المؤلفون: Xiaoshan Min, Junming Yie, Jinghong Wang, Ben C. Chung, Ching-Shin Huang, Haoda Xu, Jie Yang, Liying Deng, Joanne Lin, Qing Chen, Christina M. Abbott, Caroline Gundel, Stephen A. Thibault, Tina Meng, Darren L. Bates, David J. Lloyd, Murielle M. Véniant, Zhulun Wang

المصدر: mAbs, Vol 12, Iss 1 (2020)

مصطلحات موضوعية: GIPR, antagonistic antibody, crystallography, structure, Therapeutics. Pharmacology, RM1-950, Immunologic diseases. Allergy, RC581-607

الوصف: Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone involved in regulating glucose and lipid metabolism. GIP receptor (GIPR) antagonism is believed to offer therapeutic potential for various metabolic diseases. Pharmacological intervention of GIPR, however, has limited success due to lack of effective antagonistic reagents. Previously we reported the discovery of two mouse anti-murine GIPR monoclonal antibodies (mAbs) with distinctive properties in rodent models. Here, we report the detailed structural and biochemical characterization of these two antibodies, mAb1 and mAb2. In vitro and in vivo characterizations demonstrated mAb2 is a full GIPR antagonistic antibody and mAb1 is a non-neutralizing GIPR binder. To understand the molecular basis of these two antibodies, we determined the co-crystal structures of GIPR extracellular domain in complex with mAb1 and with mAb2 at resolutions of 2.1 and 2.6 Å, respectively. While the non-neutralizing mAb1 binds to GIPR without competing with the ligand peptide, mAb2 not only partially occludes the ligand peptide binding, but also recognizes the GIPR C-terminal stalk region in a helical conformation that acts as a molecular mimic of the ligand peptide and locks GIPR in a novel auto-inhibited state. Furthermore, administration of mAb2 in diet-induced obesity mice for 7 weeks leads to both reduction in body weight gain and improvement of metabolic profiles. In contrast, mAb1 has no effect on body weight or other metabolic improvement. Together, our studies reveal the unique molecular mechanism of action underlying the superior antagonistic activity of mAb2 and signify the promising therapeutic potential of effective GIPR antagonism for the treatment of metabolic disorders.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/1942-0862Test; https://doaj.org/toc/1942-0870Test

الوصول الحر: https://doaj.org/article/968f0a3d32354e02bad2c843bdc326daTest

المؤلفون: Yongheng Chen, Darren L. Bates, Raja Dey, Po-Han Chen, Ana Carolina Dantas Machado, Ite A. Laird-Offringa, Remo Rohs, Lin Chen

المصدر: Cell Reports, Vol 2, Iss 5, Pp 1197-1206 (2012)

مصطلحات موضوعية: Biology (General), QH301-705.5

الوصف: GATA transcription factors regulate transcription during development and differentiation by recognizing distinct GATA sites with a tandem of two conserved zinc fingers, and by mediating long-range DNA looping. However, the molecular basis of these processes is not well understood. Here, we determined three crystal structures of the full DNA-binding domain (DBD) of human GATA3 protein, which contains both zinc fingers, in complex with different DNA sites. In one structure, both zinc fingers wrap around a palindromic GATA site, cooperatively enhancing the binding affinity and kinetic stability. Strikingly, in the other two structures, the two fingers of GATA DBD bind GATA sites on different DNA molecules, thereby bridging two separate DNA fragments. This was confirmed in solution by an in-gel fluorescence resonance energy transfer analysis. These findings not only provide insights into the structure and function of GATA proteins but also shed light on the molecular basis of long-range gene regulation.

وصف الملف: electronic resource

العلاقة: http://www.sciencedirect.com/science/article/pii/S2211124712003634Test; https://doaj.org/toc/2211-1247Test

الوصول الحر: https://doaj.org/article/736900e0c8384678923d76d0433d55ebTest

المؤلفون: Kip P. Conner, Min-Zu Wu, Jiamiao Lu, Aazam Ghelani, Yi-Ling Hu, Darren L. Bates, Ashutosh Chaudhry, Sue J. Sohn, Chi-Ming Li

المصدر: Frontiers in Immunology
Frontiers in Immunology, Vol 11 (2020)

