Ubiquitination of GlyRs subunit on plasma
Ubiquitination of GlyRs-α1 subunit on plasma membrane has been shown to initiate the receptor Oxaprozin and subsequent degradation in lysosomes (Buttner et al., 2001). However, the specific E3 ligase responsible for GlyRs-α1 ubiquitination is largely unknown as yet. Also, the functional consequence of GlyRs-α1 ubiquitination in pain-related spinal cord dorsal horn is far from clear. The current study demonstrated that HUWE1 (HECT, UBA, WWE domain containing 1), a 482-kDa E3 ubiquitin ligase with HECT domain (Khoronenkova and Dianov, 2011, Lorenz, 2018), specifically interacted with and ubiquitinated GlyRs-α1 subunit in spinal dorsal horn. We provided evidence that HUWE1-mediated ubiquitination and endocytosis of GlyRs contributed to glycinergic disinhibition during inflammatory pain.
Materials and methods
Discussion HUWE1 belongs to the E3 ubiquitin ligases that have the signature HECT domain at the C-terminal region (Sluimer and Distel, 2018). The HECT domain is responsible for the transfer of ubiquitin molecules to substrate proteins (Rape, 2018). In addition to the HECT domain, HUWE1 contains two Armadillo Repeat-Like Domains at the N terminus, whose functions remain to be elucidated. There exist a ubiquitin-associated (UBA) domain, a WWE domain and a Bcl-2 Homology 3 domain in the middle region of HUWE1, which are proposed to mediate protein-protein interaction and determine the substrate specificity. HUWE1 has been shown to ubiquitinate a line of proteins (Dominguez-Brauer et al., 2017, Hao et al., 2017, Yang et al., 2010). In the developing brain, HUWE1 is implicated in the neural differentiation and proliferation (Zhao et al., 2008, Zhao et al., 2009). However, the synaptic function of HUWE1 in adulthood is yet largely unknown. The major finding in the current study was the identification of GlyRs-α1 subunit as a novel substrate of HUWE1. We provided evidence that HUWE1-mediated GlyRs-α1 ubiquitination contributed to the reduced glycinergic inhibition during inflammatory pain. Our data showed that HUWE1 was located in puncta throughout spinal neurons, some of which were present at excitatory synapses under resting conditions. NMDAR and AMPAR are two principle mediators of excitatory synaptic transmission. The E3 ligases targeting NMDAR include Mind bomb-2 (Jurd et al., 2008), while those ubiquitinating AMPAR include neural-precursor cell-expressed developmentally downregulated gene 4-1 and RING finger protein 167 (Lussier et al., 2012, Schwarz et al., 2010, Scudder et al., 2014). These E3 ligases reside at excitatory synapses and form complexes with glutamate receptors. Our data showed that HUWE1 didn't interact with glutamate receptors, suggesting that synaptic HUWE1 might modulate other substrates than glutamate receptors. The significance of HUWE1 distribution at excitatory synapses requires further investigation. In C. elegans, the HUWE1 ortholog EEL-1 has been shown to localize at the presynaptic terminals of GABAergic motor neurons (Opperman et al., 2017). EEL-1 mutants alter the frequencies and amplitudes of GABAergic mIPSCs, suggesting that EEL-1 regulates GABAergic transmission presynaptically and to a modest extent, postsynaptically. The β2/β3 subunits-containing GABAA receptors are widely distributed in mammalian nervous system and important for the modification of nociceptive signals (Masocha, 2015, Woll et al., 2018). Our data showed that β2/β3 subunits didn't interact with HUWE1, suggesting that GABAAR were not the good substrates for HUWE1 ubiquitination. Glycinergic synaptic transmission onto excitatory interneurons within spinal cord dorsal horn negatively controls the output of action potentials to lamina I projection neurons (Foster et al., 2015). Pharmacological or genetic removal of glycinergic inhibition is sufficient to evoke pain hypersensitivity in intact animals (Foster et al., 2015). GlyRs are composed of functional α1-α4 subunits and/or regulatory β subunit. The β subunit is able to interact with gephyrin and stabilize GlyRs at inhibitory synapses (Lynch, 2009). The four α subunits share high similarity in the amino acid sequences but exhibit distinct expression patterns. The α2 subunit is prenatally expressed in central nervous system and becomes dispersible for glycinergic transmission by postnatal 2–3 weeks (Lynch, 2009). Therefore, the majority of glycinergic synaptic currents in adult spinal cords are generated by GlyRs containing α1, while few are produced by those with α3 subunit (Graham et al., 2011). Mutation or genetic downregulation of α1 subunit yields severe motor and sensory disorders such as hyperekplexia and pain sensitization (Duan et al., 2014). The α3 subunit deletion, however, generates no detectable phenotypes (Harvey et al., 2004). Although the expression of α3 subunit is less abundant than that of α1 subunit in spinal dorsal horn, a great deal of attention has been drawn to the activity-dependent modification of α3 function during pathological pain. Inflammatory factor prostaglandin E2 can phosphorylate α3 subunit at Ser346 through cAMP-dependent protein kinase signaling (Harvey et al., 2004). This phosphorylation event blunts glycinergic transmission and accounts for the development of inflammatory pain (Harvey et al., 2004). Regardless of the predominant role played by GlyRs-α1 subunit in pain modification, it remains largely unknown whether and how peripheral injuries regulate the function of this key GlyRs subunit.