Computational reconstruction of multidomain proteins using atomic force microscopy data

Classical structural biology techniques face a great challenge to determine the structure at the atomic level of large and flexible macromolecules. We present a novel methodology that combines high-resolution AFM topographic images with atomic coordinates of proteins to assemble very large macromolecules or particles. Our method uses a two-step protocol: atomic coordinates of individual domains are docked beneath the molecular surface of the large macromolecule, and then each domain is assembled using a combinatorial search. The protocol was validated on three test cases: a simulated system of antibody structures; and two experimentally based test cases: Tobacco mosaic virus, a rod-shaped virus; and Aquaporin Z, a bacterial membrane protein. We have shown that AFM-intermediate resolution topography and partial surface data are useful constraints for building macromolecular assemblies. The protocol is applicable to multicomponent structures connected in the polypeptide chain or as disjoint molecules. The approach effectively increases the resolution of AFM beyond topographical information down to atomic-detail structures.     

Empathy beyond the conceptual level: core nonspecific factors of psychotherapy

The human mind contains much more than concepts. By only taking into account the conceptual level, a cared-for person may feel utterly lonely and abandoned, not deeply in contact with the caregiver, not deeply understood for who he or she really is. A chronic pain patient, for instance, may react to a purely conceptual-level communication, with its lack of deeper contact, by an increasing sense of loneliness. This in itself may substantially contribute to the suffering of chronic functional pain or even functional disorders in general. In dealing with chronic pain patients, as with any patients, it is therefore very important to develop a sense of empathy that goes beyond this, towards deeply understanding the patient as complete person. This sheds a profound light on the all-important nonspecific factors of psychotherapy, which according to many researchers form the only profoundly active principle in psychotherapy.     

Differential effects of Bcl-2 and caspases on mitochondrial permeabilization during endogenous or exogenous reactive oxygen species-induced cell death

In this study, we have compared several features of cell death triggered by classical inducers of apoptotic pathways (etoposide and tumour necrosis factor (TNF)-α) versus exogenous reactive oxygen species (ROS; hydrogen peroxide (H?O?), tert-butyl hydroperoxide (t-BHP)) or a ROS generator (paraquat). Our aim was to characterize relationships that exist between ROS, mitochondrial perturbations, Bcl-2 and caspases, depending on source and identity of ROS. First, we have found that these five inducers trigger oxidative stress, mitochondrial membrane permeabilization (MMP), cytochrome c (cyt c) release from mitochondria and cell death. In each case, cell death could be inhibited by several antioxidants, showing that it is primarily ROS dependent. Second, we have highlighted that during etoposide or TNF-α treatments, intracellular ROS level, MMP and cell death are all regulated by caspases and Bcl-2, with caspases acting early in the process. Third, we have demonstrated that H?O?-induced cell death shares many of these characteristics with etoposide and TNF-α, whereas t-BHP induces both caspase-dependent and caspase-independent cell death. Surprisingly, paraquat-induced cell death, which harbours some characteristics of apoptosis such as cyt c release and caspase-3 activation, is not modulated by Bcl-2 and caspase inhibitors, suggesting that paraquat also triggers non-apoptotic cell death signals. On the one hand, these results show that endogenous or exogenous ROS can trigger multiple cell death pathways with Bcl-2 and caspases acting differentially. On the other hand, they suggest that H?O? could be an important mediator of etoposide and TNF-α-dependent cell death since these inducers trigger similar phenotypes.     

The 22nd ion channel meeting, september 2011, france

The 22(nd) Ion Channel Meeting was organized by the French Ion Channel Society (Association Canaux Ioniques) from the 25(th) to the 28(th) of September 2011 on the French Riviera (Giens). This year again, more than one hundred researchers from France, Europe and extra-European countries gathered to present and discuss their recent advances and future challenges in the ion channels and transporters field. The scientific committee organized a plenary lecture and five thematic symposia by inviting international researchers to present their recent outstanding work on themes as diverse as muscular channelopathies, regulation of channels by extracellular matrix, receptor-channels interactions, localization and distribution of ion channels, their involvement in the cell life and death, and finally how they participate in the evolution and adaptability of cellular excitability. These presentations are summarized in this meeting report. Two sessions of oral communications selected from submitted abstracts and two poster sessions were also organized to present the ongoing work of young researchers worldwide.     

