21 results on '"Qin, W"'
Search Results
2. RNA-dependent RNA polymerase activity of the soluble recombinant hepatitis C virus NS5B protein truncated at the C-terminal region.
- Author
-
Yamashita, T, Kaneko, S, Shirota, Y, Qin, W, Nomura, T, Kobayashi, K, and Murakami, S
- Abstract
The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRP), which is the central catalytic enzyme of HCV replicase. We established a new method to purify soluble HCV NS5B in the glutathione S-transferase-fused form NS5Bt from Escherichia coli which lacks the C-terminal 21 amino acid residues encompassing a putative anchoring domain (anino acids 2990-3010). The recombinant soluble protein exhibited RdRP activity in vitro which was dependent upon the template and primer, but it did not exhibit the terminal transferase activity that has been reported to be associated with the recombinant NS5B protein from insect cells. The RdRP activity of purified glutathione S-transferase-NS5Bt and thrombin-cleavaged non-fused NS5Bt shares most of the properties. Substitution mutations of NS5Bt at the GDD motif, which is highly conserved among viral RdRPs, and at the clustered basic residues (amino acids 2919-2924 and 2693-2699) abolished the RdRP activity. The C-terminal region of NS5B, which is dispensable for the RdRP activity, dramatically affected the subcellular localization of NS5B retaining it in perinuclear sites in transiently overexpressed mammalian cells. These results may provide some clues to dissecting the molecular mechanism of the HCV replication and also act as a basis for developing new anti-viral drugs.
- Published
- 1998
3. Elements regulating cardiomyocyte expression of the human sarcomeric mitochondrial creatine kinase gene in transgenic mice.
- Author
-
Qin, W, Khuchua, Z, Klein, S C, and Strauss, A W
- Abstract
Sarcomeric mitochondrial creatine kinase (sMtCK) is one component of a multiprotein, high energy channeling complex consisting of porin, mitochondrial creatine kinase, and adenine nucleotide translocase. To study the transcriptional mechanisms specifying sMtCK gene expression to the heart, transgenic mice were created carrying the 5'-flanking sequences of the human sMtCK gene ligated upstream of the human growth hormone (hGH) reporter gene. RNA blot hybridization demonstrated that the human sMtCK sequence, -485 to +6 base pair (bp), did not activate reporter gene expression to a detectable level. However, the human sMtCK sequence, -921 to +6 bp, expressed the hGH reporter gene at a high level in heart and skeletal muscle and at a very low level in esophagus and kidney, and it did not express the hGH gene in other organs tested (brain, lung, liver, spleen, bladder, uterus, and stomach). In situ hybridization revealed that reporter gene transcription was specified to cardiac and skeletal myocytes, recapitulating precisely the expression pattern of the endogenous gene. Sequence analysis identified several consensus binding sites between -921 and -757 bp, including four GATT motifs, one E box, and one MEF2 site. Further analysis of a third transgenic mouse strain demonstrated that the human sMtCK sequence, -757 to +6 bp, did not direct detectable expression of the hGH reporter gene. We conclude that this 160-bp genomic sequence, from -921 to -757 bp, is necessary in specifying expression of the human sMtCK gene to the oxidative and highly metabolically active heart tissue.
- Published
- 1997
4. Functional organization of saposin C. Definition of the neurotrophic and acid beta-glucosidase activation regions.
- Author
-
Qi, X, Qin, W, Sun, Y, Kondoh, K, and Grabowski, G A
- Abstract
Saposin C is an essential co-factor for the hydrolysis of glucosylceramide by acid beta-glucosidase in mammals. In addition, prosaposin promotes neurite outgrowth in vitro via sequences in saposin C. The regional organization of these neurotrophic and activation properties of saposin C was elucidated using recombinant or chemically synthesized saposin Cs from various regions of the molecule. Unreduced and reduced proteins were analyzed by electrospray-mass spectrometry to establish the complement of disulfide bonds in selected saposin Cs. Using saposin B as a unreactive backbone, chimeric saposins containing various length segments of saposin B and C localized the neurotrophic and acid beta-glucosidase activation properties to the carboxyl- and NH2-terminal 50% of saposin C, respectively. The peptide spanning residues 22-31 had neurotrophic effects. Molecular modeling and site-directed mutagenesis localized the activation properties of saposin C to the region spanning residues 47-62. Secondary structure was needed for retention of this property. Single substitutions of R and S at the conserved cysteines at 47 or 78 diminished but did not obliterate the activation properties. These results indicate the segregation of neurotrophic and activation properties of saposin C to two different faces of the molecule and suggest a topographic sequestration of the activation region of prosaposin for protection of the cell from adverse hydrolytic activity of acid beta-glucosidase.
- Published
- 1996
5. Molecular cloning and functional analysis of polyphosphoinositide-dependent phospholipase D, PLDbeta, from Arabidopsis.
