STRUCTURAL AND FUNCTIONAL INSIGHTS ON THE DROSOPHILA TNFR GRINDELWALD, COUPLING LOSS OF CELL POLARITY AND NEOPLASTIC GROWTH

Disruption of polarity and impairment of programmed cell death are signalling pathways of advanced epithelial tumors, whose progression often involves the activation of signalling controlled by TNF receptors, such as the JNK pathway. However, the link between loss of polarity and JNK pathway still remains elusive. In a disc-specific rn>avl RNAi screen aimed at finding new genes involved in neoplastic onset, a novel Drosophila TNFR was identified, named Grindelwald (Grnd). Grnd and the unique fly TNF Eiger activate the JNK pathway, and induce apoptosis or malignancy in a cell context-dependent manner. My PhD project focused on the characterization of the molecular mechanisms underlying Grnd activation and transduction of the JNK pathway. Upon binding to Eiger, Grnd recruits to the intracellular membrane Traf2, the most upstream component of the JNK pathway, and activates the JNK cascade. To shed light on the mechanism of Grnd activation by Eiger, I determined the crystal structure of the extracellular domain (ECD) of Grnd, in isolation and in complex with Eiger. Grnd-ECD comprises a single cysteine-rich domain, organized in two modules, named X2 and C2, and stabilized by disulphide bridges. The crystal structure of the Eiger:Grnd complex showed that the Eiger-TNF domain folds in a typical “jelly-roll” antiparallel -sheet, and forms hetero-hexamers with three Grnd molecules. To map the interacting surface between Grnd and Eiger, we engineered point mutations and performed binding assays revealing that Thr51, His66-Asn67 of Grnd and Asp337, His388-Arg391, and Arg396-Arg401 of Eiger are essential for the nanomolar affinity between the ligand and the receptor. In vitro studies suggest that glycosylation on Asn332 of Eiger lowers the affinity between Eiger and Grnd. We are currently generating collaboratively Grnd mutant flies to assess the relevance of the Eiger-Grnd interaction in activating the JNK pathway and inducing the small-eye phenotype, or in promoting malignancy in scrib-/-;rasV12 clones. Previous results showed that the JNK pathway was activated in polarity-deficient cells to drive neoplastic growth. In the rn>avl RNAi screen, Grnd, but not Eiger, was necessary for the JNK pathway to be induced. I showed that Grnd directly interacts with the polarity protein Veli, to promote hyper-proliferation and invasiveness. Collectively, these findings depict Grnd as the first TNFR able to couple cell polarity with tumor overgrowth. One major goal of my project was also the identification of a Grnd orthologue in human, able to couple polarity and signal transduction. Biochemical analyses revealed that Fas is the only TNFR interacting with Veli and with the basolateral polarity protein Dlg1, and provided evidence that Dlg1 compete with Veli for binding to Fas. The relevance of these interactions for the Fas biology is currently under investigation.