مصطلحات موضوعية: lcsh:Immunologic diseases. Allergy, 0301 basic medicine, Immunology, Lymphocyte Activation, Protein Engineering, T-Lymphocytes, Regulatory, law.invention, 03 medical and health sciences, Mice, Structure-Activity Relationship, 0302 clinical medicine, law, In vivo, Immunology and Allergy, Animals, Humans, IL-2 receptor, Receptor, STAT5, Original Research, tolerance, biology, Chemistry, IL-2, autoimmunity, In vitro, Recombinant Proteins, regulatory T, Cell biology, Treg, 030104 developmental biology, inflammation, Humanized mouse, biology.protein, Suppressor, Phosphorylation, Interleukin-2, mutein, lcsh:RC581-607, 030215 immunology, Protein Binding

الوصف: Among all T and NK cell subsets, regulatory T (Treg) cells typically respond to the lowest concentrations of IL-2 due to elevated surface expression of the IL-2R alpha chain (IL2RA; CD25) and the high affinity IL-2 receptor (IL-2R) complex. This enhanced sensitivity forms the basis for low-dose (LD) IL-2 therapy for the treatment of inflammatory diseases, where efficacy correlates with increased Treg cell number and expression of functional markers. Despite strong preclinical support for this approach, moderate and variable clinical efficacy has raised concerns that adequate Treg selectivity still cannot be achieved with LD IL-2, and/or that doses are too low to stimulate effective Treg-mediated suppression within tissues. This has prompted development of IL-2 variants with greater Treg selectivity, achieved through attenuated affinity for the signaling chains of the IL-2R complex (IL2RB or CD122 and IL2RG or CD132) and, consequently, greater reliance on high CD25 levels for full receptor binding and signaling. While certain IL-2 variants have advanced to the clinic, it remains unknown if the full range of IL-2R signaling potency and Treg-selectivity observed with low concentrations of wildtype IL-2 can be sufficiently recapitulated with attenuated IL-2 muteins at high concentrations. Using a panel of engineered IL-2 muteins, we investigated how a range of IL-2R signaling intensity, benchmarked by the degree of STAT5 phosphorylation, relates to biologically relevant Treg cell responses such as proliferation, lineage and phenotypic marker expression, and suppressor function. Our results demonstrate that a surprisingly wide dynamic range of IL-2R signaling intensity leads to productive biological responses in Treg cells, with negligible STAT5 phosphorylation associating with nearly complete downstream effects such as Treg proliferation and suppressor activity. Furthermore, we show with both in vitro and humanized mouse in vivo systems that different biological responses in Treg cells require different minimal IL-2R signaling thresholds. Our findings suggest that more than minimal IL-2R signaling, beyond that capable of driving Treg cell proliferation, may be required to fully enhance Treg cell stability and suppressor function in vivo.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::2c5300a7e9e52e6055f1cfa65656b9afTest
https://pubmed.ncbi.nlm.nih.gov/32582190Test

المؤلفون: Ching-Shin Huang, Caroline Gundel, Ben C. Chung, Joanne Lin, Qing Chen, Liying Deng, Darren L. Bates, Zhulun Wang, Jinghong Wang, Jie Yang, Murielle M. Véniant, David Lloyd, Xiaoshan Min, Stephen A. Thibault, Haoda Xu, Christina Abbott, Tina Meng, Junming Yie

المصدر: mAbs

مصطلحات موضوعية: Male, endocrine system, medicine.medical_specialty, Protein Conformation, Immunology, Diet, High-Fat, Weight Gain, Receptors, Gastrointestinal Hormone, Mice, Immune system, Report, Internal medicine, medicine, Animals, Immunology and Allergy, structure, Obesity, crystallography, Receptor, GIPR, biology, Chemistry, Antibodies, Monoclonal, Lipid metabolism, Mice, Inbred C57BL, Endocrinology, Incretin Hormone, Molecular mechanism, biology.protein, antagonistic antibody, Glucose-dependent insulinotropic polypeptide, Antibody, Antagonism, hormones, hormone substitutes, and hormone antagonists

