Introduction

Asthma is a heterogeneous airway inflammation with no certain cause, unlike chronic obstructive pulmonary disease (COPD), for which smoking is the main cause.1 It is often driven by Th2 cell activation and associated cytokines, leading to eosinophil recruitment, mast cell activation, and excessive mucus production by airway epithelial cells.2 This chronic inflammation is associated with hyperresponsiveness of the respiratory tract, which usually results in generalized and variable limitation of reversible expiratory circulation, leading to repeated occurrences of wheezing, breathlessness, chest tightness, and coughing.3 According to the WHO, a significant proportion of asthma-related deaths occurs in low- and lower-middle-income countries, where the challenges of under-diagnosis and under-treatment persist.4 Bronchospasm is an important pathophysiological feature of asthma attacks, with histamine playing a pivotal mediating role in the process of bronchospasm caused by asthma.5 On the one hand, it causes bronchospasm or bronchial hyperreactivity through both direct and indirect effects, and on the other hand, it participates in the inflammatory response of asthma, which makes airway inflammation persist and aggravate, and consequently increases the frequency and severity of bronchospasm.5,6 In recent years, with the deepening of our understanding of the pathogenesis of asthma, targeted therapy and specific signaling pathway therapy have gradually become the new direction of clinical asthma treatment. For instance, omalizumab is suitable for allergic asthma patients with elevated serum IgE levels and clear allergens by antagonizing IgE.7 In addition to omalizumab, other asthma biologics, such as mepolizumab, reslizumab, and dupilumab have been employed to target specific inflammatory pathways in patients exhibiting a suboptimal response to conventional therapies such as inhaled glucocorticoids and long-acting β2-agonists.8,9 However, asthma biologics still face serious challenges in terms of cost, delivery methods, and variability in response. Furthermore, studies on the abnormally activated inflammatory pathways such as Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) and Phosphoinositide 3-Kinase (PI3K) in the pathogenesis of asthma have also become popular.10 Currently commonly used treatments are palliative and do not address the root cause of asthma. Therefore, we want to explore the specific attack mechanism of asthma and carry out targeted therapy from the root to relieve asthma symptoms. To study the pathogenesis and development basis of asthma, scientists need to establish in vitro animal asthma models that can reflect the pathophysiology of human asthma, including IgE mediated antigen sensitivity, acute bronchoconstriction, increased airway resistance, chronic airway inflammation, etc.11 The current common approach is to induce mice using ovalbumin (OVA) or house dust mite (HDM).12 The scientific findings introduced in this paper are basically based on these two induction methods.

Ubiquitin-proteasome system (UPS) is a complex cascade reaction process in eukaryotic cells that ubiquitinates the corresponding protein, leading to its recognition and subsequent degradation by the proteasome.13 This highly specific process consumes cellular energy and serves as a crucial regulator of various biological processes implicated in the development of conditions like cardiovascular disease, cancer, and inflammation-related disorders.14 The UPS comprises key components including ubiquitin, E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, ubiquitin-protein ligase E3, proteasomes, and deubiquitinating enzymes deubiquitinating enzymes (DUBs).15 Notably, extensive research has focused on elucidating the roles of E3 ubiquitin ligases and DUBs in the occurrence and development of those diseases. Therefore, this review will primarily emphasize the functions of E3 enzymes and DUBs.

Ubiquitination is a multistep post-translational modification governed by the ubiquitin ligases that successively activates, conjugates, and ligates 76-amino acid proteins ubiquitin onto substrate proteins.16 This adjustment alters important properties of the substrate protein, including its intracellular activity and localization, reaction with other proteins, and extensive half-life within the cell.17 Therefore, ubiquitination could regulate a large cohort of crucial cellular processes and a deficiency in the ubiquitin function has been shown in several diseases, such as inflammatory diseases, immune disorders, and susceptibility to infections.18 There are three types of ligases, E1, E2, and E3 enzymes. Of all the enzymes involved in ubiquitination, the E3 enzyme is the most abundant. E3 ubiquitin ligases fall into three families: Really Interesting New Gene (RING E3s), homologous to E6-AP C-terminus E3s (HECT E3s), and RING-between RING-RING E3s (RBR E3s) according to their structural characteristics and mechanism of action. After binding to the E2 Ub complex and substrate, RING E3s catalyzes the direct transfer of ubiquitin from E2 to the lysine residue of the substrate, while HECT and RBR E3 ligase catalyze the transfer of ubiquitin from E2 to cysteine, the active site of E3 ligase, followed by the transfer of ubiquitin to the substrate protein.19 E3 ligase plays a vital role in the onset and progression of chronic human inflammation, including asthma, COPD, arteriosclerosis, and arthritis.20

Scientists have made extensive achievements in the research on the regulation of ubiquitination and its coupling and the transmission of downstream signals, but the research on deubiquitination has not made great progress until recent years. Deubiquitination is the removal of ubiquitin from modified substrates mediated by DUBs against ubiquitin couplings and ligase induced signals.21 Human genes encode almost 100 different DUBs that break down the ubiquitin chains and their signals while recycling ubiquitin for further coupling.22 This can be done by breaking the entire ubiquitin chain by cutting the bond between the near-end ubiquitin and the substrate, or by cutting a single ubiquitin at the far end of the chain.22 Dysregulation or dysfunction of DUBs is associated with many major human diseases, including chronic inflammation, cancer and neurological disorders, which is often caused by abnormal signals within cells.23 With the further elucidations of the mechanism of DUBs, its importance in clinical diagnosis and treatment has been gradually explored. In the future, DUBs may be used as new targets for the treatment of some major human diseases and improve prognosis. In this review, we will focus on the E3 ubiquitin ligases, deubiquitinases, and their roles in regulating asthma.

E3 Ligases and Their Mechanisms Involved in Asthma Pathology E3 Ubiquitin Ligases Promote the Advancement and Course of Asthma

Only a limited subset of E3 enzymes, including autocrine motor factor receptor (AMFR), Midline 1 (MID1), Parkin, Chromobox 4 (Cbx4) and tripartite motif-containing 27 (TRIM27), positively contribute to the pathogenesis and progression of asthma as shown in Figure 1 and Table 1. These enzymes play a pivotal role in orchestrating widespread airway inflammation by regulating the production of inflammatory mediators and promoting the proliferation of immune cells. The mechanisms of these E3 ligases will be explained in more detail below in this section.

