Structural origins of constitutive activation in rhodopsin: Role of the K296/E113 salt bridge

The intramolecular interactions that stabilize the inactive conformation of rhodopsin are of primary importance in elucidating the mechanism of activation of this and other G protein-coupled receptors. In the present study, site-directed spin labeling is used to explore the role of a buried salt bridge between the protonated Schiff base at K296 in TM7 and its counterion at E113 in TM3. Spin-label sensors are placed at positions in the cytoplasmic surface of rhodopsin to monitor changes in the structure of the helix bundle caused by point mutations that disrupt the salt bridge. The single point mutations E113Q, G90D, and A292E, which were previously reported to cause constitutive activation of the apoprotein opsin, are found to cause profound movements of both TM3 and TM6 in the dark state, the latter of which is similar to that caused by light activation. The mutant M257Y, which constitutively activates opsin but does not disrupt the salt bridge, is shown to cause related but distinguishable structural changes. The double mutants E113Q/M257Y and G90D/M257Y produce strong activation of the receptor in the dark state. In the E113Q/M257Y mutant investigated with spin labeling, the movement of TM6 and other changes are exaggerated relative to either E113Q or M257Y alone. Collectively, the results provide structural evidence that the salt bridge is a key constraint maintaining the resting state of the receptor, and that the disruption of the salt bridge is the cause, rather than a consequence, of the TM6 motion that occurs upon activation.     

Structure and function in rhodopsin: Mass spectrometric identification of the abnormal intradiscal disulfide bond in misfolded retinitis pigmentosa mutants

Retinitis pigmentosa (RP) point mutations in both the intradiscal (ID) and transmembrane domains of rhodopsin cause partial or complete misfolding of rhodopsin, resulting in loss of 11-cis-retinal binding. Previous work has shown that misfolding is caused by the formation of a disulfide bond in the ID domain different from the native Cys-110-Cys-187 disulfide bond in native rhodopsin. Here we report on direct identification of the abnormal disulfide bond in misfolded RP mutants in the transmembrane domain by mass spectrometric analysis. This disulfide bond is between Cys-185 and Cys-187, the same as previously identified in misfolded RP mutations in the ID domain. The strategy described here should be generally applicable to identification of disulfide bonds in other integral membrane proteins.     

Sequence of late molecular events in the activation of rhodopsin

Activation of the G protein-coupled receptor rhodopsin involves both the motion of transmembrane helix 6 (TM6) and proton exchange events. To study how these activation steps relate to each other, spin-labeled rhodopsin in solutions of dodecyl maltoside was used so that time-resolved TM6 motion and proton exchange could each be monitored as a function of pH and temperature after an activating light flash. The results reveal that the motion of TM6 is not synchronized with deprotonation of the Schiff base that binds the chromophore to the protein but is an order of magnitude slower at 30 degrees C. However, TM6 motion and the uptake of a proton from solution in the neutral pH range follow the same time course. Importantly, the motion of TM6 is virtually independent of pH, as is Schiff base deprotonation under the conditions used, whereas proton uptake titrates with a pK of 6.5. This finding shows that proton uptake is a consequence rather than a cause of helix motion. Activated rhodopsin binds to and subsequently activates the cognate G protein, transducin. It has been shown that peptides derived from the C terminus of the transducin alpha-subunit mimic in part binding of the intact G protein. These peptides are found to bind to rhodopsin after TM6 movement, resulting in the release of protons. Collectively, the data suggest the following temporal sequence of events involved in activation: (i) internal Schiff base proton transfer; (ii) TM6 movement; and (iii) proton uptake from solution and binding of transducin.     

The product of uncI gene in F1Fo-ATP synthase operon plays a chaperone-like role to assist c-ring assembly

Bacterial operons for F₁F_o-ATP synthase typically include an uncI gene that encodes a function-unknown small hydrophobic protein. When we expressed a hybrid F₁F_o (F₁ from thermophilic Bacillus PS3 and Na⁺-translocating F_o from Propionigenium modestum) in Escherchia coli cells, we found that uncI derived from P. modestum was indispensable to produce active enzyme; without uncI, c-subunits in F₁F_o existed as monomers but not as functional c₁₁-ring. When uncI was expressed from another plasmid at the same time, active F₁F_o with c₁₁-ring was produced. A plasmid containing only uncI and c-subunit gene produced c₁₁-ring, but a plasmid containing only c-subunit gene did not. Direct interaction of UncI protein with c-subunits was suggested from copurification of His-tagged UncI protein and c-subunits, both in the state of c₁₁-ring and c-monomers. Na⁺ induced dissociation of His-tagged UncI protein from c₁₁-ring but not from c-monomers. These results show that UncI is a chaperone-like protein that assists c₁₁-ring assembly from c-monomers in the membrane.     

