Journal articles: 'Per Arnt Sim (PAS)'

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Hirose, K., M. Morita, M. Ema, J. Mimura, H. Hamada, H. Fujii, Y. Saijo, O. Gotoh, K. Sogawa, and Y. Fujii-Kuriyama. "cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt)." Molecular and Cellular Biology 16, no. 4 (April 1996): 1706–13. http://dx.doi.org/10.1128/mcb.16.4.1706.

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We isolated mouse cDNA clones (Arnt2) that are highly similar to but distinct from the aryl hydrocarbon receptor (AhR) nuclear translocator (Arnt). The composite cDNA covered a 2,443-bp sequence consisting of a putative 2,136-bp open reading frame encoding a polypeptide of 712 amino acids. The predicted Arnt2 polypeptide carries a characteristic basic helix-loop-helix (bHLH)/PAS motif in its N-terminal region with close similarity (81% identity) to that of mouse Arnt and has an overall sequence identity of 57% with Arnt. Biochemical properties and interaction of Arnt2 with other bHLH/PAS proteins were investigated by coimmunoprecipitation assays, gel mobility shift assays, and the yeast two-hybrid system. Arnt2 interacted with AhR and mouse Sim as efficiently as Arnt, and the Arnt2-AhR complex recognized and bound specifically the xenobiotic responsive element (XRE) sequence. Expression of Arnt2 successfully rescued XRE-driven reporter gene activity in the Arnt-defective c4 mutant of Hepa-1 cells. RNA blot analysis revealed that expression of Arnt2 mRNA was restricted to the brains and kidneys of adult mice, while Arnt mRNA was expressed ubiquitously. In addition, whole-mount in situ hybridization of 9.5-day mouse embryos showed that Arnt2 mRNA was expressed in the dorsal neural tube and branchial arch 1, while Arnt transcripts were detected broadly in various tissues of mesodermal and endodermal origins. These results suggest that Arnt2 may play different roles from Arnt both in adult mice and in developing embryos. Finally, sequence comparison of the currently known bHLH/PAS proteins indicates a division into two phylogenetic groups: the Arnt group, containing Arnt, Arnt2, and Per, and the AhR group, consisting of AhR, Sim, and Hif-1alpha.

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Yang, Jinsong, Lei Zhang, Paul J. A. Erbel, Kevin H. Gardner, Kan Ding, Joseph A. Garcia, and Richard K. Bruick. "Functions of the Per/ARNT/Sim Domains of the Hypoxia-inducible Factor." Journal of Biological Chemistry 280, no. 43 (August 29, 2005): 36047–54. http://dx.doi.org/10.1074/jbc.m501755200.

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The heterodimeric transcription factor hypoxia-inducible factor (HIF) plays an important role in the progression of a number of processes in which O2 availability is compromised and, as such, has become an increasingly attractive therapeutic target. Although tremendous progress has been made in recent years in unraveling the mechanisms underlying O2-dependent regulation of HIF through its O2-dependent degradation domain and C-terminal transactivation domain, our understanding of the contributions of other structural elements, particularly the Per/ARNT/Sim (PAS)-A and PAS-B domains, to the activity of HIF is incomplete. Using insights derived from the recently determined solution structures of the HIF PAS-B domains as a starting point, we have explored the function(s) of the HIF-2α PAS domains via mutational analysis. In contrast to recent models, our data reveal that both PAS domains of the HIF-α subunit are necessary for heterodimer formation but are not required to mediate other HIF functions in which PAS domains have been implicated. Because disruption of individual PAS domains compromise HIF function independent of the mechanism of HIF induction, these data demonstrate the potential utility of targeting these domains for therapeutic applications.

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de Souza, João V., Sylvia Reznikov, Ruidi Zhu, and Agnieszka K. Bronowska. "Druggability assessment of mammalian Per–Arnt–Sim [PAS] domains using computational approaches." MedChemComm 10, no. 7 (2019): 1126–37. http://dx.doi.org/10.1039/c9md00148d.

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Moffett, P., M. Reece, and J. Pelletier. "The murine Sim-2 gene product inhibits transcription by active repression and functional interference." Molecular and Cellular Biology 17, no. 9 (September 1997): 4933–47. http://dx.doi.org/10.1128/mcb.17.9.4933.

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The Drosophila single-minded (Dsim) gene encodes a master regulatory protein involved in cell fate determination during midline development. This protein is a member of a rapidly expanding family of gene products possessing basic helix-loop-helix (bHLH) and hydrophobic PAS (designated a conserved region among PER, ARNT [aryl hydrocarbon receptor nuclear translocator] and SIM) protein association domains. Members of this family function as central transcriptional regulators in cellular differentiation and in the response to environmental stimuli such as xenobiotics and hypoxia. We have previously identified a murine member of this family, called mSim-2, showing sequence homology to the bHLH and PAS domains of Dsim. Immunoprecipitation experiments with recombinant proteins indicate that mSIM-2 associates with the arnt gene product. In the present work, by using fine-structure mapping we found that the HLH and PAS motifs of both proteins are required for optimal association. Forced expression of GAL4/mSIM-2 fusion constructs in mammalian cells demonstrated the presence of two separable repression domains within the carboxy terminus of mSIM-2. We found that mSIM-2 is capable of repressing ARNT-mediated transcriptional activation in a mammalian two-hybrid system. This effect (i) is dependent on the ability of mSIM-2 and ARNT to heterodimerize, (ii) is dependent on the presence of the mSIM-2 carboxy-terminal repression domain, and (iii) is not specific to the ARNT activation domain. These results suggest that mSIM-2 repression activity can dominantly override the activation potential of adjacent transcription factors. We also demonstrated that mSIM-2 can functionally interfere with hypoxia-inducible factor 1alpha (HIF-1alpha)/ARNT transcription complexes, providing a second mechanism by which mSIM-2 may inhibit transcription.

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Reisz-Porszasz, S., M. R. Probst, B. N. Fukunaga, and O. Hankinson. "Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT)." Molecular and Cellular Biology 14, no. 9 (September 1994): 6075–86. http://dx.doi.org/10.1128/mcb.14.9.6075-6086.1994.

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The activated aryl hydrocarbon receptor (AHR) and the AHR nuclear translocator (ARNT) bind DNA as a heterodimer. Both proteins represent a novel class of basic helix-loop-helix (bHLH)-containing transcription factors in that (i) activation of AHR requires the binding of ligand (e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]), (ii) the xenobiotic responsive element (XRE) recognized by the AHR/ARNT heterodimer differs from the recognition sequence for nearly all other bHLH proteins, and (iii) both proteins contain a PAS homology region, which in the Drosophila PER and SIM proteins functions as a dimerization domain. A cDNA for mouse ARNT has been cloned, and potential functional domains of ARNT were investigated by deletion analysis. A mutant lacking all regions of ARNT other than the bHLH and PAS regions is unimpaired in TCDD-dependent dimerization and subsequent XRE binding and only modestly reduced in ability to complement an ARNT-deficient mutant cell line, c4, in vivo. Both the first and second alpha helices of the bHLH region are required for dimerization. The basic region is required for XRE binding but not for dimerization. Deletion of either the A or B segments of the PAS region slightly affects TCDD-induced heterodimerization, while deletion of the complete PAS region severely affects (but does not eliminate) dimerization. Thus, ARNT possesses multiple domains required for maximal heterodimerization. Mutants deleted for PAS A, PAS B, and the complete PAS region all retain some degree of XRE binding, yet none can rescue the c4 mutant. Therefore, both the PAS A and PAS B segments, besides contributing to dimerization, apparently fulfill additional, unknown functions required for biological activity of ARNT.

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Reisz-Porszasz, S., M. R. Probst, B. N. Fukunaga, and O. Hankinson. "Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT)." Molecular and Cellular Biology 14, no. 9 (September 1994): 6075–86. http://dx.doi.org/10.1128/mcb.14.9.6075.

