46 results on '"Chen, Chen"'
Search Results
2. Methyltransferase METTL8 is required for 3-methylcytosine modification in human mitochondrial tRNAs
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Lentini, Jenna M., Bargabos, Rachel, Chen, Chen, and Fu, Dragony
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- 2022
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3. Induced Expression of FcγRIIIa (CD16a) on CD4+ T Cells Triggers Generation of IFN-γhigh Subset.
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Chauhan, Anil K., Chen Chen, Moore, Terry L., and DiPaolo, Richard J.
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T cell differentiation , *IMMUNE response , *IMMUNE complexes , *CELL differentiation , *INFLAMMATION , *AUTOIMMUNITY - Abstract
Whether or not CD4+ T-cells express low affinity receptor FcγRIIIa (CD16a) in disease pathology has not been examined in great detail. In this study, we show that a subset of activated CD4+ T-cells in humans express FcγRIIIa. The ligation of FcγRIIIa by immune complexes (ICs) in human CD4+ T-cells produced co-stimulatory signal like CD28 that triggered IFN-γ production. The induced expression of FcγRIIIa on CD4+ helper T-cells is an important finding since these receptors via ITAM contribute to intracellular signaling. The induced expression of FcγRIIIa on CD4+ T helper cells and their ability to co-stimulate T-cell activation are important and novel findings that may reveal new pathways to regulate adaptive immune responses during inflammation and in autoimmunity. [ABSTRACT FROM AUTHOR]
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- 2015
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4. Secreted Proteases Control Autolysin-mediated Biofilm Growth of Staphylococcus aureus.
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Chen, Chen, Krishnan, Vengadesan, Macon, Kevin, Manne, Kartik, Narayana, Sthanam V. L., and Schneewind, Olaf
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PROTEOLYTIC enzymes , *PEPTIDOGLYCAN hydrolase , *AUTOLYSINS , *STAPHYLOCOCCUS aureus , *BIOFILMS , *X-ray crystallography , *PROTEOLYSIS , *STAPHYLOCOCCUS epidermidis - Abstract
Staphylococcus epidermidis, a commensal of humans, secretes Esp protease to prevent Staphylococcus aureus biofilm formation and colonization. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases; however, the mechanism where by Esp disrupts biofilms is unknown. We show here that Esp cleaves autolysin (Atl)-derived murein hydrolases and prevents staphylococcal release of DNA, which serves as extracellular matrix in biofilms. The three-dimensional structure of Espwas revealed by x-ray crystallography and shown to be highly similar to that of S. aureus V8 (SspA). Both atl and sspA are necessary for biofilm formation, and purified SspA cleaves Atl-derived murein hydrolases. Thus, S. aureus biofilms are formed via the controlled secretion and proteolysis of autolysin, and this developmental programappears to be perturbed by the Esp protease of S. epidermidis. [ABSTRACT FROM AUTHOR]
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- 2013
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5. Coenzyme Q10 Rescues Ethanol-induced Corneal Fibroblast Apoptosis through the Inhibition of Caspase-2 Activation.
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Chun-Chen Chen, Shiow-Wen Liou, Chi-Chih Chen, Wen-Chung Chen, Fung-Rong Hu, I-Jong Wang, and Shing-Jong Lin
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UBIQUINONES , *CASPASES , *FIBROBLASTS , *APOPTOSIS , *BIOCHEMISTRY - Abstract
Recent studies indicate that caspase-2 is involved in the early stages of apoptosis, particularly before the occurrence of mitochondrial damage. Here we report the important role of the coenzyme Q10 (CoQ10) on the activity of caspase-2 upstream of mitochondria in ethanol (EtOH)-treated corneal fibroblasts. After EtOH exposure, cells produce excessive reactive oxygen species formation, p53 expression, and most importantly, caspase-2 activation. After the activation of the caspase-2, the cells exhibited hallmarks of apoptotic pathway, such as mitochondrial damage and translocation of Bax and cytochrome c, which were then followed by caspase-3 activation. By pretreating the cells with a cell-permeable, biotinylated pan-caspase inhibitor, we identified caspase-2 as an initiator caspase in EtOH-treated corneal fibroblasts. Loss of caspase-2 inhibited EtOH-induced apoptosis. We further found that caspase-2 acts upstream of mitochondria to mediate EtOH-induced apoptosis. The loss of caspase-2 significantly inhibited EtOH-induced mitochondrial dysfunction, Bax translocation, and cytochrome c release from mitochondria. The pretreatment of CoQ10 prevented EtOH-induced caspase-2 activation and mitochondria-mediated apoptosis. Our data demonstrated that by blocking caspase-2 activity, CoQ10 can protect the cells from mitochondrial membrane change, apoptotic protein translocation, and apoptosis. Taken together, EtOH-induced mitochondria-mediated apoptosis is initiated by caspase-2 activation, which is regulated by CoQ10. [ABSTRACT FROM AUTHOR]
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- 2013
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6. Identification of the E3 Deubiquitinase Ubiquitin-specific Peptidase 21 (USP21) as a Positive Regulator of the Transcription Factor GATA3.
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Jing Zhang, Chen Chen, Xiaoxia Hou, Yayi Gao, Fang Lin, Jing Yang, Zhimei Gao, Pan, Lina, Lianqin Tao, Chijun Wen, Zhengju Yao, Tsun, Andy, Guochao Shi, and Bin Li
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TRANSCRIPTION factors , *PROTEOLYTIC enzymes , *LYMPHOCYTES , *CELL culture , *T cell receptors , *GENE expression - Abstract
The expression of the transcription factor GATA3 in FOXP3+ regulatory T (Treg) cells is crucial for their physiological function in limiting inflammatory responses. Although other studies have shown how T cell receptor (TcR) signals induce the up-regulation of GATA3 expression in Treg cells, the underlying mechanism that maintains GATA3 expression in Treg cells remains unclear. Here, we show how USP21 interacts with and stabilizes GATA3 by mediating its deubiquitination. In a T cell line model, we found that TcR stimulation promoted USP21 expression, which was further up-regulated in the presence of FOXP3. The USP21 mutant C221A reduced its capacity to stabilize GATA3 expression, and its knockdown led to the down-regulation of GATA3 protein expression in Treg cells. Furthermore, we found that FOXP3 could directly bind to the USP21 gene promoter and activated its transcription upon TcR stimulation. Finally, USP21, GATA3, and FOXP3 were found up-regulated in Treg cells that were isolated from asthmatic subjects. In summary, we have identified a USP21-mediated pathway that promotes GATA3 stabilization and expression at the post-translational level. We propose that this pathway forms an important signaling loop that stabilizes the expression of GATA3 in Treg cells. [ABSTRACT FROM AUTHOR]
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- 2013
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7. Pliable DNA Conformation of Response Elements Bound to Transcription Factor p63.
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Chen Chen, Gorlatova, Natalia, and Herzberg, Osnat
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DNA , *TRANSCRIPTION factors , *CARRIER proteins , *NUCLEOTIDE sequence , *HELIX (Mollusks) - Abstract
We show that changes in the nucleotide sequence alter the DNA conformation in the crystal structures of p63 DNA-binding domain (p63DBD) bound to its response element. The conformation of a 22-bp canonical response element containing an AT spacer between the two half-sites is unaltered compared with that containing a TA spacer, exhibiting superhelical trajectory. In contrast, a GC spacers abolishes the DNA superhelical trajectory and exhibits less bent DNA, suggesting that increased GC content accompanies increased double helix rigidity. A 19-bp DNA, representing an AT-rich response element with overlapping half-sites, maintains superhelical trajectory and reveals two interacting p63DBD dimers crossing one another at 120°. p63DBD binding assays to response elements of increasing length complement the structural studies. We propose that DNA deformation may affect promoter activity, that the ability of p63DBD to bind to superhelical DNA suggests that it is capable of binding to nucleosomes, and that overlapping response elements may provide a mechanism to distinguish between p63 and p53 promoters. [ABSTRACT FROM AUTHOR]
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- 2012
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8. Structure of Oxalacetate Acetylhydrolase, a Virulence Factor of the Chestnut Blight Fungus.
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Chen Chen, Qihong Sun, Narayanan, Buvaneswari, Nuss, Donald L., and Herzberg, Osnat
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PYRUVATE kinase , *OXALIC acid , *CHESTNUT diseases & pests , *FUNGI , *ENZYMES - Abstract
Oxalacetate acetylhydrolase (OAH), a member of the phosphoenolpyruvate mutase/isocitrate lyase superfamily, catalyzes the hydrolysis of oxalacetate to oxalic acid and acetate. This study shows that knock-out of the oah gene in Cryphonectria parasitica, the chestnut blight fungus, reduces the ability of the fungus to form cankers on chestnut trees, suggesting that OAH plays a key role in virulence. OAH was produced in Escherichia coli and purified, and its catalytic rates were determined. Oxalacetate is the main OAH substrate, but the enzyme also acts as a lyase of (2R,3S)-dimethyl malate with ~1000-fold lower efficacy. The crystal structure of OAH was determined alone, in complex with a mechanism-based inhibitor, 3,3-difluorooxalacetate (DFOA), and in complex with the reaction product, oxalate, to a resolution limit of 1.30, 1.55, and 1.65 Å, respectively. OAH assembles into a dimer of dimers with each subunit exhibiting an (α/β)8 barrel fold and each pair swapping the 8th α-helix. An active site "gating loop" exhibits conformationai disorder in the ligand-free structure. To obtain the structures of the OAH⋅ligand complexes, the ligand-free OAH crystals were soaked briefly with DFOA or oxalacetate. DFOA binding leads to ordering of the gating loop in a conformation that sequesters the ligand from the solvent. DFOA binds in a gem-diol form analogous to the oxalacetate intermediate/transition state. Oxalate binds in a planar conformation, but the gating loop is largely disordered. Comparison between the OAH structure and that of the closely related enzyme, 2,3-dimethylmalate lyase, suggests potential determinants of substrate preference. [ABSTRACT FROM AUTHOR]
- Published
- 2010
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9. Gangliosides as High Affinity Receptors for Tetanus Neurotoxin.
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Chen Chen, Zhuji Fu, Jung-Ja P. Kim, Barbieri, Joseph T., and Baldwin, Michael R.
