A unique fold of phospholipase C-beta mediates dimerization and interaction with G alpha q.

GTP-bound subunits of the Gq family of G alpha subunits directly activate phospholipase C-beta (PLC-beta) isozymes to produce the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. PLC-betas are GTPase activating proteins (GAPs) that also promote the formation of GDP-bound, inactive G beta subunits. Both phospholipase activation by G alpha-GTP subunits and GAP activity require a C-terminal region unique to PLC-beta isozymes. The crystal structure of the C-terminal region from an avian PLC-beta, determined at 2.4 A resolution, reveals a novel fold composed almost entirely of three long helices forming a coiled-coil that dimerizes along its long axis in an antiparallel orientation. The dimer interface is extensive ( approximately 3,200 A(2)), and, based on gel exclusion chromatography, full length PLC-betas are dimeric, indicating that PLC-betas likely function as dimers. Sequence conservation, mutational data and molecular modeling show that an electrostatically positive surface of the dimer contains the major determinants for binding G beta q. Effector dimerization, as highlighted by PLC-betas, provides a viable mechanism for regulating signaling cascades linked to heterotrimeric G proteins.     

A family of small cyclic amphipathic peptides (SCAmpPs) genes in citrus

Background

Citrus represents a crop of global importance both in economic impact and significance to nutrition. Citrus production worldwide is threatened by the disease Huanglongbing (HLB), caused by the phloem-limited pathogen Candidatus Liberibacter spp.. As a source of stable HLB-resistance has yet to be identified, there is considerable interest in characterization of novel disease-associated citrus genes.

Results

A gene family of Small Cyclic Amphipathic Peptides (SCAmpPs) in citrus is described. The citrus genomes contain 100–150 SCAmpPs genes, approximately 50 of which are represented in the citrus EST database. These genes encode small ~50 residue precursor proteins that are post-translationally processed, releasing 5–10 residue cyclic peptides. The structures of the SCAmpPs genes are highly conserved, with the small coding domains interrupted by a single intron and relatively extended untranslated regions. Some family members are very highly transcribed in specific citrus tissues, as determined by representation in tissue-specific cDNA libraries. Comparison of the ESTs of related SCAmpPs revealed an unexpected evolutionary profile, consistent with targeted mutagenesis of the predicted cyclic peptide domain. The SCAmpPs genes are displayed in clusters on the citrus chromosomes, with apparent association with receptor leucine-rich repeat protein arrays. This study focused on three SCAmpPs family members with high constitutive expression in citrus phloem. Unexpectedly high sequence conservation was observed in the promoter region of two phloem-expressed SCAmpPs that encode very distinct predicted cyclic products. The processed cyclic product of one of these phloem SCAmpPs was characterized by LC-MS-MS analysis of phloem tissue, revealing properties consistent with a K⁺ ionophore.

Conclusions

The SCAmpPs amino acid composition, protein structure, expression patterns, evolutionary profile and chromosomal distribution are consistent with designation as ribosomally synthesized defense-related peptides.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1486-4) contains supplementary material, which is available to authorized users.     

Synthesis and screening of a random dimeric peptide library using the one-bead-one-dimer combinatorial approach

Large combinatorial libraries of random peptides have been used for a variety of applications that include analysis of protein-protein interactions, epitope mapping, and drug targeting. The major obstacle in screening these libraries is the loss of specific but low affinity binding peptides during washing steps. Loss of these specific binders often results in isolation of peptides that bind nonspecifically to components used in the selection process. Previously, it has been demonstrated that dimerizing or multimerizing a peptide can remarkably improve its binding kinetics by 10- to 1000-fold due to an avidity effect. To take advantage of this observation, we constructed a random library of 12 amino acid dimeric peptides on polyethylene glycol acrylamide (PEGA) beads by modifying the 'one-bead-one-compound' approach. The chemical synthesis of 100,000 peptides as dimers can be problematic due to steric and aggregation effects and the presence of many peptide sequences that are difficult to synthesize. We have designed a method, described in detail here, to minimize the problems inherent in the synthesis of a dimeric library by modifying the existing 'split and pool' synthetic method. Using this approach the dimeric library was used to isolate a series of peptides that bound selectively to epithelial cancer cells. One peptide with the amino acid sequence QMARIPKRLARH bound as a dimer to prostate cancer cells spiked into the blood but did not bind to circulating hematopoeitic cells. The monomeric form of this peptide, however, did not bind well to the same LNCaP cell line. These data demonstrate that "hits" obtained from such a 'one-bead-one-dimer' library can be used directly for the final application or used as leads for construction of second generation libraries.     

