Prostaglandins as Candidate Ligands for a Per-ARNT-Sim (PAS) Domain of Steroid Receptor Coactivator 1 (SRC1).

Steroid receptor coactivators (SRCs) comprise a family of three paralogous proteins commonly recruited by eukaryotic transcription factors. Each SRC harbors two tandem Per-ARNT-Sim (PAS) domains that are broadly distributed that bind small molecules and regulate interactions. Using computational docking, solution NMR, mass spectrometry, and molecular dynamics simulations, we show that the SRC1 PAS-B domain can bind to certain prostaglandins (PGs) either non-covalently to a surface that overlaps with the site used to engage transcription factors or covalently to a single, specific, conserved cysteine residue next to a solvent accessible hydrophobic pocket. This pocket is in proximity to the canonical transcription factor binding site, but on the opposite side of the domain, suggesting a potential mode of regulating transcriptional activator-coactivator interactions.

Photoactive Yellow Protein Represents a Distinct, Evolutionarily Novel Family of PAS Domains.

Photoactive yellow protein (PYP) is a model photoreceptor. It binds a p-coumaric acid as a chromophore, thus enabling blue light sensing. The first discovered single-domain PYP from Halorhodospira halophila has been studied thoroughly in terms of its structural dynamics and photochemical properties. However, the evolutionary origins and biological role of PYP homologs are not well understood. Here, we show that PYP is an evolutionarily novel domain family of the ubiquitous PAS (Per-Arnt-Sim) superfamily. It likely originated from the phylum Myxococcota and was then horizontally transferred to representatives of a few other bacterial phyla. We show that PYP is associated with signal transduction either by domain fusion or by genome context. Key cellular functions modulated by PYP-initiated signal transduction pathways likely involve gene expression, motility, and biofilm formation. We identified three clades of the PYP family, one of which is poorly understood and potentially has novel functional properties. The Tyr42, Glu46, and Cys69 residues that are involved in p-coumaric acid binding in the model PYP from H. halophila are well conserved in the PYP family. However, we also identified cases where substitutions in these residues might have led to neofunctionalization, such as the proposed transition from light to redox sensing. Overall, this study provides definition, a newly built hidden Markov model, and the current genomic landscape of the PYP family and sets the stage for the future exploration of its signaling mechanisms and functional diversity. IMPORTANCE Photoactive yellow protein is a model bacterial photoreceptor. For many years, it was considered a prototypical model of the ubiquitous PAS domain superfamily. Here, we show that, in fact, the PYP family is evolutionarily novel, restricted to a few bacterial phyla and distinct from other PAS domains. We also reveal the diversity of PYP-containing signal transduction proteins and their potential mechanisms.

A Novel Mechanism of Coactivator Recruitment by the Nurr1 Nuclear Receptor.

Nuclear receptors constitute one of the largest families of transcription factors that regulate genes in metazoans in response to small molecule ligands. Many receptors harbor two transactivation domains, one at each end of the protein sequence. Whereas the molecular mechanisms of transactivation mediated by the ligand-binding domain at the C-terminus of the protein are generally well established, the mechanism involving the N-terminal domain called activation function 1 (AF1) has remained elusive. Previous studies implicated the AF1 domain as a significant contributor towards the overall transcriptional activity of the NR4A family of nuclear receptors and suggested that the steroid receptor coactivators (SRCs) play an important role in this process. Here we show that a short segment within the AF1 domain of the NR4A receptor Nurr1 can directly engage with the SRC1 PAS-B domain. We also show that this segment forms a helix upon binding to a largely hydrophobic groove on PAS-B, overlapping with the surface engaged by the STAT6 transcription factor, suggesting that this mode of recruitment could be shared by diverse transcription factors including other nuclear receptors.

Diurnal control of intracellular distributions of PAS-Histidine kinase 1 and its interactions with partner proteins in the moss Physcomitrium patens.

In multi-step phosphorelay (MSP) signaling, upon reception of various environmental signals, histidine kinases (HKs) induce autophosphorylation and subsequent phosphotransfer to partner histidine-containing phosphotransfer proteins (HPts). Recently, we reported that (i) two Per-Arnt-Sim (PAS) domain-containing HKs (PHK1 and PHK2) of the moss Physcomitrium (Physcomitrella) patens suppressed red light-induced branching of protonema tissue, and (ii) they interacted with partner HPts (HPt1 and HPt2) in the nucleus in the dark while cytoplasmic interactions also occurred under red light. Here we demonstrate that PHK1 is diurnally regulated, i.e., it is localized and interacts with HPt1 and HPt2 in the nucleus at night whereas these activities reversibly expand and become nucleocytoplasmic in the day. In the dark, PHK1 interacts with HPts only in the nucleus, even in subjective daytime, indicating that endogenous regulation by the circadian clock is not involved. These results suggest that PHK1 is a regulator of moss' adaptation to a light environment on a daily timescale. We discuss a possible regulatory mechanism for the branching of protonema.

