60 results on '"Shih-Che Sue"'
Search Results
2. Fig.S4 from Dual Roles for CXCL4 Chemokines and CXCR3 in Angiogenesis and Invasion of Pancreatic Cancer
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Andreas Bikfalvi, Hervé Prats, Martin Hagedorn, Abdel-Majid Khatib, Abderrahim Lomri, Kim Clarke, Shih-Che Sue, Vincent Castronovo, Martin Schilling, Raphael Pineau, Anne Couvelard, Harald Wodrich, Fabienne Rayne, Thomas Daubon, Renaud Grepin, Marie Sire, Laurent Dumartin, Florence Darlot, Fabienne Soulet, Clotilde Billottet, Alexandre Dubrac, Kevin Boyé, Jessica Baud, and Cathy Quemener
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CXCR3 gene expression analysis
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- 2023
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3. Supplemental Material and Methods from Dual Roles for CXCL4 Chemokines and CXCR3 in Angiogenesis and Invasion of Pancreatic Cancer
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Andreas Bikfalvi, Hervé Prats, Martin Hagedorn, Abdel-Majid Khatib, Abderrahim Lomri, Kim Clarke, Shih-Che Sue, Vincent Castronovo, Martin Schilling, Raphael Pineau, Anne Couvelard, Harald Wodrich, Fabienne Rayne, Thomas Daubon, Renaud Grepin, Marie Sire, Laurent Dumartin, Florence Darlot, Fabienne Soulet, Clotilde Billottet, Alexandre Dubrac, Kevin Boyé, Jessica Baud, and Cathy Quemener
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Supplemental Material and Method section
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- 2023
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4. Data from Dual Roles for CXCL4 Chemokines and CXCR3 in Angiogenesis and Invasion of Pancreatic Cancer
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Andreas Bikfalvi, Hervé Prats, Martin Hagedorn, Abdel-Majid Khatib, Abderrahim Lomri, Kim Clarke, Shih-Che Sue, Vincent Castronovo, Martin Schilling, Raphael Pineau, Anne Couvelard, Harald Wodrich, Fabienne Rayne, Thomas Daubon, Renaud Grepin, Marie Sire, Laurent Dumartin, Florence Darlot, Fabienne Soulet, Clotilde Billottet, Alexandre Dubrac, Kevin Boyé, Jessica Baud, and Cathy Quemener
- Abstract
The CXCL4 paralog CXCL4L1 is a less studied chemokine that has been suggested to exert an antiangiogenic function. However, CXCL4L1 is also expressed in patient tumors, tumor cell lines, and murine xenografts, prompting a more detailed analysis of its role in cancer pathogenesis. We used genetic and antibody-based approaches to attenuate CXCL4L1 in models of pancreatic ductal adenocarcinoma (PDAC). Mechanisms of expression were assessed in cell coculture experiments, murine, and avian xenotransplants, including through an evaluation of CpG methylation and mutation of critical CpG residues. CXCL4L1 gene expression was increased greatly in primary and metastatic PDAC. We found that myofibroblasts triggered cues in the tumor microenvironment, which led to induction of CXCL4L1 in tumor cells. CXCL4L1 expression was also controlled by epigenetic modifications at critical CpG islands, which were mapped. CXCL4L1 inhibited angiogenesis but also affected tumor development more directly, depending on the tumor cell type. In vivo administration of an mAb against CXCL4L1 demonstrated a blockade in the growth of tumors positive for CXCR3, a critical receptor for CXCL4 ligands. Our findings define a protumorigenic role in PDAC development for endogenous CXCL4L1, which is independent of its antiangiogenic function. Cancer Res; 76(22); 6507–19. ©2016 AACR.
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- 2023
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5. Fig.S8 from Dual Roles for CXCL4 Chemokines and CXCR3 in Angiogenesis and Invasion of Pancreatic Cancer
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Andreas Bikfalvi, Hervé Prats, Martin Hagedorn, Abdel-Majid Khatib, Abderrahim Lomri, Kim Clarke, Shih-Che Sue, Vincent Castronovo, Martin Schilling, Raphael Pineau, Anne Couvelard, Harald Wodrich, Fabienne Rayne, Thomas Daubon, Renaud Grepin, Marie Sire, Laurent Dumartin, Florence Darlot, Fabienne Soulet, Clotilde Billottet, Alexandre Dubrac, Kevin Boyé, Jessica Baud, and Cathy Quemener
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Characterization of CXCR3 overexpression in MIA PaCa-2 cells in vitro
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- 2023
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6. Fig.S5 from Dual Roles for CXCL4 Chemokines and CXCR3 in Angiogenesis and Invasion of Pancreatic Cancer
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Andreas Bikfalvi, Hervé Prats, Martin Hagedorn, Abdel-Majid Khatib, Abderrahim Lomri, Kim Clarke, Shih-Che Sue, Vincent Castronovo, Martin Schilling, Raphael Pineau, Anne Couvelard, Harald Wodrich, Fabienne Rayne, Thomas Daubon, Renaud Grepin, Marie Sire, Laurent Dumartin, Florence Darlot, Fabienne Soulet, Clotilde Billottet, Alexandre Dubrac, Kevin Boyé, Jessica Baud, and Cathy Quemener
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Effect of CXCL4L1 and CXCL4 on apoptosis in pancreatic adenocarcinoma cells and endothelial cells
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- 2023
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7. Fig.S9 from Dual Roles for CXCL4 Chemokines and CXCR3 in Angiogenesis and Invasion of Pancreatic Cancer
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Andreas Bikfalvi, Hervé Prats, Martin Hagedorn, Abdel-Majid Khatib, Abderrahim Lomri, Kim Clarke, Shih-Che Sue, Vincent Castronovo, Martin Schilling, Raphael Pineau, Anne Couvelard, Harald Wodrich, Fabienne Rayne, Thomas Daubon, Renaud Grepin, Marie Sire, Laurent Dumartin, Florence Darlot, Fabienne Soulet, Clotilde Billottet, Alexandre Dubrac, Kevin Boyé, Jessica Baud, and Cathy Quemener
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Impact of CXCR3 overexpression in MIA PaCa-2 cells in vitro and in vivo
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- 2023
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8. Fig.S7 from Dual Roles for CXCL4 Chemokines and CXCR3 in Angiogenesis and Invasion of Pancreatic Cancer
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Andreas Bikfalvi, Hervé Prats, Martin Hagedorn, Abdel-Majid Khatib, Abderrahim Lomri, Kim Clarke, Shih-Che Sue, Vincent Castronovo, Martin Schilling, Raphael Pineau, Anne Couvelard, Harald Wodrich, Fabienne Rayne, Thomas Daubon, Renaud Grepin, Marie Sire, Laurent Dumartin, Florence Darlot, Fabienne Soulet, Clotilde Billottet, Alexandre Dubrac, Kevin Boyé, Jessica Baud, and Cathy Quemener
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Characterization of PECAM-1 expression in pancreatic tumours
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- 2023
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9. Fig.S1 from Dual Roles for CXCL4 Chemokines and CXCR3 in Angiogenesis and Invasion of Pancreatic Cancer
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Andreas Bikfalvi, Hervé Prats, Martin Hagedorn, Abdel-Majid Khatib, Abderrahim Lomri, Kim Clarke, Shih-Che Sue, Vincent Castronovo, Martin Schilling, Raphael Pineau, Anne Couvelard, Harald Wodrich, Fabienne Rayne, Thomas Daubon, Renaud Grepin, Marie Sire, Laurent Dumartin, Florence Darlot, Fabienne Soulet, Clotilde Billottet, Alexandre Dubrac, Kevin Boyé, Jessica Baud, and Cathy Quemener
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Expression Analysis of CXCL4L1 in human pancreatic tissue
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- 2023
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10. Fig.S3 from Dual Roles for CXCL4 Chemokines and CXCR3 in Angiogenesis and Invasion of Pancreatic Cancer
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Andreas Bikfalvi, Hervé Prats, Martin Hagedorn, Abdel-Majid Khatib, Abderrahim Lomri, Kim Clarke, Shih-Che Sue, Vincent Castronovo, Martin Schilling, Raphael Pineau, Anne Couvelard, Harald Wodrich, Fabienne Rayne, Thomas Daubon, Renaud Grepin, Marie Sire, Laurent Dumartin, Florence Darlot, Fabienne Soulet, Clotilde Billottet, Alexandre Dubrac, Kevin Boyé, Jessica Baud, and Cathy Quemener
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Cocultures of Panc-1 cells with myofibroblasts and expression analysis
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- 2023
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11. Fig.S6 from Dual Roles for CXCL4 Chemokines and CXCR3 in Angiogenesis and Invasion of Pancreatic Cancer
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Andreas Bikfalvi, Hervé Prats, Martin Hagedorn, Abdel-Majid Khatib, Abderrahim Lomri, Kim Clarke, Shih-Che Sue, Vincent Castronovo, Martin Schilling, Raphael Pineau, Anne Couvelard, Harald Wodrich, Fabienne Rayne, Thomas Daubon, Renaud Grepin, Marie Sire, Laurent Dumartin, Florence Darlot, Fabienne Soulet, Clotilde Billottet, Alexandre Dubrac, Kevin Boyé, Jessica Baud, and Cathy Quemener
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In vivo characterization of the MabL1 antibody
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- 2023
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12. Fig.S2 from Dual Roles for CXCL4 Chemokines and CXCR3 in Angiogenesis and Invasion of Pancreatic Cancer
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Andreas Bikfalvi, Hervé Prats, Martin Hagedorn, Abdel-Majid Khatib, Abderrahim Lomri, Kim Clarke, Shih-Che Sue, Vincent Castronovo, Martin Schilling, Raphael Pineau, Anne Couvelard, Harald Wodrich, Fabienne Rayne, Thomas Daubon, Renaud Grepin, Marie Sire, Laurent Dumartin, Florence Darlot, Fabienne Soulet, Clotilde Billottet, Alexandre Dubrac, Kevin Boyé, Jessica Baud, and Cathy Quemener
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CXCL4L1 and CXCL4 gene expression analysis
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- 2023
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13. High Level Expression and Purification of Cecropin-like Antimicrobial Peptides in
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Chih-Lung, Wu, Ya-Han, Chih, Hsin-Ying, Hsieh, Kuang-Li, Peng, Yi-Zong, Lee, Bak-Sau, Yip, Shih-Che, Sue, and Jya-Wei, Cheng
- Abstract
Cecropins are a family of antimicrobial peptides (AMPs) that are widely found in the innate immune system of Cecropia moths. Cecropins exhibit a broad spectrum of antimicrobial and anticancer activities. The structures of Cecropins are composed of 34-39 amino acids with an N-terminal amphipathic α-helix, an AGP hinge and a hydrophobic C-terminal α-helix. KR12AGPWR6 was designed based on the Cecropin-like structural feature. In addition to its antimicrobial activities, KR12AGPWR6 also possesses enhanced salt resistance, antiendotoxin and anticancer properties. Herein, we have developed a strategy to produce recombinant KR12AGPWR6 through a salt-sensitive, pH and temperature dependent intein self-cleavage system. The His6-Intein-KR12AGPWR6 was expressed by
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- 2022
14. Oligomerization State of CXCL4 Chemokines Regulates G Protein-Coupled Receptor Activation
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Yi-Chen Chen, Kevin Boyé, Hsin-Li Wu, Isabel D. Alves, Nadège Pujol, Shih-Che Sue, Chun-Wei Lin, Clotilde Billottet, Ya-Ping Chen, Liang-Yuan Chiu, Chen-Ya Pan, and Andreas Bikfalvi
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Models, Molecular ,0301 basic medicine ,Receptors, CXCR3 ,Protein Conformation ,Plasma protein binding ,Platelet Factor 4 ,Biochemistry ,Cell Line ,03 medical and health sciences ,Sulfation ,Protein structure ,Tetramer ,Humans ,Tyrosine ,Receptor ,G protein-coupled receptor ,030102 biochemistry & molecular biology ,Chemistry ,General Medicine ,Hydrogen-Ion Concentration ,Kinetics ,030104 developmental biology ,Helix ,Biophysics ,Molecular Medicine ,Protein Multimerization ,Protein Binding - Abstract
CXCL4 chemokines have antiangiogenic properties, mediated by different mechanisms, including CXCR3 receptor activation. Chemokines have distinct oligomerization states that are correlated with their biological functions. CXCL4 exists as a stable tetramer under physiological conditions. It is unclear whether the oligomerization state impacts CXCL4-receptor interaction. We found that the CXCL4 tetramer is sensitive to pH and salt concentration. Residues Glu28 and Lys50 were important for tetramer formation, and the first β-strand and the C-terminal helix are critical for dimerization. By mutating the critical residues responsible for oligomerization, we generated CXCL4 mutants that behave as dimers or monomers under neutral/physiological conditions. The CXCL4 monomer acts as the minimal active unit for interacting CXCR3A, and sulfation of N-terminal tyrosine residues on the receptor is important for binding. Noticeably, CXCL4L1, a CXCL4 variant that differs by three residues in the C-terminal helix, could activate CXCR3A. CXCL4L1 showed a higher tendency to dissociate into monomers, but native CXCL4 did not. This result indicates that monomeric CXCL4 behaves like CXCL4L1. Thus, in this chemokine family, being in the monomeric state seems critical for interaction with CXCR3A.