Figure 1 E3 ubiquitin ligases promote asthma pathology. Autocrine motor factor receptor (AMFR) can promote Granulocyte macrophage-colony stimulating factor (GM-CSF) production by targeting Cytokine Inducible SH2-containing protein (CIS) and facilitating its ubiquitination degradation. E3 enzyme Midline 1 (MID1) can mediate the ubiquitinating degradation of Protein Phosphatase 2A (PP2A) to inhibit the secretion of inflammatory Cytokines Interleukin-25 (IL-25) and Interleukin-13 (IL-13). E3 enzyme Parkin has exerted its function in two pathways. One is mediating the ubiquitinating degradation of P53, the other is increasing the production of inflammatory substrate Nuclear Factor Kappa-light-chain-enhancer (NF-κB). CBX4 functions as an E3 enzyme but it increases the secretion of Interleukin-9 (IL-9) through the SUMOysation of Hypoxia-inducible factor alpha (HIF-α). All these reactions promote inflammation, which ultimately leads to an increase in the course and progression of asthma.

Abbreviations: AMFR, Autocrine motor factor receptor; GM-CSF, Granulocyte macrophage-colony stimulating factor; CIS, Cytokine Inducible SH2-containing protein; MID1, Midline 1; PP2A, Protein Phosphatase 2A; IL-25, Interleukin-25; IL-13, Interleukin-13; NF-κB, Nuclear Factor Kappa-light-chain-enhancer; IL-9, Interleukin-9; HIF-α, Hypoxia-inducible factor alpha.

Table 1 E3 Ubiquitin Ligases promoting the progression of Asthma

AMFR

Alveolar macrophages (AMs), an important kind of airway immune cells, play crucial roles in lung homeostasis, immune response, and airway remodeling.31 In the mouse models of asthma induced by OVA and papain respectively, AMs can mediate the presentation of OVA-related allergic antigen and stimulate the proliferation and differentiation of Th2 cells through the production of Interleukin-1β (IL-1β), Transforming growth factor-β (TGF-β), Granulocyte macrophage-colony stimulating factor (GM-CSF) and other cellular inflammatory factors to regulate Th2 cell and eosinophilic inflammation in asthma.32 RNA sequencing of purified AMs in these two types of OVA- and papain-induced asthmatic mouse models revealed that the E3 ubiquitin ligase AMFR was upregulated in both types of asthmatic mice.24 AMFR, an endoplasmic reticulum resident E3 ubiquitination enzyme, recognizes misfolded proteins for ubiquitination and subsequent proteasome degradation.33 AMFR can positively regulate the production of GM-CSF by targeting Cytokine Inducible SH2-containing protein (CIS) and facilitating its ubiquitination degradation in UPS.24 Thus, the overproduction of GM-CSF will drive the aggregation of eosinophils and the proliferation and differentiation of Th2 cells in asthma, intensifying the airway inflammation.24 These studies demonstrate a new mechanism of communication between AMs and Th2 cells and eosinophils in the context of OVA- and papain-induced asthma, and AMFR selective inhibitors may become novel pharmacological targets for future asthma treatment related to these allergen-induced asthma models.

Cbx4

Cbx4 is a member of the HP1 proteins, comprising 560 amino acids.34 In addition, Cbx4 is the sole enzymatically active member identified within the Cbx family so far. It possesses various distinctive domains and is involved in post-translational modification of substrates such as Homeodomain Interacting Protein Kinase 2 (HIPK2), septin interacting protein 1 (SIP1) and hypoxia-inducing factor-1α (HIF-1α) as a small ubiquitin-like modifier (SUMO) E3 ubiquitination enzyme.35 In a recent study, it was reported that Cbx4 can enhance the deactivation of HIF-1α through interacting with HIF-1α and promote transcription of Interleukin-9 (IL-9), ultimately promoting T helper 9 (Th9) cell differentiation, thereby promoting the progression of asthma.25 This study introduces a novel concept for the clinical management of asthma. Targeting Cbx4 and HIF-1α through SUMO E3 ubiquitin ligase activity inhibitors can effectively alleviate chronic lung inflammation caused by Th9 cell activation. In the context of house dust mite (HDM)-induced asthma, Cbx4 can aggravate epithelial barrier dysfunction by mediating SUMOylation of β-catenin. Knockdown of Cbx4 in vivo in the HDM-induced asthma model promotes membrane localization of β-catenin and inhibits inflammatory Wnt/β-catenin signaling, improving airway epithelial barrier function, and thereby reducing HDM-induced allergic airway inflammation and asthma epithelial barrier dysfunction.36

Midline-1

Midline 1 (MID) is a microtubule binding E3 ubiquitin ligase that is known to be involved in organ development and diseases such as cancer and allergic inflammation.37,38 The occurrence and attack of asthma are often closely related to allergic airway inflammation caused by the activation of innate immune pathways by allergens. In the mouse models of acute asthma attack induced by HDM or rhinovirus infection, MID1 exhibited increased expression in mouse bronchial epithelium.26 MID1 was found to decrease the activity of protein phosphatase 2A (PP2A) by interacting with its catalytic subunit PP2Ac, thereby promoting the expression of airway hyperreactivity and inflammation-related factors such as Interleukin-25 (IL-25), Interleukin-33 (IL-33), and C-C Motif Chemokine Ligand 20 (CCL20), and the release of Interleukin-5 (IL-5) and Interleukin-13 (IL-13) in the HDM- and rhinovirus-induced asthma models. Further studies showed that the inhibition of MID1 on PP2A inflammatory signaling pathway was regulated by upstream TNF-related apoptosis-inducing ligand (TRAIL), and TRAIL promoted asthma by up-regulating MID1. This gives us a more complete understanding of how MID1 functions in asthma.2 In addition, it can promote the accumulation of eosinophils, T lymphocytes and dendritic cells, further promoting the development of HDM- and rhinovirus-induced asthma.26 Specifically inhibiting MID1 or activating PP2A pharmacologically can limit the progression of allergic airway disease caused by rhinovirus or HDM.26 These findings identify the key role and certain action sites of MID1 in allergic airway inflammation such as HDM- and rhinovirus-induced asthma and establish a bridge between the development of asthma and the activation of immune pathways in the context of these allergen-induced asthma models.

