Ubiquitin Ligases in Control: Regulating NLRP3 Inflammasome Activation

Abstract

Ubiquitin ligases play pivotal roles in the regulation of NLR family pyrin domain containing 3 (NLRP3) inflammasome activation, a critical process in innate immunity and inflammatory responses. This review explores the intricate mechanisms by which various E3 ubiquitin ligases exert both positive and negative influences on NLRP3 inflammasome activity through diverse post-translational modifications. Negative regulation of NLRP3 inflammasome assembly is mediated by several E3 ligases, including F-box and leucine-rich repeat protein 2 (FBXL2), tripartite motif-containing protein 31 (TRIM31), and Casitas B-lineage lymphoma b (Cbl-b), which induce K48-linked ubiquitination of NLRP3, targeting it for proteasomal degradation. Membrane-associated RING-CH 7 (MARCH7) similarly promotes K48-linked ubiquitination leading to autophagic degradation, while RING finger protein (RNF125) induces K63-linked ubiquitination to modulate NLRP3 function. Ariadne homolog 2 (ARIH2) targets the nucleotide-binding domain (NBD) domain of NLRP3, inhibiting its activation, and tripartite motif-containing protein (TRIM65) employs dual K48 and K63-linked ubiquitination to suppress inflammasome assembly. Conversely, Pellino2 exemplifies a positive regulator, promoting NLRP3 inflammasome activation through K63-linked ubiquitination. Additionally, ubiquitin ligases influence other components critical for inflammasome function. TNF receptor-associated factor 3 (TRAF3) mediates K63 polyubiquitination of apoptosis-associated speck-like protein containing a CARD (ASC), facilitating its degradation, while E3 ligases regulate caspase-1 activation and DEAH-box helicase 33 (DHX33)-NLRP3 complex formation through specific ubiquitination events. Beyond direct inflammasome regulation, ubiquitin ligases impact broader innate immune signaling pathways, modulating pattern-recognition receptor responses and dendritic cell maturation. Furthermore, they intricately control NOD1/NOD2 signaling through K63-linked polyubiquitination of receptor-interacting protein 2 (RIP2), crucial for nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) activation. Furthermore, we explore how various pathogens, including bacteria, viruses, and parasites, have evolved sophisticated strategies to hijack the host ubiquitination machinery, manipulating NLRP3 inflammasome activation to evade immune responses. This comprehensive analysis provides insights into the molecular mechanisms underlying inflammasome regulation and their implications for inflammatory diseases, offering potential avenues for therapeutic interventions targeting the NLRP3 inflammasome. In conclusion, ubiquitin ligases emerge as key regulators of NLRP3 inflammasome activation, exhibiting a complex array of functions that finely tune immune responses. Understanding these regulatory mechanisms not only sheds light on fundamental aspects of inflammation but also offers potential therapeutic avenues for inflammatory disorders and infectious diseases.

Graphical abstract

Keywords

ubiquitin ligases / NLRP3 inflammasome / innate immunity / inflammation / post-translational modifications

1. Introduction

Inflammasomes are sophisticated multiprotein complexes that play a crucial role in innate immunity, acting as critical sensors and integrators of danger signals [1, 2, 3]. These complexes are essential for the body’s initial defense against both pathogenic microorganisms pathogen-associated molecular patterns (PAMPs) and sterile incursions damage-associated molecular patterns (DAMPs) such as trauma, cancer, ischemia, and metabolic perturbations [4, 5]. Typically, an inflammasome consists of three main components: a sensor protein (e.g., NOD-like receptors (NLR) family members or Pyrin and HIN domain-containing (PYHIN) proteins), an adaptor protein (usually ASC - apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD), and an effector protein (typically pro-caspase-1) [6, 7, 8].

Inflammasome activation is a tightly regulated process involving two distinct steps. The initial step, known as priming, involves the upregulation of inflammasome components and pro-forms of inflammatory cytokines, typically through nuclear factor kappa-light-chain-enhancer of activated B cells (NF-\kappaB) signaling [9, 10, 11, 12]. The second step triggers the assembly of the inflammasome complex, leading to the activation of caspase-1, which then processes inactive precursors of the cytokines interleukin-1\beta (IL-1\beta) and interleukin-18 (pro-IL-1\beta and pro-IL-18) into their mature, bioactive forms [9, 12, 13].

Several types of inflammasomes have been identified, each responding to specific stimuli and playing distinct roles in immune responses [14, 15, 16, 17]. The NLR family pyrin domain containing 3 (NLRP3) inflammasome, for instance, is activated by a diverse range of stimuli including ATP, pore-forming toxins, crystalline substances, and cellular stress signals, and is involved in responses to a wide array of pathogens and sterile inflammatory conditions [18]. The NLR family pyrin domain containing 1 (NLRP1) inflammasome, activated by Bacillus anthracis lethal toxin and muramyl dipeptide, plays a significant role in anthrax infection and certain autoimmune disorders [6, 19]. The NLR family caspase activation and recruitment domain (CARD) domain-containing protein (NLRC4) inflammasome, often in conjunction with Neuronal Apoptosis Inhibitory Protein (NAIP) proteins, detects bacterial flagellin and components of type III secretion systems, making it critical for responding to intracellular bacterial pathogens [20, 21]. The absent in melanoma 2 (AIM2) inflammasome responds to cytosolic double-stranded DNA, playing an important role in host defense against DNA viruses and intracellular bacteria [22, 23]. Additionally, the Pyrin inflammasome, activated by bacterial toxins that modify Ras homolog gene family member A (RhoA) GTPases, is involved in autoinflammatory diseases and responses to certain bacterial infections [24, 25].

Other inflammasomes, including Interferon-Inducible Protein 16 (IFI16), NLRP6, and NLRP7, have unique activators and roles in immune responses [17, 26, 27, 28]. The activation of inflammasomes results in the proteolytic activation of inflammatory caspases, leading to the maturation and secretion of pro-inflammatory cytokines IL-1\beta and IL-18 [29]. Inflammasome activation can also trigger pyroptosis, a form of inflammatory cell death characterized by cellular swelling, membrane rupture, and the release of intracellular contents [30, 31].

While inflammasome activation is critical for host defense, it requires tight regulation to prevent excessive inflammation and tissue damage [32, 33]. Dysregulation of inflammasome activity has been associated with various inflammatory disorders, including autoinflammatory diseases, cardiometabolic diseases, infections, cancer, and neurological disorders [34, 35]. In the context of intracerebral hemorrhage (ICH), edaravone has emerged as a promising neuroprotective agent due to its multifaceted mechanisms of action [36, 37, 38]. As a potent free radical scavenger, neutralizes reactive oxygen species generated from hemoglobin breakdown, potentially mitigating oxidative stress-induced neuronal damage [39]. Additionally, edaravone exhibits anti-inflammatory properties by suppressing microglial activation and reducing pro-inflammatory cytokine production [40]. Preclinical study demonstrated edaravone’s ability to preserve blood-brain barrier integrity, inhibit neuronal apoptosis in the perihematomal region, and attenuate early brain injury following ICH. In animal models, edaravone treatment has been associated with reduced brain edema, decreased neurological deficits, and improved long-term functional outcomes [41]. While clinical evidence remains limited, a small randomized controlled trial showed promising results, with early edaravone administration linked to reduced hematoma growth and improved functional outcomes in ICH patients [38, 42]. The diversity of inflammasome types allows for a nuanced and targeted immune response to a wide range of potential threats [43]. Understanding the specific activation mechanisms and regulatory pathways of each inflammasome type is crucial for developing targeted therapeutic strategies for various inflammatory and infectious diseases [44]. Ongoing research continues to unravel the complex interplay between different inflammasomes and their roles in health and disease.