Investigating the effect of an identified mutation within a critical site of PAS domain of WalK protein in a vancomycin-intermediate resistant Staphylococcus aureus by computational approaches.

BACKGROUND: Vancomycin-intermediate resistant Staphylococcus aureus (VISA) is becoming a common cause of nosocomial infections worldwide. VISA isolates are developed by unclear molecular mechanisms via mutations in several genes, including walKR. Although studies have verified some of these mutations, there are a few studies that pay attention to the importance of molecular modelling of mutations. METHOD: For genomic and transcriptomic comparisons in a laboratory-derived VISA strain and its parental strain, Sanger sequencing and reverse transcriptase quantitative PCR (RT-qPCR) methods were used, respectively. After structural protein mapping of the detected mutation, mutation effects were analyzed using molecular computational approaches and crystal structures of related proteins. RESULTS: A mutation WalK-H364R was occurred in a functional zinc ion coordinating residue within the PAS domain in the VISA strain. WalK-H364R was predicted to destabilize protein and decrease WalK interactions with proteins and nucleic acids. The RT-qPCR method showed downregulation of walKR, WalKR-regulated autolysins, and agr locus. CONCLUSION: Overall, WalK-H364R mutation within a critical metal-coordinating site was presumably related to the VISA development. We assume that the WalK-H364R mutation resulted in deleterious effects on protein, which was verified by walKR gene expression changes.. Therefore, molecular modelling provides detailed insight into the molecular mechanism of VISA development, in particular, where allelic replacement experiments are not readily available.

Differential modulation of Ahr and Arid5a: A promising therapeutic strategy for autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune disease that involves demyelination of axons in the central nervous system (CNS) and affects patients worldwide. It has been demonstrated that ligand-activated aryl hydrocarbon receptor (Ahr) ameliorates experimental autoimmune encephalomyelitis (EAE), a murine model of MS, by increasing CD4+FoxP3+ T cells. Recent evidence indicates that AT-rich interactive domain-containing protein 5a (Arid5a) is required for EAE pathogenesis by stabilizing Il6 and OX40 mRNAs. However, the differential modulation of Ahr and Arid5a in autoimmunity as a therapeutic strategy is unexplored. Herein, an in silico, in vitro and in vivo approach identified Flavipin (3,4,5-trihydroxy-6-methylphthalaldehyde) as an Ahr agonist that induces the expression of Ahr downstream genes in mouse CD4+ T cells and CD11b+ macrophages. Interestingly, Flavipin inhibited the stabilizing function of Arid5a and its counteracting effects on Regnase-1 on the 3' untranslated region (3'UTR) of target mRNAs. Furthermore, it inhibited the stabilizing function of Arid5a on Il23a 3'UTR, a newly identified target mRNA. In EAE, Flavipin ameliorated disease severity, with reduced CD4+IL-17+ T cells, IL-6 and TNF-α and increased CD4+FoxP3+ T cells. Moreover, EAE amelioration was concomitant with reduced CD4+OX40+ and CD4+CD45+ T cells in the CNS. RNA interference showed that the modulatory effects of Flavipin on pro- and anti-inflammatory mediators in CD4+ T cells and macrophages were Ahr- and/or Arid5a-dependent. In conclusion, our findings reveal differential modulation of Ahr and Arid5a as a new therapeutic strategy for MS.

Treadmill training increases the motor activity and neuron survival of the cerebellum in a mouse model of spinocerebellar ataxia type 1.

