28 results on '"T. Tran"'
Search Results
2. Manipulation of a spider peptide toxin alters its affinity for lipid bilayers and potency and selectivity for voltage-gated sodium channel subtype 1.7
- Author
-
David J. Craik, Christina I. Schroeder, Akello J. Agwa, Mathilde R. Israel, Alexander Mueller, Irina Vetter, Jennifer R. Deuis, Hue N. T. Tran, Kirsten L. McMahon, and Poanna Tran
- Subjects
Male ,Nociception ,0301 basic medicine ,Lipid Bilayers ,Scorpion Venoms ,Spider Venoms ,Peptide ,Gating ,Biochemistry ,Mice ,03 medical and health sciences ,Neurobiology ,In vivo ,Animals ,Potency ,Lipid bilayer ,Molecular Biology ,Ion channel ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,Chemistry ,Sodium channel ,Bilayer ,NAV1.7 Voltage-Gated Sodium Channel ,Cell Biology ,Peptide Fragments ,Mice, Inbred C57BL ,030104 developmental biology ,Biophysics - Abstract
Huwentoxin-IV (HwTx-IV) is a gating modifier peptide toxin from spiders that has weak affinity for the lipid bilayer. As some gating modifier toxins have affinity for model lipid bilayers, a tripartite relationship among gating modifier toxins, voltage-gated ion channels, and the lipid membrane surrounding the channels has been proposed. We previously designed an HwTx-IV analogue (gHwTx-IV) with reduced negative charge and increased hydrophobic surface profile, which displays increased lipid bilayer affinity and in vitro activity at the voltage-gated sodium channel subtype 1.7 (Na(V)1.7), a channel targeted in pain management. Here, we show that replacements of the positively-charged residues that contribute to the activity of the peptide can improve gHwTx-IV's potency and selectivity for Na(V)1.7. Using HwTx-IV, gHwTx-IV, [R26A]gHwTx-IV, [K27A]gHwTx-IV, and [R29A]gHwTx-IV variants, we examined their potency and selectivity at human Na(V)1.7 and their affinity for the lipid bilayer. [R26A]gHwTx-IV consistently displayed the most improved potency and selectivity for Na(V)1.7, examined alongside off-target Na(V)s, compared with HwTx-IV and gHwTx-IV. The lipid affinity of each of the three novel analogues was weaker than that of gHwTx-IV, but stronger than that of HwTx-IV, suggesting a possible relationship between in vitro potency at Na(V)1.7 and affinity for lipid bilayers. In a murine Na(V)1.7 engagement model, [R26A]gHwTx-IV exhibited an efficacy comparable with that of native HwTx-IV. In summary, this study reports the development of an HwTx-IV analogue with improved in vitro selectivity for the pain target Na(V)1.7 and with an in vivo efficacy similar to that of native HwTx-IV.
- Published
- 2020
3. Catalytically Relevant Electrostatic Interactions of Cytochrome P450c17 (CYP17A1) and Cytochrome b5
- Author
-
Sean M. Anderson, Richard J. Auchus, Hwei Ming Peng, Sarah E. Forsberg, Hong T. Tran, and JJ Liu
- Subjects
Models, Molecular ,endocrine system ,Cytochrome ,Stereochemistry ,Molecular Sequence Data ,Static Electricity ,Gene Expression ,Peptide ,Crystallography, X-Ray ,Biochemistry ,Protein Structure, Secondary ,chemistry.chemical_compound ,Ethyldimethylaminopropyl Carbodiimide ,Catalytic Domain ,Cytochrome b5 ,Escherichia coli ,Humans ,Macromolecular docking ,Amino Acid Sequence ,Amino Acids ,Molecular Biology ,Peptide sequence ,chemistry.chemical_classification ,biology ,Chemistry ,Steroid 17-alpha-Hydroxylase ,Cytochrome P450 ,Cell Biology ,Recombinant Proteins ,Amino acid ,Kinetics ,Cross-Linking Reagents ,Cytochromes b5 ,Mutagenesis, Site-Directed ,Enzymology ,biology.protein ,Thermodynamics ,1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide ,Protein Multimerization ,Protein Binding - Abstract
Two acidic residues, Glu-48 and Glu-49, of cytochrome b5 (b5) are essential for stimulating the 17,20-lyase activity of cytochrome P450c17 (CYP17A1). Substitution of Ala, Gly, Cys, or Gln for these two glutamic acid residues abrogated all capacity to stimulate 17,20-lyase activity. Mutations E49D and E48D/E49D retained 23 and 38% of wild-type activity, respectively. Using the zero-length cross-linker ethyl-3-(3-dimethylaminopropyl)carbodiimide, we obtained cross-linked heterodimers of b5 and CYP17A1, wild-type, or mutations R347K and R358K. In sharp contrast, the b5 double mutation E48G/E49G did not form cross-linked complexes with wild-type CYP17A1. Mass spectrometric analysis of the CYP17A1-b5 complexes identified two cross-linked peptide pairs as follows: CYP17A1-WT: (84)EVLIKK(89)-b5: (53)EQAGGDATENFEDVGHSTDAR(73) and CYP17A1-R347K: (341)TPTISDKNR(349)-b5: (40)FLEEHPGGEEVLR(52). Using these two sites of interaction and Glu-48/Glu-49 in b5 as constraints, protein docking calculations based on the crystal structures of the two proteins yielded a structural model of the CYP17A1-b5 complex. The appositional surfaces include Lys-88, Arg-347, and Arg-358/Arg-449 of CYP17A1, which interact with Glu-61, Glu-42, and Glu-48/Glu-49 of b5, respectively. Our data reveal the structural basis of the electrostatic interactions between these two proteins, which is critical for 17,20-lyase activity and androgen biosynthesis.
- Published
- 2014
4. Mucin-type O-Glycosylation during Development
- Author
-
Duy T. Tran and Kelly G. Ten Hagen
- Subjects
Acetylgalactosamine ,Glycosylation ,Disease ,Biology ,Models, Biological ,Biochemistry ,chemistry.chemical_compound ,symbols.namesake ,Polysaccharides ,Animals ,Humans ,Secretion ,Molecular Biology ,Membrane Glycoproteins ,Mucin ,Mucins ,Glycosyltransferases ,Minireviews ,Cell Biology ,Golgi apparatus ,Cell biology ,carbohydrates (lipids) ,Membrane glycoproteins ,Secretory protein ,chemistry ,biology.protein ,symbols ,N-Acetylgalactosaminyltransferases ,lipids (amino acids, peptides, and proteins) ,Function (biology) - Abstract
Mucin-type O-glycosylation is an evolutionarily conserved protein modification present on membrane-bound and secreted proteins. Aberrations in O-glycosylation are responsible for certain human diseases and are associated with disease risk factors. Recent studies have demonstrated essential roles for mucin-type O-glycosylation in protein secretion, stability, processing, and function. Here, we summarize our current understanding of the diverse roles of mucin-type O-glycosylation during eukaryotic development. Appreciating how this conserved modification operates in developmental processes will provide insight into its roles in human disease and disease susceptibilities.
