Erinacine C Activates Transcription from a Consensus ETS DNA Binding Site in Astrocytic Cells in Addition to NGF Induction.

Medicinal mushrooms of the genus Hericium are known to produce secondary metabolites with homeostatic properties for the central nervous system. We and others have recently demonstrated that among these metabolites cyathane diterpenoids and in particular erinacine C possess potent neurotrophin inducing properties in astrocytic cells. Yet, the signaling events downstream of erinacine C induced neurotrophin acitivity in neural-like adrenal phaeochromocytoma cells (PC12) cells have remained elusive. Similar, signaling events activated by erinacine C in astrocytic cells are unknown. Using a combination of genetic and pharmacological inhibitors we show that erinacine C induced neurotrophic activity mediates PC12 cell differentiation via the TrkA receptor and likely its associated PLCγ-, PI3K-, and MAPK/ERK pathways. Furthermore, a small library of transcriptional activation reporters revealed that erinacine C induces transcriptional activation mediated by DNA consensus binding sites of selected conserved transcription factor families. Among these, transcription is activated from an ETS consensus in a concentration dependent manner. Interestingly, induced ETS-consensus transcription occurs in parallel and independent of neurotrophin induction. This finding helps to explain the many pleiotropic functions of cyathane diterpenoids. Moreover, our studies provide genetic access to cyathane diterpenoid functions in astrocytic cells and help to mechanistically understand the action of cyathanes in glial cells.

Intracellular Binding Site for a Positive Allosteric Modulator of the Dopamine D1 Receptor.

The binding site for DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one], a positive allosteric modulator (PAM) of the dopamine D1 receptor, was identified and compared with the binding site for CID 2886111 [N-(6-tert-butyl-3-carbamoyl-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)pyridine-4-carboxamide], a reference D1 PAM. From D1/D5 chimeras, the site responsible for potentiation by DETQ of the increase in cAMP in response to dopamine was narrowed down to the N-terminal intracellular quadrant of the receptor; arginine-130 in intracellular loop 2 (IC2) was then identified as a critical amino acid based on a human/rat species difference. Confirming the importance of IC2, a ß2-adrenergic receptor construct in which the IC2 region was replaced with its D1 counterpart gained the ability to respond to DETQ. A homology model was built from the agonist-state ß2-receptor structure, and DETQ was found to dock to a cleft created by IC2 and adjacent portions of transmembrane helices 3 and 4 (TM3 and TM4). When residues modeled as pointing into the cleft were mutated to alanine, large reductions in the potency of DETQ were found for Val119 and Trp123 (flanking the conserved DRY sequence in TM3), Arg130 (located in IC2), and Leu143 (TM4). The D1/D5 difference was found to reside in Ala139; changing this residue to methionine as in the D5 receptor reduced the potency of DETQ by approximately 1000-fold. None of these mutations affected the activity of CID 2886111, indicating that it binds to a different allosteric site. When combined, DETQ and CID 2886111 elicited a supra-additive response in the absence of dopamine, implying that both PAMs can bind to the D1 receptor simultaneously.

Potent influenza A virus entry inhibitors targeting a conserved region of hemagglutinin.

Influenza A viruses (IAVs) induce acute respiratory disease and cause significant morbidity and mortality throughout the world. With the emergence of drug-resistant viral strains, new and effective anti-IAV drugs with different modes of action are urgently needed. In this study, by conjugating cholesterol to the N-terminus of the short peptide KKWK, a lipopeptide named S-KKWK was created. The anti-IAV test indicated that S-KKWK and its derivatives displayed potent antiviral activities against a broad variety of influenza A viral strains including oseltamivir-resistant strains and clinically relevant isolates with IC50 values ranging from 0.7 to 3.0µM. An extensive mechanistic study showed that these peptides functioned as viral "entry blockers" by inhibiting the conformational rearrangements of HA2 subunit, thereby interrupting the fusion of virus-host cell membranes. Significantly, a computer-aided docking simulation and protein sequence alignment identified conserved residues in the stem region of HA2 as the possible binding site of S-KKWK, which may be employed as a potential drug target for designing anti-IAVs with a broad-spectrum of activity. By targeting this region, a potent anti-IAV agent was subsequently created. In addition, the anti-IAV activity of S-KKWK was assessed by experiments with influenza A virus-infected mice, in which S-KKWK reduced the mortality of infected animals and extended survival time significantly. Overall, in addition to providing a strategy for designing broad-spectrum anti-IAV agents, these results indicate that S-KKWK and its derivatives are prospective candidates for potent antivirals.

