ABSTRACT
GRIM-19 (Gene associated with Retinoid-IFN-induced Mortality-19) was originally isolated as a growth suppressor in a genome-wide knockdown screen with antisense libraries. Like classical tumor suppressors, mutations, and/or loss of GRIM-19 expression occur in primary human tumors; and it is inactivated by viral gene products. Our search for potential GRIM-19-binding proteins, using mass spectrometry, that permit its antitumor actions led to the inhibitor of cyclin-dependent kinase 4, CDKN2A. The GRIM-19/CDKN2A synergistically suppressed cell cycle progression via inhibiting E2F1-driven gene expression. The N terminus of GRIM-19 and the fourth ankyrin repeat of CDKN2A are crucial for their interaction. The biological relevance of these interactions is underscored by observations that GRIM-19 promotes the inhibitory effect of CDKN2A on CDK4; and mutations from primary tumors disrupt its ability to interact with GRIM-19 and suppress E2F1-driven gene expression.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Cell Cycle , Cyclin-Dependent Kinase Inhibitor p16/metabolism , E2F1 Transcription Factor/metabolism , Gene Expression Regulation, Neoplastic , NADH, NADPH Oxidoreductases/metabolism , Amino Acid Sequence , Animals , Ankyrin Repeat , Apoptosis Regulatory Proteins/chemistry , Apoptosis Regulatory Proteins/deficiency , Apoptosis Regulatory Proteins/genetics , Cell Line, Tumor , Cyclin D1/metabolism , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase Inhibitor p16/chemistry , Cyclin-Dependent Kinase Inhibitor p16/genetics , G1 Phase , Gene Knockdown Techniques , Humans , Mice , Molecular Sequence Data , Mutation , NADH, NADPH Oxidoreductases/chemistry , NADH, NADPH Oxidoreductases/deficiency , NADH, NADPH Oxidoreductases/genetics , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Protein Structure, TertiaryABSTRACT
Scaling is a major problem in the thermal desalination operation which is mainly attributed to the deposition of salts on the tube, thereby increasing the resistance to heat transfer. To reduce or prevent the formation of scale on heat transfer surfaces, treating desalination concentrates and precipitating sparingly soluble salts can be a promising method. In the present work, the effect of ultrasound pretreatment to the synthetically prepared sea water as desalination feed has been investigated with an objective of intensifying salt removal process and avoiding scale formation leading to better heat transfer rates. A lab scale double pipe heat exchanger setup was designed and operated under simulated conditions of the thermal desalination operation. Total operational volume of 2000 ml was used for all experiments with a fixed flow rate of 5 ml/s. To understand the process of scaling, synthetic seawater was prepared as per the ASTM D 1141-98 and was used for scale deposition experiments. The experiments conducted using untreated synthetic seawater confirmed substantial scaling and drop in the heat transfer coefficient from an initial value of 776 W/m2 K to 603 W/m2 K (about 22%) after 24 h operation as compared to deionized water. SEM-EDX analysis was performed to investigate the morphology and main components of the scale. Subsequently, synthetic seawater was treated with ultrasound under continuous flow condition for removal of salts responsible for scaling. It was demonstrated that pretreatment resulted into salt crystallization, after which, the crystals were separated and the filtered solution was passed through the heat exchanger to check the effects on heat transfer rate. It was confirmed that the heat transfer rate was found to be higher with a value of 797 W/m2 K. Overall an effective approach based on ultrasound to remove the scale forming components has been demonstrated with established best conditions as 70% amplitude for 30 min of irradiation at fixed frequency of 20 kHz and 50% duty cycle.
ABSTRACT
Annexin A7 (synexin, annexin VII), a member of the annexin family of proteins, causes aggregation of membranes in a Ca2+-dependent manner and has been suggested to promote membrane fusion during exocytosis of lung surfactant, catecholamines, and insulin. Although annexin A7 (A7) was one of the first annexin proteins described, limited studies of its physical characteristics or of structural domains affecting any of its proposed functions have been conducted. As postulated for other annexin proteins, the unique NH2-domain possibly determines the functional specificity of A7. Therefore, we evaluated the effects of segmental deletions in the NH2-terminus on several characteristics associated with the COOH-terminus of A7. The COOH-terminus contains the only tryptophan residue, and all potential trypsin sites, and the Ca2+ and phospholipid binding sites. Recombinant rat A7 and its deletion mutants were expressed using constructs based on the cDNA sequence obtained by screening a rat lung cDNA library. Ca2+ increased the tryptophan fluorescence of A7 and caused a small red shift in the emission maximum (lambdamax), which was further increased in presence of phospholipid vesicles (PLV). NH2-terminal deletions of 29, 51, and 109 residues affected the peak width of fluorescence and lambdamax, surface-exposure of tryptophan residue, and caused a smaller Ca2+-dependent red shift in lambdamax of membrane-bound protein in comparison to A7. Limited proteolysis with trypsin showed that Ca2+ increased the proteolysis of all proteins, but the deletions also affected the pattern of proteolysis. The presence of PLV protected against Ca2+-dependent increase in proteolysis of all proteins. The deletion of first 29 residues also caused decreased membrane binding, aggregation, and fusion, when compared with A7. Collectively, these results suggest that specific NH2-terminus domains can alter those properties of A7 that are normally associated with the COOH-terminus. We speculate that interactions between the NH2- and COOH-termini are required for membrane binding, and aggregation and fusion properties of annexin A7.
