APL are known to activate monocytes, leading to the expression of TF, IL-1β and TNF-α, thus triggering a cascade of thrombosis, inflammation and autoimmune responses, primarily through the NF-κB signaling pathway. Studies have demonstrated that the UPS in monocytes have an obligatory role in regulating pathogenesis of APS (13) ( Table 2 ). Mononuclear cells isolated from patients with the APS exhibit increased expression of caspase-1 and NLRP3 indicating the chronic inflammation activation in APS patients.

The UPS plays a pivotal role in controlling inflammatory signals by modulating the activity of NLRP3 and NF-κB pathway. UBE2L3 (ubiquitin-conjugating enzyme E2 L3), an E2 ubiquitin-conjugating enzyme, is essential for the LUBAC-mediated activation of NF-κB in monocytes (14). The anti-β2GPI/β2GPI complex has been shown to simultaneously and acutely enhance mRNA levels of TRAF6 (TNF receptor associated factor 6) and TRAF4. It has been demonstrated that TRAFs, which possess E3 ligase activity, play a role in the activation of the NLRP3 inflammasome, which is crucial for regulating the TLR4/TF signaling pathway in activated THP-1 cells (15). The TRIM (tripartite motif) proteins are a versatile family of E3 ligases, are composed of over 80 distinct members in human and are recognized for their roles in antiviral responses. Numerous studies have demonstrated the regulatory roles of TRIM proteins in mediating inflammation (16). TRIM65 has the ability to mediate NLRP3 ubiquitination, thereby inhibiting caspase-1 activation and IL-1β secretion, which negatively regulates inflammatory responses in monocytes (17). J. S. Bednash et al. (18) identified STAMBP (STAM-binding protein), a JAMM (Jab1/MPN domain associated metalloisopeptidase) metalloprotease in the DUBs family, as a negative regulatory factor that helps maintain the inflammatory balance. Cellular depletion of STAMBP increases NLRP3 K63 chain polyubiquitination, resulting in enhanced NLRP3 inflammasome activation. The specific proteasome inhibitor MG132 can attenuate the expression of TRAFs and TF that are induced by anti-β2GPI/β2GPI complex (19). In brief, the UPS plays a significant role in the anti-β2GPI/β2GPI-stimulated TLRs signaling pathway in THP-1 cells and contributes to the pathological processes of APS. These findings provide a robust foundation for the development of targeted therapies for APS.

Neutrophils constitute the majority of white blood cells in peripheral blood, accounting for approximately 50% to 70% of the total, and they play a critical role in immune responses, particularly in the context of APS. Anti-β2GPI can stimulate neutrophils to release NETs (neutrophil extracellular traps), which are extracellular web-like scaffolds of decondensed chromatin adorned with microbicidal proteins such as PAD4 (peptidylarginine deiminase 4) and MPO (myeloperoxidase). NETs have capacity to initiate thrombosis in situ through various mechanisms, including activation of coagulation factors, the endothelium and platelets, thereby exacerbating autoimmune diseases (1). Meanwhile, APS patients experience increased NETs generation accompanied by decreased clearance, leading to acute thrombosis, occlusive vascular disease, and adverse pregnancy outcomes (20).

Research on NETs has primarily focused on their main components and regulatory mechanisms. For instance, ALDH2 (aldehyde dehydrogenase 2) has been found to inhibit NETosis by promoting K48-linked polyubiquitination and degradation of PAD4, thus maintaining innate immune system homeostasis. Although ALDH2 is not a component of the UPS, it can facilitate the binding of PAD4 to the E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70-interacting protein). Pharmacological activation of ALDH2 using Alda-1 has been shown to significantly alleviate septic acute respiratory distress syndrome by inhibiting NETs (21). In vitro studies have demonstrated that the RING (really interesting new gene) finger protein RNF128 (RING finger protein 128) decreases the level of MPO, restrains NF-κB signaling, and reduces the expression of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6, thereby monitoring neutrophil activation (22). A20 is a cytoplasmic zinc finger protein that inhibits NF-κB activity through its deubiquitinating activity, which is vital for limiting inflammation in neutrophils. Research has revealed that mutant A20 cells exhibit heightened inflammatory characteristics, as evidenced by increased levels of IL-1β, IL-9, and IL-17A (23). A20 C103A mutation or A20 rs2230926 polymorphism has been associated with upregulation of PADI4 expression. Consequently, A20 C103A cells display enhanced protein citrullination and NETs formation, thereby exacerbating susceptibility to SLE. Mutations in the A20 locus have been linked to a variety of autoimmune diseases, including RA, psoriasis, SLE, celiac disease, Crohn’s disease and diabetes, likely through the deregulation of the NF-κB pathway (5).