Preparation of unilamellar lipid vesicles at 37°C by vaporization methods

We have developed a method utilizing low boiling solvents to prepare large, unilamellar vesicles at physiologic temperatures. Solutions of ethyl methyl ether or dichlorofluoromethane (Freon-21) at 4°C containing solubilized lipids were injected into a column of a aqueous buffer at 37°C. Vesicles prepared in this manner have been examined by freeze-fracture, negative stain electron microscopy, and fluorescence microscopy. The principal advantages of this technique are: (1) heat labile substances may be more readily entrapped in the internal vesicle volume without thermal denaturation, and (2) the range of lipids which are soluble in dichlorofluoromethane is greater than that of many other solvents, e.g. diethyl ether.     

Guanine nucleotides modulate the affinity of antagonists at beta-adrenergic receptors

Investigation of the properties of the binding of the radiolabelled antagonists (125I)-iodohydroxybenzylpindolol, (125I)-iodopindolol, and (125I)-iodocyanopindolol to beta-adrenergic receptors of L6 myoblast membranes revealed that guanine nucleotides caused a 2 to 4.5 fold increase in the apparent affinity of these antagonists. No significant effects of GTP were observed on the density of binding sites determined with each radioligand. GTP, GDP, and GMPPNP were of similar high affinity in producing this effect, while GMP was much less potent, and ATP was without effect. Under similar assay conditions GTP reduced the apparent binding affinity of the agonist isoproterenol for the beta-adrenergic receptors of L6 cells. The results indicate that, contrary to previous observations, guanine nucleotides affect not only the interactions of agonists with beta-adrenergic receptors, but also the interaction of antagonists with these adenylate cyclase-linked receptors.     

A receptor and G-protein-regulated polyphosphoinositide-specific phospholipase C from turkey erythrocytes. I. Purification and properties

Eighty-three percent of polyphosphoinositide-specific phospholipase C activity was recovered in a cytosolic fraction after nitrogen cavitation of turkey erythrocytes. This activity has been purified approximately 50,000-fold when compared to the starting cytosol with a yield of 1.7-5.0%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phospholipase C preparation revealed a major polypeptide of 150 kDa. The specific activity of the purified enzyme was 6.7-14.0 mumol/min/mg of protein with phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 4-phosphate as substrate. Phospholipase C activity was markedly dependent on the presence of Ca2+. The phospholipase C showed an acidic pH optimum (pH 4.0). At neutral pH, noncyclic inositol phosphates were the major products formed by the phospholipase C, while at pH 4.0, substantial formation of inositol 1:2-cyclic phosphate derivatives occurred. Properties of the purified 150-kDa turkey erythrocyte phospholipase C were compared with the approximately 150-kDa phospholipase C-beta and -gamma isoenzymes previously purified from bovine brain (Ryu, S. H., Cho, K. S., Lee, K. Y., Suh, P. G., and Rhee, S. G. (1987) J. Biol. Chem. 262, 12511-12518). The turkey erythrocyte phospholipase C differed from the two mammalian phospholipases with respect to the effect of sodium cholate on the rate of polyphosphoinositide hydrolysis observed. Moreover, when presented with dispersions of pure inositol lipids, phospholipases C-beta and -gamma displayed comparable maximal rates of polyphosphoinositide and phosphatidylinositol hydrolysis. By contrast, the turkey erythrocyte phospholipase C displays a marked preference for polyphosphoinositide substrates.     

Inhibition of Gene Transfer by Antiserum and Identification of Serotypes of Sex Pili

Gene transfer by conjugation due to F or R (drug resistance) factors is inhibited by antibody to the sex pili. Serological analysis is able to distinguish between the sex pili determined by closely related sex factors, and the specificity of inhibition of transfer agrees with that previously determined by direct electron microscopical observation of antibody bound to the sex pili (10). Inhibition of transfer can therefore be applied to the identification of wild-type R factors with repressed sex factors that determine too few pili to be examined directly. It can also be used to differentiate the activities of two unrelated sex factors in the same donor bacterium.     