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- 2017
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15. Integrative model to coordinate the oligomerization and aggregation mechanisms of CCL5
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Kun-Mou Li, Siou-Pei Chen, Yi-Zong Lee, Raz Zarivach, Pei-Chun Chen, Jin-Ye Li, Yi-Chen Chen, Yuh-Ju Sun, and Shih-Che Sue
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Magnetic Resonance Spectroscopy ,Dimer ,Mutant ,Sequence (biology) ,Trimer ,Oligomer ,Protein Structure, Secondary ,CCL5 ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,stomatognathic system ,Structural Biology ,parasitic diseases ,Animals ,Humans ,Amino Acid Sequence ,Chemokine CCL5 ,Molecular Biology ,Glycosaminoglycans ,030304 developmental biology ,Inflammation ,0303 health sciences ,Mechanism (biology) ,Chemistry ,virus diseases ,hemic and immune systems ,Ligand (biochemistry) ,stomatognathic diseases ,Mutation ,Biophysics ,030217 neurology & neurosurgery ,Protein Binding - Abstract
CC-type chemokine ligand 5 (CCL5) is involved in the pathogenesis of many inflammatory conditions. Under physiological conditions, CCL5 oligomerization and aggregation are considered to be responsible for its inflammatory properties. The structural basis of CCL5 oligomerization remains controversial because the current oligomer models contain no consensus interactions. In this study, NMR and biophysical analyses proposed evidence that the CC-type CCL5 dimer acts as the basic unit to constitute the oligomer and that CCL5 oligomerizes alternatively through E66-K25 and E66-R44/K45 interactions. In addition, a newly determined trimer structure, constituted by CCL5 and the E66S mutant, reported an interfacial interaction through the N-terminal 12FAY14 sequence. The interaction contributes to CCL5 aggregation and precipitation but not to oligomerization. In accordance with the observations, an integrative model explains the CCL5 oligomerization and aggregation mechanism in which CCL5 assembly consists of two types of dimer-dimer interactions and one aggregation mechanism. For full-length CCL5, the molecular accumulation triggers oligomerization through the E66-K25 and E66-R44/K45 interactions, and the 12FAY14 interaction acts as a secondary effect to derive aggregation and precipitation. In contrast, the E66-R44/K45 interaction might dominate in CCL5 N-terminal truncations, and the interaction would lead to filament-like formation in solution.
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- 2019
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16. Salt-sensitive intein for large-scale polypeptide production
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Yi-Zong, Lee and Shih-Che, Sue
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Models, Molecular ,Bacteria ,Recombinant Fusion Proteins ,Osmolar Concentration ,Salts ,Peptides ,Inteins - Abstract
In this chapter, we propose to use salt-sensitive intein as a fusion protein to promote polypeptide expression; the removal of intein from the target sequence requires no enzyme, only a buffer change. The method will be particularly helpful for large-scale polypeptide preparations. Intein is an enzyme that can perform N- and C-terminal self-cleavage. Upon introduction of a mutation to eliminate the N-terminal cleavage activity, the C-terminal cleavage function can still be preserved. This feature was used to develop intein as a fusion protein through conjugation with a given sequence to promote protein expression in a biosynthesis system. Fused intein could later be separated from the target sequence through a C-terminal self-cleavage reaction. Here, a type of salt-sensitive intein is characterized in which ionic strength becomes an effector to control the self-cleavage activity. Low salt concentrations favor the cleavage reaction. Thus, using salt-sensitive intein as a fusion protein simply requires a buffer change to activate the self-cleavage mechanism, which makes it an enzyme-free process. This process has many advantages, including low cost, no extra residue remaining after cleavage, feasibility for preparing proteins starting from a non-Met codon and a special benefit for producing isotope-labeled peptides.
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- 2019
17. Heparin-Promoted Cellular Uptake of the Cell-Penetrating Glycosaminoglycan Binding Peptide, GBPECP, Depends on a Single Tryptophan
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Ping-Hsieh Kuo, Shih-Che Sue, Jia-Yin Lu, Chien-Jung Chen, Ingjye Jiang, Yi-Lin Hung, Li-Chun Hung, Margaret Dah-Tsyr Chang, Yi-Fen Hsieh, and Lily Hui-Ching Wang
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0301 basic medicine ,chemistry.chemical_classification ,Glycan ,Glycosaminoglycan binding ,030102 biochemistry & molecular biology ,biology ,Peptide ,General Medicine ,Heparin ,Plasma protein binding ,Biochemistry ,Micelle ,03 medical and health sciences ,Residue (chemistry) ,030104 developmental biology ,chemistry ,biology.protein ,medicine ,Molecular Medicine ,Binding site ,medicine.drug - Abstract
A 10-residue, glycosaminoglycan-binding peptide, GBPECP, derived from human eosinophil cationic protein has been recently designated as a potent cell-penetrating peptide. A model system containing peptide, glycan, and lipid was monitored by nuclear magnetic resonance (NMR) spectroscopy to determine the cell-penetrating mechanism. Heparin octasaccharide with dodecylphosphocholine (DPC) lipid micelle was titrated into the GBPECP solution. Our data revealed substantial roles for the charged residues Arg5 and Lys7 in recognizing heparin, whereas Arg3 had less effect. The aromatic residue Trp4 acted as an irreplaceable moiety for membrane insertion, as the replacement of Trp4 with Arg4 abolished cell penetration, although it significantly improved the heparin-binding ability. GBPECP bound either heparin or lipid in the presence or absence of the other ligand indicating that the peptide has two alternative binding sites: Trp4 is responsible for lipid insertion, and Arg5 and Lys7 are for GAG binding. We develope...
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- 2016
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18. Type IIb Heat Labile Enterotoxin B Subunit as a Mucosal Adjuvant to Enhance Protective Immunity against H5N1 Avian Influenza Viruses
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Chun-Yi Lu, Bor-Luen Chiang, Ming-Hsi Huang, Chung-Chu Chen, Li-Min Huang, Ting-Hsuan Chen, Suh-Chin Wu, Chung-Hsiung Huang, Jia-Tsrong Jan, Neos Tang, and Shih-Che Sue
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0301 basic medicine ,H5N1 vaccine ,medicine.drug_class ,medicine.medical_treatment ,Immunology ,lcsh:Medicine ,Heat-labile enterotoxin ,Monoclonal antibody ,medicine.disease_cause ,Article ,Virus ,03 medical and health sciences ,0302 clinical medicine ,Drug Discovery ,medicine ,Pharmacology (medical) ,Neutralizing antibody ,Pharmacology ,biology ,lcsh:R ,mucosal adjuvant ,intranasal immunization ,Virology ,Influenza A virus subtype H5N1 ,Vaccination ,030104 developmental biology ,Infectious Diseases ,LTIIb-B5 ,biology.protein ,Adjuvant ,030215 immunology - Abstract
Human infections with highly pathogenic avian influenza H5N1 viruses persist as a major global health concern. Vaccination remains the primary protective strategy against H5N1 and other novel avian influenza virus infections. We investigated the use of E. coli type IIb heat labile enterotoxin B subunit (LTIIb-B5) as a mucosal adjuvant for intranasal immunizations with recombinant HA proteins against H5N1 avian influenza viruses. Use of LTIIb-B5 adjuvant elicited more potent IgG, IgA, and neutralizing antibody titers in both sera and bronchoalveolar lavage fluids, thus increasing protection against lethal virus challenges. LTIIb-B5 mucosal adjuvanticity was found to trigger stronger Th17 cellular response in spleen lymphocytes and cervical lymph nodes. Studies of anti-IL-17A monoclonal antibody depletion and IL-17A knockout mice also suggest the contribution from Th17 cellular response to anti-H5N1 protective immunity. Our results indicate a link between improved protection against H5N1 live virus challenges and increased Th17 response due to the use of LTIIb-B5 mucosal adjuvant with HA subunit proteins.
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- 2020
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19. Salt-sensitive intein for large-scale polypeptide production
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Shih-Che Sue and Yi-Zong Lee
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chemistry.chemical_classification ,chemistry.chemical_compound ,Enzyme ,Biosynthesis ,Biochemistry ,Chemistry ,Effector ,Salt sensitivity ,Intein ,Cleavage (embryo) ,Fusion protein ,Protein expression - Abstract
In this chapter, we propose to use salt-sensitive intein as a fusion protein to promote polypeptide expression; the removal of intein from the target sequence requires no enzyme, only a buffer change. The method will be particularly helpful for large-scale polypeptide preparations. Intein is an enzyme that can perform N- and C-terminal self-cleavage. Upon introduction of a mutation to eliminate the N-terminal cleavage activity, the C-terminal cleavage function can still be preserved. This feature was used to develop intein as a fusion protein through conjugation with a given sequence to promote protein expression in a biosynthesis system. Fused intein could later be separated from the target sequence through a C-terminal self-cleavage reaction. Here, a type of salt-sensitive intein is characterized in which ionic strength becomes an effector to control the self-cleavage activity. Low salt concentrations favor the cleavage reaction. Thus, using salt-sensitive intein as a fusion protein simply requires a buffer change to activate the self-cleavage mechanism, which makes it an enzyme-free process. This process has many advantages, including low cost, no extra residue remaining after cleavage, feasibility for preparing proteins starting from a non-Met codon and a special benefit for producing isotope-labeled peptides.