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CLOSTRIDIUM diseases , *TETANUS , *NEUROTOXIC agents , *NEUROTRANSMITTERS , *NERVOUS system , *ANAEROBIC infections - Abstract
Tetanus neurotoxin (TeNT) is an exotoxin produced by Clostridium tetani that causes paralytic death to hundreds of thousands of humans annually. TeNT cleaves vesicle-associated membrane protein-2, which inhibits neurotransmitter release in the central nervous system to elicit spastic paralysis, but the molecular basis for TeNT entry into neurons remains unclear. TeNT is a ~150-kDa protein that has AB structure-function properties; the A domain is a zinc metalloprotease, and the B domain encodes a translocation domain and C-terminal receptor-binding domain (HCR/T). Earlier studies showed that HCR/T bound gangliosides via two carbohydrate-binding sites, termed the lactose-binding site (the "W" pocket) and the sialic acid-binding site (the "R" pocket). Here we report that TeNT high affinity binding to neurons is mediated solely by gangliosides. Glycan array and solid phase binding analyses identified gangliosides that bound exclusively to either the W pocket or the R pocket of TeNT; GM1a bound to the W pocket, and GD3 bound to the R pocket. Using these gangliosides and mutated forms of HCR/T that lacked one or both carbohydrate-binding pocket, gangliosides binding to both of the W and R pockets were shown to be necessary for high affinity binding to neuronal and non-neuronal cells. The crystal structure of a ternary complex of HCR/T with sugar components of two gangliosides bound to the W and R supported the binding of gangliosides to both carbohydrate pockets. These data show that gangliosides are functional dual receptors for TeNT. [ABSTRACT FROM AUTHOR]
- Published
- 2009
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10. Mechanistic Studies of the Phytochromobilin Synthase HY2 from Arabidopsis.
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Shih-Long Tu, Hsiu-Chen Chen, and Li-Wen Ku
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TETRAPYRROLES , *PHYTOCHROMES , *ELECTRON paramagnetic resonance spectroscopy , *ASPARTATE aminotransferase , *ARABIDOPSIS - Abstract
Phytochromobilin (PΦB) is an open chain tetrapyrrole molecule that functions as the chromophore of light-sensing phytochromes in plants. Derived from heme, PΦB is synthesized through an open chain tetrapyrrole intermediate, biliverdin IXα (BV), in the biosynthesis pathway. BV is subsequently reduced by the PΦB synthase HY2 in plants. HY2 is a ferredoxin-dependent bilin reductase that catalyzes the reduction of the A-ring 2,3,3¹,3²-diene system to produce an ethylidene group for assembly with apophytochromes. In this study, we sought to determine the catalytic mechanism of HY2. Data from UV-visible and EPR spectroscopy showed that the HY2-catalyzed BV reaction proceeds via a transient radical intermediate. Site-directed mutagenesis showed several ionizable residues that are involved in the catalytic steps. Detailed analysis of these site-directed mutants highlighted a pair of aspartate residues central to proton donation and substrate positioning. A mechanistic prediction for the HY2 reaction is proposed. These results support the hypothesis that ferredoxin-dependent bilin reductases reduce BV through a radical mechanism, but their double bond specificity is decided by strategic placement of different protondonating residues surrounding the bilin substrate in the active sites. [ABSTRACT FROM AUTHOR]
- Published
- 2008
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11. p21 Ras/Impedes Mitogenic Signal Propagation Regulates Cytokine Production and Migration in CD4 T Cells.
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Czyzyk, Jan, Hui-Chen Chen, BottomIy, Kim, and FIaveII, Richard A.
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LYMPHOCYTES , *LEUCOCYTES , *CELLULAR immunity , *T cells , *TH1 cells , *CYTOKINES , *BIOLOGICAL transport - Abstract
The propensity of T cells to generate coordinated cytokine responses is critical for the host to develop resistance to pathogens while maintaining the state of immunotolerance to self-antigens. The exact mechanisms responsible for preventing the overproduction of proinflammatory cytokines including interferon (IFN)-γ are not fully understood, however. In this study, we examined the role of a recently described Ras GTPase effector and repressor of the Raf/MEK/ERK cascade called impedes mitogenic signal propagation (Imp) in limiting the induction of T-cell cytokines. We found that stimulation of the T cell receptor complex leads to the rapid development of a physical association between Ras and Imp. Consistent with the hypothesis that Imp inhibits signal transduction, we also found that disengagement of this molecule by the RasV12G37 effector loop mutant or RNA interference markedly enhances the activation of the NFAT transcription factor and IFN-γ secretion. A strong output of IFN-γ is responsible for the distinct lymphocyte traffic pattern observed in vivo because the transgenic or retroviral expression of RasV12G37 caused T cells to accumulate preferentially in the lymph nodes and delayed their escape from the lymphoid tissue, respectively. Together, our results describe a hitherto unrecognized negative regulatory role for Imp in the production of IFN-γ in T cells and point to Ras-Imp binding as an attractive target for therapeutic interventions in conditions involving the production of this inflammatory cytokine. [ABSTRACT FROM AUTHOR]
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- 2008
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12. Mitochondrial ATP Synthase.
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Chen Chen, Saxena, Ajay K., Simcoke, William N., Garboczi, David N., Pedersen, Peter L., and Ko, Young H.
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ADENOSINE triphosphate , *MITOCHONDRIA , *BILIARY tract , *ALANINE , *AMINO acids - Abstract
ATP synthesis from ADP, Pi, and Mg2+ takes place in mitochondria on the catalytic F1 unit (α3β3γδϵ) of the ATP synthase complex (F0F1), a remarkable nanomachine that interconverts electrochemical and mechanical energy, producing the high energy terminal bond of ATP. In currently available structural models of F1, the P-loop (amino acid residues 156GGAGVGKT163) contributes to substrate binding at the β subunit catalytic sites. Here, we report the first transition state-like structure of F1 (ADP·Vi·Mg·F1) from rat liver that was crystallized with the phosphate (Pi) analog vanadate (VO43- or Vi). Compared with earlier "ground state" structures, this new F1 structure reveals that the active site region has undergone significant remodeling. P-loop residue alanine 158 is located much closer to Vi than it is to Pi in a previous structural model. No significant movements of P-loop residues of the a subunit were observed at its analogous but noncatalytic sites. Under physiological conditions, such active site remodeling involving the small hydrophobic alanine residue may promote ATP synthesis by lowering the local dielectric constant, thus facilitating the dehydration of ADP and Pi. This new crystallographic study provides strong support for the catalytic mechanism of ATP synthesis deduced from earlier biochemical studies of liver F1 conducted in the presence of Vi (Ko, Y. H., Bianchet, M., Amzel, L. M., and Pedersen, P. L. (1997)1 Biol. Che,n. 272, 18875-18881; Ko, Y. H., Hong, S., and Pedersen, P. L. (1999) J. Blot. Chern. 274, 28853-28856). [ABSTRACT FROM AUTHOR]
- Published
- 2006
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13. Functional Analysis of Oxygenase in Jadomycin Biosynthesis and Identification of JadH as a Bifuntional Oxygenase/Dehydrase.
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Yi-Hua Chen, Chen-Chen Wang, Greenwell, Lisa, Rix, Uwe, Hoffmeister, Dirk, Vining, Leo C., Rohr, Jürgen, and Ke-Qian Yang
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OXYGENASES , *STREPTOMYCES , *METABOLITES , *BIOSYNTHESIS , *POLYKETIDES , *POLYMERS , *ORGANIC synthesis - Abstract
A novel angucycline metabolite, 2,3-dehydro-UWM6, was identified in ajadH mutant of Streptomyces venezuelae ISP5230. Both UWM6 and 2,3-dehydro-UWM6 could be converted to jadomycin A or B by a ketosynthase α (jadA) mutant of S. venezuelae. These angucycline intermediates were also converted to jadomycin A by transformant of the heterologous host Streptomyces lividans expressing the jadFGH oxygenases in vivo and by its cell-free extracts in vitro; thus the three gene products JadFGH are implicated in catalysis of the post-polyketide synthase biosynthetic reactions converting UWM6 to jadomycin aglycone. Genetic and biochemical analyses indicate that JadH possesses dehydrase activity, not previously associated with polyketide-modifying oxygenase. Since the formation of aromatic polyketides often requires multiple dehydration steps, bifunctionality of oxygenases modifying aromatic polyketides may be a general phenomenon. [ABSTRACT FROM AUTHOR]
- Published
- 2005
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14. Mitochondrial ATP Synthasome.
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Chen Chen, Young Ko, Delannoy, Michael, Ludtke, Steven J., Wah Chiu, and Pedersen, Peter L.
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MITOCHONDRIA , *ADENINE nucleotides , *ELECTRON microscopy , *PHOSPHATES , *ADENOSINE triphosphate , *BIOCHEMISTRY - Abstract
The terminal steps involved in making ATP in mitochondria require an ATP synthase (F0F1) comprised of two motors, a phosphate carrier (PIC), and an adenine nucleotide carrier (ANC). Under mild conditions, these entities sub-fractionate as an ATP synthase/PIC/ANC complex or "ATP synthasome" (Ko, Y.H., Delannoy, M, Hullihen, J., Chiu, W., and Pedersen, P.L. (2003) J. Biol. Chem. 278, 12305-12309). As a first step toward obtaining three-dimensional information about this large complex or "metabolon" and the locations of PIC and ANC therein, we dispersed ATP synthasomes into single complexes and visualized negatively stained images by electron microscopy (EM) that showed clearly the classical headpiece, central stalk, and basepiece. Parallel immuno-EM studies revealed the presence of PIC and ANC located non-centrally in the basepiece, and other studies implicated an ATP synthase/PIC/ANC stoichiometry near 1:1:1. Single ATP synthasome images (7506) were boxed, and, using EMAN software, a three-dimensional model was obtained at a resolution of 23 Å. Significantly, the basepiece is oblong and contains two domains, the larger of which connects to the central stalk, whereas the smaller appears as an extension. Docking studies with known structures together with the immuno-EM studies suggest that PIC or ANC may be located in the smaller domain, whereas the other transporter resides nearby in the larger domain. Collectively, these finding support a mechanism in which the entry of the substrates ADP and Pi into mitochondria, the synthesis of ATP on F1, and the release and exit of ATP are very localized and highly coordinated events. [ABSTRACT FROM AUTHOR]
- Published
- 2004
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15. Impact of the Mitogen-activated Protein Kinase Pathway on Parathyroid Hormone-related Protein Actions in Osteoblasts.
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Chen Chen, Koh, Amy J., Datta, Nabanita S., Jian Zhang, Keller, Evan T., Guozhi Xiao, Franceschi, Renny T., D'Silva, Nisha J., and McCauley, Laurie K.