- Published
- 2013
5. Glycosylation of α-Dystroglycan
- Author
-
Jae-Min Lim, Kelly G. Ten Hagen, David Live, Mian Liu, Duy T. Tran, Stephanie H. Stalnaker, and Lance Wells
- Subjects
chemistry.chemical_classification ,Enzyme complex ,Glycosylation ,Endoplasmic reticulum ,Mannose ,Cell Biology ,Golgi apparatus ,Biochemistry ,carbohydrates (lipids) ,chemistry.chemical_compound ,symbols.namesake ,N-linked glycosylation ,chemistry ,symbols ,O-linked glycosylation ,lipids (amino acids, peptides, and proteins) ,Glycoprotein ,Molecular Biology - Abstract
O-Linked glycosylation is a functionally and structurally diverse type of protein modification present in many tissues and across many species. α-Dystroglycan (α-DG), a protein linked to the extracellular matrix, whose glycosylation status is associated with human muscular dystrophies, displays two predominant types of O-glycosylation, O-linked mannose (O-Man) and O-linked N-acetylgalactosamine (O-GalNAc), in its highly conserved mucin-like domain. The O-Man is installed by an enzyme complex present in the endoplasmic reticulum. O-GalNAc modifications are initiated subsequently in the Golgi apparatus by the UDP-GalNAc polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) enzymes. How the presence and position of O-Man influences the action of the ppGalNAc-Ts on α-DG and the distribution of the two forms of glycosylation in this domain is not known. Here, we investigated the interplay between O-Man and the addition of O-GalNAc by examining the activity of the ppGalNAc-Ts on peptides and O-Man-containing glycopeptides mimicking those found in native α-DG. These synthetic glycopeptides emulate intermediate structures, not otherwise readily available from natural sources. Through enzymatic and mass spectrometric methods, we demonstrate that the presence and specific location of O-Man can impact either the regional exclusion or the site of O-GalNAc addition on α-DG, elucidating the factors contributing to the glycosylation patterns observed in vivo. These results provide evidence that one form of glycosylation can influence another form of glycosylation in α-DG and suggest that in the absence of proper O-mannosylation, as is associated with certain forms of muscular dystrophy, aberrant O-GalNAc modifications may occur and could play a role in disease presentation.
- Published
- 2012
6. Multiple Members of the UDP-GalNAc: Polypeptide N-Acetylgalactosaminyltransferase Family Are Essential for Viability in Drosophila
- Author
-
Kelly G. Ten Hagen, Liping Zhang, Lesley A. Earl, Duy T. Tran, Ying Zhang, and E Tian
- Subjects
Male ,Mesoderm ,animal structures ,Glycosylation ,Cellular differentiation ,Glycobiology and Extracellular Matrices ,macromolecular substances ,medicine.disease_cause ,Biochemistry ,chemistry.chemical_compound ,RNA interference ,Cell Adhesion ,medicine ,Animals ,Wings, Animal ,Cell adhesion ,Molecular Biology ,Gene ,Genetics ,Mutation ,biology ,Cell Biology ,Hydrogen-Ion Concentration ,biology.organism_classification ,carbohydrates (lipids) ,Oxygen ,Drosophila melanogaster ,medicine.anatomical_structure ,chemistry ,Organ Specificity ,N-Acetylgalactosaminyltransferases ,lipids (amino acids, peptides, and proteins) ,Female ,RNA Interference ,Digestive System - Abstract
Mucin-type O-glycosylation represents a major form of post-translational modification that is conserved across most eukaryotic species. This type of glycosylation is initiated by a family of enzymes (GalNAc-Ts in mammals and PGANTs in Drosophila) whose members are expressed in distinct spatial and temporal patterns during development. Previous work from our group demonstrated that one member of this family is essential for viability and another member modulates extracellular matrix composition and integrin-mediated cell adhesion during development. To investigate whether other members of this family are essential, we employed RNA interference (RNAi) to each gene in vivo. Using this approach, we identified 4 additional pgant genes that are required for viability. Ubiquitous RNAi to pgant4, pgant5, pgant7, or the putative glycosyltransferase CG30463 resulted in lethality. Tissue-specific RNAi was also used to define the specific organ systems and tissues in which each essential family member is required. Interestingly, each essential pgant had a unique complement of tissues in which it was required. Additionally, certain tissues (mesoderm, digestive system, and tracheal system) required more than one pgant, suggesting unique functions for specific enzymes in these tissues. Expanding upon our RNAi results, we found that conventional mutations in pgant5 resulted in lethality and specific defects in specialized cells of the digestive tract, resulting in loss of proper digestive system acidification. In summary, our results highlight essential roles for O-glycosylation and specific members of the pgant family in many aspects of development and organogenesis.
- Published
- 2012
7. Adenoviral Overexpression of the Glutamylcysteine Ligase Catalytic Subunit Protects Pancreatic Islets against Oxidative Stress
- Author
-
Jamie S. Harmon, Elizabeth Oseid, Sarah M. Parker, Christopher C. Franklin, R. Paul Robertson, Huarong Zhou, Tao Zhang, Eric LeRoy, Terrance J. Kavanagh, Phuong Oanh T. Tran, and Portia A. Vliet
- Subjects
medicine.medical_specialty ,Glutamate-Cysteine Ligase ,p38 mitogen-activated protein kinases ,Protein subunit ,Genetic Vectors ,Gene Expression ,Biology ,Transfection ,medicine.disease_cause ,p38 Mitogen-Activated Protein Kinases ,Biochemistry ,Gene Expression Regulation, Enzymologic ,Adenoviridae ,Islets of Langerhans ,chemistry.chemical_compound ,Internal medicine ,Insulin Secretion ,medicine ,Animals ,Humans ,Insulin ,Rats, Wistar ,Protein kinase A ,Molecular Biology ,chemistry.chemical_classification ,Reactive oxygen species ,Pancreatic islets ,NF-kappa B ,Cell Biology ,Glutathione ,Flow Cytometry ,Rats ,Oxidative Stress ,Glucose ,Endocrinology ,medicine.anatomical_structure ,GCLC ,chemistry ,Reactive Oxygen Species ,Oxidation-Reduction ,Oxidative stress ,Interleukin-1 - Abstract
The catalytic subunit of glutamylcysteine ligase (GCLC) primarily regulates de novo synthesis of glutathione (GSH) in mammalian cells and is central to the antioxidant capacity of the cell. However, GCLC expression in pancreatic islets has not been previously examined. We designed experiments to ascertain whether GCLC is normally expressed in islets and whether it is up-regulated by interleukin-1 beta (IL-1 beta). GCLC expression levels were intermediate compared with other metabolic tissues (kidney, liver, muscle, fat, and lung). IL-1 beta up-regulated GCLC expression (10 ng/ml IL-1 beta, 3.76 +/- 0.86; 100 ng/ml IL-1 beta, 4.22 +/- 0.68-fold control) via the p38 form of mitogen-activated protein kinase and NF kappa B and also increased reactive oxygen species levels (10 ng/ml IL-1 beta, 5.41 +/- 1.8-fold control). This was accompanied by an increase in intraislet GSH/GSSG ratio (control, 7.1 +/- 0.1; 10 ng/ml IL-1 beta, 8.0 +/- 0.5; 100 ng/ml IL-1 beta, 8.2 +/- 0.5-fold control; p0.05). To determine whether overexpression of GCLC increases the antioxidant capacity of the islet and prevents the adverse effects of IL-1 beta on glucose-induced insulin secretion, islets were infected with an adenovirus encoding GCLC. IL-1 beta significantly decreased glucose-stimulated insulin secretion (control, 123.8 +/- 17.7; IL-1 beta, 40.2 +/- 3.9 microunits/ml insulin/islet). GCLC overexpression increased intraislet GSH levels and partially prevented the decrease in glucose-stimulated insulin secretion caused by IL-1 beta. These data provide the first report of GCLC expression in the islet and demonstrate that adenoviral overexpression of GCLC increases intracellular GSH levels and protects the beta cell from the adverse effects of IL-1 beta.