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The activated aryl hydrocarbon receptor (AHR) and the AHR nuclear translocator (ARNT) bind DNA as a heterodimer. Both proteins represent a novel class of basic helix-loop-helix (bHLH)-containing transcription factors in that (i) activation of AHR requires the binding of ligand (e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]), (ii) the xenobiotic responsive element (XRE) recognized by the AHR/ARNT heterodimer differs from the recognition sequence for nearly all other bHLH proteins, and (iii) both proteins contain a PAS homology region, which in the Drosophila PER and SIM proteins functions as a dimerization domain. A cDNA for mouse ARNT has been cloned, and potential functional domains of ARNT were investigated by deletion analysis. A mutant lacking all regions of ARNT other than the bHLH and PAS regions is unimpaired in TCDD-dependent dimerization and subsequent XRE binding and only modestly reduced in ability to complement an ARNT-deficient mutant cell line, c4, in vivo. Both the first and second alpha helices of the bHLH region are required for dimerization. The basic region is required for XRE binding but not for dimerization. Deletion of either the A or B segments of the PAS region slightly affects TCDD-induced heterodimerization, while deletion of the complete PAS region severely affects (but does not eliminate) dimerization. Thus, ARNT possesses multiple domains required for maximal heterodimerization. Mutants deleted for PAS A, PAS B, and the complete PAS region all retain some degree of XRE binding, yet none can rescue the c4 mutant. Therefore, both the PAS A and PAS B segments, besides contributing to dimerization, apparently fulfill additional, unknown functions required for biological activity of ARNT.

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Wang, Feng, Shengli Shi, Ruixue Zhang, and Oliver Hankinson. "Identifying target genes of the aryl hydrocarbon receptor nuclear translocator (Arnt) using DNA microarray analysis." Biological Chemistry 387, no. 9 (September 1, 2006): 1215–18. http://dx.doi.org/10.1515/bc.2006.150.

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Abstract The aryl hydrocarbon receptor nuclear translocator (Arnt) is a basic helix-loop-helix (bHLH) protein that also contains a Per-Arnt-Sim (PAS) domain. In addition to forming heterodimers with many other bHLH-PAS proteins, including the aryl hydrocarbon receptor (AhR) and hypoxia-inducible factors 1α, 2α and 3α, Arnt can also form homodimers when expressed from its cDNA in vitro or in vivo. However, target genes of the Arnt/Arnt homodimer remain to be identified. In this study, we have elucidated the profile of genes responsive to the reintroduction of Arnt expression in an Arnt-deficient mouse hepatoma cell line (c4), using DNA microarray analysis. The expression of 27 genes was upregulated by 1.5-fold or more in c4 cells infected with a retroviral vector expressing mouse Arnt, while no genes were found to be downregulated. Among the upregulated genes, BCL2/adenovirus E1B 19 kDa-interacting protein 1 (NIP3), serine (or cysteine) proteinase inhibitor, clade E, member 1 (PAI1), and N-myc downstream regulated-like (NDR1), were confirmed to be induced by Arnt using real-time PCR. We also found that the 5′ promoter region of 15 out of 20 upregulated genes contain the type 2 E-box 5′-CACGTG-3′ Arnt/Arnt binding sequence, consistent with the notion that they represent target genes for Arnt.

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Pongratz, Ingemar, Camilla Antonsson, Murray L. Whitelaw, and Lorenz Poellinger. "Role of the PAS Domain in Regulation of Dimerization and DNA Binding Specificity of the Dioxin Receptor." Molecular and Cellular Biology 18, no. 7 (July 1, 1998): 4079–88. http://dx.doi.org/10.1128/mcb.18.7.4079.

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ABSTRACT The dioxin receptor is a ligand-regulated transcription factor that mediates signal transduction by dioxin and related environmental pollutants. The receptor belongs to the basic helix-loop-helix (bHLH)–Per-Arnt-Sim (PAS) family of factors, which, in addition to the bHLH motif, contain a PAS region of homology. Upon activation, the dioxin receptor dimerizes with the bHLH-PAS factor Arnt, enabling the receptor to recognize xenobiotic response elements in the vicinity of target genes. We have studied the role of the PAS domain in dimerization and DNA binding specificity of the dioxin receptor and Arnt by monitoring the abilities of the individual bHLH domains and different bHLH-PAS fragments to dimerize and bind DNA in vitro and recognize target genes in vivo. The minimal bHLH domain of the dioxin receptor formed homodimeric complexes, heterodimerized with full-length Arnt, and together with Arnt was sufficient for recognition of target DNA in vitro and in vivo. In a similar fashion, only the bHLH domain of Arnt was necessary for DNA binding specificity in the presence of the dioxin receptor bHLH domain. Moreover, the bHLH domain of the dioxin receptor displayed a broad dimerization potential, as manifested by complex formation with, e.g., the unrelated bHLH-Zip transcription factor USF. In contrast, a construct spanning the dioxin receptor bHLH domain and an N-terminal portion of the PAS domain failed to form homodimers and was capable of dimerizing only with Arnt. Thus, the PAS domain is essential to confer dimerization specificity of the dioxin receptor.

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Zhao, J. M., H. Lee, R. A. Nome, S. Majid, N. F. Scherer, and W. D. Hoff. "Single-molecule detection of structural changes during Per-Arnt-Sim (PAS) domain activation." Proceedings of the National Academy of Sciences 103, no. 31 (July 19, 2006): 11561–66. http://dx.doi.org/10.1073/pnas.0601567103.

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MacDonald, P. E., and P. Rorsman. "Per-arnt-sim (PAS) domain kinase (PASK) as a regulator of glucagon secretion." Diabetologia 54, no. 4 (February 17, 2011): 719–21. http://dx.doi.org/10.1007/s00125-011-2072-1.

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Adaixo, Ricardo, and João Henrique Morais-Cabral. "Crystallization and preliminary crystallographic characterization of the PAS domains of EAG and ELK potassium channels." Acta Crystallographica Section F Structural Biology and Crystallization Communications 66, no. 9 (August 26, 2010): 1056–59. http://dx.doi.org/10.1107/s1744309110027880.

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Per–Arnt–Sim (PAS) domains are ubiquitous in nature; they are ∼130-amino-acid protein domains that adopt a fairly conserved three-dimensional structure despite their low degree of sequence homology. These domains constitute the N-terminus or, less frequently, the C-terminus of a number of proteins, where they exert regulatory functions. PAS-containing proteins generally display two or more copies of this motif. In this work, the crystallization and preliminary analysis of the PAS domains of two eukaryotic potassium channels from the ether-à-go-go (EAG) family are reported.

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Philip, A. F., M. Kumauchi, and W. D. Hoff. "Robustness and evolvability in the functional anatomy of a PER-ARNT-SIM (PAS) domain." Proceedings of the National Academy of Sciences 107, no. 42 (October 1, 2010): 17986–91. http://dx.doi.org/10.1073/pnas.1004823107.

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de Souza, João Victor, Piotr Zaborniak, Sylvia Reznikov, Matthew Kondal, Ruidi Zhu, and Agnieszka K. Bronowska. "Molecular Forces Governing the Biological Function of Per-Arnt-Sim-B (PAS-B) Domains: A Comparative Computational Study." Biophysica 1, no. 1 (February 5, 2021): 1–14. http://dx.doi.org/10.3390/biophysica1010001.

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Per-Arnt-Sim (PAS) domains are evolutionarily-conserved regions found in proteins in all living systems, involved in transcriptional regulation and the response to hypoxic and xenobiotic stress. Despite having low primary sequence similarity, they show an impressively high structural conservation. Nonetheless, understanding the underlying mechanisms that drive the biological function of the PAS domains remains elusive. In this work, we used molecular dynamics simulations and bioinformatics tools in order the investigate the molecular characteristics that govern the intrinsic dynamics of five PAS-B domains (human AhR receptor, NCOA1, HIF1α, and HIF2α transcription factors, and Drosophila Suzukii (D. Suzukii) juvenile hormone receptor JHR). First, we investigated the effects of different length of N and C terminal regions of the AhR PAS-B domain, showing that truncation of those segments directly affects structural stability and aggregation propensity of the domain. Secondly, using the recently annotated PAS-B located in the methoprene-tolerant protein/juvenile hormone receptor (JHR) from D. Suzukii, we have shown that the mutation of the highly conserved “gatekeeper” tyrosine to phenylalanine (Y322F) does not affect the stability of the domain. Finally, we investigated possible redox-regulation of the AhR PAS-B domain by focusing on the cysteinome residues within PAS-B domains. The cysteines in AhR PAS-B are directly regulating the dynamics of the small molecule ligand-gating loop (residues 305 to 326). In conclusion, we comprehensibly described several molecular features governing the behaviour of PAS-B domains in solution, which may lead to a better understanding of the forces driving their biological functions.

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Xu, Xingjian, Igor Dikiy, Matthew R. Evans, Leandro P. Marcelino, and Kevin H. Gardner. "Fragile protein folds: sequence and environmental factors affecting the equilibrium of two interconverting, stably folded protein conformations." Magnetic Resonance 2, no. 1 (March 10, 2021): 63–76. http://dx.doi.org/10.5194/mr-2-63-2021.