- Author
-
Pappan, K, Qin, W, Dyer, J H, Zheng, L, and Wang, X
- Abstract
A novel plant phospholipase D (PLD; EC 3.1.4.4) activity, which is dependent on phosphatidylinositol 4,5-bisphosphate (PIP2) and nanomolar concentrations of calcium, has been identified in Arabidopsis. This report describes the cloning, expression, and characterization of an Arabidopsis cDNA that encodes this PLD. We have designated names of PLDbeta for this PIP2-dependent PLD and PLDalpha for the previously characterized PIP2-independent PLD that requires millimolar Ca2+ for optimal activity. The PLDbeta cDNA contains an open reading frame of 2904 nucleotides coding for a 968-amino acid protein of 108,575 daltons. Expression of this PLDbeta cDNA clone in Escherichia coli results in the accumulation of a functional PLD having PLDbeta, but not PLDalpha, activity. The activity of the expressed PLDbeta is dependent on PIP2 and submicromolar amounts of Ca2+, inhibited by neomycin, and stimulated by a soluble factor from plant extracts. Sequence analysis reveals that PLDbeta is evolutionarily divergent from PLDalpha and that its N terminus contains a regulatory Ca2+-dependent phospholipid-binding (C2) domain that is found in a number of signal transducing and membrane trafficking proteins.
- Published
- 1997
6. Octamer formation and coupling of cardiac sarcomeric mitochondrial creatine kinase are mediated by charged N-terminal residues.
- Author
-
Khuchua, Z A, Qin, W, Boero, J, Cheng, J, Payne, R M, Saks, V A, and Strauss, A W
- Abstract
Mitochondrial creatine kinases form octameric structures composed of four active and stable dimers. Octamer formation has been postulated to occur via interaction of the charged amino acids in the N-terminal peptide of the mature enzyme. We altered codons for charged amino acids in the N-terminal region of mature sarcomeric mitochondrial creatine kinase (sMtCK) to those encoding neutral amino acids. Transfection of normal sMtCK cDNA or those with the mutations R42G, E43G/H45G, and K46G into rat neonatal cardiomyocytes resulted in enzymatically active sMtCK expression in all. After hypoosmotic treatment of isolated mitochondria, mitochondrial inner membrane-associated and soluble sMtCK from the intermembranous space were measured. The R42G and E43G/H45G double mutation caused destabilization of the octameric structure of sMtCK and a profound reduction in binding of sMtCK to the inner mitochondrial membrane. The other mutant sMtCK proteins had modest reductions in binding. Creatine-stimulated respiration was markedly reduced in mitochondria isolated from cells transfected with the R42G mutant cDNA as compared with those transfected with normal sMtCK cDNA. We conclude that neutralization of charges in N-terminal peptide resulted in destabilization of octamer structure of sMtCK. Thus, charged amino acids at the N-terminal moiety of mature sMtCK are essential for octamer formation, binding of sMtCK with inner mitochondrial membrane, and coupling of sMtCK to oxidative phosphorylation.
- Published
- 1998
7. PTBP1 suppresses porcine epidemic diarrhea virus replication via inducing protein degradation and IFN production.
- Author
-
Qin W, Kong N, Zhang Y, Wang C, Dong S, Zhai H, Zhai X, Yang X, Ye C, Ye M, Tong W, Liu C, Yu L, Zheng H, Yu H, Zhang W, Lan D, Tong G, and Shan T
- Subjects
- Animals, Cell Line, Chlorocebus aethiops, Signal Transduction, Swine, Vero Cells, Coronavirus Infections genetics, Coronavirus Infections veterinary, Interferon Type I metabolism, Porcine epidemic diarrhea virus physiology, Proteolysis, Swine Diseases genetics, Swine Diseases virology, Virus Replication, Polypyrimidine Tract-Binding Protein metabolism
- Abstract
Porcine epidemic diarrhea virus (PEDV) causes severe morbidity and mortality among newborn piglets. It significantly threatens the porcine industry in China and around the globe. To accelerate the developmental pace of drugs or vaccines against PEDV, a deeper understanding of the interaction between viral proteins and host factors is crucial. The RNA-binding protein, polypyrimidine tract-binding protein 1 (PTBP1), is crucial for controlling RNA metabolism and biological processes. The present work focused on exploring the effect of PTBP1 on PEDV replication. PTBP1 was upregulated during PEDV infection. The PEDV nucleocapsid (N) protein was degraded through the autophagic and proteasomal degradation pathways. Moreover, PTBP1 recruits MARCH8 (an E3 ubiquitin ligase) and NDP52 (a cargo receptor) for N protein catalysis and degradation through selective autophagy. Furthermore, PTBP1 induces the host innate antiviral response via upregulating the expression of MyD88, which then regulates TNF receptor-associated factor 3/ TNF receptor-associated factor 6 expression and induces the phosphorylation of TBK1 and IFN regulatory factor 3. These processes activate the type Ⅰ IFN signaling pathway to antagonize PEDV replication. Collectively, this work illustrates a new mechanism related to PTBP1-induced viral restriction, where PTBP1 degrades the viral N protein and induces type Ⅰ IFN production to suppress PEDV replication., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
8. Nuclear ribonucleoprotein RALY targets virus nucleocapsid protein and induces autophagy to restrict porcine epidemic diarrhea virus replication.