Pyrimidine sulfonylacetanilides with improved potency against key mutant viruses of HIV-1 by specific targeting of a highly conserved residue.

Based on molecular simulation, the etravirine-VRX-480773 hybrids previously disclosed by our group were optimized to yield novel pyrimidine sulfonylacetanilides 8 with improved activity against a panel of seven clinically relevant single and double mutant strains of HIV-1. The improvement in potency in this in vitro model of HIV RNA replication partly validates the mechanism by which this class of allosteric pyrimidine derivatives inhibits the reverse transcriptase (RT), and represents a remarkable step forward in the development of anti-HIV drugs.

6-Methoxyflavone inhibits NFAT translocation into the nucleus and suppresses T cell activation.

NFAT plays a crucial role in the immune system by regulating the transcription of inducible genes during immune responses. In T cells, NFAT proteins govern various cellular events related to T cell development, activation, tolerance induction, and differentiation. We previously reported the NFAT1-dependent enhancer activity of conserved noncoding sequence (CNS)-9, a distal cis-acting element, in the regulation of IL-10 transcription in T cells. In this study, we developed a T cell-based reporter system to identify compounds that modulate the regulatory activity of CNS-9. Among the identified candidates, 6-methoxyflavone (6-MF) significantly inhibited the enhancer activity of CNS-9, thereby reducing IL-10 expression in T cells without affecting cell viability. 6-MF also downregulated the transcription of NFAT1 target genes such as IL-4, IL-13, and IFN-γ. Treatment of 6-MF inhibited the translocation of NFAT1 into the nucleus, which consequently interrupted NFAT1 binding to the target loci, without affecting the expression or dephosphorylation of NFAT1. Treatment of 6-MF to CD4(+) T cells or B cells isolated from mice with atopic dermatitis significantly reduced disease-associated cytokine production, as well as the levels of IgE. In addition, oral administration of 6-MF to atopic dermatitis mice ameliorated disease symptoms by reducing serum IgE levels and infiltrating lymphocytes. Conclusively, our results suggest that 6-MF can be a potential candidate for the development of an effective immunomodulator via the suppression of NFAT-mediated T cell activation.

Suppression of bovine viral diarrhea virus replication by single and dual short hairpin RNA-mediated RNA interference.

Bovine viral diarrhea virus (BVDV) is one of the most important pathogens to the cattle industry, causing a significant economic loss throughout the world. Despite the wide use of various control measures for BVDV, the disease remains prevalent. In this study, we achieved an efficient inhibition of NADL strain replication by plasmid-mediated shRNA targeting conserved regions of the viral genome. To further enhance the inhibiting efficiency, a dual shRNA expression plasmid, which could simultaneously express two different shRNA, was established and showed stronger inhibitory effects on virus replication. Moreover, the antiviral activity induced by the dual shRNA expression system was also evident on other BVDV-1 subgenotypes (BVDV-1a, BVDV-1b and BVDV-1c). Therefore, the dual shRNA system provides a more powerful strategy for inhibiting BVDV replication in a cross-resistance manner.

Ligand-binding domain of an α7-nicotinic receptor chimera and its complex with agonist.

