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Kevin H. Gardner - One of the best experts on this subject based on the ideXlab platform.
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Structural basis for PAS Domain heterodimerization in the basic helix-loop-helix-PAS transcription factor hypoxia-inducible factor
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Paul J. A. Erbel, Paul B. Card, Ozgur Karakuzu, Richard K. Bruick, Kevin H. GardnerAbstract:Biological responses to oxygen availability play important roles in development, physiological homeostasis, and many disease processes. In mammalian cells, this adaptation is mediated in part by a conserved pathway centered on the hypoxia-inducible factor (HIF). HIF is a heterodimeric protein complex composed of two members of the basic helix-loop-helix Per-ARNT-Sim (PAS) (ARNT, aryl hydrocarbon receptor nuclear translocator) Domain family of transcriptional activators, HIFalpha and ARNT. Although this complex involves protein-protein interactions mediated by basic helix-loop-helix and PAS Domains in both proteins, the role played by the PAS Domains is poorly understood. To address this issue, we have studied the structure and interactions of the C-terminal PAS Domain of human HIF-2alpha by NMR spectroscopy. We demonstrate that HIF-2alpha PAS-B binds the analogous ARNT Domain in vitro, showing that residues involved in this interaction are located on the solvent-exposed side of the HIF-2alpha central beta-sheet. Mutating residues at this surface not only disrupts the interaction between isolated PAS Domains in vitro but also interferes with the ability of full-length HIF to respond to hypoxia in living cells. Extending our findings to other PAS Domains, we find that this beta-sheet interface is widely used for both intra- and intermolecular interactions, suggesting a basis of specificity and regulation of many types of PAS-containing signaling proteins.
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Structural basis for PAS Domain heterodimerization in the basic helix-loop-helix-PAS transcription factor hypoxia-inducible factor
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Paul J. A. Erbel, Paul B. Card, Ozgur Karakuzu, Richard K. Bruick, Kevin H. GardnerAbstract:Biological responses to oxygen availability play important roles in development, physiological homeostasis, and many disease processes. In mammalian cells, this adaptation is mediated in part by a conserved pathway centered on the hypoxia-inducible factor (HIF). HIF is a heterodimeric protein complex composed of two members of the basic helix–loop–helix Per-ARNT-Sim (PAS) (ARNT, aryl hydrocarbon receptor nuclear translocator) Domain family of transcriptional activators, HIFα and ARNT. Although this complex involves protein–protein interactions mediated by basic helix–loop–helix and PAS Domains in both proteins, the role played by the PAS Domains is poorly understood. To address this issue, we have studied the structure and interactions of the C-terminal PAS Domain of human HIF-2α by NMR spectroscopy. We demonstrate that HIF-2α PAS-B binds the analogous ARNT Domain in vitro, showing that residues involved in this interaction are located on the solvent-exposed side of the HIF-2α central β-sheet. Mutating residues at this surface not only disrupts the interaction between isolated PAS Domains in vitro but also interferes with the ability of full-length HIF to respond to hypoxia in living cells. Extending our findings to other PAS Domains, we find that this β-sheet interface is widely used for both intra- and intermolecular interactions, suggesting a basis of specificity and regulation of many types of PAS-containing signaling proteins.
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PAS Domain mediated wc 1 wc 2 interaction is essential for maintaining the steady state level of wc 1 and the function of both proteins in circadian clock and light responses of neurospora
Molecular and Cellular Biology, 2002Co-Authors: Ping Cheng, Kevin H. Gardner, Yuhong Yang, Yi LiuAbstract:In the frq-wc-based circadian feedback loops of Neurospora, two PAS Domain-containing transcription factors, WHITE COLLAR-1 (WC-1) and WC-2, form heterodimeric complexes that activate the transcription of frequency (frq). FRQ serves two roles in these feedback loops: repressing its own transcription by interacting with the WC complex and positively upregulating the levels of WC-1 and WC-2 proteins. We report here that the steady-state level of WC-1 protein is independently regulated by both FRQ and WC-2 through different posttranscriptional mechanisms. The WC-1 level is extremely low in wc-2 knockout strains, and this low level of expression is independent of wc-1 transcription and FRQ protein expression. In addition, our data show that the PAS Domain of WC-2 mediates the interactions of this protein with both WC-1 and FRQ in vivo. Such interactions are essential for maintaining the steady-state level of WC-1 and the proper function of WC-1 and WC-2 in circadian clock and light responses.
