Characterization of Ca2+-triggered (de)ubiquitination events in depolarized synaptosomes by mass spectrometry
by Sofia Ainatzi
Date of Examination:2023-09-28
Date of issue:2024-08-15
Advisor:Prof. Dr. Henning Urlaub
Referee:Dr. Alexander Stein
Referee:Prof. Dr. Reinhard Jahn
Persistent Address:
http://dx.doi.org/10.53846/goediss-10653
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Abstract
English
Communication between neurons takes place at synapses, where the transmission of information from the presynaptic neuron to the postsynaptic cell involves the Ca2+-regulated fusion of synaptic vesicles (SVs) with the presynaptic plasma membrane, leading to the release of neurotransmitters (NTs). While much progress has been made in understanding the molecular mechanism mediating SV fusion and recycling, the intricate fine-tuning of this process remains an area of intense research. In this context, post-translational modifications (PTMs) of proteins, such as phosphorylation have been implicated in the fine-tuning of NT release. In contrast to phosphorylation and glycosylation, other PTMs, such as ubiquitination in the presynapse remain largely unexplored. Like phosphorylation, non-degradative ubiquitination can serve as a rapid molecular switch to modulate protein activity and location and is therefore amenable to signalling cascades. Indeed, altered non-degradative protein ubiquitination has recently been associated with membrane depolarization and NT release. However, we still do not know the identity of the synaptic proteins that are de- or ubiquitinated nor the exact amino acid residues that are targeted for ubiquitination, in response to depolarization and Ca2+-influx. This information will help us to understand the role of non-degradative ubiquitination in modulating signalling cascades in the synapse. In this work, we have focused on identifying the synaptic proteins and their exact ubiquitination sites that alter in response to membrane depolarization, as they may regulate non-proteolytic processes associated with signal transduction. To address our questions, we used a well-established model system of the synapse, synaptosomes, which were chemically depolarized either in the presence of Ca2+ or in the presence of EGTA. Having differentiated between these two conditions, we have optimized a mass spectrometric, proteomic workflow to systematically identify and quantify ubiquitinated proteins from differently treated synaptosomes. To boost the sensitivity required for PTM analysis, antibody-based enrichment of formerly ubiquitinated proteins coupled with isobaric labelling (tandem mass tags, TMT) was employed. By comparative quantitative analysis of ubiquitination sites in differently treated synaptosomes, we were able to show that a small fraction of the quantified ubiquitination sites changed in response to Ca2+ -influx. Many of these significantly altered ubiquitination sites were mapped to key synaptic proteins; while a few proteins involved in clathrin mediated endocytocis (CME) appeared to be significantly deubiquitinated, AZ proteins appeared to undergo ubiquitination in “stimulated” synaptosomes. To provide further support to our observations, we developed additional validation assays to monitor and quantify selected ubiquitination sites. Validated ubiquitination sites mapped to evolutionarily conserved lysine residues were predicted to be functionally significant. One of these, namely K291 mapped to the autoinhibitory domain of CaMKIIα was further functionally characterized in non-neuronal cells and cultured hippocampal neurons. To investigate the functional importance of CaMKIIα K291 ubiquitination, we used a FRET probe (Camuiα-CR) and generated a non-ubiquitinated variant, in which K291 was replaced by an R residue. Comparative analysis of the two forms suggested that K291 ubiquitination negatively affects enzyme activity and NT release. Overall, this study was the first to identify the synaptic proteins whose ubiquitination state alters in response to Ca2+-influx in depolarized synaptosomes. These events are likely to be non-degradative and to be associated with signal transduction in the synapse. Indeed, in this work we showed that K291 ubiquitination of CaMKII negatively affects CaMKII activity and NT release.
Keywords: synapse; signaling; ubiquitination; mass spectrometry; CaMKII; proteomics
