Hydration-mediated G-protein-coupled receptor activation.

The Rhodopsin family of G-protein­coupled receptors (GPCRs) comprises the targets of nearly a third of all pharmaceuticals. Despite structural water present in GPCR X-ray structures, the physiological relevance of these solvent molecules to rhodopsin signaling remains unknown. Here, we show experimental results consistent with the idea that rhodopsin activation in lipid membranes is coupled to bulk water movements into the protein. To quantify hydration changes, we measured reversible shifting of the metarhodopsin equilibrium due to osmotic stress using an extensive series of polyethylene glycol (PEG) osmolytes. We discovered clear evidence that light activation entails a large influx of bulk water (∼80­100 molecules) into the protein, giving insight into GPCR activation mechanisms. Various size polymer osmolytes directly control rhodopsin activation, in which large solutes are excluded from rhodopsin and dehydrate the protein, favoring the inactive state. In contrast, small osmolytes initially forward shift the activation equilibrium until a quantifiable saturation point is reached, similar to gain-of-function protein mutations. For the limit of increasing osmolyte size, a universal response of rhodopsin to osmotic stress is observed, suggesting it adopts a dynamic, hydrated sponge-like state upon photoactivation. Our results demand a rethinking of the role of water dynamics in modulating various intermediates in the GPCR energy landscape. We propose that besides bound water, an influx of bulk water plays a necessary role in establishing the active GPCR conformation that mediates signaling.

Membrane Curvature Revisited-the Archetype of Rhodopsin Studied by Time-Resolved Electronic Spectroscopy.

G-protein-coupled receptors (GPCRs) comprise the largest and most pharmacologically targeted membrane protein family. Here, we used the visual receptor rhodopsin as an archetype for understanding membrane lipid influences on conformational changes involved in GPCR activation. Visual rhodopsin was recombined with lipids varying in their degree of acyl chain unsaturation and polar headgroup size using 1-palmitoyl-2-oleoyl-sn-glycero- and 1,2-dioleoyl-sn-glycerophospholipids with phosphocholine (PC) or phosphoethanolamine (PE) substituents. The receptor activation profile after light excitation was measured using time-resolved ultraviolet-visible spectroscopy. We discovered that more saturated POPC lipids back shifted the equilibrium to the inactive state, whereas the small-headgroup, highly unsaturated DOPE lipids favored the active state. Increasing unsaturation and decreasing headgroup size have similar effects that combine to yield control of rhodopsin activation, and necessitate factors beyond proteolipid solvation energy and bilayer surface electrostatics. Hence, we consider a balance of curvature free energy with hydrophobic matching and demonstrate how our data support a flexible surface model (FSM) for the coupling between proteins and lipids. The FSM is based on the Helfrich formulation of membrane bending energy as we previously first applied to lipid-protein interactions. Membrane elasticity and curvature strain are induced by lateral pressure imbalances between the constituent lipids and drive key physiological processes at the membrane level. Spontaneous negative monolayer curvature toward water is mediated by unsaturated, small-headgroup lipids and couples directly to GPCR activation upon light absorption by rhodopsin. For the first time to our knowledge, we demonstrate this modulation in both the equilibrium and pre-equilibrium evolving states using a time-resolved approach.

Native Mass Spectrometry Reveals the Simultaneous Binding of Lipids and Zinc to Rhodopsin.

Rhodopsin, a prototypical G-protein-coupled receptor, is responsible for scoptic vision at low-light levels. Although rhodopsin's photoactivation cascade is well understood, it remains unclear how lipid and zinc binding to the receptor are coupled. Using native mass spectrometry, we developed a novel data analysis strategy to deconvolve zinc and lipid bound to the proteoforms of rhodopsin and investigated the allosteric interaction between lipids and zinc binding. We discovered that phosphatidylcholine bound to rhodopsin with a greater affinity than phosphatidylserine or phosphatidylethanolamine, and that binding of all lipids was influenced by zinc but with different effects. In contrast, zinc binding was relatively unperturbed by lipids. Overall, these data reveal that lipid binding can be strongly and differentially influenced by metal ions.

Activation of the G-Protein-Coupled Receptor Rhodopsin by Water.