- Published
- 2004
8. Promyelocytic Leukemia Protein 4 Induces Apoptosis by Inhibition of Survivin Expression
- Author
-
Zhi-Xiang Xu, Tian Ding, Thanh T. Tran, Rui Xun Zhao, Kun Sang Chang, Pier Paolo Pandolfi, and Wei Zhang
- Subjects
Transcriptional Activation ,Receptors, Retinoic Acid ,Survivin ,viruses ,Retinoic Acid ,Down-Regulation ,Apoptosis ,Caspase 3 ,Promyelocytic Leukemia Protein ,Biology ,Biochemistry ,Caspase 7 ,Inhibitor of Apoptosis Proteins ,Cell Line ,Promoter Regions ,Transactivation ,Promyelocytic leukemia protein ,Genetic ,Receptors ,Humans ,Promoter Regions, Genetic ,Molecular Biology ,Gene knockdown ,Retinoic Acid Receptor alpha ,Tumor Suppressor Proteins ,Cell Cycle ,Nuclear Proteins ,virus diseases ,Cell Biology ,Cell cycle ,Neoplasm Proteins ,Repressor Proteins ,Gene Expression Regulation ,Cancer research ,biology.protein ,Microtubule-Associated Proteins ,Transcription Factors - Abstract
The promyelocytic leukemia protein (PML) plays an essential role in multiple pathways of apoptosis. Our previous study showed that PML enhances tumor necrosis factor-induced apoptosis by inhibiting the NFkappaB survival pathway. To continue exploring the mechanism of PML-induced apoptosis, we performed a DNA microarray screening of PML target genes using a PML-inducible stable cell line. We found that Survivin was one of the downstream target genes of PML. Cotransfection experiments demonstrated that PML4 repressed transactivation of the Survivin promoter in an isoform-specific manner. Western blot analysis demonstrated that induced PML expression down-regulated Survivin. Inversely, PML knockdown by siRNA up-regulated Survivin expression. A substantial increase in Survivin expression was found in PML-deficient cells. Re-expression of PML in PML-/- mouse embryo fibroblasts down-regulated the expression of Survivin. Furthermore, cells arrested at the G2/M cell cycle phase expressed a high level of Survivin and a significantly lower level of PML. Overexpression of PML in A549 cells reduced Survivin expression leading to massive apoptotic cell death associated with activation of procaspase 9, caspase 3, and caspase 7. Together, our results demonstrate a novel mechanism of PML-induced apoptosis by down-regulation of Survivin.
- Published
- 2004
9. A UDP-GalNAc:PolypeptideN-Acetylgalactosaminyltransferase Is Essential for Viability in Drosophila melanogaster
- Author
-
Duy T. Tran and Kelly G. Ten Hagen
- Subjects
Male ,DNA, Complementary ,Cell Survival ,Recombinant Fusion Proteins ,Green Fluorescent Proteins ,Molecular Sequence Data ,Mutant ,Mutagenesis (molecular biology technique) ,Biology ,Arginine ,Biochemistry ,Open Reading Frames ,Animals ,Protein Isoforms ,Coding region ,Gene family ,Amino Acid Sequence ,Codon ,Molecular Biology ,Gene ,Alleles ,Genetics ,Base Sequence ,Sequence Homology, Amino Acid ,Homozygote ,Tryptophan ,Cell Biology ,Blotting, Northern ,biology.organism_classification ,Molecular biology ,Stop codon ,Luminescent Proteins ,Open reading frame ,Drosophila melanogaster ,Phenotype ,COS Cells ,Mutation ,N-Acetylgalactosaminyltransferases ,Electrophoresis, Polyacrylamide Gel ,Female - Abstract
We report the first demonstration that the activity of a member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase gene family is necessary for viability in Drosophila melanogaster. Expression of the wild-type recombinant pgant35A gene in COS7 cells resulted in in vitro activity against peptide and glycopeptide substrates, demonstrating that this gene encodes a biochemically active transferase. Previous mutagenesis studies identified recessive lethal mutations that were rescued by a genomic fragment containing the pgant35A gene; however, the presence of additional open reading frames within this fragment left open the possibility that another gene was responsible for rescue of the observed lethality. Here, we have determined the molecular nature of the mutations in three independent mutant alleles. Two of the mutant alleles contain premature stop codons within the coding region of pgant35A. The third mutant contains an arginine to tryptophan amino acid change, which, when expressed in COS7 cells, resulted in a dramatic reduction of transferase activity in vitro. PCR amplification of this gene from Drosophila cDNA panels and Northern analysis revealed that it is expressed throughout embryonic, larval, and pupal stages as well as in adult males and females. This study provides the first direct evidence for the involvement of a member of this conserved multigene family in eukaryotic development and viability.
- Published
- 2002
10. Dissociable Rpb4-Rpb7 Subassembly of RNA Polymerase II Binds to Single-strand Nucleic Acid and Mediates a Post-recruitment Step in Transcription Initiation
- Author
-
Stephen Orlicky, Aled M. Edwards, Phan T. Tran, and Michael H. Sayre
- Subjects
Models, Molecular ,Insecta ,Saccharomyces cerevisiae Proteins ,Transcription, Genetic ,Amino Acid Motifs ,Molecular Sequence Data ,DNA, Single-Stranded ,RNA polymerase II ,Biochemistry ,Cell Line ,Sulfolobus ,Escherichia coli ,Animals ,Amino Acid Sequence ,Promoter Regions, Genetic ,Molecular Biology ,Polymerase ,Dose-Response Relationship, Drug ,Sequence Homology, Amino Acid ,biology ,General transcription factor ,Cell Biology ,Protein Structure, Tertiary ,Phenotype ,Mutation ,Transcription preinitiation complex ,biology.protein ,Transcription factor II F ,RNA Polymerase II ,Transcription factor II E ,Transcription factor II D ,Baculoviridae ,Transcription factor II B ,Gene Deletion ,Protein Binding - Abstract
The Rpb4 and Rpb7 subunits of yeast RNA polymerase II form a heterodimeric complex essential for promoter-directed transcription initiation in a reconstituted system. Results of template competition experiments indicate that the Rpb4-Rpb7 complex is not required for stable recruitment of polymerase to active preinitiation complexes, suggesting that Rpb4-Rpb7 mediates an essential step subsequent to promoter binding. Sequence and structure-based alignments revealed a possible OB-fold single-strand nucleic acid-binding motif in Rpb7. Purified Rpb4-Rpb7 complex exhibited both single-strand DNA- and RNA-binding activities, and a small deletion in the putative OB-fold nucleic acid-binding surface of Rpb7 abolished binding activity without affecting the stability of the Rpb4-Rpb7 complex or its ability to associate with polymerase. The same mutation destroyed the transcription activity of the Rpb4-Rpb7 complex. A separate deletion elsewhere in the OB-fold motif of Rpb7 also blocked transcription but did not affect nucleic acid binding, suggesting that the OB-fold of Rpb7 mediates both DNA-protein and protein-protein interactions required for productive initiation.
- Published
- 2001
11. Müllerian Inhibiting Substance Inhibits Ovarian Cell Growth through an Rb-independent Mechanism
- Author
-
David Dombkowski, Shyamala Maheswaran, Patricia K. Donahoe, Trent R. Clarke, Trinh T. Tran, Michelle Glander, Hans K. Lorenzo, Thanh U. Ha, David A. Barbie, Peter T. Masiakos, and Dorry L. Segev
- Subjects
Anti-Mullerian Hormone ,Male ,medicine.medical_specialty ,endocrine system diseases ,Cellular differentiation ,Ligands ,Biochemistry ,Mice ,chemistry.chemical_compound ,Internal medicine ,Tumor Cells, Cultured ,otorhinolaryngologic diseases ,medicine ,Animals ,Humans ,E2F1 ,Molecular Biology ,Cyclin-Dependent Kinase Inhibitor p16 ,Glycoproteins ,Ovarian Neoplasms ,Retinoblastoma-Like Protein p130 ,biology ,Ovary ,Proteins ,Cell Differentiation ,Anti-Müllerian hormone ,Blood Proteins ,Cell Biology ,Cell cycle ,Rubidium ,medicine.disease ,Growth Inhibitors ,Testicular Hormones ,Endocrinology ,chemistry ,Cell culture ,Apoptosis ,biology.protein ,Cancer research ,Calmodulin-Binding Proteins ,Female ,Growth inhibition ,Ovarian cancer - Abstract
Müllerian inhibiting substance (MIS), a transforming growth factor-beta family member, causes regression of the Müllerian duct in male embryos. MIS overexpression in transgenic mice ablates the ovary, and MIS inhibits the growth of ovarian cancer cell lines in vitro, suggesting a key role for this hormone in postnatal development of the ovary. This report describes a mechanism for MIS-mediated growth inhibition in both a human epithelial ovarian cancer cell line and a cell line derived from normal ovarian surface epithelium, which is the origin of human epithelial ovarian cancers. MIS-treated cells accumulated in the G(1) phase of the cell cycle and subsequently underwent apoptosis. MIS up-regulated the cyclin-dependent kinase inhibitor p16 through an MIS type II receptor-mediated mechanism and inhibited growth in the absence of detectable or inactive Rb protein. Prolonged treatment with MIS down-regulated the Rb-related protein p130 and increased the Rb family-regulated transcription factor E2F1, overexpression of which inhibited growth. These findings demonstrate that p16 is required for MIS-mediated growth inhibition in ovarian epithelial cells and tumor cells and suggest that up-regulation of E2F1 also plays a role in this process.
