Regulation of Gi and Go by mastoparan, related amphiphilic peptides, and hydrophobic amines. Mechanism and structural determinants of activity

Mastoparan (MP), a cationic, amphiphilic tetradecapeptide, stimulates guanine nucleotide exchange by GTP-binding regulatory proteins (G proteins) in a manner similar to that of G protein-coupled receptors. 1) MP stimulated exchange by isolated G protein alpha subunits and alpha beta gamma trimers. Relative stimulation was greater with alpha beta gamma trimers and beta gamma subunits could increase net MP-stimulated activity. 2) MP action was enhanced by reconstitution of trimeric G protein into phospholipid vesicles. Hill coefficients for activation were 2-4. The membrane-bound alpha-helical conformation of MP appeared to be the activating species. 3) MP blocked the ability of Go to increase the affinity of muscarinic receptors for agonist ligands, suggesting that MP and the receptor may compete for a common binding site on Go. 4) MP stimulated steady state GTPase activity at less than 1 microM Mg2+ and stimulated the dissociation of both GDP and guanosine 5'-O-(3-thiotriphosphate) at less than 1 nM Mg2+. Millimolar Mg2+ blocked the stimulatory effect of MP. Both high and low affinity Mg2+ binding sites are on the alpha subunit. 5) Increasing the amphiphilicity or hydrophobicity of MP enhanced its regulatory activity more than 2-fold and lowered the EC50 more than 10-fold. Several natural amphiphilic peptides also displayed modest stimulatory activity. 6) Benzalkonium chloride competitively antagonized the stimulation of Gi by MP but potently stimulated nucleotide exchange on Go. Because cationic, amphiphilic sequences on the cytoplasmic faces of receptors are required for G protein regulation, these findings suggest that nucleotide exchange on G proteins is regulated by the presentation of multiple cationic structures on the inner face of the plasma membrane.     

Different combinations of Notch ligands and receptors regulate V2 interneuron progenitor proliferation and V2a/V2b cell fate determination

The broad diversity of neurons is vital to neuronal functions. During vertebrate development, the spinal cord is a site of sensory and motor tasks coordinated by interneurons and the ongoing neurogenesis. In the spinal cord, V2-interneuron (V2-IN) progenitors (p2) develop into excitatory V2a-INs and inhibitory V2b-INs. The balance of these two types of interneurons requires precise control in the number and timing of their production. Here, using zebrafish embryos with altered Notch signaling, we show that different combinations of Notch ligands and receptors regulate two functions: the maintenance of p2 progenitor cells and the V2a/V2b cell fate decision in V2-IN development. Two ligands, DeltaA and DeltaD, and three receptors, Notch1a, Notch1b, and Notch3 redundantly contribute to p2 progenitor maintenance. On the other hand, DeltaA, DeltaC, and Notch1a mainly contribute to the V2a/V2b cell fate determination. A ubiquitin ligase Mib, which activates Notch ligands, acts in both functions through its activation of DeltaA, DeltaC, and DeltaD. Moreover, p2 progenitor maintenance and V2a/V2b fate determination are not distinct temporal processes, but occur within the same time frame during development. In conclusion, V2-IN cell progenitor proliferation and V2a/V2b cell fate determination involve signaling through different sets of Notch ligand–receptor combinations that occur concurrently during development in zebrafish.     

Structural basis of the initial binding of tRNA(Ile) lysidine synthetase TilS with ATP and L-lysine

In the bacterial genetic-code system, the codon AUA is decoded as isoleucine by tRNA(Ile)(2) with the lysidine residue at the wobble position. Lysidine is derived from cytidine, with ATP and L-lysine, by tRNA(Ile) lysidine synthetase (TilS), which is an N-type ATP pyrophosphatase. In this study, we determined the crystal structure of Aquifex aeolicus TilS, complexed with ATP, Mg2+, and L-lysine, at 2.5 A resolution. The presence of the TilS-specific subdomain causes the active site to have two separate gateways, a large hole and a narrow tunnel on the opposite side. ATP is bound inside the hole, and L-lysine is bound at the entrance of the tunnel. The conserved Asp36 in the PP-motif coordinates Mg2+. In these initial binding modes, the ATP, Mg2+, and L-lysine are held far apart from each other, but they seem to be brought together for the reaction upon cytidine binding, with putative structural changes of the complex.     