Parkin

Parkin is a cytoplasmic E3 ubiquitination enzyme expressed in the airway epithelium.39 Parkin can ubiquitinate and activate the pro-inflammatory nuclear factor kappa-light-chain-enhancer (NF-κB) of activated B cells.40 Parkin also promotes lipopolysaccharide (LPS)-induced lung inflammation such as lung injury by inhibiting p53 and promoting the activation of NF-κB.41,42 Under high Interferon-γ (IFN-γ) environment, IFN-γ induced Parkin to promote the production of neutrophil chemokines (LIX and IL-8) and airway neutrophilic inflammation by decreasing the expression of Parkin inhibitor Thapp11.27 These studies predict an important role for Parkin as a proinflammatory factor in pulmonary inflammation. In Kris genelyn Dimasuay’s study of the asthmatic airway, it was found that the mRNA levels of Parkin were significantly up-regulated in the HDM-induced asthmatic airway epithelium, which was positively correlated with mitochondrial DNA (mtDNA) release in bronchoalveolar lavage fluid (BALF).27 Earlier reports have demonstrated mitochondrial dysfunction in mouse models of allergic asthma28,29 The pro-inflammatory factor, IL-13, only induces mtDNA release in Parkin sufficient human tracheobronchial epithelial (HTBE) cells to aggravate mitochondrial damage but not in Parkin deficient conditions,27 while Kris genelyn Dimasuay’s study suggested that Parkin could mediate the exacerbation of airway inflammation as an E3 ubiquitin ligase induced by mtDNA release in asthmatic airway epithelium. However, the exact role of Parkin in mitochondrial dysfunction in asthma has not been fully elucidated.

TRIM27

The expression of tripartite motif-containing 27 (TRIM27) has been found to exacerbate airway hyperresponsiveness (AHR) and the pathological changes of lung tissue, as well as to significantly increase airway inflammation and oxidative stress in asthmatic mice.30 TRIM27 knockdown effectively alleviated OVA-induced airway hyperresponsiveness (AHR) and lung pathological changes.30 In addition, TRIM27 knockdown significantly reduced airway inflammation and oxidative stress in asthmatic mice, and in vitro analysis confirmed the favorable effects of TRIM27 deletion on inflammation and oxidative stress in mouse airway epithelial cells.30 Further studies found that the loss of TRIM27 significantly reduced the activation of NOD-like receptor protein 3 (NLRP3) inflammasome, which provided ideas for exploring the pathological mechanism of TRIM27 promoting asthma.30

E3 Ubiquitin Ligases Inhibit the Development and Progression of Asthma

Most of the reported E3 enzymes can inhibit asthma pathogenesis rather than promote asthma progression shown in Figure 2 and Table 2, which may be due to the degradation of key substrates in the pathogenesis and progression of asthma by E3 enzyme-mediated ubiquitination. The mechanisms are discussed in more detail below.

Figure 2 E3 ubiquitin ligases inhibit asthma pathology. The E3 enzyme F-box and leucine-rich repeat protein 19 (FBXL19) plays a pivotal role in orchestrating the ubiquitinating degradation of ST2L, thereby effectively suppressing Interleukin-8 (IL-8) secretion. Concurrently, Tripartite -motif protein 21 (TRIM21) acts as an essential mediator in the ubiquitinating degradation of Transient Receptor Potential Cation Channel Subfamily M Member 2 (TRPM2), leading to the inhibition of inflammatory cytokine secretion and macrophage production. Notably, E3 enzymes peroxisome proliferators-activated receptor γ (PPARγ), Casitas B lymphoma-b (Cbl-b), and Cullin-5 (CUL5) collaborate to mediate the ubiquitinating degradation of Signal Transducer and Activator of Transcription 6 (STAT6). The activation of the PPARγ signaling pathway results in the downregulation of IgE secretion, while Cbl-b and CUL5contribute to the reduction of T helper 2 (Th2) cell differentiation. ITCH, another crucial E3 enzyme, exerts its influence by facilitating the ubiquitinating degradation of both STAT6 and GATA Binding Protein 3 (GATA3). This dual degradation process leads to a decrease in Interleukin-4 (IL-4) secretion and Th2 differentiation. Furthermore, the E3 enzyme Cullin 4b (CUL4B) intervenes in the ubiquitinating degradation of H2Ak19, effectively inhibiting Th2 cell differentiation. Additionally, F-box and WD repeat domain-containing 7 (FBW7), acting as an E3 enzyme, engages in the ubiquitinating degradation of GATA Binding Protein (GATA), thereby suppressing Th2 cell differentiation and eosinophil production. The regulatory protein, An E3 ubiquitin ligase gene related to anergy in lymphocytes (GRAIL) assumes a central role in mediating the ubiquitinating degradation of CUL5. This action promotes Interleukin-2 (IL-2) activation mediated by phosphorylated Janus kinase (pJAK1), ultimately enhancing Treg activation and, consequently, inhibiting inflammation. The E3 enzyme Suppressor of cytokine signaling (SOCS1) plays a crucial role in mediating the ubiquitinating degradation of Insulin Receptor Substrate 2 (IRS-2), leading to a reduction in phosphorylated IRS-2 levels and ultimately inhibiting M2 macrophage polarization. Moreover, the E3 enzyme PARK exerts inhibitory effects on the secretion of inflammatory cytokines Interleukin-1β (IL-1β) and Interleukin-13 (IL-13), mediated by House Dust Mite (HDM). This inhibition is achieved through the ubiquitinating degradation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3). Collectively, these intricate molecular interactions culminate in the inhibition of inflammation, contributing to the attenuation of asthma’s course and progression.