Visual rhodopsin is an important archetype for G-protein-coupled receptors, which are membrane proteins implicated in cellular signal transduction. Herein, we show experimentally that approximately 80 water molecules flood rhodopsin upon light absorption to form a solvent-swollen active state. An influx of mobile water is necessary for activating the photoreceptor, and this finding is supported by molecular dynamics (MD) simulations. Combined force-based measurements involving osmotic and hydrostatic pressure indicate the expansion occurs by changes in cavity volumes, together with greater hydration in the active metarhodopsin-II state. Moreover, we discovered that binding and release of the C-terminal helix of transducin is coupled to hydration changes as may occur in visual signal amplification. Hydration-dehydration explains signaling by a dynamic allosteric mechanism, in which the soft membrane matter (lipids and water) has a pivotal role in the catalytic G-protein cycle.

Osmotic stress studies of G-protein-coupled receptor rhodopsin activation.

We summarize and critically review osmotic stress studies of the G-protein-coupled receptor rhodopsin. Although small amounts of structural water are present in these receptors, the effect of bulk water on their function remains uncertain. Studies of the influences of osmotic stress on the GPCR archetype rhodopsin have given insights into the functional role of water in receptor activation. Experimental work has discovered that osmolytes shift the metarhodopsin equilibrium after photoactivation, either to the active or inactive conformations according to their molar mass. At least 80 water molecules are found to enter rhodopsin in the transition to the photoreceptor active state. We infer that this movement of water is both necessary and sufficient for receptor activation. If the water influx is prevented, e.g., by large polymer osmolytes or by dehydration, then the receptor functional transition is back shifted. These findings imply a new paradigm in which rhodopsin becomes solvent swollen in the activation mechanism. Water thus acts as an allosteric modulator of function for rhodopsin-like receptors in lipid membranes.

Reactive Oxygen Species/Reactive Nitrogen Species as Messengers in the Gut: Impact on Physiology and Metabolic Disorders.

Significance: The role of reactive oxygen/nitrogen species as "friend" or "foe" messengers in the whole body is well characterized. Depending on the concentration in the tissue considered, these molecular actors exert beneficial or deleterious impacts leading to a pathological state, as observed in metabolic disorders such as type 2 diabetes and obesity. Recent Advances: Among the tissues impacted by oxidation and inflammation in this pathological state, the intestine is a site of dysfunction that can establish diabetic symptoms, such as alterations in the intestinal barrier, gut motility, microbiota composition, and gut/brain axis communication. In the intestine, reactive oxygen/nitrogen species (from the host and/or microbiota) are key factors that modulate the transition from physiological to pathological signaling. Critical Issues: Controlling the levels of intestinal reactive oxygen/nitrogen species is a complicated balance between positive and negative impacts that is in constant equilibrium. Here, we describe the synthesis and degradation of intestinal reactive oxygen/nitrogen species and their interactions with the host. The development of novel redox-based therapeutics that alter these processes could restore intestinal health in patients with metabolic disorders. Future Directions: Deciphering the mode of action of reactive oxygen/nitrogen species in the gut of obese/diabetic patients could result in a future therapeutic strategy that combines nutritional and pharmacological approaches. Consequently, preventive and curative treatments must take into account one of the first sites of oxidative and inflammatory dysfunctions in the body, that is, the intestine. Antioxid. Redox Signal. 37, 394-415.

Solvent Organization and Electrostatics Tuned by Solute Electronic Structure: Amide versus Non-Amide Carbonyls.

The ability to exploit carbonyl groups to measure electric fields in enzymes and other complex reactive environments by using the vibrational Stark effect has inspired growing interest in how these fields can be measured, tuned, and ultimately designed. Previous studies have concentrated on the role of the solvent in tuning the fields exerted on the solute. Here, we explore instead the role of the solute electronic structure in modifying the local solvent organization and electric field exerted on the solute. By measuring the infrared absorption spectra of amide-containing molecules, as prototypical peptides, and contrasting them with non-amide carbonyls in a wide range of solvents, we show that these solutes experience notable differences in their frequency shifts in polar solvents. Using vibrational Stark spectroscopy and molecular dynamics simulations, we demonstrate that while some of these differences can be rationalized by using the distinct intrinsic Stark tuning rates of the solutes, the larger frequency shifts for amides and dimethylurea primarily result from the larger solvent electric fields experienced by their carbonyl groups. These larger fields arise due to their stronger p-π conjugation, which results in larger C═O bond dipole moments that further induce substantial solvent organization. Using electronic structure calculations, we decompose the electric fields into contributions from solvent molecules that are in the first solvation shell and those from the bulk and show that both of these contributions are significant and become larger with enhanced conjugation in solutes. These results show that structural modifications of a solute can be used to tune both the solvent organization and electrostatic environment, indicating the importance of a solute-centric paradigm in modulating and designing the electrostatic environment in condensed-phase chemical processes.