2. NLRP3 Inflammasome Activation

The NLRP3 inflammasome is a cornerstone of the innate immune system, serving as a sophisticated sentinel that detects a wide array of pathogenic threats and cellular stresses [10]. This multiprotein complex, comprising the NLRP3 sensor protein, the ASC adaptor, and pro-caspase 1, orchestrates the initiation and amplification of inflammatory responses crucial for host defense [45].

The NLRP3 protein itself is an architectural marvel, featuring three distinct domains: a C-terminal leucine-rich repeat (LRR) domain involved in ligand sensing, a central NACHT domain that facilitates oligomerization, and an N-terminal pyrin (PYD) domain responsible for protein-protein interactions during inflammasome assembly [46]. This intricate structure allows NLRP3 to function as a molecular switch, transitioning from an inactive to an active state in response to cellular danger signals [47].

Activation of the NLRP3 inflammasome is a meticulously regulated process that typically unfolds in two stages: priming and activation. The priming phase, initiated by pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), activates NF-\kappaB signaling through various pattern recognition receptors such as Toll-like receptors (TLRs), NOD2, IL-1R, or tumor necrosis factor receptor (TNFR) [45, 48, 49]. This signaling cascade leads to the transcriptional upregulation of NLRP3 itself, as well as its substrate pro-inflammatory cytokines, pro-IL-1\beta and pro-IL-18 [50, 51, 52, 53]. Beyond transcriptional effects, priming also induces critical post-translational modifications of NLRP3, including ubiquitination and phosphorylation, which fine-tune its activation potential [54].

The activation phase, triggered by a remarkably diverse array of stimuli ranging from extracellular ATP and pore-forming toxins to particulate matter and various pathogens, initiates the assembly of the inflammasome complex [1]. While the precise mechanism by which NLRP3 recognizes such a broad spectrum of activators remains a subject of intense research, emerging evidence suggests that NLRP3 likely senses common cellular perturbations induced by these stimuli rather than directly binding to them [55]. One proposed mechanism involves the disassembly of the trans-Golgi network (TGN), which serves as a cellular stress indicator that can recruit and activate NLRP3 [47, 56].

Upon activation, NIMA-related kinase 7 (NEK7) emerges as a critical player, binding to NLRP3 and inducing a conformational change that disrupts its inactive double-ring structure [57]. This structural rearrangement exposes the PYD domains, allowing the NACHT domain to oligomerize [58]. The exposed PYD domain then recruits the adaptor molecule ASC, forming a remarkable filamentous structure known as the ASC pyroptosome or “speck” [59]. This speck serves as a molecular platform, with the caspase recruitment domain (CARD) of ASC binding to pro-caspase-1 and facilitating its conversion to active caspase-1 through proximity-induced autoproteolysis [60, 61].

Activated caspase-1 functions as the effector arm of the inflammasome, processing the pro-forms of IL-1\beta and IL-18 into their bioactive states [62]. Additionally, caspase-1 cleaves gasdermin D (GSDMD), a key mediator of pyroptosis, a highly inflammatory form of programmed cell death [63]. The N-terminal domain of cleaved GSDMD oligomerizes to form pores in the cell membrane, leading to cellular swelling, lysis, and the release of intracellular contents, including the mature inflammatory cytokines IL-1\beta and IL-18 [64].

The NLRP3 inflammasome is tightly regulated through multiple mechanisms to maintain immune homeostasis. Autophagy plays a crucial role in limiting NLRP3 activation by promoting its degradation [65]. MicroRNAs, particularly miR-223, negatively regulate NLRP3 expression by targeting its mRNA [50, 65]. Anti-inflammatory cytokines like IL-10 suppress NLRP3 activity [45], while endogenous inhibitors such as CARD8 and pyrin-only proteins provide additional control [66, 67]. Species-specific differences in NLRP3 activation exist between humans and mice, highlighting the importance of careful interpretation in translational research [68]. Recent structural biology insights have revealed conformational changes during NLRP3 activation, enhancing our understanding of its molecular mechanisms [67]. Cellular metabolism, especially glycolysis and mitochondrial function, significantly influences NLRP3 activation [47]. NLRP3 function varies across different tissue contexts, with distinct roles in immune cells versus epithelial cells [69]. From an evolutionary perspective, NLRP3 is conserved across species, suggesting its fundamental importance in innate immunity [70].

Complementing this canonical pathway, NLRP3 inflammasome activation can also proceed through a non-canonical route mediated by caspase-4 and caspase-5 in humans, or caspase-11 in mice [71, 72]. This pathway is particularly relevant in the context of intracellular LPS detection from Gram-negative bacteria, further expanding the surveillance capacity of the inflammasome system [50, 73].

The exquisite regulation of NLRP3 inflammasome activation is paramount for maintaining immune homeostasis [66]. Dysregulation of this process has been implicated in a spectrum of inflammatory disorders, ranging from autoinflammatory diseases to metabolic syndromes and neurodegenerative conditions [74]. This central role in health and disease has positioned the NLRP3 inflammasome as a prime target for therapeutic interventions, with ongoing research exploring various strategies to modulate its activity in inflammatory diseases [75].

3. Post Translational Modifications of NLRP3

Post-translational modifications (PTMs) play a pivotal role in regulating the activity of the NLRP3 inflammasome, a critical component of innate immunity responsible for detecting cellular stress and pathogen invasion [76, 77]. These PTMs exert precise control over NLRP3 function, influencing its activation, assembly into the inflammasome complex, and subsequent cytokine secretion [56, 78].

Phosphorylation plays a critical and multifaceted role in regulating NLRP3 inflammasome activation, with different phosphorylation sites exerting diverse and sometimes opposing effects [79]. At Ser295 (human)/Ser293 (mouse), phosphorylation by protein kinase D (PKD) at the Golgi apparatus promotes activation. In contrast, phosphorylation by protein kinase A (PKA) inhibits activation by suppressing the ATPase activity of the NLRP3 NACHT domain. At Ser5 (human)/Ser3 (mouse), phosphorylation by AKT has a dual role: it impedes inflammasome activation by inhibiting NLRP3 oligomerization and prevents proteasome-mediated degradation of NLRP3 during lipopolysaccharide (LPS) priming. Dephosphorylation of this site by protein phosphatase 2A (PP2A) enhances activation by promoting NLRP3-ASC interaction [45, 80, 81]. Additionally, phosphorylation at Ser198 (human)/Ser194 (mouse) by c-Jun N-terminal kinase 1 (JNK1) facilitates NLRP3 homo-oligomerization and is essential for inflammasome activation [82]. In contrast, enhanced tyrosine phosphorylation at Tyr861 negatively regulates inflammasome activation through autophagy induction, with protein tyrosine phosphatase nonreceptor 22 (PTPN22) promoting activation by dephosphorylating this site [77, 83].