Crystal structure of Sulfolobus tokodaii Sua5 complexed with L-threonine and AMPPNP

The hypermodified nucleoside N⁶‐threonylcarbamoyladenosine resides at position 37 of tRNA molecules bearing U at position 36 and maintains translational fidelity in the three kingdoms of life. The N⁶‐threonylcarbamoyl moiety is composed of L ‐threonine and bicarbonate, and its synthesis was genetically shown to require YrdC/Sua5. YrdC/Sua5 binds to tRNA and ATP. In this study, we analyzed the L ‐threonine‐binding mode of Sua5 from the archaeon Sulfolobus tokodaii. Isothermal titration calorimetry measurements revealed that S. tokodaii Sua5 binds L ‐threonine more strongly than L ‐serine and glycine. The Kd values of Sua5 for L ‐threonine and L ‐serine are 9.3 μM and 2.6 mM, respectively. We determined the crystal structure of S. tokodaii Sua5, complexed with AMPPNP and L ‐threonine, at 1.8 Å resolution. The L ‐threonine is bound next to AMPPNP in the same pocket of the N‐terminal domain. Thr118 and two water molecules form hydrogen bonds with AMPPNP in a unique manner for adenine‐specific recognition. The carboxyl group and the side‐chain hydroxyl and methyl groups of L ‐threonine are buried deep in the pocket, whereas the amino group faces AMPPNP. The L ‐threonine is located in a suitable position to react together with ATP for the synthesis of N⁶‐threonylcarbamoyladenosine. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

V2a and V2b neurons are generated by the final divisions of pair-producing progenitors in the zebrafish spinal cord

The p2 progenitor domain in the ventral spinal cord gives rise to two interneuron subtypes: V2a and V2b. Delta-Notch-mediated cell-cell interactions between postmitotic immature neurons have been implicated in the segregation of neuron subtypes. However, lineage relationships between V2a and V2b neurons have not been reported. We address this issue using Tg[vsx1:GFP] zebrafish, a model system in which high GFP expression is initiated near the final stage of p2 progenitors. Cell fates were followed in progeny using time-lapse microscopy. Results indicate that the vast majority, if not all, of GFP-labeled p2 progenitors divide once to produce V2a/V2b neuron pairs, indicating that V2a and V2b neurons are generated by the asymmetric division of pair-producing progenitor cells. Together with evidence that Notch signaling is involved in the cell fate specification process, our results strongly suggest that Delta-Notch interactions between sister cells play a crucial role in the final outcome of these asymmetric divisions. This mechanism for determining cell fate is similar to asymmetric divisions that occur during Drosophila neurogenesis, where ganglion mother cells divide once to produce distinct neurons. However, unlike in Drosophila, the divisional axes of p2 progenitors in zebrafish were not fixed. We report that the terminal division of pair-producing progenitor cells in vertebrate neurogenesis can reproducibly produce two distinct neurons through a mechanism that may not depend on the orientation of the division axis.     

Exogastrulation and interference with the expression of major yolk protein by estrogens administered to sea urchins

Although estrogens have been detected in some echinoderm species, their role is not clearly understood; so we examined the effects of estrogens administered to sea urchin embryos and larvae. A typical malformation was exogastrulation, induced by the exposure to ethynylestradiol (EER) in a defined period of 12 h from 12 h after fertilization (HAF). Morphogenesis for gastrulation was delayed in the treated embryos: protrusion of the archenteron started at 30 HAF when gastrulation had already finished in normal embryos. Exogastrulation induced by EER was cancelled by the antiestrogen chemical, ICI182,780. Feeding larvae were less sensitive to estrogens than those in early embryogenesis and, at certain concentrations, developed without abnormal morphology. The effect of estrogens was examined at the level of gene expression of the major yolk protein (MYP). MYP expression started during the larval stage and was suppressed by estrone at the six-armed stage, but not by β-estradiol, and in later stage larvae, the expression was not affected by treatment with either estrogen. Estrogens affect sea urchins in the early stage of embryogenesis, leading to abnormal morphogenesis and interference with gene expression.     

Reconstitution of agonist-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis using purified m1 muscarinic receptor, Gq/11, and phospholipase C-beta 1.