Subject(s)
Annexin A7 , Cell Membrane/metabolism , Membrane Fusion/physiology , Amino Acid Sequence , Animals , Annexin A7/chemistry , Annexin A7/genetics , Annexin A7/metabolism , Base Sequence , Calcium/metabolism , Gene Library , Lung/metabolism , Molecular Sequence Data , Phospholipids/chemistry , Phospholipids/metabolism , Protein Binding , Protein Structure, Tertiary , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Tryptophan/chemistryABSTRACT
A combination of interferon-beta (IFN-beta) and all-trans retinoic acid (IFN/RA) induces tumor cell apoptosis via some unknown mechanisms. Apoptosis is a gene-directed process that limits the proliferation of undesired cells. Several genes are required to regulate cell death in the higher-order animals. Earlier, we employed a gene expression knockout technique to isolate cell death-related genes. A novel gene, the gene associated with retinoid-interferon-induced mortality-19 (GRIM-19), was found to be essential for tumor cell death induced by IFN/RA. Here, we describe the development and characterization of three monoclonal antibodies (mAbs) against GRIM-19. GRIM-19 is present in the nucleus and cytoplasm. Its expression is induced by the IFN/RA combination. We also show that GRIM-19 inhibits the cell-transforming property of viral oncogenic protein viral IFN regulatory factor-1 (vIRF-1) via a physical interaction. mAbs developed in this study should be useful for studying the other physiologic roles of GRIM-19 and serve as a potent tool for studying tumor responses to IFN/RA therapy.
Subject(s)
Antibodies, Monoclonal/immunology , Apoptosis/drug effects , Gene Expression Regulation/drug effects , Genes, Tumor Suppressor , Interferon-beta/pharmacology , NADH, NADPH Oxidoreductases/immunology , 3T3 Cells , Animals , Apoptosis Regulatory Proteins , Humans , Immunologic Factors/pharmacology , Mice , Mice, Mutant Strains , NADH, NADPH Oxidoreductases/metabolism , Nuclear Proteins/immunology , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Tretinoin/pharmacology , Tumor Cells, CulturedABSTRACT
Transcription factor CCAAT/enhancer-binding protein (C/EBP)-beta is crucial for regulating transcription of genes involved in a number of diverse cellular processes, including those involved in some cytokine-induced responses. However, the mechanisms that contribute to its diverse transcriptional activity are not yet fully understood. To gain an understanding into its mechanisms of action, we took a proteomic approach and identified cellular proteins that associate with C/EBP-beta in an interferon (IFN)-gamma-dependent manner. Transcriptional mediator (Mediator) is a multisubunit protein complex that regulates signal-induced cellular gene transcription from enhancer-bound transcription factor(s). Here, we report that the Med1 subunit of the Mediator as a C/EBP-beta-interacting protein. Using gene knock-out cells and mutational and RNA interference approaches, we show that Med1 is critical for IFN-induced expression of certain genes. Med1 associates with C/EBP-beta through a domain located between amino acids 125 and 155 of its N terminus. We also show that the MAPK, ERK1/2, and an ERK phosphorylation site within regulatory domain 2, more specifically the Thr(189) residue, of C/EBP-beta are essential for it to bind to Med1. Last, an ERK-regulated site in Med1 protein is also essential for up-regulating IFN-induced transcription although not critical for binding to C/EBP-beta.