- Author
-
Qin W, Kong N, Zhang Y, Dong S, Zhai H, Zhai X, Yang X, Ye C, Ye M, Liu C, Yu L, Zheng H, Yu H, Zhang W, Tong G, Lan D, Tong W, and Shan T
- Subjects
- Animals, Autophagy, Chlorocebus aethiops, Nucleocapsid Proteins, Ribonucleoproteins, Swine, Vero Cells, Virus Replication, Coronavirus Infections veterinary, Porcine epidemic diarrhea virus physiology, Swine Diseases
- Abstract
Porcine epidemic diarrhea virus (PEDV) causes diarrhea and dehydration in pigs and leads to great economic losses in the commercial swine industry. However, the underlying molecular mechanisms of host response to viral infection remain unclear. In the present study, we investigated a novel mechanism by which RALY, a member of the heterogeneous nuclear ribonucleoprotein family, significantly promotes the degradation of the PEDV nucleocapsid (N) protein to inhibit viral replication. Furthermore, we identified an interaction between RALY and the E3 ubiquitin ligase MARCH8 (membrane-associated RING-CH 8), as well as the cargo receptor NDP52 (nuclear dot protein 52 kDa), suggesting that RALY could suppress PEDV replication by degrading the viral N protein through a RALY-MARCH8-NDP52-autophagosome pathway. Collectively, these results suggest a preventive role of RALY against PEDV infection via the autophagy pathway and open up the possibility of inducing RALY in vivo as an effective prophylactic and preventive treatment for PEDV infection., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
- Full Text
- View/download PDF
9. The long noncoding RNA LOC105374325 causes podocyte injury in individuals with focal segmental glomerulosclerosis.
- Author
-
Hu S, Han R, Shi J, Zhu X, Qin W, Zeng C, Bao H, and Liu Z
- Subjects
- CCAAT-Enhancer-Binding Protein-beta metabolism, Cells, Cultured, Doxorubicin pharmacology, Female, Gene Expression Regulation drug effects, Glomerulosclerosis, Focal Segmental pathology, Humans, Male, MicroRNAs biosynthesis, Podocytes pathology, Signal Transduction drug effects, bcl-2 Homologous Antagonist-Killer Protein metabolism, bcl-2-Associated X Protein metabolism, Glomerulosclerosis, Focal Segmental metabolism, Podocytes metabolism, RNA, Long Noncoding biosynthesis
- Abstract
Focal segmental glomerulosclerosis (FSGS) is a common kidney disease that results in nephrotic syndrome. FSGS arises from dysfunction and apoptosis of podocytes in the glomerulus of the kidney, leading to podocytopathy. The molecular mechanisms underlying podocyte apoptosis remain incompletely understood. Using an array of gene expression profiling, PCR, and in situ hybridization assay, we found here that the levels of the long noncoding RNA LOC105374325 were elevated in the renal podocytes of individuals with FSGS. We also observed that the microRNAs miR-34c and miR-196a/b down-regulated the expression of the apoptosis regulators BCL2-associated X, apoptosis regulator (Bax), and BCL2 antagonist/killer 1 (Bak) in podocytes. Competitive binding between LOC105374325 and miR-34c or miR-196a/b increased Bax and Bak levels and caused podocyte apoptosis. Of note, the mitogen-activated protein kinase P38 and the transcription factor CCAAT enhancer-binding protein β (C/EBPβ) up-regulated LOC105374325 expression. P38 inhibition or C/EBPβ silencing decreased LOC105374325 levels and inhibited apoptosis in adriamycin-treated podocytes. LOC105374325 overexpression decreased miR-34c and miR-196a/b levels, increased Bax and Bak levels, and induced proteinuria and focal segmental lesions in mice. In conclusion, activation of the P38/C/EBPβ pathway stimulates the expression of LOC105374325, which, in turn, increases Bax and Bak levels and causes apoptosis by competitively binding to miR-34c and miR-196a/b in the podocytes of individuals with FSGS., (© 2018 Hu et al.)
- Published
- 2018
- Full Text
- View/download PDF
10. Myostatin inhibits osteoblastic differentiation by suppressing osteocyte-derived exosomal microRNA-218: A novel mechanism in muscle-bone communication.
- Author
-
Qin Y, Peng Y, Zhao W, Pan J, Ksiezak-Reding H, Cardozo C, Wu Y, Divieti Pajevic P, Bonewald LF, Bauman WA, and Qin W
- Subjects
- Adaptor Proteins, Signal Transducing, Animals, Cell Line, Exosomes genetics, Glycoproteins genetics, Glycoproteins metabolism, Intercellular Signaling Peptides and Proteins genetics, Intercellular Signaling Peptides and Proteins metabolism, Mice, MicroRNAs genetics, Myostatin genetics, RANK Ligand genetics, RANK Ligand metabolism, Cell Differentiation, Exosomes metabolism, MicroRNAs metabolism, Muscle, Skeletal metabolism, Myostatin metabolism, Osteocytes metabolism, Wnt Signaling Pathway physiology
- Abstract
Muscle and bone are closely associated in both anatomy and function, but the mechanisms that coordinate their synergistic action remain poorly defined. Myostatin, a myokine secreted by muscles, has been shown to inhibit muscle growth, and the disruption of the myostatin gene has been reported to cause muscle hypertrophy and increase bone mass. Extracellular vesicle-exosomes that carry microRNA (miRNA), mRNA, and proteins are known to perform an important role in cell-cell communication. We hypothesized that myostatin may play a crucial role in muscle-bone interactions and may promote direct effects on osteocytes and on osteocyte-derived exosomal miRNAs, thereby indirectly influencing the function of other bone cells. We report herein that myostatin promotes expression of several bone regulators such as sclerostin (SOST), DKK1, and RANKL in cultured osteocytic (Ocy454) cells, concomitant with the suppression of miR-218 in both parent Ocy454 cells and derived exosomes. Exosomes produced by Ocy454 cells that had been pretreated with myostatin could be taken up by osteoblastic MC3T3 cells, resulting in a marked reduction of Runx2, a key regulator of osteoblastic differentiation, and in decreased osteoblastic differentiation via the down-regulation of the Wnt signaling pathway. Importantly, the inhibitory effect of myostatin-modified osteocytic exosomes on osteoblast differentiation is completely reversed by expression of exogenous miR-218, through a mechanism involving miR-218-mediated inhibition of SOST. Together, our findings indicate that myostatin directly influences osteocyte function and thereby inhibits osteoblastic differentiation, at least in part, through the suppression of osteocyte-derived exosomal miR-218, suggesting a novel mechanism in muscle-bone communication., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2017
- Full Text
- View/download PDF
11. The central nervous system (CNS)-independent anti-bone-resorptive activity of muscle contraction and the underlying molecular and cellular signatures.