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Abstract. Recent research on fold-switching metamorphic proteins has revealed some notable exceptions to Anfinsen's hypothesis of protein folding. We have previously described how a single point mutation can enable a well-folded protein domain, one of the two PAS (Per-ARNT-Sim) domains of the human ARNT (aryl hydrocarbon receptor nuclear translocator) protein, to interconvert between two conformers related by a slip of an internal β strand. Using this protein as a test case, we advance the concept of a “fragile fold”, a protein fold that can reversibly rearrange into another fold that differs by a substantial number of hydrogen bonds, entailing reorganization of single secondary structure elements to more drastic changes seen in metamorphic proteins. Here we use a battery of biophysical tests to examine several factors affecting the equilibrium between the two conformations of the switching ARNT PAS-B Y456T protein. Of note is that we find that factors which impact the HI loop preceding the shifted Iβ strand affect both the equilibrium levels of the two conformers and the denatured state which links them in the interconversion process. Finally, we describe small molecules that selectively bind to and stabilize the wild-type conformation of ARNT PAS-B. These studies form a toolkit for studying fragile protein folds and could enable ways to modulate the biological functions of such fragile folds, both in natural and engineered proteins.

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Gradin, K., J. McGuire, R. H. Wenger, I. Kvietikova, M. L. fhitelaw, R. Toftgård, L. Tora, M. Gassmann, and L. Poellinger. "Functional interference between hypoxia and dioxin signal transduction pathways: competition for recruitment of the Arnt transcription factor." Molecular and Cellular Biology 16, no. 10 (October 1996): 5221–31. http://dx.doi.org/10.1128/mcb.16.10.5221.

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Hypoxia-inducible factor 1 alpha (HIF-1 alpha) and the intracellular dioxin receptor mediate hypoxia and dioxin signalling, respectively. Both proteins are conditionally regulated basic helix-loop-helix (bHLH) transcription factors that, in addition to the bHLH motif, share a Per-Arnt-Sim (PAS) region of homology and form heterodimeric complexes with the common bHLH/PAS partner factor Arnt. Here we demonstrate that HIF-1 alpha required Arnt for DNA binding in vitro and functional activity in vivo. Both the bHLH and PAS motifs of Arnt were critical for dimerization with HIF-1 alpha. Strikingly, HIF-1 alpha exhibited very high affinity for Arnt in coimmunoprecipitation assays in vitro, resulting in competition with the ligand-activated dioxin receptor for recruitment of Arnt. Consistent with these observations, activation of HIF-1 alpha function in vivo or overexpression of HIF-1 alpha inhibited ligand-dependent induction of DNA binding activity by the dioxin receptor and dioxin receptor function on minimal reporter gene constructs. However, HIF-1 alpha- and dioxin receptor-mediated signalling pathways were not mutually exclusive, since activation of dioxin receptor function did not impair HIF-1 alpha-dependent induction of target gene expression. Both HIF-1 alpha and Arnt mRNAs were expressed constitutively in a large number of human tissues and cell lines, and these steady-state expression levels were not affected by exposure to hypoxia. Thus, HIF-1 alpha may be conditionally regulated by a mechanism that is distinct from induced expression levels, the prevalent model of activation of HIF-1 alpha function. Interestingly, we observed that HIF-1 alpha was associated with the molecular chaperone hsp90. Given the critical role of hsp90 for ligand binding activity and activation of the dioxin receptor, it is therefore possible that HIF-1 alpha is regulated by a similar mechanism, possibly by binding an as yet unknown class of ligands.

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Aitola, Marjo, Christine M. Sadek, Jan-Åke Gustafsson, and Markku Pelto-Huikko. "Aint/Tacc3 Is Highly Expressed in Proliferating Mouse Tissues During Development, Spermatogenesis, and Oogenesis." Journal of Histochemistry & Cytochemistry 51, no. 4 (April 2003): 455–69. http://dx.doi.org/10.1177/002215540305100407.

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Aint was originally identified on the basis of its interaction in vitro with the aryl hydrocarbon nuclear receptor translocator (Arnt). Arnt is a common heterodimerization partner in the basic helix-loop–helix (bHLH)-PER-ARNT-SIM (PAS) protein family and is involved in diverse biological functions. These include xenobiotic metabolism, hypoxic response, and circadian rhythm. In addition, Arnt has a crucial role during development. Aint is a member of a growing family of transforming acidic coiled-coil (TACC) proteins and is the murine homologue of human TACC3. Here we report the spatiotemporal expression of Tacc3 mRNA and protein in embryonic, postnatally developing, and adult mouse tissues using in situ hybridization and immunocytochemistry. Tacc3 mRNA was highly expressed in proliferating cells of several organs during murine development. However, the only adult tissues expressing high levels were testis and ovary. Immunocytochemistry revealed that Tacc3 is a nuclear protein. Our results suggest that Tacc3 has an important role in murine development, spermatogenesis, and oogenesis.

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Seok, Seung-Hyeon, Woojong Lee, Li Jiang, Kaivalya Molugu, Aiping Zheng, Yitong Li, Sanghyun Park, Christopher A. Bradfield, and Yongna Xing. "Structural hierarchy controlling dimerization and target DNA recognition in the AHR transcriptional complex." Proceedings of the National Academy of Sciences 114, no. 21 (April 10, 2017): 5431–36. http://dx.doi.org/10.1073/pnas.1617035114.

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The aryl hydrocarbon receptor (AHR) belongs to the PAS (PER-ARNT-SIM) family transcription factors and mediates broad responses to numerous environmental pollutants and cellular metabolites, modulating diverse biological processes from adaptive metabolism, acute toxicity, to normal physiology of vascular and immune systems. The AHR forms a transcriptionally active heterodimer with ARNT (AHR nuclear translocator), which recognizes the dioxin response element (DRE) in the promoter of downstream genes. We determined the crystal structure of the mammalian AHR–ARNT heterodimer in complex with the DRE, in which ARNT curls around AHR into a highly intertwined asymmetric architecture, with extensive heterodimerization interfaces and AHR interdomain interactions. Specific recognition of the DRE is determined locally by the DNA-binding residues, which discriminates it from the closely related hypoxia response element (HRE), and is globally affected by the dimerization interfaces and interdomain interactions. Changes at the interdomain interactions caused either AHR constitutive nuclear localization or failure to translocate to nucleus, underlying an allosteric structural pathway for mediating ligand-induced exposure of nuclear localization signal. These observations, together with the global higher flexibility of the AHR PAS-A and its loosely packed structural elements, suggest a dynamic structural hierarchy for complex scenarios of AHR activation induced by its diverse ligands.

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Antonsson, C., M. L. Whitelaw, J. McGuire, J. A. Gustafsson, and L. Poellinger. "Distinct roles of the molecular chaperone hsp90 in modulating dioxin receptor function via the basic helix-loop-helix and PAS domains." Molecular and Cellular Biology 15, no. 2 (February 1995): 756–65. http://dx.doi.org/10.1128/mcb.15.2.756.

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The intracellular dioxin receptor mediates signal transduction by dioxin and functions as a ligand-activated transcription factor. It contains a basic helix-loop-helix (bHLH) motif contiguous with a Per-Arnt-Sim (PAS) homology region. In extracts from nonstimulated cells the receptor is recovered in an inducible cytoplasmic form associated with the 90-kDa heat shock protein (hsp90), a molecular chaperone. We have reconstituted ligand-dependent activation of the receptor to a DNA-binding form by using the dioxin receptor and its bHLH-PAS partner factor Arnt expressed by in vitro translation in reticulocyte lysate. Deletion of the PAS domain of the receptor resulted in constitutive dimerization with Arnt. In contrast, this receptor mutant showed low levels of xenobiotic response element-binding activity, indicating that the PAS domain may be important for DNA-binding affinity and/or specificity of the receptor. It was not possible to reconstitute dioxin receptor function with proteins expressed in wheat germ lysate. In line with these observations, reticulocyte lysate but not wheat germ lysate promoted the association of de novo synthesized dioxin receptor with hsp90. At least two distinct domains of the receptor mediated interaction with hsp90: the ligand-binding domain located within the PAS region and, surprisingly, the bHLH domain. Whereas ligand-binding activity correlated with association with hsp90, bHLH-hsp90 interaction appeared to be important for DNA-binding activity but not for dimerization of the receptor. Several distinct roles for hsp90 in modulating dioxin receptor function are therefore likely: correct folding of the ligand-binding domain, interference with Arnt heterodimerization, and folding of a DNA-binding conformation of the bHLH domain. Thus, the dioxin receptor system provides a complex and interesting model of the regulation of transcription factors by hsp90.

