Neuropeptide FF (FLFQPQRF-NH2) and its Fragments Bind to α2δ Subunit of Voltage-Gated Calcium Channels.

PURPOSE: Gabapentin, a drug for neuropathic pain, exerts its therapeutic effect via inhibition of the a2d subunit of N-type Ca2+ channels. Thus, finding peptides that specifically displace gabapentin from its binding site may lead to the development of new drugs. METHODS: Displacement of bound [3H]-gabapentin in membrane preparations of rat cerebral cortex and of human Cav2.2/ß3/α2δ1 expressed in CHO cell line. RESULTS: Neuropeptide FLFQPQRF-NH2 specifically displaced bound [3H]-gabapentin in membrane preparations from rats and CHO cells. Truncation of the C-terminus of FLFQPQRF-NH2 by three amino acid residues to produce FLFQP-NH2 improved the displacement of gabapentin. FLFQP-NH2 displaced bound  [3H]-gabapentin with IC50 and Ki values of 2.7 µM and 1.7 µM, respectively. Deletion of two amino acid residues (FQ) in the middle of the FLFQP-NH2 sequence yielded FLP-NH2 that displaced bound [3H]-gabapentin with a lower affinity.  IC50 and Ki values were 11.9 µM and 7.8 µM, respectively. Neutral binding cooperativity existed when of FLFQP-NH2, FLP- NH2 and gabapentin when incubated together. FLFQPQRF-NH2 but not FLFQP-NH2 displaced bound [3H]- gabapentin to membrane preparations of human Cav2.2/b3/a2d1 expressed in CHO cells. CONCLUSION: FLFQPQRF-NH2, FLFQP-NH2 and FLP-NH2 displace bound gabapentin in membrane preparations of rat cerebral cortex. Binding cooperativity was detected when GBP/FLFQP-NH2/FLP-NH2 were incubated together. These novel binding sites may provide new approaches to modulate L-type Ca2+ channels.

Crystal and molecular structure of the analgesic tetrapeptide, L-Phe-L-Leu-L-Pro-L-Ser.

The tetrapeptide, L-Phe-L-Leu-L-Pro-L-Ser (FLPS), alleviates pain in a rat model of post-surgery pain. The crystal structure of the tetrapeptide is solved at high resolution (0.54 Å). The asymmetric unit contains two FLPS molecules, one Zn ion, and four molecules of water with a formula of [Zn(C23H33N4O6)2(H2O)4]. Each Zn ion is octahedrally coordinated with Phe and Ser residues from four peptide molecules [2N+4O]. The linking of Phe and Ser residues of one FLPS molecule to three other FLPS molecules by Zn ion forms a complex consisting of chains of metal ions and FLPS molecules oriented along the b axis. Analysis of molecular packing reveals the coexistence of two FLPS conformers in the same crystal. The crystallographic parameters for [Zn(C23H33N4O6)2(H2O)4] are as follows: space group P21 21 21 , a = 9.8698(2) Å, α = 90°, b = 20.1844(4) Å, ß = 90°, c = 25.9302(6) Å, γ = 90°. Volume = 5165.71(19) Å(3), Z = 4, density (calc) = 1.364 Mg/cm(3), and agreement factor R1 = 4.13%.

Thermoregulation in the appendix of the Sauromatum guttatum (Schott) inflorescence.