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Structure and interactions of PAS kinase N-terminal PAS Domain: model for intramolecular kinase regulation.
Structure (London England : 1993), 2002Co-Authors: Carlos A. Amezcua, Shannon M. Harper, Jared Rutter, Kevin H. GardnerAbstract:PAS Domains are sensory modules in signal-transducing proteins that control responses to various environmental stimuli. To examine how those Domains can regulate a eukaryotic kinase, we have studied the structure and binding interactions of the N-terminal PAS Domain of human PAS kinase using solution NMR methods. While this Domain adopts a characteristic PAS fold, two regions are unusually flexible in solution. One of these serves as a portal that allows small organic compounds to enter into the core of the Domain, while the other binds and inhibits the kinase Domain within the same protein. Structural and functional analyses of point mutants demonstrate that the compound and ligand binding regions are linked, suggesting that the PAS Domain serves as a ligand-regulated switch for this eukaryotic signaling system.
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PAS kinase an evolutionarily conserved PAS Domain regulated serine threonine kinase
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Jared Rutter, Kevin H. Gardner, C. H. Michnoff, Shannon M. Harper, Steven L McknightAbstract:PAS Domains regulate the function of many intracellular signaling pathways in response to both extrinsic and intrinsic stimuli. PAS Domain-regulated histidine kinases are common in prokaryotes and control a wide range of fundamental physiological processes. Similarly regulated kinases are rare in eukaryotes and are to date completely absent in mammals. PAS kinase (PASK) is an evolutionarily conserved gene product present in yeast, flies, and mammals. The amino acid sequence of PASK specifies two PAS Domains followed by a canonical serine/threonine kinase Domain, indicating that it might represent the first mammalian PAS-regulated protein kinase. We present evidence that the activity of PASK is regulated by two mechanisms. Autophosphorylation at two threonine residues located within the activation loop significantly increases catalytic activity. We further demonstrate that the N-terminal PAS Domain is a cis regulator of PASK catalytic activity. When the PAS Domain-containing region is removed, enzyme activity is significantly increased, and supplementation of the purified PAS-A Domain in trans selectively inhibits PASK catalytic activity. These studies define a eukaryotic signaling pathway suitable for studies of PAS Domains in a purified in vitro setting.
Steven L Mcknight - One of the best experts on this subject based on the ideXlab platform.
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the neuronal PAS Domain protein 3 transcription factor controls fgf mediated adult hippocampal neurogenesis in mice
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Andrew A Pieper, James A. Richardson, Tina W Han, Sandi Jo Estill, Quyen Dang, Sarah Reecefincanon, Carol A Dudley, Daniel J Brat, Steven L McknightAbstract:The neuronal PAS Domain protein 3 (NPAS3) gene encoding a brain-enriched transcription factor was recently found to be disrupted in a family suffering from schizophrenia. Mice harboring compound disruptions in the NPAS3 and related NPAS1 genes manifest behavioral and neuroanatomical abnormalities reminiscent of schizophrenia. Herein we demonstrate that NPAS3-/- mice are deficient in expression of hippocampal FGF receptor subtype 1 mRNA, most notably in the dentate gyrus. In vivo BrdUrd-labeling shows that basal neural precursor cell proliferation in the dentate gyrus of NPAS3-/- mice is reduced by 84% relative to wild-type littermates. We propose that a deficiency in adult neurogenesis may cause the behavioral and neuroanatomical abnormalities seen in NPAS3-/- mice, and we speculate that impaired neurogenesis may be involved in the pathophysiology of schizophrenia.