Abbreviations: FBXL19, F-box and leucine-rich repeat protein 19; IL-8, Interleukin-8; TRIM21, Tripartite -motif protein 21; TRPM2, Transient Receptor Potential Cation Channel Subfamily M Member 2; PPARγ, peroxisome proliferators-activated receptor γ; Cbl-b, Casitas B lymphoma-b; CUL5, Cullin-5; STAT6, Signal Transducer and Activator of Transcription 6; Th2, T helper 2; GATA3, GATA Binding Protein 3; IL-4, Interleukin-4; CUL4B, Cullin 4b; FBW7, F-box and WD repeat domain-containing 7; GATA, GATA Binding Protein; GRAIL, An E3 ubiquitin ligase gene related to anergy in lymphocyte; IL-2, Interleukin-2; pJAK1, phosphorylated Janus kinase; SOCS1, Suppressor of cytokine signaling; IRS-2, Insulin Receptor Substrate 2; IL-1β, Interleukin-1β; IL-13, Interleukin-13; HDM, House Dust Mite; NLRP3, NOD-like receptor thermal protein domain associated protein 3.

Table 2 E3 Ubiquitin Ligases suppressing the progression of Asthma

FBXL19

The Skp1-Cullin-1-F-box protein (SCF) ligase complex is one of the largest families of E3 ubiquitin ligases,59 which is involved in the ubiquitination process. In this complex, the F-box contains two main domains for substrate recognition. The F-box motif binds to S-phase kinase-associated protein 1 (Skp1) to create the SCF ligase complex. Another substrate-binding motif recognizes and interacts with phosphorylated substrates.60 The F-box protein, F-Box And Leucine Rich Repeat Protein 19 (FBXL19), has been shown to share considerable sequence similarity to the SCF protein family.61

In a study using ovalbumin (OVA)-induced asthma mouse models, investigators first applied MG-132 proteasome or lysosomal inhibitors to mice lung epithelial cells MLE12. They found that only MG-132 could attenuate Suppression of Tumorigenicity 2 (ST2L) degradation, but not the lysosomal inhibitors. Moreover, ST2L was found to be polyubiquitinated via co-IP assays.43 These results indicated that ST2L degradation in lung epithelial cell lines was mediated by the ubiquitin-proteasome machinery. When FBXL19 selectively mediated the ubiquitination and degradation of ST2L, the expression of pro-inflammatory factor IL-33 is also decreased, and IL-33-induced pulmonary inflammation was alleviated. Overexpression of FBXL19 eliminated the pro-inflammatory and pro-apoptotic effects of IL-33 and effectively alleviate the severity of lung injury in OVA-induced asthmatic mice.43 This study demonstrated that targeting the IL-33-ST2L axis via the E3 ubiquitin ligase FBXL19 was a potential strategy to alleviate OVA-induced asthma.

TRIM Family Proteins

TRIM proteins are one of the largest families of E3 ubiquitinating enzymes, which consists of more than 80 proteins. Tripartite motif-containing 21 (TRIM21, Ro52), as a member of the TRIM family, contains PRY and SPRY domains at the c-terminus.62 As a common E3 ubiquitin ligase, TRIM21 holds significance in pathogenesis and progression of inflammation,63,64 cancer,65,66 and autoimmunity.67

In the immune microenvironment, macrophages play crucial roles in asthma pathogenesis and development.68,69 In OVA-induced asthma mouse models and asthmatic patients, the number of alveolar macrophages is increased, and the level of alternative activation (M2) polarization of macrophages is also increased.70 Recent studies have found that TRIM21 can interact with transient receptor potential cation channel, subfamily M, member 2 (TRPM2) protein, an encoded protein is activated by oxidative stress and stressed susceptibility to cell death.44,71 The interaction between TRIM21 and TRPM2 will promote the apoptosis of pro-inflammatory macrophages and inhibit the production of inflammatory cytokines such as Interleukin-1β (IL-1β), IL-4, IL-6, IL-10, TNF-α, and TGF-β, thus alleviating asthma symptoms.44 The specific mechanism is that TRIM21 relies on the key site TRPM2 K1218 to degrade TRPM2 via ubiquitination, thereby reducing intracellular calcium, Reactive oxygen species (ROS) levels and the production of inflammatory factors, thus promoting the apoptosis of macrophages. The long non-coding RNA lncTRPM2-AS blocks this ubiquitination process and exacerbates macrophage inflammation in OVA-induced asthma models.44

Recently, with the deepening of the research on TRIM family, more and more TRIM proteins have been found to play a significant role in the pathological mechanism of asthma. TRIM31 expression can alleviate the pathological changes of OVA-induced asthma and reduce the infiltration of inflammatory cells.45 TRIM31 deficiency exacerbates NLRP3 inflammasome activation in OVA-induced asthmatic mice and HDM-stimulated airway epithelial cells.45 In this extensive TRIM family, distinct proteins play varied roles in the pathological mechanism of asthma. Further exploration of the TRIM family will facilitate a more comprehensive understanding of the pathological mechanism of asthma.

PPARγ

Peroxisome proliferator-activated receptor-γ (PPAR-γ), first identified in rodents 30 years ago, belonged to the recipient family of nuclear hormones, acting on fat metabolism, insulin sensitivity, and glucose stability.72 Recent studies indicate that PPARγ can function as an E3 ubiquitin ligase. In OVA-induced asthma models, PPARγ binds to the phosphorylated signal transducer and activator of transcription 6 (STAT6) and initiates its ubiquitination and degradation, thereby inhibiting the synthesis of serum IgE downstream.47 It is well known that most asthma cases are linked to IgE-mediated responses, and elevated serum IgE levels are one of the hallmarks of allergic asthma.73 PPARγ-induced downregulation of IgE would alleviate symptoms associated with allergic asthma. Additionally, a signaling pathway upstream of PPAR-γ was also explored in this study. The researchers found that serum IgE levels were significantly increased in E-prostanoid 4 (EP4) receptor deficient mice, and bioinformatics analysis showed that the inhibitory effect of EP4 signaling on IgE was dependent on activation of the PI3K-AKT pathway.47 In conclusion, the EP4-PI3K-AKT-PPARγ-STAT6-IgE pathway significantly contributes to the induction and pathogenesis of OVA-induced allergic asthma.