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Semplici, Francesca, Martine Vaxillaire, Sarah Fogarty, Meriem Semache, Amélie Bonnefond, Ghislaine Fontés, Julien Philippe, et al. "Human Mutation within Per-Arnt-Sim (PAS) Domain-containing Protein Kinase (PASK) Causes Basal Insulin Hypersecretion." Journal of Biological Chemistry 286, no. 51 (November 7, 2011): 44005–14. http://dx.doi.org/10.1074/jbc.m111.254995.

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Chapman-Smith, Anne, Jodi K. Lutwyche, and Murray L. Whitelaw. "Contribution of the Per/Arnt/Sim (PAS) Domains to DNA Binding by the Basic Helix-Loop-Helix PAS Transcriptional Regulators." Journal of Biological Chemistry 279, no. 7 (November 24, 2003): 5353–62. http://dx.doi.org/10.1074/jbc.m310041200.

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Dardente, Hugues, Erin E. Fortier, Vincent Martineau, and Nicolas Cermakian. "Cryptochromes impair phosphorylation of transcriptional activators in the clock: a general mechanism for circadian repression." Biochemical Journal 402, no. 3 (February 26, 2007): 525–36. http://dx.doi.org/10.1042/bj20060827.

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CLOCK and BMAL1 [brain and muscle ARNT (arylhydrocarbon receptor nuclear translocator)-like protein 1] are central components of the molecular clock in mammals and belong to the bHLH (basic helix–loop–helix)/PAS [PER (Period)/ARNT/SIM (single-minded)] family. Features of their dimerization have never been investigated. Here, we demonstrate that PAS domain function requires regions extending over the short PAS core repeats. Strikingly, while deleting PAS core repeats does not overtly affect dimerization, it abolishes the transcriptional activity of the heterodimer. Interestingly, these deletions also abolish co-dependent phosphorylation of CLOCK and BMAL1, suggesting a link between the phosphorylation status of the heterodimer and its transactivation potential. We demonstrate that NPAS2 (neuronal PAS domain protein 2) and BMAL2 also undergo similar posttranslational modifications, thereby establishing the mechanism proposed for CLOCK–BMAL1 as a common feature of transcriptional activators in the circadian clock. The discovery of two novel splice variants of BMAL2 confirms the crucial role of the PAS domain and further strengthens the view that co-dependent phosphorylation is of functional significance. In agreement with this, we demonstrate that CRY1–2 (cryptochromes 1–2) affect transactivation and phosphorylation of transcriptional activators of the clock. Furthermore, CRY proteins stabilize the unphosphorylated forms of BMAL1(BMAL2) thereby shifting the phosphorylated/unphosphorylated ratio towards a predominantly unphosphorylated (transcriptionally inactive) form. In contrast, PER proteins, which are weak repressors, are without effect. From these results, we propose a general mechanism for the inhibition of CLOCK(NPAS2)–BMAL1(BMAL2) circadian transcriptional activation by CRY1–2.

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Brody, Stuart. "A Comparison of the Neurospora and Drosophila Clocks." Journal of Biological Rhythms 35, no. 2 (December 26, 2019): 119–33. http://dx.doi.org/10.1177/0748730419892434.

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In Neurospora and other fungi, the protein frequency (FRQ) is an integral part and a negative element in the fungal circadian oscillator. In Drosophila and many other higher organisms, the protein period (PER) is an integral part and a negative element of their circadian oscillator. Employing bioinformatic techniques, such as BLAST, CLUSTAL, and MEME (Multiple Em for Motif Elicitation), 11 regions (sequences) of potential similarity were found between the fungal FRQ and the Drosophila PER. Many of these FRQ regions are conserved in many fungal FRQ(s). Many of these PER regions are conserved in many insects. In addition, these regions are also of biological significance since mutations in these regions lead to changes in the circadian clock of Neurospora and Drosophila. Many of these regions of similarity between FRQ and PER are also conserved between the Drosophila PER and the mouse PER (mPER2). This suggests conserved and important regions for all 3 proteins and a common ancestor, possibly in those amoeba, such as Capsaspora, that sits at the base of the phylogenetic tree where fungi and animals diverged. Two additional examples of a possible common ancestor between Neurospora and Drosophila were found. One, the white collar (WC-1) protein of Neurospora and the Drosophila PER, shows significant similarity in its Per/Arnt/Sim (PAS) motifs to the PAS motif of an ARNT-like protein found in the amoeba, Capsaspora. Two, both of the positive elements in each system (i.e., WC-1 in Neurospora and cycle [CYC] in Drosophila), show significant similarity to this Capsaspora ARNT protein. A discussion of these findings centers on the long-time debate about the origins of the many different clock systems (i.e., independent evolution or common ancestor as well as to the question of how new genes are formed).

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Bersten, David C., John B. Bruning, Daniel J. Peet, and Murray L. Whitelaw. "Human Variants in the Neuronal Basic Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS) Transcription Factor Complex NPAS4/ARNT2 Disrupt Function." PLoS ONE 9, no. 1 (January 17, 2014): e85768. http://dx.doi.org/10.1371/journal.pone.0085768.

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Yun, Jaesuk, Taku Nagai, Yoko Furukawa-Hibi, Keisuke Kuroda, Kozo Kaibuchi, Michael E. Greenberg, and Kiyofumi Yamada. "Neuronal Per Arnt Sim (PAS) Domain Protein 4 (NPAS4) Regulates Neurite Outgrowth and Phosphorylation of Synapsin I." Journal of Biological Chemistry 288, no. 4 (November 21, 2012): 2655–64. http://dx.doi.org/10.1074/jbc.m112.413310.

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Coban, Mathew A., Patrick R. Blackburn, Murray L. Whitelaw, Mieke M. van Haelst, Paldeep S. Atwal, and Thomas R. Caulfield. "Structural Models for the Dynamic Effects of Loss-of-Function Variants in the Human SIM1 Protein Transcriptional Activation Domain." Biomolecules 10, no. 9 (September 12, 2020): 1314. http://dx.doi.org/10.3390/biom10091314.

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Single-minded homologue 1 (SIM1) is a transcription factor with numerous different physiological and developmental functions. SIM1 is a member of the class I basic helix-loop-helix-PER-ARNT-SIM (bHLH–PAS) transcription factor family, that includes several other conserved proteins, including the hypoxia-inducible factors, aryl hydrocarbon receptor, neuronal PAS proteins, and the CLOCK circadian regulator. Recent studies of HIF-a-ARNT and CLOCK-BMAL1 protein complexes have revealed the organization of their bHLH, PASA, and PASB domains and provided insight into how these heterodimeric protein complexes form; however, experimental structures for SIM1 have been lacking. Here, we describe the first full-length atomic structural model for human SIM1 with its binding partner ARNT in a heterodimeric complex and analyze several pathogenic variants utilizing state-of-the-art simulations and algorithms. Using local and global positional deviation metrics, deductions to the structural basis for the individual mutants are addressed in terms of the deleterious structural reorganizations that could alter protein function. We propose new experiments to probe these hypotheses and examine an interesting SIM1 dynamic behavior. The conformational dynamics demonstrates conformational changes on local and global regions that represent a mechanism for dysfunction in variants presented. In addition, we used our ab initio hybrid model for further prediction of variant hotspots that can be engineered to test for counter variant (restoration of wild-type function) or basic research probe.

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Beischlag, Timothy V., Song Wang, David W. Rose, Joseph Torchia, Suzanne Reisz-Porszasz, Khurshid Muhammad, Walter E. Nelson, Markus R. Probst, Michael G. Rosenfeld, and Oliver Hankinson. "Recruitment of the NCoA/SRC-1/p160 Family of Transcriptional Coactivators by the Aryl Hydrocarbon Receptor/Aryl Hydrocarbon Receptor Nuclear Translocator Complex." Molecular and Cellular Biology 22, no. 12 (June 15, 2002): 4319–33. http://dx.doi.org/10.1128/mcb.22.12.4319-4333.2002.