الوصف: Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone involved in regulating glucose and lipid metabolism. GIP receptor (GIPR) antagonism is believed to offer therapeutic potential for various metabolic diseases. Pharmacological intervention of GIPR, however, has limited success due to lack of effective antagonistic reagents. Previously we reported the discovery of two mouse anti-murine GIPR monoclonal antibodies (mAbs) with distinctive properties in rodent models. Here, we report the detailed structural and biochemical characterization of these two antibodies, mAb1 and mAb2. In vitro and in vivo characterizations demonstrated mAb2 is a full GIPR antagonistic antibody and mAb1 is a non-neutralizing GIPR binder. To understand the molecular basis of these two antibodies, we determined the co-crystal structures of GIPR extracellular domain in complex with mAb1 and with mAb2 at resolutions of 2.1 and 2.6 Å, respectively. While the non-neutralizing mAb1 binds to GIPR without competing with the ligand peptide, mAb2 not only partially occludes the ligand peptide binding, but also recognizes the GIPR C-terminal stalk region in a helical conformation that acts as a molecular mimic of the ligand peptide and locks GIPR in a novel auto-inhibited state. Furthermore, administration of mAb2 in diet-induced obesity mice for 7 weeks leads to both reduction in body weight gain and improvement of metabolic profiles. In contrast, mAb1 has no effect on body weight or other metabolic improvement. Together, our studies reveal the unique molecular mechanism of action underlying the superior antagonistic activity of mAb2 and signify the promising therapeutic potential of effective GIPR antagonism for the treatment of metabolic disorders.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::c5ec036e73660230e011396dc0673377Test
https://doi.org/10.1080/19420862.2019.1710047Test

المؤلفون: Sebastiaan H. Meijsing, Miles A. Pufall, Alex Y. So, Darren L. Bates, Lin Chen, Keith R. Yamamoto

المساهمون: The Pennsylvania State University CiteSeerX Archives

المصدر: http://www.ucsf.edu/krylab/PDF/Meijsing%20et%20al%20Science%202009.pdfTest.

الوصف: The following resources related to this article are available online at

وصف الملف: application/pdf

العلاقة: http://citeseerx.ist.psu.edu/viewdoc/summary?doi=10.1.1.158.1965Test; http://www.ucsf.edu/krylab/PDF/Meijsing%20et%20al%20Science%202009.pdfTest

الإتاحة: http://www.ucsf.edu/krylab/PDF/Meijsing%20et%20al%20Science%202009.pdfTest

المؤلفون: Yongqing Wu, Madhuri Borde, Vigo Heissmeyer, Markus Feuerer, Ariya D Lapan, James C Stroud, Darren L Bates, Liang Guo, Aidong Han, Steven F Ziegler, Diane Mathis, Christophe Benoist, Lin Chen, Anjana Rao

المساهمون: The Pennsylvania State University CiteSeerX Archives

المصدر: http://scheid.blog.yorku.ca/files/2013/02/FoxP3-and-NFAT.pdfTest.

الوصف: SUMMARY Antigen stimulation of immune cells activates the transcription factor NFAT, a key regulator of T cell activation and anergy. NFAT forms cooperative complexes with the AP-1 family of transcription factors and regulates T cell activation-associated genes. Here we show that regulatory T cell (Treg) function is mediated by an analogous cooperative complex of NFAT with the forkhead transcription factor FOXP3, a lineage specification factor for Tregs. The crystal structure of an NFAT:FOXP2:DNA complex reveals an extensive protein-protein interaction interface between NFAT and FOXP2. Structure-guided mutations of FOXP3, predicted to progressively disrupt its interaction with NFAT, interfere in a graded manner with the ability of FOXP3 to repress expression of the cytokine IL2, upregulate expression of the Treg markers CTLA4 and CD25, and confer suppressor function in a murine model of autoimmune diabetes. Thus by switching transcriptional partners, NFAT converts the acute T cell activation program into the suppressor program of Tregs.

وصف الملف: application/pdf

العلاقة: http://citeseerx.ist.psu.edu/viewdoc/summary?doi=10.1.1.1082.7265Test; http://scheid.blog.yorku.ca/files/2013/02/FoxP3-and-NFAT.pdfTest

الإتاحة: http://scheid.blog.yorku.ca/files/2013/02/FoxP3-and-NFAT.pdfTest

المؤلفون: Brandon C. P. Clavette, Xiaoshan Min, Joanne Lin, Elizabeth A. Killion, Robert J.M. Kurzeja, David Lloyd, Liying Deng, Zhulun Wang, Clarence Hale, Christopher Murawsky, Stone D.-H. Shi, Murielle M. Véniant, Jinghong Wang, James B. Rottman, Christina Abbott, Todd Hager, Lu Shu Chen, Junming Yie, Ian Foltz, Qing Chen, Stephen A. Thibault, Darren L. Bates, Renee Komorowski, Larissa Atangan, Glenn Sivits, Tina Meng