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PAS kinase an evolutionarily conserved PAS Domain regulated serine threonine kinase
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Jared Rutter, Kevin H. Gardner, C. H. Michnoff, Shannon M. Harper, Steven L McknightAbstract:PAS Domains regulate the function of many intracellular signaling pathways in response to both extrinsic and intrinsic stimuli. PAS Domain-regulated histidine kinases are common in prokaryotes and control a wide range of fundamental physiological processes. Similarly regulated kinases are rare in eukaryotes and are to date completely absent in mammals. PAS kinase (PASK) is an evolutionarily conserved gene product present in yeast, flies, and mammals. The amino acid sequence of PASK specifies two PAS Domains followed by a canonical serine/threonine kinase Domain, indicating that it might represent the first mammalian PAS-regulated protein kinase. We present evidence that the activity of PASK is regulated by two mechanisms. Autophosphorylation at two threonine residues located within the activation loop significantly increases catalytic activity. We further demonstrate that the N-terminal PAS Domain is a cis regulator of PASK catalytic activity. When the PAS Domain-containing region is removed, enzyme activity is significantly increased, and supplementation of the purified PAS-A Domain in trans selectively inhibits PASK catalytic activity. These studies define a eukaryotic signaling pathway suitable for studies of PAS Domains in a purified in vitro setting.
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PAS kinase: An evolutionarily conserved PAS Domain-regulated serine/threonine kinase
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Jared Rutter, Kevin H. Gardner, C. H. Michnoff, Shannon M. Harper, Steven L McknightAbstract:PAS Domains regulate the function of many intracellular signaling pathways in response to both extrinsic and intrinsic stimuli. PAS Domain-regulated histidine kinases are common in prokaryotes and control a wide range of fundamental physiological processes. Similarly regulated kinases are rare in eukaryotes and are to date completely absent in mammals. PAS kinase (PASK) is an evolutionarily conserved gene product present in yeast, flies, and mammals. The amino acid sequence of PASK specifies two PAS Domains followed by a canonical serine/threonine kinase Domain, indicating that it might represent the first mammalian PAS-regulated protein kinase. We present evidence that the activity of PASK is regulated by two mechanisms. Autophosphorylation at two threonine residues located within the activation loop significantly increases catalytic activity. We further demonstrate that the N-terminal PAS Domain is a cis regulator of PASK catalytic activity. When the PAS Domain-containing region is removed, enzyme activity is significantly increased, and supplementation of the purified PAS-A Domain in trans selectively inhibits PASK catalytic activity. These studies define a eukaryotic signaling pathway suitable for studies of PAS Domains in a purified in vitro setting.
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Molecular characterization of two mammalian bHLH-PAS Domain proteins selectively expressed in the central nervous system.
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Yu Dong Zhou, Mary Barnard, Hui Tian, Huijun Z. Ring, Uta Francke, John M. Shelton, James A. Richardson, David W. Russell, Steven L McknightAbstract:Here we describe two mammalian transcription factors selectively expressed in the central nervous system. Both proteins, neuronal PAS Domain protein (NPAS) 1 and NPAS2, are members of the basic helix–loop–helix-PAS family of transcription factors. cDNAs encoding mouse and human forms of NPAS1 and NPAS2 have been isolated and sequenced. RNA blotting assays demonstrated the selective presence of NPAS1 and NPAS2 mRNAs in brain and spinal cord tissues of adult mice. NPAS1 mRNA was first detected at embryonic day 15 of mouse development, shortly after early organogenesis of the brain. NPAS2 mRNA was first detected during early postnatal development of the mouse brain. In situ hybridization assays using brain tissue of postnatal mice revealed an exclusively neuronal pattern of expression for NPAS1 and NPAS2 mRNAs. The human NPAS1 gene was mapped to chromosome 19q13.2–q13.3, and the mouse NPAS1 gene to chromosome 7 at 2 centimorgans. Similarly, the human NPAS2 gene was assigned to chromosome 2p11.2–2q13, and the mouse NPAS2 gene to chromosome 1 at 21–22 centimorgans. The chromosomal regions to which human NPAS1 and NPAS2 map are syntenic with those containing the mouse NPAS1 and NPAS2 genes, indicating that the mouse and human genes are true homologs.
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Endothelial PAS Domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells.
Genes & development, 1997Co-Authors: Hui Tian, Steven L Mcknight, David W. RussellAbstract:Here we describe the cloning and characterization of a PAS Domain transcription factor termed endothelial PAS-1 (EPAS1). This protein shares 48% sequence identity with hypoxia inducible factor (HIF-1alpha) and lesser similarity with other members of the basic helix-loop-helix/PAS Domain family of transcription factors. Like HIF-1alpha, EPAS1 binds to and activates transcription from a DNA element originally isolated from the erythropoietin gene and containing the sequence 5'-GCCCTACGTGCTGTCTCA-3'. Activation by both HIF-1alpha and EPAS1 is stimulated by hypoxic conditions. EPAS1 forms a heterodimeric complex with the aryl hydrocarbon nuclear transporter prior to transcriptional activation of target genes. EPAS1 expression is limited to the endothelium of mouse embryos and, in agreement with its cell type-specific expression pattern, is capable of specifically activating the transcription of the endothelial tyrosine kinase gene Tie-2. These observations raise the possibility that EPAS1 may represent an important regulator of vascularization, perhaps involving the regulation of endothelial cell gene expression in response to hypoxia.
