Cedric Viero

Oxytocin: Crossing the Bridge between Basic Science and Pharmacotherapy

Is oxytocin the hormone of happiness? Probably not. However, this small nine amino acid peptide is involved in a wide variety of physiological and pathological functions such as sexual activity, penile erection, ejaculation, pregnancy, uterus contraction, milk ejection, maternal behavior, osteoporosis, diabetes, cancer, social bonding, and stress, which makes oxytocin and its receptor potential candidates as targets for drug therapy. In this review, we address the issues of drug design and specificity and focus our discussion on recent findings on oxytocin and its heterotrimeric G protein‐coupled receptor OTR. In this regard, we will highlight the following topics: (i) the role of oxytocin in behavior and affectivity, (ii) the relationship between oxytocin and stress with emphasis on the hypothalamo–pituitary–adrenal axis, (iii) the involvement of oxytocin in pain regulation and nociception, (iv) the specific action mechanisms of oxytocin on intracellular Ca2+ in the hypothalamo neurohypophysial system (HNS) cell bodies, (v) newly generated transgenic rats tagged by a visible fluorescent protein to study the physiology of vasopressin and oxytocin, and (vi) the action of the neurohypophysial hormone outside the central nervous system, including the myometrium, heart and peripheral nervous system. As a short nine amino acid peptide, closely related to its partner peptide vasopressin, oxytocin appears to be ideal for the design of agonists and antagonists of its receptor. In addition, not only the hormone itself and its binding to OTR, but also its synthesis, storage and release can be endogenously and exogenously regulated to counteract pathophysiological states. Understanding the fundamental physiopharmacology of the effects of oxytocin is an important and necessary approach for developing a potential pharmacotherapy.

CLIC2-RyR1 Interaction and Structural Characterization by Cryo-electron Microscopy

Chloride intracellular channel 2 (CLIC2), a newly discovered small protein distantly related to the glutathione transferase (GST) structural family, is highly expressed in cardiac and skeletal muscle, although its physiological function in these tissues has not been established. In the present study, [3H]ryanodine binding, Ca2+ efflux from skeletal sarcoplasmic reticulum (SR) vesicles, single channel recording, and cryo-electron microscopy were employed to investigate whether CLIC2 can interact with skeletal ryanodine receptor (RyR1) and modulate its channel activity. We found that: (1) CLIC2 facilitated [3H]ryanodine binding to skeletal SR and purified RyR1, by increasing the binding affinity of ryanodine for its receptor without significantly changing the apparent maximal binding capacity; (2) CLIC2 reduced the maximal Ca2+ efflux rate from skeletal SR vesicles; (3) CLIC2 decreased the open probability of RyR1 channel, through increasing the mean closed time of the channel; (4) CLIC2 bound to a region between domains 5 and 6 in the clamp-shaped region of RyR1; (5) and in the same clamp region, domains 9 and 10 became separated after CLIC2 binding, indicating CLIC2 induced a conformational change of RyR1. These data suggest that CLIC2 can interact with RyR1 and modulate its channel activity. We propose that CLIC2 functions as an intrinsic stabilizer of the closed state of RyR channels.

Neurosteroids are excitatory in supraoptic neurons but inhibitory in the peripheral nervous system: it is all about oxytocin and progesterone receptors

Neuroactive steroids synthesized from the brain or peripheral sources are called neurosteroids. Beside their common nuclear effects, they are considered to be potent neuromodulators, acting rapidly mainly in a non-genomic manner, either through allosteric regulation of ionic channels, or through membrane-bound steroid receptors. In contrast to the situation in the adult, the neurotransmitter GABA is excitatory during development and plays a trophic role, in particular inducing calcium signals necessary for the regulation of excitability and neuronal maturation. We demonstrated that the primary metabolite of progesterone (Proges), allopregnanolone (Allo), evoked a robust Ca(2+) influx in foetal hypothalamic neurons and in postnatal supraoptic nucleus (SON) neurons. In the latter, this led to oxytocin and arginine vasopressin release. Interestingly, these responses were GABA(A) and oxytocin-receptor-dependent. Allo is a well-known positive allosteric modulator of GABA(A) receptors. It is noteworthy that two other steroids, Proges and 17-beta-estradiol, displayed the same effect on Ca(2+) and oxytocin release but to a lesser extent. Importantly, no effect was observed in adult neurons from the SON, or in neurohypophysial axon terminals, regardless of the stage. The molecular mechanisms of the neurosteroid actions are multifaceted and depend on the type of cells, and are thus extremely interesting and challenging. In the peripheral nervous system, Allo and Proges surprisingly inhibited the GABA-induced Ca(2+) increases in embryonic dorsal root ganglion neurons. We propose that this rapid, reversible and dose-dependent phenomenon (at very low concentrations) was mediated by membrane Proges receptors, since transcripts for a newly discovered receptor protein, 25-Dx, were detected in our model. Recently, novel families of membrane steroid receptors, activating intracellular-signalling pathways such as MAP kinases, have been identified and described. This opens new perspectives to understand the intracellular machinery involved in the interaction between neuropeptides and neurosteroids, two major regulators of hypothalamo-neurohypophysial system development.

