Sterol Carrier Protein-2: Binding Protein for Endocannabinoids

The endocannabinoid (eCB) system, consisting of eCB ligands and the type 1 cannabinoid receptor (CB1R), subserves retrograde, activity-dependent synaptic plasticity in the brain. eCB signaling occurs “on-demand,” thus the processes regulating synthesis, mobilization and degradation of eCBs are also primary mechanisms for the regulation of CB1R activity. The eCBs, N-arachidonylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), are poorly soluble in water. We hypothesize that their aqueous solubility, and, therefore, their intracellular and transcellular distribution, are facilitated by protein binding. Using in silico docking studies, we have identified the nonspecific lipid binding protein, sterol carrier protein 2 (SCP-2), as a potential AEA binding protein. The docking studies predict that AEA and AM404 associate with SCP-2 at a putative cholesterol binding pocket with ?G values of ?3.6 and ?4.6 kcal/mol, respectively. These values are considerably higher than cholesterol (?6.62 kcal/mol) but consistent with a favorable binding interaction. In support of the docking studies, SCP-2-mediated transfer of cholesterol in vitro is inhibited by micromolar concentrations of AEA; and heterologous expression of SCP-2 in HEK 293 cells increases time-related accumulation of AEA in a temperature-dependent fashion. These results suggest that SCP-2 facilitates cellular uptake of AEA. However, there is no effect of SCP-2 transfection on the cellular accumulation of AEA determined at equilibrium or the IC50 values for AEA, AM404 or 2-AG to inhibit steady state accumulation of radiolabelled AEA. We conclude that SCP-2 is a low affinity binding protein for AEA that can facilitate its cellular uptake but does not contribute significantly to intracellular sequestration of AEA.     

WNK2 kinase is a novel regulator of essential neuronal cation-chloride cotransporters

NKCC1 and KCC2, related cation-chloride cotransporters (CCC), regulate cell volume and γ-aminobutyric acid (GABA)-ergic neurotranmission by modulating the intracellular concentration of chloride [Cl(-)]. These CCCs are oppositely regulated by serine-threonine phosphorylation, which activates NKCC1 but inhibits KCC2. The kinase(s) that performs this function in the nervous system are not known with certainty. WNK1 and WNK4, members of the WNK (with no lysine [K]) kinase family, either directly or via the downstream SPAK/OSR1 Ste20-type kinases, regulate the furosemide-sensitive NKCC2 and the thiazide-sensitive NCC, kidney-specific CCCs. What role the novel WNK2 kinase plays in this regulatory cascade, if any, is unknown. Here, we show that WNK2, unlike other WNKs, is not expressed in kidney; rather, it is a neuron-enriched kinase primarily expressed in neocortical pyramidal cells, thalamic relay cells, and cerebellar granule and Purkinje cells in both the developing and adult brain. Bumetanide-sensitive and Cl(-)-dependent (86)Rb(+) uptake assays in Xenopus laevis oocytes revealed that WNK2 promotes Cl(-) accumulation by reciprocally activating NKCC1 and inhibiting KCC2 in a kinase-dependent manner, effectively bypassing normal tonicity requirements for cotransporter regulation. TiO(2) enrichment and tandem mass spectrometry studies demonstrate WNK2 forms a protein complex in the mammalian brain with SPAK, a known phosphoregulator of NKCC1. In this complex, SPAK is phosphorylated at Ser-383, a consensus WNK recognition site. These findings suggest a role for WNK2 in the regulation of CCCs in the mammalian brain, with implications for both cell volume regulation and/or GABAergic signaling.     

Reversible covalent inhibition of a phenol sulfotransferase by coenzyme A

Phenol sulfotransferases (SULTs), which normally bind 3'-phosphoadenosine-5'-phosphosulfate as the donor substrate, are inhibited by CoA and its thioesters. Here, we report that inhibition of bovine SULT1A1 by CoA is time-dependent at neutral pH under non-reducing conditions. The rates of inactivation by CoA indicate an initial reversible SULT:CoA complex with a dissociation constant of 5.7 microM and an inactivation rate constant of 0.07 min(-1). Titrations with CoA and prolonged incubations reveal that inactivation of the dimeric enzyme is stoichiometric, consistent with the observation of complete conversion of the protein to a slightly decreased electrophoretic mobility. Both activity and normal electrophoretic migration are restored by 2-mercaptoethanol. Mutagenesis demonstrated that Cys168 is the site of CoA adduction, and a consistent model was constructed that reveals a new SULT molecular dynamic. Cysteine reaction kinetics with Ellman's reagent revealed a PAPS-induced structural change consistent with the model that accounts for binding of CoA.     

TGF-β1 is an organizer of responses to neurodgeneration

TGF-β1 mRNA and protein were recently found to increase in animal brains after experimental lesions that cause local deafferentation or neuron death. Elevations of TGF-β1 mRNA after lesions are prominent in microglia but are also observed in neurons and astrocytes. Moreover, TGF-β1 mRNA autoinduces its own mRNA in the brain. These responses provide models for studying the increases of TGF-β1 protein observed in βA/amyloid-containing extracellular plaques of Alzheimer's disease (AD) and Down's syndrome (DS) and in brain cells of AIDS victims. Involvement of TGF-β1 in these human brain disorders is discussed in relation to the potent effects of TGF-β1 on wound healing and inflammatory responses in peripheral tissues. We hypothesize that TGF-β1 and possibly other TGF-β peptides have organizing roles in responses to neurodegeneration and brain injury that are similar to those observed in non-neural tissues. Work from many laboratories has shown that activities of TGF-β peptides on brain cells include chemotaxis, modification of extracellular matrix, and regulation of cytoskeletal gene expression and of neurotrophins. Similar activities of the TGF-β's are well established in other tissues.     

Comparative phylogeography of West African amphibians and reptiles

Comparative phylogeographic studies often support shared divergence times for co-distributed species with similar life histories and habitat specializations. During the late Holocene, West Africa experienced aridification and the turnover of rain forest habitats into savannas. These fragmented rain forests harbor impressive numbers of endemic and threatened species. In this setting, populations of co-distributed rain forest species are expected to have diverged simultaneously, whereas divergence events for species adapted to savanna and forest-edge habitats should be absent or idiosyncratic. We conducted a Bayesian analysis of shared evolutionary events to test models of population divergence for 20 species of anurans (frogs) and squamates (lizards and snakes) that are distributed across the Dahomey Gap, a climate change-induced savanna barrier responsible for fragmenting previously contiguous rain forests of Ghana into two regions: the Togo-Volta Hills and the Southwestern Forests. A model of asynchronous diversification is supported for anurans and squamates, suggesting that drivers of diversification are not specifically related to ecological and life history associations with habitat types. Instead, the wide variability of genetic divergence histories exhibited by these species suggests that biodiversity in this region has been shaped by diversification events that extend beyond the Holocene. Comparisons of the genealogical divergence index, a measure of the genetic divergence between populations due to the combined effects of genetic isolation and gene flow, illustrate that these populations represent a broad sampling of the speciation continuum.     

Mesoionic pyrido[1,2-a]pyrimidinones: Discovery of dicloromezotiaz as a lepidoptera insecticide acting on nicotinic acetylcholine receptors1,2

A novel class of mesoionic pyrido[1,2-a]pyrimidinones has been discovered with exceptional insecticidal activity controlling a number of insect species. In this communication, we report the part of the optimization program that led to the identification of dicloromezotiaz as a potent insecticide to control a broad range of lepidoptera. Our efforts in discovery, synthesis, structure-activity relationship elucidation, and biological activity evaluation are also presented.