In brief, the role of ubiquitination and neutrophils in the pathogenesis of autoimmune diseases has attracted considerable scholarly attention ( Table 2 ). Although advancements have been achieved, there is an imperative need for further in-depth research to elucidate the intricate interactions between neutrophils and the UPS in APS, which is fundamental to precisely designing targeted treatments and improving the overall prognosis for patients.

Emerging research suggests that the UPS is intricately involved in endothelial barrier function, endothelial activation, cell apoptosis, and autophagy (37). Disruptions in endothelial barrier function not only boost platelet adhesion and aggregation but are also correlated with inflammation in APS. Here we focus on the role of ubiquitin and deubiquitination events in ECs implicated in APS pathophysiology ( Table 2 ).

MARCH3 (membrane associated RING-CH 3), a key enzyme identified through siRNA library screening, is essential for preserving endothelial permeability. MARCH3 silencing in ECs exacerbates cell-cell contacts and disrupts the endothelial barrier, as evidenced by the upregulation of the tight junction-encoding gene OCLN (occludin) (38). Another E3 ligase CHFR (checkpoint with forkhead-associated and RING finger domains), mediates ubiquitylation-dependent degradation of VE-cadherin, which is vital for maintaining vascular integrity and endothelial homeostasis. Both processes involve the activation of the FoxO signaling pathway (38, 39). Additionally, the deubiquitinating enzyme UCHL1 (ubiquitin carboxyl terminal hydrolase L1) has been shown to have a protective effect in models of increased pulmonary vascular permeability. Knockdown of UCHL1 or its pharmacological inhibition LDN results in an increase in vascular leakage both in vitro and in vivo (40). Endothelial barrier compromise results in augmented expression of adhesive molecules and intensified leukocyte adhesion, consequently precipitating an exacerbated inflammatory cascade and the pathogenesis of thrombosis.

Tight regulation of cell death and NF-κB responses in the ECs is important for homeostasis and pathology of the immune system, as demonstrated in genetic mouse models and in patients with APS (20). YAP (yes-associated protein) promotes the ubiquitination and proteasomal clearance of TRAF6 to inhibit the activation of NF-κB signaling. Consuming TRAF6 in ECs can rescue the inflammatory phenotype observed in endothelial-specific YAP knockout mice (41). Another RING-type E3 ligase, FBXW7, is involved in regulating ECs function as well. It recognizes phosphorylation degradation domains on KLF2 (krüppel-like factor 2), leading to KLF2 ubiquitination and subsequent degradation via the 26S proteasome pathway (42). FBXW7 can also degrade KPNA2 (karyopherin subunit alpha 2) through ubiquitination, thereby alleviating endothelial dysfunction and inflammatory responses by inhibiting p65 and interferon regulatory factor 3 (43). TRIM47, a novel ECs modulatory factor, has been shown to augment inflammation and monocyte adhesion when overexpressed, manifested as a significant upregulation in the levels of IL-1β, IL-6, and IL-8 mRNA and a potentiation of ICAM-1 (intercellular cell adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), E-selectin, and MCP-1 (monocyte chemotactic protein-1). Mechanistically, TRIM47 facilitates K63-linked ubiquitination of TRAF2 thus activating NF-κB and MAPK signaling pathways (44).

