Quantitative Proteomic Analysis of the Central Amygdala in Neuropathic Pain Model Rats.

Pain and emotional distress have a reciprocal relation. The amygdala has been implicated in emotional processing. The central nucleus of the amygdala (CeA) receives nociceptive information from the dorsal horn of spinal cord and is responsible for the central plasticity in chronic pain. Neuropathic pain is a type of severe chronic pain and can be strongly influenced by emotional components. Plastic changes in the CeA may play a key role in the development or maintenance or both of neuropathic pain. We studied the expression levels of proteins in the CeA of spinal nerve transection (SNT) model rats. Total tissue lysate proteins were separated by two-dimensional-gel electrophoresis (2D-PAGE). Gels from different time points were compared using Progenesis SameSpot software, and the spots with Fold Change greater than 2 were excised for protein identification by mass spectrometry. We identified more than 50 cytosolic proteins as significantly altered in their expression levels in the CeA of SNT rats, and most of these changes have been validated at mRNA levels by qRT-PCR. We also identified more than 40 membrane proteins as notably up- or down-regulated in the CeA of SNT model rats relative to a control using stable isotope dimethyl labeling nano-LC-MS/MS based proteomics and found that one such protein, doublecortin (DCX), a microtubule-associated protein expressed by neuronal precursor cells during development, is specifically localized in the membrane fraction without changes in total amount of the protein. Immunohistochemistry showed that doublecortin is expressed in processes in the CeA of rats 7 and 21 days after SNT surgery, suggesting that doublecortin is one of the proteins that may contribute to the plastic changes, namely, redevelopment or rewiring of neural networks, in the CeA in the neuropathic pain model. These dysregulated proteins may play roles in reciprocal relationships between pain and psychological distress in the amygdala and contribute to central sensitization. Data are available via ProteomeXchange with identifier PXD017473.

Identification of a receptor for neuropeptide VGF and its role in neuropathic pain.

VGF (nonacronymic) is a neuropeptide precursor that plays multiple roles in regulation of energy balance, reproduction, hippocampal synaptic plasticity, and pain. Data from a number of pain models showed significant up-regulation of VGF in sensory neurons. TLQP-21, one of the VGF-derived neuropeptides, has been shown to induce a hyperalgesic response when injected subcutaneously into the hind paw of mice. However, the precise role of VGF-derived neuropeptides in neuropathic pain and the molecular identity of the receptor for VGF-derived peptides are yet to be investigated. Here we identified gC1qR, the globular heads of the C1q receptor, as the receptor for TLQP-21 using chemical cross-linking combined with mass spectrometry analysis. TLQP-21 caused an increase in intracellular Ca(2+) levels in rat macrophages and microglia. Inoculation of TLQP-21-stimulated macrophages into rat hind paw caused mechanical hypersensitivity. The increase in intracellular Ca(2+) levels in macrophages was attenuated by either siRNA or neutralizing antibodies against gC1qR. Furthermore, application of the gC1qR-neutralizing antibody to rats with partial sciatic nerve ligation resulted in a delayed onset of nerve injury-associated mechanical hypersensitivity. These results indicate that gC1qR is the receptor for TLQP-21 and plays an important role in chronic pain through activation of macrophages. Because direct association between TLQP-21 and gC1qR is required for activation of macrophages and causes hypersensitivity, disrupting this interaction may be a useful new approach to develop novel analgesics.

The trafficking of Na(V)1.8.

The α-subunit of tetrodotoxin-resistant voltage-gated sodium channel Na(V)1.8 is selectively expressed in sensory neurons. It has been reported that Na(V)1.8 is involved in the transmission of nociceptive information from sensory neurons to the central nervous system in nociceptive [1] and neuropathic [24] pain conditions. Thus Na(V)1.8 has been a promising target to treat chronic pain. Here we discuss the recent advances in the study of trafficking mechanism of Na(V)1.8. These pieces of information are particularly important as such trafficking machinery could be new targets for painkillers.

Highly site-selective stability increases by glycosylation of dihydrofolate reductase.