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- 2019
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20. Understanding the Biomineralization Role of Magnetite-Interacting Components (MICs) From Magnetotactic Bacteria
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Hila Nudelman, Yi-Zong Lee, Yi-Lin Hung, Sofiya Kolusheva, Alexander Upcher, Yi-Chen Chen, Jih-Ying Chen, Shih-Che Sue, and Raz Zarivach
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0301 basic medicine ,Microbiology (medical) ,Magnetotactic bacteria ,MamC ,Magnetosome ,lcsh:QR1-502 ,Nucleation ,02 engineering and technology ,Microbiology ,lcsh:Microbiology ,magnetite-associated proteins ,03 medical and health sciences ,chemistry.chemical_compound ,Ion binding ,protein–mineral interactions ,Organelle ,Mms6 ,Mms7 ,Original Research ,Magnetite ,magnetotactic bacteria ,biomineralization ,021001 nanoscience & nanotechnology ,030104 developmental biology ,chemistry ,Biophysics ,Particle size ,0210 nano-technology ,Biomineralization - Abstract
Biomineralization is a process that takes place in all domains of life and which usually helps organisms to harden soft tissues by creating inorganic structures that facilitate their biological functions. It was shown that biominerals are under tight biological control via proteins that are involved in nucleation initiation and/or which act as structural skeletons. Magnetotactic bacteria (MTB) use iron biomineralization to create nano-magnetic particles in a specialized organelle, the magnetosome, to align to the geomagnetic field. A specific set of magnetite-associated proteins (MAPs) is involved in regulating magnetite nucleation, size, and shape. These MAPs are all predicted to contain specific 17 to 22 residue-long sequences involved in magnetite formation. To understand the mechanism of magnetite formation, we focused on three different MAPs, MamC, Mms6 and Mms7, and studied the predicted iron-binding sequences. Using nuclear magnetic resonance (NMR), we differentiated the recognition mode of each MAP based on ion specificity, affinity, and binding residues. The significance of critical residues in each peptide was evaluated by mutation followed by an iron co-precipitation assay. Among the peptides, MamC showed weak ion binding but created the most significant effect in enhancing magnetite particle size, indicating the potency in controlling magnetite particle shape and size. Alternatively, Mms6 and Mms7 had strong binding affinities but less effect in modulating magnetite particle size, representing their major role potentially in initiating nucleation by increasing local metal concentration. Overall, our results explain how different MAPs affect magnetite synthesis, interact with Fe2+ ions and which residues are important for the MAPs functions.
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- 2018
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21. The First Residue of the PWWP Motif Modulates HATH Domain Binding, Stability, and Protein–Protein Interaction
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Yi Lin Hung, Wei-Cheng Lo, Hsia Ju Lee, Ingjye Jiang, Shang Chi Lin, Yi Jan Lin, and Shih Che Sue
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Models, Molecular ,Helix bundle ,Circular dichroism ,Heparin ,Protein Conformation ,Protein Stability ,Chemistry ,Molecular Sequence Data ,DNA ,Nuclear Overhauser effect ,Plasma protein binding ,Biochemistry ,Protein–protein interaction ,Crystallography ,Protein structure ,Humans ,Intercellular Signaling Peptides and Proteins ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,Structural motif ,Sequence Alignment ,Peptide sequence ,Protein Binding - Abstract
Hepatoma-derived growth factor (hHDGF) and HDGF-related proteins (HRPs) contain conserved N-terminal HATH domains with a characteristic structural motif, namely the PWWP motif. The HATH domain has attracted attention because of its ability to bind with heparin/heparan sulfate, DNA, and methylated histone peptide. Depending on the sequence of the PWWP motif, HRP HATHs are classified into P-type (Pro-His-Trp-Pro) and A-type (Ala-His-Trp-Pro) forms. A-type HATH is highly unstable and tends to precipitate in solution. We replaced the Pro residue in P-type HATHHDGF with Ala and evaluated the influence on structure, dynamics, and ligand binding. Nuclear magnetic resonance (NMR) hydrogen/deuterium exchange and circular dichroism (CD) measurements revealed reduced stability. Analysis of NMR backbone (15)N relaxations (R1, R2, and nuclear Overhauser effect) revealed additional backbone dynamics in the interface between the β-barrel and the C-terminal helix bundle. The β1-β2 loop, where the AHWP sequence is located, has great structural flexibility, which aids HATH-HATH interaction through the loop. A-type HATH, therefore, shows a stronger tendency to aggregate when binding with heparin and DNA oligomers. This study defines the role of the first residue of the PWWP motif in modulating HATH domain stability and oligomer formation in binding.
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- 2015
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22. NMR characterization of the electrostatic interaction of the basic residues in HDGF and FGF2 during heparin binding
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Yun-Wei Chiang, Shih-Che Sue, Siu-Cin Tjong, Liang-Yuan Chiu, and Kuo-Wei Hung
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Chemistry ,Stereochemistry ,Chemical shift ,Mutant ,Biophysics ,Pulse sequence ,Hepatoma-derived growth factor ,Electrostatics ,Biochemistry ,Analytical Chemistry ,Residue (chemistry) ,Side chain ,Titration ,Molecular Biology - Abstract
Electrostatic interaction is a major driving force in the binding of proteins to highly acidic glycosaminoglycan, such as heparin. Although NMR backbone chemical shifts have generally been used to identify the heparin-binding site on a protein, however, there is no correlation between the binding free energies and the perturbed backbone chemical shifts for individual residues. The binding event occurs at the end of a side chain of basic residue, and does not require causing significant alterations in the backbone environment at a distance of multiple bonds. We used the H2CN NMR pulse sequence to detect heparin binding through the side-chain resonances He–Ce–Nζ of Lys and Hδ–Cδ–Ne of Arg in the two proteins of hepatoma-derived growth factor (HDGF) and basic fibroblast growth factor (FGF2). H2CN titration experiments revealed chemical shift perturbations in the side chains, which were correlated with the free energy changes in various mutants. The residues K19 in HDGF and K125 in FGF2 demonstrated the most significant perturbations, consistent with our previous observation that the two residues are crucial for binding. The result suggests that H2CN NMR provides a precise evaluation for the electrostatic interactions. The discrepancy observed between backbone and side chain chemical shifts is correlated to the solvent accessibility of residues that the K19 and K125 backbones are highly buried with the restricted backbone conformation and are not strongly affected by the events at the end of the side chains.
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- 2014
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23. Correlations between membrane immersion depth, orientation, and salt-resistance of tryptophan-rich antimicrobial peptides
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Jya-Wei Cheng, Hsi-Tsung Cheng, Hung-Lun Chu, Chih-Hsiang Tu, Ya-Han Chih, Bak-Sau Yip, Heng-Li Chen, Shih-Che Sue, and Hui-Yuan Yu
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Models, Molecular ,Magnetic Resonance Spectroscopy ,Protein Conformation ,Antimicrobial peptides ,Cell ,Biophysics ,Salt (chemistry) ,Peptide ,Microbial Sensitivity Tests ,Sodium Chloride ,Biochemistry ,medicine ,Immersion (virtual reality) ,Micelles ,chemistry.chemical_classification ,Chemistry ,Electron Spin Resonance Spectroscopy ,Tryptophan ,Salt-resistance ,Cell Biology ,Antimicrobial ,NMR ,Membrane ,medicine.anatomical_structure ,Tryptophan-rich ,Antimicrobial peptide ,PRE ,Antimicrobial Cationic Peptides - Abstract
The efficacies of many antimicrobial peptides are greatly reduced in the presence of high salt concentrations therefore limiting their development as pharmaceutical compounds. PEM-2-W5K/A9W, a short Trp-rich antimicrobial peptide developed based on the structural studies of PEM-2, has been shown to be highly active against various bacterial strains with less hemolytic activity. Here, correlations between membrane immersion depth, orientation, and salt-resistance of PEM-2 and PEM-2-W5K/A9W have been investigated via solution structure and paramagnetic resonance enhancement studies. The antimicrobial activities of PEM-2-W5K/A9W and PEM-2 against various bacterial and fungal strains including multidrug-resistant and clinical isolates under high salt conditions were tested. The activities of the salt-sensitive peptide PEM-2 were reduced and diminished at high salt concentrations, whereas the activities of PEM-2-W5K/A9W were less affected. The results indicated that the strong salt-resistance of PEM-2-W5K/A9W may arise from the peptide positioning itself deeply into microbial cell membranes and thus able to disrupt the membranes more efficiently.
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- 2013
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24. Heparin-Promoted Cellular Uptake of the Cell-Penetrating Glycosaminoglycan Binding Peptide, GBP
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Li-Chun, Hung, Ingjye, Jiang, Chien-Jung, Chen, Jia-Yin, Lu, Yi-Fen, Hsieh, Ping-Hsieh, Kuo, Yi-Lin, Hung, Lily Hui-Ching, Wang, Margaret Dah-Tsyr, Chang, and Shih-Che, Sue
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Microscopy, Fluorescence ,Heparin ,Cell Line, Tumor ,Proton Magnetic Resonance Spectroscopy ,Tryptophan ,Animals ,Humans ,Cell-Penetrating Peptides ,Carbon-13 Magnetic Resonance Spectroscopy ,Glycosaminoglycans ,Protein Binding - Abstract
A 10-residue, glycosaminoglycan-binding peptide, GBP
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- 2016
25. Computational Prediction of New Intein Split Sites
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Yi-Zong, Lee, Wei-Cheng, Lo, and Shih-Che, Sue
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Sequence Analysis, Protein ,Protein Splicing ,Protein Engineering ,Software ,Inteins - Abstract
Split inteins have emerged as a powerful tool in protein engineering. We describe a reliable in silico method to predict viable split sites for the design of new split inteins. A computational circular permutation (CP) prediction method facilitates the search for internal permissive sites to create artificial circular permutants. In this procedure, the original amino- and carboxyl-termini are connected and new termini are created. The identified new terminal sites are promising candidates for the generation of new split sites with the backbone opening being tolerated by the structural scaffold. Here we show how to integrate the online usage of the CP predictor, CPred, in the search of new split intein sites.