The α(7) acetylcholine receptor (AChR) mediates pre- and postsynaptic neurotransmission in the central nervous system and is a potential therapeutic target in neurodegenerative, neuropsychiatric and inflammatory disorders. We determined the crystal structure of the extracellular domain of a receptor chimera constructed from the human α(7) AChR and Lymnaea stagnalis acetylcholine binding protein (AChBP), which shares 64% sequence identity and 71% similarity with native α(7). We also determined the structure with bound epibatidine, a potent AChR agonist. Comparison of the structures revealed molecular rearrangements and interactions that mediate agonist recognition and early steps in signal transduction in α(7) AChRs. The structures further revealed a ring of negative charge within the central vestibule, poised to contribute to cation selectivity. Structure-guided mutational studies disclosed distinctive contributions to agonist recognition and signal transduction in α(7) AChRs. The structures provide a realistic template for structure-aided drug design and for defining structure-function relationships of α(7) AChRs.

A conserved long noncoding RNA affects sleep behavior in Drosophila.

Metazoan genomes encode an abundant collection of mRNA-like, long noncoding (lnc)RNAs. Although lncRNAs greatly expand the transcriptional repertoire, we have a limited understanding of how these RNAs contribute to developmental regulation. Here, we investigate the function of the Drosophila lncRNA called yellow-achaete intergenic RNA (yar). Comparative sequence analyses show that the yar gene is conserved in Drosophila species representing 40-60 million years of evolution, with one of the conserved sequence motifs encompassing the yar promoter. Further, the timing of yar expression in Drosophila virilis parallels that in D. melanogaster, suggesting that transcriptional regulation of yar is conserved. The function of yar was defined by generating null alleles. Flies lacking yar RNAs are viable and show no overt morphological defects, consistent with maintained transcriptional regulation of the adjacent yellow (y) and achaete (ac) genes. The location of yar within a neural gene cluster led to the investigation of effects of yar in behavioral assays. These studies demonstrated that loss of yar alters sleep regulation in the context of a normal circadian rhythm. Nighttime sleep was reduced and fragmented, with yar mutants displaying diminished sleep rebound following sleep deprivation. Importantly, these defects were rescued by a yar transgene. These data provide the first example of a lncRNA gene involved in Drosophila sleep regulation. We find that yar is a cytoplasmic lncRNA, suggesting that yar may regulate sleep by affecting stabilization or translational regulation of mRNAs. Such functions of lncRNAs may extend to vertebrates, as lncRNAs are abundant in neural tissues.

Parathyroid hormone controls receptor activator of NF-kappaB ligand gene expression via a distant transcriptional enhancer.

RANKL, a protein essential for osteoclast development and survival, is stimulated by parathyroid hormone (PTH) via a PTH receptor 1/cyclic AMP (cAMP)/protein kinase A (PKA)/CREB cascade, exclusively in osteoblastic cells. We report that a bacterial artificial chromosome-based transcriptional reporter construct containing 120 kb of RANKL 5'-flanking region was stimulated by dibutyryl-cAMP in stromal/osteoblastic cells, but not other cell types. Full cAMP responsiveness was dependent upon a conserved 715-bp region located 76 kb upstream from the transcription start site, which we identified by sequential deletion analysis and by comparison of human and mouse genomic sequences in silico. This region contained conserved consensus sequences which bound CREB and the osteoblast-specific transcription factor Runx2, and when mutated blunted cAMP responsiveness. Overexpression of Runx2 potentiated cAMP responsiveness of the endogenous RANKL gene in a cell-type-specific manner. Lastly, PTH responsiveness of the endogenous RANKL gene was abrogated in mice from which we deleted this conserved upstream region. Thus, PTH responsiveness of the RANKL gene is determined by a distant regulatory region that responds to cAMP in a cell-type-specific manner and Runx2 may contribute to such cell-type specificity.

Neuronal transmission stimulates the phosphorylation of Kv1.4 channel at Ser229 through protein kinase A1.