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ABSTRACT The aryl hydrocarbon receptor complex heterodimeric transcription factor, comprising the basic helix-loop-helix-Per-ARNT-Sim (bHLH-PAS) domain aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) proteins, mediates the toxic effects of TCDD (2,3,7,8 tetrachlorodibenzo-p-dioxin). The molecular events underlying TCDD-inducible gene activation, beyond the activation of the AHRC, are poorly understood. The SRC-1/NCoA-1, NCoA-2/GRIP-1/TIF-2, and p/CIP/AIB/ACTR proteins have been shown to act as mediators of transcriptional activation. In this report, we demonstrate that SRC-1, NCoA-2, and p/CIP are capable of independently enhancing TCDD-dependent induction of a luciferase reporter gene by the AHR/ARNT dimer. Furthermore, injection of anti-SRC-1 or anti-p/CIP immunoglobulin G into mammalian cells abolishes the transcriptional activity of a TCDD-dependent reporter gene. We demonstrate by coimmunoprecipitation and by a reporter gene assay that SRC-1 and NCoA-2 but not p/CIP are capable of interacting with ARNT in vivo after transient transfection into mammalian cells, while AHR is capable of interacting with all three coactivators. We confirm the interactions of ARNT and AHR with SRC-1 with immunocytochemical techniques. Furthermore, SRC-1, NCoA-2, and p/CIP all associate with the CYP1A1 enhancer region in a TCDD-dependent fashion, as demonstrated by chromatin immunoprecipitation assays. We demonstrate by yeast two-hybrid, glutathione S-transferase pulldown, and mammalian reporter gene assays that ARNT requires its helix 2 domain but not its transactivation domain to interact with SRC-1. This indicates a novel mechanism of action for SRC-1. SRC-1 does not require its bHLH-PAS domain to interact with ARNT or AHR, but utilizes distinct domains proximal to its p300/CBP interaction domain. Taken together, these data support a role for the SRC family of transcriptional coactivators in TCDD-dependent gene regulation.

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Yamashita, Toshiharu, Osamu Ohneda, Masumi Nagano, Motoyuki Iemitsu, Yuichi Makino, Hirotoshi Tanaka, Takashi Miyauchi, et al. "Abnormal Heart Development and Lung Remodeling in Mice Lacking the Hypoxia-Inducible Factor-Related Basic Helix-Loop-Helix PAS Protein NEPAS." Molecular and Cellular Biology 28, no. 4 (December 10, 2007): 1285–97. http://dx.doi.org/10.1128/mcb.01332-07.

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ABSTRACT Hypoxia-inducible factors (HIFs) are crucial for oxygen homeostasis during both embryonic development and postnatal life. Here we show that a novel HIF family basic helix-loop-helix (bHLH) PAS (Per-Arnt-Sim) protein, which is expressed predominantly during embryonic and neonatal stages and thereby designated NEPAS (neonatal and embryonic PAS), acts as a negative regulator of HIF-mediated gene expression. NEPAS mRNA is derived from the HIF-3α gene by alternative splicing, replacing the first exon of HIF-3α with that of inhibitory PAS. NEPAS can dimerize with Arnt and exhibits only low levels of transcriptional activity, similar to that of HIF-3α. NEPAS suppressed reporter gene expression driven by HIF-1α and HIF-2α. By generating mice with a targeted disruption of the NEPAS/HIF-3α locus, we found that homozygous mutant mice (NEPAS/HIF-3α− / −) were viable but displayed enlargement of the right ventricle and impaired lung remodeling. The expression of endothelin 1 and platelet-derived growth factor β was increased in the lung endothelial cells of NEPAS/HIF-3α-null mice. These results demonstrate a novel regulatory mechanism in which the activities of HIF-1α and HIF-2α are negatively regulated by NEPAS in endothelial cells, which is pertinent to lung and heart development during the embryonic and neonatal stages.

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Liu, Yu C., Mayra A. Machuca, Simone A. Beckham, Menachem J. Gunzburg, and Anna Roujeinikova. "Structural basis for amino-acid recognition and transmembrane signalling by tandem Per–Arnt–Sim (tandem PAS) chemoreceptor sensory domains." Acta Crystallographica Section D Biological Crystallography 71, no. 10 (September 30, 2015): 2127–36. http://dx.doi.org/10.1107/s139900471501384x.

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Chemotaxis, mediated by methyl-accepting chemotaxis protein (MCP) receptors, plays an important role in the ecology of bacterial populations. This paper presents the first crystallographic analysis of the structure and ligand-induced conformational changes of the periplasmic tandem Per-Arnt-Sim (PAS) sensing domain (PTPSD) of a characterized MCP chemoreceptor. Analysis of the complex of theCampylobacter jejuniTlp3 PTPSD with isoleucine (a chemoattractant) revealed that the PTPSD is a dimer in the crystal. The two ligand-binding sites are located in the membrane-distal PAS domains on the faces opposite to the dimer interface. Mutagenesis experiments show that the five strongly conserved residues that stabilize the main-chain moiety of isoleucine are essential for binding, suggesting that the mechanism by which this family of chemoreceptors recognizes amino acids is highly conserved. Although the fold and mode of ligand binding of the PTPSD are different from the aspartic acid receptor Tar, the structural analysis suggests that the PTPSDs of amino-acid chemoreceptors are also likely to signal by a piston displacement mechanism. The PTPSD fluctuates between piston (C-terminal helix) `up' and piston `down' states. Binding of an attractant to the distal PAS domain locks it in the closed form, weakening its association with the proximal domain and resulting in the transition of the latter into an open form, concomitant with a downward (towards the membrane) 4 Å piston displacement of the C-terminal helix.In vivo, this movement would generate a transmembrane signal by driving a downward displacement of the transmembrane helix 2 towards the cytoplasm.

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Tang, Xue, Juan Shao, and Xiaohong Qin. "Crystal structure of the PAS domain of the hEAG potassium channel." Acta Crystallographica Section F Structural Biology Communications 72, no. 8 (July 13, 2016): 578–85. http://dx.doi.org/10.1107/s2053230x16009419.

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KCNH voltage-gated potassium channels play critical roles in regulating cellular functions. The channel is composed of four subunits, each of which contains six transmembrane helices forming the central pore. The cytoplasmic parts of the subunits present a Per–Arnt–Sim (PAS) domain at the N-terminus and a cyclic nucleotide-binding homology domain at the C-terminus. PAS domains are conserved from prokaryotes to eukaryotes and are involved in sensing signals and cellular responses. To better understand the functional roles of PAS domains in KCNH channels, the structure of this domain from the humanether-à-go-gochannel (hEAG channel) was determined. By comparing it with the structures of theHomo sapiensEAG-related gene (hERG) channel and theDrosophilaEAG-like K+(dELK) channel and analyzing the structural features of the hEAG channel, it was identified that a hydrophobic patch on the β-sheet may mediate interaction between the PAS domain and other regions of the channel to regulate its functions.

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An, R., G. da Silva Xavier, H. X. Hao, F. Semplici, J. Rutter, and G. A. Rutter. "Regulation by Per-Arnt-Sim (PAS) kinase of pancreatic duodenal homeobox-1 nuclear import in pancreatic β-cells." Biochemical Society Transactions 34, no. 5 (October 1, 2006): 791–93. http://dx.doi.org/10.1042/bst0340791.

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The transcription factor PDX-1 (pancreatic duodenal homeobox-1) is required for normal pancreatic development and for the function of insulin-producing islet β-cells in mammals. We have shown previously that glucose regulates insulin gene expression in part through the activation and translocation of PDX-1 from the nuclear periphery to the nucleoplasm. We have also found that PASK [PAS (Per-Arnt-Sim) kinase], a member of the nutrient-regulated family of protein kinases, is activated in response to glucose challenge in β-cells and is involved in the regulation of expression of PDX-1. Purified PASK efficiently phosphorylated recombinant PDX-1 in vitro on a single site (Thr-152). To determine the impact of phosphorylation at this site, we generated wild-type and mutant (T152A, T152D and T152E) forms of PDX-1 and examined the distribution of each of these in clonal MIN6 β-cells by immunocytochemical analysis. Unexpectedly, only the T152D mutation significantly affected subcellular distribution, increasing the ratio of nuclear/cytosolic labelling at low and high glucose concentrations, suggesting that phosphorylation at Thr-152 inhibits nuclear uptake in response to glucose. Based on these results, experiments to examine the contribution of Thr-152 to the overall phosphorylation of PDX-1 in intact cells will be undertaken.

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Gilles-Gonzalez, Marie-Alda, and Gonzalo Gonzalez. "Signal transduction by heme-containing PAS-domain proteins." Journal of Applied Physiology 96, no. 2 (February 2004): 774–83. http://dx.doi.org/10.1152/japplphysiol.00941.2003.