Barry L. Taylor - One of the best experts on this subject based on the ideXlab platform.
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Gain-of-function mutations cluster in distinct regions associated with the signalling pathway in the PAS Domain of the aerotaxis receptor, Aer.
Molecular microbiology, 2010Co-Authors: Asharie J. Campbell, Mark S. Johnson, Kylie J. Watts, Barry L. TaylorAbstract:Summary The Aer receptor monitors internal energy (redox) levels in Escherichia coli with an FAD-containing PAS Domain. Here, we randomly mutagenized the region encoding residues 14–119 of the PAS Domain and found 72 aerotaxis-defective mutants, 24 of which were gain-of-function, signal-on mutants. The mutations were mapped onto an Aer homology model based on the structure of the PAS–FAD Domain in NifL from Azotobacter vinlandii. Signal-on lesions clustered in the FAD binding pocket, the β-scaffolding and in the N-cap loop. We suggest that the signal-on lesions mimic the ‘signal-on’ state of the PAS Domain, and therefore may be markers for the signal-in and signal-out regions of this Domain. We propose that the reduction of FAD rearranges the FAD binding pocket in a way that repositions the β-scaffolding and the N-cap loop. The resulting conformational changes are likely to be conveyed directly to the HAMP Domain, and on to the kinase control module. In support of this hypothesis, we demonstrated disulphide band formation between cysteines substituted at residues N98C or I114C in the PAS β-scaffold and residue Q248C in the HAMP AS-2 helix.
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Function of the N-Terminal Cap of the PAS Domain in Signaling by the Aerotaxis Receptor Aer
Journal of bacteriology, 2006Co-Authors: Kylie J. Watts, Mark S. Johnson, Kirsten Sommer, Sheena L. Fry, Barry L. TaylorAbstract:Aer, the Escherichia coli receptor for behavioral responses to oxygen (aerotaxis), energy, and redox potential, contains a PAS sensory-input Domain. Within the PAS superfamily, the N-terminal segment (N-cap) is poorly conserved and its role is not well understood. We investigated the role of the N-cap (residues 1 to 19) in the Aer PAS Domain by missense and truncation mutagenesis. Aer-PAS N-cap truncations and an Aer-M21P substitution resulted in low cellular levels of the mutant proteins, suggesting that the N-terminal region was important for stabilizing the structure of the PAS Domain. The junction of the N-cap and PAS core was critical for signaling in Aer. Mutations and truncations in the sequence encoding residues 15 to 21 introduced a range of phenotypes, including defects in FAD binding, constant tumbling motility, and an inverse response in which E. coli cells migrated away from oxygen concentrations to which they are normally attracted. The proximity of two N-cap regions in an Aer dimer was assessed in vivo by oxidatively cross-linking serial cysteine substitutions. Cross-linking of several cysteine replacements at 23°C was attenuated at 10°C, indicating contact was not at a stable dimer interface but required lateral mobility. We observed large multimers of Aer when we combined cross-linking of N-cap residues with a cysteine replacement that cross-links exclusively at the Aer dimer interface. This suggests that the PAS N-cap faces outwards in a dimer and that PAS-PAS contacts can occur between adjacent dimers.
