E3 Ubiquitin Ligases and Asthma Relief

Asthma is marked by airway hyperresponsiveness, inflammation, mucus secretion and tissue remodeling. These pathological changes in the lungs are principally orchestrated by the actions of multiple exogenous and endogenous stimuli, including cytokines, pathogen- and damage-associated molecular patterns that can activate receptors on immune and non-immune cells in the lung and transduce complex intracellular signaling cascades resulting in modulation of effector protein expression and/or functions. These pathways are finely regulated by post-translational modifications such as phosphorylation, ubiquitination, and sumoylation. Ubiquitination of proteins includes sequential actions of E1-activating, E2-conjugating and E3-ligase enzymes that ultimately insert ubiquitin moieties on target proteins. E3 ubiquitin ligases confer substrate specificity in ubiquitination process and mark target proteins for degradation, altering their location within cellular compartments or modulating their physiological activity. Consequently, E3 ligases are attractive targets in manipulating ubiquitination that plays a critical role in the initiation and progression of chronic inflammatory diseases.

In murine models of asthma, E3 ligases can act as both promoters and inhibitors of asthma progression by modulating cellular function via modifications to specific target proteins (1, 2). For example, MID1, TRIM27 and Parkin ubiquitinate PP2A, NLRP3 and NF-κB, respectively, and promote airway hyperresponsiveness, epithelial barrier dysfunction and inflammation. In contrast, respective ubiquitination of STAT6, pJAK1 and NLRP3 by PPARγ, CUL5 and TRIM31 inhibits IgE synthesis and Th2 differentiation. Diverse E3 ligases can modulate asthma progression by targeting specific substrates to repattern complex and interconnected signaling pathways, thus targeting specific E3 ligases is an intriguing therapeutic concept in asthma.

Here, Wang and colleagues (3) detail the role of Pellinos 1 (PELI1), an E3 ubiquitin ligase, in a murine model of asthma. PELI1 is an E3 ligase belonging to the Pellinos family that regulates multiple signaling networks (4). PELI1 is consistently activated by diverse receptor-driven signal transduction pathways. In this regard, TLR3- and TLR4-dependent signaling are critical regulators of PELI1 (5). PELI mediates ubiquitination of effector proteins through K48 or K63 linkage of the ubiquitin moiety (6). Overexpression of PELI1 also drives sumoylation, a post-translational modification involving addition of SUMO-1 (small ubiquitin-related modifier 1) to target proteins (7). PELI1 is highly expressed in AECs (airway epithelial cells) (8), but in the current work from Wang et al (3) demonstrate that AECs from asthmatic children show reduced PELI1 transcript levels compared to healthy donors. They show that this can be modeled effectively in newborn mice repeatedly challenged with house dust mite (HDM) allergen, a protocol that significantly reduced PELI1 expression in lung tissues. This phenomenon is preserved in human bronchial epithelial cells challenged with HDM. Importantly, overexpression of PELI1 using adenoviral delivery was able to rescue mice from HDM-induced inflammation, thus establishing a consequential role for PELI1. Further, to establish AECs as the primary cell type for PELI1 actions, the authors treated 16HBE cell line with HDM, which compromised cellular viability and promoted apoptosis. This observation was reproducible in 16HBE cells by knocking down endogenously expressed PELI1 using the CRISPR/Cas9 approach. Importantly, overexpression of PELI1 restores epithelial cellular viability. Collectively, these studies unequivocally establish the role of PELI1 in maintaining epithelial cell integrity and inhibiting the progression of asthma pathology.

To establish the precise mechanisms by which PELI1 suppresses the development of asthma features, the authors probed for potential substrate targets of PELI1. Among various potential targets for PELI1, IRAK2 (interleukin-1 receptor-associated kinase-like 2) emerged as a major candidate. Further, dual-labeling immunofluorescence of lung tissues from HDM-challenged mice shows a strong inverse correlation between PELI1 and IRAK2 expression. Transgenic overexpression of PELI1 also significantly reduces IRAK2 expression and mitigates the development of asthma features. Conversely, Wang and colleagues show that overexpression of IRAK2 in miceexacerbates multiple features of HDM-induced asthma pathology, including production of pro-inflammatory cytokines, and upregulation of necroptosis-related proteins RIPK3 and phospho-MLKL.

To explore physical interactions between PELI1 and IRAK2, the authors co-expressed these proteins in HEK293T cells. Co-immunoprecipitation (co-IP) assay confirmed protein-protein interaction between PELI1 and IRAK2, and increased IRAK2 ubiquitination in the presence of PELI1. Additional co-IP studies showed that a PELI1 mutant lacking the forkhead-associated (FHA) domain does not associate with IRAK2. Using K63R mutants, the authors also showed that the mutation leads to loss of IRAK2 ubiquitination, underscoring the relevance of the K63 site for PELI1. Finally, IRAK2 ubiquitination by PELI1 inhibits signaling of MyD88-IRAK4-IRAK2, which is essential for activation of pro-inflammatory NF-κB and MAPK pathways. Collectively, these studies establish PELI1 regulation of IRAK2 through K63 ubiquitination, thereby contributing to inflammation in this asthma model.

Study by Wang et al (3) raises some interesting questions about the role of PELI1 in asthma progression. It would be interesting to see if PELI1 knockout mice and/or mice lacking ligase activity for PELI1 exhibit increased asthma severity, or whether they are protected through a compensatory action of other E3 ligases. Herein, the authors demonstrate that co-expression of IRAK2 and PELI1 in mice exacerbates features of asthma, possibly through regulation of PELI1 activity or expression, as shown previously (9). Indeed, PELI1 expresses phosphorylation sites for IRAK proteins, can undergo dephosphorylation and autoubiquitination, which can regulate PELI1 expression and function (4).

PELI1 actions appear to be contextual and differ among disease models. Mice lacking PELI1 show reduced severity of experimental autoimmune encephalomyelitis (10) and sepsis (5). While PELI1 can negatively regulate asthma development via suppression of IRAK2 (3), it may yet exacerbate inflammation in other diseases through modulation of target proteins such as STAT3 (intestinal inflammation and tumor development) (11), RIP1 (TRIF-dependent TLR signaling and promotion of sepsis) (5), PKCθ (TCR signaling and pro-tumor growth) (12) and BCL6 (lymphomagenesis) (13). PELI1 has complex interactions with other proteins, and its own regulation is unclear as noted above. Nevertheless, PELI1 is highly expressed in airway epithelial cells and has a profound effect on cellular signaling and function, underscoring its potential as a therapeutic target.

In summary, the studies by Wang and colleagues add to existing literature implicating E3 ligases in asthma pathobiology, furthering interest in these proteins as targets for asthma relief. While the development of targeted protein degradation strategies (e.g., PROTACs) for E3 ligases has been promising, the development of small molecules that target E3 ligases has been challenging (14). E3 ligases exhibit structural heterogeneity among members of the same family (15). The human genome codes for more than 600 E3 ligases, and the mechanisms by which these distinct ligases regulate the physiological actions of target proteins remain unclear. Further, individual E3 ligases modulate the course of inflammation differently in cell- and disease-specific manner (as noted earlier for PELI1), underscoring a critical knowledge gap. Nevertheless, substrate selectivity, an intrinsic feature of E3 ligases, presents exciting opportunities for the development of novel and precise (presumably reduced off-target effects) therapeutic strategies for asthma control.