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PROTEIN kinases , *MITOGENS , *PARATHYROID hormone , *CYCLIC adenylic acid , *GENE expression , *MESSENGER RNA - Abstract
Parathyroid hormone-related protein (PTHrP) regulates proliferation and differentiation of osteoblastic cells via binding to the parathyroid hormone receptor (PTH-1R). The cAMP-dependent protein kinase A pathway governs the majority of these effects, but recent evidence also implicates the MAPK pathway. MC3T3-E1 subclone 4 cells (MC4) were treated with the MAPK inhibitor U0126 and PTHrP. In differentiated MC4 cells, osteocalcin and bone sialoprotein gene expression were both down-regulated by PTHrP and also by inhibition of the MAPK pathway. PTHrP-mediated down-regulation of PTH-1R mRNA and up-regulation of c-fos mRNA were MAPK-independent, whereas PTHrP stimulation of fra-2 and interleukin-6 (IL-6) mRNA was MAPK-dependent. Luciferase promoter assays revealed that regulation of IL-6 involved the cAMP-dependent protein kinase A and MAPK pathways with a potential minor role of the protein kinase C pathway, and a promoter region containing an activator protein-1 site was necessary for PTHrP-induced IL-6 gene transcription. An alternative pathway, through cAMP/Epac/Rap1/MAPK, mediated ERK phosphorylation but was not sufficient for IL-6 promoter activation. Phosphorylation of the transcription factor CREB was also necessary but not sufficient for PTHrP-mediated IL-6 promoter activity. Most interesting, a bidirectional effect was found with PTHrP increasing phosphorylated ERK in undifferentiated MC4 cells but decreasing phosphorylated ERK in differentiated cells. These data indicate that inactivation of the MAPK pathway shows differential regulation of PTHrP-stimulated activator protein-1 members, blocks PTHrPstimulated IL-6, and synergistically down-regulates certain osteoblastic markers associated with differentiation. These novel findings indicate that the MAPK pathway plays a selective but important role in the actions of PTHrP. [ABSTRACT FROM AUTHOR]
- Published
- 2004
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16. When Escherichia coli doesn't fit the mold: A pertussis-like toxin with altered specificity.
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Chen Chen and Barbieri, Joseph T.
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ESCHERICHIA coli , *PERTUSSIS toxin , *ADP-ribosylation , *TOXINS , *EUKARYOTIC cells - Abstract
Bacterial toxins introduce protein modifications such as ADP-ribosylation to manipulate host cell signaling and physiology. Several general mechanisms for toxin function have been established, but the extent to which previously uncharacterized toxins utilize these mechanisms is unknown. A study of an Escherichia coli pertussis-like toxin demonstrates that this protein acts on a known toxin substrate but displays distinct and dual chemoselectivity, suggesting this E. coli pertussis-like toxin may serve as a unique tool to study G-protein signaling in eukaryotic cells. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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17. Caveolin-1 promotes the tumor suppressor properties of oncogene-induced cellular senescence.
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Volonte, Daniela, Vyas, Avani R., Chen Chen, Dacic, Sanja, Stabile, Laura P., Kurland, Brenda F., Abberbock, Shira R., Burns, Timothy F., Herman, James G., Di, Yuanpu Peter, and Galbiati, Ferruccio
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CANCER treatment , *LUNG cancer , *PRECANCEROUS conditions , *SCAFFOLD proteins , *EPITHELIAL cells - Abstract
Oncogene-induced senescence (OIS) is considered a powerful tumor suppressor mechanism. Caveolin-1 acts as a scaffolding protein to functionally regulate signaling molecules. We demonstrate that a lack of caveolin-1 expression inhibits oncogenic K-Ras (K-RasG12V)-induced premature senescence in mouse embryonic fibroblasts and normal human bronchial epithelial cells. Oncogenic K-Ras induces senescence by limiting the detoxification function of MTH1. We found that K-RasG12V promotes the interaction of caveolin-1 with MTH1, which results in inhibition of MTH1 activity. Lung cancer cells expressing oncogenic K-Ras have bypassed the senescence barrier. Interestingly, overexpression of caveolin-1 restores cellular senescence in both A549 and H460 lung cancer cells and inhibits their transformed phenotype. In support of these findings, our in vivo data demonstrate that overexpression of oncogenic K-Ras (K-RasG12D) induces cellular senescence in the lung of wildtype but not caveolin-1-null mice. A lack of K-RasG12D- induced premature senescence in caveolin-1-null mice results in the formation of more abundant lung tumors. Consistent with these data, caveolin-1-null mice overexpressing K-RasG12D display accelerated mortality. Finally, our animal data were supported by human sample analysis in which we show that caveolin-1 expression is dramatically down-regulated in lung adenocarcinomas from lung cancer patients, both at the mRNA and protein levels, and that low caveolin-1 expression is associated with poor survival. Together, our data suggest that lung cancer cells escape oncogene-induced premature senescence through down-regulation of caveolin-1 expression to progress from premalignant lesions to cancer. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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18. Identification of a distal enhancer that determines the expression pattern of acute phase marker C-reactive protein.
- Author
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Ming-Yu Wang, Chun-Miao Zhang, Hai-Hong Zhou, Zhong-Bo Ge, Chen-Chen Su, Zi-Hao Lou, Xin-Yun Zhang, Tao-Tao Xu, Si-Yi Li, Li Zhu, Ya-Li Zhou, Yi Wu, and Shang-Rong Ji
- Subjects
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ACUTE phase proteins , *C-reactive protein , *GENE enhancers , *TRANSCRIPTION factors , *MOLECULAR evolution , *GENETIC regulation - Abstract
C-reactive protein (CRP) is a major acute phase protein and inflammatory marker, the expression of which is largely liver specific and highly inducible. Enhancers are regulatory elements critical for the precise activation of gene expression, yet the contributions of enhancers to the expression pattern of CRP have not been well defined. Here, we identify a constitutively active enhancer (E1) located 37.7 kb upstream of the promoter of human CRP in hepatocytes. By using chromatin immunoprecipitation, luciferase reporter assay, in situ genetic manipulation, CRISPRi, and CRISPRa, we show that E1 is enriched in binding sites for transcription factors STAT3 and C/EBP-β and is essential for the full induction of human CRP during the acute phase. Moreover, we demonstrate that E1 orchestrates with the promoter of CRP to determine its varied expression across tissues and species through surveying activities of E1-promoter hybrids and the associated epigenetic modifications. These results thus suggest an intriguing mode of molecular evolution wherein expression-changing mutations in distal regulatory elements initiate subsequent functional selection involving coupling among distal/proximal regulatory mutations and activity-changing coding mutations. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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19. Augmented Phosphorylation of Cardiac Troponin I in Hypertensive Heart Failure.
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Xintong Dong, Sumandea, C. Amelia, Yi-Chen Chen, Garcia-Cazarin, Mary L., Jiang Zhang, Balke, C. William, Sumandea, Marius P., and Ying Ge
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CYTOPLASMIC filaments , *HEART failure , *PHOSPHORYLATION , *TROPONIN I , *PROTEIN kinases - Abstract
An altered cardiac myofilament response to activating Ca2+ is a hallmark of human heart failure. Phosphorylation of cardiac troponin I (cTnI) is critical in modulating contractility and Ca2+ sensitivity of cardiac muscle. cTnI can be phosphorylated by protein kinaseA(PKA) at Ser22/23 and protein kinase C (PKC) at Ser22/23, Ser42/44, and Thr143. Whereas the functional significance of Ser22/23 phosphorylation is well understood, the role of other cTnI phosphorylation sites in the regulation of cardiac contractility remains a topic of intense debate, in part, due to the lack of evidence of in vivo phosphorylation. In this study, we utilized top-down high resolution mass spectrometry (MS) combined with immunoaffinity chromatography to determine quantitatively the cTnI phosphorylation changes in spontaneously hypertensive rat (SHR) model of hypertensive heart disease and failure. Our data indicate that cTnI is hyperphosphorylated in the failing SHR myocardium compared with age-matched normotensive Wistar-Kyoto rats. The top-down electron capture dissociation MS unambiguously localized augmented phosphorylation sites to Ser22/23 and Ser42/44 in SHR. Enhanced Ser22/23 phosphorylation was verified by immunoblotting with phospho-specific antibodies. Immunoblot analysis also revealed up-regulation of PKC-α and -δ, decreased PKCϵ, but no changes in PKA or PKC-β levels in the SHR myocardium. This provides direct evidence of in vivo phosphorylation of cTnI-Ser42/44 (PKC-specific) sites in an animal model of hypertensive heart failure, supporting the hypothesis that PKC phosphorylation of cTnI may be maladaptive and potentially associated with cardiac dysfunction. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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20. Akt Activates the Mammalian Target of Rapamycin by Regulating Cellular ATP Level and AMPK Activity.
- Author
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Hahn-Windgassen, Annett, Nogueira, Veronique, Chia-Chen Chen, Skeent, Jennifer E., Sonenberg, Nahum, and Hay, Nissim
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PROTEIN kinases , *FOCAL adhesion kinase , *SERINE , *ADENOSINE monophosphate , *RAPAMYCIN , *TUBEROUS sclerosis - Abstract
The serine/threonine kinase Akt is an upstream positive regulator of the mammalian target of rapamycin (mTOR). However, the mechanism by which Akt activates mTOR is not fully understood. The known pathway by which Akt activates mTOR is via direct phosphorylation and inhibition of tuberous sclerosis complex 2 (TSC2), which is a negative regulator of mTOR. Here we establish an additional pathway by which Akt inhibits TSC2 and activates mTOR. We provide for the first time genetic evidence that Akt regulates intracellular ATP level and demonstrate that Akt is a negative regulator of the AMP-activated protein kinase (AMPK), which is an activator of TSC2. We show that in Akt1/Akt2 DKO cells AMP/ATP ratio is markedly elevated with concomitant increase in AMPK activity, whereas in cells expressing activated Akt there is a dramatic decrease in AMP/ATP ratio and a decline in AMPK activity. Currently, the Akt-mediated phosphorylation of TSC2 and the inhibition of AMPK-mediated phosphorylation of TSC2 are viewed as two separate pathways, which activate mTOR. Our results demonstrate that Akt lies upstream of these two pathways and induces full inhibition of TSC2 and activation of mTOR both through direct phosphorylation and by inhibition of AMPK-mediated phosphorylation of TSC2. We propose that the activation of mTOR by Akt-mediated cellular energy and inhibition of AMPK is the predominant pathway by which Akt activates mTOR in vivo. [ABSTRACT FROM AUTHOR]
- Published
- 2005
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21. FcγRllla-Syk Co-signal Modulates CD4+ T-cell Response, and Up-regulates Toll-like Receptor (TLR) Expression and Up-regulates Toll-like Receptor (TLR) Expression FcγRllla-Syk Co-signal Modulates CD4+ T-cell Response and Up-regulates Toll-like Receptor (TLR) Expression
- Author
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Chauhan, Anil K., Moore, Terry L., Bi, Ye, and Chen Chen
- Subjects
- *
T cells , *CELLULAR signal transduction , *CD4 antigen , *TOLL-like receptors , *GENE expression , *SYSTEMIC lupus erythematosus , *PATIENTS - Abstract
CD4+ T-cells in systemic lupus erythematosus (SLE) patients show altered T-cell receptor signaling, which utilizes Fc-receptor γ-chain FcRγ-Syk. A role for FcγRIIIa activation from immune complex (IC) ligation and sublytic terminal complement complex (C5b-9) in CD4+T-cell responses is not investigated. In this study, we show that the ICs present in SLE patients by ligating to FcγRIIIa on CD4+ T-cells phosphorylate Syk and provide a co-stimulatory signal to CD4+ T-cells in the absence of CD28 signal. This led to the development of pathogenic IL-17A+ and IFN-γhigh CD4+ T-cells in vitro. Cytokines IL-1β, IL-6, TGF-β1, and IL-23 were the only requirement for the development of both populations. SLE patients CD4+ T-cells that expressed CD25, CD69, and CD98 bound to ICs showed pSyk and produced IFN-γ and IL-17A. This FcγRIIIa-mediated co-signal differentially up-regulated the expression of IFN pathway genes compared with CD28 co-signal. FcγRIIIa-pSyk upregulated several toll-like receptor genes as well as the HMGB1 and MyD88 gene transcripts. ICs co-localized with these tolllike receptor pathway proteins. These results suggest a role for the FcγRIIIa-pSyk signal in modulating adaptive immune responses. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
22. Metabolic Control of Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)-mediated Caspase-2 Suppression by the B55β/Protein Phosphatase 2A (PP2A).