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- 2016
26. Computational Prediction of New Intein Split Sites
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Shih Che Sue, Yi Zong Lee, and Wei-Cheng Lo
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0301 basic medicine ,030103 biophysics ,03 medical and health sciences ,Scaffold ,030104 developmental biology ,Computer science ,Protein splicing ,In silico ,Computational biology ,Protein engineering ,Circular permutation in proteins ,Intein - Abstract
Split inteins have emerged as a powerful tool in protein engineering. We describe a reliable in silico method to predict viable split sites for the design of new split inteins. A computational circular permutation (CP) prediction method facilitates the search for internal permissive sites to create artificial circular permutants. In this procedure, the original amino- and carboxyl-termini are connected and new termini are created. The identified new terminal sites are promising candidates for the generation of new split sites with the backbone opening being tolerated by the structural scaffold. Here we show how to integrate the online usage of the CP predictor, CPred, in the search of new split intein sites.
- Published
- 2016
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27. Stress-induced phosphoprotein-1 maintains the stability of JAK2 in cancer cells
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Shih-Che Sue, Hsin-Shih Wang, Chi-Neu Tsai, Shun-Hua Chen, Chyong-Huey Lai, Chia-Lung Tsai, Shih-Ming Jung, Chiao-Yun Lin, Tzu-Hao Wang, and Angel Chao
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0301 basic medicine ,Oncology ,Gerontology ,STAT3 Transcription Factor ,medicine.medical_specialty ,Peptide fragment ,Cell Survival ,Mice, Nude ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Combined treatment ,Animal model ,Protein Domains ,Internal medicine ,hemic and lymphatic diseases ,Cell Line, Tumor ,medicine ,Genomic medicine ,Animals ,Humans ,cancer ,Tumor growth ,RNA, Small Interfering ,Heat-Shock Proteins ,Ovarian Neoplasms ,business.industry ,Gene Expression Profiling ,STIP1 ,Janus Kinase 2 ,Endometrial Neoplasms ,Gene Expression Regulation, Neoplastic ,030104 developmental biology ,HEK293 Cells ,JAK2 ,030220 oncology & carcinogenesis ,Cancer cell ,Female ,Core laboratory ,business ,Gene Deletion ,Neoplasm Transplantation ,Protein Binding ,Research Paper - Abstract
// Chia-Lung Tsai 1, * , Angel Chao 1, 2, * , Shih-Ming Jung 3 , Chi-Neu Tsai 4 , Chiao-Yun Lin 2 , Shun-Hua Chen 2, 5 , Shih-Che Sue 6 , Tzu-Hao Wang 1, 2, 7 , Hsin-Shih Wang 2 , Chyong-Huey Lai 2 1 Genomic Medicine Research Core Laboratory, Chang Gung Memorial Hospital, Taoyuan, Taiwan 2 Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital and Chang Gung University, Taoyuan, Taiwan 3 Department of Pathology, Chang Gung Memorial Hospital and Chang Gung University, Taoyuan, Taiwan 4 Graduate Institute of Clinical Medical Sciences, Chang Gung University, Taoyuan, Taiwan 5 Graduate Institute of Biomedical Science, School of Medicine, Chang Gung University, Taoyuan, Taiwan 6 Department of Life Sciences, Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Taiwan 7 School of Traditional Chinese Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan * These authors have contributed equally to this work Correspondence to: Tzu-Hao Wang, email: knoxtn@cgmh.org.tw Keywords: JAK2, STIP1, cancer Received: March 08, 2016 Accepted: June 17, 2016 Published: July 8, 2016 ABSTRACT Overexpression of stress-induced phosphoprotein 1 (STIP1) − a co-chaperone of heat shock protein (HSP) 70/HSP90 – and activation of the JAK2-STAT3 pathway occur in several tumors. Combined treatment with a HSP90 inhibitor and a JAK2 inhibitor exert synergistic anti-cancer effects. Here, we show that STIP1 stabilizes JAK2 protein in ovarian and endometrial cancer cells. Knock-down of endogenous STIP1 decreased JAK2 and phospho-STAT3 protein levels. The N-terminal fragment of STIP1 interacts with the N-terminus of JAK2, whereas the C-terminal DP2 domain of STIP1 mediates the interaction with HSP90 and STAT3. A peptide fragment in the DP2 domain of STIP1 (peptide 520) disrupted the interaction between STIP1 and HSP90 and induced cell death through JAK2 suppression. In an animal model, treatment with peptide 520 inhibited tumor growth. In summary, STIP1 modulates the function of the HSP90-JAK2-STAT3 complex. Peptide 520 may have therapeutic potential in the treatment of JAK2-overexpressing tumors.
- Published
- 2016
28. Detection of a ternary complex of NF-κB and IκBα with DNA provides insights into how IκBα removes NF-κB from transcription sites
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Elizabeth A. Komives, H. Jane Dyson, Vera Alverdi, and Shih-Che Sue
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Multidisciplinary ,NF-κB ,Biology ,Cell biology ,IκBα ,chemistry.chemical_compound ,PEST sequence ,Protein structure ,chemistry ,Biochemistry ,Ankyrin repeat ,Binding site ,Ternary complex ,DNA - Abstract
It has been axiomatic in the field of NF-κB signaling that the formation of a stable complex between NF-κB and the ankyrin repeat protein IκBα precludes the interaction of NF-κB with DNA. Contradicting this assumption, we present stopped-flow fluorescence and NMR experiments that give unequivocal evidence for the presence of a ternary DNA–NF-κB–IκBα complex in solution. Stepwise addition of a DNA fragment containing the κB binding sequence to the IκBα–NF-κB complex results in changes in the IκBα NMR spectrum that are consistent with dissociation of the region rich in proline, glutamate, serine, and threonine (PEST) and C-terminal ankyrin repeat sequences of IκBα from the complex. However, even at high concentrations of DNA, IκBα remains associated with NF-κB, indicated by the absence of resonances of the free N-terminal ankyrin repeats of IκBα. The IκBα-mediated release of NF-κB from its DNA-bound state may be envisioned as the reverse of this process. The initial step would consist of the coupled folding and binding of the intrinsically disordered nuclear localization sequence of the p65 subunit of NF-κB to the well-structured N-terminal ankyrin repeats of IκBα. Subsequently the poorly folded C-terminal ankyrin repeats of IκBα would fold upon binding to the p50 and p65 dimerization domains of NF-κB, permitting the negatively charged C-terminal PEST sequence of IκBα to displace the bound DNA through a process of local mass action.
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- 2011
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29. The RelA Nuclear Localization Signal Folds upon Binding to IκBα
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Magnus Kjaergaard, Gerard Kroon, H. Jane Dyson, Elizabeth A. Komives, Carla F. Cervantes, Simon Bergqvist, and Shih-Che Sue
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Models, Molecular ,Protein Folding ,Amino Acid Motifs ,Molecular Sequence Data ,Nuclear Localization Signals ,Transcription Factor RelA ,Article ,Protein Structure, Secondary ,NF-KappaB Inhibitor alpha ,Structural Biology ,Animals ,Humans ,NLS ,Amino Acid Sequence ,Molecular Biology ,Cell Nucleus ,RELA ,Chemistry ,Isothermal titration calorimetry ,IκBα ,Biochemistry ,Biophysics ,I-kappa B Proteins ,Protein folding ,Protein Multimerization ,Nuclear localization sequence ,Heteronuclear single quantum coherence spectroscopy ,Protein Binding - Abstract
The nuclear localization signal (NLS) polypeptide of RelA, the canonical nuclear factor-κB family member, is responsible for regulating the nuclear localization of RelA-containing nuclear factor-κB dimers. The RelA NLS polypeptide also plays a crucial role in mediating the high affinity and specificity of the interaction of RelA-containing dimers with the inhibitor IκBα, forming two helical motifs according to the published X-ray crystal structure. In order to define the nature of the interaction between the RelA NLS and IκBα under solution conditions, we conducted NMR and isothermal titration calorimetry studies using a truncated form of IκBα containing residues 67-206 and a peptide spanning residues 293-321 of RelA. The NLS peptide, although largely unfolded, has a weak tendency toward helical structure when free in solution. Upon addition of the labeled peptide to unlabeled IκBα, the resonance dispersion in the NMR spectrum is significantly greater, providing definitive evidence that the RelA NLS polypeptide folds upon binding IκBα. Isothermal titration calorimetry studies of single-point mutants reveal that residue F309, which is located in the middle of the more C-terminal of the two helices (helix 4) in the IκBα-bound RelA NLS polypeptide, is critical for the binding of the RelA NLS polypeptide to IκBα. These results help to explain the role of helix 4 in mediating the high affinity of RelA for IκBα.
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- 2011
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30. Cell surface heparan sulfates mediate internalization of the PWWP/HATH domain of HDGF via macropinocytosis to fine-tune cell signalling processes involved in fibroblast cell migration
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Chiaho Shih, Tai Huang Huang, Chia-Hui Wang, Wen-guey Wu, Fan Mei Tang, Po Long Wu, Shao Chen Lee, Shih Che Sue, and Fabian Davamani
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Cell signaling ,media_common.quotation_subject ,Biology ,Biochemistry ,3T3 cells ,Cell membrane ,Mice ,Cell Movement ,Cell surface receptor ,medicine ,Animals ,Internalization ,Molecular Biology ,Cell Proliferation ,media_common ,Cell growth ,Cell Membrane ,3T3 Cells ,Cell Biology ,Fibroblasts ,Matrix Metalloproteinases ,Protein Structure, Tertiary ,Cell biology ,medicine.anatomical_structure ,Intercellular Signaling Peptides and Proteins ,Pinocytosis ,Heparan sulfate binding ,Heparitin Sulfate ,Signal transduction ,Signal Transduction - Abstract
HDGF (hepatoma-derived growth factor) stimulates cell proliferation by functioning on both sides of the plasma membrane as a ligand for membrane receptor binding to trigger cell signalling and as a stimulator for DNA synthesis in the nucleus. Although HDGF was initially identified as a secretory heparin-binding protein, the biological significance of its heparin-binding ability remains to be determined. In the present study we demonstrate that cells devoid of surface HS (heparan sulfate) were unable to internalize HDGF, HATH (N-terminal domain of HDGF consisting of amino acid residues 1–100, including the PWWP motif) and HATH(K96A) (single-site mutant form of HATH devoid of receptor binding activity), suggesting that the binding of HATH to surface HS is important for HDGF internalization. We further demonstrate that both HATH and HATH(K96A) could be internalized through macropinocytosis after binding to the cell surface HS. Interestingly, HS-mediated HATH(K96A) internalization is found to exhibit an inhibitory effect on cell migration and proliferation in contrast with that observed for HATH action on NIH 3T3 cells, suggesting that HDGF exploits the innate properties of both cell surface HS and membrane receptor via the HATH domain to affect related cell signalling processes. The present study indicates that MAPK (mitogen-activated protein kinase) signalling pathways could be affected by the HS-mediated HATH internalization to regulate cell migration in NIH 3T3 fibroblasts, as judged from the differential effect of HATH and HATH(K96A) treatment on the expression level of matrix metalloproteases.
