CHARACTERIZATION OF THE E3 LIGASE DHECW, A NOVEL MEMBER OF THE DROSOPHILA MELANOGASTER NEDD4 FAMILY

Abstract

Ubiquitination is one of the most abundant and versatile post-translation modifications in eukaryotes, and it plays an important role in many biological processes by affecting protein activity, interactions, localization and stability. E3 ligases (E3s) have a key function as molecular ubiquitin-substrate matchmakers, providing specificity to the reaction, yet little is known about the targets and functions of the majority of E3s. In this thesis, we identified and characterized dHecw, a novel member of the Drosophila melanogaster Nedd4 family of ubiquitin E3s. dHecw is the single ortholog of the human HECT ligases HECW1 and HECW2, which are the less characterized members of the family. We proved that dHecw is a catalytically active enzyme, whose expression is tightly regulated in the central nervous system and in the ovary, and is down-modulated during aging. To investigate dHecw function in vivo, we generated catalytic inactive dHecw and KO fly mutants by CRISPR/Cas9 technology. Both types of mutants are viable in homozygosis but presented signs of neurodegeneration, such as short lifespan, limited motor function and brain tissue vacuolarization. They also showed premature decline in fertility due to germline specific defects in oogenesis, including aberrant number of nurse cells, compound egg chambers and misspecification of additional oocytes. The interactome of dHecw was identified by mass spectrometry analysis and includes several ribonucleoparticles (RNPs) components, including dFmr1, a translational repressor that controls localized mRNA translation in developing fly egg chambers. Interestingly, dfmr1 loss of function flies presented ovarian phenotypes that closely resemble the ones of dHecw mutants and KO flies. We demonstrated that dFmrp is a dHecw substrate in vitro, and we found a genetic interaction among the two proteins. Our investigation of the functional outcome of dFmrp ubiquitination suggests that it does not cause dFmrp degradation but, instead, it impacts on its function/interaction network. Indeed, we found that the expression of Orb, a known target of dFmrp repression, is upregulated in dHecw fly mutants. On the contrary, Orb levels are down-modulated upon dHecw overexpression in the germline tissue. Altogether, our data suggest that dHecw is a novel player involved in the dynamic regulation of RNPs required for neuronal health and fertility