- Author
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Bofu Huang, Chih-Sheng Yang, Wojton, Jeffrey, Nai-Jia Huang, Chen Chen, Soderblom, Erik J., Liguo Zhang, and Kornbluth, Sally
- Subjects
- *
CASPASES , *CALMODULIN , *PROTEIN kinases , *PHOSPHOPROTEIN phosphatases , *DEPHOSPHORYLATION - Abstract
High levels of metabolic activity confer resistance to apoptosis. Caspase-2, an apoptotic initiator, can be suppressed by high levels of nutrient flux through the pentose phosphate pathway. This metabolic control is exerted via inhibitory phosphorylation of the caspase-2 prodomain by activated Ca2+/calmodulin-dependent protein kinase II (CaMKII). We show here that this activation of CaMKII depends, in part, on dephosphorylation of CaMKII at novel sites (Thr393/Ser395) and that this is mediated by metabolic activation of protein phosphatase 2A in complex with the B55β targeting subunit. This represents a novel locus of CaMKII control and also provides a mechanism contributing to metabolic control of apoptosis. These findings may have implications for metabolic control of the many CaMKII-controlled and protein phosphatase 2A-regulated physiological processes, because both enzymes appear to be responsive to alterations in glucose metabolized via the pentose phosphate pathway. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
23. The Inducible Nitric-oxide Synthase (iNOS)/Src Axis Mediates Toll-like Receptor 3 Tyrosine 759 Phosphorylation and Enhances Its Signal Transduction, Leading to Interferon-β Synthesis in Macrophages.
- Author
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Ming-Yu Hsieh, Miao Ying Chang, Yen-Jen Chen, Yung Kuo Li, Tsung-Hsien Chuang, Guann-Yi Yu, Chun Hei Antonio Cheung, Hui-Chen Chen, Ming-Chei Maa, and Tzeng-Horng Leu
- Subjects
- *
DOUBLE-stranded RNA , *PHOSPHORYLATION , *TYROSINE , *TRANSCRIPTION factors , *NITRIC-oxide synthases - Abstract
Double-stranded RNA (dsRNA) induces phosphorylation of Toll-like receptor 3 (TLR3) at tyrosine 759 and subsequently triggers signaling pathways to promote interferon-β (IFN-β) production. In this study, we found that dsRNA stimulation induces biphasic TLR3 Tyr-759 phosphorylation in macrophages. In addition to the immediate TLR3 Tyr-759 phosphorylation, we identified a second wave of Tyr-759 phosphorylation accompanied by an increase of both Src and ifn-β transcription in the later phase of dsRNA stimulation. Interestingly, Src phosphorylated TLR3 Tyr-759 in vitro and in vivo. However, knockdown of Src abolished the late phase of TLR3 Tyr-759 phosphorylation and decreased the nuclear accumulation of interferon regulatory factors 3 and 7 (IRF3 and -7) and IFN-β production. Reintroduction of Src restored all of these molecular changes. Notably, via down-regulation of Src, dsRNA-elicited TLR3 Tyr-759 phosphorylation, the nuclear accumulation of IRF3/IRF7, and IFN-β generation were inhibited in inducible nitric-oxide synthase (iNOS)-null macrophages. TLR3 knockdown destabilized Src and reduced the nuclear level of IRF3/IRF7 and IFN-β production in macrophages exposed to LPS (a TLR4 ligand known to induce Src and IFN-β expression). Ectopic expression of wild type TLR3, but not its 759-phenylalanine mutant, restored Src activity and ifn-β transcription. Taken together, these results suggested an essential role of the iNOS/Src/ TLR3 axis in IFN-β production in macrophages. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
24. Eps8 Protein Facilitates Phagocytosis by Increasing TLR4-MyD88 Protein Interaction in Lipopolysaccharide-stimulated Macrophages.
- Author
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Yen-Jen Chen, Ming-Yu Hsieh, Miao Ying Chang, Hui-Chen Chen, Ming-Shiou Jan, Ming-Chei Maa, and Tzeng-Horng Leu
- Subjects
- *
PROTEIN-protein interactions , *LIPOPOLYSACCHARIDES , *MACROPHAGES , *IMMUNE response , *FOCAL adhesion kinase - Abstract
Toll-like receptors (TLRs) are crucial in macrophage phagocytosis, which is pivotal in host innate immune response. However, the detailed mechanism is not fully defined. Here, we demonstrated that the induction of Src and Eps8 in LPS-treated macrophages was TLR4- and MyD88-dependent, and their attenuation reduced LPS-promoted phagocytosis. Confocal microscopy indicated the colocalization of Eps8 and TLR4 in the cytosol and at the phagosome. Consistently, both Eps8 and TLR4 were present in the same immunocomplex regardless of LPS stimulation. Inhibition of this complex formation by eps8 siRNA or overexpression of pleckstrin homology domain-truncated Eps8 (i.e.261-p97Eps8) decreased LPS-induced TLR4-MyD88 interaction and the following activation of Src, focal adhesion kinase, and p38 MAPK. Importantly, attenuation of Eps8 impaired the bacterium-killing ability of macrophages. Thus, Eps8 is a key regulator of the LPS-stimulated TLR4-MyD88 interaction and contributes to macrophage phagocytosis. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
- View/download PDF
25. Structural and Enzymatic Characterization of the Streptococcal ATP/Diadenosine Polyphosphate and Phosphodiester Hydrolase Spr1479/SapH.
- Author
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Yong-Liang Jiang, Jun-Wei Zhang, Wei-Li Yu, Wang Cheng, Chen-Chen Zhang, Frolet, Cecile, Di Guilmi, Anne-Marie, Vernet, Thierry, Cong-Zhao Zhou, and Yuxing Chen
- Subjects
- *
STREPTOCOCCUS pneumoniae , *CRYSTAL structure , *ENZYMATIC analysis , *HYDROLASES , *POLYPHOSPHATES - Abstract
Spr1479 from Streptococcus pneumoniae R6 is a 33-kDa hypothetical protein of unknown function. Here, we determined the crystal structures of its apo-form at 1.90 Å and complex forms with inorganic phosphate and AMP at 2.30 and 2.20 Å, respectively. The core structure of Spr1479 adopts a four-layer αββα-sandwich fold, with Fe3+ and Mn2+ coordinated at the binuclear center of the active site (similar to metallophosphoesterases). Enzymatic assays showed that, in addition to phosphodiesterase activity for bis(p-nitrophenyl) phosphate, Spr1479 has hydrolase activity for diadenosine polyphosphate (ApnA) and ATP. Residues that coordinate with the two metals are indispensable for both activities. By contrast, the streptococcus-specific residue Trp-67, which binds to phosphate in the two complex structures, is indispensable for the ATP/ApnA hydrolase activity only. Moreover, the AMP-binding pocket is conserved exclusively in all streptococci. Therefore, we named the protein SapH for streptococcal ATP/ApnA and phosphodiester hydrolase. [ABSTRACT FROM AUTHOR]
- Published
- 2011
- Full Text
- View/download PDF
26. Trio Is a Key Guanine Nucleotide Exchange Factor Coordinating Regulation of the Migration and Morphogenesis of Granule Cells in the Developing Cerebellum.
- Author
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Ya-Jing Peng, Wei-Qi He, Jing Tang, Tao Tao, Chen Chen, Yun-Qian Gao, Wen-Cheng Zhang, Xue-Yan He, Yu-Yuan Dai, Nian-Chun Zhu, Ning Lv, Cheng-Hai Zhang, Yan-Ning Qiao, Li-Ping Zhao, Xiang Gao, and Min-Sheng Zhu
- Subjects
- *
G proteins , *NEURONS , *CELLS , *CEREBELLUM , *CYTOSKELETON , *GUANOSINE triphosphatase - Abstract
Orchestrated regulation of neuronal migration and morphogenesis is critical for neuronal development and establishment of functional circuits, but its regulatory mechanism is incompletely defined. We established and analyzed mice with neural-specific knock-out of Trio, a guanine nucleotide exchange factor with multiple guanine nucleotide exchange factor domains. Knock-out mice showed defective cerebella and severe signs ataxia. Mutant cerebella had no granule cells in the internal granule cell layer due to aberrant granule cell migration as well as abnormal neurite growth. Trio-deficient granule cells showed reduced extension of neurites and highly branched and misguided processes with perturbed stabilization of actin microtubules. Trio deletion caused down-regulation of the activation of Rac1, RhoA, and Cdc42, and mutant granule appeared to be unresponsive to neurite growth-promoting molecules such as Netrin-1 and Semaphorin 6A. These results suggest that Trio may be a key signal module for the orchestrated regulation of neuronal migration and morphogenesis during cerebellar development. Trio may serve as a signal integrator decoding extrinsic signals to Rho GTPases for cytoskeleton organization. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
27. Akt Activation Emulates Chk1 Inhibition and Bcl2 Overexpression and Abrogates G2 Cell Cycle Checkpoint by Inhibiting BRCA1 Foci.