Phosphorylation of voltage-gated K+ channels (Kv) is involved in regulation of neuronal excitability, synaptic plasticity and neuronal survival. Among Kv channels expressed in the CNS, Kv1.4 is located in the soma, dendrite and axon terminus of neurones in most regions of the brain. Here, we show that Ser229 found within the highly conserved T1 domain of Kv1.4 in cultured rat cortical neurones is phosphorylated by protein kinase A (PKA), as demonstrated by in vitro protein kinase assay and Western blotting with a polyclonal antibody specific against phosphorylated Ser229. Glutamate, high concentrations of K+ or K+ channel blockers known to increase neurotransmission all stimulated the phosphorylation of Kv1.4 at Ser229 via N-methyl-D-aspartate (NMDA), but not alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptor, whereas tetradotoxin (TTX), known to block neuronal transmission, and depletion of extracellular Ca2+ inhibited phosphorylation induced by tetraethylammonium (TEA), a non-selective K+ channel blocker. Mutation of Ser229 to Ala229 enhanced the current density. Taken together, elevation of the neuronal transmission stimulates the phosphorylation of Kv1.4 at Ser229 via the Ca2+ influx through NMDA receptor. Thus, it is possible that neuronal transmission regulates neuronal excitability partially through the phosphorylation of Kv1.4S229.

The permissive cue laminin is essential for growth cone turning in vivo.

The proper guidance of migrating growth cones relies on the balance of multiple guidance cues in the embryonic environment. In addition to guidance cues, growth cones are in contact with other substrates that may contribute to the pathfinding of neurons. For example, in the developing insect peripheral nervous system, pioneer neurons migrate on and between layers of the basal lamina. Previous studies have demonstrated that one basal lamina molecule, laminin, promotes outgrowth of many classes of neurons in vitro. In this study, the simple grasshopper nervous system was used to investigate the role of laminin in neuronal pathfinding. Laminin expression precedes axonogenesis of the Tibial (Ti1) pioneer neurons in the developing limb bud, and expression continues during outgrowth and guidance of the pioneer neurons. The role of a nidogen-binding motif on laminin was investigated using subunit-specific antibodies and peptides as blocking reagents in vivo. Antibodies and peptides that block the nidogen-binding site on laminin resulted in stalled Ti1 axon migration, predominantly at the precise location where they normally turn ventrally. After prolonged culturing, Ti1 axons remained stalled at the same location. Therefore, although Ti1 axons were capable of outgrowth in the presence of blocking reagents, they were not able to navigate an essential turn. This study indicates that the interaction of the Ti1 growth cone with the nidogen-binding site on laminin is vital for neuronal pathfinding in vivo and suggests that permissive cues may be essential for growth cone steering.

A yeast gene, MGS1, encoding a DNA-dependent AAA(+) ATPase is required to maintain genome stability.

Changes in DNA superhelicity during DNA replication are mediated primarily by the activities of DNA helicases and topoisomerases. If these activities are defective, the progression of the replication fork can be hindered or blocked, which can lead to double-strand breaks, elevated recombination in regions of repeated DNA, and genome instability. Hereditary diseases like Werner's and Bloom's Syndromes are caused by defects in DNA helicases, and these diseases are associated with genome instability and carcinogenesis in humans. Here we report a Saccharomyces cerevisiae gene, MGS1 (Maintenance of Genome Stability 1), which encodes a protein belonging to the AAA(+) class of ATPases, and whose central region is similar to Escherichia coli RuvB, a Holliday junction branch migration motor protein. The Mgs1 orthologues are highly conserved in prokaryotes and eukaryotes. The Mgs1 protein possesses DNA-dependent ATPase and single-strand DNA annealing activities. An mgs1 deletion mutant has an elevated rate of mitotic recombination, which causes genome instability. The mgs1 mutation is synergistic with a mutation in top3 (encoding topoisomerase III), and the double mutant exhibits severe growth defects and markedly increased genome instability. In contrast to the mgs1 mutation, a mutation in the sgs1 gene encoding a DNA helicase homologous to the Werner and Bloom helicases suppresses both the growth defect and the increased genome instability of the top3 mutant. Therefore, evolutionarily conserved Mgs1 may play a role together with RecQ family helicases and DNA topoisomerases in maintaining proper DNA topology, which is essential for genome stability.

The conserved cysteine 7.38 residue is differentially accessible in the binding-site crevices of the mu, delta, and kappa opioid receptors.