- Published
- 2000
12. Müllerian Inhibiting Substance Inhibits Breast Cancer Cell Growth through an NFκB-mediated Pathway
- Author
-
Patricia K. Donahoe, Mira Jung, Mary K. Kenneally, Paul Harkin, Shyamala Maheswaran, Dorry L. Segev, Trinh T. Tran, David T. MacLaughlin, and Thanh U. Ha
- Subjects
Anti-Mullerian Hormone ,medicine.medical_specialty ,Receptors, Peptide ,Receptor expression ,Estrogen receptor ,Antineoplastic Agents ,Breast Neoplasms ,Biochemistry ,Immediate early protein ,Immediate-Early Proteins ,Internal medicine ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,skin and connective tissue diseases ,Molecular Biology ,Glycoproteins ,Membrane Glycoproteins ,biology ,NF-kappa B ,Membrane Proteins ,Cell Biology ,Transforming growth factor beta ,Cell cycle ,Growth Inhibitors ,Neoplasm Proteins ,Testicular Hormones ,IκBα ,Endocrinology ,Receptors, Estrogen ,COS Cells ,biology.protein ,Cancer research ,Female ,Signal transduction ,Apoptosis Regulatory Proteins ,Receptors, Transforming Growth Factor beta ,Transforming growth factor - Abstract
Müllerian inhibiting substance (MIS), a member of the transforming growth factor-beta superfamily, induces regression of the Müllerian duct in male embryos. In this report, we demonstrate MIS type II receptor expression in normal breast tissue and in human breast cancer cell lines, breast fibroadenoma, and ductal adenocarcinomas. MIS inhibited the growth of both estrogen receptor (ER)-positive T47D and ER-negative MDA-MB-231 breast cancer cell lines, suggesting a broader range of target tissues for MIS action. Inhibition of growth was manifested by an increase in the fraction of cells in the G(1) phase of the cell cycle and induction of apoptosis. Treatment of breast cancer cells with MIS activated the NFkappaB pathway and selectively up-regulated the immediate early gene IEX-1S, which, when overexpressed, inhibited breast cancer cell growth. Dominant negative IkappaBalpha expression ablated both MIS-mediated induction of IEX-1S and inhibition of growth, indicating that activation of the NFkappaB signaling pathway was required for these processes. These results identify the NFkappaB-mediated signaling pathway and a target gene for MIS action and suggest a putative role for the MIS ligand and its downstream interactors in the treatment of ER-positive as well as negative breast cancers.
- Published
- 2000
13. Prostaglandin E2 Mediates Inhibition of Insulin Secretion by Interleukin-1β
- Author
-
Vincent Poitout, R. Paul Robertson, Phuong Oanh T. Tran, and Catherine E. Gleason
- Subjects
Male ,medicine.medical_specialty ,Prostaglandin E2 receptor ,medicine.medical_treatment ,Biochemistry ,Dinoprostone ,Adenylyl cyclase ,Islets of Langerhans ,chemistry.chemical_compound ,Internal medicine ,Insulin Secretion ,medicine ,Animals ,Insulin ,Receptors, Prostaglandin E ,Cyclooxygenase Inhibitors ,Rats, Wistar ,Prostaglandin E2 ,Molecular Biology ,Nitrobenzenes ,Sulfonamides ,Cyclooxygenase 2 Inhibitors ,biology ,Pancreatic islets ,Interleukin ,Cell Biology ,Rats ,Isoenzymes ,Glucose ,Endocrinology ,medicine.anatomical_structure ,chemistry ,Cyclooxygenase 2 ,Prostaglandin-Endoperoxide Synthases ,Receptors, Prostaglandin E, EP3 Subtype ,biology.protein ,Pyrazoles ,lipids (amino acids, peptides, and proteins) ,Cyclooxygenase ,Autacoid ,Adenylyl Cyclases ,Interleukin-1 ,medicine.drug - Abstract
Interleukin-1beta (IL-1beta) and prostaglandin E(2) (PGE(2)), frequently co-participants in inflammatory states, are two well recognized inhibitors of glucose-induced insulin secretion. Previous reports have concluded that the inhibitory effects of these two autacoids on pancreatic beta cell function are not related because indomethacin, a potent prostaglandin synthesis inhibitor, does not prevent IL-1beta effects. However, indomethacin is not a specific cyclooxygenase inhibitor, and its other pharmacologic effects are likely to inhibit insulin secretion independently. Since we recently observed that IL-1beta induces cyclooxygenase-2 (COX-2) gene expression and PGE(2) synthesis in islet beta cells, we have reassessed the possibility that PGE(2) mediates IL-1beta effects on beta function. By using two cell lines (HIT-T15 and betaHC13) as well as Wistar rat isolated pancreatic islets, we examined the ability of two COX-2-specific antagonists, NS-398 and SC-236, to prevent IL-1beta inhibition of insulin secretion. Both drugs prevented IL-1beta from inducing PGE(2) synthesis and inhibiting insulin secretion; adding back exogenous PGE(2) re-established inhibition of insulin secretion in the presence of IL-1beta. We also found that EP3, the PGE(2) receptor subtype whose post-receptor effect is to decrease adenylyl cyclase activity and, thereby, insulin secretion, is the dominant mRNA subtype expressed. We conclude that endogenous PGE(2) mediates the inhibitory effects of exogenous IL-1beta on beta cell function.
- Published
- 1999
14. Characterization of low potassium-resistant mutants of the Madin-Darby canine kidney cell line with defects in NaCl/KCl symport
- Author
-
C T Tran, Milton H. Saier, and J A McRoberts
- Subjects
Chemistry ,Potassium ,Mutant ,Madin Darby canine kidney cell ,chemistry.chemical_element ,Furosemide ,Cell Biology ,Biochemistry ,Molecular biology ,Cell culture ,Symporter ,medicine ,Tonicity ,Efflux ,Molecular Biology ,medicine.drug - Abstract
Three independent mutants of the Madin-Darby canine kidney cell line (MDCK) have been isolated which were capable of growth in media containing low concentrations of potassium. All three mutants were deficient to varying extents in furosemide- and bumetanide-sensitive 22Na+, 86+b+, and 36Cl- uptake. The two mutants most resistant to low K+ media had lost essentially all of the 22Na+, 86Rb+, and 36Cl- uptake activities of this system. The third mutant was partially resistant to low K+ media and had reduced levels of bumetanide-sensitive uptake for all three ions. Extrapolated initial uptake rates for 22Na+, 86Rb+, and 36Cl- revealed that the partial mutant exhibited approximately 50% of the parental uptake rates for all three ions. The stoichiometries of bumetanide-sensitive uptake in both the parental cell line and the partial mutant approximated 1 Rb+:1 Na+:2 Cl-. The results of this study provide genetic evidence for a single tightly-coupled NaCl/KCl symporter in MDCK cells. The correlation between the ability to grow in low K+ media and decreased activity of the bumetanide-sensitive co-transport system suggests that the bumetanide-sensitive transport system catalyzes net K+ efflux from cells in low K+ media. The results of 86Rb+ efflux studies conducted on ouabain-pretreated mutant and parental cells are consistent with this interpretation. Cell volume measurements made on cells at different densities in media containing normal K+ concentrations showed that none of the mutants differed significantly in volume from the parental strain at a similar cell density. Furthermore, all three mutants were able to readjust their volume after suspension in hypotonic media. These results suggest that in the MDCK cell line, the bumetanide-sensitive NaCl/KCl symport system does not function in the regulation of cell volume under the conditions employed.