Ubiquitination plays a crucial role in regulating NLRP3 inflammasome function through various linkage types, with the balance between ubiquitination and deubiquitination being essential for controlling inflammasome activation [84]. K48-linked ubiquitination targets NLRP3 for proteasomal degradation, inhibiting inflammasome activation [85]. Several E3 ubiquitin ligases mediate this process, including TRIM31, which promotes K48-linked polyubiquitination of NLRP3 [45]. Other E3 ligases such as MARCH7, ARIH2, and FBXL2 also attenuate NLRP3 inflammasome activation through protein degradation [86]. K63-linked ubiquitination, present in resting macrophages, has been associated with NLRP3 regulation. The E3 ubiquitin ligase TRIM65 can induce both K48- and K63-linked ubiquitination of NLRP3, with a higher efficiency for K63-linked ubiquitination. Deubiquitination enhances inflammasome activation, with BRCA1/BRCA2-containing complex 3 (BRCC3), a component of the BRCC36 isopeptidase complex (BRISC), acting as a positive regulator by promoting deubiquitination of the Leucine Rich Repeat (LRR) region of NLRP3 [86]. The USP1-Associated Factor 1/Ubiquitin-Specific Peptidase 1 UAF1/USP1 deubiquitinase complex specifically removes K48-linked ubiquitination of NLRP3, stabilizing its expression and facilitating inflammasome activation [84].

Other post-translational modifications (PTMs) intricately regulate NLRP3 inflammasome activity through diverse mechanisms. SUMOylation, mediated by the E3 ligase MAPL (MUL1), inhibits NLRP3 by preventing its oligomerization and interaction with ASC, thereby blocking inflammasome assembly and activation [45, 87, 88]. S-nitrosylation at cysteine residues, such as Cys3 and Cys394, by nitric oxide (NO) donors, impedes NLRP3 oligomerization and inflammasome formation [89]. ADP-ribosylation mediated by Poly (ADP-ribose) polymerase 1 (PARP1) suppresses NLRP3 activation by disrupting its interaction with NIMA-related kinase 7 (NEK7), which is crucial for inflammasome assembly [90]. Acetylation at lysine residues by p300/CBP enhances NLRP3 activation, promoting its interaction with ASC and facilitating IL-1\beta secretion [91]. O-GlcNAcylation at Ser198 inhibits NLRP3 inflammasome activation by disrupting its association with ASC and caspase-1, thus reducing IL-1\beta production [66]. Nitration of Tyr861 by reactive nitrogen species (RNS) attenuates NLRP3 activation, lowering IL-1\beta secretion and inflammation [89]. Glycosylation at Asn451 stabilizes NLRP3, enhancing inflammasome activity, while methylation of specific residues can alter NLRP3’s interaction dynamics and affect its assembly and activation [13]. Palmitoylation of cysteine residues regulates NLRP3 membrane association, potentially modulating its activation state and influencing inflammasome function [92]. These PTMs collectively orchestrate the precise regulation of NLRP3 inflammasome responses to various stimuli, crucial for maintaining immune homeostasis and responding appropriately to pathogens and cellular stress.

4. Roles of Key Ubiquitin Ligases in NLRP3 Inflammasome Regulation

The intricate regulation of the NLRP3 inflammasome is heavily influenced by various post-translational modifications (PTMs), with ubiquitination playing a pivotal role [93]. Understanding these modifications is crucial for developing targeted therapies to modulate inflammasome activity in inflammatory and immune-related diseases [78]. Ubiquitin ligases, through the addition of ubiquitin molecules, exert precise control over NLRP3’s stability, localization, and activity, ultimately modulating inflammasome activation [94]. These modifications can either tag NLRP3 for degradation or facilitate its assembly and activation, thus balancing the immune response to prevent both insufficient and excessive inflammation [56]. Understanding the specific ubiquitin ligases involved in these processes is crucial for comprehending the broader regulatory mechanisms at play. Each ubiquitin ligase, including TRIM31, MARCH7, FBXL2, Pellino2, and TRAF6, contributes uniquely to the modulation of NLRP3 activity through distinct ubiquitination pathways [1, 77]. Below, we delve into the detailed roles of these key ubiquitin ligases and their impact on NLRP3 inflammasome regulation (Fig. 1) created using Biorender (https://biorender.com/).

5. Negative Regulators of NLRP3 Inflammasome

Understanding the negative regulation of the NLRP3 inflammasome is crucial for comprehending how the body maintains immune homeostasis and prevents excessive inflammation. Several ubiquitin ligases act as key negative regulators, mediating the ubiquitination and subsequent proteasomal degradation of NLRP3, thus limiting its activation. These ubiquitin ligases ensure that the NLRP3 inflammasome is tightly controlled, preventing unwarranted inflammatory responses that can lead to chronic inflammation and autoimmune diseases [94].

5.1 TRIM31

TRIM31, a member of the TRIM protein family with E3 ubiquitin ligase activity, intricately regulates NLRP3 inflammasome activation through multifaceted mechanisms [95]. It directly binds to NLRP3 via its PYD domain, facilitating both K48-linked ubiquitination for proteasomal degradation and K63-linked ubiquitination that enhances NLRP3 oligomerization and assembly with ASC, thereby promoting inflammasome activation [85]. This dual regulatory role of TRIM31 is pivotal in modulating immune responses: it negatively regulates NLRP3 by promoting degradation to prevent hyperinflammation, while positively influencing inflammatory responses through assembly promotion. Experimental evidence demonstrates that TRIM31 overexpression decreases NLRP3 levels and IL-1\beta production, whereas its deficiency increases inflammasome activation, exacerbating inflammatory conditions such as DSS-induced colitis [85]. These findings underscore TRIM31’s complex role in fine-tuning NLRP3 inflammasome activity, suggesting potential therapeutic avenues for modulating inflammation in various diseases.

5.2 MARCH7

Membrane-Associated Ring-CH-Type Finger 7 (MARCH7) is a member of the MARCH family of E3 ubiquitin ligases, characterized by the presence of a Really Interesting New Gene-Cysteine/Histidine-rich (RING-CH) domain essential for their ubiquitin ligase activity [96]. MARCH7 specifically targets the NLRP3 protein for ubiquitination, catalyzing the addition of K48-linked polyubiquitin chains to NLRP3, typically signaling it for degradation [97]. Unlike other E3 ligases that promote proteasomal degradation, MARCH7 mediates the autophagic degradation of NLRP3 [98]. This distinct autophagic pathway helps regulate NLRP3 levels and activity. MARCH7 binds to the Leucine-Rich Repeat (LRR) and NACHT domains of NLRP3, which are critical for its function in inflammasome formation. This specific interaction does not affect other inflammasome components such as ASC or Pro-Caspase-1, highlighting MARCH7’s targeted regulation [66].

By promoting the degradation of NLRP3, MARCH7 acts as a negative regulator, suppressing the formation and activation of the NLRP3 inflammasome [66]. This process reduces NLRP3 protein levels, preventing unnecessary or excessive inflammasome activation and maintaining cellular homeostasis under basal conditions. This process is intricately linked to the dopamine D1 receptor pathway, providing a fascinating connection between neurotransmitter signaling and innate immunity [50]. By promoting NLRP3 degradation in response to dopaminergic stimulation, MARCH7 contributes to the anti-inflammatory effects of dopamine, effectively inhibiting NLRP3 inflammasome activation. This dopamine-MARCH7-NLRP3 axis represents a sophisticated regulatory mechanism that highlights the intricate crosstalk between the nervous system and inflammatory processes [99]. A Study showed that overexpression of MARCH7 decreases NLRP3 protein levels and IL-1\beta production, while its knockdown results in increased NLRP3 levels and enhanced inflammasome activation [98]. Enhancing MARCH7 activity or expression could be a strategy for dampening excessive NLRP3-mediated inflammation, beneficial in treating diseases like gout, atherosclerosis, and certain autoimmune conditions. Understanding MARCH7’s interplay with other NLRP3 regulators, such as TRIM31 and FBXL2, and further research into the dopamine-MARCH7-NLRP3 axis, could provide new therapeutic approaches for managing NLRP3-associated inflammatory disorders.