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PAS Domain of the Aer Redox Sensor Requires C-Terminal Residues for Native-Fold Formation and Flavin Adenine Dinucleotide Binding
Journal of bacteriology, 2004Co-Authors: Sarah Herrmann, Mark S. Johnson, Alexandre V. Repik, Barry L. TaylorAbstract:The Aer protein in Escherichia coli is a membrane-bound, FAD-containing aerotaxis and energy sensor that putatively monitors the redox state of the electron transport system. Binding of FAD to Aer requires the N-terminal PAS Domain and residues in the F1 region and C-terminal HAMP Domain. The PAS Domains of other PAS proteins are soluble in water. To investigate properties of the PAS Domain, we subcloned segments of the aer gene from E. coli that encode the PAS Domain with and without His6 tags and expressed the PAS peptides in E. coli. The 20-kDa His6-Aer2-166 PAS-F1 fragment was purified as an 800-kDa complex by gel filtration chromatography, and the associating protein was identified by N-terminal sequencing as the chaperone protein GroEL. None of the N-terminal fragments of Aer found in the soluble fraction was released from GroEL, suggesting that these peptides do not fold correctly in an aqueous environment and require a motif external to the PAS Domain for proper folding. Consistent with this model, peptide fragments that included the membrane binding region and part (Aer2-231) or all (Aer2-285) of the HAMP Domain inserted into the membrane, indicating that they were released by GroEL. Aer2-285, but not Aer2-231, bound FAD, confirming the requirement for the HAMP Domain in stabilizing FAD binding. The results raise an interesting possibility that residues outside the PAS Domain that are required for FAD binding are essential for formation of the PAS native fold.
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The FAD-PAS Domain as a sensor for behavioral responses in Escherichia coli.
Antioxidants & redox signaling, 2001Co-Authors: Barry L. Taylor, Anuradha Rebbapragada, Mark S. JohnsonAbstract:Aer, the aerotaxis receptor in Escherichia coli, is a member of a novel class of flavoproteins that act as redox sensors. The internal energy of the cell is coupled to the redox state of the electron transport system, and this status is sensed by Aer(FAD). This is a more versatile sensory response system than if E. coli sensed oxygen per se. Energy-depleting conditions that decrease electron transport also alter the redox state of the electron transport system. Aer responds by sending a signal to the flagellar motor to change direction. The output of other sensory systems that utilize redox sensors is more commonly transcriptional regulation than a behavioral response. Analysis in silico showed Aer to be part of a superfamily of PAS Domain proteins that sense the intracellular environment. In Aer, FAD binds to the PAS Domain. By using site-specific mutagenesis, residues critical for FAD binding and sensory transduction were identified in the PAS Domain. The PAS Domain appears to interact with a linker region in the C-terminus. The linker region is a member of a HAMP Domain family, which has signal transduction roles in other systems.
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PAS Domain residues involved in signal transduction by the Aer redox sensor of Escherichia coli
Molecular microbiology, 2000Co-Authors: Alexandre V. Repik, Anuradha Rebbapragada, Mark S. Johnson, Joshua Ö. Haznedar, Igor B. Zhulin, Barry L. TaylorAbstract:PAS Domains sense oxygen, redox potential and light, and are implicated in behaviour, circadian rhythmicity, development and metabolic regulation. Although PAS Domains are widespread in archaea, bacteria and eukaryota, the mechanism of signal transduction has been elucidated only for the bacterial photo sensor PYP and oxygen sensor FixL. We investigated the signalling mechanism in the PAS Domain of Aer, the redox potential sensor and aerotaxis transducer in Escherichia coli. Forty-two residues in Aer were substituted using cysteine-replacement mutagenesis. Eight mutations resulted in a null phenotype for aerotaxis, the behavioural response to oxygen. Four of them also led to the loss of the non-covalently bound FAD cofactor. Three mutant Aer proteins, N34C, F66C and N85C, transmitted a constant signal-on bias. One mutation, Y111C, inverted signalling by the transducer so that positive stimuli produced negative signals and vice versa. Residues critical for signalling were mapped onto a three-dimensional model of the Aer PAS Domain, and an FAD-binding site and 'active site' for signal transduction are proposed.
Gail A Robertson - One of the best experts on this subject based on the ideXlab platform.
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Long QT Syndrome 2 PAS Domain Variant Induces hERG1a/1b Subunit Imbalance in Patient-specific iPSC-cardiomyocytes.