- Author
-
Qin W, Sun L, Cao J, Peng Y, Collier L, Wu Y, Creasey G, Li J, Qin Y, Jarvis J, Bauman WA, Zaidi M, and Cardozo C
- Subjects
- Animals, Bone Marrow Cells physiology, Bone Resorption blood, Bone Resorption pathology, Cell Differentiation, Cells, Cultured, Central Nervous System physiopathology, Collagen Type I blood, Electric Stimulation, Female, Femur metabolism, Femur pathology, Hindlimb innervation, Hindlimb physiopathology, Muscle, Skeletal innervation, Muscle, Skeletal pathology, Muscle, Skeletal physiopathology, Organ Size, Osteoblasts metabolism, Osteoblasts physiology, Osteocalcin blood, Osteoclasts metabolism, Rats, Rats, Wistar, Signal Transduction, Bone Resorption physiopathology, Muscle Contraction, Transcriptome
- Abstract
Background: Mechanisms by which muscle regulates bone are poorly understood., Results: Electrically stimulated muscle contraction reversed elevations in bone resorption and increased Wnt signaling in bone-derived cells after spinal cord transection., Conclusion: Muscle contraction reduced resorption of unloaded bone independently of the CNS, through mechanical effects and, potentially, nonmechanical signals (e.g. myokines)., Significance: The study provides new insights regarding muscle-bone interactions. Muscle and bone work as a functional unit. Cellular and molecular mechanisms underlying effects of muscle activity on bone mass are largely unknown. Spinal cord injury (SCI) causes muscle paralysis and extensive sublesional bone loss and disrupts neural connections between the central nervous system (CNS) and bone. Muscle contraction elicited by electrical stimulation (ES) of nerves partially protects against SCI-related bone loss. Thus, application of ES after SCI provides an opportunity to study the effects of muscle activity on bone and roles of the CNS in this interaction, as well as the underlying mechanisms. Using a rat model of SCI, the effects on bone of ES-induced muscle contraction were characterized. The SCI-mediated increase in serum C-terminal telopeptide of type I collagen (CTX) was completely reversed by ES. In ex vivo bone marrow cell cultures, SCI increased the number of osteoclasts and their expression of mRNA for several osteoclast differentiation markers, whereas ES significantly reduced these changes; SCI decreased osteoblast numbers, but increased expression in these cells of receptor activator of NF-κB ligand (RANKL) mRNA, whereas ES increased expression of osteoprotegerin (OPG) and the OPG/RANKL ratio. A microarray analysis revealed that ES partially reversed SCI-induced alterations in expression of genes involved in signaling through Wnt, FSH, parathyroid hormone (PTH), oxytocin, and calcineurin/nuclear factor of activated T-cells (NFAT) pathways. ES mitigated SCI-mediated increases in mRNA levels for the Wnt inhibitors DKK1, sFRP2, and sclerostin in ex vivo cultured osteoblasts. Our results demonstrate an anti-bone-resorptive activity of muscle contraction by ES that develops rapidly and is independent of the CNS. The pathways involved, particularly Wnt signaling, suggest future strategies to minimize bone loss after immobilization.
- Published
- 2013
- Full Text
- View/download PDF
12. Missense mutation in APOC3 within the C-terminal lipid binding domain of human ApoC-III results in impaired assembly and secretion of triacylglycerol-rich very low density lipoproteins: evidence that ApoC-III plays a major role in the formation of lipid precursors within the microsomal lumen.
