LEYRAT, C. | Search bioRxiv

bioRxiv2025

E(spl)m4 Directly Antagonizes Traf4 to Inhibit JNK Signaling in Drosophila

TRAF proteins are adaptor proteins that participate in signal transduction downstream of the Toll or TNF receptors and could elicit E3-Ubiquitin Ligase activity. They have been implicated in multiple processes during signal transduction, inflammation, and morphogenesis. In Drosophila, Traf4 has been implicated in the regulation of JNK signaling and cell death as well as Adherens Junctions regulation. Using overexpression approaches, we show here that Traf4 promotes JNK and caspase activation, as well as junctional E-Cadherin/ {beta}-Catenin depletion, resulting in epithelial cell delamination. Using biochemical, modelling, and functional genetics approaches, we further show that the Bearded-type small protein E(spl)m4 binds to Traf4, and inhibits its downstream signaling towards JNK activation and cell delamination, without affecting the effects of Traf4 on Adherens Junctions. Thus, this study identifies an endogenous peptide inhibiting Traf4 signaling, potentially by blocking Traf4 trimerization.

developmental biology↗

bioRxiv2021

Discovery and mechanism of action of small molecule inhibitors of ceramidases

Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes such as energy utilisation and cell proliferation. The central metabolite is ceramide, a pro-apoptotic lipid catabolized by ceramidase enzymes to ultimately produce pro-proliferative sphingosine-1-phosphate. Human ceramidases can be soluble proteins (acid and neutral ceramidase) or integral membrane proteins (alkaline ceramidases). Increasing ceramide levels to increase apoptosis has shown efficacy as a cancer treatment using small molecules inhibiting a soluble ceramidase. Due to the transmembrane nature of alkaline ceramidases, no specific small molecule inhibitors have been reported. Here, we report novel fluorescent substrates (FRETceramides) of ceramidases that can be used to monitor enzyme activity in real-time. We use FRETceramides to discover the first drug-like inhibitors of alkaline ceramidase 3 (ACER3) which are active in cell-based assays. Biophysical characterization of enzyme:inhibitor interactions reveal a new paradigm for inhibition of lipid metabolising enzymes with non-lipidic small molecules. Table of contents summaryUse of synthetic fluorescent ceramide molecules allows the discovery of the first selective drug-like small molecule inhibitors for alkaline ceramidase 3, an intra-membrane enzyme involved in sphingolipid metabolism in health and disease.

biochemistry↗