USP9X promotes lipopolysaccharide‐stimulated acute lung injury by deubiquitination of NLRP3

Abstract

Alveolar epithelial cells (AECs) function as a vital defense barrier avoiding the invasion of exogenous agents and preserving the functional and structural integrity of lung tissues, while damage/breakdown of this airway epithelial barrier is frequently associated with the pathogenesis of acute lung injury (ALI). NOD‐like receptor family, pyrindomain‐containing 3 (NLRP3) inflammasome activation‐associated pyroptosis is involved in the development of ALI. Yet, how the activity of NLRP3 inflammasome is regulated in the context of ALI remains unknown. Herein we hypothesized that USP9X, an important deubiquitinase, participates in modulating the activation of NLRP3 inflammasome, thereby affecting the phenotypes in a lipopolysaccharide (LPS)‐stimulated AEC model. Human pulmonary AECs were subjected to LPS/adenosine triphosphate (ATP) treatment to induce NLRP3 inflammasome activation and cell pyroptosis. Knockdown and overexpression of USP9X were applied to validate the function of USP9X. Inhibitors of proteinase and protein synthesis, as well as approach of co‐immunoprecipitation coupled with Western blot, were utilized to explore the molecular mechanism. LPS/ATP challenge resulted in pronouncedly increased pyroptosis of AECs, activation of NLRP3 inflammasome and release of interleukin (IL)‐1β and IL‐18 cytokines, while downregulation of USP9X could reverse these alterations. USP9X was found to have marked impact on NLRP3 protein instead of mRNA level. Furthermore, increased ubiquitination of NLRP3 was observed upon downregulating USP9X. Additionally, the inhibitory effect of USP9X downregulation was reversed by NLRP3 overexpression, while the promoting impact of USP9X overexpression was dampened by NLRP3 inhibitor in terms of cell pyroptosis and cytokine secretion. USP9X modulated the activity of NLRP3 inflammasome and pyroptosis of AECs via its deubiquitination function.

Proximity proteomics reveals UCH-L1 as an NLRP3 interactor that modulates IL-1β production in human macrophages and microglia

“…A combination of proximity proteomics and RNAi screening revealed 7 DUBs showing effects in both assays (USP9X, USP8, USP14, USP10, UCH-L1, OTUB1 and OTUD4) ( Figure 4H ). USP9X, USP14, and OTUB1 have already been reported to modulate the NLRP3 inflammasome (Hai et al, 2022; Xiang et al, 2023), suggesting additional NLRP3-associated DUBs (USP8, USP10, OTUD4 and UCH-L1).…”

“…In addition, in mice, BRCC3, a DUB reflecting a component of the BRISC complex, was shown to deubiquitylate NLRP3, thereby regulating inflammasome activity (Py et al, 2013). Furthermore, the ubiquitin-specific proteases USP9X (Xiang et al, 2023), USP14 (Hai et al, 2022) and UCHL5 (Kummari et al, 2015; Ramachandran et al, 2021) have been proposed to deubiquitylate NLRP3 and thereby modulate inflammasome activation in alveolar epithelial cells (AECs) and annulus fibrosus cells derived from patients with intervertebral disc degeneration, respectively. In addition, UAF1 deubiquitylase complexes also promote NLRP3 inflammasome expression (Song et al, 2020) and factors such as ABRO1 regulate activation through NLRP3 deubiquitylation (Ren et al, 2019).…”

The roles of ubiquitination and deubiquitination of NLRP3 inflammasome in inflammation-related diseases: A review

“…This process enhanced the stability of the NLRP3 inflammasome, increased the expression of its relevant component proteins, and facilitated the DSS-induced inflammatory response [ 47 ]. Additionally, in an in vitro model using LPS-induced human lung epithelial cells, the USP9X DUB induced deubiquitination modification of NLRP3 inflammasomes, which activated NLRP3 inflammasomes, induced IL-1β and IL-18 maturation, and enhanced LPS-induced pyroptosis and inflammation [ 48 ]. Furthermore, in the annulus fibrosus cells derived from patients with intervertebral disc degeneration (IDD), overexpression of the USP14 DUB promoted the deubiquitination of NLRP3 inflammasomes.…”

Posttranslational modifications of NLRP3 and their regulatory roles in inflammasome activation

“…The DUB complex ubiquitin‐specific processing protease 1 (USP1)/USP1‐associated factor 1(UAF1) removes the K48‐linked polyubiquitination of NLRP3 to stabilize its protein expression, thereby promoting NLRP3 inflammasome activation [46]. Although many DUBs, such as USP7 [47], USP47 [47], USP30 [48], USP14 [49], and USP9X [50], promote NLRP3 inflammasome activation by reducing NLRP3 ubiquitination, the specific underlying mechanisms remain unclear. STAM‐binding protein (STAMBP), a negative regulator of NLRP3 inflammasome activation, removes NLRP3 K63‐linked ubiquitination [51].…”