Binding pockets of the opioid receptors are presumably formed among the transmembrane domains (TMDs) and are accessible from the extracellular medium. In this study, we determined the sensitivity of binding of [(3)H]diprenorphine, an antagonist, to mu, delta, and kappa opioid receptors to charged methanethiosulfonate (MTS) derivatives and identified the cysteine residues within the TMDs that conferred the sensitivity. Incubation of the mu opioid receptor expressed in HEK293 cells with MTS ethylammonium (MTSEA), MTS ethyltrimethylammonium (MTSET), or MTS ethylsulfonate (MTSES) inhibited [(3)H]diprenorphine binding with the potency order of MTSEA > MTSET > MTSES. Pretreatment of mu, delta, and kappa opioid receptors with MTSEA dose-dependently inhibited [(3)H]diprenorphine binding with MTSEA sensitivity in the order of kappa > mu >> delta. The effects of MTSEA occurred rapidly, reaching the maximal inhibition in 10 min. (-)-Naloxone, but not (+)-naloxone, prevented the MTSEA effect, demonstrating that the reaction occurs within or in the vicinity of the binding pockets. Each cysteine residue in the TMDs of the three receptors was mutated singly, and the effects of MTSEA treatment were examined. The mutants had similar affinities for [(3)H]diprenorphine, and C7. 38(321)S, C7.38(303)S, and C7.38(315)S mutations rendered mu, delta, and kappa opioid receptors less sensitive to the effect of MTSEA, respectively. These results indicate that the conserved Cys7.38 is differentially accessible in the binding-site crevice of these receptors. The second extracellular loop of the kappa receptor, which contains several acidic residues, appears to play a role, albeit small, in its higher sensitivity to MTSEA, whereas the negative charge of Glu6.58(297) did not. To the best of our knowledge, this is the first report to show that a conserved residue among highly homologous G protein-coupled receptors is differentially accessible in the binding-site crevice. In addition, this represents the first successful generation of MTSEA-insensitive mutants of mu, delta, and kappa opioid receptors, which will allow determination of residues accessible in the binding-site crevices of these receptors by the substituted cysteine accessibility method.

Oct-1 enhances the in vitro replication of a mammalian autonomously replicating DNA sequence.

A 186-base pair fragment of ors8, a mammalian autonomously replicating DNA sequence isolated by extrusion of nascent monkey DNA in early S phase, has previously been identified as the minimal sequence required for replication function in vitro and in vivo. This 186-base pair fragment contains, among other sequence characteristics, an imperfect consensus binding site for the ubiquitous transcription factor Oct-1. We have investigated the role of Oct-1 protein in the in vitro replication of this mammalian origin. Depletion of the endogenous Oct-1 protein, by inclusion of an oligonucleotide comprising the Oct-1 binding site,inhibited the in vitro replication of p186 to approximately 15-20% of the control, whereas a mutated Oct-1 and a nonspecific oligonucleotide had no effect. Furthermore, immunodepletion of the Oct-1 protein from the HeLa cell extracts by addition of an anti-POU antibody to the in vitro replication reactioninhibited p186 replication to 25% of control levels. This inhibition of replication could be partially reversed to 50-65% of control levels, a two- to threefold increase, upon the addition of exogenous Oct-1 POU domain protein. Site-directed mutagenesis of the octamer binding site in p186 resulted in a mutant clone, p186-MutOct, which abolished Oct-1 binding but was still able to replicate as efficiently as the wild-type p186. The results suggest that Oct-1 protein is an enhancing component in the in vitro replication of p186 but that its effect on replication is not caused through direct binding to the octamer motif.

Molecular basis of rifampin resistance in Mycobacterium leprae.

Rifampin is currently the most potent drug used in leprosy control programs. We show that the rifampin resistance which emerged in nine patients with lepromatous leprosy, who had received rifampin monotherapy, stemmed from mutations in the rpoB gene, which encodes the beta subunit of RNA polymerase of Mycobacterium leprae. In eight cases missense mutations were found to affect a serine residue, Ser-425, while in the remaining mutant a small insertion was found close to this site. These findings will be of use for the development of a rapid screening procedure, involving the polymerase chain reaction, for monitoring the emergence of rifampin-resistant M. leprae strains.