Analysis of hyperekplexia mutations identifies transmembrane domain rearrangements that mediate glycine receptor activation

Pentameric ligand-gated ion channels (pLGICs) mediate numerous physiological processes and are therapeutic targets for a wide range of clinical indications. Elucidating the structural differences between their closed and open states may help in designing improved drugs that bias receptors toward the desired conformational state. We recently showed that two new hyperekplexia mutations, Q226E and V280M, induced spontaneous activity in α1 glycine receptors. Gln-226, located near the top of transmembrane (TM) 1, is closely apposed to Arg-271 at the top of TM2 in the neighboring subunit. Using mutant cycle analysis, we inferred that Q226E induces activation via an enhanced electrostatic attraction to Arg-271. This would tilt the top of TM2 toward TM1 and hence away from the pore axis to open the channel. We also concluded that the increased side chain volume of V280M, in the TM2-TM3 loop, exerts a steric repulsion against Ile-225 at the top of TM1 in the neighboring subunit. We infer that this steric repulsion would tilt the top of TM3 radially outwards against the stationary TM1 and thus provide space for TM2 to relax away from the pore axis to create an open channel. Because the transmembrane domain movements inferred from this functional analysis are consistent with the structural differences evident in the x-ray atomic structures of closed and open state bacterial pLGICs, we propyose that the model of pLGIC activation as outlined here may be broadly applicable across the eukaryotic pLGIC receptor family

C-Fos expression is a molecular predictor of progression and survival in epithelial ovarian carcinoma

Members of the Fos protein family dimerise with Jun proteins to form the AP-1 transcription factor complex. They have a central function in proliferation and differentiation of normal tissue as well as in oncogenic transformation and tumour progression. We analysed the expression of c-Fos, FosB, Fra-1 and Fra-2 to investigate the function of Fos transcription factors in ovarian cancer. A total of 101 patients were included in the study. Expression of Fos proteins was determined by western blot analysis, quantified by densitometry and verified by immunohistochemistry. Reduced c-Fos expression was independently associated with unfavourable progression-free survival (20.6, 31.6 and 51.2 months for patients with low, moderate and high c-Fos expression; P=0.003) as well as overall survival (23.8, 46.0 and 55.5 months for low, moderate and high c-Fos levels; P=0.003). No correlations were observed for FosB, Fra-1 and Fra-2. We conclude that loss of c-Fos expression is associated with tumour progression in ovarian carcinoma and that c-Fos may be a prognostic factor. These results are in contrast to the classic concept of c-Fos as an oncogene, but are supported by the recently discovered tumour-suppressing and proapoptotic function of c-Fos in various cancer types

Ivermectin, an unconventional agonist of the glycine receptor chloride channel

The effects of the antihelmintic, ivermectin, were investigated in recombinantly expressed human alpha (1) homomeric and alpha (1)beta heteromeric glycine receptors (GlyRs), At low (0.03 muM) concentrations ivermectin potentiated the response to sub-saturating glycine concentrations, and at higher (greater than or equal to0.03 muM) concentrations it irreversibly activated both alpha (1) homomeric and alpha (1)beta heteromeric GlyRs. Relative to glycine-gated currents, ivermectin-gated currents exhibited a dramatically reduced sensitivity to inhibition by strychnine, picrotoxin, and zinc. The insensitivity to strychnine could not be explained by ivermectin preventing the access of strychnine to its binding site. Furthermore, the elimination of a known glycine- and strychnine-binding site by site-directed mutagenesis had little effect on ivermectin sensitivity, demonstrating that the ivermectin- and glycine-binding sites were not identical. Ivermectin strongly and irreversibly activated a fast-desensitizing mutant GlyR after it had been completely desensitized by a saturating concentration of glycine. Finally, a mutation known to impair dramatically the glycine signal transduction mechanism had little effect on the apparent affinity or efficacy of ivermectin, Together, these findings indicate that ivermectin activates the GlyR by a novel mechanism

RHYTHM—a server to predict the orientation of transmembrane helices in channels and membrane-coils