Post-translational glycosylation is one of the most abundant forms of covalent protein modification in eukaryotic cells. It plays an important role in determining the properties of proteins, and stabilizes many proteins against thermal denaturation. Protein glycosylation may establish a surface microenvironment that resembles that of unglycosylated proteins in concentrated solutions of sugars and other polyols. We have used site-directed mutagenesis to introduce a series of unique cysteine residues into a cysteine-free double mutant (DM, C85A/C152S) of dihydrofolate reductase from Escherichia coli (EcDHFR). The resulting triple mutants, DM-N18C, DM-R52C, DM-D87C and DM-D132C EcDHFR, were alkylated with glucose, N-acetylglucosamine, lactose and maltotriose iodoacetamides. We found little effect on catalysis or stability in three cases. However, when DM-D87C EcDHFR is glycosylated, stability is increased by between 1.5 and 2.6 kcal.mol(-1) in a sugar-dependent manner. D87 is found in a hinge region of EcDHFR that loses structure early in the thermal denaturation process, whereas the other glycosylation sites are found in regions involved in the later stages of temperature-induced unfolding. Glycosylation at this site may improve the stability of EcDHFR by protecting a region of the enzyme that is particularly prone to denaturation.

Different reaction mechanisms for mesophilic and thermophilic dihydrofolate reductases.

We report here solvent kinetic isotope effects for two dihydrofolate reductases, namely the monomeric, mesophilic enzyme from E. coli (EcDHFR) and the dimeric, thermophilic enzyme from Thermotoga maritima (TmDHFR). Multiple isotope effects reveal mechanistic differences between the two enzymes. EcDHFR follows a stepwise mechanism in which proton transfer precedes hydride transfer, whereas the two steps are concerted in TmDHFR. At elevated pH, EcDHFR also follows a concerted reaction pathway. TmDHFR at pH 7 behaves more like EcDHFR at elevated pH suggesting that the restricted motions of TmDHFR resulting from dimerization preclude it from modulating the pK(a) of its substrate as efficiently as EcDHFR. The reduced reaction rates of TmDHFR therefore appear to be a consequence of its quaternary structure, which is required for increased thermostability but which also prevents active modulation of the reactivity of the active site bound substrate observed in EcDHFR.

Effect of dimerization on the stability and catalytic activity of dihydrofolate reductase from the hyperthermophile Thermotoga maritima.

In contrast to all other chromosomally encoded dihydrofolate reductases characterized so far, dihydrofolate reductase (DHFR) from the hyperthermophile Thermotoga maritima forms a highly stable dimer. The dimer interface involves residues whose mobility is important for catalysis in monomeric DHFRs. Here, we report the generation of a variant of DHFR from T. maritima, TmDHFR-V11D, in which a single amino acid replacement was sufficient to favor the monomeric form of the enzyme in the presence of the nondenaturing zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The free energy of stabilization of monomeric TmDHFR-V11D was 15 kJ mol(-1) lower than that of the wild-type dimer, while the melting temperature of monomeric TmDHFR-V11D was comparable to that of monomeric DHFR from the thermophile Bacillus stearothermophilus, supporting the hypothesis that oligomerization is required to achieve the thermal stabilities necessary for activity at temperatures optimal for growth of hyperthermophiles. Both the steady-state turnover numbers and rates of hydride transfer were reduced in TmDHFR-V11D. However, a similar reduction of the rate constants was observed in a different variant, TmDHFR-V126E, which remained as a dimer under all experimental conditions used here. Monomeric TmDHFR-V11D had a similar rate of hydride transfer to the dimeric form, but a reduced steady-state turnover rate. Intersubunit motions therefore appear to be less important than correlated motions within individual subunits for TmDHFR-catalyzed hydride transfer, but are critical to the overall progression of the catalytic cycle. Hence, the reduced catalytic activity of TmDHFR relative to the monomeric Escherichia coli enzyme is not caused by rigidity resulting from dimerization, but is a subtle consequence of the sequence and structure of its subunits, which appear to have evolved to allow thermostability at the expense of catalysis.

Protein motions during catalysis by dihydrofolate reductases.

Dihydrofolate reductase (DHFR) maintains the intracellular pool of tetrahydrofolate through catalysis of hydrogen transfer from reduced nicotinamide adenine dinucleotide to 7,8-dihydrofolate. We report results for pre-steady-state kinetic studies of the temperature dependence of the rates and the hydrogen/deuterium-kinetic isotope effects for the reactions catalysed by the enzymes from the mesophilic Escherichia coli and the hyperthermophilic Thermatoga maritima. We propose an evolutionary pattern in which catalysis progressed from a relatively rigid active site structure in the ancient thermophilic DHFR to a more flexible and kinetically more efficient structure in E. coli that actively promotes hydrogen transfer at physiological pH by modulating the tunnelling distance. The E. coli enzyme appeared relatively robust, in that kinetically severely compromised mutants still actively propagated the reaction. The reduced hydrogen transfer rates of the extensively studied Gly121Val mutant of DHFR from E. coli were most likely due to sterically unfavourable long-range effects from the introduction of the bulky isopropyl group.