Gas secretion and resorption in the swimbladder of the codGadus morhua

Summary The codGadus morhua has a closed, compliant swimbladder which occupies 5% of its volume. Pressure changes caused by vertical movements lead to the expansion and compression of the swimbladder gas, and the fish responds to the accompanying changes in density with compensatory swimming movements and the resorption or secretion of gas. A simple physiological model, based on estimates of cardiac output, the blood supply to the swimbladder and the oxygen-carrying capacity of the blood, is developed to set limits to these processes.An apparatus is described for making observations on the rate of buoyancy adaptation by cod subjected to pressure changes within the range 1.15 to 7.50 ATA at temperatures from 0 to 17°C. One hundred and twenty eight experiments were made with 38 cod ranging in length from 25 to 50 cm and in weight from 138 to 1440 g.The results showed that the rate of gas resorption increased markedly with the pressure to which the fish were adapted (from 0.14 ml kg^–1 min^–1 at 1.5 ATA to 1.54 ml kg^–1 min^–1 at 7.0 ATA), but not significantly with the weight of the fish or with temperature. In contrast, the rate of gas secretion increased markedly with temperature (from 0.02 ml kg^–1 min^–1 at 0°C to 0.11 ml kg^–1 min^–1 at 17°C), increased slightly with pressure, and decreased with the weight of the fish.The rate of gas resorption was much faster than that of gas secretion, and the difference between the two rates increased with pressure. The difference in performance is discussed in relation to the restriction that the swimbladder might impose to the speed and extent of vertical movements. It is suggested that when a closed swimbladder has a hydrostatic function it may be advantageous if neutral buoyancy is maintained only at the upper limit to the diurnal vertical range.     

Effects of dopaminergic agonists and antagonists on cerebellar guanosine-3',5'-monophosphate (cGMP).

Apomorphine was found to cause an increase in cerebellar cGMP content. Bromocriptine, at a dose that caused stereotypies, neither elevated cGMP, nor blocked the apomorphine- induced rise in cGMP. The apomorphine-induced rise in cGMP was effectively blocked by haloperidol and some other neuroleptics, but not by sulpiride. These actions of the neuroleptics correlated with their ability to displace ³H-spiroperidol from striatal membranes, suggesting that dopamine receptor interactions were important in the cGMP changes noted. Based on the observation that haloperidol antagonized the increase induced by restraint, it is suggested that dopaminergic systems are involved in the reaction to stress.     

Organic carbon and carbon isotopes in modern and 100-year-old-soil archives of the Russian steppe

Archived soils can provide valuable information about changes in the carbon and carbon isotope content of soils during the past century. We characterized soil carbon dynamics in a Russian steppe preserve using a 100‐year‐old‐soil archive and modern samples collected from the same site. The site has been protected since 1885 to the present, during which time the region has experienced widespread conversion to cultivation, a decrease in fire frequency, and a trend of increasing precipitation. In the preserve, the amount of organic carbon did not change appreciably between the 1900 and 1997 sampling dates, with 32 kg C/m² in the top meter and a third of that in the top 20 cm. Carbon and nitrogen stocks varied by less than 6% between two replicate modern soil pits or between the modern sites and the archive. Radiocarbon content decreased with depth in all sites and the modern SOM had positive Δ values near the surface due to nuclear weapons testing in the early 1960s. In the upper 10 cm, most of the SOM had a turnover time of 6–10 years, according to a model fit to the radiocarbon content. Below about 10 cm, the organic matter was almost all passive material with long (millennial) turnover times. Soil respiration Δ¹⁴CO₂ on a summer day was 106–109‰, an isotopic disequilibrium of about 9‰ relative to atmospheric ¹⁴CO₂. In both the modern and archive soil, the relative abundance of ¹³C in organic matter increased with depth by 2‰ in the upper meter from δ¹³C = ‐‐26‰ at 5 cm to ‐‐24‰ below a meter. In addition, the slope of δ¹³C vs. depth below 5 cm was the same for both soils. Given the age of the soil archive, these results give clear evidence that the depth gradients are not due to depletion of atmospheric ¹³CO₂ by fossil fuel emissions but must instead be caused by isotopic fractionation between plant litter inputs and preservation of SOM. Overall, the data show that these soils have a large reservoir of recalcitrant C and stocks had not changed between sampling dates 100 years apart.