Inositol hexakisphosphate suppresses excitatory neurotransmission via synaptotagmin-1 C2B domain in the hippocampal neuron

Inositol hexakisphosphate (InsP(6)) levels rise and fall with neuronal excitation and silence, respectively, in the hippocampus, suggesting potential signaling functions of this inositol polyphosphate in hippocampal neurons. We now demonstrate that intracellular application of InsP(6) caused a concentration-dependent inhibition of autaptic excitatory postsynaptic currents (EPSCs) in cultured hippocampal neurons. The treatment did not alter the size and replenishment rate of the readily releasable pool in autaptic neurons. Intracellular exposure to InsP(6) did not affect spontaneous EPSCs or excitatory amino acid-activated currents in neurons lacking autapses. The InsP(6)-induced inhibition of autaptic EPSCs was effectively abolished by coapplication of an antibody to synaptotagmin-1 C2B domain. Importantly, preabsorption of the antibody with a GST-WT synaptotagmin-1 C2B domain fragment but not with a GST-mutant synaptotagmin-1 C2B domain fragment that poorly reacted with the antibody impaired the activity of the antibody on the InsP(6)-induced inhibition of autaptic EPSCs. Furthermore, K(+) depolarization significantly elevated endogenous levels of InsP(6) and occluded the inhibition of autaptic EPSCs by exogenous InsP(6). These data reveal that InsP(6) suppresses excitatory neurotransmission via inhibition of the presynaptic synaptotagmin-1 C2B domain-mediated fusion via an interaction with the synaptotagmin Ca(2+)-binding sites rather than via interference with presynaptic Ca(2+) levels, synaptic vesicle trafficking, or inactivation of postsynaptic ionotropic glutamate receptors. Therefore, elevated InsP(6) in activated neurons serves as a unique negative feedback signal to control hippocampal excitatory neurotransmission.     

Binding affinity of lactose permease is not altered by the H+ electrochemical gradient

The x-ray structure of lactose permease of Escherichia coli (LacY) exhibits a single sugar-binding site at the apex of a hydrophilic cavity open to the cytoplasm, and it has been postulated that the binding site has alternating access to either side of the membrane during turnover. Here, the affinity of LacY for ligand in right-side-out or inside-out membrane vesicles is measured in the absence or presence of an H(+) electrochemical gradient (Deltamicro(H(+))) by utilizing ligand protection against alkylation. Right-side-out or inside-out membrane vesicles containing LacY with a single cysteine residue at position 148 exhibit K(D) values for lactose or beta-d-galactopyranosyl 1-thio-beta-d-galactopyranoside of approximately 1.0 mM or 40 microM, respectively, and no systematic change is observed in the presence of Deltamicro(H(+)) under conditions in which there is little or no accumulation of ligand. The results are consistent with a mechanism in which the major effect of Deltamicro(H(+)) on sugar accumulation is caused by an increased rate of deprotonation on the inner face of the membrane, leading to an increase in the rate of return of the unloaded symporter to the outer face of the membrane.     

Insertion domain within mammalian mitochondrial translation initiation factor 2 serves the role of eubacterial initiation factor 1

Mitochondria have their own translational machineries for the synthesis of thirteen polypeptide chains that are components of the complexes that participate in the process of oxidative phosphorylation (or ATP generation). Translation initiation in mammalian mitochondria requires two initiation factors, IF2(mt) and IF3(mt), instead of the three that are present in eubacteria. The mammalian IF2(mt) possesses a unique 37 amino acid insertion domain, which is known to be important for the formation of the translation initiation complex. We have obtained a three-dimensional cryoelectron microscopic map of the mammalian IF2(mt) in complex with initiator fMet-tRNA(iMet) and the eubacterial ribosome. We find that the 37 amino acid insertion domain interacts with the same binding site on the ribosome that would be occupied by the eubacterial initiation factor IF1, which is absent in mitochondria. Our finding suggests that the insertion domain of IF2(mt) mimics the function of eubacterial IF1, by blocking the ribosomal aminoacyl-tRNA binding site (A site) at the initiation step.     