A novel in silico reverse-transcriptomics-based identification and blood-based validation of a panel of sub-type specific biomarkers in lung cancer

Lung cancer accounts for the highest number of cancer-related deaths worldwide. Early diagnosis significantly increases the disease-free survival rate and a large amount of effort has been expended in screening trials and the development of early molecular diagnostics. However, a gold standard diagnostic strategy is not yet available. Here, based on miRNA expression profile in lung cancer and using a novel in silico reverse-transcriptomics approach, followed by analysis of the interactome; we have identified potential transcription factor (TF) markers that would facilitate diagnosis of subtype specific lung cancer. A subset of seven TF markers has been used in a microarray screen and was then validated by blood-based qPCR using stage-II and IV non-small cell lung carcinomas (NSCLC). Our results suggest that overexpression of HMGA1, E2F6, IRF1, and TFDP1 and downregulation or no expression of SUV39H1, RBL1, and HNRPD in blood is suitable for diagnosis of lung adenocarcinoma and squamous cell carcinoma sub-types of NSCLC. Here, E2F6 was, for the first time, found to be upregulated in NSCLC blood samples. The miRNA-TF-miRNA interaction based molecular mechanisms of these seven markers in NSCLC revealed that HMGA1 and TFDP1 play vital roles in lung cancer tumorigenesis. The strategy developed in this work is applicable to any other cancer or disease and can assist in the identification of potential biomarkers.

N-terminus oligomerization regulates the function of cardiac ryanodine receptors.

Summary The ryanodine receptor (RyR) is an ion channel composed of four identical subunits mediating calcium efflux from the endo/sarcoplasmic reticulum of excitable and non-excitable cells. We present several lines of evidence indicating that the RyR2 N-terminus is capable of self-association. A combination of yeast two-hybrid screens, co-immunoprecipitation analysis, chemical crosslinking and gel filtration assays collectively demonstrate that a RyR2 N-terminal fragment possesses the intrinsic ability to oligomerize, enabling apparent tetramer formation. Interestingly, N-terminus tetramerization mediated by endogenous disulfide bond formation occurs in native RyR2, but notably not in RyR1. Disruption of N-terminal inter-subunit interactions within RyR2 results in dysregulation of channel activation at diastolic Ca2+ concentrations from ryanodine binding and single channel measurements. Our findings suggest that the N-terminus interactions mediating tetramer assembly are involved in RyR channel closure, identifying a crucial role for this structural association in the dynamic regulation of intracellular Ca2+ release.

miReg: a resource for microRNA regulation

MicroRNAs (miRNAs/miRs) are important cellular components that regulate gene expression at posttranscriptional level. Various upstream components regulate miR expression and any deregulation causes disease conditions. Therefore, understanding of miR regulatory network both at upstream and downstream level is crucial and a resource on this aspect will be helpful. Currently available miR databases are mostly related to downstream targets, sequences, or diseases. But as of now, no database is available that provides a complete picture of miR regulation in a specific condition. Our miR regulation web resource (miReg) is a manually curated one that represents validated upstream regulators (transcription factor, drug, physical, and chemical) along with downstream targets, associated biological process, experimental condition or disease state, up or down regulation of the miR in that condition, and corresponding PubMed references in a graphical and user friendly manner, browseable through 5 browsing options. We have presented exact facts that have been described in the corresponding literature in relation to a given miR, whether its a feed-back/feed-forward loop or inhibition/activation. Moreover we have given various links to integrate data and to get a complete picture on any miR listed. Current version (Version 1.0) of miReg contains 47 important human miRs with 295 relations using 190 absolute references. We have also provided an example on usefulness of miReg to establish signalling pathways involved in cardiomyopathy. We believe that miReg will be an essential miRNA knowledge base to research community, with its continuous upgrade and data enrichment. This HTML based miReg can be accessed from: www.iioab-mireg.webs.com or www.iioab.webs.com/mireg.htm.

Investigations of the contribution of a putative glycine hinge to ryanodine receptor channel gating.