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- 2010
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31. Interaction of the IκBα C-terminal PEST Sequence with NF-κB: Insights into the Inhibition of NF-κB DNA Binding by IκBα
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H. Jane Dyson and Shih-Che Sue
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Protein subunit ,Mutagenesis ,NF-κB ,Inhibitor protein ,Biology ,Molecular biology ,Cell biology ,PEST sequence ,chemistry.chemical_compound ,IκBα ,chemistry ,Structural Biology ,Molecular Biology ,Transcription factor ,DNA - Abstract
The transcription factor NF-κB (p50/p65) binds either a κB DNA element or its inhibitor protein, IκBα, but these two binding events are mutually exclusive. The reason for this exclusivity is not obvious from the available crystal structure data. The C-terminal PEST-like sequence of IκBα appears to be involved in the process, but it is located in both of the published X-ray structures of the IκBα/NF-κB complex at a significant distance away from the DNA contact loop in the NF-κB DNA-binding domain. We have used nuclear magnetic resonance spectroscopy and differential isotopic labeling to probe the interactions between the p50/p65 NF-κB heterodimer and IκBα in solution. Our measurements are able to resolve a local structural discrepancy between the two crystal structures, and we confirm that the primary interaction of the IκBα PEST domain is with the DNA-binding domain of the p65 subunit. Mutagenesis of key arginine residues in the DNA contact sequence results in the loss of specific interaction of the PEST sequence with the p65 subdomain. We conclude that the local structure of the IκBα/NF-κB complex in the region of the PEST sequence is consistent with a direct interaction of this acidic sequence with the basic DNA contact sequence in p65, thus reducing the affinity of NF-κB for DNA by a competitive mechanism that is still to be elucidated fully.
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- 2009
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32. Transfer of Flexibility between Ankyrin Repeats in IκBα upon Formation of the NF-κB Complex
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Carla F. Cervantes, Elizabeth A. Komives, Shih-Che Sue, and H. Jane Dyson
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Models, Molecular ,Protein Folding ,Protein subunit ,Amino Acid Motifs ,Molecular Sequence Data ,Nuclear Localization Signals ,Protein domain ,Plasma protein binding ,Article ,Mass Spectrometry ,Protein Structure, Secondary ,NF-KappaB Inhibitor alpha ,Structural Biology ,Amino Acid Sequence ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Binding Sites ,Chemistry ,NF-kappa B ,Deuterium Exchange Measurement ,Ankyrin Repeat ,Protein Structure, Tertiary ,Molecular Weight ,Protein Subunits ,IκBα ,Biochemistry ,Biophysics ,I-kappa B Proteins ,Protein folding ,Ankyrin repeat ,Dimerization ,Heteronuclear single quantum coherence spectroscopy ,Protein Binding ,Binding domain - Abstract
The mechanism of inhibition of the transcriptional activator nuclear factor kappaB (NF-kappaB) by the inhibitor IkappaB* is central to the understanding of the control of transcriptional activity via this widely employed pathway. Previous studies suggested that IkappaB* , a modular protein with an NF-kappaB binding domain consisting of six ankyrin repeat domains (ANKs), shows differential flexibility, with ANK 1-4 apparently more rigid in solution in the absence of NF-kappaB than ANK 5 and 6. Here we report NMR studies that confirm the enhanced flexibility of ANK 5 and 6 in free IkappaB* . Upon binding of NF-kappaB, ANK 5 and 6 become well structured and rigid, but, somewhat surprisingly, other domains of the IkappaB* , which were relatively rigid in the free protein, become significantly more flexible. Due to the high molecular masses of the component proteins and the complexes, we employ a hierarchical experimental plan to maximize the available information on local flexibility in the ankyrin repeat domains. Backbone resonances of the 221-residue IkappaB* protein were assigned firstly in a smaller construct consisting of ankyrin repeats 1-4. These assignments could be readily transferred to the spectra of the construct containing six repeats, both free and complexed with various combinations of the NF-kappaB p50 and p65 domains. Transverse relaxation optimized spectroscopy-type NMR experiments on differentially labeled proteins enabled information on backbone structure and dynamics to be obtained, even in complexes with molecular masses approaching 100 kDa. Changes in the flexibility and stability of the various ankyrin repeat domains of IkappaB* complex formation take a variety of forms depending on the position of the domain in the complex, providing a variety of examples of the structural and functional utility of intrinsically unstructured or partly folded protein domains.
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- 2008
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33. Structure and function of chicken interleukin-1 beta mutants: uncoupling of receptor binding and in vivo biological activity
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Lee-Wei Yang, Emmanuel Oluwatobi Salawu, Ting Chen, Xinquan Wang, Wen-Ting Chen, Yuan-Yu Chang, Shih-Che Sue, Hsien-Sheng Yin, Dongli Wang, Yi-Zong Lee, and Wen-Yang Huang
- Subjects
0301 basic medicine ,Models, Molecular ,Mutant ,Interleukin-1beta ,Plasma protein binding ,Biology ,Molecular Dynamics Simulation ,medicine.disease_cause ,Crystallography, X-Ray ,01 natural sciences ,Protein Structure, Secondary ,Article ,Cell Line ,03 medical and health sciences ,Protein structure ,0103 physical sciences ,medicine ,Animals ,Binding site ,Receptor ,Mutation ,Multidisciplinary ,Binding Sites ,010304 chemical physics ,Receptors, Interleukin-1 ,Biological activity ,In vitro ,Cell biology ,030104 developmental biology ,Biochemistry ,Gene Expression Regulation ,Interleukin-1 Receptor Accessory Protein ,Chickens ,Protein Binding - Abstract
Receptor-binding and subsequent signal-activation of interleukin-1 beta (IL-1β) are essential to immune and proinflammatory responses. We mutated 12 residues to identify sites important for biological activity and/or receptor binding. Four of these mutants with mutations in loop 9 (T117A, E118K, E118A, E118R) displayed significantly reduced biological activity. Neither T117A nor E118K mutants substantially affected receptor binding, whereas both mutants lack the IL-1β signaling in vitro but can antagonize wild-type (WT) IL-1β. Crystal structures of T117A, E118A and E118K revealed that the secondary structure or surface charge of loop 9 is dramatically altered compared with that of wild-type chicken IL-1β. Molecular dynamics simulations of IL-1β bound to its receptor (IL-1RI) and receptor accessory protein (IL-1RAcP) revealed that loop 9 lies in a pocket that is formed at the IL-1RI/IL-1RAcP interface. This pocket is also observed in the human ternary structure. The conformations of above mutants in loop 9 may disrupt structural packing and therefore the stability in a chicken IL-1β/IL-1RI/IL-1RAcP signaling complex. We identify the hot spots in IL-1β that are essential to immune responses and elucidate a mechanism by which IL-1β activity can be inhibited. These findings should aid in the development of new therapeutics that neutralize IL-1 activity.
- Published
- 2016
34. Structure of the SARS Coronavirus Nucleocapsid Protein RNA-binding Dimerization Domain Suggests a Mechanism for Helical Packaging of Viral RNA
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Tai Huang Huang, Chung-ke Chang, Shih Che Sue, Hsin I. Bai, Chwan-Deng Hsiao, Chun-Yuan Chen, Lilianty Riang, and Yi-Wei Chang
- Subjects
Models, Molecular ,SeMet, selenomethionine ,viral packaging ,Viral protein ,viruses ,Molecular Sequence Data ,SARS-CoV, severe acute respiratory syndrome coronavirus ,IBV, infectious bronchitis virus ,nucleocapsid ,RNA-binding protein ,Biology ,medicine.disease_cause ,Article ,oligomerization ,Protein structure ,Structural Biology ,medicine ,Coronavirus Nucleocapsid Proteins ,Amino Acid Sequence ,Histone octamer ,Molecular Biology ,Coronavirus ,severe acute respiratory syndrome coronavirus ,Oligonucleotide ,15N-HSQC, 15N-edited heteronuclear single-quantum coherence ,RNA-Binding Proteins ,RNA ,Nucleocapsid Proteins ,RNA-binding domain ,Molecular biology ,Protein Structure, Tertiary ,MAD, multiwavelengh anomalous dispersion ,Severe acute respiratory syndrome-related coronavirus ,CTD, C-terminal domain containing residues 248–365 ,NP270-370, fragment of SARS-CoV nucleocapsid protein containing residues 270–370 ,Nucleic acid ,Biophysics ,Nucleic Acid Conformation ,RNA, Viral ,NTD, N-terminal domain containing residues 45–181 ,Dimerization - Abstract
Coronavirus nucleocapsid proteins are basic proteins that encapsulate viral genomic RNA to form part of the virus structure. The nucleocapsid protein of SARS-CoV is highly antigenic and associated with several host-cell interactions. Our previous studies using nuclear magnetic resonance revealed the domain organization of the SARS-CoV nucleocapsid protein. RNA has been shown to bind to the N-terminal domain (NTD), although recently the C-terminal half of the protein has also been implicated in RNA binding. Here, we report that the C-terminal domain (CTD), spanning residues 248–365 (NP248-365), had stronger nucleic acid-binding activity than the NTD. To determine the molecular basis of this activity, we have also solved the crystal structure of the NP248-365 region. Residues 248–280 form a positively charged groove similar to that found in the infectious bronchitis virus (IBV) nucleocapsid protein. Furthermore, the positively charged surface area is larger in the SARS-CoV construct than in the IBV. Interactions between residues 248–280 and the rest of the molecule also stabilize the formation of an octamer in the asymmetric unit. Packing of the octamers in the crystal forms two parallel, basic helical grooves, which may be oligonucleotide attachment sites, and suggests a mechanism for helical RNA packaging in the virus.