Spinocerebellar ataxia (SCA) type 1 (SCA1) is a rare autosomal dominant disorder that is characterized by worsening of disordered coordination, ataxia of the trunk, and other neurological symptoms. Physical activity improves both mobility and the daily living activities of patients with SCA. Intervention with daily regular treadmill exercise may slow the deterioration of cerebellar neurons in SCA1. Therefore, the signal changes and performance of cerebellar neurons after exercise in SCA1 was investigated in this study. We employed a transgenic mouse model of SCA1, generated by amplifying the cytosine-adenine-guanine trinucleotide repeat expansions, and the mice underwent 1 month of moderate daily treadmill exercise for 1 hour. The rotarod test revealed that the motor function of the SCA1 mice that underwent training was superior to that of the control SCA1 mice, which did not undergo training. Moreover, the cerebellar pathology revealed preserved Purkinje neurons stained by carbindin with an increase of the neuronal Per Arnt Sim domain protein 4, a key regulation in the structural and functional plasticity of neurons, in the excised SCA1 mice relative to the controls. The mechanism was related to an increase of phosphorylation of ribosomal protein S6, a downstream target of the mammalian target of rapamycin pathway, but not to autophagy activation. This study determined that regular treadmill exercise may play a crucial role in the viable support of cerebellar neurons in SCA1.

Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins.

The discovery of light-inducible protein-protein interactions has allowed for the spatial and temporal control of a variety of biological processes. To be effective, a photodimerizer should have several characteristics: it should show a large change in binding affinity upon light stimulation, it should not cross-react with other molecules in the cell, and it should be easily used in a variety of organisms to recruit proteins of interest to each other. To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa. In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB. Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation. Here, we describe the use of computational protein design, phage display, and high-throughput binding assays to create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation. A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark. We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.

Decreased expression of neuronal Per-Arnt-Sim domain protein 4 gene in the hippocampus of a post-stroke depression rat model.

Neuronal Per-Arnt-Sim domain protein 4 (NPAS4) is important in regulating transcription and function in the limbic system and in brain development. Post-stroke depression (PSD) is a common complication following a stroke. Furthermore, organic damage as a result of a stroke affects the restoration of nerve function and indicates that hippocampal neural activity may be associated with PSD. A PSD rat model was established via a middle cerebral artery occlusion procedure, which was combined with isolation and chronic unexpected mild stress, and was used to investigate the expression of the NPAS4 gene in the hippocampus. The neurological deficit and behavior were evaluated and NPAS4 mRNA expression was measured by semi-quantitative reverse transcription-polymerase chain reaction; furthermore, the association with cognitive impairment was analyzed. The PSD rats displayed neuropsychopathic disorders and the NPAS4 mRNA expression levels in the hippocampus were significantly lower in the depression and PSD groups compared with the control group. Therefore, the present study identified that NPAS4 expression was decreased in the hippocampus of PSD rats.

The variant hERG/R148W associated with LQTS is a mutation that reduces current density on co-expression with the WT.

BACKGROUND: A variant of the ether-à-go-go related channel (hERG), p.Arg148Trp (R148W) was found at heterozygous state in two infants who died from sudden infant death syndrome (SIDS), one with documented prolonged QTc and Torsade de Pointes (TdP), and in an adult woman with QTc >500 ms, atrioventricular block and TdP. This variant was previously reported in cases of severe ventricular arrhythmia but very rarely in control subjects. Its classification as mutation or polymorphism awaited electrophysiological characterization. METHODS: The properties of this N-terminal, proximal domain, hERG variant were explored in Xenopus oocytes injected with the same amount of RNA encoding for either hERG/WT or hERG/R148W or their equimolar mixture. The human ventricular cell (TNNP) model was used to test the effects of changes in hERG current. RESULTS: R148W alone produced a current similar to the WT (369 ± 76 nA (mean ± SEM), n=13 versus 342 ± 55 nA in WT, n=13), while the co-expression of 1/2 WT+1/2 R148W lowered the current by 29% versus WT (243 ± 35 nA, n=13, p<0.05). The voltage dependencies of steady-state activation and inactivation were not changed in the variant alone or in co-expression with the WT. The time constants of fast recovery from inactivation and of fast and slow deactivation analyzed between -120 and +20 mV were not changed. The voltage-dependent distribution of the current amplitudes among fast-, slow- and non-deactivating fractions was unaltered. A 6.6% increase in APD90 from 323.5 ms to 345 ms was observed using the human cardiac ventricular myocyte model. CONCLUSIONS: Such a decrease in hERG current as evidenced here when co-expressing the hERG/R148W variant with the WT may have predisposed to the observed long QT syndrome and associated TdP. Therefore, the heterozygous carriers of hERG/R148W may be at risk of cardiac sudden death.

Sesquiterpene action, and morphogenetic signaling through the ortholog of retinoid X receptor, in higher Diptera.