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The most common physiological strategy for detecting the gases oxygen, carbon monoxide, and nitric oxide is signal transduction by heme-based sensors, a broad class of modular proteins in which a heme-binding domain governs the activity of a neighboring transmitter domain. Different structures are possible for the heme-binding domains in these sensors, but, so far, the Per-ARNT-Sim motif, or PAS domain, is the one most commonly encountered. Heme-binding PAS (heme-PAS) domains can accomplish ligand-dependent switching of a variety of partner domains, including histidine kinase, phosphodiesterase, and basic helix-loop-helix (bHLH) DNA-binding modules. Proteins with heme-PAS domains occur in all kingdoms of life and are quite diverse in their physiological roles. Examples include the neuronal bHLH-PAS carbon monoxide sensor NPAS2 that is implicated in the mammalian circadian clock, the acetobacterial oxygen sensor AxPDEA1 that directs cellulose production, and the rhizobial oxygen sensor FixL, which governs nitrogen fixation. What factors determine the range of detection of these sensors? How do they transduce their signal? This review examines the recent advances in answering these questions.

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Militi, Stefania, Elizabeth S. Maywood, Colby R. Sandate, Johanna E. Chesham, Alun R. Barnard, Michael J. Parsons, Jennifer L. Vibert, et al. "Early doors (Edo) mutant mouse reveals the importance of period 2 (PER2) PAS domain structure for circadian pacemaking." Proceedings of the National Academy of Sciences 113, no. 10 (February 22, 2016): 2756–61. http://dx.doi.org/10.1073/pnas.1517549113.

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The suprachiasmatic nucleus (SCN) defines 24 h of time via a transcriptional/posttranslational feedback loop in which transactivation of Per (period) and Cry (cryptochrome) genes by BMAL1–CLOCK complexes is suppressed by PER–CRY complexes. The molecular/structural basis of how circadian protein complexes function is poorly understood. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced mutation, early doors (Edo), in the PER-ARNT-SIM (PAS) domain dimerization region of period 2 (PER2) (I324N) that accelerates the circadian clock of Per2Edo/Edo mice by 1.5 h. Structural and biophysical analyses revealed that Edo alters the packing of the highly conserved interdomain linker of the PER2 PAS core such that, although PER2Edo complexes with clock proteins, its vulnerability to degradation mediated by casein kinase 1ε (CSNK1E) is increased. The functional relevance of this mutation is revealed by the ultrashort (<19 h) but robust circadian rhythms in Per2Edo/Edo; Csnk1eTau/Tau mice and the SCN. These periods are unprecedented in mice. Thus, Per2Edo reveals a direct causal link between the molecular structure of the PER2 PAS core and the pace of SCN circadian timekeeping.

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Kolonko, Marta, and Beata Greb-Markiewicz. "bHLH–PAS Proteins: Their Structure and Intrinsic Disorder." International Journal of Molecular Sciences 20, no. 15 (July 26, 2019): 3653. http://dx.doi.org/10.3390/ijms20153653.

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The basic helix–loop–helix/Per-ARNT-SIM (bHLH–PAS) proteins are a class of transcriptional regulators, commonly occurring in living organisms and highly conserved among vertebrates and invertebrates. These proteins exhibit a relatively well-conserved domain structure: the bHLH domain located at the N-terminus, followed by PAS-A and PAS-B domains. In contrast, their C-terminal fragments present significant variability in their primary structure and are unique for individual proteins. C-termini were shown to be responsible for the specific modulation of protein action. In this review, we present the current state of knowledge, based on NMR and X-ray analysis, concerning the structural properties of bHLH–PAS proteins. It is worth noting that all determined structures comprise only selected domains (bHLH and/or PAS). At the same time, substantial parts of proteins, comprising their long C-termini, have not been structurally characterized to date. Interestingly, these regions appear to be intrinsically disordered (IDRs) and are still a challenge to research. We aim to emphasize the significance of IDRs for the flexibility and function of bHLH–PAS proteins. Finally, we propose modern NMR methods for the structural characterization of the IDRs of bHLH–PAS proteins.

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DeMille, Desiree, Benjamin T. Bikman, Andrew D. Mathis, John T. Prince, Jordan T. Mackay, Steven W. Sowa, Tacie D. Hall, and Julianne H. Grose. "A comprehensive protein–protein interactome for yeast PAS kinase 1 reveals direct inhibition of respiration through the phosphorylation of Cbf1." Molecular Biology of the Cell 25, no. 14 (July 15, 2014): 2199–215. http://dx.doi.org/10.1091/mbc.e13-10-0631.

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Per-Arnt-Sim (PAS) kinase is a sensory protein kinase required for glucose homeostasis in yeast, mice, and humans, yet little is known about the molecular mechanisms of its function. Using both yeast two-hybrid and copurification approaches, we identified the protein–protein interactome for yeast PAS kinase 1 (Psk1), revealing 93 novel putative protein binding partners. Several of the Psk1 binding partners expand the role of PAS kinase in glucose homeostasis, including new pathways involved in mitochondrial metabolism. In addition, the interactome suggests novel roles for PAS kinase in cell growth (gene/protein expression, replication/cell division, and protein modification and degradation), vacuole function, and stress tolerance. In vitro kinase studies using a subset of 25 of these binding partners identified Mot3, Zds1, Utr1, and Cbf1 as substrates. Further evidence is provided for the in vivo phosphorylation of Cbf1 at T211/T212 and for the subsequent inhibition of respiration. This respiratory role of PAS kinase is consistent with the reported hypermetabolism of PAS kinase–deficient mice, identifying a possible molecular mechanism and solidifying the evolutionary importance of PAS kinase in the regulation of glucose homeostasis.

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Chen, Jun, Anrou Zou, Igor Splawski, Mark T. Keating, and Michael C. Sanguinetti. "Long QT Syndrome-associated Mutations in the Per-Arnt-Sim (PAS) Domain of HERG Potassium Channels Accelerate Channel Deactivation." Journal of Biological Chemistry 274, no. 15 (April 9, 1999): 10113–18. http://dx.doi.org/10.1074/jbc.274.15.10113.

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Ke, Ying, Chai Ann Ng, Mark J. Hunter, Stefan A. Mann, Juliane Heide, Adam P. Hill, and Jamie I. Vandenberg. "Trafficking defects in PAS domain mutant Kv11.1 channels: roles of reduced domain stability and altered domain–domain interactions." Biochemical Journal 454, no. 1 (July 26, 2013): 69–77. http://dx.doi.org/10.1042/bj20130328.

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Loss of Kv11.1 potassium channel function is the underlying cause of pathology in long-QT syndrome type 2, one of the commonest causes of sudden cardiac death in the young. Previous studies have identified the cytosolic PAS (Per/Arnt/Sim) domain as a hotspot for mutations that cause Kv11.1 trafficking defects. To investigate the underlying basis of this observation, we have quantified the effect of mutants on domain folding as well as interactions between the PAS domain and the remainder of the channel. Apart from R56Q, all mutants impaired the thermostability of the isolated PAS domain. Six mutants, located in the vicinity of a hydrophobic patch on the PAS domain surface, also affected binding of the isolated PAS domain to an N-terminal truncated hERG (human ether-a-go-go-related gene) channel. Conversely, four other surface mutants (C64Y, T65P, A78P and I96T) and one buried mutant (L86R) did not prevent the isolated PAS domain binding to the truncated channels. Our results highlight a critical role for interactions between the PAS domain and the remainder of the channel in the hERG assembly and that mutants that affect PAS domain interactions with the remainder of the channel have a more severe trafficking defect than that caused by domain unfolding alone.

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Harley, Carol A., Greg Starek, David K. Jones, Andreia S. Fernandes, Gail A. Robertson, and João H. Morais-Cabral. "Enhancement of hERG channel activity by scFv antibody fragments targeted to the PAS domain." Proceedings of the National Academy of Sciences 113, no. 35 (August 11, 2016): 9916–21. http://dx.doi.org/10.1073/pnas.1601116113.

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The human human ether-à-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.

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Pape, Jenny, Colleen Newey, Haley Burrell, Audrey Workman, Katelyn Perry, Benjamin Bikman, Laura Bridgewater, and Julianne Grose. "Per-Arnt-Sim Kinase (PASK) Deficiency Increases Cellular Respiration on a Standard Diet and Decreases Liver Triglyceride Accumulation on a Western High-Fat High-Sugar Diet." Nutrients 10, no. 12 (December 15, 2018): 1990. http://dx.doi.org/10.3390/nu10121990.

