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. 2012;7(9):e41278.
doi: 10.1371/journal.pone.0041278. Epub 2012 Sep 13.

Proteomic and systems biology analysis of monocytes exposed to securinine, a GABA(A) receptor antagonist and immune adjuvant

Matt Shipman  1 , Kirk Lubick, David Fouchard, Rajani Guram, Paul Grieco, Mark Jutila, Edward A Dratz
Affiliations

Proteomic and systems biology analysis of monocytes exposed to securinine, a GABA(A) receptor antagonist and immune adjuvant

Matt Shipman et al. PLoS One. 2012.

Abstract

Securinine, a GABA(A) receptor antagonist, has been reported to enhance monocyte cell killing of Coxiella burnetii without obvious adverse effects in vivo. We employed multiplex 2D gel electrophoresis using Zdyes, a new generation of covalently linked fluorescent differential protein detection dyes to analyze changes in the monocyte proteome in response to Securinine. Securinine antagonism of GABA(A) receptors triggers the activation of p38. We used the differential protein expression results to guide a search of the literature and network analysis software to construct a systems biology model of the effect of Securinine on monocytes. The model suggests that various metabolic modulators (fatty acid binding protein 5, inosine 5'-monophosphate dehydrogenase, and thioredoxin) are at least partially reshaping the metabolic landscape within the monocytes. The actin bundling protein L-plastin, and the Ca(2+) binding protein S100A4 also appear to have important roles in the immune response stimulated by Securinine. Fatty acid binding protein 5 (FABP5) may be involved in effecting lipid raft composition, inflammation, and hormonal regulation of monocytes, and the model suggests that FABP5 may be a central regulator of metabolism in activated monocytes. The model also suggests that the heat shock proteins have a significant impact on the monocyte immune response. The model provides a framework to guide future investigations into the mechanisms of Securinine action and with elaboration may help guide development of new types of immune adjuvants.

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Conflict of interest statement

Competing Interests: EAD and PG are founders and share holders in Zdye LLC that licenses patents issued to Montana State University for the Zdye detection reagents used in the present study. Zdye LLC provides research grants to Montana State University for development of Zdyes and related technology. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. The presence of GABAA receptors in monocytes and showing that GABAA receptros are expressed on the cell surface of MonoMac1 cells.
A) GABAA 1 α receptors detected on MonoMac-1 cells and MNK cells by western blot after stimulation with securinine. B) MonoMac-1 cells were collected by Cytospin followed by fixation (75% Acetone/25% Ethanol). Cells were blocked in PBS containing 10% goat serum and 0.025% Tween 20. Slides were then stained with anti-GABAA 1 α receptor mAb (20 ug/ml) for 30 min, washed with PBS containing 0.025% Tween 20, and then stained with AlexaFluor 488 goat anti-rabbit antibody. Slides were washed and treated with prolong Gold antifade reagent. DAPI was used to detected nuclei and the cells were analyzed by immunofluorescence microscopy. Green = GABAA 1 α receptor and blue = DAPI. Top panel is DAPI alone. C) Higher magnification of the anti GABAA 1 α stained Monomac I cells in the lower panel of B.
Figure 2
Figure 2. 2D gel image of the soluble fraction from Securinine stimulated monocytes.
Progenesis master image following image analysis and statistical analysis. Numbered spot areas indicate statistically significant differentially expressed spots, as determined by nested ANOVA analysis. The pI separation range (3-11NL) is the horizontal direction and the polyacrylamide gradient (9.5–18%) is the vertical direction. Spots 13 and and 23 are indicated by the arrows.
Figure 3
Figure 3. 2D gel of the membrane fraction from Securinine stimulated monocytes.
Progenesis master image, following image analysis and statistical analysis. Numbered spot areas indicate statistically significant differentially expressed spots, as determined by nested ANOVA analysis. The pI separation range (3-11NL) is in the horizontal direction and the polyacrylamide gradient (9.5–16%) is in the vertical direction.
Figure 4
Figure 4. Schematic diagram of proposed functions of Hsp70 in Securinine stimulated monocytes, based on the literature.
Proposed activities include increased monocyte maturation, inhibition of apoptosis, intracellular signaling (Ca2+, p38, NF-κB), and extracellular signaling (danger signal).
Figure 5
Figure 5. Schematic of antigen processing and presentation pathway for Hsp70 and Hsp90 generated by DAVID.
DAVID utilized the KEGG pathways to diagram the role of Hsp70 and Hsp90 for antigen processing and presentation. Both Hsp70 and Hsp90 (red stars) are involved in MHC class I antigen processing. Hsp60 (not shown) is involved in MHC class II processing. The proteins and pathways proposed are in agreement with our manual search of the literature, which suggested that Hsps are involved in antigen presentation.
Figure 6
Figure 6. Schematic of metabolic pathways related to inosine 5′-monophosphate generated by DAVID.
DAVID utilized KEGG pathways to depict metabolic pathways related to purine metabolism. Inosine 5′-monophosphate dehydrogenase (IMPDH, red star near the center of the figure) catalyzes the conversion of inosine 5′-monophosphate to xanthosine 5′-monophosphate which is thought to be the rate-limiting step for GMP and GTP production. Although the biological consequences of IMPDH are not detailed here, this diagram does offer an indicat-on of potential metabolites to monitor in targeted metabolomic experiments. The numbers in the boxes are enzyme (EC) numbers.
Figure 7
Figure 7. Securinine induces rapid phosphorylation of p38 MAPK in MonoMac-1 cells.
MonoMac-1 cells were treated with DMSO, 50 uM securinine or 20 ug/mL anisomycin for the indicated times. Lysates were prepared and subjected to Western blot with anti-phospho-p38 MAPK (top panel) or anti-p38 MAPK (bottom panel). Blots were developed with ECL (GE Healthcare) and exposed to film for autoradiography. Anisomycin was used as a positive control.
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