BACKGROUND: Three phenolic compounds are capable of activating the process that simultaneously leads to temperature rise and odor-production in the Sauromatum appendix. These compounds are salicylic acid, aspirin, and 2,6 dihydroxybenzoic acid. The objectives of the present study were to examine the effect of various concentrations of the these inducers on the temperature rise and to study the effect of mitochondrial inhibitors (KCN and SHAM) and an uncoupler (DNP) on the temperature rise. RESULTS: In sections of the Sauromatum appendix two successive temperature rate maxima were detected in the presence of the three inducers. Two temperature maxima were also detected in appendices of intact inflorescences. The temperature profiles demonstrated a considerable variability within sections of one appendix in both magnitude and time of reaching a peak. When the Sauromatum temperature decreased it returned either to the same temperature baseline or to a slightly different baseline. The temperature rise was blocked by KCN (20 mM) and SHAM (40 mM) alone or when added together. DNP, an uncoupler, at 2.5 mM also blocked the rise in temperature. The thermogenic inducers also triggered a temperature rise in Arum appendix. CONCLUSIONS: The presence of two rate maxima may indicate different heat-generating sources. The blockage of the temperature rise in the presence of KCN or SHAM implies that the activity of the cyanide-resistant and -sensitive pathways is required for generating heat. The variability in temperature profiles maybe related to changes in cellular control factors. This study provides the basis for investigating thermoregulation in plants.

Human ortholog of a plant salicylic acid receptor found in SK-N-SH cell line.

Our previous studies have described the purification and characterization of a novel plant NAD(P)-reductase like protein (RL) from the thermogenic appendix of the Sauromatum guttatum inflorescence. RL is mainly located in cytoplasm of thermogenic plants and it can act like a bistable switch. It adopts a compact conformation during heat-production and a more expanded conformation when heat is not generated. Addition of salicylic acid, a natural thermogenic inducer, at picomolar concentration to a solution of purified RL induced a discontinuous volume phase transition in which the volume of RL in the oligomeric form expanded and shrunk repeatedly every 4-5 min. In the present study using ESI-MS analysis we have demonstrated the existence of RL in the human SK-N-SH cell line and in mouse brain tissue. The molecular mass of human RL is in the same range as of its plant counterpart, 34,140 ± 34 Da. The charge state distribution of the human RL is identical to its plant counterpart from the Sauromatum appendix during heat-production. Human RL was present in the compact state when it was purified from the SK-N-SH cell line When these cells were treated with salicylic acid (10 µM) a shift to a much more compact conformation was observed. It seems that the potential of RL to respond to salicylic acid was conserved. These results may reveal the existence of a thermoregulation system that is evolutionarily conserved and is operating by conformational changes. This discovery may also represent an opportunity for a better understanding of some of the diverse functions of salicylic acid and aspirin in plants and humans.

Two global conformation states of a novel NAD(P) reductase like protein of the thermogenic appendix of the Sauromatum guttatum inflorescence.

A novel NAD(P) reductase like protein (RL) belonging to a class of reductases involved in phenylpropanoid synthesis was previously purified to homogeneity from the Sauromatum guttatum appendix. The Sauromatum appendix raises its temperature above ambient temperature to ~30 °C on the day of inflorescence opening (D-day). Changes in the charge state distribution of the protein in electrospray ionization-mass spectrometry spectra were observed during the development of the appendix. RL adopted two conformations, state A (an extended state) that appeared before heat-production (D - 4 to D - 2), and state B (a compact state) that began appearing on D - 1 and reached a maximum on D-day. RL in healthy leaves of Arabidopsis is present in state A, whereas in thermogenic sporophylls of male cones of Encephalartos ferox is present in state B. These conformational changes strongly suggest an involvement of RL in heat-production. The biophysical properties of this protein are remarkable. It is self-assembled in aqueous solutions into micrometer sizes of organized morphologies. The assembly produces a broad range of cyclic and linear morphologies that resemble micelles, rods, lamellar micelles, as well as vesicles. The assemblies could also form network structures. RL molecules entangle with each other and formed branched, interconnected networks. These unusual assemblies suggest that RL is an oligomer, and its oligomerization can provide additional information needed for thermoregulation. We hypothesize that state A controls the plant basal temperature and state B allows a shift in the temperature set point to above ambient temperature.

Purification of a NAD(P) reductase-like protein from the thermogenic appendix of the Sauromatum guttatum inflorescence.