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Diabetes and the related disease metabolic syndrome are epidemic in the United States, in part due to a shift in diet and decrease in physical exercise. PAS kinase is a sensory protein kinase associated with many of the phenotypes of these diseases, including hepatic triglyceride accumulation and metabolic dysregulation in male mice placed on a high-fat diet. Herein we provide the first characterization of the effects of western diet (high-fat high-sugar, HFHS) on Per-Arnt-Sim kinase mice (PASK−/−) and the first characterization of both male and female PASK−/− mice. Soleus muscle from the PASK−/− male mice displayed a 2-fold higher oxidative phosphorylation capacity than wild type (WT) on the normal chow diet. PASK−/− male mice were also resistant to hepatic triglyceride accumulation on the HFHS diet, displaying a 2.7-fold reduction in hepatic triglycerides compared to WT mice on the HFHS diet. These effects on male hepatic triglyceride were further explored through mass spectrometry-based lipidomics. The absence of PAS kinase was found to affect many of the 44 triglycerides analyzed, preventing hepatic triglyceride accumulation in response to the HFHS diet. In contrast, the female mice showed resistance to hepatic triglyceride accumulation on the HFHS diet regardless of genotype, suggesting the effects of PAS kinase may be masked.

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Soshilov, Anatoly A., Stefano Motta, Laura Bonati, and Michael S. Denison. "Transitional States in Ligand-Dependent Transformation of the Aryl Hydrocarbon Receptor into Its DNA-Binding Form." International Journal of Molecular Sciences 21, no. 7 (April 2, 2020): 2474. http://dx.doi.org/10.3390/ijms21072474.

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The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological and toxicological effects of an AhR lacking the entire PASB structurally diverse chemicals, including halogenated aromatic hydrocarbons. Ligand-dependent transformation of the AhR into its DNA binding form involves a ligand-dependent conformational change, heat shock protein 90 (hsp90), dissociation from the AhR complex and AhR dimerization with the AhR nuclear translocator (ARNT) protein. The mechanism of AhR transformation was examined using mutational approaches and stabilization of the AhR:hsp90 complex with sodium molybdate. Insertion of a single mutation (F281A) in the hsp90-binding region of the AhR resulted in its constitutive (ligand-independent) transformation/DNA binding in vitro. Mutations of AhR residues within the Arg-Cys-rich region (R212A, R217A, R219A) and Asp371 (D371A) impaired AhR transformation without a significant effect on ligand binding. Stabilization of AhR:hsp90 binding with sodium molybdate decreased transformation/DNA binding of the wild type AhR but had no effect on constitutively active AhR mutants. Interestingly, transformation of the AhR in the presence of molybdate allowed detection of an intermediate transformation ternary complex containing hsp90, AhR, and ARNT. These results are consistent with a stepwise transformation mechanism in which binding of ARNT to the liganded AhR:hsp90 complex results in a progressive displacement of hsp90 and conversion of the AhR into its high affinity DNA binding form. The available molecular insights into the signaling mechanism of other Per-ARNT-Sim (PAS) domains and structural information on hsp90 association with other client proteins are consistent with the proposed transformation mechanism of the AhR.

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Kolonko-Adamska, Marta, Vladimir N. Uversky, and Beata Greb-Markiewicz. "The Participation of the Intrinsically Disordered Regions of the bHLH-PAS Transcription Factors in Disease Development." International Journal of Molecular Sciences 22, no. 6 (March 11, 2021): 2868. http://dx.doi.org/10.3390/ijms22062868.

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The basic helix–loop–helix/Per-ARNT-SIM (bHLH-PAS) proteins are a family of transcription factors regulating expression of a wide range of genes involved in different functions, ranging from differentiation and development control by oxygen and toxins sensing to circadian clock setting. In addition to the well-preserved DNA-binding bHLH and PAS domains, bHLH-PAS proteins contain long intrinsically disordered C-terminal regions, responsible for regulation of their activity. Our aim was to analyze the potential connection between disordered regions of the bHLH-PAS transcription factors, post-transcriptional modifications and liquid-liquid phase separation, in the context of disease-associated missense mutations. Highly flexible disordered regions, enriched in short motives which are more ordered, are responsible for a wide spectrum of interactions with transcriptional co-regulators. Based on our in silico analysis and taking into account the fact that the functions of transcription factors can be modulated by posttranslational modifications and spontaneous phase separation, we assume that the locations of missense mutations inducing disease states are clearly related to sequences directly undergoing these processes or to sequences responsible for their regulation.

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Burton, Mark J., Joel Cresser-Brown, Morgan Thomas, Nicola Portolano, Jaswir Basran, Samuel L. Freeman, Hanna Kwon, et al. "Discovery of a heme-binding domain in a neuronal voltage-gated potassium channel." Journal of Biological Chemistry 295, no. 38 (July 28, 2020): 13277–86. http://dx.doi.org/10.1074/jbc.ra120.014150.

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The EAG (ether-à-go-go) family of voltage-gated K+ channels are important regulators of neuronal and cardiac action potential firing (excitability) and have major roles in human diseases such as epilepsy, schizophrenia, cancer, and sudden cardiac death. A defining feature of EAG (Kv10–12) channels is a highly conserved domain on the N terminus, known as the eag domain, consisting of a Per–ARNT–Sim (PAS) domain capped by a short sequence containing an amphipathic helix (Cap domain). The PAS and Cap domains are both vital for the normal function of EAG channels. Using heme-affinity pulldown assays and proteomics of lysates from primary cortical neurons, we identified that an EAG channel, hERG3 (Kv11.3), binds to heme. In whole-cell electrophysiology experiments, we identified that heme inhibits hERG3 channel activity. In addition, we expressed the Cap and PAS domain of hERG3 in Escherichia coli and, using spectroscopy and kinetics, identified the PAS domain as the location for heme binding. The results identify heme as a regulator of hERG3 channel activity. These observations are discussed in the context of the emerging role for heme as a regulator of ion channel activity in cells.

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Ke, Ying, Mark J. Hunter, Chai Ann Ng, Matthew D. Perry, and Jamie I. Vandenberg. "Role of the Cytoplasmic N-terminal Cap and Per-Arnt-Sim (PAS) Domain in Trafficking and Stabilization of Kv11.1 Channels." Journal of Biological Chemistry 289, no. 20 (April 2, 2014): 13782–91. http://dx.doi.org/10.1074/jbc.m113.531277.

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Wang, Ze-Jun, Stephanie M. Soohoo, Purushottam B. Tiwari, Grzegorz Piszczek, and Tinatin I. Brelidze. "Chlorpromazine binding to the PAS domains uncovers the effect of ligand modulation on EAG channel activity." Journal of Biological Chemistry 295, no. 13 (February 11, 2020): 4114–23. http://dx.doi.org/10.1074/jbc.ra119.012377.

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Ether-a-go-go (EAG) potassium selective channels are major regulators of neuronal excitability and cancer progression. EAG channels contain a Per–Arnt–Sim (PAS) domain in their intracellular N-terminal region. The PAS domain is structurally similar to the PAS domains in non-ion channel proteins, where these domains frequently function as ligand-binding domains. Despite the structural similarity, it is not known whether the PAS domain can regulate EAG channel function via ligand binding. Here, using surface plasmon resonance, tryptophan fluorescence, and analysis of EAG currents recorded in Xenopus laevis oocytes, we show that a small molecule chlorpromazine (CH), widely used as an antipsychotic medication, binds to the isolated PAS domain of EAG channels and inhibits currents from these channels. Mutant EAG channels that lack the PAS domain show significantly lower inhibition by CH, suggesting that CH affects currents from EAG channels directly through the binding to the PAS domain. Our study lends support to the hypothesis that there are previously unaccounted steps in EAG channel gating that could be activated by ligand binding to the PAS domain. This has broad implications for understanding gating mechanisms of EAG and related ERG and ELK K+ channels and places the PAS domain as a new target for drug discovery in EAG and related channels. Up-regulation of EAG channel activity is linked to cancer and neurological disorders. Our study raises the possibility of repurposing the antipsychotic drug chlorpromazine for treatment of neurological disorders and cancer.

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da Silva Xavier, G., J. Rutter, and G. A. Rutter. "Involvement of Per-Arnt-Sim (PAS) kinase in the stimulation of preproinsulin and pancreatic duodenum homeobox 1 gene expression by glucose." Proceedings of the National Academy of Sciences 101, no. 22 (May 17, 2004): 8319–24. http://dx.doi.org/10.1073/pnas.0307737101.