- Author
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Tonic, Ivana, Wan-Ni Yu, Park, Youngku, Chia-Chen Chen, and Hay, Nissim
- Subjects
- *
MYC proteins , *CANCER genetics , *APOPTOSIS , *GENE expression , *GENETIC mutation - Abstract
Akt is perhaps the most frequently activated oncoprotein human cancers. Overriding cell cycle checkpoint in combination with the inhibition of apoptosis are two principal requirements for predisposition to cancer. Here we show that the activation of Akt is sufficient to promote these two principal processes, by inhibiting Chk1 activation with concomitant inhibition of apoptosis. These activities of Akt cannot be recapitulated by the knockdown of Chk1 alone or by overexpression Bcl2. Rather the combination of Chk1 knockdown and Bcl2 overexpression is required to recapitulate Akt activities. Akt was shown to directly phosphorylate Chk1. However, we found Chk1 mutants in the Akt phosphorylation sites behave like wild-type Chk1 in mediating G2 arrest, suggesting that the phosphorylation of Chk1 by Akt is either dispensable for Chk1 activity insufficient by itself to exert an effect on Chk1 activity. Here report a new mechanism by which Akt affects G2 cell cycle arrest. We show that Akt inhibits BRCA1 function that induces G2 cell cycle arrest. Akt prevents the translocation of BRCA1 DNA damage foci and, thereby, inhibiting the activation of Chk1 following DNA damage. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
28. Myosin Light Chain Kinase Is Necessary for Tonic Airway Smooth Muscle Contraction.
- Author
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Wen-Cheng Zhang, Ya-Jing Peng, Gen-Sheng Zhang, Wei-Qi He, Yan-Ning Qiao, Ying-Ying Dong, Yun-Qian Gao, Chen Chen, Cheng-Hai Zhang, Wen Li, Hua-Hao Shen, Wen Ning, Kamm, Kristine E., Stull, James T., Xiang Gao, and Min-Sheng Zhu
- Subjects
- *
MYOSIN , *CALMODULIN , *PHOSPHORYLATION , *MUSCLE contraction , *LABORATORY mice - Abstract
Different interacting signaling modules involving Ca2+/calmodulin-dependent myosin light chain kinase, Ca2+-independent regulatory light chain phosphorylation, myosin phosphatase inhibition, and actin filament-based proteins are proposed as specific cellular mechanisms involved in the regulation of smooth muscle contraction. However, the relative importance of specific modules is not well defined. By using tamoxifen-activated and smooth muscle-specific knock-out of myosin light chain kinase in mice, we analyzed its role in tonic airway smooth muscle contraction. Knock-out of the kinase in both tracheal and bronchial smooth muscle significantly reduced contraction and myosin phosphorylation responses to K+-depolarization and acetylcholine. Kinase-deficient mice lacked bronchial constrictions in normal and asthmatic airways, whereas the asthmatic inflammation response was not affected. These results indicate that myosin light chain kinase acts as a central participant in the contractile signaling module of tonic smooth muscle. Importantly, contractile airway smooth muscles are necessary for physiological and asthmatic airway resistance. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
29. Nitro-fatty Acid Metabolome: Saturation, Desaturation, β-Oxidation, and Protein Adduction*.
- Author
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Rudolph, Volker, Schopfer, Francisco J., Khoo, Nicholas K. H., Rudolph, Tanja K., Cole, Marsha P., Woodcock, Steven R., Bonacci, Gustavo, Groeger, Alison L., Golin-Bisello, Franca, Chen, Chen-Shan, Baker, Paul R. S., and Freeman, Bruce A.
- Subjects
- *
NITRO compounds , *FATTY acids , *METABOLISM , *OXIDATION , *BLOOD plasma , *LIQUID chromatography , *LABORATORY mice - Abstract
Nitrated derivatives of fatty acids (NO[sub2]-FA) are pluripotent cell-signaling mediators that display anti-inflammatory properties. Current understanding of NO[sub2]-FA signal transduction lacks insight into how or if NO[sub2]-FA are modified or metabolized upon formation or administration in vivo. Here the disposition and metabolism of nitro-9-cis-octadecenoic (18:1-NO[sub2]) acid was investigated in plasma and liver after intravenous injection in mice. High performance liquid chromatography-tandem mass spectrometry analysis showed that no 18:1-NO[sub2] or metabolites were detected under basal conditions, whereas administered 18:1-NO[sub2] is rapidly adducted to plasma thiol-containing proteins and glutathione. NO[sub2]-FA are also metabolized via β-oxidation, with high performance liquid chromatographytandem mass spectrometry analysis of liver lipid extracts of treated mice revealing nitro-7-cis-hexadecenoic acid, nitro-5-cis-tetradecenoic acid, and nitro-3-cis-dodecenoic acid and corresponding coenzyme A derivatives of 18:1-NO[sub2] as metabolites. Additionally, a significant proportion of 18:1-NO[sub2] and its metabolites are converted to nitroalkane derivatives by saturation of the double bond, and to a lesser extent are desaturated to diene derivatives. There was no evidence of the formation of nitrohydroxyl or conjugated ketone derivatives in organs of interest, metabolites expected upon 18:1-NO[sub2] hydration or nitric oxide (∙NO) release. Plasma samples from treated mice had significant extents of protein-adducted 18:1-NO[sub2] detected by exchange to added β-mercaptoethanol. This, coupled with the observation of 18:1-NO[sub2] release from glutathione-18:1-NO[sub2] adducts, supports that reversible and exchangeable NO[sub2]-FAthiol adducts occur under biological conditions. After administration of [3H]18:1-NO[sub2], 64% of net radiolabel was recovered 90 min later in plasma (0.2%), liver (18%), kidney (2%), adipose tissue (2%), muscle (31%), urine (6%), and other tissue compartments, and may include metabolites not yet identified. In aggregate, these findings show that electrophilic FA nitroalkene derivatives (a) acquire an extended half-life by undergoing reversible and exchangeable electrophilic reactions with nucleophilic targets and (b) are metabolized predominantly via saturation of the double bond and β-oxidation reactions that terminate at the site of acyl-chain nitration. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
30. Species Selectivity of Nonpeptide Antagonists of the Gonadotropin-releasing Hormone Receptor Is Determined by Residues in Extracellular Loops II and III and the Amino Terminus.
- Author
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Reinhart, Greg J., Qiu Xie, Xin-Jun Liu, Yun-Fei Zhu, Jun Fan, Chen Chen, and Struthers, R. Scott
- Subjects
- *
GONADOTROPIN releasing hormone , *PITUITARY hormone releasing factors , *HYPOTHALAMIC hormones , *HYPOTHALAMO-hypophyseal system , *BIOCHEMISTRY , *MOLECULAR biology - Abstract
Efforts to develop orally available gonadotropin-releasing hormone (GnRH) receptor antagonists have led to the discovery of several classes of potent nonpeptide antagonists. Here we investigated molecular interactions of three classes of nonpeptide antagonists with human, rat, and macaque GnRH receptors. Although all are high affinity ligands of the human receptor (Ki <5 nM), these compounds show reduced affinity for the macaque receptor and bind only weakly (Ki > 1 μM) to the rat receptor. To identify residues responsible for this selectivity, a series of chimeric receptors and mutant receptors was constructed and evaluated for nonpeptide binding. Surprisingly, 4 key residues located in the amino terminus (Met-24) and extracellular loops II (Ser-203, Gln-208) and III (Leu-300) of the GnRH receptor appear to be primarily responsible for species-selective binding. Comparisons of reciprocal mutations suggest that these may not be direct contacts but rather may be involved in organizing extracellular portions of the receptor. These data are novel because most previous reports of residues involved in binding of nonpeptide ligands to peptide-activated G protein-coupled receptors, including the GnRH receptor as well as mono-amine receptors, have identified binding sites in the transmembrane regions. [ABSTRACT FROM AUTHOR]
- Published
- 2004
- Full Text
- View/download PDF
31. Src Phosphorylates Grb2-associated Binder 1 upon Hepatocyte Growth Factor Stimulation.
- Author
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Po-Chao Chan, J. Pablo, Yen-Ling Chen, J. Pablo, Chi-Hung Cheng, Kuo-Ching Yu, J. Pablo, Cary, Leslie A., Kuo-Hsiung Shu, Ho, William L., and Hong-Chen Chen, William L.
- Subjects
- *
PHOSPHORYLATION , *HEPATOCYTE growth factor , *CELLULAR signal transduction - Abstract
Grb2-associated binder 1 (Gab1) is known to play an important role in hepatocyte growth factor (HGF) signaling, which rapidly becomes tyrosine-phosphorylated upon HGF stimulation. In this study, we found that the tyrosine phosphorylation of Gab1 in the cells derived from Src/Yes/Fyn null mouse embryos was ∼40% lower than that in their wild type counterparts upon HGF stimulation. Increased expression of wild-type Src enhanced HGF-induced phosphorylation of Gab1, and, in contrast, expression of the Src kinase-deficient mutant or treatment of the specific Src inhibitor PPI suppressed it. Expression of a constitutively active Src mutant (Y527F) or oncogenic v-Src led to a prominent increase in Gab1 phosphorylation independent of HGF stimulation. Moreover, Src interacted with Gab1 via both its Src homology 2 and 3 domains and was capable of phosphorylating purified Gab1 in vitro. Finally, the increased phosphorylation of Gab1 by Src selectively potentiated HGF-induced activation of ERK and AKT. Taken together, our results establish a new role for Src in HGF-induced Gab1 phosphorylation. [ABSTRACT FROM AUTHOR]
- Published
- 2003
- Full Text
- View/download PDF
32. Genome-wide CRISPR-based gene knockout screens reveal cellular factors and pathways essential for nasopharyngeal carcinoma.
