hGBP2 is a 67 kDa GTPase that is comprised of three distinct domains (13). The N-terminal region contains a large GTPase domain (LG; residues 1–309), followed by the middle domain (MD; residues 310–480) in the central region and the α12/13 helical domain (HD; residues 480–580) at the C-terminus. In its crystalline state, hGBP2 adopts a bi-molecular asymmetric unit configuration.

The LG domain serves as the nucleotide-binding site and is characterized by five conserved motifs involved in GTP binding and hydrolysis (Table I) (14). The G1/P-loop motif has a GxxxxGKS/T sequence (15) that encloses the β-phosphate of GTP. By contrast, the G2/SWITCH I motif serves a critical role in magnesium ion binding, which is a key feature for all GTPases, since it stabilizes the GTP molecule through coordination bonds with the phosphate groups. The G3/SWITCH II motif, with its conserved DTEG residue sequence, forms hydrogen bonds with the phosphate, aiding in its release. The G4 motif interacts specifically with the guanine base with its acidic Asp residue in the RDF residue sequence, facilitating the binding of GTP to the domain. The primary non-conserved region, located between residue 240 and 280, corresponds to a flexible region involved in membrane interactions and is situated within the G5 region. The G1-G5 motifs collectively constitute the ‘nucleotide-binding pocket’ for GTP (Table I).

The MD domain consists of 5 α-helices. The first helix group is formed of the N-terminal halves of α7, α8 and α9, where the second helix group is formed of the C-terminal half of α9, α10 and α11. By contrast, the α12/α13 domain contains a conserved CaaX sequence, where isoprenylation occurs, promoting the localization of hGBP2 on the membrane as a hydrophobic group. Another feature of α12/α13 domain is its interaction with LG domain. The α12/α13 domain is mainly negatively charged, while the LG domain mainly carries positive charge. This enables the two domains to contact through electrostatic interactions (13,16), where the α12/α13 domain is in a tightened state.

The hydrolase function of hGBP2 is essential for a number of mechanisms and processes. The membrane localization of hGBP2 requires homodimerization, which is dependent on the transition state of GTP hydrolysis (17).

The hGBP2 hydrolysate, which contains >75% GDP (18,19), yields only a small amount of GMP as the final product (13,16), which is obtained by the hydrolysis of GTP instead of GDP. This distinct characteristic, differing from hGBP1, is primarily attributed to the LG domain of hGBP2 rather than changes in the MD or HD domains. Specifically, this is due to differences in the adjustment of the active site in the LG domain of hGBP2 following the first hydrolysis event. In terms of tetramerization, hGBP2, unlike hGBP1 but like mGBP2 (18), can extensively tetramerize, although the tetramerization of hGBP2 does not contribute to the formation of GMP (20,21).

When hGBP2 binds GTP, GTP enters the nucleotide binding pocket formed by the G1-G5 motif, where hGBP2 undergoes homodimerization at the same time with the participation of LG and MD domains (22,23). The LG domain of hGBP2 is dimerized only 50% of the time in the presence of GTP, suggesting that the C-terminal domain of hGBP2 also participates in dimerization by providing a dimerization interface or otherwise stabilizing hGBP2 dimers in the GTP state (16). In addition, residue T75 in the G2/SWITCH motif and S52 in the G1 motif are involved in Mg²⁺ coordination (13), implying that Mg²⁺ may enter the nucleotide binding pocket and bind to the phosphate group and surrounding amino acid residues at this stage, which are necessary for hGBP2 activity. It has previously been reported that Mg²⁺ is necessary for the activation of hGBP2 at Ph 8.0, but not pH 6.0 (16), which suggests that Mg²⁺ and H₃O⁺ may have a functional substitution relationship. However, this interaction requires further validation.