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da Silva Xavier, G., H. Farhan, H. Kim, S. Caxaria, P. Johnson, S. Hughes, M. Bugliani, et al. "Per-arnt-sim (PAS) domain-containing protein kinase is downregulated in human islets in type 2 diabetes and regulates glucagon secretion." Diabetologia 54, no. 4 (December 23, 2010): 819–27. http://dx.doi.org/10.1007/s00125-010-2010-7.

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Heintz, Udo, Anton Meinhart, and Andreas Winkler. "Multi-PAS domain-mediated protein oligomerization of PpsR fromRhodobacter sphaeroides." Acta Crystallographica Section D Biological Crystallography 70, no. 3 (February 27, 2014): 863–76. http://dx.doi.org/10.1107/s1399004713033634.

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Per–ARNT–Sim (PAS) domains are essential modules of many multi-domain signalling proteins that mediate protein interaction and/or sense environmental stimuli. Frequently, multiple PAS domains are present within single polypeptide chains, where their interplay is required for protein function. Although many isolated PAS domain structures have been reported over the last decades, only a few structures of multi-PAS proteins are known. Therefore, the molecular mechanism of multi-PAS domain-mediated protein oligomerization and function is poorly understood. The transcription factor PpsR fromRhodobacter sphaeroidesis such a multi-PAS domain protein that, in addition to its three PAS domains, contains a glutamine-rich linker and a C-terminal helix–turn–helix DNA-binding motif. Here, crystal structures of two N-terminally and C-terminally truncated PpsR variants that comprise a single (PpsRQ-PAS1) and two (PpsRN-Q-PAS1) PAS domains, respectively, are presented and the multi-step strategy required for the phasing of a triple PAS domain construct (PpsRΔHTH) is illustrated. While parts of the biologically relevant dimerization interface can already be observed in the two shorter constructs, the PpsRΔHTHstructure reveals how three PAS domains enable the formation of multiple oligomeric states (dimer, tetramer and octamer), highlighting that not only the PAS cores but also their α-helical extensions are essential for protein oligomerization. The results demonstrate that the long helical glutamine-rich linker of PpsR results from a direct fusion of the N-cap of the PAS1 domain with the C-terminal extension of the N-domain that plays an important role in signal transduction.

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Torii, Satoru, Shuya Kasai, Tatsushi Yoshida, Ken-ichi Yasumoto, and Shigeomi Shimizu. "Mitochondrial E3 Ubiquitin Ligase Parkin: Relationships with Other Causal Proteins in Familial Parkinson’s Disease and Its Substrate-Involved Mouse Experimental Models." International Journal of Molecular Sciences 21, no. 4 (February 11, 2020): 1202. http://dx.doi.org/10.3390/ijms21041202.

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Parkinson’s disease (PD) is a common neurodegenerative disorder. Recent identification of genes linked to familial forms of PD has revealed that post-translational modifications, such as phosphorylation and ubiquitination of proteins, are key factors in disease pathogenesis. In PD, E3 ubiquitin ligase Parkin and the serine/threonine-protein kinase PTEN-induced kinase 1 (PINK1) mediate the mitophagy pathway for mitochondrial quality control via phosphorylation and ubiquitination of their substrates. In this review, we first focus on well-characterized PINK1 phosphorylation motifs. Second, we describe our findings concerning relationships between Parkin and HtrA2/Omi, a protein involved in familial PD. Third, we describe our findings regarding inhibitory PAS (Per/Arnt/Sim) domain protein (IPAS), a member of PINK1 and Parkin substrates, involved in neurodegeneration during PD. IPAS is a dual-function protein involved in transcriptional repression of hypoxic responses and the pro-apoptotic activities.

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Karakkat, Jimsheena V., Suneesh Kaimala, Sreejisha P. Sreedharan, Princy Jayaprakash, Ernest A. Adeghate, Suraiya A. Ansari, Ernesto Guccione, Eric P. K. Mensah-Brown, and Bright Starling Emerald. "The metabolic sensor PASK is a histone 3 kinase that also regulates H3K4 methylation by associating with H3K4 MLL2 methyltransferase complex." Nucleic Acids Research 47, no. 19 (September 16, 2019): 10086–103. http://dx.doi.org/10.1093/nar/gkz786.

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Abstract The metabolic sensor Per-Arnt-Sim (Pas) domain-containing serine/threonine kinase (PASK) is expressed predominantly in the cytoplasm of different cell types, although a small percentage is also expressed in the nucleus. Herein, we show that the nuclear PASK associates with the mammalian H3K4 MLL2 methyltransferase complex and enhances H3K4 di- and tri-methylation. We also show that PASK is a histone kinase that phosphorylates H3 at T3, T6, S10 and T11. Taken together, these results suggest that PASK regulates two different H3 tail modifications involving H3K4 methylation and H3 phosphorylation. Using muscle satellite cell differentiation and functional analysis after loss or gain of Pask expression using the CRISPR/Cas9 system, we provide evidence that some of the regulatory functions of PASK during development and differentiation may occur through the regulation of these histone modifications.

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Kimura, Satoshi, Yoshihiro Shiraiwa, and Iwane Suzuki. "Function of the N-terminal region of the phosphate-sensing histidine kinase, SphS, in Synechocystis sp. PCC 6803." Microbiology 155, no. 7 (July 1, 2009): 2256–64. http://dx.doi.org/10.1099/mic.0.028514-0.

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In Synechocystis sp. PCC 6803 the histidine kinase SphS (sll0337) is involved in transcriptional activation of the phosphate (Pi)-acquisition system which includes alkaline phosphatase (AP). The N-terminal region of SphS contains both a hydrophobic region and a Per-Arnt-Sim (PAS) domain. The C-terminal region has a highly conserved transmitter domain. Immunological localization studies on heterologously expressed SphS in Escherichia coli indicate that the hydrophobic region is important for membrane localization. In order to evaluate the function of the N-terminal region of SphS, deletion mutants under the control of the native promoter were analysed for in vivo AP activity. Deletion of the N-terminal hydrophobic region resulted in loss of AP activity under both Pi-deficient and Pi-sufficient conditions. Substitution of the hydrophobic region of SphS with that from the Ni2+-sensing histidine kinase, NrsS, resulted in the same induction characteristics as SphS. Deletion of the PAS domain resulted in the constitutive induction of AP activity regardless of Pi availability. To characterize the PAS domain in more in detail, four amino acid residues conserved in the PAS domain were substituted with Ala. Among the mutants R121A constitutively expressed AP activity, suggesting that R121 is important for the function of the PAS domain. Our observations indicated that the presence of a transmembrane helix in the N-terminal region of SphS is critical for activity and that the PAS domain is involved in perception of Pi availability.

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Katschinski, Dörthe M., Hugo H. Marti, Klaus F. Wagner, Junpei Shibata, Katrin Eckhardt, Falk Martin, Reinhard Depping, et al. "Targeted Disruption of the Mouse PAS Domain Serine/Threonine Kinase PASKIN." Molecular and Cellular Biology 23, no. 19 (October 1, 2003): 6780–89. http://dx.doi.org/10.1128/mcb.23.19.6780-6789.2003.

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ABSTRACT PASKIN is a novel mammalian serine/threonine kinase containing two PAS (Per-Arnt-Sim) domains. PASKIN is related to the Rhizobium oxygen sensor protein FixL and to AMP-regulated kinases. Like FixL, the sensory PAS domain of PASKIN controls the kinase activity by autophosphorylation in a (unknown) ligand-dependent manner. In Saccharomyces cerevisiae, the two PASKIN orthologues PSK1 and PSK2 phosphorylate three translation factors and two enzymes involved in glycogen synthesis, thereby coordinately regulating protein synthesis and glycolytic flux. To elucidate the function of mammalian PASKIN, we inactivated the mouse Paskin gene by homologous recombination in embryonic stem cells. Paskin −/− mice showed normal development, growth, and reproduction. The targeted integration of a lacZ reporter gene allowed the identification of the cell types expressing mouse PASKIN. Surprisingly, PASKIN expression is strongly upregulated in postmeiotic germ cells during spermatogenesis. However, fertility and sperm production and motility were not affected by the PASKIN knockout. The Ppp1r7 gene encoding Sds22, a regulatory subunit of protein phosphatase 1, shares the promoter region with the Paskin gene, pointing towards a common transcriptional regulation. Indeed, Sds22 colocalized with the cell types expressing PASKIN in vivo, suggesting a functional role of protein phosphatase-1 in the regulation of PASKIN autophosphorylation.

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Journal articles on the topic 'Per Arnt Sim (PAS)'

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