- Author
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Wang C, Jiang S, Ke L, Zhang L, Li D, Liang J, Narita Y, Hou I, Chen CH, Wang L, Zhong Q, Ling Y, Lv X, Xiang Y, Guo X, Teng M, Tsao SW, Gewurz BE, Zeng MS, and Zhao B
- Subjects
- Biomarkers, Tumor antagonists & inhibitors, Humans, Nasopharyngeal Carcinoma pathology, Nasopharyngeal Neoplasms pathology, Proto-Oncogene Mas, Signal Transduction, Tumor Cells, Cultured, Biomarkers, Tumor genetics, CRISPR-Cas Systems, Cell Proliferation, Gene Knockout Techniques methods, Genome, Human, Nasopharyngeal Carcinoma genetics, Nasopharyngeal Neoplasms genetics
- Abstract
Early diagnosis of nasopharyngeal carcinoma (NPC) is difficult because of a lack of specific symptoms. Many patients have advanced disease at diagnosis, and these patients respond poorly to treatment. New treatments are therefore needed to improve the outcome of NPC. To better understand the molecular pathogenesis of NPC, here we used an NPC cell line in a genome-wide CRISPR-based knockout screen to identify the cellular factors and pathways essential for NPC ( i.e. dependence factors). This screen identified the Moz, Ybf2/Sas3, Sas2, Tip60 histone acetyl transferase complex, NF-κB signaling, purine synthesis, and linear ubiquitination pathways; and MDM2 proto-oncogene as NPC dependence factors/pathways. Using gene knock out, complementary DNA rescue, and inhibitor assays, we found that perturbation of these pathways greatly reduces the growth of NPC cell lines but does not affect growth of SV40-immortalized normal nasopharyngeal epithelial cells. These results suggest that targeting these pathways/proteins may hold promise for achieving better treatment of patients with NPC., (© 2019 Wang et al.)
- Published
- 2019
- Full Text
- View/download PDF
33. FcγRIIIa-Syk Co-signal Modulates CD4+ T-cell Response and Up-regulates Toll-like Receptor (TLR) Expression.
- Author
-
Chauhan AK, Moore TL, Bi Y, and Chen C
- Subjects
- Adaptive Immunity, Antigen-Antibody Complex analysis, Antigen-Antibody Complex isolation & purification, Antigen-Antibody Complex metabolism, Biomarkers blood, Biomarkers metabolism, CD4-Positive T-Lymphocytes immunology, CD4-Positive T-Lymphocytes pathology, Cells, Cultured, Complement Membrane Attack Complex analysis, Complement Membrane Attack Complex isolation & purification, Complement Membrane Attack Complex metabolism, HMGB1 Protein agonists, HMGB1 Protein genetics, HMGB1 Protein metabolism, Humans, Interferon-gamma metabolism, Interleukin-17 metabolism, Intracellular Signaling Peptides and Proteins blood, Lupus Erythematosus, Systemic blood, Lupus Erythematosus, Systemic immunology, Lupus Erythematosus, Systemic pathology, Membrane Glycoproteins agonists, Membrane Glycoproteins genetics, Membrane Glycoproteins metabolism, Myeloid Differentiation Factor 88 agonists, Myeloid Differentiation Factor 88 genetics, Myeloid Differentiation Factor 88 metabolism, Phosphorylation, Protein Processing, Post-Translational, Protein-Tyrosine Kinases blood, Receptors, IgG blood, Receptors, Interleukin-1 agonists, Receptors, Interleukin-1 genetics, Receptors, Interleukin-1 metabolism, Syk Kinase, Toll-Like Receptors genetics, Toll-Like Receptors metabolism, Up-Regulation, CD4-Positive T-Lymphocytes metabolism, Intracellular Signaling Peptides and Proteins metabolism, Lupus Erythematosus, Systemic metabolism, Lymphocyte Activation, Protein-Tyrosine Kinases metabolism, Receptors, IgG metabolism, Toll-Like Receptors agonists
- Abstract
CD4(+) T-cells in systemic lupus erythematosus (SLE) patients show altered T-cell receptor signaling, which utilizes Fc-receptor γ-chain FcRγ-Syk. A role for FcγRIIIa activation from immune complex (IC) ligation and sublytic terminal complement complex (C5b-9) in CD4(+) T-cell responses is not investigated. In this study, we show that the ICs present in SLE patients by ligating to FcγRIIIa on CD4(+) T-cells phosphorylate Syk and provide a co-stimulatory signal to CD4(+) T-cells in the absence of CD28 signal. This led to the development of pathogenic IL-17A(+) and IFN-γ(high) CD4(+) T-cells in vitro. Cytokines IL-1β, IL-6, TGF-β1, and IL-23 were the only requirement for the development of both populations. SLE patients CD4(+) T-cells that expressed CD25, CD69, and CD98 bound to ICs showed pSyk and produced IFN-γ and IL-17A. This FcγRIIIa-mediated co-signal differentially up-regulated the expression of IFN pathway genes compared with CD28 co-signal. FcγRIIIa-pSyk up-regulated several toll-like receptor genes as well as the HMGB1 and MyD88 gene transcripts. ICs co-localized with these toll-like receptor pathway proteins. These results suggest a role for the FcγRIIIa-pSyk signal in modulating adaptive immune responses., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
- Full Text
- View/download PDF
34. Induced expression of FcγRIIIa (CD16a) on CD4+ T cells triggers generation of IFN-γhigh subset.
- Author
-
Chauhan AK, Chen C, Moore TL, and DiPaolo RJ
- Subjects
- Autoimmune Diseases immunology, Autoimmune Diseases metabolism, Autoimmune Diseases pathology, CD28 Antigens biosynthesis, CD28 Antigens immunology, Female, Humans, Interferon-gamma metabolism, Jurkat Cells, Male, Receptors, IgG biosynthesis, Receptors, IgG immunology, T-Lymphocytes, Helper-Inducer pathology, Gene Expression Regulation immunology, Interferon-gamma immunology, Lymphocyte Activation, T-Lymphocytes, Helper-Inducer immunology
- Abstract
Whether or not CD4(+) T-cells express low affinity receptor FcγRIIIa (CD16a) in disease pathology has not been examined in great detail. In this study, we show that a subset of activated CD4(+) T-cells in humans express FcγRIIIa. The ligation of FcγRIIIa by immune complexes (ICs) in human CD4(+) T-cells produced co-stimulatory signal like CD28 that triggered IFN-γ production. The induced expression of FcγRIIIa on CD4(+) helper T-cells is an important finding since these receptors via ITAM contribute to intracellular signaling. The induced expression of FcγRIIIa on CD4(+) T helper cells and their ability to co-stimulate T-cell activation are important and novel findings that may reveal new pathways to regulate adaptive immune responses during inflammation and in autoimmunity., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
- Full Text
- View/download PDF
35. Metabolic control of Ca2+/calmodulin-dependent protein kinase II (CaMKII)-mediated caspase-2 suppression by the B55β/protein phosphatase 2A (PP2A).
- Author
-
Huang B, Yang CS, Wojton J, Huang NJ, Chen C, Soderblom EJ, Zhang L, and Kornbluth S
- Subjects
- Animals, Enzyme Activation, Glucose-6-Phosphate physiology, HEK293 Cells, Humans, Phosphorylation, Protein Binding, Protein Processing, Post-Translational, Xenopus laevis, Calcium-Calmodulin-Dependent Protein Kinase Type 2 physiology, Caspase 2 metabolism, Protein Phosphatase 2 metabolism, Xenopus Proteins metabolism
- Abstract
High levels of metabolic activity confer resistance to apoptosis. Caspase-2, an apoptotic initiator, can be suppressed by high levels of nutrient flux through the pentose phosphate pathway. This metabolic control is exerted via inhibitory phosphorylation of the caspase-2 prodomain by activated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). We show here that this activation of CaMKII depends, in part, on dephosphorylation of CaMKII at novel sites (Thr(393)/Ser(395)) and that this is mediated by metabolic activation of protein phosphatase 2A in complex with the B55β targeting subunit. This represents a novel locus of CaMKII control and also provides a mechanism contributing to metabolic control of apoptosis. These findings may have implications for metabolic control of the many CaMKII-controlled and protein phosphatase 2A-regulated physiological processes, because both enzymes appear to be responsive to alterations in glucose metabolized via the pentose phosphate pathway., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
- Full Text
- View/download PDF
36. Myosin light chain kinase (MLCK) regulates cell migration in a myosin regulatory light chain phosphorylation-independent mechanism.
- Author
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Chen C, Tao T, Wen C, He WQ, Qiao YN, Gao YQ, Chen X, Wang P, Chen CP, Zhao W, Chen HQ, Ye AP, Peng YJ, and Zhu MS
- Subjects
- Actin Cytoskeleton chemistry, Actins chemistry, Actins genetics, Adenoviridae genetics, Amino Acid Motifs, Animals, Cell Membrane chemistry, Cell Movement, Gene Expression Regulation, Genetic Vectors, Jejunum cytology, Mice, Mice, Knockout, Molecular Sequence Data, Myocytes, Smooth Muscle cytology, Myosin-Light-Chain Kinase chemistry, Myosin-Light-Chain Kinase genetics, Phosphorylation, Primary Cell Culture, Protein Binding, Signal Transduction, Surface Tension, Transfection, Actin Cytoskeleton metabolism, Actins metabolism, Cell Membrane metabolism, Jejunum metabolism, Myocytes, Smooth Muscle metabolism, Myosin-Light-Chain Kinase metabolism
- Abstract
Myosin light chain kinase (MLCK) has long been implicated in the myosin phosphorylation and force generation required for cell migration. Here, we surprisingly found that the deletion of MLCK resulted in fast cell migration, enhanced protrusion formation, and no alteration of myosin light chain phosphorylation. The mutant cells showed reduced membrane tether force and fewer membrane F-actin filaments. This phenotype was rescued by either kinase-dead MLCK or five-DFRXXL motif, a MLCK fragment with potent F-actin-binding activity. Pull-down and co-immunoprecipitation assays showed that the absence of MLCK led to attenuated formation of transmembrane complexes, including myosin II, integrins and fibronectin. We suggest that MLCK is not required for myosin phosphorylation in a migrating cell. A critical role of MLCK in cell migration involves regulating the cell membrane tension and protrusion necessary for migration, thereby stabilizing the membrane skeleton through F-actin-binding activity. This finding sheds light on a novel regulatory mechanism of protrusion during cell migration., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