5.3 RNF125

Ring Finger Protein 125 (RNF125) is an E3 ubiquitin ligase that plays a crucial role in the regulation of the NLRP3 inflammasome. RNF125 initiates K63-linked polyubiquitination of the NLRP3 leucine-rich repeat (LRR) domain, a modification that facilitates the recruitment of another E3 ubiquitin ligase, Cbl-b [100]. By targeting NLRP3 for K63-linked ubiquitination, RNF125 sets the stage for a two-step process that ultimately controls NLRP3 protein levels and inflammasome activity [100]. This mechanism creates a negative feedback loop to control inflammation, particularly in the context of viral infections where type I interferons are prominently induced. By reducing NLRP3 protein levels, RNF125 suppresses inflammasome assembly and activation, leading to decreased production of pro-inflammatory cytokines like IL-1\beta and IL-18 [100]. This function is particularly important in preventing excessive or prolonged NLRP3 inflammasome activation, helping to maintain immune homeostasis [77].

5.4 CBL-b

Casitas B-lineage Lymphoma-b (Cbl-b) is another E3 ubiquitin ligase involved in the regulation of the NLRP3 inflammasome [100]. Cbl-b specifically targets the LRR domain of NLRP3 for K48-linked polyubiquitination, also leading to proteasomal degradation. By targeting NLRP3 for degradation, Cbl-b acts as a negative regulator, preventing the excessive activation of the inflammasome [100]. This regulation is critical for balancing immune responses and avoiding hyperinflammation. Research indicates that Cbl-b deficiency leads to increased NLRP3 levels and heightened inflammasome activity, contributing to exacerbated inflammatory conditions [45]. The role of Cbl-b in inflammasome regulation underscores the importance of ubiquitin-mediated pathways in controlling innate immune responses. Furthermore, the negative regulation of NLRP3 inflammasome activation by Cbl has been associated with the inhibition of glycolysis through GLUT1-dependent mechanisms [101]. By inhibiting Cbl, there is an increase in GLUT1 expression, leading to enhanced glycolytic capacity and subsequent NLRP3 inflammasome activation [101]. This demonstrates the diverse pathways through which proteins like Cbl modulate NLRP3 activity.

5.5 FBXL2

F-box and Leucine-rich repeat protein 2 (FBXL2) is a critical regulator of the NLRP3 inflammasome, functioning as part of the Skp1-Cullin-F-box (SCF) E3 ubiquitin ligase complex [102]. This complex consists of Skp1, Cullin1, Rbx1, and FBXL2 as the F-box protein that determines substrate specificity. FBXL2 directly interacts with NLRP3 through its leucine-rich repeats (LRRs), specifically targeting Lysine689 of NLRP3 for ubiquitination [77, 103]. It facilitates the addition of K48-linked polyubiquitin chains to NLRP3, marking it for proteasomal degradation by the 26S proteasome [104]. This process effectively reduces cellular levels of NLRP3, thereby inhibiting inflammasome activation and decreasing production of pro-inflammatory cytokines like IL-1\beta and IL-18 [104].

FBXL2’s activity is modulated by various cellular signals, including JNK1-mediated phosphorylation, which enhances its ability to target NLRP3 [10]. Interestingly, FBXL2 itself is regulated by another F-box protein, FBXO3, creating a regulatory loop where TLR signaling can indirectly increase NLRP3 levels by reducing FBXL2-mediated degradation [105]. This mechanism helps maintain homeostasis under basal conditions and plays a crucial role in resolving inflammation after the initial immune response.

Experimental evidence underscores FBXL2’s importance, showing that its overexpression decreases NLRP3 levels and reduces inflammasome activation, while its knockdown has the opposite effect [103]. Dysregulation of FBXL2 could potentially contribute to excessive NLRP3 activation in various inflammatory disorders, making it a promising therapeutic target [45].

5.6 ARIH2

Ariadne RBR E3 Ubiquitin Protein Ligase 2 (ARIH2), a member of the Ring-Between-Ring (RBR) family of E3 ubiquitin ligases, plays a pivotal role in modulating NLRP3 inflammasome activation through its distinctive ubiquitin ligase activity [106, 107]. It specifically targets NLRP3 for ubiquitination, catalyzing both K48-linked and K63-linked polyubiquitination. While K48-linked ubiquitination typically marks proteins for proteasomal degradation, the exact consequences of ARIH2-mediated ubiquitination on NLRP3 function are still under investigation [108]. This dual mode of ubiquitination by ARIH2 highlights its nuanced regulatory role in fine-tuning inflammasome activity, potentially impacting the formation and activation of the NLRP3 complex [109]. This mechanism is crucial during the priming phase of NLRP3 activation, influencing the expression and assembly of inflammasome components in response to inflammatory cues.

ARIH2’s interaction with NLRP3 is pivotal in macrophages, where it negatively regulates inflammasome activation. By modifying NLRP3 through ubiquitination, ARIH2 likely modulates its stability or activity, thereby affecting downstream inflammatory responses mediated by IL-1\beta and IL-18 [50]. ARIH2 as a potential therapeutic target in inflammatory diseases characterized by NLRP3 hyperactivation.

5.7 Cullin1

Cullin1, as an essential component of the Skp1-Cullin1-F-box (SCF) E3 ubiquitin ligase complex, plays a critical role in regulating NLRP3 inflammasome activation through its distinct mechanism. Structurally, Cullin1 acts as a scaffold within the SCF complex, facilitating the assembly of Skp1, an F-box protein, and a RING protein (typically Rbx1). This complex formation is crucial for its function as an E3 ubiquitin ligase. Interaction studies have revealed that Cullin1 directly interacts with the PYD domain of NLRP3 [45, 110]. This interaction facilitates the specific ubiquitination of NLRP3, particularly through K63-linked polyubiquitin chains [111]. Unlike K48-linked ubiquitination, which typically targets proteins for proteasomal degradation, K63-linked ubiquitination serves primarily as a signaling mechanism involved in protein-protein interactions and cellular signaling pathways.

Specifically, Cullin1 promotes K63-linked ubiquitination at lysine 689 (K689) of NLRP3. This modification plays a pivotal role in preventing NLRP3 inflammasome assembly and activation under basal conditions [112]. By ubiquitinating NLRP3 in this manner, Cullin1 interferes with the assembly of the NLRP3 inflammasome complex or its responsiveness to activating signals, thereby maintaining NLRP3 in an inactive state [13]. This regulatory role of Cullin1 is distinct from other E3 ligases such as TRIM31 or FBXL2, which primarily induce proteasomal degradation of NLRP3 [105]. The non-degradative nature of K63-linked ubiquitination by Cullin1 suggests a mechanism for finely tuning NLRP3 activity without the need for protein degradation, enabling rapid activation or inactivation as needed by the cell [77]. Understanding the intricate regulatory mechanisms involving Cullin1 and NLRP3 provides insights into how cells tightly control inflammatory responses. This knowledge could potentially lead to new therapeutic strategies for managing NLRP3-associated inflammatory disorders by modulating Cullin1 activity or its interaction with NLRP3 to influence inflammasome activation appropriately.