ITCH

The E3 ubiquitin ligase ITCH was named based on the genetic analysis of mutant mice showing abnormal immune phenotypes and severe skin scratching.74 ITCH interacts with signal molecules like Jun protein, Smad2, Notch, and p73, contributing significantly to DNA damage response and cell cycle regulation.74 Mutation in the ITCH gene is a cause of syndromic multisystem autoimmune diseases.75 Allergic asthma is a complex inflammatory disease, which is a type II immune disease characterized by the increased number of TH2 cell-mediated inflammatory factors IL-4, IL-5, IL-13, and eosinophils.76

Itch−/− mice have increased Th2-type inflammation in the lungs and digestive tract.74 To explore the role of ITCH in Treg cells, researchers generated Treg-specific ITCH knockout mice and challenged them with OVA in an allergic asthma model. They found that ITCH abolished Treg-specific mice had more severe lung inflammation than control mice, with specific IgE and Th2 cytokines were significantly increased.48 With further study, ITCH-deficient Treg cells have Th2 properties and produce Interleukin-4 (IL-4) to guide the development of Th2 inflammatory responses.48 GATA binding protein 3 (GATA3) expression in Tregs is essential for the maintenance of Treg suppressive function and stability.77,78 However, the molecular pathway through which ITCH selectively inhibits GATA3-mediated Th2 response is crucial for controlling the progression of allergic asthma.48 In addition, ITCH has been shown to inhibit inflammatory signaling via the nucleotide binding oligomerization domain containing 2 (NOD2) pathway through ubiquitination and degradation of inhibitor of apoptosis proteins (IAP), especially cIAP1.79 Furthermore, ITCH has been demonstrated to inhibit asthma, Crohn’s disease, sarcoidosis and other inflammatory diseases that are highly associated with the NOD2 signaling pathway.80,81

Cbl-b

Casitas B lineage lymphoma b (Cbl-b) is an E3 ubiquitin ligase containing multiple domains such as the protein tyrosine kinase binding (TKB) domain, RING-finger domain, and proline-rich domain.82 These domains are required for the Cbl-b protein to recruit ubiquitin-binding enzymes, recognize ubiquitin-coupled target proteins and degrade proteins. In addition, Cbl-b is involved in T cell differentiation, B cell antigen receptor signaling, and peripheral tolerance regulation.83,84 Cbl-b was known to affect immune/anergy switching points at multiple levels.85

In OVA-induced allergic asthma mouse models, compared to the normal mice, the Cbl-b−/− allergic asthma mice had more severe inflammatory cell infiltration in the blood vessels and around the bronchus and increased mucus secretion. In addition, respiratory resistance responses to methylamine choline (methch) aerosols showed that Cbl-b−/− mice remained highly responsive to methyl groups 24 hours after OVA attack, indicating that severe airway inflammation in Cbl-b−/− mice led to increased airway hyperreactivity (AHR), which is a classic sign of asthma.49 Cbl-b deficiency led to elevated IL-4, IL-5, IL-9, and IL-13 production by T cells in vitro and in the bronchoalveolar of mice in vivo. Higher levels of these cytokines in lavage compared to the WT group suggested that Cbl-b negatively regulates the differentiation of Th2 and Th9 cells.49 Further investigation showed that Cbl-b could target STAT6 for ubiquitination degradation, thereby inhibiting Th2 reaction and IL-4-STAT6 signal transduction.49

CUL Family

Cullin-RING E3 ubiquitin ligases (CRLs), the largest family of E3 enzymes, are responsible for the post-translational modification of nearly 20% of intracellular proteins and controlling numerous important cellular physiological and biochemical functions.86 However, Cullin5 (CUL5) is a scaffold protein nucleating CRLs complex.87 Nedd8, a ubiquitin-like protein, covalently attaches to CUL5 to activate its E3 ubiquitin ligase activity.88 The Suppressor of cytokine signaling (SOCS) box domain in the SOCS protein allows it to act as a substrate receptor for the CRL5 complex, facilitating ubiquitination of the substrate.89 It was demonstrated in a previous study that a cytokine induced CIS, a member of the SOCS protein family, restricts Th2 and Th9 differentiation, as well as lung inflammation in OVA-induced experimental asthma.90 Mice with low CUL5 expression in T cells exhibit Th2 inflammation that will further worsen with age.50 CUL5-deficient T cells show a propensity for Th2 and Th9 differentiation and heightened STAT6 inflammatory signaling.50 The specific mechanism is that CUL5 forms a complex with CIS and phospho-Jak1 to promote the ubiquitination and degradation of phospho-Jak1. CUL5 deletion will inhibit this process and increase the intracellular levels of phospho-Jak1 and phospho-Stat6, thereby reducing the threshold of IL-4 receptor signaling.50 Thus, these CUL5-deficient CD4+T cells can differentiate from Treg to Th9 even under conditions of low levels of IL-4, thereby increasing susceptibility to OVA-induced allergic asthma.50 In addition, a recent study showed that in a mouse model, preexisting allergic insult induces CUL5 expression, impairs antiviral immunity and promotes neutrophil inflammation that exacerbates asthma.91

Like CUL5, Cullin 4B (CUL4B) is also a scaffold protein of its corresponding complex CRL4B, which plays an important role in various physiological, biochemical, and developmental degradations.92 Disruption of the CUL4B gene results in X-linked mental retardation in humans and severely inhibits a range of developmental processes such as cell proliferation, hematopoiesis, and neurogenesis in mice.93–96 In addition, CUL4B also plays a key role in Th cell differentiation.51 In OVA-induced mouse models of asthma, CUL4B-deficient mice have a more severe Th2 response than controls.51 CUL4B depletion enhances CD4+ T cell differentiation into Th1 and Th2 cells in vitro.51 In addition to classical transcription factors, several epigenetic factors have also emerged as key regulators in CD4+T cell differentiation.97,98 For example, IL-4 in Th1 cells and Ifng in Th2 cells are marked by the repressive mark trimethylated histone H3 at lysine 27 (H3K27me3).99 Polycom repression complex 2 (PRC2) plays a key role in catalyzing H3K27 in this process.100 Thus, when PRC2-mediated trimethylation of H3K27me3 is inhibited, CD4+T cells will enhance their differentiation into Th1 and Th2 cells.100 In addition, polycomb repression complex 1 (PRC1) recognizes H3K27me3 established by PRC2 to enhance PRC2 action, and PRC1 catalyzes H2AK119ub1, which is essential for the efficient recruitment and activity of PRC2 on its target genes.101 CRL4B during this series of compounds by promoting H2AK119 single ubiquitin (H2AK119ub1) and PRC2 mediated H3K27me3 three methylation, which inhibits their expression in the process of Th cell differentiation.51