Circulation. Arrhythmia and electrophysiology, 2021Co-Authors: Li Feng, Gina Kim, Jianhua Zhang, Changhwan Lee, Fang Liu, Andrew J Petersen, Evi Lim, Corey L Anderson, Kate M Orland, Gail A RobertsonAbstract:Background - Inherited long QT syndrome type 2 (LQT2) results from variants in the KCNH2 gene encoding the hERG1 potassium channel. Two main isoforms, hERG1a and hERG1b, assemble to form tetrameric channel. The N-terminal Per-Arnt-Sim (PAS) Domain, present only on hERG1a subunits, is a hotspot for pathogenic variants, but it is unknown whether PAS Domain variants impact hERG1b expression to contribute to the LQT2 phenotype. We aimed to use patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to investigate the pathogenesis of the hERG1a PAS Domain variant hERG1-H70R. Methods - Human iPSCs were derived from a LQT2 patient carrying the PAS Domain variant hERG1-H70R. CRISPR/Cas9 gene editing produced isogenic control iPSC lines. Differentiated iPSC-CMs were evaluated for their electrophysiology, hERG1a/1b mRNA expression, and hERG1a/1b protein expression. Results - Action potentials from single hERG1-H70R iPSC-CMs were prolonged relative to controls, and voltage clamp studies showed an underlying decrease in IKr with accelerated deactivation. In hERG1-H70R iPSC-CMs, transcription of hERG1a and hERG1b mRNA was unchanged compared to controls based on nascent nuclear transcript analysis, but hERG1b mRNA was significantly increased as was the ratio of hERG1b/hERG1a in mRNA complexes, suggesting post-transcriptional changes. Expression of complex glycosylated hERG1a in hERG1-H70R iPSC-CMs was reduced due to impaired protein trafficking, whereas the expression of the complex glycosylated form of hERG1b was unchanged. Conclusions - Patient-specific hERG1-H70R iPSC-CMs reveal a newly appreciated mechanism of pathogenesis of the LQT2 phenotype due to both impaired trafficking of hERG1a and maintained expression of hERG1b that produces subunit imbalance and reduced IKr with accelerated deactivation.
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long qt syndrome 2 PAS Domain variant induces herg1a 1b subunit imbalance in patient specific ipsc cardiomyocytes
Circulation-arrhythmia and Electrophysiology, 2021Co-Authors: Li Feng, Gina Kim, Jianhua Zhang, Changhwan Lee, Fang Liu, Andrew J Petersen, Evi Lim, Corey L Anderson, Kate M Orland, Gail A RobertsonAbstract:Background - Inherited long QT syndrome type 2 (LQT2) results from variants in the KCNH2 gene encoding the hERG1 potassium channel. Two main isoforms, hERG1a and hERG1b, assemble to form tetrameric channel. The N-terminal Per-Arnt-Sim (PAS) Domain, present only on hERG1a subunits, is a hotspot for pathogenic variants, but it is unknown whether PAS Domain variants impact hERG1b expression to contribute to the LQT2 phenotype. We aimed to use patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to investigate the pathogenesis of the hERG1a PAS Domain variant hERG1-H70R. Methods - Human iPSCs were derived from a LQT2 patient carrying the PAS Domain variant hERG1-H70R. CRISPR/Cas9 gene editing produced isogenic control iPSC lines. Differentiated iPSC-CMs were evaluated for their electrophysiology, hERG1a/1b mRNA expression, and hERG1a/1b protein expression. Results - Action potentials from single hERG1-H70R iPSC-CMs were prolonged relative to controls, and voltage clamp studies showed an underlying decrease in IKr with accelerated deactivation. In hERG1-H70R iPSC-CMs, transcription of hERG1a and hERG1b mRNA was unchanged compared to controls based on nascent nuclear transcript analysis, but hERG1b mRNA was significantly increased as was the ratio of hERG1b/hERG1a in mRNA complexes, suggesting post-transcriptional changes. Expression of complex glycosylated hERG1a in hERG1-H70R iPSC-CMs was reduced due to impaired protein trafficking, whereas the expression of the complex glycosylated form of hERG1b was unchanged. Conclusions - Patient-specific hERG1-H70R iPSC-CMs reveal a newly appreciated mechanism of pathogenesis of the LQT2 phenotype due to both impaired trafficking of hERG1a and maintained expression of hERG1b that produces subunit imbalance and reduced IKr with accelerated deactivation.
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Enhancement of hERG channel activity by scFv antibody fragments targeted to the PAS Domain.