- Author
-
Qin W, Sundaram M, Wang Y, Zhou H, Zhong S, Chang CC, Manhas S, Yao EF, Parks RJ, McFie PJ, Stone SJ, Jiang ZG, Wang C, Figeys D, Jia W, and Yao Z
- Subjects
- Apolipoprotein C-III chemistry, Apolipoprotein C-III genetics, Chromatography, Gel, Humans, Microsomes, Liver metabolism, Reverse Transcriptase Polymerase Chain Reaction, Subcellular Fractions metabolism, Tandem Mass Spectrometry, Apolipoprotein C-III metabolism, Lipoproteins, LDL metabolism, Mutation, Missense, Triglycerides metabolism
- Abstract
Hepatic assembly of triacylglycerol (TAG)-rich very low density lipoproteins (VLDL) is achieved through recruitment of bulk TAG (presumably in the form of lipid droplets within the microsomal lumen) into VLDL precursor containing apolipoprotein (apo) B-100. We determined protein/lipid components of lumenal lipid droplets (LLD) in cells expressing recombinant human apoC-III (C3wt) or a mutant form (K58E, C3KE) initially identified in humans that displayed hypotriglyceridemia. Although expression of C3wt markedly stimulated secretion of TAG and apoB-100 as VLDL(1), the K58E mutation (located at the C-terminal lipid binding domain) abolished the effect in transfected McA-RH7777 cells and in apoc3-null mice. Metabolic labeling studies revealed that accumulation of TAG in LLD was decreased (by 50%) in cells expressing C3KE. A Fat Western lipid protein overlay assay showed drastically reduced lipid binding of the mutant protein. Substituting Lys(58) with Arg demonstrated that the positive charge at position 58 is crucial for apoC-III binding to lipid and for promoting TAG secretion. On the other hand, substituting both Lys(58) and Lys(60) with Glu resulted in almost entire elimination of lipid binding and loss of function in promoting TAG secretion. Thus, the lipid binding domain of apoC-III plays a key role in the formation of LLD for hepatic VLDL assembly and secretion.
- Published
- 2011
- Full Text
- View/download PDF
13. Neuronal SIRT1 activation as a novel mechanism underlying the prevention of Alzheimer disease amyloid neuropathology by calorie restriction.
- Author
-
Qin W, Yang T, Ho L, Zhao Z, Wang J, Chen L, Zhao W, Thiyagarajan M, MacGrogan D, Rodgers JT, Puigserver P, Sadoshima J, Deng H, Pedrini S, Gandy S, Sauve AA, and Pasinetti GM
- Subjects
- Alzheimer Disease prevention & control, Amyloid analysis, Amyloid Precursor Protein Secretases, Animals, Aspartic Acid Endopeptidases, Endopeptidases metabolism, Enzyme Activation, Female, Humans, Intracellular Signaling Peptides and Proteins genetics, Intracellular Signaling Peptides and Proteins physiology, Mice, Mice, Inbred Strains, Mice, Transgenic, Neurons pathology, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases physiology, Sirtuin 1, rho-Associated Kinases, Alzheimer Disease diet therapy, Caloric Restriction, Neurons enzymology, Sirtuins metabolism
- Abstract
Nicotinamide adenine dinucleotide (NAD)+-dependent sirtuins have been identified to be key regulators in the lifespan extending effects of calorie restriction (CR) in a number of species. In this study we report for the first time that promotion of the NAD+-dependent sirtuin, SIRT1-mediated deacetylase activity, may be a mechanism by which CR influences Alzheimer disease (AD)-type amyloid neuropathology. Most importantly, we report that the predicted attenuation of beta-amyloid content in the brain during CR can be reproduced in mouse neurons in vitro by manipulating cellular SIRT1 expression/activity through mechanisms involving the regulation of the serine/threonine Rho kinase ROCK1, known in part for its role in the inhibition of the non-amyloidogenic alpha-secretase processing of the amyloid precursor protein. Conversely, we found that the expression of constitutively active ROCK1 in vitro cultures significantly prevented SIRT1-mediated response, suggesting that alpha-secretase activity is required for SIRT1-mediated prevention of AD-type amyloid neuropathology. Consistently we found that the expression of exogenous human (h) SIRT1 in the brain of hSIRT1 transgenics also resulted in decreased ROCK1 expression and elevated alpha-secretase activity in vivo. These results demonstrate for the first time a role for SIRT1 activation in the brain as a novel mechanism through which CR may influence AD amyloid neuropathology. The study provides a potentially novel pharmacological strategy for AD prevention and/or treatment.