Interactions between the microbiota and enteric nervous system during gut-brain disorders.

For the last 20 years, researchers have focused their intention on the impact of gut microbiota in healthy and pathological conditions. This year (2021), more than 25,000 articles can be retrieved from PubMed with the keywords "gut microbiota and physiology", showing the constant progress and impact of gut microbes in scientific life. As a result, numerous therapeutic perspectives have been proposed to modulate the gut microbiota composition and/or bioactive factors released from microbes to restore our body functions. Currently, the gut is considered a primary site for the development of pathologies that modify brain functions such as neurodegenerative (Parkinson's, Alzheimer's, etc.) and metabolic (type 2 diabetes, obesity, etc.) disorders. Deciphering the mode of interaction between microbiota and the brain is a real original option to prevent (and maybe treat in the future) the establishment of gut-brain pathologies. The objective of this review is to describe recent scientific elements that explore the communication between gut microbiota and the brain by focusing our interest on the enteric nervous system (ENS) as an intermediate partner. The ENS, which is known as the "second brain", could be under the direct or indirect influence of the gut microbiota and its released factors (short-chain fatty acids, neurotransmitters, gaseous factors, etc.). Thus, in addition to their actions on tissue (adipose tissue, liver, brain, etc.), microbes can have an impact on local ENS activity. This potential modification of ENS function has global repercussions in the whole body via the gut-brain axis and represents a new therapeutic strategy. This article is part of the special Issue on 'Cross Talk between Periphery and the Brain'.

Differential anti-thrombotic benefit and bleeding risk profiles of antagonists of protease-activated receptor 1 and 4 in Cynomolgus Macaques.

Platelet activation plays a crucial role in hemostasis and thrombosis. Thrombin, the most potent stimulus of platelet activation, mediates platelet activation via the protease activated receptors (PARs). The platelet PAR repertoire in mediating thrombin's action differs across species. Only nonhuman primate (NHP) platelet activation is known to be similar to humans, mediated by PAR1 and PAR4, hence limiting translational in vivo studies of PAR's role in thrombosis and hemostasis to NHPs. Earlier studies have demonstrated a range of distinct in vitro activities of PAR1 and 4 in platelet activation yet the implications of these events in vivo is unclear. The objective of this study is to investigate and compare the roles of PAR1 and PAR4 in hemostasis and thrombosis in a relevant animal species. NHP models for pharmacokinetic, ex vivo platelet aggregation responses, FeCI3 injury-mediated arterial thrombosis and template bleeding were developed in Cynomolgus Macaques. Potent and selective small molecule antagonists of PAR1 and PAR4 were characterized in an array of in vitro assays, and subsequently examined head-to-head in the NHP models. Treatment of NHPs with antagonists of PAR1 or PAR4 both resulted in strong inhibition of ex vivo platelet aggregation. At doses that led to similar inhibition of platelet aggregation, animals treated with the PAR4 antagonist showed similar levels of anti-thrombotic efficacy, but longer bleeding times, compared to animals treated with the PAR1 antagonist. These findings suggest that PAR1 antagonism will likely produce a larger therapeutic index (ie. a larger anti-thrombotic efficacy over bleeding risk margin) than PAR4 antagonism.

Cloning and characterization of rhesus monkey MCH-R1 and MCH-R2.

Rhesus monkey MCH-R1 and MCH-R2 receptors were cloned. Amino acid homology is 98.8% between monkey and human MCH-R1, while monkey and human MCH-R2 are 98% homologous. Binding and intracellular signaling characteristics of the monkey receptors were compared with the human homologues. The results demonstrate that MCH binds to the monkey MCH-R1 receptor with a K(d) of 6.5 nM and monkey MCH-R2 with a K(d) of 2.2 nM similar to K(d) values for human MCH-R1 and MCH-R2. Additionally, monkey MCH-R1 couples through G(i)/G(o) and G(q)-type G proteins similar to human MCH-R1 whereas monkey and human MCH-R2 utilize the G(q) signaling pathway.