- Published
- 1983
15. A Lettuce (Lactuca sativa) Homolog of Human Nogo-B Receptor Interacts with cis-Prenyltransferase and Is Necessary for Natural Rubber Biosynthesis.
- Author
-
Yang Qu, Chakrabarty, Romit, Hue T. Tran, Eun-Joo G. Kwon, Moonhyuk Kwon, Trinh-Don Nguyen, and Dae-Kyun Ro
- Subjects
- *
RUBBER , *LETTUCE research , *HEVEA , *DIMETHYLALLYLTRANSTRANSFERASE , *LATEX - Abstract
Natural rubber (cis-1,4-polyisoprene) is an indispensable biopolymer used to manufacture diverse consumer products. Although a major source of natural rubber is the rubber tree (Hevea brasiliensis), lettuce (Lactuca sativa) is also known to synthesize natural rubber. Here, we report that an unusual cis-prenyltransferase-like 2 (CPTL2) that lacks the conserved motifs of conventional cis-prenyltransferase is required for natural rubber biosynthesis in lettuce. CPTL2, identified from the lettuce rubber particle proteome, displays homology to a human NogoB receptor and is predominantly expressed in latex. Multiple transgenic lettuces expressing CPTL2-RNAi constructs showed that a decrease of CPTL2 transcripts (3-15% CPTL2 expression relative to controls) coincided with the reduction of natural rubber as low as 5%. We also identified a conventional cis-prenyltransferase 3 (CPT3), exclusively expressed in latex. In subcellular localization studies using fluorescent proteins, cytosolic CPT3 was relocalized to endoplasmic reticulum by co-occurrence of CPTL2 in tobacco and yeast at the log phase. Furthermore, yeast two-hybrid data showed that CPTL2 and CPT3 interact. Yeast microsomes containing CPTL2/CPT3 showed enhanced synthesis of short cis-polyisoprenes, but natural rubber could not be synthesized in vitro. Intriguingly, a homologous pair CPTL1/CPT1, which displays ubiquitous expressions in lettuce, showed a potent dolichol biosynthetic activity in vitro. Taken together, our data suggest that CPTL2 is a scaffolding protein that tethers CPT3 on endoplasmic reticulum and is necessary for natural rubber biosynthesis in planta, but yeast-expressed CPTL2 and CPT3 alone could not synthesize high molecular weight natural rubber in vitro. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
16. Catalytically Relevant Electrostatic Interactions of Cytochrome P450c17 (CYP17A1) and Cytochrome b5.
- Author
-
Hwei-Ming Peng, Jiayan Liu, Forsberg, Sarah E., Hong T. Tran, Anderson, Sean M., and Auchus, Richard J.
- Subjects
- *
CYTOCHROME P-450 structure , *CROSSLINKING (Polymerization) , *HETERODIMERS , *GENETIC mutation , *ANDROGENS - Abstract
Two acidic residues, Glu-48 and Glu-49, of cytochrome b5 (b5) are essential for stimulating the 17,20-lyase activity of cytochrome P450c17 (CYP17A1). Substitution of Ala, Gly, Cys, or Gln for these two glutamic acid residues abrogated all capacity to stimulate 17,20-lyase activity. Mutations E49D and E48D/E49D retained 23 and 38% of wild-type activity, respectively. Using the zero-length cross-linker ethyl-3-(3-dimethylaminopropyl) carbodiimide, we obtained cross-linked heterodimers of b5 and CYP17A1, wild-type, or mutations R347K and R358K. In sharp contrast, the b5 double mutation E48G/E49G did not form cross-linked complexes with wild-type CYP17A1. Mass spectrometric analysis of the CYP17A1-b5 complexes identified two cross-linked peptide pairs as follows: CYP17A1-WT: 84EVLIKK89-b5: 53EQAGG-DATENFEDVGHSTDAR73 and CYP17A1-R347K: 341TPTISDKNR349- b5: 40FLEEHPGGEEVLR52. Using these two sites of interaction and Glu-48/Glu-49 in b5 as constraints, protein docking calculations based on the crystal structures of the two proteins yielded a structural model of the CYP17A1-b5 complex. The appositional surfaces include Lys-88, Arg-347, and Arg-358/Arg-449 of CYP17A1, which interact with Glu-61, Glu-42, and Glu-48/Glu-49 of b5, respectively. Our data reveal the structural basis of the electrostatic interactions between these two proteins, which is critical for 17,20-lyase activity and androgen biosynthesis. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
17. Increasing kinase domain proximity promotes MST2 autophosphorylation during Hippo signaling.
- Author
-
Tran T, Mitra J, Ha T, and Kavran JM
- Subjects
- Enzyme Activation, HEK293 Cells, Hippo Signaling Pathway, Humans, Phosphorylation, Protein Domains, Protein Serine-Threonine Kinases genetics, Serine-Threonine Kinase 3, Signal Transduction, Protein Multimerization, Protein Serine-Threonine Kinases metabolism
- Abstract
The Hippo pathway plays an important role in developmental biology, mediating organ size by controlling cell proliferation through the activity of a core kinase cassette. Multiple upstream events activate the pathway, but how each controls this core kinase cassette is not fully understood. Activation of the core kinase cassette begins with phosphorylation of the kinase MST1/2 (also known as STK3/4). Here, using a combination of in vitro biochemistry and cell-based assays, including chemically induced dimerization and single-molecule pulldown, we revealed that increasing the proximity of adjacent kinase domains, rather than formation of a specific protein assembly, is sufficient to trigger autophosphorylation. We validate this mechanism in cells and demonstrate that multiple events associated with the active pathway, including SARAH domain-mediated homodimerization, membrane recruitment, and complex formation with the effector protein SAV1, each increase the kinase domain proximity and autophosphorylation of MST2. Together, our results reveal that multiple and distinct upstream signals each utilize the same common molecular mechanism to stimulate MST2 autophosphorylation. This mechanism is likely conserved among MST2 homologs. Our work also highlights potential differences in Hippo signal propagation between each activating event owing to differences in the dynamics and regulation of each protein ensemble that triggers MST2 autophosphorylation and possible redundancy in activation., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Tran et al.)
- Published
- 2020
- Full Text
- View/download PDF
18. Correction: Tumor progression locus 2 (Tpl2) kinase promotes chemokine receptor expression and macrophage migration during acute inflammation.