Coupling of protein motions and hydrogen transfer during catalysis by Escherichia coli dihydrofolate reductase.

The enzyme DHFR (dihydrofolate reductase) catalyses hydride transfer from NADPH to, and protonation of, dihydrofolate. The physical basis of the hydride transfer step catalysed by DHFR from Escherichia coli has been studied through the measurement of the temperature dependence of the reaction rates and the kinetic isotope effects. Single turnover experiments at pH 7.0 revealed a strong dependence of the reaction rates on temperature. The observed relatively large difference in the activation energies for hydrogen and deuterium transfer led to a temperature dependence of the primary kinetic isotope effects from 3.0+/-0.2 at 5 degrees C to 2.2+/-0.2 at 40 degrees C and an inverse ratio of the pre-exponential factors of 0.108+/-0.04. These results are consistent with theoretical models for hydrogen transfer that include contributions from quantum mechanical tunnelling coupled with protein motions that actively modulate the tunnelling distance. Previous work had suggested a coupling of a remote residue,Gly121, with the kinetic events at the active site. However, pre-steady-state experiments at pH 7.0 with the mutant G121V-DHFR, in which Gly121 was replaced with valine, revealed that the chemical mechanism of DHFR catalysis was robust to this replacement. The reduced catalytic efficiency of G121V-DHFR was mainly a consequence of the significantly reduced pre-exponential factors, indicating the requirement for significant molecular reorganization during G121V-DHFR catalysis. In contrast, steady-state measurements at pH 9.5, where hydride transfer is rate limiting, revealed temperature-independent kinetic isotope effects between 15 and 35 degrees C and a ratio of the pre-exponential factors above the semi-classical limit, suggesting a rigid active site configuration from which hydrogen tunnelling occurs. The mechanism by which hydrogen tunnelling in DHFR is coupled with the environment appears therefore to be sensitive to pH.

Synthesis of homogenous site-selectively glycosylated proteins.

Apparently homogenous glycoproteins can be synthesised in good yield by a combination of site directed mutagenesis, a highly flexible but selective chemical derivatisation and efficient purification through the use of glycosyl thiosulfonates such as 2-((biotinoyl)-amino)-ethyl methanethiosulfonate.

Pivotal role of Gly 121 in dihydrofolate reductase from Escherichia coli: the altered structure of a mutant enzyme may form the basis of its diminished catalytic performance.

The structure and folding of dihydrofolate reductase (DHFR) from Escherichia coli and the mutant G121V-DHFR, in which glycine 121 in the exterior FG loop was replaced with valine, were studied by molecular dynamics simulations and CD and fluorescence spectroscopy. The importance of residue 121 for the chemical step during DHFR catalysis had been demonstrated previously. High-temperature MD simulations indicated that while DHFR and G121V-DHFR followed similar unfolding pathways, the strong contacts between the M20 loop and the FG loop in DHFR were less stable in the mutant. These contacts have been proposed to be involved in a coupled network of interactions that influence the protein dynamics and promote catalysis [Benkovic, S. J., and Hammes-Schiffer, S. (2003) Science 301, 1196-1202]. CD spectroscopy of DHFR and G121V-DHFR indicated that the two proteins existed in different conformations at room temperature. While the thermally induced unfolding of DHFR was highly cooperative with a midpoint at 51.6 +/- 0.7 degrees C, G121V-DHFR exhibited a gradual decrease in its level of secondary structure without a clear melting temperature. Temperature-induced unfolding and renaturation from the urea-denatured state revealed that both proteins folded via highly fluorescent intermediates. The formation of these intermediates occurred with relaxation times of 149 +/- 4.5 and 256 +/- 13 ms for DHFR and G121V-DHFR, respectively. The fluorescence intensity for the intermediates formed during refolding of G121V-DHFR was approximately twice that of the wild-type. While the fluorescence intensity then slowly decayed for DHFR toward a state representing the native protein, G121V-DHFR appeared to be trapped in a highly fluorescent state. These results suggest that the reduced catalytic activity of G121V-DHFR is the consequence of nonlocal structural effects that may result in a perturbation of the network of promoting motions.