MDM2 (murine double minute 2) is an E3 ligase containing a RING domain, which acts as a negative regulatory factor of p53 protein. MDM2 binds to p53, inhibiting its interaction with DNA and promoting its monoubiquitination and nuclear export, or facilitating its polyubiquitination and degradation via the proteasome (45). Dysregulated MDM2 is implicated in cardiovascular impairments as well such as atherosclerosis. In endothelial dysfunction and atherosclerosis associated with APS-related features, overexpression of MDM2 exacerbates mitochondrial damage and activates TLR9/NF-κB and NLRP3/caspase-1 pathways (46). MDM2 is a recently identified E3 ubiquitin ligase, regulates endothelial function by modulating mitochondrial energy metabolism. APLs purified from the serum of APS patients with thrombosis and pregnancy complications increase mitochondrial hyperpolarization in ECs, thereby supporting a mechanism by which aPLs induce oxidative stress (47). When mitochondria are damaged, elevated mitochondrial reactive oxygen species contributes to the release of mitochondrial DNA, which in turn activates the TLR9-MyD88-NF-κB pathway, induces inflammasome activation, and exacerbates the inflammatory response (47). To summarize, these findings support the link between the UPS and mitochondrial dysfunction in APS, offering a new perspective on the mechanisms underlying rheumatic diseases.

Anti-β2GPI bind to platelets to promote the release of procoagulant factors, including thromboxane B2, TF, and platelet factor 4, which in turn promote thrombotic manifestations and pregnancy loss. Most importantly, the UPS is a key participant in maintaining platelet homeostasis ( Table 2 ). Resting platelets exhibit extensive ubiquitination, with 1,634 ubiquitylated peptides derived from 691 proteins and over 900 of these peptides showing an increase of more than twofold following stimulation with collagen-related peptide. Multiple sites of ubiquitylation were identified on spleen tyrosine kinase and FcRγ (Fc receptor γ) chain, which are involved in the glycoprotein VI signaling pathway. Collagen-related peptide triggers platelet adhesion and dense granule secretion indicating that ubiquitination plays a role downstream of platelet collagen receptor glycoprotein VI (48). The adapter molecule SHARPIN which is also expressed by human platelets can directly bind to the cytoplasmic tail of αIIbβ3 and promote the activation of the NF-κB pathway as part of the Met-1 LUBAC. Mice with platelet-specific deficiency of SHARPIN exhibit prolonged bleeding time (49). α-subunit KVGFFKR motif exerts a vital influence on the regulation of integrin activation. RNF181, an emerging platelet protein with E3 ligase capabilities, interacts directly with the platelet-specific αIIbβ3 cytoplasmic tail via its highly conserved KVGFFKR regulatory motif, which is implicated in platelet aggregation and thrombotic disorders (50). Deubiquitinating enzymes USP14 and UCHL5, expressed by platelets, play a role in platelet function. Inhibition of deubiquitination reduces platelet adhesion and impairs occlusive thrombosis. More recent next-generation RNA-sequencing and proteomic profiling studies confirmed that platelets express key 20S and 26S proteasomal components, perform chymotrypsin-, trypsin-, and caspase-like proteolytic activities which largely ascribe to the proteasome a regulatory role in platelet production, viability, and function (51).

Individuals with APS frequently present with diminished levels or a dysfunction of antithrombin, a circumstance that can precipitate clot formation (52). Capitalizing on its capacity to bind with AT and enhance anticoagulant efficacy exponentially, heparin is routinely utilized to manage and preempt thrombotic episodes in APS and analogous thrombotic disorders (53). Cullin2 and USP2 have been identified as novel regulators of antithrombin expression. Cullin2 and its interacting partner RBX1 are associated with antithrombin, leading to its ubiquitination and protease-dependent clearance, while USP2 overexpression inhibits this ubiquitination. USP2 inhibitor ML364 is capable of promoting antithrombin degradation (54). The role of the ubiquitin proteasome system in the coagulation cascade still needs further research to elucidate its detailed mechanism.

The downregulation of long non-coding RNA MALAT1 is associated with miscarriage, as well as a reduction in trophoblast cell proliferation and invasiveness. MALAT1 recruits FBXW7 to trigger the ubiquitin-mediated degradation of cyptochrome circadian regulator 2 protein, while overexpression of cyptochrome circadian regulator 2 significantly inhibits trophoblast cell migration and invasion by repressing the MMP2/9 (matrix metalloproteinase 2/9). Scaffold protein CUL1 of FBXW7 ubiquitin ligase complex has the ability to promote the proliferation and migration of trophoblast cells, whose downregulation has been observed in PE (preeclampsia) placental tissue (60) ( Table 2 ). Similarly, Dedicator of cytokinesis 1 is downregulated in placental villi of patients with recurrent spontaneous abortion. DOCK1 participates in regulating DUSP4 (dual specificity phosphatase 4) ubiquitin levels through the E3 ligase HUWE1 (HECT, UBA And WWE domain containing E3 ubiquitin protein ligase 1), and its expression determines the invasive characteristics of EVT (61).