- Full Text
- View/download PDF
37. Modulation of nitro-fatty acid signaling: prostaglandin reductase-1 is a nitroalkene reductase.
- Author
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Vitturi DA, Chen CS, Woodcock SR, Salvatore SR, Bonacci G, Koenitzer JR, Stewart NA, Wakabayashi N, Kensler TW, Freeman BA, and Schopfer FJ
- Subjects
- Alcohol Oxidoreductases genetics, Animals, Heme Oxygenase-1 genetics, Heme Oxygenase-1 metabolism, Hep G2 Cells, Humans, Intracellular Signaling Peptides and Proteins genetics, Intracellular Signaling Peptides and Proteins metabolism, Kelch-Like ECH-Associated Protein 1, NF-E2-Related Factor 2 genetics, NF-E2-Related Factor 2 metabolism, Oleic Acid genetics, Rats, Alcohol Oxidoreductases metabolism, Liver metabolism, Nitro Compounds metabolism, Oleic Acid metabolism, Signal Transduction physiology, Stearic Acids metabolism
- Abstract
Inflammation, characterized by the activation of both resident and infiltrated immune cells, is accompanied by increased production of oxidizing and nitrating species. Nitrogen dioxide, the proximal nitrating species formed under these conditions, reacts with unsaturated fatty acids to yield nitroalkene derivatives. These electrophilic products modulate protein function via post-translational modification of susceptible nucleophilic amino acids. Nitroalkenes react with Keap1 to instigate Nrf2 signaling, activate heat shock response gene expression, and inhibit NF-κB-mediated signaling, inducing net anti-inflammatory and tissue-protective metabolic responses. We report the purification and characterization of a NADPH-dependent liver enzyme that reduces the nitroalkene moiety of nitro-oleic acid, yielding the inactive product nitro-stearic acid. Prostaglandin reductase-1 (PtGR-1) was identified as a nitroalkene reductase by protein purification and proteomic studies. Kinetic measurements, inhibition studies, immunological and molecular biology approaches as well as clinical analyses confirmed this identification. Overexpression of PtGR-1 in HEK293T cells promoted nitroalkene metabolism to inactive nitroalkanes, an effect that abrogated the Nrf2-dependent induction of heme oxygenase-1 expression by nitro-oleic acid. These results situate PtGR-1 as a critical modulator of both the steady state levels and signaling activities of fatty acid nitroalkenes in vivo.
- Published
- 2013
- Full Text
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38. Identification of the E3 deubiquitinase ubiquitin-specific peptidase 21 (USP21) as a positive regulator of the transcription factor GATA3.
- Author
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Zhang J, Chen C, Hou X, Gao Y, Lin F, Yang J, Gao Z, Pan L, Tao L, Wen C, Yao Z, Tsun A, Shi G, and Li B
- Subjects
- Adolescent, Adult, Asthma metabolism, Cell Line, Tumor, Forkhead Transcription Factors biosynthesis, HEK293 Cells, Humans, Lymphocyte Activation, Middle Aged, Promoter Regions, Genetic, Protein Processing, Post-Translational, T-Lymphocytes metabolism, GATA3 Transcription Factor metabolism, Gene Expression Regulation, Ubiquitin Thiolesterase metabolism
- Abstract
The expression of the transcription factor GATA3 in FOXP3(+) regulatory T (Treg) cells is crucial for their physiological function in limiting inflammatory responses. Although other studies have shown how T cell receptor (TcR) signals induce the up-regulation of GATA3 expression in Treg cells, the underlying mechanism that maintains GATA3 expression in Treg cells remains unclear. Here, we show how USP21 interacts with and stabilizes GATA3 by mediating its deubiquitination. In a T cell line model, we found that TcR stimulation promoted USP21 expression, which was further up-regulated in the presence of FOXP3. The USP21 mutant C221A reduced its capacity to stabilize GATA3 expression, and its knockdown led to the down-regulation of GATA3 protein expression in Treg cells. Furthermore, we found that FOXP3 could directly bind to the USP21 gene promoter and activated its transcription upon TcR stimulation. Finally, USP21, GATA3, and FOXP3 were found up-regulated in Treg cells that were isolated from asthmatic subjects. In summary, we have identified a USP21-mediated pathway that promotes GATA3 stabilization and expression at the post-translational level. We propose that this pathway forms an important signaling loop that stabilizes the expression of GATA3 in Treg cells.
- Published
- 2013
- Full Text
- View/download PDF
39. Structural and enzymatic characterization of the streptococcal ATP/diadenosine polyphosphate and phosphodiester hydrolase Spr1479/SapH.
- Author
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Jiang YL, Zhang JW, Yu WL, Cheng W, Zhang CC, Frolet C, Di Guilmi AM, Vernet T, Zhou CZ, and Chen Y
- Subjects
- Apoenzymes chemistry, Binding Sites, Crystallography, X-Ray, Protein Structure, Secondary, Acid Anhydride Hydrolases chemistry, Adenosine Triphosphatases chemistry, Bacterial Proteins chemistry, Protein Folding, Streptococcus pneumoniae enzymology
- Abstract
Spr1479 from Streptococcus pneumoniae R6 is a 33-kDa hypothetical protein of unknown function. Here, we determined the crystal structures of its apo-form at 1.90 Å and complex forms with inorganic phosphate and AMP at 2.30 and 2.20 Å, respectively. The core structure of Spr1479 adopts a four-layer αββα-sandwich fold, with Fe(3+) and Mn(2+) coordinated at the binuclear center of the active site (similar to metallophosphoesterases). Enzymatic assays showed that, in addition to phosphodiesterase activity for bis(p-nitrophenyl) phosphate, Spr1479 has hydrolase activity for diadenosine polyphosphate (Ap(n)A) and ATP. Residues that coordinate with the two metals are indispensable for both activities. By contrast, the streptococcus-specific residue Trp-67, which binds to phosphate in the two complex structures, is indispensable for the ATP/Ap(n)A hydrolase activity only. Moreover, the AMP-binding pocket is conserved exclusively in all streptococci. Therefore, we named the protein SapH for streptococcal ATP/Ap(n)A and phosphodiester hydrolase.
- Published
- 2011
- Full Text
- View/download PDF
40. Trio is a key guanine nucleotide exchange factor coordinating regulation of the migration and morphogenesis of granule cells in the developing cerebellum.
- Author
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Peng YJ, He WQ, Tang J, Tao T, Chen C, Gao YQ, Zhang WC, He XY, Dai YY, Zhu NC, Lv N, Zhang CH, Qiao YN, Zhao LP, Gao X, and Zhu MS
- Subjects
- Animals, Cell Movement, Chromosomes, Artificial, Bacterial metabolism, Cytoskeleton metabolism, Glial Fibrillary Acidic Protein metabolism, Guanine Nucleotide Exchange Factors genetics, Guanine Nucleotide Exchange Factors physiology, Intermediate Filament Proteins metabolism, Mice, Mice, Knockout, Morphogenesis, Nerve Tissue Proteins metabolism, Nestin, Neurons metabolism, Phosphoproteins genetics, Protein Serine-Threonine Kinases genetics, Signal Transduction, rho GTP-Binding Proteins metabolism, Cerebellum embryology, Gene Expression Regulation, Developmental, Guanine Nucleotide Exchange Factors chemistry, Phosphoproteins physiology, Protein Serine-Threonine Kinases physiology
- Abstract
Orchestrated regulation of neuronal migration and morphogenesis is critical for neuronal development and establishment of functional circuits, but its regulatory mechanism is incompletely defined. We established and analyzed mice with neural-specific knock-out of Trio, a guanine nucleotide exchange factor with multiple guanine nucleotide exchange factor domains. Knock-out mice showed defective cerebella and severe signs of ataxia. Mutant cerebella had no granule cells in the internal granule cell layer due to aberrant granule cell migration as well as abnormal neurite growth. Trio-deficient granule cells showed reduced extension of neurites and highly branched and misguided processes with perturbed stabilization of actin and microtubules. Trio deletion caused down-regulation of the activation of Rac1, RhoA, and Cdc42, and mutant granule cells appeared to be unresponsive to neurite growth-promoting molecules such as Netrin-1 and Semaphorin 6A. These results suggest that Trio may be a key signal module for the orchestrated regulation of neuronal migration and morphogenesis during cerebellar development. Trio may serve as a signal integrator decoding extrinsic signals to Rho GTPases for cytoskeleton organization.
- Published
- 2010
- Full Text
- View/download PDF
41. Covalent peroxisome proliferator-activated receptor gamma adduction by nitro-fatty acids: selective ligand activity and anti-diabetic signaling actions.
- Author
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Schopfer FJ, Cole MP, Groeger AL, Chen CS, Khoo NK, Woodcock SR, Golin-Bisello F, Motanya UN, Li Y, Zhang J, Garcia-Barrio MT, Rudolph TK, Rudolph V, Bonacci G, Baker PR, Xu HE, Batthyany CI, Chen YE, Hallis TM, and Freeman BA
- Subjects
- 3T3-L1 Cells, Amino Acid Sequence, Amino Acid Substitution, Animals, Base Sequence, Blood Glucose metabolism, Cell Line, DNA Primers metabolism, Diabetes Mellitus drug therapy, Diabetes Mellitus metabolism, Fatty Acids, Unsaturated chemistry, Humans, Hypoglycemic Agents chemistry, In Vitro Techniques, Insulin blood, Ligands, Lipid Metabolism, Male, Mice, Mice, Inbred C57BL, Mice, Obese, Molecular Sequence Data, Mutagenesis, Site-Directed, Nitro Compounds chemistry, Oleic Acid chemistry, Oleic Acid pharmacology, PPAR gamma chemistry, PPAR gamma genetics, Protein Binding, Protein Processing, Post-Translational, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Rosiglitazone, Signal Transduction, Tandem Mass Spectrometry, Thiazolidinediones pharmacology, Fatty Acids, Unsaturated pharmacology, Hypoglycemic Agents pharmacology, Nitro Compounds pharmacology, PPAR gamma agonists, PPAR gamma metabolism
- Abstract
The peroxisome proliferator-activated receptor-gamma (PPARgamma) binds diverse ligands to transcriptionally regulate metabolism and inflammation. Activators of PPARgamma include lipids and anti-hyperglycemic drugs such as thiazolidinediones (TZDs). Recently, TZDs have raised concern after being linked with increased risk of peripheral edema, weight gain, and adverse cardiovascular events. Most reported endogenous PPARgamma ligands are intermediates of lipid metabolism and oxidation that bind PPARgamma with very low affinity. In contrast, nitro derivatives of unsaturated fatty acids (NO(2)-FA) are endogenous products of nitric oxide ((*)NO) and nitrite (NO(2)(-))-mediated redox reactions that activate PPARgamma at nanomolar concentrations. We report that NO(2)-FA act as partial agonists of PPARgamma and covalently bind PPARgamma at Cys-285 via Michael addition. NO(2)-FA show selective PPARgamma modulator characteristics by inducing coregulator protein interactions, PPARgamma-dependent expression of key target genes, and lipid accumulation is distinctively different from responses induced by the TZD rosiglitazone. Administration of this class of signaling mediators to ob/ob mice revealed that NO(2)-FA lower insulin and glucose levels without inducing adverse side effects such as the increased weight gain induced by TZDs.