Background: In many K+ channels, gating requires flexing of inner helices at glycine residues within a “hinge motif.” An analogous motif is present in the ryanodine receptor channel. Results: Mutation of ryanodine receptor “hinge” glycines produces varying effects on gating. Conclusion: Ryanodine receptors do not contain a typical glycine hinge. Significance: This work provides new insights into mechanisms of ryanodine receptor function. Ryanodine receptor channels (RyR) are key components of striated muscle excitation-contraction coupling, and alterations in their function underlie both inherited and acquired disease. A full understanding of the disease process will require a detailed knowledge of the mechanisms and structures involved in RyR function. Unfortunately, high-resolution structural data, such as exist for K+-selective channels, are not available for RyR. In the absence of these data, we have used modeling to identify similarities in the structural elements of K+ channel pore-forming regions and postulated equivalent regions of RyR. This has identified a sequence of residues in the cytosolic cavity-lining transmembrane helix of RyR (G4864LIIDA4869 in RyR2) analogous to the glycine hinge motif present in many K+ channels. Gating in these K+ channels can be disrupted by substitution of residues for the hinge glycine. We investigated the involvement of glycine 4864 in RyR2 gating by monitoring properties of recombinant human RyR2 channels in which this glycine is replaced by residues that alter gating in K+ channels. Our data demonstrate that introducing alanine at position 4864 produces no significant change in RyR2 function. In contrast, function is altered when glycine 4864 is replaced by either valine or proline, the former preventing channel opening and the latter modifying both ion translocation and gating. Our studies reveal novel information on the structural basis of RyR gating, identifying both similarities with, and differences from, K+ channels. Glycine 4864 is not absolutely required for channel gating, but some flexibility at this point in the cavity-lining transmembrane helix is necessary for normal RyR function.

The contribution of hydrophobic residues in the pore-forming region of the ryanodine receptor channel to block by large tetraalkylammonium cations and Shaker B inactivation peptides

Although no high-resolution structural information is available for the ryanodine receptor (RyR) channel pore-forming region (PFR), molecular modeling has revealed broad structural similarities between this region and the equivalent region of K+ channels. This study predicts that, as is the case in K+ channels, RyR has a cytosolic vestibule lined with predominantly hydrophobic residues of transmembrane helices (TM10). In K+ channels, this vestibule is the binding site for blocking tetraalkylammonium (TAA) cations and Shaker B inactivation peptides (ShBPs), which are stabilized by hydrophobic interactions involving specific residues of the lining helices. We have tested the hypothesis that the cytosolic vestibule of RyR fulfils a similar role and that TAAs and ShBPs are stabilized by hydrophobic interactions with residues of TM10. Both TAAs and ShBPs block RyR from the cytosolic side of the channel. By varying the composition of TAAs and ShBPs, we demonstrate that the affinity of both species is determined by their hydrophobicity, with variations reflecting alterations in the dissociation rate of the bound blockers. We investigated the role of TM10 residues of RyR by monitoring block by TAAs and ShBPs in channels in which the hydrophobicity of individual TM10 residues was lowered by alanine substitution. Although substitutions changed the kinetics of TAA interaction, they produced no significant changes in ShBP kinetics, indicating the absence of specific hydrophobic sites of interactions between RyR and these peptides. Our investigations (a) provide significant new information on both the mechanisms and structural components of the RyR PFR involved in block by TAAs and ShBPs, (b) highlight important differences in the mechanisms and structures determining TAA and ShBP block in RyR and K+ channels, and (c) demonstrate that although the PFRs of these channels contain analogous structural components, significant differences in structure determine the distinct ion-handling properties of the two species of channel.

The Ryanodine Receptor Pore: Is There a Consensus View?

Publisher Summary The intracellular Ca 2+ -release channel referred to as the “ryanodine receptor (RyR)” is a key factor in a plethora of biological and pathophysiological processes. This chapter reviews a fundamental aspect of RyR function—the structures and mechanisms that controls ion flow through the open channel and underpins the unusual ion handling characteristics of RyR and its great efficiency as a Ca 2+ -release channel. The chapter focuses on a small region of the channel that provides the pathway for ion movement across the membrane: the pore-forming region (PFR). The likely structure of the pore-forming region (PFR) has been provided by high-resolution cryo-electron microscopy (cryo-EM) and by molecular modeling, following the identification of structural analogies between RyR and other ion channels. The mechanisms involved in discrimination and translocation have emerged from detailed characterization of channel function, theoretical models, and the interpretation of the consequences of residue mutation in the PFR. Together, these approaches have been used to identify regions of RyR that are critical for ion movement and may contribute to the binding site for ryanodine.

Alteration of Sarcoplasmic Reticulum Ca2+ Release in Skeletal Muscle from Calpain 3-Deficient Mice

Mutations of Ca2+-activated proteases (calpains) cause muscular dystrophies. Nevertheless, the specific role of calpains in Ca2+ signalling during the onset of dystrophies remains unclear. We investigated Ca2+ handling in skeletal cells from calpain 3-deficient mice. [Ca2+]i responses to caffeine, a ryanodine receptor (RyR) agonist, were decreased in −/− myotubes and absent in −/− myoblasts. The −/− myotubes displayed smaller amplitudes of the Ca2+ transients induced by cyclopiazonic acid in comparison to wild type cells. Inhibition of L-type Ca2+ channels (LCC) suppressed the caffeine-induced [Ca2+]i responses in −/− myotubes. Hence, the absence of calpain 3 modifies the sarcoplasmic reticulum (SR) Ca2+ release, by a decrease of the SR content, an impairment of RyR signalling, and an increase of LCC activity. We propose that calpain 3-dependent proteolysis plays a role in activating support proteins of intracellular Ca2+ signalling at a stage of cellular differentiation which is crucial for skeletal muscle regeneration.