Toponomics Analysis of Drug-Induced Changes in Arachidonic Acid-Dependent Signaling Pathways during Spinal Nociceptive Processing

Abstract

Multi-Epitope-Ligand-Carthography (MELC) allows consecutive immunohistochemical visualization of up to 100 proteins on the same tissue sample. Subsequent biomathematical analysis of these images allows a quantitative description of changes in protein networks. We used the MELC technology to study the effect of the nonopioid analgesic drug dipyrone on protein network profiles associated with arachidonic acid-dependent signaling pathways. MELC analysis with 31 different fluorescence-labeled tags was used to compare the effect of dipyrone on protein networks in spinal cords of mice with zymosan-induced hyperalgesia, a common model for inflammatory pain. We found that the number of motifs which describe the colocalization of 5-lipoxygenase (5-LO) or 12-LO with other proteins increased disproportionally after dipyrone treatment. Activation of 5-LO and 12-LO induces their translocation to membrane compartments which was also reflected by MELC results. Although no changes in 5-LO or 12-LO expression were seen by Western blot analysis or by immunohistochemistry in spinal cords of dipyrone-treated mice, the activation of both enzymes was verified by determining LO-products. Spinal amounts of 5(S)-hydroxyeicosatetraenoic acid (HETE) and 12(S)-HETE, which are generated by 5-LO and 12-LO, respectively, were significantly increased in spinal cords of dipyrone-treated animals. In primary spinal cord neurons, dipyrone selectively and dose-dependently increased 5(S)-(HETE) and 12(S)-HETE synthesis. Thus, we show for the first time that monitoring protein network profiles by topological proteomic analysis is a useful tool to identify mechanisms of drug actions.

Lack of Microsomal Prostaglandin E 2 Synthase-1 in Bone Marrow–Derived Myeloid Cells Impairs Left Ventricular Function and Increases Mortality After Acute Myocardial Infarction

“…Multiple epitope ligand cartography enables sequential immunofluorescence-based visualization of multiple proteins in a tissue sample and can be used to identify the cell type that an individual protein is expressed in, and to determine which proteins colocalize in individual cells. 19 In Ptges ϩ/ϩ mice, mPGES-1 localized with COX-1 and COX-2 in cardiac fibroblasts but was not expressed in cardiomyocytes after MI (online-only Data Supplement Figure VIII). As expected, there was no mPGES-1 staining in the LV of Ptges Ϫ/Ϫ mice, which served as a negative control, after MI (online-only Data Supplement Figure IX).…”

Multimolecular Analysis of Stable Immunological Synapses Reveals Sustained Recruitment and Sequential Assembly of Signaling Clusters

“…We tested the frequencies of co-localization of pZAP70 and pSLP76 in synaptic areas that were also positive for pLAT and CD3 (pCD3[Y5]). The use of binarized imaging data resulting from the thresholding of fluorescent signals was not only useful in evaluating low-intensity signals of individual markers, but could also be used to generate and quantify CMP motifs (25,42,43), patterns of localizations of several markers within one spot (pixel). Although it is conceptually easier to think of a CMP motif as a protein complex, MELC analysis does not provide data on actual binding between proteins-only on shared locations.…”

Toponome imaging system: multiplex biomarkers in oncology

“…Most drugs are designed to influence protein interactions (e.g., analgesic drugs influence the protein network structure of synapses [77]). TIS may enhance our ability to design drugs that are disease-specific by assessing their effects on disease-specific protein networks.…”