GRAIL

Allergic asthma can develop due to defects in peripheral regulatory T cells (Tregs).102 In previous studies, the use of low-dose Interleukin-2 (IL-2) has been reported to successfully treat autoimmune diseases caused by Tregs.103,104 Researchers have isolated equal numbers of Tregs from patients with allergic asthma and patients with autoimmune diseases and compared their responses to low doses of IL-2 in vitro. The results showed that it is not a defect in Treg numbers that influences disease development but that Tregs in these patients lose their suppressive regulatory role in IL-2R desensitization.105 In this process, Tregs from healthy people can inhibit IL-2R desensitization and prolong the sustained expression of key factors (eg pSTAT5 and Deptor) of its downstream pathways, thereby maintaining Treg stability by promoting STAT5 transcription as well as mTOR inhibition.106–108 However, when activated by IL-2, CRL degradation of pJAK1 and Deptor associated with the IL-2Rβ chain occurs, and sustained expression of pSTAT5 and Deptor is suppressed, resulting in a loss of Treg stability.105

An E3 ubiquitin ligase gene related to anergy in lymphocytes (GRAIL) was abundantly expressed in healthy controls.52 GRAIL can ubiquitinate the lysine (K724) on CUL5 protein, which is required for CRL activation to degrade Deptor and pJAK1 by SUMOylating.52,108 Therefore, GRAIL can be used as a competitive inhibitor of this SUMOylating modification to block the activation of CUL5 CRLs complex, thereby preserving the protein activity and expression of pJAK1 and Deptor.50,87,109 At this point, the treatment idea for allergic asthma can be transformed from immunosuppression to self-tolerance recovery.

FBW7

Allergic asthma involves persistent eosinophilic airway inflammation due to Th2 cells released-cytokines like IL-4 and IL-5.110 Therefore, inhibiting the development of Th2 cells and the differentiation of CD4+ T cells into Th2 cells are beneficial measures to treat or relieve the symptoms of allergic asthma. However, the classical JAK/STAT6 signal transduction pathway is closely linked to T cell development, Th2 differentiation, and the regulation of Th1/Th2 balance.111,112 The mechanism is that activated STAT6 initiates transcription in the downstream region of Th2-specific genes by up-regulating GATA3 to induce the functional differentiation of CD4+ T cells into Th2 cells.113 The E3 ubiquitin ligase F-box and WD repeat domain-containing 7 (FBW7), part of the F-box protein family, plays a crucial role in tumor development.114,115 In addition, FBW7 can regulate the ubiquitination of GATA3, thereby disrupting its structure, and conditionally inactivating FBW7 in the T cell lineage, which results in the reduction of thymic CD4 single-positive cells and splenic CD4+ and CD8+ T cells.53,54 Therefore, enhancing FBW7 function can effectively inhibit Th2 cell development and prevent the development of allergic asthma. This conclusion has been demonstrated experimentally. Ken-Ichi Suehiro’s group found that SRY-Box Transcription Factor 12 (Sox12−/−) mice showed increased eosinophil infiltration into the lungs and exacerbated Th2 cell differentiation in response to HDM.55 Moreover, Sox12 enhanced FBW7-mediated ubiquitination of GATA3, a process demonstrated by the elimination of Sox12 repression of GATA3 upon FBW7 knockdown.55

SOCS1

SOCS family proteins can act as inhibitory signals in a negative feedback loop in the JAK-STAT pathway.116 These proteins feature a central SH2 domain and a C-terminal SOCS box domain and interact with RING finger proteins, Culin proteins, and elongin B and C to exert E3 ubiquitinating enzyme activity.89,117–120 As one of the most potent signaling suppressors of the SOCS family, SOCS1 can regulate the polarization of the M2 phenotype.121 Numerous studies in asthmatic patients have shown that the presence of M2 macrophages in the lungs and airways correlates with the severity of pneumonia as well as poor lung function.121,122 Inhibiting the polarization of M2 macrophages will be the key to reducing allergic pulmonary inflammation. In macrophages, IL-4 activates insulin receptor substrate (IRS)-2 upon binding to its corresponding IL-4 receptor, thereby inducing differentiation of M2 macrophages.123–125 SOCS1 was found to be highly induced in human monocytes upon IL-4 receptor activation, and knockdown of SOCS1 by siRNA resulted in extended IRS-2 tyrosine phosphorylation and heightened M2 differentiation.56 In addition, healthy monocytes with higher SOCS1 content showed higher IRS-2 ubiquitination and lower M2 polarization than allergic monocytes upon IL-4 stimulation.56 In this process, SOCS1 shortens IRS-2 tyrosine phosphorylation by promoting ubiquitination of IRS-2, which in turn inhibits the transduction of downstream signals and ultimately inhibits M2 differentiation.56

RNF125

RING finger protein 125 (RNF125) is an E3 ubiquitin ligase in the RING domain family,126 interacting with the high mobility group (HMG) B-box domain of high mobility group box 1 protein (HMGB1) and degrade it through UPS system, thereby inhibiting autophagy and oxidative stress in airway epithelium and alleviating the progression of asthma.57 RNF125 expression was significantly decreased in asthmatic patients and mice, and further studies showed that RNF125 hypermethylation was the cause of RNF125 low expression in primary airway epithelial cells of OVA-treated mice.57 Therefore, demethylation therapy targeting RNF125 may be one of the methods for precision treatment of OVA-induced asthma in the future.