The deubiquitinating enzyme USP7 regulates trophoblast cell functions by interacting with EZH2 (enhancer of zeste homolog 2) and modulating the Wnt/β-catenin signaling pathway. In the villous tissue of women with recurrent miscarriage, decreased USP7 expression is associated with reduced trophoblast cell invasion and migration (62). USP17 is significantly downregulated in PE patients. Overexpression of USP17 promotes proliferation, migration, and invasion of the trophoblast layer. Upregulation of USP17 enhances the level of HDAC2 protein, strengthens the interaction between HDAC2 and signal transducer and activator of transcription 1, resulting in increased STAT1 activity and inhibition of NF-κB signaling (63). Conversely, the expression of USP14 in placental tissue of patients with preeclampsia was significantly upregulated, knocking down or inhibiting USP14 significantly eliminated upregulation of NF-κB activation and production of pro-inflammatory cytokines (TNF-α and IL-1β) (64). In PE, USP22 mRNA was highly elevated and removes ubiquitination on ADAM9 (a disintegrin and metalloprotease 9) and increasing its stability, therefore weakening trophoblast cell proliferation and invasion (65).

Anti-β2GPI antibodies can inhibit the secretion of MMP2 and MMP9 by human trophoblast cells, suggesting essential roles in obstetric APS. Large multifunctional peptidase 2 (LMP2), a proteasome subunit critical for proteasome activity, influences the activity of MMP2 and MMP9, which are zinc-dependent proteases that degrade the extracellular matrix and are implicated in inflammation, trophoblast invasion, and vascular remodeling during pregnancy. Mechanistically, LMP2 contributes to the degradation of NF-κB inhibitor α and the generation of p50. Inhibiting LMP2 can block the transfer of active NF-κB heterodimers into the nucleus, thereby inhibiting the expression and activity of MMP (66).

Upon the invasion of EVT into the decidua, macrophages tend to adopt the M2 phenotype, which is essential for pregnancy maintenance. An imbalance between the M1 and M2 macrophage phenotypes can lead to adverse pregnancy outcomes in APS. Single-cell sequencing transcriptome analysis has revealed that in obstetrics APS patients, macrophages with heightened glycolytic activity are more prevalent within the decidual immune cell population (59). Macrophage polarization is regulated by UPS ( Table 2 ). Notably, S. Wu et al. (67) discovered an abnormal increase in the AOC4P (amine oxidase copper containing 4 pseudogene) of amine oxidase in the villi of RSA (recurrent spontaneous abortion) patients. AOC4P interacts with TRAF6, leading to the upregulation of TRAF6 expression and promoting M1 macrophage polarization, which could be implicated in the pathogenesis of pregnancy morbidity (67). Knockdown of the E3 ubiquitin ligase MARCH7 reduces the ubiquitination levels of NLRP3, which contributes to the subsequent assembly of NLRP3 inflammasome and promotes macrophage polarization toward the M1-type (68). UHRF1 (ubiquitin like with PHD and RING finger domains 1) with E3 ligase function can promote M1 macrophage polarization by activating the MyD88/NF-κB signaling pathway and CXCR2/IL-1R1 receptor, further influencing pregnancy outcomes (55). Interestingly, TGF-β secreted by M2 macrophages, can induce trophoblast migration and invasion, while anti-TGF-β antibodies can mitigate this process (69). TGF-β also upregulates the expression of USP2a in trophoblast cells, interacts with TGFBR1, and promotes the transcription of epithelial-mesenchymal transition-related genes. USP2a activates the PI3K/Akt/GSK3β signaling pathway, facilitating the nuclear translocation of β-catenin and activating EMT in trophoblast cells (69). In conclusion, the UPS plays a significant role in the pathological mechanisms of obstetric APS, partly by influencing macrophage polarization. Targeted alteration of macrophage polarity may be a future therapeutic direction for OAPS.

