMN and FMN.NADH complexes of an NADH:FMN oxidoreductase were determined. Two stacked isoalloxazine rings and nicotinamide/isoalloxazine rings were at a proper distance for hydride transfer. The structures indicated a ping-pong reaction mechanism, which was confirmed by activity assays. Thus, the structural data offer detailed mechanistic information for hydride transfer between NADH to an enzyme-bound FMN and between the bound FMNH2 and a diffusible FMN.
Journal of Biological Chemistry 09/2008; 283(42):28710-20. · 4.77 Impact Factor
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ABSTRACT: Mutations of conserved residues of human cardiac calsequestrin (hCSQ2), a high-capacity, low-affinity Ca2+-binding protein in the sarcoplasmic reticulum, have been associated with catecholamine-induced polymorphic ventricular tachycardia (CPVT). In order to understand the molecular mechanism and pathophysiological link between these CPVT-related missense mutations of hCSQ2 and the resulting arrhythmias, we generated three CPVT-causing mutants of hCSQ2 (R33Q, L167H, and D307H) and two non-pathological mutants (T66A and V76M) and investigated the effect of these mutations. In addition, we determined the crystal structure of the corresponding wild-type hCSQ2 to gain insight into the structural effects of those mutations. Our data show clearly that all three CPVT-related mutations lead to significant reduction in Ca2+-binding capacity in spite of the similarity of their secondary structures to that of the wild-type hCSQ2. Light-scattering experiments indicate that the Ca2+-dependent monomer-polymer transitions of the mutants are quite different, confirming that the linear polymerization behavior of CSQ is linked directly to its high-capacity Ca2+ binding. R33Q and D307H mutations result in a monomer that appears to be unable to form a properly oriented dimer. On the other hand, the L167H mutant has a disrupted hydrophobic core in domain II, resulting in high molecular aggregates, which cannot respond to Ca2+. Although one of the non-pathological mutants, T66A, shares characteristics with the wild-type, the other null mutant, V76M, shows significantly altered Ca2+-binding and polymerization behaviors, calling for careful reconsideration of its status.
Journal of Molecular Biology 12/2007; 373(4):1047-57. · 4.00 Impact Factor
