AFM observation of single,functioning ionotropic glutamate receptors reconstituted in lipid bilayers

Background

Ionotropic glutamate receptors (iGluRs) are responsible for extracellular signaling in the central nervous system. However, the relationship between the overall structure of the protein and its function has yet to be resolved. Atomic force microscopy (AFM) is an important technique that allows nano-scale imaging in liquid. In the present work we have succeeded in imaging by AFM of the external features of the most common iGluR, AMPA-R (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor), in a physiological environment.

Methods

Homomeric GluR3 receptors were over-expressed in insect cells, purified and reconstituted into lipid membranes. AFM images were obtained in a buffer from membranes immobilized on a mica substrate.

Results

Using Au nanoparticle-conjugated antibodies, we show that proteins reconstitute predominantly with the N-terminal domain uppermost on the membrane. A tetrameric receptor structure is clearly observed, but it displays considerable heterogeneity, and the dimensions differ considerably from cryo-electron microscopy measurements.

Conclusions

Our results indicate that the extracellular domains of AMPA-R are highly flexible in a physiological environment.

General significance

AFM allows us to observe the protein surface structure, suggesting the possibility of visualizing real time conformational changes of a functioning protein. This knowledge may be useful for neuroscience as well as in pharmaceutical applications.     

Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding

Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to d-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2′-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.     

Overexpression of monocyte chemoattractant protein-1 in adipose tissues causes macrophage recruitment and insulin resistance

Adipose tissue expression and circulating concentrations of monocyte chemoattractant protein-1 (MCP-1) correlate positively with adiposity. To ascertain the roles of MCP-1 overexpression in adipose, we generated transgenic mice by utilizing the adipocyte P2 (aP2) promoter (aP2-MCP-1 mice). These mice had higher plasma MCP-1 concentrations and increased macrophage accumulation in adipose tissues, as confirmed by immunochemical, flow cytometric, and gene expression analyses. Tumor necrosis factor-alpha and interleukin-6 mRNA levels in white adipose tissue and plasma non-esterified fatty acid levels were increased in transgenic mice. aP2-MCP-1 mice showed insulin resistance, suggesting that inflammatory changes in adipose tissues may be involved in the development of insulin resistance. Insulin resistance in aP2-MCP-1 mice was confirmed by hyperinsulinemic euglycemic clamp studies showing that transgenic mice had lower rates of glucose disappearance and higher endogenous glucose production than wild-type mice. Consistent with this, insulin-induced phosphorylations of Akt were significantly decreased in both skeletal muscles and livers of aP2-MCP-1 mice. MCP-1 pretreatment of isolated skeletal muscle blunted insulin-stimulated glucose uptake, which was partially restored by treatment with the MEK inhibitor U0126, suggesting that circulating MCP-1 may contribute to insulin resistance in aP2-MCP-1 mice. We concluded that both paracrine and endocrine effects of MCP-1 may contribute to the development of insulin resistance in aP2-MCP-1 mice.     

Solution structure of agelenin, an insecticidal peptide isolated from the spider Agelena opulenta, and its structural similarities to insect-specific calcium channel inhibitors

Agelenin, isolated from the Agelenidae spider Agelena opulenta, is a peptide composed of 35 amino acids. We determined the three-dimensional structure of agelenin using two-dimensional NMR spectroscopy. The structure is composed of a short antiparallel beta-sheet and four beta-turns, which are stabilized by three disulfide bonds. Agelenin has characteristic residues, Phe9, Ser28 and Arg33, which are arranged similarly to the pharmacophore of the insect channel inhibitor, omega-atracotoxin-Hv1a. These observations suggest that agelenin and omega-atracotoxin-Hv1a bind to insect calcium channels in a similar manner. We also suggest that another mode of action may operate in the channel inhibition by omega-agatoxin-IVA and omega-atracotoxin-Hv2a.     

A thromboxane mimetic, U-46619, produces plasma exudation in airways of the guinea pig.

Thromboxane A2 (TxA2) has been implicated in airway responses to allergen and in the bronchial hyperresponsiveness observed in asthma. Furthermore a TxA2 receptor antagonist and a TxA2 synthase inhibitor inhibit plasma exudation in airways induced by inhaled platelet-activating factor. To evaluate whether TxA2 has any direct effect on plasma exudation in the airways, we studied the effect of a stable TxA2 mimetic (U-46619; 2, 20, and 200 nmol/kg iv) on lung resistance (RL) and Evans blue dye extravasation (marker of plasma albumin; 20 mg/kg iv) at the airway levels of trachea, main bronchi, and proximal and distal intrapulmonary airways in anesthetized, tracheostomized, and mechanically ventilated guinea pigs. Injection of U-46619 produced an immediate and marked dose-dependent increase in RL, which peaked at approximately 30 s. At the highest dose of U-46619, we also observed a later increase in RL, starting at approximately 3 min and reaching a second peak at approximately 8 min. Mean systemic blood pressure increased in a dose-dependent manner [maximum 82 +/- 8 (SE) mmHg]. U-46619 also produces dose-dependent plasma exudation, measured as Evans blue dye extravasation, at all airway levels as well as into the tracheal lumen. Airway responses to U-46619 (200 nmol/kg iv) were abolished in animals pretreated with the TxA2 receptor antagonist ICI-192605 (0.5 mg/kg iv). We conclude that U-46619, despite being a vasoconstrictor, is potent in inducing plasma exudation in airways and that this effect is mediated via a TxA2 receptor.     