- Published
- 2006
- Full Text
- View/download PDF
14. Green tea polyphenols modulate insulin secretion by inhibiting glutamate dehydrogenase.
- Author
-
Li C, Allen A, Kwagh J, Doliba NM, Qin W, Najafi H, Collins HW, Matschinsky FM, Stanley CA, and Smith TJ
- Subjects
- Adenosine Diphosphate chemistry, Adenosine Triphosphate chemistry, Animals, Cattle, Dose-Response Relationship, Drug, Glutamate Dehydrogenase metabolism, Guanosine Triphosphate chemistry, Hyperammonemia metabolism, Insulin Secretion, Insulin-Secreting Cells metabolism, Islets of Langerhans metabolism, Kinetics, Leucine chemistry, Male, Models, Biological, Models, Chemical, Models, Molecular, Oxygen Consumption, Perfusion, Polyphenols, Protein Conformation, Rats, Rats, Wistar, Tea, Time Factors, Enzyme Inhibitors pharmacology, Flavonoids chemistry, Glutamate Dehydrogenase antagonists & inhibitors, Insulin metabolism, Phenols chemistry
- Abstract
Insulin secretion by pancreatic beta-cells is stimulated by glucose, amino acids, and other metabolic fuels. Glutamate dehydrogenase (GDH) has been shown to play a regulatory role in this process. The importance of GDH was underscored by features of hyperinsulinemia/hyperammonemia syndrome, where a dominant mutation causes the loss of inhibition by GTP and ATP. Here we report the effects of green tea polyphenols on GDH and insulin secretion. Of the four compounds tested, epigallocatechin gallate (EGCG) and epicatechin gallate were found to inhibit GDH with nanomolar ED(50) values and were therefore found to be as potent as the physiologically important inhibitor GTP. Furthermore, we have demonstrated that EGCG inhibits BCH-stimulated insulin secretion, a process that is mediated by GDH, under conditions where GDH is no longer inhibited by high energy metabolites. EGCG does not affect glucose-stimulated insulin secretion under high energy conditions where GDH is probably fully inhibited. We have further shown that these compounds act in an allosteric manner independent of their antioxidant activity and that the beta-cell stimulatory effects are directly correlated with glutamine oxidation. These results demonstrate that EGCG, much like the activator of GDH (BCH), can facilitate dissecting the complex regulation of insulin secretion by pharmacologically modulating the effects of GDH.
- Published
- 2006
- Full Text
- View/download PDF
15. Cyclooxygenase (COX)-2 and COX-1 potentiate beta-amyloid peptide generation through mechanisms that involve gamma-secretase activity.
- Author
-
Qin W, Ho L, Pompl PN, Peng Y, Zhao Z, Xiang Z, Robakis NK, Shioi J, Suh J, and Pasinetti GM
- Subjects
- Alzheimer Disease etiology, Amyloid Precursor Protein Secretases, Animals, Aspartic Acid Endopeptidases, Cell Line, Cyclooxygenase 1, Cyclooxygenase 2, Cyclooxygenase 2 Inhibitors, Cyclooxygenase Inhibitors pharmacology, Dinoprostone pharmacology, Endopeptidases drug effects, Humans, Ibuprofen pharmacology, Membrane Proteins, Transfection, Amyloid beta-Peptides biosynthesis, Endopeptidases metabolism, Isoenzymes physiology, Peptide Fragments biosynthesis, Prostaglandin-Endoperoxide Synthases physiology
- Abstract
In previous studies we found that overexpression of the inducible form of cyclooxygenase, COX-2, in the brain exacerbated beta-amyloid (A beta) neuropathology in a transgenic mouse model of Alzheimer's disease. To explore the mechanism through which COX may influence A beta amyloidosis, we used an adenoviral gene transfer system to study the effects of human (h)COX-1 and hCOX-2 isoform expression on A beta peptide generation. We found that expression of hCOXs in human amyloid precursor protein (APP)-overexpressing (Chinese hamster ovary (CHO)-APPswe) cells or human neuroglioma (H4-APP751) cells resulting in 10-25 nM prostaglandin (PG)-E2 concentration in the conditioned medium coincided with an approximately 1.8-fold elevation of A beta-(1-40) and A beta-(1-42) peptide generation and an approximately 1.8-fold induction of the C-terminal fragment (CTF)-gamma cleavage product of the APP, an index of gamma-secretase activity. Treatment of APP-overexpressing cells with the non-selective COX inhibitor ibuprofen (1 microM, 48 h) or with the specific gamma-secretase inhibitor L-685,458 significantly attenuated hCOX-1- and hCOX-2-mediated induction of A beta peptide generation and CTF-gamma cleavage product formation. Based on this evidence, we next tested the hypothesis that COX expression might promote A beta peptide generation via a PG-E2-mediated mechanism. We found that exposure of CHO-APPswe or human embryonic kidney (HEK-APPswe) cells to PG-E2 (11-deoxy-PG-E2) at a concentration (10 nM) within the range of PG-E2 found in hCOX-expressing cells similarly promoted (approximately 1.8-fold) the generation of the CTF-gamma cleavage product of APP and commensurate A beta-(1-40) and A beta-(1-42) peptide elevation. The study suggests that expression of COXs may influence A beta peptide generation through mechanisms that involve PG-E2-mediated potentiation of gamma-secretase activity, further supporting a role for COX-2 and COX-1 in Alzheimer's disease neuropathology.
