Allosteric activation mechanism of the alpha1beta2gamma2 gamma-aminobutyric acid type A receptor revealed by mutation of the conserved M2 leucine

A conserved leucine residue in the midpoint of the second transmembrane domain (M2) of the ligand-activated ion channel family has been proposed to play an important role in receptor activation. In this study, we assessed the importance of this leucine in the activation of rat alpha1beta2gamma2 GABA receptors expressed in Xenopus laevis oocytes by site-directed mutagenesis and two-electrode voltage clamp. The hydrophobic conserved M2 leucines in alpha1(L263), beta2(L259), and gamma2(L274) subunits were mutated to the hydrophilic amino acid residue serine and coexpressed in all possible combinations with their wild-type and/or mutant counterparts. The mutation in any one subunit decreased the EC(50) and created spontaneous openings that were blocked by picrotoxin and, surprisingly, by the competitive antagonist bicuculline. The magnitudes of the shifts in GABA EC(50) and picrotoxin IC(50) as well as the degree of spontaneous openings were all correlated with the number of subunits carrying the leucine mutation. Simultaneous mutation of the GABA binding site (beta2Y157S; increased the EC(50)) and the conserved M2 leucine (beta2L259S; decreased the EC(50)) produced receptors with the predicted intermediate agonist sensitivity, indicating the two mutations affect binding and gating independently. The results are discussed in light of a proposed allosteric activation mechanism.     

Novel mutations in TARDBP (TDP-43) in patients with familial amyotrophic lateral sclerosis

The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V, p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in 24 patients with sporadic ALS or 180 patients with other TDP-43-positive neurodegenerative diseases. The presence of p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved fragments, including the approximately 25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or intracellular accumulation of TDP-43 fragments leading to apoptosis.     

Ranaspumin-2: Structure and Function of a Surfactant Protein from the Foam Nests of a Tropical Frog

Ranaspumin-2 (Rsn-2) is a monomeric, 11 kDa surfactant protein identified as one of the major foam nest components of the túngara frog (Engystomops pustulosus), with an amino acid sequence unlike any other protein described so far. We report here on its structure in solution as determined by high-resolution NMR analysis, together with investigations of its conformation and packing at the air-water interface using a combination of infrared and neutron reflectivity techniques. Despite the lack of any significant sequence similarity, Rsn-2 in solution adopts a compact globular fold characteristic of the cystatin family, comprising a single helix over a four-stranded sheet, in a motif not previously associated with surfactant activity. The NMR structure of Rsn-2 shows no obvious amphiphilicity that might be anticipated for a surfactant protein. This suggests that it must undergo a significant conformational change when incorporated into the air-water interface that may involve a hinge-bending, clamshell opening of the separate helix and sheet segments to expose hydrophobic faces to air while maintaining the highly polar surfaces in contact with the underlying water layer. This model is supported by direct observation of the relative orientations of secondary structure elements at the interface by infrared reflection absorption spectroscopy, and by protein packing densities determined from neutron reflectivity profiles.     

Stress-related hormones modulate cytokine expression in the head kidney of gilthead seabream (Sparus aurata)

Neuro-endocrine and immune systems closely interact in fish, and their regulation is crucial for the maintenance of good health of cultured fish. We have used the seabream head kidney to study whether stress-related hormones can modulate the immune response. For this purpose, the effects of adrenaline, adrenocorticotropic hormone (ACTH) and cortisol on the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and the anti-inflammatory cytokine TGF-β1 were determined by means of quantitative real-time PCR on isolated head kidney cells. ACTH (150 ng mL⁻¹) caused an acute increase of TNF-α and IL-6 mRNA levels as well as an inhibition of IL-1β expression. The expression of the anti-inflammatory cytokine TGF-β1 was also increased, although in a lower extent. Adrenaline (1 μM) early effects were only clear inhibiting IL-1β expression but not TNF-α, IL-6 or TGF-β1 mRNA levels, while a longer exposure to the hormone inhibited all cytokines. Moreover, cortisol (50 and 100 ng mL⁻¹) reduced the expression of all cytokines in a dose-dependent manner. Bacterial lipopolysaccharide (LPS) stimulated IL-1β expression and inhibited that of the anti-inflammatory TGF-β1, although it was ineffective on TNF-α and IL-6. In addition, adrenaline and cortisol decreased the LPS-stimulated IL-1β expression, further demonstrating their previously reported anti-inflammatory effects. The combination of ACTH and LPS, on the other hand, did not affect LPS-stimulated IL-1β expression but was effective increasing TNF-α expression. Taking all these results in consideration, we conclude that the expression of pro- and anti-inflammatory cytokines in the seabream head kidney is highly influenced by stress-related hormones, thus indicating an important role for the endocrine system in the modulation of the immune response in teleost fish.     

Vector competence of Australian Culex gelidus Theobald (Diptera: Culicidae) for endemic and exotic arboviruses

The recent recognition of established populations of the mosquito, Culex gelidus Theobald, in Australia has raised concerns about local transmission of arboviruses. The vector competence of a mainland population of Cx. gelidus was investigated for two local alphaviruses, Ross River (RRV) and Barmah Forest (BFV) viruses, and three flaviviruses, Japanese encephalitis (JEV), Kunjin (KUNV) and Murray Valley encephalitis (MVEV) viruses. Colonised mosquitoes were exposed to virus via blood-soaked pledgets and transmission was tested using a capillary-tube method. The important Australian vectors, Aedes vigilax (Skuse) and Culex annulirostris Skuse, were used as internal controls for the alphaviruses and flaviviruses, respectively. Overall, Cx. gelidus was a more efficient vector of flaviviruses than alphaviruses. Culex gelidus was refractory to infection with BFV, and nearly 25% transmitted RRV, which was comparable to Ae. vigilax . Culex gelidus was susceptible to all three flaviviruses, with transmission rates of 96%, 95% and 41% for JEV, KUNV and MVEV, respectively. JEV transmission rates in Cx. annulirostris were unexpectedly low and this was possibly due to differences in susceptibility to JEV genotypes I and II. Considering the high susceptibility to the flaviviruses demonstrated here, and the natural infections with RRV and JEV that have been detected from northern Australian populations, the establishment of the exotic mosquito, Cx. gelidus , in Australia is potentially a significant public health concern.