Evaluation of Molecular Test for the Discrimination of “Naked” DNA from Infectious Parvovirus B19 Particles in Serum and Bone Marrow Samples

Low levels of parvovirus B19 (B19V) DNA can be detected in the circulation and in different tissue of immunocompetent individuals for months or years, which has been linked to inflammatory diseases such as cardiomyopathy, rheumatoid arthritis, hepatitis, and vasculitis. However, the detection of B19V DNA does not necessarily imply that infectious virions are present. This study aimed to evaluate the method based on the Benzonase^® treatment for differentiation between the infectious virions from “naked” DNA in serum and bone marrow (BM) samples to be useful for the B19V routine diagnosis. In addition, we estimated the period of viremia and DNAemia in the sera and bone marrow of nonhuman primates experimentally infected with B19V. Serum samples from ten patients and from four cynomolgus monkeys experimentally infected with B19V followed up for 60 days were used. Most of the human serum samples became negative after pretreatment; however, only decreased viral DNA loads were observed in four patients, indicating that these samples still contained the infectious virus. Reduced B19V DNA levels were observed in animals since 7th dpi. At approximately 45th dpi, B19V DNA levels were below 10⁵ IU/mL after Benzonase^® pretreatment, which was not a consequence of active B19V replication. The test based on Benzonase^® pretreatment enabled the discrimination of “naked DNA” from B19V DNA encapsidated in virions. Therefore, this test can be used to clarify the role of B19V as an etiological agent associated with atypical clinical manifestations.     

SNAP-25, a SNARE protein, inhibits two types of K channels in esophageal smooth muscle

BACKGROUND & AIMS: The plasma membrane-associated soluble N-ethylmaleimide-sensitive factors attachment protein receptors (SNAREs), synaptosome-associated protein of 25 kilodaltons (SNAP-25), and syntaxin 1A, have been found to physically interact with and functionally modify membrane-spanning ion channels. Studies were performed in cat esophageal body and lower esophageal sphincter (LES) smooth muscle to (1) show the presence of SNAP-25, and (2) determine whether SNAP-25 affects K+ channel activity. METHODS: Single circular muscle cells from the esophageal body and sphincter were studied. Cellular localization of SNAP-25 and K+ channel activity were assessed. RESULTS: SNAP-25 was found in the plasma membrane of all regions examined. Outward K+ currents in body circular muscle were mainly composed of large conductance Ca2+-activated channel currents (K(Ca), 40.1%) and delayed rectifier K+ channel currents (K(V), 54.2%). Microinjection of SNAP-25 into muscle cells caused a dose-dependent inhibition of both outward K+ currents, maximal 44% at 10(-8) mol/L. Cleavage of endogenous SNAP-25 by dialyzing botulinum neurotoxin A into the cell interior resulted in a 35% increase in outward currents. CONCLUSIONS: SNAP-25 protein is present in esophageal smooth muscle cells, and inhibits both K(V) and K(Ca) currents in circular muscle cells. The findings suggest a role for SNAP-25 in regulation of esophageal muscle cell excitability and contractility, and point to potential new targets for treatment of esophageal motor disorders.     

NMR Structure of the Myristylated Feline Immunodeficiency Virus Matrix Protein

Membrane targeting by the Gag proteins of the human immunodeficiency viruses (HIV types-1 and -2) is mediated by Gag’s N-terminally myristylated matrix (MA) domain and is dependent on cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂]. To determine if other lentiviruses employ a similar membrane targeting mechanism, we initiated studies of the feline immunodeficiency virus (FIV), a widespread feline pathogen with potential utility for development of human therapeutics. Bacterial co-translational myristylation was facilitated by mutation of two amino acids near the amino-terminus of the protein (Q5A/G6S; myrMA^Q5A/G6S). These substitutions did not affect virus assembly or release from transfected cells. NMR studies revealed that the myristyl group is buried within a hydrophobic pocket in a manner that is structurally similar to that observed for the myristylated HIV-1 protein. Comparisons with a recent crystal structure of the unmyristylated FIV protein [myr(-)MA] indicate that only small changes in helix orientation are required to accommodate the sequestered myr group. Depletion of PI(4,5)P₂ from the plasma membrane of FIV-infected CRFK cells inhibited production of FIV particles, indicating that, like HIV, FIV hijacks the PI(4,5)P₂ cellular signaling system to direct intracellular Gag trafficking during virus assembly.     