- Author
-
Rowley SM, Kuriakose T, Dockery LM, Tran-Nguyen T, Gingerich AD, Wei L, and Watford WT
- Published
- 2020
- Full Text
- View/download PDF
19. Salvador has an extended SARAH domain that mediates binding to Hippo kinase.
- Author
-
Cairns L, Tran T, Fowl BH, Patterson A, Kim YJ, Bothner B, and Kavran JM
- Subjects
- Animals, Drosophila melanogaster, HEK293 Cells, Humans, Phosphorylation genetics, Protein Domains, Protein Structure, Quaternary, Serine-Threonine Kinase 3, Cell Cycle Proteins chemistry, Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, Drosophila Proteins chemistry, Drosophila Proteins genetics, Drosophila Proteins metabolism, Intracellular Signaling Peptides and Proteins chemistry, Intracellular Signaling Peptides and Proteins genetics, Intracellular Signaling Peptides and Proteins metabolism, Multiprotein Complexes chemistry, Multiprotein Complexes genetics, Multiprotein Complexes metabolism, Protein Serine-Threonine Kinases chemistry, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism, Signal Transduction
- Abstract
The Hippo pathway controls cell proliferation and differentiation through the precisely tuned activity of a core kinase cassette. The activity of Hippo kinase is modulated by interactions between its C-terminal coiled-coil, termed the SARAH domain, and the SARAH domains of either dRassF or Salvador. Here, we wanted to understand the molecular basis of SARAH domain-mediated interactions and their influence on Hippo kinase activity. We focused on Salvador, a positive effector of Hippo activity and the least well-characterized SARAH domain-containing protein. We determined the crystal structure of a complex between Salvador and Hippo SARAH domains from Drosophila This structure provided insight into the organization of the Salvador SARAH domain including a folded N-terminal extension that expands the binding interface with Hippo SARAH domain. We also found that this extension improves the solubility of the Salvador SARAH domain, enhances binding to Hippo, and is unique to Salvador. We therefore suggest expanding the definition of the Salvador SARAH domain to include this extended region. The heterodimeric assembly observed in the crystal was confirmed by cross-linked MS and provided a structural basis for the mutually exclusive interactions of Hippo with either dRassF or Salvador. Of note, Salvador influenced the kinase activity of Mst2, the mammalian Hippo homolog. In co-transfected HEK293T cells, human Salvador increased the levels of Mst2 autophosphorylation and Mst2-mediated phosphorylation of select substrates, whereas Salvador SARAH domain inhibited Mst2 autophosphorylation in vitro These results suggest Salvador enhances the effects of Hippo kinase activity at multiple points in the Hippo pathway., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2018
- Full Text
- View/download PDF
20. Ligands for FKBP12 increase Ca2+ influx and protein synthesis to improve skeletal muscle function.
- Author
-
Lee CS, Georgiou DK, Dagnino-Acosta A, Xu J, Ismailov II, Knoblauch M, Monroe TO, Ji R, Hanna AD, Joshi AD, Long C, Oakes J, Tran T, Corona BT, Lorca S, Ingalls CP, Narkar VA, Lanner JT, Bayle JH, Durham WJ, and Hamilton SL
- Subjects
- Animals, Calcium Signaling drug effects, Dose-Response Relationship, Drug, Mechanistic Target of Rapamycin Complex 1, Mice, Multiprotein Complexes, Muscle Contraction drug effects, Muscle, Skeletal metabolism, Protein Binding, Protein Biosynthesis drug effects, Sarcoplasmic Reticulum drug effects, Sarcoplasmic Reticulum metabolism, TOR Serine-Threonine Kinases, Tacrolimus Binding Protein 1A chemistry, Tacrolimus Binding Protein 1A genetics, Ligands, Muscle, Skeletal growth & development, Sirolimus administration & dosage, Tacrolimus Binding Protein 1A metabolism
- Abstract
Rapamycin at high doses (2-10 mg/kg body weight) inhibits mammalian target of rapamycin complex 1 (mTORC1) and protein synthesis in mice. In contrast, low doses of rapamycin (10 μg/kg) increase mTORC1 activity and protein synthesis in skeletal muscle. Similar changes are found with SLF (synthetic ligand for FKBP12, which does not inhibit mTORC1) and in mice with a skeletal muscle-specific FKBP12 deficiency. These interventions also increase Ca(2+) influx to enhance refilling of sarcoplasmic reticulum Ca(2+) stores, slow muscle fatigue, and increase running endurance without negatively impacting cardiac function. FKBP12 deficiency or longer treatments with low dose rapamycin or SLF increase the percentage of type I fibers, further adding to fatigue resistance. We demonstrate that FKBP12 and its ligands impact multiple aspects of muscle function., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
- Full Text
- View/download PDF
21. Tumor progression locus 2 (Tpl2) kinase promotes chemokine receptor expression and macrophage migration during acute inflammation.
- Author
-
Rowley SM, Kuriakose T, Dockery LM, Tran-Nguyen T, Gingerich AD, Wei L, and Watford WT
- Subjects
- Acute Disease, Adaptor Proteins, Vesicular Transport genetics, Animals, Cell Movement immunology, Chemokines metabolism, Female, Lipopolysaccharides pharmacology, MAP Kinase Kinase Kinases genetics, MAP Kinase Kinase Kinases metabolism, MAP Kinase Signaling System immunology, Macrophages cytology, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Myeloid Differentiation Factor 88 genetics, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins metabolism, Receptors, CCR1 genetics, Receptors, CCR1 immunology, Receptors, CCR1 metabolism, Receptors, CCR2 genetics, Receptors, CCR2 immunology, Receptors, CCR2 metabolism, Receptors, CCR5 genetics, Receptors, CCR5 immunology, Receptors, CCR5 metabolism, Receptors, Chemokine genetics, Receptors, Chemokine metabolism, Chemokines immunology, Inflammation immunology, MAP Kinase Kinase Kinases immunology, Macrophages immunology, Proto-Oncogene Proteins immunology, Receptors, Chemokine immunology
- Abstract
In autoimmune diseases, the accumulation of activated leukocytes correlates with inflammation and disease progression, and, therefore, the disruption of leukocyte trafficking is an active area of research. The serine/threonine protein kinase Tpl2 (MAP3K8) regulates leukocyte inflammatory responses and is also being investigated for therapeutic inhibition during autoimmunity. Here we addressed the contribution of Tpl2 to the regulation of macrophage chemokine receptor expression and migration in vivo using a mouse model of Tpl2 ablation. LPS stimulation of bone marrow-derived macrophages induced early CCR1 chemokine receptor expression but repressed CCR2 and CCR5 expression. Notably, early induction of CCR1 expression by LPS was dependent upon a signaling pathway involving Tpl2, PI3K, and ERK. On the contrary, Tpl2 was required to maintain the basal expression of CCR2 and CCR5 as well as to stabilize CCR5 mRNA expression. Consistent with impairments in chemokine receptor expression, tpl2(-/-) macrophages were defective in trafficking to the peritoneal cavity following thioglycollate-induced inflammation. Overall, this study demonstrates a Tpl2-dependent mechanism for macrophage expression of select chemokine receptors and provides further insight into how Tpl2 inhibition may be used therapeutically to disrupt inflammatory networks in vivo., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
- Full Text
- View/download PDF
22. Protein kinase G and focal adhesion kinase converge on Src/Akt/β-catenin signaling module in osteoblast mechanotransduction.