المصدر: Science Translational Medicine. 10

مصطلحات موضوعية: Primates, 0301 basic medicine, endocrine system, medicine.medical_specialty, Recombinant Fusion Proteins, Glucagon-Like Peptides, Mice, Obese, Genome-wide association study, Gastric Inhibitory Polypeptide, Weight Gain, Antibodies, Glucagon-Like Peptide-1 Receptor, Receptors, Gastrointestinal Hormone, 03 medical and health sciences, 0302 clinical medicine, Weight loss, Insulin-Secreting Cells, Internal medicine, Weight Loss, Conditional gene knockout, Adipocytes, Animals, Humans, Medicine, Obesity, Receptor, Respiratory exchange ratio, biology, business.industry, Respiration, Feeding Behavior, General Medicine, Liraglutide, medicine.disease, Diet, Immunoglobulin Fc Fragments, 030104 developmental biology, Endocrinology, 030220 oncology & carcinogenesis, Knockout mouse, biology.protein, Drug Therapy, Combination, medicine.symptom, Antibody, business, hormones, hormone substitutes, and hormone antagonists

الوصف: Glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) has been identified in multiple genome-wide association studies (GWAS) as a contributor to obesity, and GIPR knockout mice are protected against diet-induced obesity (DIO). On the basis of this genetic evidence, we developed anti-GIPR antagonistic antibodies as a potential therapeutic strategy for the treatment of obesity and observed that a mouse anti-murine GIPR antibody (muGIPR-Ab) protected against body weight gain, improved multiple metabolic parameters, and was associated with reduced food intake and resting respiratory exchange ratio (RER) in DIO mice. We replicated these results in obese nonhuman primates (NHPs) using an anti-human GIPR antibody (hGIPR-Ab) and found that weight loss was more pronounced than in mice. In addition, we observed enhanced weight loss in DIO mice and NHPs when anti-GIPR antibodies were codosed with glucagon-like peptide-1 receptor (GLP-1R) agonists. Mechanistic and crystallographic studies demonstrated that hGIPR-Ab displaced GIP and bound to GIPR using the same conserved hydrophobic residues as GIP. Further, using a conditional knockout mouse model, we excluded the role of GIPR in pancreatic β-cells in the regulation of body weight and response to GIPR antagonism. In conclusion, these data provide preclinical validation of a therapeutic approach to treat obesity with anti-GIPR antibodies.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::4219a5cd6927493203264b2541e2f88bTest
https://doi.org/10.1126/scitranslmed.aat3392Test

المؤلفون: Ite A. Laird-Offringa, Po Han Chen, Lin Chen, Ana Carolina Dantas Machado, Yongheng Chen, Remo Rohs, Darren L. Bates, Raja Dey

المصدر: Cell Reports, Vol 2, Iss 5, Pp 1197-1206 (2012)

مصطلحات موضوعية: Molecular Sequence Data, Biology, Crystallography, X-Ray, GATA Transcription Factors, Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, chemistry.chemical_compound, Transcription (biology), Fluorescence Resonance Energy Transfer, Humans, Amino Acid Sequence, lcsh:QH301-705.5, 030304 developmental biology, Zinc finger, 0303 health sciences, Binding Sites, Base Sequence, 030302 biochemistry & molecular biology, GATA2, GATA3, Zinc Fingers, DNA, DNA-binding domain, Molecular biology, Protein Structure, Tertiary, Cell biology, DNA binding site, Gene Expression Regulation, lcsh:Biology (General), chemistry, GATA transcription factor, Sequence Alignment, Protein Binding

الوصف: SummaryGATA transcription factors regulate transcription during development and differentiation by recognizing distinct GATA sites with a tandem of two conserved zinc fingers, and by mediating long-range DNA looping. However, the molecular basis of these processes is not well understood. Here, we determined three crystal structures of the full DNA-binding domain (DBD) of human GATA3 protein, which contains both zinc fingers, in complex with different DNA sites. In one structure, both zinc fingers wrap around a palindromic GATA site, cooperatively enhancing the binding affinity and kinetic stability. Strikingly, in the other two structures, the two fingers of GATA DBD bind GATA sites on different DNA molecules, thereby bridging two separate DNA fragments. This was confirmed in solution by an in-gel fluorescence resonance energy transfer analysis. These findings not only provide insights into the structure and function of GATA proteins but also shed light on the molecular basis of long-range gene regulation.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::f279d5e267bf9c3088e357f4944b9b35Test
https://doi.org/10.1016/j.celrep.2012.10.012Test