Morphogenetic signaling by small terpenoid hormones is a common feature of both vertebrate and invertebrate development. Most attention on insect developmental signaling by small terpenoids has focused on signaling by juvenile hormone through bHLH-PAS proteins (e.g., the MET protein), especially as that signaling axis intersects with ecdysteroid action through the receptor EcR. However, a series of endocrine and pharmacological studies on pupariation in cyclorrhaphous Diptera have remained persistently refractory to explanation with the above two-axis model. Recently, the terpenoid compound methyl farnesoate has been physicochemically demonstrated to exist in circulation at physiological concentrations, in several mecopterid orders, including Diptera. In addition, it has also been recently demonstrated that the receptor to which methyl farnesoate binds with nanomolar affinity (ultraspiracle, an ortholog of retinoid X receptor) requires a functioning ligand binding pocket to sustain the morphogenetic transition to puparium formation. This review evaluates endocrine and pharmacological evidence for developmental pathways reached by methyl farnesoate action, and assesses the participation of the retinoid X receptor ligand pocket in signal transduction to those developmental endpoints.

LQT2 nonsense mutations generate trafficking defective NH2-terminally truncated channels by the reinitiation of translation.

The human ether-a-go-go-related gene (hERG) encodes a voltage-activated K(+) channel that contributes to the repolarization of the cardiac action potential. Long QT syndrome type 2 (LQT2) is an autosomal dominant disorder caused by mutations in hERG, and patients with LQT2 are susceptible to severe ventricular arrhythmias. We have previously shown that nonsense and frameshift LQT2 mutations caused a decrease in mutant mRNA by the nonsense-mediated mRNA decay (NMD) pathway. The Q81X nonsense mutation was recently found to be resistant to NMD. Translation of Q81X is reinitiated at Met(124), resulting in the generation of NH2-terminally truncated hERG channels with altered gating properties. In the present study, we identified two additional NMD-resistant LQT2 nonsense mutations, C39X and C44X, in which translation is reinitiated at Met(60). Deletion of the first 59 residues of the channel truncated nearly one-third of the highly structured Per-Arnt-Sim domain and resulted in the generation of trafficking-defective proteins and a complete loss of hERG current. Partial deletion of the Per-Arnt-Sim domain also resulted in the accelerated degradation of the mutant channel proteins. The coexpression of mutant and wild-type channels did not significantly disrupt the function and trafficking properties of wild-type hERG. Our present findings indicate that translation reinitiation may generate trafficking-defective as well as dysfunctional channels in patients with LQT2 premature termination codon mutations that occur early in the coding sequence.

Crystal structure of the Alpha subunit PAS domain from soluble guanylyl cyclase.

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein of ≈ 150 kDa and the primary nitric oxide receptor. Binding of NO stimulates cyclase activity, leading to regulation of cardiovascular physiology and providing attractive opportunities for drug discovery. How sGC is stimulated and where candidate drugs bind remains unknown. The α and ß sGC chains are each composed of Heme-Nitric Oxide Oxygen (H-NOX), Per-ARNT-Sim (PAS), coiled-coil and cyclase domains. Here, we present the crystal structure of the α1 PAS domain to 1.8 Å resolution. The structure reveals the binding surfaces of importance to heterodimer function, particularly with respect to regulating NO binding to heme in the ß1 H-NOX domain. It also reveals a small internal cavity that may serve to bind ligands or participate in signal transduction.

Novel alternative splice variants of chicken NPAS3 are expressed in the developing central nervous system.

We report isolation of novel splice variants of chicken Neuronal Per-Arnt-Sim domain protein 3 (cNPAS3) gene distinct from the previously predicted cNPAS3 at the 5' end. Newly identified cNPAS3 splice variants feature N-terminus coding sequences with high degrees of homology to human NPAS3 (hNAPS3). We also show that the alternative splicing pattern of NPAS3 is conserved between chicken and human. RNA in situ hybridization indicated that the expression of cNPAS3 in the developing central nervous system (CNS) is limited to the ventricular zone and only partially overlaps with that of chicken Reelin (cReelin), the only known regulatory target gene of NPAS3 in the adult brain. Overexpression of cNPAS3 by in ovo electroporation had little effect on the expression of Sox2, a marker for neural precursors, or of Isl1/2, a marker for early differentiating motor neurons. Taken together with the little effect of cNPAS3 overexpression on cReelin, it is noted that the function of NPAS3 in the developing CNS remains to be determined. Still, identification of proper cDNA sequences for cNPAS3 should represent a solid beginning of the understanding process.