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- 2007
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35. Dual Roles for CXCL4 Chemokines and CXCR3 in Angiogenesis and Invasion of Pancreatic Cancer
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Marie Sire, Alexandre Dubrac, Abderrahim Lomri, Cathy Quemener, Kevin Boyé, Martin K. Schilling, Abdel-Majid Khatib, Shih-Che Sue, Anne Couvelard, Renaud Grépin, Jessica Baud, Thomas Daubon, Fabienne Rayne, Clotilde Billottet, Florence Darlot, Hervé Prats, Harald Wodrich, Raphael Pineau, Kim Clarke, Andreas Bikfalvi, Fabienne Soulet, Martin Hagedorn, Laurent Dumartin, Vincenzo Castronovo, Laboratoire Angiogenèse et Micro-environnement des Cancers (LAMC), Université Sciences et Technologies - Bordeaux 1-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherches en Cancérologie de Toulouse (CRCT), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM), Microbiologie cellulaire et moléculaire et pathogénicité (MCMP), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), AP-HP - Hôpital Bichat - Claude Bernard [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Homburg University, Université de Liège, National Tsing Hua University [Hsinchu] (NTHU), and University of Liverpool
- Subjects
0301 basic medicine ,Cancer Research ,Receptors, CXCR3 ,Angiogenesis ,[SDV]Life Sciences [q-bio] ,Angiogenesis Inhibitors ,Biology ,CXCR3 ,Platelet Factor 4 ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Pancreatic cancer ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Cell Proliferation ,Tumor microenvironment ,Neovascularization, Pathologic ,Cell growth ,Cancer ,medicine.disease ,Survival Analysis ,Xenograft Model Antitumor Assays ,3. Good health ,Pancreatic Neoplasms ,030104 developmental biology ,Oncology ,CpG site ,030220 oncology & carcinogenesis ,Immunology ,DNA methylation ,Cancer research ,Chemokines - Abstract
The CXCL4 paralog CXCL4L1 is a less studied chemokine that has been suggested to exert an antiangiogenic function. However, CXCL4L1 is also expressed in patient tumors, tumor cell lines, and murine xenografts, prompting a more detailed analysis of its role in cancer pathogenesis. We used genetic and antibody-based approaches to attenuate CXCL4L1 in models of pancreatic ductal adenocarcinoma (PDAC). Mechanisms of expression were assessed in cell coculture experiments, murine, and avian xenotransplants, including through an evaluation of CpG methylation and mutation of critical CpG residues. CXCL4L1 gene expression was increased greatly in primary and metastatic PDAC. We found that myofibroblasts triggered cues in the tumor microenvironment, which led to induction of CXCL4L1 in tumor cells. CXCL4L1 expression was also controlled by epigenetic modifications at critical CpG islands, which were mapped. CXCL4L1 inhibited angiogenesis but also affected tumor development more directly, depending on the tumor cell type. In vivo administration of an mAb against CXCL4L1 demonstrated a blockade in the growth of tumors positive for CXCR3, a critical receptor for CXCL4 ligands. Our findings define a protumorigenic role in PDAC development for endogenous CXCL4L1, which is independent of its antiangiogenic function. Cancer Res; 76(22); 6507–19. ©2016 AACR.
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- 2015
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36. Solution Structure of the Arabidopsis thaliana Telomeric Repeat-binding Protein DNA Binding Domain: A New Fold with an Additional C-terminal Helix
- Author
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Ben C. Chung, Tai Huang Huang, Shih Che Sue, Chia Hsing Ho, Ying Hsien Cheng, Hsin Hao Hsiao, Chung Mong Chen, and Kuang Lung Hsueh
- Subjects
Models, Molecular ,Protein Folding ,DNA, Plant ,HMG-box ,Stereochemistry ,Molecular Sequence Data ,Arabidopsis ,Sequence alignment ,Biology ,Protein Structure, Secondary ,chemistry.chemical_compound ,Protein structure ,Structural Biology ,Animals ,Humans ,Amino Acid Sequence ,Telomeric Repeat Binding Protein 1 ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,Peptide sequence ,Sequence Homology, Amino Acid ,DNA-binding domain ,Molecular biology ,Protein Structure, Tertiary ,DNA binding site ,chemistry ,Hydrophobic and Hydrophilic Interactions ,Sequence Alignment ,DNA ,Protein Binding ,Binding domain - Abstract
The double-stranded telomeric repeat-binding protein (TRP) AtTRP1 is isolated from Arabidopsis thaliana. Using gel retardation assays, we defined the C-terminal 97 amino acid residues, Gln464 to Val560 (AtTRP1(464-560)), as the minimal structured telomeric repeat-binding domain. This region contains a typical Myb DNA-binding motif and a C-terminal extension of 40 amino acid residues. The monomeric AtTRP1(464-560) binds to a 13-mer DNA duplex containing a single repeat of an A.thaliana telomeric DNA sequence (GGTTTAG) in a 1:1 complex, with a K(D) approximately 10(-6)-10(-7) M. Nuclear magnetic resonance (NMR) examination revealed that the solution structure of AtTRP1(464-560) is a novel four-helix tetrahedron rather than the three-helix bundle structure found in typical Myb motifs and other TRPs. Binding of the 13-mer DNA duplex to AtTRP1(464-560) induced significant chemical shift perturbations of protein amide resonances, which suggests that helix 3 (H3) and the flexible loop connecting H3 and H4 are essential for telomeric DNA sequence recognition. Furthermore, similar to that in hTRF1, the N-terminal arm likely contributes to or stabilizes DNA binding. Sequence comparisons suggested that the four-helix structure and the involvement of the loop residues in DNA binding may be features unique to plant TRPs.
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- 2006
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37. Modular organization of SARS coronavirus nucleocapsid protein
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Chung-ke Chang, Wen-Jin Wu, Tai Huang Huang, Yen Chieh Chiang, Tsan Hung Yu, Chun-Hung Lin, Cheng Kun Tsai, Wei Lun Chang, Shih Che Sue, Shin Jye Lee, Chiu Min Hsieh, and Hsin Hao Hsiao
- Subjects
viruses ,Endocrinology, Diabetes and Metabolism ,Molecular Sequence Data ,Clinical Biochemistry ,Protein domain ,coronavirus ,domain arrangement ,Sequence alignment ,Computational biology ,Biology ,medicine.disease_cause ,Article ,Protein Structure, Secondary ,oligomerization ,Protein structure ,Viral structural protein ,medicine ,Animals ,Coronavirus Nucleocapsid Proteins ,Humans ,Pharmacology (medical) ,Amino Acid Sequence ,Antigens, Viral ,Molecular Biology ,Peptide sequence ,Ribonucleoprotein ,Coronavirus ,SARS ,Sequence Homology, Amino Acid ,Biochemistry (medical) ,Cell Biology ,General Medicine ,Nucleocapsid Proteins ,intrinsically disordered protein ,Virology ,capsid protein ,NMR ,Peptide Fragments ,Protein Structure, Tertiary ,Sequence Alignment ,Binding domain - Abstract
The SARS-CoV nucleocapsid (N) protein is a major antigen in severe acute respiratory syndrome. It binds to the viral RNA genome and forms the ribonucleoprotein core. The SARS-CoV N protein has also been suggested to be involved in other important functions in the viral life cycle. Here we show that the N protein consists of two non-interacting structural domains, the N-terminal RNA-binding domain (RBD) (residues 45-181) and the C-terminal dimerization domain (residues 248-365) (DD), surrounded by flexible linkers. The C-terminal domain exists exclusively as a dimer in solution. The flexible linkers are intrinsically disordered and represent potential interaction sites with other protein and protein-RNA partners. Bioinformatics reveal that other coronavirus N proteins could share the same modular organization. This study provides information on the domain structure partition of SARS-CoV N protein and insights into the differing roles of structured and disordered regions in coronavirus nucleocapsid proteins.
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- 2005
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38. Challenges in NMR-based structural genomics
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Yao Te Huang, Chi-Fon Chang, Tai Huang Huang, Ching Yu Chou, and Shih Che Sue
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Statistics and Probability ,Nuclear magnetic resonance ,Protein structure ,Computer science ,A protein ,Computational biology ,Condensed Matter Physics ,Genome ,Gene ,Function (biology) ,Structural genomics - Abstract
Understanding the functions of the vast number of proteins encoded in many genomes that have been completely sequenced recently is the main challenge for biologists in the post-genomics era. Since the function of a protein is determined by its exact three-dimensional structure it is paramount to determine the 3D structures of all proteins. This need has driven structural biologists to undertake the structural genomics project aimed at determining the structures of all known proteins. Several centers for structural genomics studies have been established throughout the world. Nuclear magnetic resonance (NMR) spectroscopy has played a major role in determining protein structures in atomic details and in a physiologically relevant solution state. Since the number of new genes being discovered daily far exceeds the number of structures determined by both NMR and X-ray crystallography, a high-throughput method for speeding up the process of protein structure determination is essential for the success of the structural genomics effort. In this article we will describe NMR methods currently being employed for protein structure determination. We will also describe methods under development which may drastically increase the throughput, as well as point out areas where opportunities exist for biophysicists to make significant contribution in this important field.
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- 2005
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39. Solution Structure and Heparin Interaction of Human Hepatoma-derived Growth Factor
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Shih Che Sue, Tai Huang Huang, Shao Chen Lee, Wen-guey Wu, and Jeou-Yuan Chen
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Magnetic Resonance Spectroscopy ,Time Factors ,Protein family ,Molecular Sequence Data ,Cell Cycle Proteins ,Biology ,Mice ,Protein structure ,Structural Biology ,Animals ,Humans ,NLS ,Amino Acid Sequence ,Binding site ,Cytoskeleton ,Molecular Biology ,Peptide sequence ,Binding Sites ,Heparin ,Intracellular Signaling Peptides and Proteins ,Nuclear Proteins ,Nuclear magnetic resonance spectroscopy ,Hepatoma-derived growth factor ,Protein Structure, Tertiary ,Cell biology ,Cytoskeletal Proteins ,Biochemistry ,Intercellular Signaling Peptides and Proteins - Abstract
Hepatoma-derived growth factor (HDGF)-related proteins (HRPs) comprise a new protein family that has been implicated in nephrogenesis, tumorigenesis, vascular development, cell proliferation, and transcriptional activation. All HRPs share a conserved N-terminal homologous to the amino terminus of HDGF (HATH) domain, but vary significantly in the C-terminal region. Here, we show that in solution the N and C termini of human HDGF form two structurally independent domains. The 100 amino acid residue N-terminal HATH domain is well-structured while the 140 amino acid residue C-terminal domain is disordered. We determined the solution structure of the HATH domain by NMR. The core structure of the HATH domain is a five-stranded beta-barrel followed by two alpha-helices, similar to those of PWWP domains of known structures. Surface plasmon resonance results showed that the HATH domain is primarily responsible for heparin binding. On the basis of the chemical shift perturbation induced by binding of heparin-derived hexasaccharide, we identified a prominent, highly positively charged region as the putative heparin-binding site. Sequence comparison and structure prediction suggest that all HRPs are likely to adapt a similar modular structure.
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- 2004
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40. Heparin Binding Stabilizes the Membrane-bound Form of Cobra Cardiotoxin
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Kuan-Ming Chen, Wei-Ning Huang, Wen-guey Wu, Joseph K. Abraham, Kun-Yi Chien, and Shih-Che Sue
- Subjects
Models, Molecular ,Conformational change ,Magnetic Resonance Spectroscopy ,Protein Conformation ,Stereochemistry ,Phospholipid ,Cobra Cardiotoxin Proteins ,Plasma protein binding ,Nuclear Overhauser effect ,Biochemistry ,Protein Structure, Secondary ,chemistry.chemical_compound ,Protein structure ,Animals ,Elapidae ,Binding site ,Molecular Biology ,Micelles ,Phospholipids ,Binding Sites ,Dose-Response Relationship, Drug ,Heparin ,Cell Membrane ,Lysophosphatidylcholines ,Cell Biology ,Nuclear magnetic resonance spectroscopy ,Lipids ,Extracellular Matrix ,Kinetics ,Spectrometry, Fluorescence ,Membrane ,chemistry ,Protein Binding - Abstract
It has been shown previously that the long chain fragments of heparin bind to the beta-strand cationic belt of the three-finger cobra cardiotoxin (or cytotoxin, CTX) and hence enhance its penetration into phospholipid monolayer under physiological ionic conditions. By taking lysophosphatidylcholine (LPC) micelles as a membrane model, we have shown by (1)H NMR study that the binding of heparin-derived hexasaccharide (Hep-6) to CTX at the beta-strand region can induce conformational changes of CTX near its membrane binding loops and promote the binding activity of CTX toward LPC. The Fourier-transform infrared spectra and NMR nuclear Overhauser effect of Hep-6.CTX and CTX.LPC complex in aqueous buffer also supplemented the aforementioned observation. Thus, the detected conformational change may presumably be the result of structural coupling between the connecting loops and its beta-strands. This is the first documentation of results showing how the association of hydrophilic carbohydrate molecules with amphiphilic proteins can promote hydrophobic protein-lipid interaction via the stabilization of its membrane-bound form. A similar mechanism involving tripartite interactions of heparin, protein, and lipid molecules may be operative near the extracellular matrix of cell membranes.