6. Positive Regulators of NLRP3 Inflammasome

While most ubiquitin ligases negatively regulate NLRP3 inflammasome activation, some promote its activation. These positive regulators play crucial roles in ensuring a timely and adequate inflammatory response, highlighting the complex and balanced nature of NLRP3 regulation [113]. Understanding the role of positive regulators in inflammasome dynamics and their implications for immune responses and disease is crucial to a better understanding of inflammasome dynamics.

6.1 Pellino2

Pellino2, a member of the Pellino family of E3 ubiquitin ligases, plays a crucial role in regulating the NLRP3 inflammasome [114]. Unlike many other E3 ligases that negatively regulate NLRP3, Pellino2 acts as a positive regulator, specifically during the priming phase of inflammasome activation [115]. It contains a RING-like domain that confers its ubiquitin ligase activity, allowing it to catalyze the addition of K63-linked polyubiquitin chains to NLRP3 [93, 114]. The K63-linked ubiquitination facilitated by Pellino2 is crucial for NLRP3 inflammasome activation. This type of ubiquitination, unlike K48-linked ubiquitination which typically targets proteins for degradation, is involved in regulating protein interactions and signaling pathways [13]. By adding these ubiquitin chains, Pellino2 enhances NLRP3’s ability to oligomerize and assemble into the inflammasome complex. This promotes the activation of NLRP3, leading to the cleavage of pro-caspase-1 and the subsequent maturation and secretion of pro-inflammatory cytokines such as IL-1\beta and IL-18 [116].

Pellino2’s activity is particularly important in response to pathogenic infections and other danger signals, ensuring a rapid and robust activation of the NLRP3 inflammasome [114]. Its expression and activity are regulated by various signaling pathways, including those activated by Toll-like receptors (TLRs) and other pattern recognition receptors (PRRs). This regulation ensures that Pellino2 activity is tightly controlled and context dependent [115].

Experimental evidence has shown that Pellino2 deficiency results in impaired NLRP3 inflammasome activation and reduced levels of IL-1\beta and IL-18, while its overexpression enhances NLRP3 activation and cytokine production [117]. Importantly, Pellino2 is not involved in TLR-induced NLRP3 and pro-IL-1\beta upregulation at the priming stage, suggesting its role is specific to the K63-linked ubiquitination of NLRP3 during the priming phase [50].

Given its role in promoting NLRP3 inflammasome activation, dysregulation of Pellino2 could contribute to inflammatory diseases characterized by excessive or chronic inflammation. Targeting Pellino2 activity might offer a novel approach to modulating NLRP3 inflammasome activation, with potential applications in boosting immune responses or reducing pathological inflammation in various disorders.

6.2 TRAF6

Tumor Necrosis Factor Receptor-Associated Factor 6 (TRAF6) is a crucial adaptor protein and E3 ubiquitin ligase that plays a key role in regulating the activation of the NLRP3 inflammasome, a multiprotein complex central to inflammatory responses [118]. TRAF6 directly interacts with NLRP3, catalyzing the addition of K63-linked ubiquitin chains, which promotes signaling processes rather than protein degradation [118, 119]. Unlike some E3 ligases that negatively regulate NLRP3, TRAF6 enhances its activation by promoting oligomerization and assembly of the inflammasome complex [77].

TRAF6 is involved in multiple aspects of NLRP3 regulation. It participates in the priming step by mediating NF-\kappaB activation, leading to increased expression of NLRP3 and pro-IL-1\beta [120]. Additionally, TRAF6 promotes the formation of ASC specks, which are crucial for inflammasome assembly and activation. It can also induce mitochondrial reactive oxygen species (ROS) production, a known activator of the NLRP3 inflammasome [121, 122].

The TRAF6-mediated activation of the NLRP3 inflammasome is particularly important in response to microbial infections and danger signals. TRAF6’s activity and expression are regulated by upstream signaling molecules and receptors, including Toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R), which recognize pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) [45]. Experimental evidence shows that knockdown or inhibition of TRAF6 impairs NLRP3 inflammasome activation, while its overexpression enhances inflammasome activity and cytokine production [123].

The positive regulation of NLRP3 inflammasome by TRAF6 is crucial for immune responses to certain pathogens and plays a role in the development of autoimmune diseases [123]. Dysregulation of TRAF6 can contribute to various inflammatory and autoimmune conditions, such as rheumatoid arthritis, atherosclerosis, and neuroinflammatory diseases [124]. Understanding the TRAF6-NLRP3 interaction holds significant therapeutic potential, as modulating TRAF6 activity could help control NLRP3-mediated inflammation in various disease contexts [125].

7. Deubiquitinating Enzymes in NLRP3 Inflammasome Regulation

While ubiquitin ligases play a crucial role in modulating NLRP3 inflammasome activity through the addition of ubiquitin chains, the removal of these chains is equally important for the precise regulation of this complex [126]. Deubiquitinating enzymes (DUBs) are specialized proteases that cleave ubiquitin moieties from substrate proteins, thereby reversing the actions of ubiquitin ligases. In the context of the NLRP3 inflammasome, DUBs can either enhance or suppress inflammasome activation by removing specific types of ubiquitin modifications [77, 105]. The dynamic interplay between ubiquitination and deubiquitination ensures a balanced and timely response to inflammatory signals, maintaining immune homeostasis and preventing excessive inflammation [127]. In the following sections, we will discuss key deubiquitinating enzymes involved in the regulation of the NLRP3 inflammasome and their mechanisms of action.

7.1 BRCC3

BRCA1/BRCA2-containing complex subunit 3 (BRCC3) is a crucial deubiquitinating enzyme that plays a significant role in regulating the NLRP3 inflammasome, a key component of the innate immune response [128]. As a member of the JAB1/MPN/Mov34 Metalloenzyme (JAMM) family of zinc-dependent metalloproteases, BRCC3 specifically cleaves K63-linked polyubiquitin chains from NLRP3, acting as a positive regulator of inflammasome activation [129]. This function is particularly important in the context of inflammatory responses and immune regulation.

BRCC3 is a component of the BRCC36 isopeptidase complex (BRISC), which is involved in various cellular processes, including DNA repair and maintenance of genomic stability [130]. In its role regulating the NLRP3 inflammasome, BRCC3 directly interacts with NLRP3, removing K63-linked ubiquitin chains from its LRR region [129]. This deubiquitination event is crucial for NLRP3 assembly and activation, facilitating the formation of the inflammasome complex and subsequent activation of caspase 1. The process leads to the production and secretion of pro-inflammatory cytokines such as IL-1\beta and IL-18 [13].

The timing of BRCC3’s action is specific, occurring after the priming step but before or during the activation step of NLRP3 inflammasome formation [48]. This mechanism ensures that NLRP3 activation is timely and appropriately scaled, preventing unwarranted inflammation while allowing for an effective immune response when needed. BRCC3’s activity has been observed in various cell types, including macrophages and dendritic cells, underscoring its importance in diverse immune contexts [131].

The regulation of NLRP3 by BRCC3 has significant implications for both physiological and pathological processes [47]. Proper functioning of BRCC3 is essential for the immune system’s ability to respond to infections and tissue damage. However, dysregulation of BRCC3 activity can lead to either insufficient or excessive inflammasome activation, potentially contributing to a range of inflammatory disorders [128]. Conditions such as autoinflammatory syndromes, chronic inflammatory diseases, and certain cancers have been associated with aberrant NLRP3 activation, highlighting the importance of BRCC3 in maintaining immune homeostasis [1].

