ABSTRACT
Cell-based screening is a powerful approach to identify novel chemical modulators and biological components of relevant biological processes. The canonical Wnt pathway is essential for normal embryonic development and tissue homeostasis, and its deregulation plays a crucial role in carcinogenesis. Therefore, the identification of new pathway members and regulators is of significant interest. By means of a cell-based assay monitoring Wnt signaling we identified the pyrrolocoumarin Pyrcoumin as inhibitor of canonical Wnt signaling. Target identification and validation revealed that Pyrcoumin is a competitive inhibitor of dCTP pyrophosphatase 1 (dCTPP1). We demonstrate a yet unknown interaction of dCTPP1 with ubiquitin carboxyl-terminal hydrolase (USP7) that is counteracted by dCTPP1 inhibitors. These findings indicate that dCTPP1 plays a role in regulation of Wnt/ß-catenin signaling most likely through a direct interaction with USP7.
Subject(s)
Pyrophosphatases/metabolism , Wnt Signaling Pathway , Enzyme Inhibitors/pharmacology , HCT116 Cells , HEK293 Cells , Humans , Protein Interaction Maps/drug effects , Pyrophosphatases/antagonists & inhibitors , Ubiquitin-Specific Peptidase 7/metabolism , Wnt Signaling Pathway/drug effectsABSTRACT
Deciphering the mechanisms of sensory neural map formation is a central aim in neurosciences. Failure to form a correct map frequently leads to defects in sensory processing and perception. The olfactory map develops in subsequent steps initially forming a rough and later a precise map of glomeruli in the antennal lobe (AL), mainly consisting of olfactory receptor neuron (ORN) axons and projection neuron (PN) dendrites. The mechanisms underpinning the later stage of class-specific glomerulus formation are not understood. Recent studies have shown that the important guidance molecule Eph and its ligand ephrin play a role in class-specific PN targeting. Here, we reveal aspects of the mechanism downstream of Eph signaling during olfactory map formation. We show that the Eph-specific RhoGEF Ephexin (Exn) is required to fine tune PN dendrite patterning within specific glomeruli. We provide the first report showing an in vivo neurite guidance defect in an exn mutant. Interestingly, the quality of the phenotypes is different between eph and exn mutants; while loss of Eph leads to strong misprojections of DM3/Or47a neurons along the medial-lateral axis of the antennal lobe (AL), loss of Exn induces ventral ectopic innervation of a neighboring glomerulus. Genetic interaction experiments suggest that differential signaling of the small GTPases Rac1 and Cdc42 mediated by Exn-dependent and -independent Eph signaling fine tunes spatial targeting of PN dendrites within the olfactory map. We propose that their distinct activities on the actin cytoskeleton are required for precise navigation of PN dendrites within the olfactory map. Taken together, our results suggest that the precise connectivity of an individual neuron can depend on different modes of signaling downstream of a single guidance receptor. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 00: 000-000, 2018.