- Published
- 2002
- Full Text
- View/download PDF
41. Case study of hydrogen bonding in a hydrophobic cavity
- Author
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Chao-Sheng Cheng, Siu-Cin Tjong, Hsien-Sheng Yin, Yi-Chen Chen, and Shih-Che Sue
- Subjects
Models, Molecular ,Circular dichroism ,Protein Conformation ,Low-barrier hydrogen bond ,Interleukin-1beta ,Molecular Dynamics Simulation ,Molecular dynamics ,Protein structure ,Materials Chemistry ,Peptide bond ,Animals ,Physical and Theoretical Chemistry ,Nuclear Magnetic Resonance, Biomolecular ,Hydrogen bond ,Chemistry ,Protein Stability ,Circular Dichroism ,Hydrogen Bonding ,Deuterium ,Surfaces, Coatings and Films ,Crystallography ,Mutation ,Thermodynamics ,Protein folding ,Chickens ,Hydrophobic and Hydrophilic Interactions - Abstract
Protein internal hydrogen bonds and hydrophobicity determine protein folding and structure stabilization, and the introduction of a hydrogen bond has been believed to represent a better interaction for consolidating protein structure. We observed an alternative example for chicken IL-1β. The native IL-1β contains a hydrogen bond between the Y157 side-chain OηH and I133 backbone CO, whereby the substitution from Tyr to Phe abolishes the connection and the mutant without the hydrogen bond is more stable. An attempt to explain the energetic view of the presence of the hydrogen bond fails when only considering the nearly identical X-ray structures. Here, we resolve the mechanism by monitoring the protein backbone dynamics and interior hydrogen bond network. IL-1β contains a hydrophobic cavity in the protein interior, and Y157 is one of the surrounding residues. The Y157 OηH group introduces an unfavorable energy in the hydrophobic cavity, therefore sequestering itself by forming a hydrogen bond with the proximate residue I133. The hydrogen bonding confines Y157 orientation but exerts a force to disrupt the hydrogen bond network surrounding the cavity. The effect propagates over the entire protein and reduces the stability, as reflected in the protein backbone dynamics observed by an NMR hydrogen-deuterium (H/D) exchange experiment. We describe the particular case in which a hydrogen bond does not necessarily confer enhanced protein stability while the disruption of hydrophobicity must be integrally considered.
- Published
- 2014
42. Dengue type four viruses with E-Glu345Lys adaptive mutation from MRC-5 cells induce low viremia but elicit potent neutralizing antibodies in rhesus monkeys
- Author
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Xiaofeng Li, Hsiao-Han Lin, Cheng-Feng Qin, Hsiang-Chi Lee, Tsai Meng-Ju, Suh-Chin Wu, Jia-Guan Peng, Shih-Che Sue, and Hung-Ju Hsiao
- Subjects
viruses ,DNA Mutational Analysis ,lcsh:Medicine ,Dengue virus ,medicine.disease_cause ,Antibodies, Viral ,Virus Replication ,Dengue ,Mice ,Viral Envelope Proteins ,Emerging Viral Diseases ,Chlorocebus aethiops ,lcsh:Science ,Vaccines ,Multidisciplinary ,Immunogenicity ,virus diseases ,Vaccination and Immunization ,Protein Binding ,Research Article ,Attenuated Vaccines ,Immunology ,Viremia ,Biology ,Microbiology ,Virus ,Cell Line ,Virology ,Vaccine Development ,medicine ,Animals ,Antibody-dependent enhancement ,Vero Cells ,Dengue vaccine ,Heparin ,lcsh:R ,Biology and Life Sciences ,Viral Vaccines ,Dengue Virus ,medicine.disease ,Antibodies, Neutralizing ,Macaca mulatta ,Disease Models, Animal ,Viral replication ,Amino Acid Substitution ,Mutation ,Vero cell ,lcsh:Q ,Immunization - Abstract
Knowledge of virulence and immunogenicity is important for development of live-attenuated dengue vaccines. We previously reported that an infectious clone-derived dengue type 4 virus (DENV-4) passaged in MRC-5 cells acquired a Glu345Lys (E-E345K) substitution in the E protein domain III (E-DIII). The same cloned DENV-4 was found to yield a single E-Glu327Gly (E-E327G) mutation after passage in FRhL cells and cause the loss of immunogenicity in rhesus monkeys. Here, we used site-directed mutagenesis to generate the E-E345K and E-E327G mutants from DENV-4 and DENV-4Δ30 infectious clones and propagated in Vero or MRC-5 cells. The E-E345K mutations were consistently presented in viruses recovered from MRC-5 cells, but not Vero cells. Recombinant E-DIII proteins of E345K and E327G increased heparin binding correlated with the reduced infectivity by heparin treatment in cell cultures. Different from the E-E327G mutant viruses to lose the immunogencity in rhesus monkeys, the E-E345K mutant viruses were able to induce neutralizing antibodies in rhesus monkeys with an almost a 10-fold lower level of viremia as compared to the wild type virus. Monkeys immunized with the E-E345K mutant virus were completely protected with no detectable viremia after live virus challenges with the wild type DENV-4. These results suggest that the E-E345K mutant virus propagated in MRC-5 cells may have potential for the use in live-attenuated DENV vaccine development.
- Published
- 2014
43. (13)C, (15)N and (1)H resonance assignments of receiver domain of ethylene receptor ETR1
- Author
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Yi-Lin Hung, Yi-Jan Lin, and Shih-Che Sue
- Subjects
Ethylene ,biology ,Stereochemistry ,Arabidopsis Proteins ,Chemical shift ,Arabidopsis ,Receptors, Cell Surface ,biology.organism_classification ,Biochemistry ,Two-component regulatory system ,Protein Structure, Secondary ,Protein Structure, Tertiary ,chemistry.chemical_compound ,Molecular recognition ,chemistry ,Structural Biology ,Cytoplasm ,Arabidopsis thaliana ,Receptor ,Protein secondary structure ,Nuclear Magnetic Resonance, Biomolecular - Abstract
Ethylene plays versatile functions in regulating plant physiology. Although the high affinity ethylene receptor and its downstream regulators have been identified, the molecular recognition of the receptor interacting domains remains to be established. It has been speculated that the cytoplasmic signaling of the ethylene receptor is a two-component regulatory system involving the conserved receiver domain (RD). Here, we report the NMR chemical shift assignments for RD from Arabidopsis thaliana ethylene receptor ETR1. Nearly complete backbone and side-chain assignments were achieved at pH 6.0 and 25 °C. The assignments and backbone dynamics revealed the secondary structure and showed that ETR1-RD is a monomer in solution. These results will make it possible to monitor downstream binding partners and elucidates our understanding of phosphotransfer in the plant two-component regulatory system in the ethylene signaling pathway.
- Published
- 2014
44. Action of Taiwan Cobra Cardiotoxin on Membranes: Binding Modes of a β-Sheet Polypeptide with Phosphatidylcholine Bilayers
- Author
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Ting-Shou Chen, Wen-guey Wu, P. K. Rajan, Chang-Huain Hsieh, and Shih-Che Sue
- Subjects
Magnetic Resonance Spectroscopy ,Calorimetry, Differential Scanning ,Chemistry ,Stereochemistry ,Lipid Bilayers ,Cobra Cardiotoxin Proteins ,Biochemistry ,Protein Structure, Secondary ,chemistry.chemical_compound ,Membrane ,Phosphatidylcholine ,Dipalmitoylphosphatidylcholine ,Phosphatidylcholines ,Peptides ,Cobra Cardiotoxin ,Protein Binding - Abstract
The interaction of Taiwan cobra cardiotoxin (CTX A3), a basic polypeptide consisting of three-fingered loops and five-strand beta-sheet structure, with zwitterionic dipalmitoylphosphatidylcholine (DPPC) has been studied by 31P and 2H NMR to understand the binding modes of CTX in membrane bilayers. The results, in conjunction with DPH fluorescence anisotropy and differential scanning calorimetry studies, show that CTX may penetrate and lyse the bilayers into small aggregates at a lipid/protein molar ratio of about 20 in the ripple Pbeta' phase. Elevating the temperature to that of the liquid crystalline Lalpha phase leads to the fusion of the small aggregates into larger ones as evidenced by the change of the isotropic signal into a magnetically aligned 31P signal with a marked reduction in the chemical shift anisotropy. 2H NMR study on deuterium-labeled DPPC in the head group and fatty acyl region as a function of temperature and CTX concentration reveals a molecular model that CTX undergoes a redistribution between penetrating and peripheral binding states depending on the temperature studied. In addition, both the conformational and dynamic states of the phosphocholine head group of DPPC bilayers are significantly perturbed in the presence of CTX. Structural consideration of the CTX molecule indicates that the penetration binding mode of CTX with the DPPC bilayer may involve a novel membrane-binding motif identified recently in the three-fingered loops of P-type CTX. CTX can only bind to DPPC membrane peripherally in the Lalpha phase due to the mismatch of their hydrophobic lengths.
- Published
- 1997
- Full Text
- View/download PDF
45. Membrane packing geometry of diphytanoylphosphatidylcholine is highly sensitive to hydration: phospholipid polymorphism induced by molecular rearrangement in the headgroup region
- Author
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Ping-Chiang Lyu, Chang-Huain Hsieh, Shih-Che Sue, and Wen-guey Wu
- Subjects
Magnetic Resonance Spectroscopy ,Membrane lipids ,Lipid Bilayers ,Phospholipid ,Molecular Conformation ,Biophysics ,Geometry ,chemistry.chemical_compound ,Membrane Lipids ,Polymorphism (biophysics) ,Desiccation ,Lipid bilayer ,Bilayer ,Membrane structure ,Hexagonal phase ,Membrane Proteins ,Phosphorus ,Nuclear magnetic resonance spectroscopy ,Models, Theoretical ,Deuterium ,Crystallography ,chemistry ,Phosphatidylcholines ,Thermodynamics ,lipids (amino acids, peptides, and proteins) ,Research Article - Abstract
Diphytanoylphosphatidylcholine (DPhPC) has often been used in the study of protein-lipid interaction and membrane channel activity, because of the general belief that it has high bilayer stability, low ion leakage, and fatty acyl packing comparable to that of phospholipid bilayers in the liquid-crystalline state. In this solid-state 31P and 2H NMR study, we find that the membrane packing geometry and headgroup orientation of DPhPC are highly sensitive to the temperature studied and its water content. The phosphocholine headgroup of DPhPC starts to change its orientation at a water content as high as approximately 16 water molecules per lipid, as evidenced by hydration-dependent 2H NMR study at room temperature. In addition, a temperature-induced structural transition in the headgroup orientation is detected in the temperature range of approximately 20–60 degrees C for lipids with approximately 8–11 water molecules per DPhPC. Dehydration of the lipid by one more water molecule leads to a nonlamellar, presumably cubic, phase formation. The lipid packing becomes a hexagonal phase at approximately 6 water molecules per lipid. A phase diagram of DPhPC in the temperature range of -40 degrees C to 80 degrees C is thus constructed on the basis of NMR results. The newly observed hydration-dependent DPhPC lipid polymorphism emphasizes the importance of molecular packing in the headgroup region in modulating membrane structure and protein-induced pore formation of the DPhPC bilayer.