The therapeutic potential of targeting BRCC3 and its regulatory pathways is significant. Modulating BRCC3 activity could be beneficial in treating diseases characterized by excessive inflammasome activation, such as gout, atherosclerosis, and autoimmune disorders. Inhibiting BRCC3 could potentially dampen NLRP3 inflammasome activation in conditions with excessive inflammation. Conversely, enhancing BRCC3 activity might improve immune responses in situations where NLRP3 activation is deficient [78].

7.2 USP7

Ubiquitin-specific peptidase 7 (USP7), also known as Herpesvirus-associated ubiquitin-specific protease (HAUSP), is a crucial deubiquitinating enzyme that plays a significant role in regulating the NLRP3 inflammasome [132]. As a member of the ubiquitin-specific protease (USP) family, USP7 removes ubiquitin moieties from substrate proteins, rescuing them from proteasomal degradation or altering their functional state [133]. This cysteine protease has diverse cellular functions, including roles in DNA damage response, apoptosis, and immune regulation.

In the context of NLRP3 inflammasome regulation, USP7 directly interacts with NLRP3 and modulates its stability and activity through deubiquitination [134]. It primarily removes K48-linked polyubiquitin chains, which are typically associated with targeting proteins for proteasomal degradation. By deubiquitinating NLRP3, USP7 stabilizes the protein and enhances its accumulation within the cell. This mechanism counteracts the effects of various E3 ubiquitin ligases, such as TRIM31, MARCH7, and FBXL2, which target NLRP3 for degradation [134].

USP7 acts as a positive regulator of the NLRP3 inflammasome [135]. By preventing the degradation of NLRP3, USP7 increases the availability of this protein, thereby promoting the formation and activation of the inflammasome complex [135]. This leads to the subsequent activation of caspase-1 and the maturation and secretion of pro-inflammatory cytokines such as IL-1\beta and IL-18 [136]. The stabilization of NLRP3 by USP7 ensures a robust inflammatory response to effectively combat infections or promote tissue repair.

The activity of USP7 in regulating NLRP3 has been observed in various cell types, including macrophages and other immune cells [13]. USP7 displays specificity for NLRP3 among various inflammasome components, underscoring its targeted regulatory role. It does not significantly affect other key inflammasome proteins like ASC or Pro-caspase-1, focusing its activity primarily on NLRP3 [137].

Experimental evidence supports USP7’s role in NLRP3 regulation. A study showed that the inhibition or knockdown of USP7 leads to decreased NLRP3 protein levels and reduced inflammasome activation [47]. Conversely, overexpression of USP7 results in increased NLRP3 stability and enhanced inflammasome activity, corroborating its role as a positive regulator.

The activity and expression of USP7 itself are subject to regulation, which can influence its effect on the NLRP3 inflammasome. Various factors, including post-translational modifications and protein-protein interactions, regulate USP7’s activity. These regulatory mechanisms can indirectly affect NLRP3 inflammasome activation [135].

Given its role in promoting NLRP3 activation, dysregulation of USP7 could contribute to inflammatory disorders characterized by excessive or chronic NLRP3 activation. Conditions such as autoinflammatory diseases, metabolic disorders, and certain infections might be influenced by alterations in USP7 activity [138]. This makes USP7 a potential therapeutic target for managing NLRP3-related inflammatory diseases. Modulating USP7 activity, possibly using USP7 inhibitors, could be a strategy to control NLRP3-mediated inflammation [139].

USP7 functions within a broader network of NLRP3 regulators, including other deubiquitinating enzymes and ubiquitin ligases. Understanding how USP7 interacts with these other factors is crucial for a comprehensive view of NLRP3 regulation. The balance between ubiquitination by E3 ligases and deubiquitination by DUBs like USP7 allows for fine-tuning of NLRP3 levels and activity, which is crucial for maintaining appropriate inflammatory responses while preventing excessive inflammation [77].

7.3 USP47

USP47, a member of the ubiquitin-specific protease family, plays a crucial role in regulating the NLRP3 inflammasome, a key component of the innate immune response. This deubiquitinating enzyme acts by removing ubiquitin moieties from NLRP3, preventing its proteasomal degradation and increasing its stability within the cell [140]. Through this mechanism, USP47 functions as a positive regulator of NLRP3 inflammasome activation, enhancing the availability of this key component and promoting its assembly into the active inflammasome complex [135].

The deubiquitinating activity of USP47 facilitates efficient inflammasome assembly by allowing NLRP3 to interact with the adaptor protein ASC and Pro-caspase 1 [45]. This process is essential for a robust inflammatory response, enabling cells to effectively respond to pathogenic infections and other danger signals [141]. USP47 displays specificity for NLRP3 among various inflammasome components, underscoring its targeted regulatory role [21]. Its activity is particularly significant in contexts where enhanced inflammasome activation is required, such as during microbial infections, tissue damage, or sterile inflammation.

Experimental study has demonstrated that inhibition or knockdown of USP47 leads to reduced NLRP3 protein levels and diminished inflammasome activation, while overexpression results in increased NLRP3 stability and enhanced inflammasome activity [47]. These findings suggest that USP47 is critical for maintaining appropriate levels of NLRP3 to ensure effective inflammasome responses. Given its role in promoting NLRP3 activation, dysregulation of USP47 could contribute to inflammatory disorders characterized by excessive or chronic NLRP3 activation, such as autoinflammatory diseases, metabolic disorders, and certain infections [142].

Targeting USP47 offers a promising therapeutic approach for managing NLRP3-related inflammatory diseases. Inhibiting USP47 might help reduce excessive inflammation in conditions where NLRP3 hyperactivation is detrimental, while enhancing its activity could boost NLRP3 responses when a stronger immune response is needed [135]. USP47 functions within a complex network of NLRP3 regulators, including other deubiquitinating enzymes and ubiquitin ligases.

7.4 A20

A20, also known as Tumor Necrosis Factor Alpha-Induced Protein 3 (TNFAIP3), is a pivotal anti-inflammatory molecule that plays a crucial role in regulating immune and inflammatory responses, including the NLRP3 inflammasome [143]. As a ubiquitin-editing enzyme with both deubiquitinating (DUB) and E3 ubiquitin ligase activities, A20 possesses a unique dual functionality that allows it to modulate protein ubiquitination status in complex ways, making it a versatile regulator of various signaling pathways [144].

In the context of NLRP3 inflammasome regulation, A20 primarily functions as a negative regulator, helping to prevent excessive or prolonged inflammatory responses. Its mechanism of action involves the removal of K63-linked polyubiquitin chains from NLRP3 and potentially other inflammasome components [143]. This deubiquitination is crucial for controlling the activation and assembly of the NLRP3 inflammasome, as it interferes with the recruitment of downstream signaling molecules necessary for inflammasome assembly. By maintaining NLRP3 in an inactive state under normal conditions, A20 prevents unwarranted inflammasome activation and excessive inflammation [145].

The importance of A20 in regulating the NLRP3 inflammasome is underscored by an experimental study showing that the absence or inhibition of A20 leads to enhanced NLRP3 inflammasome activation and increased production of pro-inflammatory cytokines such as IL-1\beta and IL-18 [146]. Conversely, overexpression of A20 results in reduced NLRP3 activity and dampened inflammatory responses. These findings highlight the critical role of A20 in maintaining the balance between activation and inhibition of the NLRP3 inflammasome [147].

