The prolyl-isomerase PIN1 is essential for nuclear Lamin-B structure and function and protects heterochromatin under mechanical stress

Chromatin organization plays a crucial role in tissue homeostasis. Heterochromatin relaxation and consequent unscheduled mobilization of transposable elements (TEs) are emerging as key contributors of aging and aging-related pathologies, including Alzheimer's disease (AD) and cancer. However, the mechanisms governing heterochromatin maintenance or its relaxation in pathological conditions remain poorly understood. Here we show that PIN1, the only phosphorylation-specific cis/trans prolyl isomerase, whose loss is associated with premature aging and AD, is essential to preserve heterochromatin. We demonstrate that this PIN1 function is conserved from Drosophila to humans and prevents TE mobilization-dependent neurodegeneration and cognitive defects. Mechanistically, PIN1 maintains nuclear type-B Lamin structure and anchoring function for heterochromatin protein 1\u3b1 (HP1\u3b1). This mechanism prevents nuclear envelope alterations and heterochromatin relaxation under mechanical stress, which is a key contributor to aging-related pathologies

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

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

Targeting HECT-type E3 ligases - insights from catalysis, regulation and inhibitors

Ubiquitination plays a pivotal role in most cellular processes and is critical for protein degradation and signalling. E3 ligases are the matchmakers in the ubiquitination cascade, responsible for substrate recognition and modification with specific polyubiquitin chains. Until recently, it was not clear how the catalytic activity of E3s is modulated, but major recent studies on HECT E3 ligases is filling this void. These enzymes appear to be held in a closed, inactive conformation, which is relieved by biochemical manoeuvres unique to each member, thus ensuring exquisite regulation and specificity of the enzymes. The new advances and their significance to the function of HECT E3s are described here, with a particular focus on the Nedd4 family members

The ubiquitin ligase Hecw controls oogenesis and neuronal homeostasis by promoting the liquid state of ribonucleoprotein particles

Specialised ribonucleoprotein (RNP) granules are a hallmark of germ cells. Among their main function is the spatial and temporal modulation ofthe activity of specific mRNA transcripts that allow specification of primary embryonic axes. While RNPs composition and role are well established, their regulation is poorly defined. Here, we demonstrate that Hecw, a newly identified Drosophila ubiquitin ligase, is a key modulator of RNPs in oogenesis. Loss of Hecw activity results in the formation of enlarged granules that transition from a liquid to a gel-like state. At the molecular level, Hecw depletion leads to reduced ubiquitination and activity of the translational repressorFmrp, resultinginprematureOrbexpression/recruitmentinnursecells. In addition to defective oogenesis, flies lacking Hecw show neurodegenerative traits with prematureagingandclimbingdefectsduetoneuronalloss thatarelinkedtoRNPscondensation. Our findings reveal an unprecedented function of ubiquitin in modulating RNP fluidity and activity