- Author
-
Rangaswami H, Schwappacher R, Tran T, Chan GC, Zhuang S, Boss GR, and Pilz RB
- Subjects
- Animals, Cell Line, Cyclic GMP-Dependent Protein Kinase Type II, Cyclic GMP-Dependent Protein Kinases genetics, Cyclooxygenase 2 genetics, Cyclooxygenase 2 metabolism, Female, Focal Adhesion Kinase 1 genetics, Humans, Male, Mice, Mice, Knockout, Nitric Oxide Synthase genetics, Nitric Oxide Synthase metabolism, Phosphatidylinositol 3-Kinases genetics, Phosphatidylinositol 3-Kinases metabolism, Proto-Oncogene Proteins c-akt genetics, Proto-Oncogene Proteins c-fos genetics, Proto-Oncogene Proteins c-fos metabolism, beta Catenin genetics, src-Family Kinases genetics, Cyclic GMP-Dependent Protein Kinases metabolism, Focal Adhesion Kinase 1 metabolism, Mechanotransduction, Cellular physiology, Proto-Oncogene Proteins c-akt metabolism, beta Catenin metabolism, src-Family Kinases metabolism
- Abstract
Mechanical loading of bone induces interstitial fluid flow, leading to fluid shear stress (FSS) of osteoblasts. FSS rapidly increases the intracellular calcium concentration ([Ca(2+)]) and nitric oxide (NO) synthesis in osteoblasts and activates the protein kinase Akt. Activated Akt stimulates osteoblast proliferation and survival, but the mechanism(s) leading to Akt activation is not well defined. Using pharmacological and genetic approaches in primary human and mouse osteoblasts and mouse MC3T3 osteoblast-like cells, we found that Akt activation by FSS occurred through two parallel pathways; one required calcium stimulation of NO synthase and NO/cGMP/protein kinase G II-dependent activation of Src, and the other required calcium activation of FAK and Src, independent of NO. Both pathways cooperated to increase PI3K-dependent Akt phosphorylation and were necessary for FSS to induce nuclear translocation of β-catenin, c-fos, and cox-2 gene expression and osteoblast proliferation. These data explain how mechanical stimulation of osteoblasts leads to increased signaling through a growth regulatory pathway essential for maintaining skeletal integrity.
- Published
- 2012
- Full Text
- View/download PDF
23. Activation-induced degradation of FLIP(L) is mediated via the phosphatidylinositol 3-kinase/Akt signaling pathway in macrophages.
- Author
-
Shi B, Tran T, Sobkoviak R, and Pope RM
- Subjects
- Amino Acid Substitution drug effects, Apoptosis drug effects, CASP8 and FADD-Like Apoptosis Regulating Protein genetics, Caspase 8 metabolism, Enzyme Activation drug effects, Gene Expression Regulation drug effects, Humans, Lipopolysaccharides pharmacology, Macrophages cytology, Macrophages drug effects, NF-kappa B metabolism, Phosphorylation drug effects, Phosphoserine metabolism, Proteasome Endopeptidase Complex metabolism, Protein Binding drug effects, RNA, Messenger genetics, RNA, Messenger metabolism, Repressor Proteins metabolism, Tumor Necrosis Factor-alpha pharmacology, Ubiquitin-Protein Ligases metabolism, CASP8 and FADD-Like Apoptosis Regulating Protein metabolism, Macrophages enzymology, Phosphatidylinositol 3-Kinases metabolism, Protein Processing, Post-Translational drug effects, Proto-Oncogene Proteins c-akt metabolism, Signal Transduction drug effects
- Abstract
Cellular FLIP (Flice-like inhibitory protein) is critical for the protection against death receptor-mediated cell apoptosis. In macrophages, FLIP long (FLIP(L)) and FLIP short (FLIP(S)) mRNA was induced by tumor necrosis factor (TNF) alpha, mediated through NF-kappaB. However, we observed TNFalpha reduced the protein level of FLIP(L), but not FLIP(S), at 1 and 2 h. Similar results were observed with lipopolysaccharide. The reduction of FLIP(L) by TNFalpha was not mediated by caspase 8, or through JNK or Itch, but was suppressed by inhibition of the phosphatidylinositol 3-kinase/Akt pathway employing chemical inhibitors, a dominant negative Akt-1, or Akt-1 small interfering RNA. The reduction of FLIP(L) resulted in the short term induction of caspase 8-like activity, which augmented NF-kappaB activation. A co-immunoprecipitation assay demonstrated that Akt-1 physically interacts with FLIP(L). Moreover, TNFalpha enhanced FLIP(L) serine phosphorylation, which was increased by activated Akt-1. Serine 273, a putative Akt-1 phosphorylation site in FLIP(L), was critical for the activation-induced reduction of FLIP(L). Thus, these observations document a novel mechanism where by TNFalpha facilitates the reduction of FLIP(L) protein, which is dependent on the phosphatidylinositol 3-kinase/Akt signaling.
- Published
- 2009
- Full Text
- View/download PDF
24. Structural determinants for phosphatidic acid regulation of phospholipase C-beta1.
- Author
-
Ross EM, Mateu D, Gomes AV, Arana C, Tran T, and Litosch I
- Subjects
- Binding Sites, Circular Dichroism, Enzyme Activation, Isoenzymes genetics, Mutation genetics, Phenotype, Phospholipase C beta, Type C Phospholipases genetics, Isoenzymes chemistry, Isoenzymes metabolism, Phosphatidic Acids chemistry, Phosphatidic Acids metabolism, Type C Phospholipases chemistry, Type C Phospholipases metabolism
- Abstract
Signaling from G protein-coupled receptors to phospholipase C-beta (PLC-beta) is regulated by coordinate interactions among multiple intracellular signaling molecules. Phosphatidic acid (PA), a signaling phospholipid, binds to and stimulates PLC-beta(1) through a mechanism that requires the PLC-beta(1) C-terminal domain. PA also modulates Galpha(q) stimulation of PLC-beta(1). These data suggest that PA may have a key role in the regulation of PLC-beta(1) signaling in cells. The present studies addressed the structural requirements and the mechanism for PA regulation of PLC-beta(1). We used a combination of enzymatic assays, PA-binding assays, and circular dichroism spectroscopy to evaluate the interaction of PA with wild-type and mutant PLC-beta(1) proteins and with fragments of the Galpha(q) binding domain. The results identify a region that includes the alphaA helix and flexible loop of the Galpha(q)-binding domain as necessary for PA regulation. A mutant PLC-beta(1) with multiple alanine/glycine replacements for residues (944)LIKEHTTKYNEIQN(957) was markedly impaired in PA regulation. The high affinity and low affinity component of PA stimulation was reduced 70% and PA binding was reduced 45% in this mutant. Relative PLC stimulation by PA increased with PLC-beta(1) concentration in a manner suggesting cooperative binding to PA. Similar concentration dependence was observed in the PLC-beta(1) mutant. These data are consistent with a model for PA regulation of PLC-beta(1) that involves cooperative interactions, probably PLC homodimerization, that require the flexible loop region, as is consistent with the dimeric structure of the Galpha(q)-binding domain. PA regulation of PLC-beta(1) requires unique residues that are not required for Galpha(q) stimulation or GTPase-activating protein activity.
- Published
- 2006
- Full Text
- View/download PDF
25. Selective inhibition of fibroblast activation protein protease based on dipeptide substrate specificity.
- Author
-
Edosada CY, Quan C, Wiesmann C, Tran T, Sutherlin D, Reynolds M, Elliott JM, Raab H, Fairbrother W, and Wolf BB
- Subjects
- Acetylcysteine analogs & derivatives, Acetylcysteine chemistry, Amino Acid Motifs, Antigens, Neoplasm chemistry, Biomarkers, Tumor chemistry, Biotin chemistry, Cell Line, Chromatography, Gel, Cloning, Molecular, DNA, Complementary metabolism, Dimerization, Dose-Response Relationship, Drug, Endopeptidases, Gelatinases, Humans, Hydrolysis, Kinetics, Light, Membrane Proteins, Models, Chemical, Models, Molecular, Peptide Hydrolases chemistry, Protein Binding, Scattering, Radiation, Serine Endopeptidases chemistry, Substrate Specificity, Time Factors, Adenosine Deaminase chemistry, Biomarkers, Tumor antagonists & inhibitors, Dipeptidyl Peptidase 4 chemistry, Fibroblasts metabolism, Glycoproteins chemistry, Peptides chemistry
- Abstract
Fibroblast activation protein (FAP) is a transmembrane serine peptidase that belongs to the prolyl peptidase family. FAP has been implicated in cancer; however, its specific role remains elusive because inhibitors that distinguish FAP from other prolyl peptidases like dipeptidyl peptidase-4 (DPP-4) have not been developed. To identify peptide motifs for FAP-selective inhibitor design, we used P(2)-Pro(1) and acetyl (Ac)-P(2)-Pro(1) dipeptide substrate libraries, where P(2) was varied and substrate hydrolysis occurs between Pro(1) and a fluorescent leaving group. With the P(2)-Pro(1) library, FAP preferred Ile, Pro, or Arg at the P(2) residue; however, DPP-4 showed broad reactivity against this library, precluding selectivity. By contrast, with the Ac-P(2)-Pro(1) library, FAP cleaved only Ac-Gly-Pro, whereas DPP-4 showed little reactivity with all substrates. FAP also cleaved formyl-, benzyloxycarbonyl-, biotinyl-, and peptidyl-Gly-Pro substrates, which DPP-4 cleaved poorly, suggesting an N-acyl-Gly-Pro motif for inhibitor design. Therefore, we synthesized and tested the compound Ac-Gly-prolineboronic acid, which inhibited FAP with a K(i) of 23 +/- 3 nm. This was approximately 9- to approximately 5400-fold lower than the K(i) values for other prolyl peptidases, including DPP-4, DPP-7, DPP-8, DPP-9, prolyl oligopeptidase, and acylpeptide hydrolase. These results identify Ac-Gly-BoroPro as a FAP-selective inhibitor and suggest that N-acyl-Gly-Pro-based inhibitors will allow testing of FAP as a therapeutic target.