A20’s regulatory effects extend beyond the NLRP3 inflammasome, as it also modulates other signaling pathways involved in immune responses, such as NF-\kappaB and mitogen-activated protein kinase (MAPK) [147]. This multifaceted regulation allows A20 to integrate various signals and coordinate an appropriate immune response [148]. By inhibiting NF-\kappaB signaling, A20 can reduce the transcription of pro-inflammatory cytokines and NLRP3 itself, further dampening the inflammatory response. This interplay between A20 and multiple signaling pathways adds another layer of complexity to its role in inflammation and immunity [149].

The impact of A20 on NLRP3 inflammasome activation may vary depending on the cell type and specific inflammatory context. This cell type-specific effect adds another layer of complexity to its regulatory role and highlights the need for further research to fully understand the nuances of A20’s function in different cellular environments [150].

Given its central role in regulating inflammation, A20 represents a potential therapeutic target for various inflammatory and autoimmune diseases. Enhancing A20 activity could be beneficial in conditions characterized by excessive NLRP3 activation, such as rheumatoid arthritis, systemic lupus erythematosus, and certain autoinflammatory syndromes [151]. Therapeutic strategies aimed at modulating A20 function may help restore immune homeostasis and alleviate disease symptoms. By controlling the ubiquitination status of NLRP3 and other signaling molecules, A20 helps maintain immune homeostasis and prevent chronic inflammatory responses [152]. Understanding the mechanisms by which A20 regulates the NLRP3 inflammasome and developing strategies to modulate its activity holds significant therapeutic potential for managing inflammatory and autoimmune diseases.

8. Degradation of Ubiquitinated NLRP3

The ubiquitination of NLRP3 plays a critical role in regulating its activation and degradation [45]. Ubiquitination involves the attachment of ubiquitin molecules to lysine residues on NLRP3, which can lead to different cellular outcomes depending on the type of ubiquitin linkage [66]. Specifically, K48-linked ubiquitination of NLRP3 typically targets it for proteasomal degradation. E3 ubiquitin ligases such as TRIM31 and MARCH7 have been shown to mediate K48-linked polyubiquitination of NLRP3, promoting its degradation via the proteasome and thereby inhibiting inflammasome activation [85, 153]. On the other hand, K63-linked ubiquitination is associated with autophagic degradation [77]. This process involves the interaction of ubiquitinated NLRP3 with autophagic adaptors like p62, which facilitate its delivery to the autophagosome for degradation [98, 154]. The balance between these ubiquitination pathways ensures that NLRP3 levels are tightly regulated, preventing excessive inflammasome activation that could lead to pathological inflammation [54].

9. Pathogens Hijack Host Ubiquitination Pathway to Regulate NLRP3 Inflammasome Activation

Pathogens have evolved sophisticated strategies to manipulate host cellular pathways to evade immune responses and establish infections. One such strategy involves hijacking the host ubiquitination pathway to regulate NLRP3 inflammasome activation [155]. Ubiquitination, a post-translational modification where ubiquitin proteins are attached to target proteins, plays a crucial role in controlling the stability, localization, and activity of many cellular proteins, including those involved in immune responses [156]. Below Table 1 (Ref. [94, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188]) depicts various pathogen strategies for modulating ubiquitination to influence inflammasome activation.

9.1 Bacterial Pathogens

Intracellular bacteria have evolved diverse strategies to manipulate the host ubiquitination pathway and regulate NLRP3 inflammasome activation (Fig. 2) [189, 190]. Mycobacterium tuberculosis employs protein tyrosine phosphatase A (PtpA) as a deubiquitinase to remove K63-linked ubiquitin chains from NLRP3, inhibiting its activation, reducing IL-1\beta production and dampening the host immune response [191, 192]. while its zinc metalloprotease 1 (Zmp1) interferes with the K63-linked ubiquitination of Pro-IL-1\beta, preventing its maturation and secretion [193]. This further suppresses the inflammatory response. Yersinia species use a type III secretion system to inject effector proteins like Yersinia outer protein J/P (YopJ/YopP) and Yersinia outer protein M (YopM) into host cells [194]. Acetyltransferases like YopJ/YopP inhibits the activation of NF-\kappaB and MAPK pathways, which are crucial for NLRP3 priming [195]. It also interferes with the ubiquitination of TRAF proteins, further suppressing inflammatory signaling. YopM recruits host cysteine protease USP7 to deubiquitinate NLRP3, leading to its destabilization and degradation [196]. Shigella flexneri uses IpaH family proteins as E3 ubiquitin ligases to target NLRP3 inflammasome components for degradation, like IpaH7.8 ubiquitinates gliomedin (GLMN), a negative regulator of NLRP3, leading to its degradation and enhancing inflammasome activation [161, 197]. Listeria monocytogenes utilizes Listeriolysin O (LLO) and InlC to modulate host ubiquitination and NF-\kappaB signaling [198]. LLO is a pore-forming toxin that can induce K⁺ efflux, activating the NLRP3 inflammasome. However, Listeria also uses LLO to manipulate host ubiquitination pathways, potentially modulating its activation [199, 200, 201]. Further its effector InlC interacts with IKK\alpha, affecting NF-\kappaB signaling and indirectly influencing NLRP3 inflammasome priming [202]. Chlamydia trachomatis employs Chlamydia deubiquitinating enzyme 1 (ChlaDub1) to deubiquitinate NF-\kappaB and CT441 to degrade p65 [203]. TIR domain-containing protein B (TcpB) from Brucella abortus mimics the host adaptor protein TIRAP/Mal, interfering with TLR signaling pathways [204]. By blocking the proper recruitment of signaling proteins and inhibiting NF-\kappaB and MAPK pathways, TcpB disrupts the transcription of pro-inflammatory genes, thereby reducing the priming and activation of the NLRP3 inflammasome [9]. This strategic interference helps Brucella abortus evade the host immune response and establish infection.

Other bacterial pathogens like Pseudomonas aeruginosa, use various effector proteins to disrupt host cell signaling and ubiquitination processes indirectly affecting NLRP3 inflammasome activation [164, 165]. Legionella pneumophila, an intracellular bacterium uses its type IV secretion system to inject effector proteins like LubX, E3 ubiquitin ligase that targets the host kinase clock homolog 1 (CLK1), affecting various cellular processes including inflammasome regulation [205]. Salmonella enterica uses different effector protein like Salmonella secreted effector L (SseL), deubiquitinase that removes K63-linked ubiquitin chains from various host proteins, potentially affecting NLRP3 inflammasome activation [206]. Francisella tularensis, a highly virulent bacterium induces prostaglandin E2 production, which inhibits NLRP3 inflammasome activation by interfering with potassium efflux [207].

Surface Cell Antigen (Sca) proteins from Rickettsia species, an obligate intracellular bacteria interact with host ubiquitin ligases, modulating cellular processes including inflammasome regulation [208]. Coxiella burnetiid, intracellular bacterium injects effector proteins via its Dot/Icm type IV secretion system to modulate host cell processes including ubiquitination pathways [209]. Streptococcus pyogenes uses Streptococcal Pyrogenic Exotoxin B (SpeB), cysteine protease that degrades host proteins, including those involved in inflammasome activation and Streptolysin O, pore-forming toxin that can modulate the NLRP3 inflammasome activation [210]. Porphyromonas gingivalis employs gingipains to degrade multiple host proteins, potentially affecting NLRP3 components [173]. Neisseria gonorrhoeae uses Opa proteins to interact with host receptors and modulate signaling pathways [211]. Borrelia burgdorferi’s, the causative agent of Lyme disease uses outer surface proteins like outer secreted protein A (OspA) and outer secreted protein B (OspB) proteins to interact with host cells, potentially modulating inflammasome activation [212].