Low-dose proteasome inhibitors are beneficial for the anti-inflammatory effects on vascular cells both in vitro and in vivo, and these effects are primarily attributed to the suppression of NF-κB activation. Researchers found that after incubating HUVECs with low concentrations of proteasome inhibitors MG132 (70nM) or MG262 (4nM) for 24 hours, this results in a reduction of reactive oxygen species and VCAM-1 production, consequently diminishing monocyte-HUVEC adhesion. Furthermore, low-dose proteasome inhibitors enhance the expression and activity of eNOS, thereby improving endothelial function. The use of proteasome inhibitors can significantly reduce IgG-APS-induced thrombus size in mice and inhibit TF upregulation in peritoneal macrophages and carotid artery homogenate (72). Bortezomib’s therapeutic potential extends beyond oncology, as evidenced by its capacity to exert beneficial anti-inflammatory and antithrombotic effects and mitigate the progression of atherosclerotic lesions in a murine model with low-density lipoprotein receptor deficiency when administered at low doses. The anti-inflammatory and antithrombotic properties of proteasome inhibitors could be harnessed for the treatment of APS (73).

Ubiquitin-activating enzyme E1 is located at the peak of the ubiquitin cascade reaction, and targeting E1 could influence subsequent enzymatic steps. PYR-41 is an irreversible, cell permeable inhibitor of ubiquitin-activating enzyme E1 UBA1 (ubiquitin-activating enzyme E1), which has been reported to inhibit the expression of IgE, IFN-γ and also reduced ear thickening and skin damage in mouse models of atopic dermatitis, associated with the inhibition of the NF-κB signaling pathway (74). The ubiquitin binding enzyme E2 determines the specific connection mode and length of the ubiquitin chain, and targeting E2s provides more selectivity compared to targeting E1 enzymes. Several E2 enzymes, including UBE2Q2, UBE2S, UBE2T2, UBEH9, UBEH10, and UBCH13, have been directly implicated in human disorders and cellular proliferation, further validating them as therapeutic targets. Notably, UBCH13 enhances NLRP3 inflammasome activation by promoting site-specific K63 ubiquitination of NLRP3. The small molecule inhibitor NSC697923 of UBCH13 inhibits NLRP3 inflammasome activation by suppressing NF-κB signaling, thereby alleviating the inflammatory response (75).

Treatment with MDM2 inhibitors JNJ-165 has been shown to alleviate oxidized low-density lipoprotein-induced mitochondrial damage and the production of pro-inflammatory cytokines in ECs (76). The NF-κB-antagonistic and p53-agonistic activities of MDM2 inhibitors elicit potent therapeutic effects in experimental lymphoproliferative autoimmune disorders such as SLE and APS (77). The novel role of MDM2 in vascular inflammation and mitochondrial bioenergetics presents a promising intervention target for the prevention and treatment of inflammation-related diseases. IL-17 is involved in chronic inflammation occurring during the pathogenesis of inflammation and autoimmune diseases. Significantly elevated IL-17 concentrations have been observed in primary APS patients who also exhibit thrombocytopenia, potentially contributing to the vascular manifestations of primary APS (78). Upon stimulation with IL-17A, the adaptor protein Act1 is recruited to IL-17R, subsequently recruiting the E3 ubiquitin ligase TRAF6 and TAK1, thereby initiating downstream signaling events, including the activation of MAPK and NF-κB pathways. MLN4924 has potential to attenuate IL-17A-induced autoimmune diseases by reducing the secretion of various inflammatory cytokines, including IL-1β, IL-6, TNF-α, and MCP-1. Mechanistically, MLN4924 promotes the ubiquitination and degradation of Act1, disrupting its interaction with IL-17R and impeding the formation of the IL-17R/Act1/TRAF6 complex, resulting in decreased expression of pro-inflammatory factors (79).