- Published
- 2006
- Full Text
- View/download PDF
26. Roles of stem cell factor/c-Kit and effects of Glivec/STI571 in human uveal melanoma cell tumorigenesis.
- Author
-
Lefevre G, Glotin AL, Calipel A, Mouriaux F, Tran T, Kherrouche Z, Maurage CA, Auclair C, and Mascarelli F
- Subjects
- Base Sequence, Benzamides, Cell Division, Cell Line, Tumor, Cell Transformation, Neoplastic, DNA Primers genetics, Gene Expression, Humans, Imatinib Mesylate, Melanocytes physiology, Melanoma drug therapy, Melanoma genetics, Melanoma physiopathology, Mitogens metabolism, Mutation, Piperazines therapeutic use, Proto-Oncogene Proteins c-kit genetics, Pyrimidines therapeutic use, RNA, Small Interfering genetics, Signal Transduction, Stem Cell Factor genetics, Uvea physiology, Uveal Neoplasms drug therapy, Uveal Neoplasms genetics, Uveal Neoplasms physiopathology, Melanoma etiology, Proto-Oncogene Proteins c-kit physiology, Stem Cell Factor physiology, Uveal Neoplasms etiology
- Abstract
The B-Raf(V599E)-mediated constitutive activation of ERK1/2 is involved in establishing the transformed phenotype of some uveal melanoma cells (Calipel, A., Lefevre, G., Pouponnot, C., Mouriaux, F., Eychene, A., and Mascarelli, F. (2003) J. Biol. Chem. 278, 42409-42418). We have shown that stem cell factor (SCF) is involved in the proliferation of normal uveal melanocytes and that c-Kit is expressed in 75% of primary uveal melanomas. This suggests that the acquisition of autonomous growth during melanoma progression may involve the SCF/c-Kit axis. We used six human uveal melanoma tumor-derived cell lines and normal uveal melanocytes to characterize the SCF/c-Kit system and to assess its specific role in transformation. We investigated the possible roles of activating mutations in c-KIT, the overexpression of this gene, and ligand-dependent c-Kit overactivation in uveal melanoma cell tumorigenesis. Four cell lines (92.1, SP6.5, Mel270, and TP31) expressed both SCF and c-Kit, and none harbored the c-KIT mutations in exons 9, 11, 13, and 17 that have been shown to induce SCF-independent c-Kit activation. Melanoma cell proliferation was strongly inhibited by small interfering RNA-mediated depletion of c-Kit in these cells, despite the presence of (V599E)B-Raf in SP6.5 and TP31 cells. We characterized the signaling pathways involved in SCF/c-Kit-mediated cell growth and survival in normal and tumoral melanocytes and found that constitutive ERK1/2 activation played a key role in both the SCF/c-Kit autocrine loop and the gain of function of (V599E)B-Raf for melanoma cell proliferation and transformation. We also provide the first evidence that Glivec/STI571, a c-Kit tyrosine kinase inhibitor, could be used to treat uveal melanomas.
- Published
- 2004
- Full Text
- View/download PDF
27. Neurofilament protein heterotetramers as assembly intermediates.
- Author
-
Cohlberg JA, Hajarian H, Tran T, Alipourjeddi P, and Noveen A
- Subjects
- Animals, Cattle, Electrophoresis, Gel, Two-Dimensional, Urea pharmacology, Neurofilament Proteins chemistry
- Abstract
Evidence is presented for the existence of a soluble heterotetramer containing the low and middle molecular weight neurofilament (NF) proteins, NF-L and NF-M, and one containing the low and high molecular weight proteins, NF-L and NF-H, and for their role in filament assembly. When a mixture of either pair of proteins was renatured in 2 M urea, 20 mM Tris, pH 7.2, a new band representing a complex was observed in native gel electrophoresis. No new band was observed with a mixture of NF-M and NF-H. Two-dimensional gel electrophoresis showed that treatment of the complexes with SDS caused them to dissociate into their constituent polypeptide chains. Native neurofilaments dissociated in 2 M urea into a mixture of LM and LH complexes. Titration of NF-L with NF-M indicated that complex formation was complete at an approximately equimolar ratio of the two proteins. The LM complex had a sedimentation coefficient, s20,w, of 4.4 S, consistent with a tetrameric structure. Dialysis of a solution of the LM complex against 50 mM 4-morpholineethanesulfonic acid, 0.17 M NaCl, pH 6.25, led to the formation of 10-nm filaments in good yield. These results suggest that NF protein heterooligomers are intermediates in NF assembly and disassembly.
- Published
- 1995
- Full Text
- View/download PDF
28. Rat liver canalicular membrane vesicles. Isolation and topological characterization.
- Author
-
Inoue M, Kinne R, Tran T, Biempica L, and Arias IM
- Subjects
- Animals, Cell Fractionation, Male, Membranes enzymology, Membranes ultrastructure, Microscopy, Electron, Osmolar Concentration, Rats, Rats, Inbred Strains, Liver ultrastructure
- Abstract
Canalicular plasma membranes were isolated from rat liver homogenates using nitrogen cavitation and calcium precipitation methods. Compared with homogenates, the membranes were enriched 55- to 56-fold in gamma-glutamyltransferase, aminopeptidase M, and alkaline phosphatase activities and showed very low enrichment in markers of other membranes. By electron microscopy, the membrane preparation contained neither junctional complexes nor contaminating organelles and consisted exclusively of vesicles. The presence of vesicles was also evident from the osmotic sensitivity of D-[6-3H]glucose uptake into the membrane preparation. Antisera obtained from rabbits immunized with highly purified rat kidney gamma-glutamyltransferase inhibited the transferase activity of intact or Triton X-100-solubilized membranes by 45-55%. Treatment of vesicles with anti-gamma-glutamyltransferase antisera and anti-rabbit IgG antisera increased the apparent density of the membranes during sucrose density gradient centrifugation. gamma-Glutamyltransferase and aminopeptidase M activities were selectively removed from the vesicles by limited proteolysis with papain without changing the intravesicular space or alkaline phosphatase activity of the membranes. Specific binding of anti-gamma-glutamyltransferase antibody to the outer surface of isolated hepatocytes was observed as measured by the antisera and 125I-labeled protein A; binding followed saturation kinetics with respect to antibody concentration. These data indicate that the isolated canalicular membrane vesicles are exclusively oriented right-side-out and that gamma-glutamyltransferase and aminopeptidase M are located on the luminal side of rat liver canalicular plasma membranes.
- Published
- 1983
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.