A NAD(P) reductase-like protein with a molecular mass of 34.146 ± 34 Da was purified to homogeneity from the appendix of the inflorescence of the Sauromatum guttatum. On-line liquid chromatography/electrospray ionization-mass spectrometry was used to isolate and quantify the protein. For the identification of the protein, liquid chromatography/electrospray ionization-tandem mass spectrometry analysis of tryptic digests of the protein was carried out. The acquired mass spectra were used for database searching, which led to the identification of a single tryptic peptide. The 12 amino acid tryptic peptide (FLPSEFGNDVDR) was found to be identical to amino acid residues at the positions 108-120 of isoflavone reductase in the Arabidopsis genome. A BLAST search identified this sequence region as unique and specific to a class of NAD(P)-dependent reductases involved in phenylpropanoid biosynthesis. Edman degradation revealed that the protein was N-terminally blocked. The amount of the protein (termed RL, NAD(P) reductase-like protein) increased 60-fold from D-4 (4 days before inflorescence-opening, designated as D-day) to D-Day, and declined the following day, when heat-production ceased. When salicylic acid, the endogenous trigger of heat-production in the Sauromatum appendix, was applied to premature appendices, a fivefold decrease in the amount of RL was detected in the treated section relative to the non-treated section. About 40 % of RL was found in the cytoplasm. Another 30 % was detected in Percoll-purified mitochondria and the rest, about 30 % was associated with a low speed centrifugation pellet due to nuclei and amyloplast localization. RL was also found in other thermogenic plants and detected in Arabidopsis leaves. The function of RL in thermogenic and non-thermogenic plants requires further investigation.

A NAD(P) reductase like protein is the salicylic acid receptor in the appendix of the Sauromatum guttatum inflorescence.

The mode of action of the thermogenic inducers (salicylic acid, aspirin, and 2,6-dihydroxybenzoic acid) in the appendix of the Sauromatum guttatum inflorescence is poorly understood. Using ESI-MS and light scattering analysis, we have demonstrated that NAD(P) reductase like protein (RL) is the salicylic acid receptor in the Sauromatum appendix. RL was self-assembled in water into a large unit with a hydrodynamic diameter of 800 nm. In the presence of 1 pM salicylic acid, RL exhibited discontinuous and reversible volume phase transitions. The volume phase changed from 800 to 300 nm diameter and vice versa. RL stayed at each volume phase for ~4-5 min with a fast relaxation time between the 2 phases. ESI-MS analysis of RL extracted from appendices treated with salicylic acid, aspirin, and 2,6-DHBA at a micromolar range demonstrated that these compounds are capable of inducing graded conformational changes that are concentration-dependent. A strong correlation between RL conformations and heat-production induced by salicylic acid was also observed. These preliminary findings reveal structural and conformational roles for RL by which plants regulate their temperature and synchronize their time keeping mechanisms.

The Sauromatum guttatum appendix as an osmophore: excretory pathways, composition of volatiles and attractiveness to insects.

This report combines chemical, electron microscopic and ecological studies on the volatiles liberated by the Sauromatum guttatum appendix on D-Day, the day of inflorescence-opening and heat-production. More than 100 compounds from at least nine different chemical classes (monoterpenes, sesquiterpenes, fatty acids, ketones, alcohols, aldehydes, indole, and phenolic and sulphur compounds) are liberated during the thermogenic activity. The volatiles were identified using gas chromatography-mass spectrometry. Electron microscopy provides additional evidence that the endoplasmic reticulum (ER) interacts with the plasma membrane, creating novel routes of excretion of the volatiles to the exterior of the cell. It seems that the fusion event creates channels from the interior to the exterior of the cell. Furthermore, a multitubular body, conceivably originating in the ER, seems to fuse with the plasma membrane and to appear only on D-day. This multitubular body is closely associated with lipid bodies during heat-production and might be involved in the oxidation of lipids to volatile products. The foul odour produced by the appendix attracts at least 30 species of insects.