Functional Characterization and Potential Applications for Enhanced Green Fluorescent Protein- and Epitope-Fused Human M1 Muscarinic Receptors

Four recombinant human M1 (hM1) muscarinic acetylcholine receptors (mAChRs) combining several modifications were designed and overexpressed in HEK293 cells. Three different fluorescent chimera were obtained through fusion of the receptor N terminus with enhanced green fluorescent protein (EGFP), potential glycosylation sites and a large part of the third intracellular (i3) loop were deleted, a hexahistidine tag sequence was introduced at the receptor C terminus, and, finally, a FLAG epitope was either fused at the receptor N terminus or inserted into its shortened i3 loop. High expression levels and ligand binding properties similar to those of the wild-type hM1 receptor together with confocal microscopy imaging demonstrated that the recombinant proteins were correctly folded and targeted to the plasma membrane, provided that a signal peptide was added to the N-terminal domain of the fusion proteins. Their functional properties were examined through McN-A-343-evoked Ca2+ release. Despite the numerous modifications introduced within the hM1 sequence, all receptors retained nearly normal abilities (EC50 values) to mediate the Ca2+ response, although reduced amplitudes (Emax values) were obtained for the i3-shortened constructs. Owing to the bright intrinsic fluorescence of the EGFP-fused receptors, their detection, quantitation, and visualization as well as the selection of cells with highest expression were straightforward. Moreover, the presence of the different epitopes was confirmed by immunocytochemistry. Altogether, this work demonstrates that these EGFP- and epitope-fused hM1 receptors are valuable tools for further functional, biochemical, and structural studies of muscarinic receptors.     

Isomerization between n-butyrate and isobutyrate in enrichment cultures

Abstract An isobutyrate-degrading methanogenic enrichment was obtained from a mesophilic anaerobic digester. Studies with growing cells and cell suspensions showed a reversible isomerization between butyrate and isobutyrate, suggesting that butyrate is an intermediate in the anaerobic degradation of isobutyrate. NMR experiments with ¹³C-labelled butyrate demonstrated that this isomerization resulted from the migration of the carboxyl group.     

Masking in Plants

Orcadian rhythms in plants are liable to masking, i.e. alterations by environmental influencing agents. Experiments have been reported for both positive and negative masking, attributed to a Zeitgeber which may either increase or decrease the amplitude of a circadian rhythm (CR). In some instances, the CR may even be unexpressed. This inhibition, however, may be alleviated by synchronizing agents. Reports are also available for changes in the shape or pattern of an oscillation. The latter may be prevented, at least in Acetabularia in certain conditions, by a phytohormone antagonist.

Masking may also be brought about by water stress, relative humidity, bacterial infection and alteration in the relative direction of the gravitational force.

Finally, subjecting plants to constant conditions, particularly continuous light, alters the physiological state of the organism.     

Implication of the First and Third Extracellular Loops of the μ-Opioid Receptor in the Formation of the Ligand Binding Site: A Study Using Chimeric μ-Opioid/Angiotensin Receptors

Abstract: Recent studies on chimeric μ/δ-, μ/κ- and δ/κ-opioid receptors have suggested that extracellular loops of the receptors were involved in the discriminatory binding of selective ligands by controlling their entry into the transmembrane binding site. Since homochimeric opioid receptors are mostly informative in terms of selectivity, the role of extracellular loops was examined here by studying heterochimeric μ receptors where the totality or parts of extracellular loops were replaced by the corresponding regions of the receptor for angiotensin II. Chimeric μ receptors with extracellular loop EL1 or EL3 originating from the angiotensin receptor had 100-fold decreased affinities for opioids; the length of the first extracellular loop, which is one residue longer in angiotensin than μ receptors, was shown to be responsible for this situation. Substitution of the μ receptor second extracellular loop by that of the angiotensin receptor diminished by ∼10-fold the affinities for opioids. Since all chimeras had altered affinities for selective and nonselective ligands, we propose that extracellular domains of the μ receptor, particularly the first and third loops, constrain the relative positioning of the connected transmembrane domains where selective as well as nonselective contact points form the opioid binding site.     

The cytosolic/nuclear HSC70 and HSP90 molecular chaperones are important for stomatal closure and modulate abscisic acid-dependent physiological responses in Arabidopsis

Cytosolic/nuclear molecular chaperones of the heat shock protein families HSP90 and HSC70 are conserved and essential proteins in eukaryotes. These proteins have essentially been implicated in the innate immunity and abiotic stress tolerance in higher plants. Here, we demonstrate that both chaperones are recruited in Arabidopsis (Arabidopsis thaliana) for stomatal closure induced by several environmental signals. Plants overexpressing HSC70-1 or with reduced HSP90.2 activity are compromised in the dark-, CO(2)-, flagellin 22 peptide-, and abscisic acid (ABA)-induced stomatal closure. HSC70-1 and HSP90 proteins are needed to establish basal expression levels of several ABA-responsive genes, suggesting that these chaperones might also be involved in ABA signaling events. Plants overexpressing HSC70-1 or with reduced HSP90.2 activity are hypersensitive to ABA in seed germination assays, suggesting that several chaperone complexes with distinct substrates might tune tissue-specific responses to ABA and the other biotic and abiotic stimuli studied. This study demonstrates that the HSC70/HSP90 machinery is important for stomatal closure and serves essential functions in plants to integrate signals from their biotic and abiotic environments.