Subject(s)
CCAAT-Enhancer-Binding Protein-beta/metabolism , Endodeoxyribonucleases/metabolism , Interferon-gamma/pharmacology , Transcription, Genetic/drug effects , Transcription, Genetic/genetics , Amino Acid Sequence , Animals , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , CCAAT-Enhancer-Binding Protein-beta/deficiency , CCAAT-Enhancer-Binding Protein-beta/genetics , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Line , Death-Associated Protein Kinases , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/genetics , Gene Expression Regulation/drug effects , Humans , Interferon-Stimulated Gene Factor 3, gamma Subunit/metabolism , Mice , Mice, Knockout , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Promoter Regions, Genetic/genetics , Protein Binding , Protein Structure, Tertiary , Protein Subunits/genetics , Protein Subunits/metabolism , Recombinant Proteins , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
IFN-gamma-activated transcriptional element (GATE)-binding factor 1 (GBF1) was identified as a transactivator that induces gene expression through GATE, a novel IFN-inducible element. Although it can induce gene expression, it is an extremely weak DNA-binding protein on its own. GATE also binds another transcription factor, C/EBP-beta. Therefore, we explored whether GBF1 physically interacts with C/EBP-beta to induce IFN-gamma-regulated transcription. In response to IFN-gamma, C/EBP-beta undergoes phosphorylation at a critical ERK1/2 phosphorylation motif. Mutational inactivation of this motif and/or interference with the ERK1/2 activation prevented the IFN-gamma-induced interactions between GBF1 and C/EBP-beta. A 37-aa long peptide derived from the GBF1 protein can associate with C/EBP-beta in an IFN-inducible manner. These results identify a converging point for two transactivators that exert their effects through a single response element. Together, our studies identify a novel regulatory mechanism that controls IFN-induced transcription.
Subject(s)
CCAAT-Enhancer-Binding Protein-beta/metabolism , Gene Expression Regulation/immunology , Interferon-gamma/physiology , Trans-Activators/metabolism , Trans-Activators/physiology , Amino Acid Motifs , Amino Acid Sequence , Animals , CCAAT-Enhancer-Binding Protein-beta/biosynthesis , CCAAT-Enhancer-Binding Protein-beta/chemistry , CCAAT-Enhancer-Binding Protein-beta/genetics , Cell Line , Humans , Immunologic Factors/genetics , Immunologic Factors/metabolism , Immunologic Factors/physiology , Intramolecular Oxidoreductases , Mice , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Molecular Sequence Data , Prostaglandin-E Synthases , Protein Interaction Mapping , Protein Structure, Tertiary , Trans-Activators/chemistry , Trans-Activators/genetics , TransfectionABSTRACT
We have previously identified a novel interferon (IFN)-stimulated cis-acting enhancer element, gamma-IFN-activated transcriptional element (GATE). GATE differs from the known IFN-stimulated elements in its primary sequence. Preliminary analysis has indicated that the GATE-dependent transcriptional response requires the binding of novel transacting factors. A cDNA expression library derived from an IFN-gamma-stimulated murine macrophage cell line was screened with a (32)P-labeled GATE probe to identify the potential GATE-binding factors. A cDNA coding for a novel transcription-activating factor was identified. Based on its discovery, we named it as GATE-binding factor-1 (GBF-1). GBF-1 homologs are present in mouse, human, monkey, and Drosophila. It activates transcription from reporter genes carrying GATE. It possesses a strong transactivating activity but has a weak DNA binding property. GBF-1 is expressed in most tissues with relatively higher steady-state levels in heart, liver, kidney, and brain. Its expression is induced by IFN-gamma treatment. GBF-1 is present in both cytosolic and nuclear compartments. These studies thus identify a novel transactivating factor in IFN signaling pathways.
Subject(s)
Gene Expression Regulation/physiology , Interferon-gamma/physiology , Trans-Activators/physiology , Amino Acid Sequence , Animals , Base Sequence , DNA Primers , DNA, Complementary , Humans , Intramolecular Oxidoreductases , Mice , Molecular Sequence Data , Prostaglandin-E Synthases , Sequence Homology, Amino Acid , Trans-Activators/chemistry , Trans-Activators/genetics , Trans-Activators/metabolismABSTRACT
The interferon-beta (IFNbeta) gene is not inducible in neuronal cells in response to measles virus (MV) due to lack of nuclear factor kappa B (NF-kappaB) activation. NF-kappaB is normally sequestered in the cytoplasm by an inhibitor (IkappaBalpha). Previously, the authors demonstrated that the failure to activate neuronal NF-kappaB by MV was due to the inability to phosphorylate and degrade its inhibitor, IkappaBalpha. Here the authors demonstrate that transient transfection of a brain cDNA library into neuronal cells restores the ability of MV to activate NF-kappaB. In addition, tumor necrosis factor-alpha (TNFalpha), but not interleukin-1 (IL-1) or lipopolysaccharide (LPS), stimulation resulted in IkappaBalpha phosphorylation and degradation in two neuronal cell lines. These results indicate that failure of MV to activate neuronal NF-kappaB is due to a signaling defect and that MV utilizes an NF-kappaB signaling pathway distinct from that of TNFalpha, but may overlap with that for IL-1 and LPS.