- Published
- 2003
- Full Text
- View/download PDF
16. Positive and negative regulation of the gamma-secretase activity by nicastrin in a murine model.
- Author
-
Li J, Fici GJ, Mao CA, Myers RL, Shuang R, Donoho GP, Pauley AM, Himes CS, Qin W, Kola I, Merchant KM, and Nye JS
- Subjects
- Adenoviridae genetics, Alleles, Amyloid Precursor Protein Secretases, Animals, Aspartic Acid Endopeptidases, Cell Membrane metabolism, Culture Media, Conditioned pharmacology, DNA, Complementary metabolism, Fibroblasts metabolism, Genes, Reporter, Genotype, Green Fluorescent Proteins, Heterozygote, Humans, Luciferases metabolism, Luminescent Proteins metabolism, Membrane Glycoproteins physiology, Membrane Proteins physiology, Mice, Mice, Knockout, Peptides chemistry, Phenotype, Presenilin-1, Presenilin-2, Protein Structure, Tertiary, RNA, Messenger metabolism, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins metabolism, Time Factors, Endopeptidases metabolism, Gene Expression Regulation, Enzymologic, Membrane Glycoproteins genetics, Membrane Glycoproteins metabolism
- Abstract
Nicastrin is a component of the gamma-secretase complex that has been shown to adhere to presenilin-1 (PS1), Notch, and APP. Here we demonstrate that Nicastrin-deficient mice showed a phenotype that is indistinguishable from PS1/PS2 double knock-out mice, whereas heterozygotes were healthy and viable. Fibroblasts derived from Nicastrin-deficient embryos were unable to generate amyloid beta-peptide and failed to release the intracellular domain of APP- or Notch1-Gal4-VP16 fusion proteins. Additionally, C- and N-terminal fragments of PS1 and the C-terminal fragments of PS2 were not detectable in Nicastrin-null fibroblasts, whereas full-length PS1 accumulated in null fibroblasts, indicating that Nicastrin is required for the endoproteolytic processing of presenilins. Interestingly, cells derived from Nicastrin heterozygotes produced relatively higher levels of amyloid beta-peptide whether the source was endogenous mouse or transfected human APP. These data demonstrate that Nicastrin is essential for the gamma-secretase cleavage of APP and Notch in mammalian cells and that Nicastrin has both positive and negative functions in the regulation of gamma-secretase activity.
- Published
- 2003
- Full Text
- View/download PDF
17. Direct interaction between nucleolin and hepatitis C virus NS5B.
- Author
-
Hirano M, Kaneko S, Yamashita T, Luo H, Qin W, Shirota Y, Nomura T, Kobayashi K, and Murakami S
- Subjects
- Animals, COS Cells, Green Fluorescent Proteins, HeLa Cells, Humans, Luminescent Proteins, Nuclear Proteins genetics, Phosphoproteins genetics, Protein Binding, RNA-Binding Proteins genetics, RNA-Dependent RNA Polymerase genetics, RNA-Dependent RNA Polymerase metabolism, Viral Nonstructural Proteins genetics, Nucleolin, Nuclear Proteins metabolism, Phosphoproteins metabolism, RNA-Binding Proteins metabolism, Viral Nonstructural Proteins metabolism
- Abstract
Hepatitis C virus (HCV) NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme in HCV replication. While studying the subcellular localization of a NS5B mutant lacking the C-terminal membrane-anchoring domain, NS5Bt, we found that expression of the green fluorescent protein (GFP)-fused form was exclusively nucleolar. Interestingly, the distribution of endogenous nucleolin changed greatly in the cells expressing GFP-NS5B, with nucleolin colocalized with GFP-NS5B in perinuclear regions in addition to the nucleolus, suggesting that NS5B retains the ability to bind nucleolin. The interaction between nucleolin and NS5B was demonstrated by GST pull-down assay. GST pull-down assay results indicated that C-terminal region of nucleolin was important for its binding to NS5B. Scanning clustered alanine substitution mutants library of NS5B revealed two sites on NS5B that binds nucleolin. NS5B amino acids 208-214 and 500-506 were both found to be indispensable for the nucleolin binding. We reported that the latter sequence is essential for oligomerization of NS5B, which is a prerequisite for the RdRP activity. C-terminal nucleolin inhibited the NS5B RdRP activity in a dose-dependent manner. Taken together, this indicates the binding ability of nucleolin may be involved in NS5B functions.
- Published
- 2003
- Full Text
- View/download PDF
18. Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP) NS5B and modulates RNA-dependent RNA polymerase activity.
- Author
-
Shirota Y, Luo H, Qin W, Kaneko S, Yamashita T, Kobayashi K, and Murakami S
- Subjects
- Animals, Base Sequence, COS Cells, DNA Primers, Plasmids, Protein Binding, Recombinant Proteins metabolism, RNA-Dependent RNA Polymerase metabolism, Viral Nonstructural Proteins metabolism
- Abstract
Hepatitis C virus (HCV) NS5B is RNA-dependent RNA polymerase (RdRP), the essential catalytic enzyme for HCV replication. Recently, NS5A has been reported to be important for the establishment of HCV replication in vitro by the adaptive mutations, although its role in viral replication remains uncertain. Here we report that purified bacterial recombinant NS5A and NS5B directly interact with each other in vitro, detected by glutathione S-transferase (GST) pull-down assay. Furthermore, complex formation of these proteins transiently coexpressed in mammalian cells was detected by coprecipitation. Using terminally and internally truncated NS5A, two discontinuous regions of NS5A (amino acids 105-162 and 277-334) outside of the adaptive mutations were identified to be independently essential for the binding both in vivo and in vitro (Yamashita, T., Kaneko, S., Shirota, Y., Qin, W., Nomura, T., Kobayashi, K., and Mkyrakami, S. (1998) J. Biol. Chem. 273, 15479-15486). We previously examined the effect of His-NS5A on RdRP activity of the soluble recombinant NS5Bt in vitro (see Yamashita et al. above). Wild NS5A weakly stimulated at first (when less than 0.1 molar ratio to NS5B) and then inhibited the NS5Bt RdRP activity in a dose-dependent manner. The internal deletion mutants defective in NS5B binding exhibited no inhibitory effect, indicating that the NS5B binding is necessary for the inhibition. Taken together, our results support the idea that NS5A modulates HCV replication as a component of replication complex.