Several DUBs have emerged as potential targets for modulating inflammation. b-AP15, a DUB inhibitor targeting UCHL5 and USP14, plays a crucial role in the anti-inflammatory and anti-thrombotic mechanisms. Treatment with b-AP15 significantly reduces levels of TNF-α and IL-6 by regulating the NF-κB pathway in LPS-stimulated THP-1 and macrophages. b-AP15 improves the survival rate of mice with sepsis induced by high-density LPS (80). Furthermore, b-AP15 inhibits platelet aggregation induced by thrombin, collagen, and ADP, while also accompanied by curtailed procaspase-activating compound-1 binding to activated IIb/IIIa and inhibition of P-selectin translocation to the platelet surface (81).

The UPS has emerged as a promising target for therapeutic intervention in various diseases, including APS. However, the clinical application of UPS inhibitors, such as bortezomib, is often hampered by the development of drug resistance. The limitations have spurred the adoption of innovative UPS-based technologies aimed at promoting protein degradation or stability. Among these, molecular glue degraders and PROTACs (proteolysis-targeting chimeras) have shown potential in inducing the targeted degradation of proteins, while DUBTACs (deubiquitinase-targeting chimeras) aim to increase protein stability (82).

PROTACs are designed as conjugates of target protein ligands, linked to E3 ligase ligands via a linker. This novel therapeutic strategy induces the degradation or reduction of pathogenic proteins without directly inhibiting their functional activity, offering a new approach to overcome the limitations associated with traditional protein inhibitors (83). As a key upstream regulator of NF-κB activation, BTK (Bruton’s tyrosine kinase) plays a significant role in modulating the inflammatory responses of innate immune cells. Inhibition of BTK has been demonstrated to attenuate the secretion of pro-inflammatory cytokines, establishing it as a viable therapeutic target for the treatment of acute and chronic inflammatory conditions. A research team discovered BTK PROTACs based on ibrutinib that recruit the cereblon ligase, suppressing the secretion of proinflammatory cytokines and reducing inflammatory responses in mouse models (83). HDAC6 (histone deacetylase 6) is implicated in the activation of the NLRP3 inflammasome, indicating that targeting HDAC6 could be beneficial for treating inflammatory diseases. Utilizing a PROTAC strategy, Z. Cao et al. (84) have reported a low cytotoxicity HDAC6 degrading agent by binding a selective HDAC6 inhibitor derived from natural product indigo carmine to the CRBN E3 ligand pomalidomide. The HDAC6 PROTAC can effectively and selectively reduce HDAC6 levels in activated THP-1 cells and diminish NLRP3 inflammasome activation induced by LPS in mice. This provides initial evidence that HDAC6 PROTACs may offer a novel therapeutic strategy for NLRP3 inflammasome-related diseases, potentially serving as an alternative treatment for APS.

Despite the potential of PROTACs, the high molecular weight of PROTACs and their requirement for target protein to have a binding pocket limit their application. Molecular glues are expected to overcome these obstacles. Thalidomide, initially developed in the 1950s for morning sickness and currently used for leprosy and multiple myeloma, is a prime example of a molecular glue. Thalidomide and its analogues can inhibit the synthesis of TNF-α by activated monocytes and have demonstrated efficacy in autoimmune diseases such as RA (85), SLE (86), and systemic sclerosis, although the precise mechanisms were previously unknown (87). In 2010, Japanese scientists revealed that thalidomide could act as a “molecular glue”, interacting with E3 ligase CRBN and IKZF1/3 (ikaros family zinc finger 1/3) proteins, inducing the two proteins to approach each other. Subsequently, CRBN ubiquitinated IKZF1/3, leading to the ubiquitination and subsequent degradation of IKZF1/3 by proteasomes, thereby exerting anti-angiogenic, anti-proliferative, and anti-inflammatory effects (88). Thus, the potential role of molecular glues in treating autoimmune diseases has been continuously explored. Chinese scientists have also established DUBTAC platforms based on the DUBs such as OTUB1 and USP7, significantly broadening the scope of targeting protein stability (89, 90). However, research on DUBTAC technology is still in its early stages, and its application in autoimmune disease treatment requires further validation through laboratory studies and clinical trials. The exploration of UPS-based therapeutic strategies, including PROTACs, molecular glues, and DUBTACs, represents a significant advancement in the treatment of APS and other inflammatory conditions. These innovative approaches offer new avenues for modulating protein stability and interactions, potentially leading to more effective and targeted therapies in the future (5).