Abstract
SUMOylation is a post-translational modification that manages numerous cellular pathways by modulating protein localisation, function and activity. SUMO-1 and SUMO-2/3 are conjugated to lysine residues generally within a consensus sequence in target proteins. SUMOylation is rapidly reversible, and most proteins are deSUMOylated by sentrin specific proteases (SENPs), therefore SENPs are crucial for spaciotemporal regulation of protein SUMOylation. Although SUMOylation mostly occurs in the nucleus, it is now clear that extranuclear SUMOylation regulates the functions of an increasing number of post-synaptic, presynaptic, mitochondrial and cytosolic proteins in neurons. However, understanding of specific functions of SENPs in neurons has not been explored in detail. There are six SENPs in mammals with different SUMO paralogue specificities.Here I investigate proteins under the control of SENP3. SENP3 mainly deconjugates SUMO-2/3 and is implicated in neuronal stress responses. I found that SENP3 interacts with the kainate receptor (KAR) subunit GluK2. KARs belong to the ionotropic glutamate receptor family together with AMPA and NMDA receptors. GluK2 contains a SUMOylation site, which is important for its trafficking, and SUMOylation of GluK2 has been shown to be enhanced by phosphorylation of its C-terminal tail. I show that SENP3 binds to WT GluK2 C-terminus as well as phosphomimetic and non-SUMOylatable mutants. These data suggest that SENP3 binds to GluK2 independently of whether GluK2 is SUMOylated. Moreover, I found that SENP3 KD in neurons does not affect GluK2 mRNA levels and hypothesise that SENP3 is directly involved in GluK2 trafficking. These results are intriguing because they suggest that rather than the conventional view that SENP3 is recruited following SUMOylation of a target protein, SENP3 can bind to the target prior to SUMOylation. This is important because it could provide a specificity mechanism to determine how rapidly individual proteins are deSUMOylated. SUMO would be immediately removed from targets already bound to SENP3 whereas targets without SENP3 bound would remain SUMOylated for longer. I propose that this SENP3-regulated duration of SUMO conjugation could determine the functional consequences of the modification.
In parallel work, I also showed that SENP3 KD dramatically increases levels of the cytoplasmic protein Rho GDP dissociation inhibitor (RhoGDI) in cortical neurons. RhoGDI supresses the activity of Rho GTPases. I demonstrated that RhoGDI is SUMOylated with both SUMO-1 and SUMO-2/3 and that SENP3 regulates RhoGDI levels transcriptionally. Since loss of SENP3 during ischemia is known to be neuroprotective, and RhoGDI has been implicated in the cellular response to hypoxia, my working hypothesis is that SENP3 regulation of RhoGDI might play a role in neuroprotective mechanisms during ischemia.
| Date of Award | 24 Mar 2020 |
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| Original language | English |
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| Supervisor | Jonathan G Hanley (Supervisor) & Jeremy Henley (Supervisor) |