9.2 Viral Pathogens

Viruses have also developed sophisticated mechanisms to manipulate host ubiquitination and NLRP3 inflammasome activation [213]. Influenza A Virus uses its NS1 protein to interact with TRIM25, an E3 ubiquitin ligase, inhibiting the ubiquitination of RIG-I and subsequent activation of antiviral signaling pathways indirectly affecting the NLRP3 inflammasome activation [214]. Human Papillomavirus employs its E6 oncoprotein to recruit the cellular E3 ubiquitin ligase E6AP, targeting p53 for degradation [180]. Epstein-Barr Virus’s BamHI fragment P leftward open reading frame 1 (BPLF1) deubiquitinates TRAF6 and NF-kappa-B Essential Modulator (NEMO), suppressing NF-\kappaB signaling [181]. Kaposi’s Sarcoma-associated Herpesvirus uses viral deubiquitinase Open Reading Frame 64 (ORF64) to suppress RIG-I-mediated signaling and viral E3 ubiquitin ligase K5 to target host immune proteins for degradation [182].

Enterovirus 71 (EV71) virus, responsible for hand, foot, and mouth disease uses its 3C protease to cleave TRIM25, inhibiting RIG-I-mediated antiviral signaling [183]. Hepatitis C Virus employs non-structural proteins 3 and 4A (NS3/4A) protease to cleave mitochondrial antiviral signaling protein (MAVS) and its core protein interacts with Parkin. Dengue Virus uses NS3 protease to cleave stimulator of interferon genes (STING) and Non-Structural Protein 2B and 3 (NS2B3) to target cyclic GMP-AMP synthase (cGAS) for degradation [215]. Human Cytomegalovirus’s protein UL26 (pUL26) interferes with NF-\kappaB activation, while pUL48 acts as a deubiquitinase [216]. Herpes Simplex Virus’s infected cell protein 0 (ICP0), E3 ubiquitin ligase targets various host proteins for degradation. Measles Virus’s V protein interacts with multiple host factors affecting ubiquitination processes [217]. Nipah Virus’s W protein inhibits RIG-I-like receptor signaling. Crimean-Congo Hemorrhagic Fever Virus’s L protein has deubiquitinase activity affecting various host processes including inflammasome regulation [218]. Chikungunya Virus’s nsP2 interferes with host interferon signaling, affecting NLRP3 inflammasome activation. Zika Virus’s NS3 can cleave STING, disrupting innate immune signaling [219].

9.3 Parasitic Pathogens

The NLRP3 inflammasome plays a crucial role in the innate immune response against various parasitic infections [45, 78, 220]. Protozoan parasites such as Plasmodium, Toxoplasma gondii, Leishmania species, and Trypanosoma cruzi, as well as helminth parasites like Schistosoma mansoni and Heligmosomoides polygyrus manipulate host ubiquitination pathways to regulate NLRP3 inflammasome activation [220, 221, 222]. In Plasmodium infections, hemozoin and parasite DNA have been identified as potent NLRP3 activators, contributing to both protective immunity and immunopathology in malaria [223, 224]. Leishmania species use Glycoprotein 63 (GP63), a surface protease, to cleave and inactivate multiple host proteins involved in innate immune signaling [225, 226]. Similarly, Toxoplasma gondii employs sophisticated mechanisms to modulate host cellular processes, including the regulation of the NLRP3 inflammasome [227, 228]. The parasite secretes Granule Protein 16 (GRA16), a dense granule protein that interacts with the host deubiquitinase Herpesvirus-Associated Ubiquitin-Specific Protease/ Ubiquitin-Specific Peptidase 7 (HAUSP/USP7), potentially affecting p53 stability and indirectly influencing NLRP3 inflammasome activation [227, 229]. This interaction highlights the complex interplay between T. gondii and host cellular pathways [224]. Concurrently, T. gondii activates the NLRP3 inflammasome through mechanisms involving potassium efflux and reactive oxygen species (ROS) production. The resulting NLRP3-dependent IL-1\beta production plays a crucial role in controlling T. gondii infection, demonstrating the dual nature of the parasite’s interaction with host innate immune responses. These findings underscore the intricate balance between parasite survival strategies and host defense mechanisms, where T. gondii both manipulates and triggers host inflammatory pathways to establish infection while the host attempts to control parasite replication [227, 230].

These diverse strategies employed by various pathogens highlight the complex interplay between host defense mechanisms and pathogen evasion tactics, centered around the manipulation of the ubiquitination system and NLRP3 inflammasome activation [189].

10. Conclusions and Future Perspective

The regulation of the NLRP3 inflammasome through ubiquitin ligases and deubiquitinating enzymes represents a sophisticated and tightly regulated system essential for maintaining immune balance [105]. This review has elucidated the complex network of both positive and negative regulators that modulate NLRP3 inflammasome activity through diverse ubiquitination and deubiquitination processes. The roles of E3 ubiquitin ligases, such as TRIM31, MARCH7, FBXL2, and Pellino2, alongside deubiquitinating enzymes like BRCC3, USP7, and A20, highlight the intricate nature of this regulatory system [105]. These enzymes collectively influence NLRP3 protein levels, stability, and activation, thereby affecting the magnitude and duration of inflammatory responses. In the context of intracerebral hemorrhage (ICH), where excessive inflammation contributes to secondary brain injury, understanding these regulatory mechanisms offers insights into potential therapeutic strategies [36]. Edaravone, for instance, may modulate the activity of specific ubiquitin ligases, potentially attenuating inflammasome-driven inflammation and providing a novel approach to managing ICH [42].

Furthermore, pathogens’ ability to exploit and manipulate the host’s ubiquitination machinery to evade immune detection adds another layer of complexity. Various bacteria, viruses, and parasites have developed advanced strategies to disrupt NLRP3 inflammasome activation by targeting key components of the ubiquitination pathway [191]. This interplay exemplifies the evolutionary battle between host defense mechanisms and pathogen evasion tactics, revolving around post-translational modifications.

Understanding these regulatory mechanisms offers valuable insights into the fundamental aspects of inflammation and presents potential therapeutic opportunities for inflammatory and infectious diseases. The delicate balance between NLRP3 inflammasome activation and inhibition, governed by ubiquitination and deubiquitination, presents both challenges and prospects for targeted interventions.

However, several questions remain for future investigation:

1.	1. How do distinct ubiquitination patterns on NLRP3 influence its function and activation kinetics?
2.	2. What are the specific molecular mechanisms by which pathogens disrupt host ubiquitination machinery to modulate NLRP3 inflammasome activation?
3.	3. How do cell type-specific variations in ubiquitin ligase and deubiquitinase expression impact NLRP3 inflammasome regulation across different tissues and disease states?
4.	4. Can targeting specific ubiquitin ligases or deubiquitinases provide effective and selective treatments for inflammatory diseases while preserving overall immune function?
5.	5. How do environmental factors and cellular stress affect the activity of ubiquitin ligases and deubiquitinases in the context of NLRP3 inflammasome regulation?
6.	6. What is the role of interactions between ubiquitination and other post-translational modifications in fine-tuning NLRP3 inflammasome activation?

Addressing these questions will enhance our understanding of NLRP3 inflammasome regulation and could lead to innovative therapeutic strategies for managing inflammatory disorders and combating infectious diseases.

Declaration of AI and AI-assisted Technologies in the Writing Process

During the preparation of this work the author used ChatGPT in order to check spell and grammar. After using this tool, the author reviewed and edited the content as needed and takes full responsibility for the content of the publication.

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