- Published
- 1997
- Full Text
- View/download PDF
46. A streamlined method for preparing split intein for NMR study
- Author
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Yi-Zong Lee, Yen-Ju Chen, Yun-Tzai Lee, Yi-Jan Lin, and Shih-Che Sue
- Subjects
chemistry.chemical_classification ,DNA ligase ,dnaE ,Stereochemistry ,Circular permutation in proteins ,Biology ,Inteins ,Residue (chemistry) ,Biochemistry ,chemistry ,Bacterial Proteins ,Protein splicing ,RNA splicing ,Side chain ,Protein Splicing ,Amino Acid Sequence ,Intein ,Nostoc ,Nuclear Magnetic Resonance, Biomolecular ,Biotechnology - Abstract
A protein ligase, intein, mediates a protein-splicing reaction. It can be split into two complementary fragments and reconstituted as a whole intein scaffold to perform protein trans-splicing. To understand the association of intein fragments and the splicing mechanism, it is necessary to produce a large quantity of split intein for structural study. Conventionally, two fragments are prepared separately and assembled in solution, but severe aggregation of intein fragments occurs, and precise control of the relative concentration of each fragment is difficult. Here, we present a streamlined method to incorporate a circular permutation concept into the production of split intein. By circular permutation of the native split Nostoc punctiforme DnaE intein (NpuInt), a new backbone opening is relocated to the native split site at residue 102. As the protein splicing activity is preserved, the expressed NpuInt can immediately self-cleave into a two-piece split NpuInt. Because of a tight association between the two complementary fragments, split NpuInt can be purified in one step. The idea is simple and applicable to other split inteins. Employing the new preparation, we use NMR spectra to assign the backbone and side chain resonances for the native split NpuInt.
- Published
- 2013
47. Long-range effects and functional consequences of stabilizing mutations in the ankyrin repeat domain of IκBα
- Author
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Carla F. Cervantes, Lindsey D. Handley, H. Jane Dyson, Elizabeth A. Komives, and Shih-Che Sue
- Subjects
Models, Molecular ,Protein Folding ,Magnetic Resonance Spectroscopy ,Mutant ,Protein domain ,Biology ,Article ,PEST sequence ,NF-KappaB Inhibitor alpha ,Structural Biology ,Humans ,Molecular Biology ,Genetics ,Wild type ,Magnetic Resonance Imaging ,Cell biology ,Ankyrin Repeat ,Protein Structure, Tertiary ,IκBα ,Mutation ,Ankyrin repeat ,Protein folding ,I-kappa B Proteins ,Heteronuclear single quantum coherence spectroscopy ,Half-Life ,Protein Binding - Abstract
Protein domains containing three or more ankyrin repeats (ARs) are ubiquitous in all phyla. Sequence alignments previously identified certain conserved positions, which have been shown to stabilize AR domains and promote their folding. Consensus mutations [Y254L/T257A (YLTA) and C186P/A220P (CPAP)] stabilize the naturally occuring AR domain of human IκBα to denaturation; however, only the YLTA mutations stabilize the protein to proteasomal degradation. We present results from NMR experiments designed to probe the roles of these consensus mutations in IκBα. According to residual dipolar coupling analysis, the gross structures of the AR domains of both mutants appear to be similar to the wild type (WT). Comparison of chemical shifts of mutant and WT proteins reveals that the YLTA and CPAP consensus mutations cause unexpected long-range effects throughout the AR domains. Backbone dynamics experiments reveal that the YLTA mutations in the sixth AR order the C-terminal PEST sequence on the picosecond-to-nanosecond timescale, compared to either the WT or the CPAP mutant IκBαs. This property is likely the mechanism by which the half-life of YLTA IκBα is extended in vivo.
- Published
- 2012
48. Inhibition of enterovirus 71 infections and viral IRES activity by Fructus gardeniae and geniposide
- Author
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Shih Che Sue, Wei Yi Hsu, Chien Hui Hung, Xiang Liu, Cheng Wen Lin, Shao Mei Huang, Ting Hsu Lin, Ni Tien, Yi Lin Hung, Chao Ling Chen, Ying Ju Lin, Fuu Jen Tsai, Chien-Chen Lai, and Chih Ho Lai
- Subjects
Viral protein ,Molecular Sequence Data ,Microbial Sensitivity Tests ,medicine.disease_cause ,Virus Replication ,Antiviral Agents ,Virus ,Chinese traditional ,Cell Line, Tumor ,Drug Discovery ,Enterovirus 71 ,medicine ,Enterovirus Infections ,Humans ,Iridoids ,Medicine, Chinese Traditional ,Pharmacology ,biology ,Organic Chemistry ,RNA ,Translation (biology) ,General Medicine ,biology.organism_classification ,Gardenia ,Virology ,Enterovirus A, Human ,Internal ribosome entry site ,Cell culture ,Protein Biosynthesis ,RNA, Viral ,Ribosomes ,Drugs, Chinese Herbal - Abstract
Fructus gardeniae has long been used by traditional Chinese medical practitioners for its anti-inflammatory, anti-oxidant, anti-tumor and anti-hyperlipidemic characteristics. Here we describe our finding that F. gardeniae greatly reduces anti-enterovirus 71 (EV71) activity, resulting in significant decreases in EV71 virus yields, EV71 infections, and internal ribosome entry site activity. We also found that geniposide, a primary F. gardeniae component, inhibited both EV71 replication and viral IRES activity. Our data suggest the presence of a mechanism that blocks viral protein translation. According to our findings, F. gardeniae and geniposide deserve a closer look as potential chemopreventive agents against EV71 infections.
- Published
- 2012
49. Significance of heparin binding to basic residues in homologous to the amino terminus of hepatoma-derived growth factor and related proteins
- Author
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Shang-Chi Lin, Yung-Jen Chuang, Fui-Fang Chen, Hsueh-Yao Chu, Wei-Chang Huang, Wei-Hsien Lin, Je-Hung Kuo, Shao-Chen Lee, and Shih-Che Sue
- Subjects
Conformational change ,Magnetic Resonance Spectroscopy ,media_common.quotation_subject ,medicine.medical_treatment ,Molecular Sequence Data ,Biochemistry ,chemistry.chemical_compound ,Mice ,Molecular recognition ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Internalization ,media_common ,Sequence Homology, Amino Acid ,Heparin ,Growth factor ,Heparan sulfate ,Hepatoma-derived growth factor ,Surface Plasmon Resonance ,Molecular biology ,Affinities ,N-terminus ,chemistry ,Intercellular Signaling Peptides and Proteins ,Protein Binding - Abstract
Hepatoma-derived growth factor (HDGF) recognizes cell surface heparan sulfate to promote its internalization though binding to its N-terminal HATH (homologous to amino terminus of HDGF) domain. HDGF-related proteins (HRPs) all have the HATH domain in their N terminus. In this study, we report on the commonality of heparin binding in all HRPs with a broad range of heparin-binding affinity: HRP-4 is the strongest binder, and the lens epithelium-derived growth factor shows a relatively weak binding, with binding affinities (K(D)) showing 30-fold difference in magnitude. With the HDGF HATH domain used as a model, residue K19 was the most critical basic residue in molecular recognition and protein internalization, and with its proximal proline-tryptophan-tryptophan-proline motif, coordinated a conformational change when binding to the heparin fragment. Other basic residues, K21, K61, K70, K72 and R79, confer added contribution in binding that the total ionic interaction from these residues represents more than 70% of the binding energy. Because the positive-charged residues are conserved in all HRP HATH domains, heparin binding outside of cells might be of equal importance for all HRPs in mediating downstream signaling; however, distinct effects and/or distribution might be associated with the varying affinities to heparin.
- Published
- 2012
50. Detection of a ternary complex of NF-kappaB and IkappaBalpha with DNA provides insights into how IkappaBalpha removes NF-kappaB from transcription sites
- Author
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Shih-Che, Sue, Vera, Alverdi, Elizabeth A, Komives, and H Jane, Dyson
- Subjects
Models, Molecular ,Binding Sites ,Magnetic Resonance Spectroscopy ,Base Sequence ,Transcription, Genetic ,NF-kappa B ,Transcription Factor RelA ,NF-kappa B p50 Subunit ,DNA ,Biological Sciences ,Binding, Competitive ,Ankyrin Repeat ,Protein Structure, Tertiary ,NF-KappaB Inhibitor alpha ,Multiprotein Complexes ,Animals ,Humans ,I-kappa B Proteins ,Amino Acid Sequence ,Protein Multimerization ,Protein Structure, Quaternary ,Protein Binding - Abstract
It has been axiomatic in the field of NF-κB signaling that the formation of a stable complex between NF-κB and the ankyrin repeat protein IκBα precludes the interaction of NF-κB with DNA. Contradicting this assumption, we present stopped-flow fluorescence and NMR experiments that give unequivocal evidence for the presence of a ternary DNA–NF-κB–IκBα complex in solution. Stepwise addition of a DNA fragment containing the κB binding sequence to the IκBα–NF-κB complex results in changes in the IκBα NMR spectrum that are consistent with dissociation of the region rich in proline, glutamate, serine, and threonine (PEST) and C-terminal ankyrin repeat sequences of IκBα from the complex. However, even at high concentrations of DNA, IκBα remains associated with NF-κB, indicated by the absence of resonances of the free N-terminal ankyrin repeats of IκBα. The IκBα-mediated release of NF-κB from its DNA-bound state may be envisioned as the reverse of this process. The initial step would consist of the coupled folding and binding of the intrinsically disordered nuclear localization sequence of the p65 subunit of NF-κB to the well-structured N-terminal ankyrin repeats of IκBα. Subsequently the poorly folded C-terminal ankyrin repeats of IκBα would fold upon binding to the p50 and p65 dimerization domains of NF-κB, permitting the negatively charged C-terminal PEST sequence of IκBα to displace the bound DNA through a process of local mass action.
- Published
- 2011
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