Rescue of familial cardiomyopathies by modifications at the level of sarcomere and Ca fluxes

Cardiomyopathies are a heterogeneous group of diseases of the myocardium associated with mechanical and/or electrical dysfunction that frequently show inappropriate ventricular hypertrophy or dilation. Current data suggest that numerous mutations in several genes can cause cardiomyopathies, and the severity of their phenotypes is also influenced by modifier genes. Two major types of inherited cardiomyopathies include familial hypertrophic cardiomyopathy (FHC) and dilated cardiomyopathy (DCM). FHC typically involves increased myofilament Ca²⁺ sensitivity associated with diastolic dysfunction, whereas DCM often results in decreased myofilament Ca²⁺ sensitivity and systolic dysfunction. Besides alterations in myofilament Ca²⁺ sensitivity, alterations in the levels of Ca²⁺-handling proteins have also been described in both diseases. Recent work in animal models has attempted to rescue FHC and DCM via modifications at the myofilament level, altering Ca²⁺ homeostasis by targeting Ca²⁺-handling proteins, such as the sarcoplasmic reticulum ATPase and phospholamban, or by interfering with the products of different modifiers genes. Although attempts to rescue cardiomyopathies in animal models have shown great promise, further studies are needed to validate these strategies in order to provide more effective and specific treatments.     

Early pregnancy induced expression of prostaglandin E2 receptors EP2 and EP4 in the ovine endometrium and regulated by interferon tau through multiple cell signaling pathways

Prostaglandin E2 (PGE₂) plays pleiotropic roles at fetal-maternal interface during establishment of pregnancy. The objectives of the study were to: (i) determine regulation of PGE2 receptors EP1, EP2, EP3, and EP4 in the endometrium during the estrous cycle and early pregnancy; and (ii) understand endometrial epithelial and stromal cell-specific hormonal regulation of EP2 and EP4 in sheep. Results indicate that: (i) early pregnancy induces expression of EP2 and EP4 but not EP1 and EP3 proteins in the endometrium on days 12-16 compared to that of estrous cycle; (ii) intrauterine infusion of interferon tau (IFNT) increases expression of EP2 and EP4 proteins in endometrium; and (iii) IFNT activates distinct epithelial and stromal cell-specific JAK, EGFR, ERK1/2, AKT, or JNK signaling module to regulate expression of EP2 and EP4 proteins in the ovine endometrium. Our results indicate a role for EP2 and EP4-mediated PGE₂ signaling in endometrial functions and establishment of pregnancy in ruminants.     

The role of endocannabinoids in gonadal function and fertility along the evolutionary axis

Endocannabinoids are natural lipids able to bind to cannabinoid and vanilloid receptors. Their biological actions at the central and peripheral level are under the tight control of the proteins responsible for their synthesis, transport and degradation. In the last few years, several reports have pointed out these lipid mediators as critical signals, together with sex hormones and cytokines, in various aspects of animal and human reproduction. The identification of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in reproductive cells and tissues of invertebrates, vertebrates and mammals highlights the key role played by these endogenous compounds along the evolutionary axis. Here, we review the main actions of endocannabinoids on female and male reproductive events, and discuss the interplay between them, steroid hormones and cytokines in regulating fertility. In addition, we discuss the involvement of endocannabinoid signalling in ensuring a correct chromatin remodeling, and hence a good DNA quality, in sperm cells.     

Revisiting the evolution of gonadotropin-releasing hormones and their receptors in vertebrates: secrets hidden in genomes

Gonadotropin-releasing hormone (GnRH) and its G protein-coupled receptor, GnRHR, play a pivotal role in the control of reproduction in vertebrates. To date, many GnRH and GnRHR genes have been identified in a large variety of vertebrate species using conventional biochemical and molecular biological tools in combination with bioinformatic tools. Phylogenetic approaches, primarily based on amino acid sequence identity, make it possible to classify these multiple GnRHs and GnRHRs into several lineages. Four vertebrate GnRH lineages GnRH1, GnRH2, GnRH3, and GnRH4 (for lamprey) are well established. Four vertebrate GnRHR lineages have also been proposed—three for nonmammalian GnRHRs and mammalian GnRHR2 as well as one for mammalian GnRHR1. However, these phylogenetic analyses cannot fully explain the evolutionary origins of each lineage and the relationships among the lineages. Rapid and vast accumulation of genome sequence information for many vertebrate species, together with advances in bioinformatic tools, has allowed large-scale genome comparison to explore the origin and relationship of gene families of interest. The present review discusses the evolutionary mechanism of vertebrate GnRHs and GnRHRs based on extensive genome comparison. In this article, we focus only on vertebrate genomes because of the difficulty in comparing invertebrate and vertebrate genomes due to their marked divergence.