PARK2

As an E3 ubiquitin ligase, PARK2 is involved in various cellular processes through ubiquitination degradation, and research on PARK2 has mainly focused on Parkinson’s disease.127 Ge and his team used HDM to induce airway epithelial cells BEAS-2B to mimic allergic asthma in vitro58 and found that the expression of PARK2 is significantly down-regulated in HDM-induced asthma models, which can effectively alleviate asthma inflammation.58 The specific mechanism by which PARK2 exerts its effects is as follows: it promotes the ubiquitination of NLRP3 inflammasome, negatively regulates NLRP3 protein, suppresses NLRP3 inflammasome activation, and reduces the secretion and release of pro-inflammatory factors IL-1β and IL-18. This ultimately protects the airway epithelial cell barrier.58 PARK2 will eventually relieve the symptoms of HDM-induced allergic asthma.

Deubiquitinases and Their Mechanisms Involved in Asthma Pathology

The reported studies on the effects of deubiquitinases like Ubiquitin-specific protease (USP) family, A20, Cylindromatosis gene (CYLD), ovarian tumor domain protease domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) and BRCA1/BRCA2-containing complex subunit 3 (BRCC3) on asthma pathology exhibit both pro- and anti-asthmatic properties, as depicted in Figure 3 and Table 3. The mechanisms of action of these deubiquitinases will be explained in more detail below.

Figure 3 Deubiquitinases functioning in asthma pathology. The deubiquitinating enzyme Ubiquitin specific protease 10 (USP10) can inhibit the degradation of T-bet by removing the ubiquitin residues connected to T-bet, thereby promoting the proliferation and differentiation of T helper 1 (Th1) cells, and ultimately promoting the development of airway inflammation. Similarly, Ubiquitin specific protease 38 (USP38) promotes T helper 2 (Th2) cell proliferation and differentiation by inhibiting JunB ubiquitination and degradation. Ubiquitin specific protease (USP4) can promote the development of airway inflammation by promoting the proliferation of Th2 and T helper 17 (Th17) cells and inhibiting the proliferation of Treg cells. Ubiquitin specific protease 25 (USP25) inhibits airway inflammation by promoting BRCA1 Associated RING Domain 1 (BARD1), which in turn inhibits the secretion of inflammatory factors Interleukin-13 (IL-13), Tumor Necrosis Factor (TNF-α), Interleukin-4 (IL-4) and Interleukin-8 (IL-8). Ubiquitin specific protease 21 (USP21) can inhibit the ubiquitination of GATA Binding Protein 3 (GATA3), thereby increasing the expression of GATA3, promoting the function of Treg, and ultimately inhibiting airway inflammation. A20, as a common deubiquitinating enzyme, can inhibit the expression of GATA3, thereby inhibiting the proliferation and differentiation of Th2 cells, and then inhibiting airway inflammation. Cylindromatosis (CYLD) can inhibit the ubiquitination and degradation of Nuclear Factor Kappa-light-chain-enhancer (NF-κB), thereby activating the NF-κB inflammatory pathway and promoting airway inflammation. OTU domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) promotes the formation of inflammasome by inhibiting the ubiquitination and degradation of Tumor necrosis factor Receptor-Associated Factors (TRAF3), which ultimately promotes airway inflammation. BRCA1/BRCA2-containing complex subunit 3 (BRCC3) inhibits the activation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome by reducing the ubiquitination level of NLRP3, thereby reducing airway inflammation in asthma.

Abbreviations: USP10, Ubiquitin specific protease 10; Th1, T helper 1; USP38, Ubiquitin specific protease 38; Th2, T helper 2; USP4, Ubiquitin specific protease; Th17, T helper 17; USP25, Ubiquitin specific protease 25; BARD1, BRCA1 Associated RING Domain 1; IL-13, Interleukin-13; TNF-α, Tumor Necrosis Factor; IL-4, Interleukin-4; IL-8, Interleukin-8; USP21, Ubiquitin specific protease 21; GATA3, GATA Binding Protein 3; CYLD, Cylindromatosis; NF-κB, Nuclear Factor Kappa-light-chain-enhancer; OTUB1, OTU domain-containing ubiquitin aldehyde-binding protein 1; TRAF3, Tumor necrosis factor Receptor-Associated Factors; BRCC3, BRCA1/BRCA2-containing complex subunit 3; NLRP3, NOD-like receptor protein 3.

Table 3 Deubiquitinases Functioning in Asthma

Ubiquitin Specific Protease Family

Ubiquitin-specific proteases (USPs) are the largest subfamily of deubiquitinating enzymes (DUBs) with 58 vertebrate members.141 USPs are cysteine proteases with three parts of the USP conservation region called the finger, thumb, and palm.142,143 To endow USPs with substrate specificity, not only the terminal extension, but also the ubiquitous associated domain (UBA), ubiquitous interacting motif (UIM) and zinc finger ubiquitous specific protease domain (ZnF-UBP) are included.142,144 USPs play a crucial role in cancer development by regulating the cell cycle and aiding in DNA damage repair.145,146 In addition to their roles in the regulation of cancer, USPs play important roles in some metabolic diseases such as obesity, diabetes, and atherosclerosis.147–149 Nowadays, the effects of USPs on asthma have also been studied increasingly. In this part of the review, we will summarize the role of USPs in asthma pathogenesis.

USP10

T-box expressed in T cells (T-bet), a transcription factor, governs the development and differentiation of naive CD4+ T cells into Th1 cells.150 It can drive Th1 immune responses by promoting the expression of IFN-γ, a hallmark cytokine of Th1 cells.151 The endogenous co-immunoprecipitation assay demonstrated the interaction between the deubiquitinating enzyme USP10 and T-bet, while their co-localization in the nucleus were confirmed by immunofluorescence assay after transfection with HA-T-bet and Myc-USP10.128 Specifically, USP10 promotes the deubiquitination of T-bet via its enzymatic activity, thereby preventing its post-ubiquitination degradation and enhancing the stability.128 Lys-313 has been reported to be a key site for the interaction of T-bet with the IFN-γ gene promoter, and the expression of T-bet is controlled by the ubiquitin-proteasome degradation pathway at the Lys-313 site.128 This provides an idea for the exploration of the interaction site between USP10 and T-bet, but the specific mechanism remains unknown. In addition, the expression of USP10 and IFN-γ and the transcriptional level of T-bet are highly up-regulated in peripheral blood mononuclear cells (PBMCS) from asthma patients, suggesting that USP10 may maintain high levels of T-bet and IFN-γ, promote Th1 responses to combat Th2-dominated asthma.128 In this study, the provenance of the samples from asthma patients was not specified with regard to the particular allergen exposure situation. However, it is speculated that the samples may in fact represent a group of asthma patients induced by various allergens.