Characterization of the Bacillus subtilis ywsC gene, involved in gamma-polyglutamic acid production.

The genes required for gamma-polyglutamic acid (PGA) production were cloned from Bacillus subtilis IFO16449, a strain isolated from fermented soybeans. There were four open reading frames in the cloned 4.2-kb DNA fragment, and they were almost identical to those in the ywsC and ywtABC genes of B. subtlis 168. Northern blot analysis showed that the four genes constitute an operon. Three genes, ywsC, ywtA, and ywtB, were disrupted to determine which gene plays a central role in PGA biosynthesis. No PGA was produced in Delta ywsC and Delta ywtA strains, indicating that both of these genes are essential for PGA production. To clarify the function of the YwsC protein, histidine-tagged YwsC (YwsC-His) was produced in the Delta ywsC strain and purified from the lysozyme-treated lysate of the transformant by Ni-nitrilotriacetic acid affinity chromatography. Western blot analysis revealed that the YwsC-His protein consists of two subunits, the 44-kDa and 33-kDa proteins, which are encoded by in-phase overlapping in the ywsC gene. (14)C-labeled PGA was synthesized by the purified proteins from L-[(14)C]-glutamate in the presence of ATP and MnCl(2), through an acylphosphate intermediate, indicating that the ywsC gene encodes PGA synthetase (EC 6.3.2), a crucial enzyme in PGA biosynthesis.     

The involvement of midbrain astrocyte in the development of morphine tolerance

Aims

Systemic administration of opiate analgesics such as morphine remains the most effective treatment for alleviating severe pain across a range of conditions including acute pain. However, chronic or repeated administration of opiate analgesics results in the development of analgesic tolerance. Glial cells such as microglia and astrocytes are known to release various inflammatory cytokines and neurotrophic factors leading to regulation of neuronal function. Recently, glial cells were reported to play important roles in the development of analgesic tolerance to morphine. Here, we focused on the involvement of midbrain glial cells, particularly astrocytes, in the development of analgesic tolerance to morphine.

Main methods

Mice were treated with morphine (10 mg/kg, s.c.) or vehicle once a day for 5 days. Pentoxifylline (an inhibitor of glial activation; 20 mg/kg, i.p. or 50 and 100 μg/mouse, i.c.v.) was administered 30 min before morphine treatment. Flavopiridol (a cyclin-dependent kinase inhibitor; 5 nmol/mouse, i.c.v.) was administered 10 min before and 10 h after morphine treatment. The analgesic effect of morphine was measured using the tail flick method.

Key findings

The development of analgesic tolerance to morphine was gradually observed during daily treatment of morphine for 5 days in mice. On days 1 and 3 after repeated morphine treatment, astrocyte marker glial fibrillary acidic protein expression levels were significantly increased, as determined by western blot analyses. These phenomena were significantly inhibited following pre-treatment with pentoxifylline or flavopiridol.

Significance

We demonstrated that midbrain astrocytes play an important role in the development of analgesic tolerance to morphine.     

Loss of a conserved 7-methylguanosine modification in 16S rRNA confers low-level streptomycin resistance in bacteria

Streptomycin has been an important drug for the treatment of tuberculosis since its discovery in 1944. But numerous strains of Mycobacterium tuberculosis, the bacterial pathogen that causes tuberculosis, are now streptomycin resistant. Although such resistance is often mediated by mutations within rrs, a 16S rRNA gene or rpsL, which encodes the ribosomal protein S12, these mutations are found in a limited proportion of clinically isolated streptomycin-resistant M. tuberculosis strains. Here we have succeeded in identifying a mutation that confers low-level streptomycin resistance to bacteria, including M. tuberculosis. We found that mutations within the gene gidB confer low-level streptomycin resistance and are an important cause of resistance found in 33% of resistant M. tuberculosis isolates. We further clarified that the gidB gene encodes a conserved 7-methylguanosine (m(7)G) methyltransferase specific for the 16S rRNA, apparently at position G527 located in the so-called 530 loop. Thus, we have identified gidB as a new streptomycin-resistance locus and uncovered a resistance mechanism that is mediated by loss of a conserved m(7)G modification in 16S rRNA. The clinical significance of M. tuberculosis gidB mutation also is noteworthy, as gidB mutations emerge spontaneously at a high frequency of 10(-6) and, once emerged, result in vigorous emergence of high-level streptomycin-resistant mutants at a frequency more than 2000 times greater than that seen in wild-type strains. Further studies on the precise function of GidB may provide a basis for developing strategies to suppress pathogenic bacteria, including M. tuberculosis.