Quantitative proteomics reveals neuronal ubiquitination of Rngo/Ddi1 and several proteasomal subunits by Ube3a, accounting for the complexity of Angelman syndrome

Abstract

Angelman syndrome is a complex neurodevelopmental disorder caused by the lack of function in the brain of a single gene, UBE3A. The E3 ligase coded by this gene is known to build K48-linked ubiquitin chains, a modification historically considered to target substrates for degradation by the proteasome. However, a change in protein abundance is not proof that a candidate UBE3A substrate is indeed ubiquitinated by UBE3A. We have here used an unbiased ubiquitin proteomics approach, the bioUb strategy, to identify 79 proteins that appear more ubiquitinated in the Drosophila photoreceptor cells when Ube3a is over-expressed. We found a significantly high number of those proteins to be proteasomal subunits or proteasome-interacting proteins, suggesting a wide proteasomal perturbation in the brain of Angelman patients. We focused on validating the ubiquitination by Ube3a of Rngo, a proteasomal component conserved from yeast (Ddi1) to humans (DDI1 and DDI2), but yet scarcely characterized. Ube3a-mediated Rngo ubiquitination in fly neurons was confirmed by immunoblotting. Using human neuroblastoma SH-SY5Y cells in culture, we also observed that human DDI1 is ubiquitinated by UBE3A, without being targeted for degradation. The novel observation that DDI1 is expressed in the developing mice brain, with a significant peak at E16.5, strongly suggests that DDI1 has biological functions not yet described that could be of relevance for Angelman syndrome clinical research.

Molecular Evolution, Neurodevelopmental Roles and Clinical Significance of HECT-Type UBE3 E3 Ubiquitin Ligases

“…Other reviews [56] highlight the mechanistic function of UBE3A, describing its association with several proteasome receptors, like DDI1 [57,58] or PSMD4 but also with proteasome itself, which is thought to control the proteolysis [59]. AS-associated point mutations in UBE3A N-terminus show impaired PSMD4 binding [60].…”

The ubiquitin ligase Ariadne-1 regulates NSF for neurotransmitter release

“…Higher levels of Ari-1 enzyme in bio ari flies should result on an enhanced ubiquitination of Ari-1 substrates relative to bio Ub flies. Therefore, quantitative proteomic experiments were carried out, following a previously described work-flow (28,29), to decipher the ubiquitinated proteome from both bio ari and bio Ub flies, and hence, detect those proteins whose ubiquitination is regulated by Ari-1. In brief, we collected 500 mg of heads of each fly genotype and subjected triplicate samples to biotin pulldown and LC-MS/MS analysis.…”

“…UAS-comt-GFP/TM6 (abbreviated throughout the text as bio comt) and GMR-Gal4, UAS-( bio Ub) 6 -BirA/CyO; UAS-ari-1/UAS-Comt-GFP flies (abbreviated throughout the text as bio ari/comt), respectively. Mixed-sex flies of 2-5 days old of each genotype ( bio Ub, bio ari, bio comt and bio ari/comt) were frozen in liquid nitrogen and their heads were collected using a pair of sieves with a nominal cut-off of 710 and 425 µm as previously described (26,29). Fly heads were then stored at -80 ºC until required.…”

Impaired proteostasis in rare neurological diseases

“…The function of UBE3A has been studied in Drosophila through the use of mass spectrometry proteomics in loss or excess of function mutants in vivo [188,189]. AS patient-derived iPSC lines and neurons derived from those lines have also given important insights on etiology of the disease.…”