RHYTHM is a web server that predicts buried versus exposed residues of helical membrane proteins. Starting from a given protein sequence, secondary and tertiary structure information is calculated by RHYTHM within only a few seconds. The prediction applies structural information from a growing data base of precalculated packing files and evolutionary information from sequence patterns conserved in a representative dataset of membrane proteins (‘Pfam-domains’). The program uses two types of position specific matrices to account for the different geometries of packing in channels and transporters (‘channels’) or other membrane proteins (‘membrane-coils’). The output provides information on the secondary structure and topology of the protein and specifically on the contact type of each residue and its conservation. This information can be downloaded as a graphical file for illustration, a text file for analysis and statistics and a PyMOL file for modeling purposes. The server can be freely accessed at: URL: http://proteinformatics.de/rhyth

Novel missense mutations in the glycine receptor β subunit gene (GLRB) in startle disease

Startle disease is a rare, potentially fatal neuromotor disorder characterized by exaggerated startle reflexes and hypertonia in response to sudden unexpected auditory, visual or tactile stimuli. Mutations in the GlyR alpha(1) subunit gene (GLRA1) are the major cause of this disorder, since remarkably few individuals with mutations in the GlyR beta subunit gene (GLRB) have been found to date. Systematic DNA sequencing of GLRB in individuals with hyperekplexia revealed new missense mutations in GLRB, resulting in M177R, L285R and W310C substitutions. The recessive mutation M177R results in the insertion of a positively-charged residue into a hydrophobic pocket in the extracellular domain, resulting in an increased EC50 and decreased maximal responses of alpha(1)beta GlyRs. The de novo mutation L285R results in the insertion of a positively-charged side chain into the pore-lining 9' position. Mutations at this site are known to destabilize the channel closed state and produce spontaneously active channels. Consistent with this, we identified a leak conductance associated with spontaneous GlyR activity in cells expressing alpha(1)beta(L285R) GlyRs. Peak currents were also reduced for alpha(1)beta(L285R) GlyRs although glycine sensitivity was normal. W310C was predicted to interfere with hydrophobic side-chain stacking between M1, M2 and M3. We found that W310C had no effect on glycine sensitivity, but reduced maximal currents in alpha(1)beta GlyRs in both homozygous (alpha(1)beta(W310C)) and heterozygous (alpha(1)beta beta(W310C)) stoichiometries. Since mild startle symptoms were reported in W310C carriers, this may represent an example of incomplete dominance in startle disease, providing a potential genetic explanation for the 'minor' form of hyperekplexia. (C) 2012 Elsevier Inc. All rights reserved

The selective post-translational processing of transcription factor Nrf1 yields distinct isoforms that dictate its ability to differentially regulate gene expression

Upon translation, the N-terminal homology box 1 (NHB1) signal anchor sequence of Nrf1 integrates it within the endoplasmic reticulum (ER) whilst its transactivation domains [TADs, including acidic domain 1 (AD1), the flanking Asn/Ser/Thr-rich (NST) domain and AD2] are transiently translocated into the ER lumen, whereupon the NST domain is glycosylated to yield an inactive 120-kDa glycoprotein. Subsequently, these TADs are retrotranslocated into extra-luminal subcellular compartments, where Nrf1 is deglycosylated to yield an active 95-kDa isoform. Herein, we report that AD1 and AD2 are required for the stability of the 120-kDa Nrf1 glycoprotein, but not that of the non-glycosylated/de-glycosylated 95-kDa isoform. Degrons within AD1 do not promote proteolytic degradation of the 120-kDa Nrf1 glycoprotein. However, repositioning of AD2-adjoining degrons (i.e. DSGLS-containing SDS1 and PEST2 sequences) into the cyto/nucleoplasm enables selective topovectorial processing of Nrf1 by the proteasome and/or calpains to generate a cleaved active 85-kDa Nrf1 or a dominant-negative 36-kDa Nrf1γ. Production of Nrf1γ is abolished by removal of SDS1 or PEST2 degrons, whereas production of the cleaved 85-kDa Nrf1 is blocked by deletion of the ER luminal-anchoring NHB2 sequence (aa 81–106). Importantly, Nrf1 activity is positively and/or negatively regulated by distinct doses of proteasome and calpain inhibitors