- Published
- 2002
- Full Text
- View/download PDF
19. Oligomeric interaction of hepatitis C virus NS5B is critical for catalytic activity of RNA-dependent RNA polymerase.
- Author
-
Qin W, Luo H, Nomura T, Hayashi N, Yamashita T, and Murakami S
- Subjects
- Amino Acids metabolism, Biopolymers chemistry, Catalysis, Models, Molecular, Mutagenesis, Site-Directed, Protein Conformation, Solutions, Viral Nonstructural Proteins chemistry, Viral Nonstructural Proteins genetics, Biopolymers metabolism, RNA-Dependent RNA Polymerase metabolism, Viral Nonstructural Proteins metabolism
- Abstract
HCV NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme for HCV replication, which has the "palm and fingers" substructure. We recently identified five novel residues critical for RdRP activity (Qin, W., Yamashita, T., Shirota, Y., Lin, Y., Wei, W., and Murakami, S. (2001) Hepatology 33, 728-737). Among them, GLU-18 and His-502, far from the catalytic center, may be involved in conformational change(s) for RdRP activity as addressed in some palm and fingers enzymes. We examined the possibility that NS5B is oligomerized, and we could detect the interaction between two different tagged NS5B proteins in vitro and transiently expressed in mammalian cells. By scanning 27 clustered and then point alanine substitutions in vivo and in vitro, Glu-18 and His-502 were found to be critical for the homomeric interaction in vivo and in vitro, strongly suggesting a close relationship between the oligomerization and RdRP activity of NS5B. All mutants with substitutions at these two residues failed to bind wild type NS5B, however E18H interacted with H502E in vitro and in vivo. Interestingly, the NS5B protein with E18H or H502E did not exhibit RdRP activity, but a mixture of the two mutant proteins did. These results clearly indicate that two residues of HCV NS5B are critical for the oligomerization that is prerequisite to RdRP activity.
- Published
- 2002
- Full Text
- View/download PDF
20. Direct interaction between the subunit RAP30 of transcription factor IIF (TFIIF) and RNA polymerase subunit 5, which contributes to the association between TFIIF and RNA polymerase II.
- Author
-
Wei W, Dorjsuren D, Lin Y, Qin W, Nomura T, Hayashi N, and Murakami S
- Subjects
- Animals, Base Sequence, Binding Sites, COS Cells, DNA Primers, Protein Binding, Transcription Factors chemistry, RNA Polymerase II metabolism, Transcription Factors metabolism, Transcription Factors, TFII
- Abstract
The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30 in vitro using purified recombinant proteins and in vivo in COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47-120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101-170) and the N-terminus (aa 1-100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding in in vitro and in vivo assays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.
- Published
- 2001
- Full Text
- View/download PDF
21. Molecular heterogeneity of phospholipase D (PLD). Cloning of PLDgamma and regulation of plant PLDgamma, -beta, and -alpha by polyphosphoinositides and calcium.
- Author
-
Qin W, Pappan K, and Wang X
- Subjects
- Amino Acid Sequence, Arabidopsis enzymology, Arabidopsis genetics, Blotting, Southern, Cloning, Molecular, DNA, Complementary chemistry, Escherichia coli, Gene Library, Molecular Sequence Data, Phosphatidylinositol 4,5-Diphosphate metabolism, Phospholipase D genetics, Phospholipase D metabolism, Sequence Alignment, Calcium metabolism, Phosphatidylinositol Phosphates metabolism, Phospholipase D chemistry
- Abstract
Phospholipase D (PLD) has emerged as an important enzyme involved in signal transduction, vesicle trafficking, and membrane metabolism. This report describes the cloning and expression of a new Arabidopsis PLD cDNA, designated PLDgamma, and the regulation of PLDgamma, -beta, and -alpha by phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+. The PLDgamma cDNA is 3.3 kilobases in length and codes for an 855-amino acid protein of 95,462 Da with a pI of 6.9. PLDgamma shares a 66% amino acid sequence identity with PLDbeta, but only a 41% identity with PLDalpha. A potential N-terminal myristoylation site is found in PLDgamma, but not in PLDalpha and -beta. Catalytically active PLDgamma was expressed in Escherichia coli, and its activity requires polyphosphoinositides. Both PLDgamma and -beta are most active at microM Ca2+ concentrations, whereas the optimal PLDalpha activity requires mM Ca2+ concentrations. Binding studies showed that the PLDs bound PIP2 in the order of PLDbeta > PLDgamma > PLDalpha. This binding ability correlates with the degree of conservation of a basic PIP2-binding motif located near the putative catalytic site. The binding of [3H]PIP2 was saturable and could be competitively decreased by addition of unlabeled PIP2. Neomycin inhibited the activities of PLDgamma and -beta, but not PLDalpha. These results demonstrate that PLD is encoded by a heterogeneous gene family and that direct polyphosphoinositide binding is required for the activities of PLDgamma and -beta, but not PLDalpha. The different structural and biochemical properties suggest that PLDalpha, -beta, and -gamma are regulated differently and may mediate unique cellular functions.
- Published
- 1997
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.