- Published
- 2010
- Full Text
- View/download PDF
42. SRC induces podoplanin expression to promote cell migration.
- Author
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Shen Y, Chen CS, Ichikawa H, and Goldberg GS
- Subjects
- Animals, Cell Line, Transformed, Cell Movement, Fibroblasts metabolism, Homozygote, Ligands, Mice, Microscopy, Fluorescence methods, Models, Biological, Oligonucleotide Array Sequence Analysis, RNA, Small Interfering metabolism, Crk-Associated Substrate Protein metabolism, Gene Expression Regulation, Enzymologic, Gene Expression Regulation, Neoplastic, Membrane Glycoproteins biosynthesis, src-Family Kinases metabolism
- Abstract
Nontransformed cells can force tumor cells to assume a normal morphology and phenotype by the process of contact normalization. Transformed cells must escape this process to become invasive and malignant. However, mechanisms underlying contact normalization have not been elucidated. Here, we have identified genes that are affected by contact normalization of Src-transformed cells. Tumor cells must migrate to become invasive and malignant. Src must phosphorylate the adaptor protein Cas (Crk-associated substrate) to promote tumor cell motility. We report here that Src utilizes Cas to induce podoplanin (Pdpn) expression to promote tumor cell migration. Pdpn is a membrane-bound extracellular glycoprotein that associates with endogenous ligands to promote tumor cell migration leading to cancer invasion and metastasis. In fact, Pdpn expression accounted for a major part of the increased migration seen in Src-transformed cells. Moreover, nontransformed cells suppressed Pdpn expression in adjacent Src-transformed cells. Of >39,000 genes, Pdpn was one of only 23 genes found to be induced by transforming Src activity and suppressed by contact normalization of Src-transformed cells. In addition, we found 16 genes suppressed by Src and induced by contact normalization. These genes encode growth factor receptors, adaptor proteins, and products that have not yet been annotated and may play important roles in tumor cell growth and migration.
- Published
- 2010
- Full Text
- View/download PDF
43. Myosin light chain kinase is necessary for tonic airway smooth muscle contraction.
- Author
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Zhang WC, Peng YJ, Zhang GS, He WQ, Qiao YN, Dong YY, Gao YQ, Chen C, Zhang CH, Li W, Shen HH, Ning W, Kamm KE, Stull JT, Gao X, and Zhu MS
- Subjects
- Acetylcholine metabolism, Airway Resistance drug effects, Airway Resistance physiology, Animals, Antineoplastic Agents, Hormonal pharmacology, Asthma enzymology, Asthma genetics, Calcium metabolism, Calmodulin metabolism, Female, Male, Mice, Mice, Transgenic, Muscle Contraction drug effects, Muscle Tonus drug effects, Myosin-Light-Chain Kinase genetics, Phosphorylation drug effects, Phosphorylation physiology, Potassium metabolism, Signal Transduction drug effects, Signal Transduction physiology, Tamoxifen pharmacology, Bronchi enzymology, Muscle Contraction physiology, Muscle Tonus physiology, Muscle, Smooth enzymology, Myosin-Light-Chain Kinase metabolism, Trachea enzymology
- Abstract
Different interacting signaling modules involving Ca(2+)/calmodulin-dependent myosin light chain kinase, Ca(2+)-independent regulatory light chain phosphorylation, myosin phosphatase inhibition, and actin filament-based proteins are proposed as specific cellular mechanisms involved in the regulation of smooth muscle contraction. However, the relative importance of specific modules is not well defined. By using tamoxifen-activated and smooth muscle-specific knock-out of myosin light chain kinase in mice, we analyzed its role in tonic airway smooth muscle contraction. Knock-out of the kinase in both tracheal and bronchial smooth muscle significantly reduced contraction and myosin phosphorylation responses to K(+)-depolarization and acetylcholine. Kinase-deficient mice lacked bronchial constrictions in normal and asthmatic airways, whereas the asthmatic inflammation response was not affected. These results indicate that myosin light chain kinase acts as a central participant in the contractile signaling module of tonic smooth muscle. Importantly, contractile airway smooth muscles are necessary for physiological and asthmatic airway resistance.
- Published
- 2010
- Full Text
- View/download PDF
44. Lysophosphatidic acid 2 receptor-mediated supramolecular complex formation regulates its antiapoptotic effect.
- Author
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E S, Lai YJ, Tsukahara R, Chen CS, Fujiwara Y, Yue J, Yu JH, Guo H, Kihara A, Tigyi G, and Lin FT
- Subjects
- ATPases Associated with Diverse Cellular Activities, Adaptor Proteins, Signal Transducing metabolism, Amino Acid Motifs, Amino Acid Sequence, Animals, Calcium Signaling drug effects, Cell Line, Tumor, Cytoprotection drug effects, Doxorubicin pharmacology, Female, GTP-Binding Proteins metabolism, Humans, Intracellular Signaling Peptides and Proteins metabolism, LIM Domain Proteins, Lipoylation drug effects, Lysophospholipids pharmacology, Mice, Molecular Sequence Data, Mutation genetics, Ovarian Neoplasms pathology, Phosphoproteins metabolism, Proteasome Endopeptidase Complex, Protein Binding drug effects, Receptors, Lysophosphatidic Acid chemistry, Sodium-Hydrogen Exchangers metabolism, Transcription Factors metabolism, Apoptosis drug effects, Receptors, Lysophosphatidic Acid metabolism
- Abstract
The G protein-coupled lysophosphatidic acid 2 (LPA(2)) receptor elicits prosurvival responses to prevent and rescue cells from apoptosis. However, G protein-coupled signals are not sufficient for the full protective effect of LPA(2). LPA(2) differs from other LPA receptor subtypes in the C-terminal tail, where it contains a zinc finger-binding motif for the interactions with LIM domain-containing TRIP6 and proapoptotic Siva-1, and a PDZ-binding motif through which it complexes with the NHERF2 scaffold protein. In this report, we identify a unique CXXC motif of LPA(2) responsible for the binding to TRIP6 and Siva-1, and demonstrate that disruption of these macromolecular complexes or knockdown of TRIP6 or NHERF2 expression attenuates LPA(2)-mediated protection from chemotherapeutic agent-induced apoptosis. In contrast, knockdown of Siva-1 expression enhances this effect. Furthermore, a PDZ-mediated direct interaction between TRIP6 and NHERF2 facilitates their interaction with LPA(2). Together, these results suggest that in addition to G protein-activated signals, the cooperation embedded in the LPA(2)-TRIP6-NHERF2 ternary complex provides a novel ligand-dependent signal amplification mechanism that is required for LPA(2)-mediated full activation of antiapoptotic signaling.
- Published
- 2009
- Full Text
- View/download PDF
45. Functional analyses of oxygenases in jadomycin biosynthesis and identification of JadH as a bifunctional oxygenase/dehydrase.
- Author
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Chen YH, Wang CC, Greenwell L, Rix U, Hoffmeister D, Vining LC, Rohr J, and Yang KQ
- Subjects
- Amino Acid Sequence, Blotting, Southern, Catalysis, Cell-Free System, Escherichia coli metabolism, Genetic Techniques, Hydro-Lyases chemistry, Isoquinolines chemistry, Models, Chemical, Models, Genetic, Molecular Sequence Data, Mutation, Oxygenases chemistry, Protein Binding, Protein Structure, Tertiary, Sequence Analysis, DNA, Species Specificity, Spectrophotometry, Streptomyces metabolism, Streptomyces lividans metabolism, Hydro-Lyases physiology, Isoquinolines metabolism, Oxygenases physiology
- Abstract
A novel angucycline metabolite, 2,3-dehydro-UWM6, was identified in a jadH mutant of Streptomyces venezuelae ISP5230. Both UWM6 and 2,3-dehydro-UWM6 could be converted to jadomycin A or B by a ketosynthase alpha (jadA) mutant of S. venezuelae. These angucycline intermediates were also converted to jadomycin A by transformant of the heterologous host Streptomyces lividans expressing the jadFGH oxygenases in vivo and by its cell-free extracts in vitro; thus the three gene products JadFGH are implicated in catalysis of the post-polyketide synthase biosynthetic reactions converting UWM6 to jadomycin aglycone. Genetic and biochemical analyses indicate that JadH possesses dehydrase activity, not previously associated with polyketide-modifying oxygenase. Since the formation of aromatic polyketides often requires multiple dehydration steps, bifunctionality of oxygenases modifying aromatic polyketides may be a general phenomenon.
- Published
- 2005
- Full Text
- View/download PDF
46. Dynamic O-glycosylation of nuclear and cytosolic proteins: further characterization of the nucleocytoplasmic beta-N-acetylglucosaminidase, O-GlcNAcase.
- Author
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Wells L, Gao Y, Mahoney JA, Vosseller K, Chen C, Rosen A, and Hart GW
- Subjects
- Acetylglucosaminidase chemistry, Acetylglucosaminidase genetics, Amino Acid Sequence, Animals, CHO Cells, COS Cells, Cricetinae, DNA, Complementary, Escherichia coli genetics, Glycosylation, Histone Acetyltransferases, Humans, Kinetics, Molecular Sequence Data, Multienzyme Complexes, Nuclear Proteins chemistry, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, beta-N-Acetylhexosaminidases, Acetylglucosaminidase metabolism, Cytosol enzymology, Nuclear Proteins metabolism
- Abstract
beta-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant and dynamic post-translational modification implicated in protein regulation that appears to be functionally more similar to phosphorylation than to classical glycosylation. There are nucleocytoplasmic enzymes for the attachment and removal of O-GlcNAc. Here, we further characterize the recently cloned beta-N-acetylglucosaminidase, O-GlcNAcase. Both recombinant and purified endogenous O-GlcNAcase rapidly release free GlcNAc from O-GlcNAc-modified peptide substrates. The recombinant enzyme functions as a monomer and has kinetic parameters (K(m) = 1.1 mm for paranitrophenyl-GlcNAc, k(cat) = 1 s(-1)) that are similar to those of lysosomal hexosaminidases. The endogenous O-GlcNAcase appears to be in a complex with other proteins and is predominantly localized to the cytosol. Overexpression of the enzyme in living cells results in decreased O-GlcNAc modification of nucleocytoplasmic proteins. Finally, we show that the enzyme is a substrate for caspase-3 but, surprisingly, the cleavage has no effect on in vitro O-GlcNAcase activity. These studies support the identification of this protein as an O-GlcNAcase and identify important interactions and modifications that may regulate the enzyme and O-GlcNAc cycling.
- Published
- 2002
- Full Text
- View/download PDF
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