We describe the reconstitution using purified proteins of the m1 muscarinic cholinergic pathway that activates phosphatidylinositol 4,5-bisphosphate-specific phospholipase C via the G protein Gq/11. Recombinant m1 muscarinic receptor was co-reconstituted in lipid vesicles with either hepatic Gq/11 or with cerebral alpha q/11 and beta gamma subunits. The rate of [35S]GTP gamma S binding to the reconstituted vesicles was stimulated 20-50-fold by agonist. Maximal receptor-catalyzed binding was 7 mol of GTP gamma S bound per mol of receptor. The m2 muscarinic receptor was a poor activator of Gq/11. The binding of [alpha-32P]GTP to [gamma-32P]GTP to m1/Gq/11 vesicles indicated that the receptor could maintain up to 40% of the total coupled Gq/11 in the GTP bound state. The rate of hydrolysis of bound GTP, 0.8 min-1, is consistent with the rate predicted from the GTP binding data but is 3-5-fold lower than rates reported for other trimeric G proteins. Agonist-stimulated photo-affinity labeling with gamma-(4-azidoanilido)-[alpha-32P]GTP indicated that the receptor catalyzed binding to both alpha q and alpha 11 with about equal efficiency. Receptor-catalyzed activation of Gq/11 by GTP gamma S, measured as the ability to activate purified phospholipase C-beta 1, paralleled receptor-catalyzed [35S]GTP gamma S binding. Co-reconstitution of receptor, Gq/11, and phospholipase C-beta 1 restored GTP gamma S-dependent carbachol-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate. The m1 receptor, Gq/11, and phospholipase C-beta 1 are thus sufficient to initiate the hormonal inositol trisphosphate/diacylglycerol signaling pathway without additional proteins.     

Mapping of the mastoparan-binding site on G proteins. Cross-linking of [125I-Tyr3,Cys11]mastoparan to Go

Mastoparan (MP) activates GTP-binding regulatory proteins (G proteins) by promoting GDP/GTP exchange through a mechanism similar to that of G protein-coupled receptors (Higashijima, T., Burnier, J., and Ross, E. M. (1990) J. Biol. Chem. 265, 14176-14186). [Tyr3, Cys11]MP was synthesized and shown to have regulatory activity similar to that of mastoparan when assayed in the presence of dithiothreitol (DTT). Activation by [Tyr3,Cys11]MP in the absence of DTT was complex in its kinetics, concentration dependence, and dependence on detergents. [125I-Tyr3,Cys11]MP bound covalently to the alpha subunit of G proteins. Cross-linking was blocked by mastoparan or [Tyr3,Cys11]MP. Cross-linking was enhanced by the addition of beta gamma subunits, but no cross-linking to beta gamma subunits was observed. Cross-linking was inhibited by incubation of Go with guanosine 5'-O-(thiotriphosphate) and Mg2+ and was reversed by incubation with DTT or 2-mercaptoethanol. Stoichiometry of labeling was consistent with the cross-linking of one molecule of [125I-Tyr3,Cys11]MP/alpha subunit, and CNBr hydrolysis of the [125I-Tyr3,Cys11]MP-alpha o adduct yielded one major labeled peptide fragment of approximately 6 kDa. Amino acid sequencing of this CNBr fragment prepared from recombinant alpha o showed that cross-linking occurred at Cys3. No alpha o sequence was obtained from the same fragment prepared from bovine brain alpha o, which is blocked by a myristoyl group at Gly2. Regulation of Go by MP was eliminated by tryptic proteolysis of the amino-terminal region. These observations suggest that the amino-terminal region of G protein alpha subunits contributes to the mastoparan-binding site, which may also be the receptor-binding site, and is involved in regulation of nucleotide exchange.     

Nuclear magnetic resonance study of side-chain conformation of phenylalanine residue in [Met5]-enkephalin: solvent, pH, and temperature dependence.

[Met5]-Enkephalin and N-acetylphenylalanine methylamide containing (2S,3S)-[2,3-2H2]Phe were synthesized 270 MHz 1H NMR spectra of the normal and selectively deuterated species were analysed. The lower-field and higher-field beta-proton signals of the Phe4 residue of [Met5]-enkephalin were unambiguously assigned to the pro-S and pro-R protons, respectively. The same assignments apply to N-acetylphenylalanine methylamide in polar organic solvents and in 2H2O, but the alternative assignments apply in C2HCl3. For [Met5]-enkephalin, the vicinal spin coupling constants 3JalphabetaS and 3 JalphabetaR and the rotamer populations around the Calpha-Cbeta bond were determined in a variety of solvents. From the pH and temperature dependences of rotamer populations of [Met5]-enkephalin, the side-chain conformation of the Phe residue in 2H2O solution was found to be considerably different from that in (C2H3)2SO solution. Rotamer populations of the Phe4 residue of [Met5]-enkephalin in organic solvents depend on solvent polarity. As compared with the reference model molecule of N-acetylphenylalanine methylamide, the rotamer populations of Phe4 of [Met5]-enkephalin are affected possibly by steric repulsion with other residues; the rotamer I is primarily favored but the rotamer II is appreciably destabilized in weakly polar solvents.