المؤلفون: Lin Chen, James C. Stroud, Aidong Han, Amy Oltman, Darren L. Bates

المصدر: Journal of Molecular Biology. 393:98-112

مصطلحات موضوعية: Models, Molecular, viruses, Transcription Factor RelA, Plasma protein binding, Biology, Crystallography, X-Ray, Article, Conserved sequence, chemistry.chemical_compound, Structural Biology, Transcription (biology), Humans, Protein Structure, Quaternary, Molecular Biology, HIV Long Terminal Repeat, NF-kappa B p50 Subunit, Molecular biology, Long terminal repeat, Cell biology, chemistry, DNA, Viral, Host-Pathogen Interactions, HIV-1, Virus Activation, Protein Multimerization, DNA, Protein Binding

الوصف: The activation and latency of human immunodeficiency virus type 1 (HIV-1) are tightly controlled by the transcriptional activity of its long terminal repeat (LTR) region. The LTR is regulated by viral proteins as well as host factors, including the nuclear factor kappaB (NF-kappaB) that becomes activated in virus-infected cells. The two tandem NF-kappaB sites of the LTR are among the most highly conserved sequence elements of the HIV-1 genome. Puzzlingly, these sites are arranged in a manner that seems to preclude simultaneous binding of both sites by NF-kappaB, although previous biochemical work suggests otherwise. Here, we have determined the crystal structure of p50:RelA bound to the tandem kappaB element of the HIV-1 LTR as a dimeric dimer, providing direct structural evidence that NF-kappaB can occupy both sites simultaneously. The two p50:RelA dimers bind the adjacent kappaB sites and interact through a protein contact that is accommodated by DNA bending. The two dimers clamp DNA from opposite faces of the double helix and form a topological trap of the bound DNA. Consistent with these structural features, our biochemical analyses indicate that p50:RelA binds the HIV-1 LTR tandem kappaB sites with an apparent anti-cooperativity but enhanced kinetic stability. The slow on and off rates we observe may be relevant to viral latency because viral activation requires sustained NF-kappaB activation. Furthermore, our work demonstrates that the specific arrangement of the two kappaB sites on the HIV-1 LTR can modulate the assembly kinetics of the higher-order NF-kappaB complex on the viral promoter. This phenomenon is unlikely restricted to the HIV-1 LTR but probably represents a general mechanism for the function of composite DNA elements in transcription.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::5fdab608386c8f926624f26291d4ea76Test
https://doi.org/10.1016/j.jmb.2009.08.023Test

المؤلفون: Lin Chen, Yongheng Chen, Darren L. Bates, Grace Kim, Liang Guo

المصدر: Journal of Molecular Biology. 381:1292-1306

مصطلحات موضوعية: Models, Molecular, Molecular Sequence Data, GATA zinc finger, GATA3 Transcription Factor, Biology, Crystallography, X-Ray, Protein Structure, Secondary, Article, Mice, Structure-Activity Relationship, Structural Biology, Transcription (biology), Animals, Amino Acid Sequence, Binding site, Molecular Biology, Transcription factor, Genetics, Zinc finger, Binding Sites, GATA2, GATA3, Zinc Fingers, DNA, Protein Structure, Tertiary, embryonic structures, GATA transcription factor, Dimerization, Protein Binding

الوصف: The GATA family of transcription factors (GATA1-6) binds selected GATA sites in vertebrate genomes to regulate specific gene expression. Although vertebrate GATA factors have two highly conserved zinc finger motifs, how the two fingers act together to recognize functional DNA elements is not well understood. Here we determined the crystal structures of the C-terminal zinc finger of mouse GATA3 bound to DNA containing two variously arranged GATA binding sites. Our structures and accompanying biochemical analyses reveal two distinct modes of DNA binding by GATA to closely arranged sites. One mode involves cooperative binding by two GATA factors that interact with each other through protein-protein interactions. The other involves simultaneous binding of the N-terminal zinc finger (N-finger) and the C-terminal zinc finger of the same GATA factor. Our studies represent the first crystallographic analysis of GATA zinc fingers bound to DNA and provide new insights into the DNA recognition mechanism by the GATA zinc finger. Our crystal structure also reveals a dimerization interface in GATA that has previously been shown to be important for GATA self-association. These findings significantly advance our understanding of the structure and function of GATA and provide an important framework for further investigating the in vivo mechanisms of GATA-dependent gene regulation.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::aa2430bf4d819b8222fca96a84441081Test
https://doi.org/10.1016/j.jmb.2008.06.072Test