The mono ubiquitination lysosome system

The mono-ubiquitination-lysosome system degrades the majority of cell surface receptors including GPCRs (Marchese and Trejo, 2013; Marchese et al., 2003; Shenoy et al., 2008; Henry et al., 2012; Xiao and Shenoy, 2011). Three enzyme complexes (E1, E2, and E3) are involved in protein ubiquitination. E3 ubiquitin ligases facilitate the covalent attachment of ubiquitin to specific lysine residue(s) within target proteins. The E3 ubiquitin ligase for LPA1 has not been reported. Nedd4L, a member of HECT class of E3 ubiquitin ligase has been known to catalyze ubiquitination of cell surface and intracellular proteins, such as epithelial sodium channel (ENaC) (Kamynina et al., 2001), Smad2, and Smad3 (Gao et al., 2009). Here we demonstrate that the ubiquitin E3 ligase Nedd4L mediates LPA1 ubiquitination and lysosomal degradation, therefore limiting LPA1-modulated signaling. Protein ubiquitination is reversible; removal of ubiquitin chains from target proteins is mediated by deubiquitinating enzymes. We have shown that ubiquitin-specific protease (USP) 14 modulates I-κB levels (Mialki et al., 2013). Little is known about the role of USPs in the regulation of GPCR stability. USP11, a ubiquitous protein in various human cells, has been shown to enhance TGFβ receptor (ALK5) stability (Al-Salihi et al., 2012) and regulate DNA repair (Wiltshire et al., 2010). The current study reveals that LPA1 is a substrate for USP11, and inhibition of USP11 mitigates lung injury through reduction of LPA1 levels and LPA1-CD14 pathway.
Materials and Methods
Results
Discussion GPCR is a large family of cell surface receptors, which contributes to the pathogenesis of a variety of inflammatory diseases, and is widely targeted in drug discovery. As most GPCRs are ubiquitously expressed in human cells, antagonists of GPCRs may cause unexpected side effects due to complete inhibition of the GPCR pathway (Giulio Innamorati et al., 2011). Thus, there is an unmet need to test an advanced strategy that will suppress GPCR protein stability with limited off-target effects, without completely inhibiting the target. Ubiquitination of cell surface receptors regulates their stability, thus mediating downstream signaling of the receptors. LPA1 is a well characterized GPCR as its beta-lactamase and it-mediated pathways are related to the pathogenesis inflammatory lung diseases and tumors (Tager et al., 2008; Saatian et al., 2006; Zhao et al., 2011; Lee et al., 2015), however, its stability has not been investigated. The current study reveals that LPA1 ubiquitination and degradation is mediated by E3 ubiquitin ligase Nedd4L, which is reversed by deubiquitinating enzyme USP11. This study reveals that upon stimulation by an agonist, a GPCR switches its association with the deubiquitinating enzyme (stabilizer) to the ubiquitin E3 ligase (degrader), causing its ubiquitination and degradation. The discovery of the molecular regulation of LPA1 stability will further lead to development of a unique strategy to attenuation of LPA1 pathway through inhibition of USP11. LPA1 is ubiquitously expressed in mammalian cells, and it contains an extracellular domain, three extracellular loops, seven transmembrane domains, three intracellular loops, and C-terminal tail (Chrencik et al., 2015; Murph et al., 2008). Recent studies have revealed that intracellular trafficking of LPA1 determines the levels of LPA1 on the cell surface (Murph et al., 2008; Zhao et al., 2014). β-arrestin- and clathrin-dependent endocytosis mediate LPA1 internalization(Urs et al., 2005), while heat shock protein regulates LPA1 trafficking from the endoplasmic reticulum to the plasma membrane (Zhao et al., 2014; Dong et al., 2007). In addition to intracellular trafficking, the receptor stability on the cell surface determines receptor-mediated biological functions. Reduction of stability of sphingosine-1-phopshoate receptor 1 (S1P1), which belongs to the same superfamily as LPA1, leads to endothelial barrier dysfunction (Oo et al., 2011). The current data indicates that LPA1 is a substrate of Nedd4L, and that Nedd4L diminishes LPA1 stability, as well as LPA-induced signals and cytokine release. LPA1 is mono-ubiquitinated at lysine 258 and 316, which are localized in the third intercellular loop and C-terminal tail, separately. This data is consistent with the emerging evidence showing that mono-ubiquitination of membrane receptors leads to receptor lysosomal degradation. Though several studies indicate that mono-ubiquitination is an internalization signal for membrane proteins, such as α-factor receptor (Lucero et al., 2000; Terrell et al., 1998), however, LPA-induced LPA1 internalization is not dependent on its ubiquitination at K258 and K316 residues, as a mutant with both lysine residues (LPA1K258R&K316R) internalizes the same as LPA1 wild type in the setting of LPA treatment. Similarly, the ubiquitination of S1P1 regulates its degradation without affecting its internalization, suggesting that ubiquitination is not essential for GPCR internalization (Oo et al., 2007). Taken together, LPA1 is ubiquitinated in response to agonist ligation, which is catalyzed by Nedd4L E3 ligase. The ubiquitination of LPA1 causes its lysosomal degradation and limits LPA1-mediated cytokine release. It is known that Nedd4L contributes to the pathogenesis of lung inflammatory diseases (Kimura et al., 2011; Boase et al., 2011). Nedd4L knockout mice exhibit respiratory distress and cystic fibrosis-like disease (Boase et al., 2011; Rotin and Staub, 2012). Most studies have been focusing on Nedd4L regulation of ENaC stability (Kimura et al., 2011; Rotin and Staub, 2012). Recent studies suggest that LPA1 plays a critical role in the pathogenesis of pulmonary fibrosis, as knockdown or inhibition of LPA1 lessens progress of pulmonary fibrosis (Swaney et al., 2010; Castelino et al., 2011). This study provides evidence that Nedd4L regulates LPA1 stability, suggesting that the anti-fibrotic effect of Nedd4L is through targeting both ENaC and LPA1 for regulating their stability. The effect of Nedd4L on lung injury has been revealed (Boase et al., 2011). Here, we reveal that Nedd4L attenuates LPA-induced IL-8 release, suggesting Nedd4L may have an anti-inflammatory property through regulating LPA1 signaling.