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Carol A. Harley, Gail A Robertson, Greg Starek, David K. Jones, Andreia S. Fernandes, João H. Morais-cabralAbstract:Abstract The human human ether-a-go-go–related gene (hERG) potassium channel plays a critical role in the repolarization of the cardiac action potential. Changes in hERG channel function underlie long QT syndrome (LQTS) and are associated with cardiac arrhythmias and sudden death. A striking feature of this channel and KCNH channels in general is the presence of an N-terminal Per-Arnt-Sim (PAS) Domain. In other proteins, PAS Domains bind ligands and modulate effector Domains. However, the PAS Domains of KCNH channels are orphan receptors. We have uncovered a family of positive modulators of hERG that specifically bind to the PAS Domain. We generated two single-chain variable fragments (scFvs) that recognize different epitopes on the PAS Domain. Both antibodies increase the rate of deactivation but have different effects on channel activation and inactivation. Importantly, we show that both antibodies, on binding to the PAS Domain, increase the total amount of current that permeates the channel during a ventricular action potential and significantly reduce the action potential duration recorded in human cardiomyocytes. Overall, these molecules constitute a previously unidentified class of positive modulators and establish that allosteric modulation of hERG channel function through ligand binding to the PAS Domain can be attained.
Rudy Antoine - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the PAS Domain in the sensor-kinase BvgS: mechanical role in signal transmission.
BMC Microbiology, 2013Co-Authors: Elian Dupré, Alexandre Wohlkonig, Julien Herrou, Camille Locht, Françoise Jacob-dubuisson, Rudy AntoineAbstract:BACKGROUND: In bacteria, signal-transduction two-component systems are major players for adaptation to environmental stimuli. The perception of a chemical or physical signal by a sensor-kinase triggers its autophosphorylation. The phosphoryl group is then transferred to the cognate response regulator, which mediates the appropriate adaptive response. Virulence of the whooping cough agent Bordetella pertussis is controlled by the two-component system BvgAS. Atypically, the sensor-kinase BvgS is active without specific stimuli at 37[degree sign]C in laboratory conditions and is inactivated by the addition of negative chemical modulators. The structure of BvgS is complex, with two tandem periplasmic Venus flytrap Domains and a cytoplasmic PAS Domain that precedes the kinase Domain, which is followed by additional phosphotransfer Domains. PAS Domains are small, ubiquitous sensing or regulatory Domains. The function of the PAS Domain in BvgS remains unknown. RESULTS: We showed that recombinant BvgS PAS proteins form dimers that are stabilized by alpha helical regions flanking the PAS core. A structural model of the PAS Domain dimer was built and probed by site-directed mutagenesis and by biochemical and functional analyses. Although we found no ligands for the PAS Domain cavity, its integrity is required for signaling. We also showed that the structural stability of the PAS core and its proper coupling to its flanking N- and C-terminal alpha helices are crucial for BvgS activity. CONCLUSIONS: We propose that a major function of the BvgS PAS Domain is to maintain conformational signals arising from mechanical strain generated by the periplasmic Domain. The tight structure of the PAS core and its connections with the upstream and downstream helices ensure signaling to the kinase Domain, which determines BvgS activity. Many mild substitutions that map to the PAS Domain keep BvgS active but make it unresponsive to negative modulators, supporting that modulation increases conformational strain in the protein.
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Characterization of the PAS Domain in the sensor-kinase BvgS: mechanical role in signal transmission
BMC microbiology, 2013Co-Authors: Elian Dupré, Alexandre Wohlkonig, Julien Herrou, Camille Locht, Françoise Jacob-dubuisson, Rudy AntoineAbstract:Background In bacteria, signal-transduction two-component systems are major players for adaptation to environmental stimuli. The perception of a chemical or physical signal by a sensor-kinase triggers its autophosphorylation. The phosphoryl group is then transferred to the cognate response regulator, which mediates the appropriate adaptive response. Virulence of the whooping cough agent Bordetella pertussis is controlled by the two-component system BvgAS. Atypically, the sensor-kinase BvgS is active without specific stimuli at 37°C in laboratory conditions and is inactivated by the addition of negative chemical modulators. The structure of BvgS is complex, with two tandem periplasmic Venus flytrap Domains and a cytoplasmic PAS Domain that precedes the kinase Domain, which is followed by additional phosphotransfer Domains. PAS Domains are small, ubiquitous sensing or regulatory Domains. The function of the PAS Domain in BvgS remains unknown.