USP38

Th2 cells, by producing signature cytokines IL-4 and IL-5, play a pivotal role in allergic asthma pathogenesis.110,152 USP38 can be induced by T-cell receptor (TCR) activation, and genome-wide association studies have indicated its presence at chromosomal loci associated with human asthma.153 In USP38-deficient allergic asthma models induced by OVA and aluminum, the total number of cells, eosinophils, and lymphocytes in BALF were significantly decreased, as well as the percentage and absolute count of Th2 cells in mediastinal lymph nodes detected by flow cytometry.129 In addition, depletion of USP38 in HDM-induced allergic asthma has similar results to OVA-induced allergic asthma,129 suggesting that USP38 is an essential regulator of allergic asthma. Mechanistically, USP38 interacts with JunB Proto-Oncogene (JunB) to maintain the stability of JunB by removing polyubiquitination of JunB.129 JunB is a TCR-activated transcription factor that plays a specific role in Th2 development by promoting IL-4 transcription.154 However, some E3 ubiquitin ligases such as ITCH participate in JunB ubiquitination and degradation, thereby disrupting JunB function in inflammatory diseases.155,156 USP38 deubiquitinates Lys-48-linked JunB polyubiquitylation, thereby blocking TCR-induced JunB turnover.129 USP38 deubiquitinase is a specific site that mediates Th2 immunity and related asthma, which provides a target for the precise diagnosis and treatment of asthma in the future.

In these two models, the researchers induced asthma with specific allergens (OVA or HDM) to simulate the pathogenesis of allergic asthma and systematically detected the changes in related cytokines, cell numbers, and transcription factors to explore the role of USP38.

USP4

As a deubiquitinase, USP4 functions as a key regulator in various cellular pathways. USP4 can inhibit the tumor suppressor effect of p53, thus acting as a potential oncogene.157 In addition to its role in cancer development, USP4 also plays an important role in autoimmune diseases, and the USP4 inhibitor Vialinin A is an important anti-inflammatory compound.158,159 In vitro, USP4 can act as a key regulator of Treg and Th17 cells, which play important roles in the pathogenesis of chronic asthma.157,160–163 In an OVA-induced mouse model of asthma, USP4 null mice have lower airway hyperresponsiveness and airway inflammation in the lung, as well as reduced production of Th2 and Th17-related cytokines and increased percentage of Forkhead Box P3 (Foxp3+) Treg cells compared with controls.164 Since FoxP3-driven gene expression patterns can largely determine Treg regulatory function,165 USP4 can lead to impaired Treg cell suppressive function by down-regulating Foxp3, thereby promoting further inflammation development. However, the USP4 inhibitor Vialinin A can decrease the inflammatory cell infiltration in the lungs of OVA-induced mice.164 Therefore, USP4 may be a distinct inhibitory target for the treatment of asthma and other chronic airway diseases.

In these studies, the OVA-induced asthma model was used to systematically observe the effects of USP4 knockout or inhibition on asthma-related inflammatory indicators, cytokines, and immune cells, thereby revealing its role and potential therapeutic value in the pathogenesis of asthma.

USP25

In allergic asthma patients, many genotoxic reactive oxygen species and reactive nitrogen species are produced in immune cells in response to allergen exposure such as HDM.166 These RONS can damage biological macromolecules such as nucleic acids and proteins, thus further aggravating the deterioration of asthma patients.167 Among them, DNA double-strand breaks are one of the most cytotoxic forms, which can lead to genomic instability and even cell death.168 It has recently been shown that USP25 can promote the stability of DNA repair-related proteins in lung epithelial cells of patients with lung lesions due to frequent smoking.130 In an in vitro allergic asthma model of HDM-induced BEAS-2B cells, USP25 inhibited HDM-induced DNA damage and reduced the production of pro-inflammatory cytokines TNF-α, IL-4, IL-8, and IL-13, while knockdown of USP25 had the opposite effect. USP25 can inhibit HDM-induced DNA damage and inflammation in vivo by enhancing BARD1 protein expression.131 BARD1 is one of the key proteins in the process of homologous recombination to repair DNA damage, and its loss will lead to genomic instability.132

The studies above focused on the HDM-induced asthma model. Both in vitro cell experiments and in vivo animal experiments were carried out in the context of HDM, a specific allergen, to thoroughly investigate the role of USP25 in mitigating HDM-induced asthma-related damage (such as DNA damage and inflammatory response) and its relationship with DNA repair-related proteins.

USP21

Treg cells play an important role in asthma disease, and their deficiency will lead to allergic inflammation in mice, which in turn will trigger the pathological features of asthma.169 As the severity of asthma increases, the proportion of Treg cells in patients decreases significantly.170 The E3 deubiquitinase USP21 can increase the stability of GATA3 and up-regulate its expression in Treg cells by removing its polyubiquitination.133,134 In another regulatory mechanism, USP21 and the serine/threonine kinase PIM2, which can phosphorylate FOXP3 and thereby activate FOXP3 function, are increased in Treg cells from asthmatic patients.135,171 GATA3 is a key transcription factor that co-works with FOXP3 to attenuate the immune effects of Treg cells, so USP21 suppresses the limiting effect of Treg cells on Th2-type inflammatory responses in asthma by stabilizing them.172,173

The present section of the study is mainly based on clinical observations of asthma patients and related cell mechanism studies. Although the specific allergen-induced situation was not explicitly delineated, it elaborated on the role of USP21 in regulating Treg cell function and asthma inflammatory response within the overall context of asthma, providing an important basis for understanding the immune imbalance in asthma.

USP17

Smoking is one of the most common causes of asthma. Studies on smoking-induced asthma have found that oxidative stress caused by cigarette smoking leads to inactivation of histone deacetylase 2 (HDAC2).174,175 HDAC2 can inhibit the activa