E3 ubiquitin ligases are the ultimate enzymes involved in the transfer of ubiquitin to substrate proteins, a process that determines the fate of the modified protein. Numerous diseases are caused by defects in the ubiquitin-proteasome machinery, including when the activity of a given E3 ligase is hampered. Thus, inactivation of E3 ligases and the resulting effects at molecular or cellular level have been the focus of many studies during the last few years. For this purpose, site-specific mutation of key residues involved in either protein interaction, substrate recognition or ubiquitin transfer have been reported to successfully inactivate E3 ligases. Nevertheless, it is not always trivial to predict which mutation(s) will block the catalytic activity of a ligase. Here we review over 250 sitespecific inactivating mutations that have been carried out in 120 human E3 ubiquitin ligases. We foresee that the information gathered here will be helpful for the design of future experimental strategies.

Angelman Syndrome (AS) is a neurodevelopmental disorder with complex symptomatology caused by the loss of maternal allele expression of one single gene in the brain, the ubiquitin E3 ligase UBE3A. The underlying genetic basis of AS, and the phenotypes observed in both humans and in animal models of AS, have previously been extensively described. However, the molecular mechanisms regulated by UBE3A ubiquitination in the brain remain highly elusive. Previous studies have reported a number of proteins whose abundance or activity are altered in AS models, implicating various signalling pathways in the physiopathology of AS. However, the identified pathways could well be altered further downstream of UBE3A ubiquitination events. We provide the first proteomic report of UBE3A-mediated ubiquitination events in a mammalian brain. For this we have combined the bioUb mouse model with a new mouse strain moderately increasing UBE3A levels. Several proteins known to be involved in the trafficking and maintenance of neurotransmitter receptors as well as proteins relaying the signals of these synaptic receptors are shown here to be ubiquitinated by UBE3A. The identified proteins have roles in higher mental function, long term potentiation, seizures and neurodevelopmental disorders, being involved in the BDNF, RAS/ERK and TSC/mTOR signalling pathways. A reduced ubiquitination of these proteins is expected when UBE3A levels are lower, so their identification could be key to opening novel therapeutic strategies for treating Angelman Syndrome. Further work will be required to characterize how UBE3A timely orchestrates each of these multiple regulatory events in different neuronal subtypes within the human brain.

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