In the hGBP2-GDP conformation following phosphate group dissociation, the N-O bond of the G1/P-loop motif interacts with the β-phosphate. Concurrently, residue S52 forms N-O bonds with both the α- and β-phosphates, facilitating nucleotide positioning and binding (24,25). The Y47 side chain undergoes movement and participates in dimer interface formation, whilst the displacement of Y47 and R48 in the G1 motif induces notable movement of the G2 motif, with some Cα atoms shifting >10 Å (26). The G4/RD motif interacts with the guanine moiety through hydrogen bonding. W238 in the G5 motif undergoes allosteric rearrangement and interacts with W238 from another hGBP2 molecule (13). Consequently, the guanine base and ribose are enclosed within the space formed by the G1, G4 and G5 motifs. At the opposite end of the GDP, the diphosphate group is tightly positioned within the pocket formed by the G1/P-loop, G2/SWITCH I and G3/SWITCH II motifs. The β-phosphate further coordinates with the conserved residues K51, S52 and E99. When the E99-K51 interaction is absent, SWITCH I remains open. However, when these residues engage through hydrogen bonding, SWITCH I closes, simultaneously positioning T75 in the G2 motif for optimal interaction with Mg²⁺ (16).

The hGBP2 gene, as an IFN-stimulated gene (ISG), contains a promoter region featuring two transcription factor binding sequences, namely the IFN-stimulated response element (ISRE), located at +42 (27) and the γ-activated sequence (GAS) (27,28), located at +53 and at −532 (27). Notably, GAS exhibits binding affinity for all STAT factors except for STAT2. Both sequences can independently respond to IFN signaling, whilst their combined presence results in enhanced transcriptional activity (29). By contrast, the mGbp2 gene is also an ISG, which contains one ISRE, located at −2 (27) and two GASs, located at −487 and −735 (27). However, Ramsauer et al (28) previously reported that the mGbpP2 gene has two ISREs, located at −30 and −440 and one GAS located at −530 (28).

Upon pathogen invasion or intracellular abnormalities, the corresponding PAMPs or DAMPs are recognized by a series of pattern recognition receptors (PRRs). This type of recognition triggers the phosphorylation and activation of IRFs, thereby promoting the expression of IFN-I and IFN-II (the former mainly includes subtypes IFN-α and IFN-β) through the classical secretory pathway. These IFNs are secreted extracellularly and bind to their corresponding respective receptors. Mitochondrial outer membrane permeabilization (MOMP) under radiation conditions and apoptosis can lead to the release of mitochondrial double-stranded (ds)DNA, which activates the cyclic GMP-AMP synthase/stimulator of IFN genes pathway, subsequently inducing IFN-β and upregulating mGBP2 expression (32).

IFN-I can bind to the ubiquitously expressed IFN-α/β receptor (consisting of the IFNAR1 and IFNAR2 subunits), leading to the dimerization of the receptor subunits. This dimerization, through juxtaposition and trans-phosphorylation, enhances the kinase activity of Janus kinase (JAK) 1 and tyrosine kinase (TYK) 2. Subsequently, JAK1 and TYK2 phosphorylate tyrosine residues on IFNAR1 and IFNAR2, which serve as binding sites for STAT1 and STAT2 (33). Phosphorylation of STAT1 at Tyr701 and STAT2 at Tyr690 then occurs. By contrast, IFN-II, specifically IFN-γ, interacts with a tetrameric receptor complex composed of two IFNGR1 subunits and two IFNGR2 subunits. This receptor is associated with JAK1 and JAK2 kinases, which exclusively phosphorylate the STAT1 protein.

Similar to the pathway under normal conditions, phosphorylated (p-)STAT1, p-STAT2 and IRF1 can form four types of oligomers: p-STAT1-p-STAT1 (also known as γ-IFN activation factor, binds to GAS), p-STAT1-p-STAT2-IRF9 (binds GAS), pSTAT1-pSTAT2-IRF9 (also known as IFN-stimulated gene factor, binds to ISRE) and IRF1-IRF1 (binds ISRE). These oligomers subsequently localize to the hGBP2 or mGbp2 gene and activate its transcription through a similar mechanism (Fig. 2), exhibiting higher activity compared with their non-phosphorylated counterparts.

The inflammasome pathway can be divided into two types, namely the canonical pathway and the non-canonical pathway. Their objective is to induce pyroptosis, which is mediated by the formation of plasma membrane pores through gasdermin-D. This leads to membrane rupture and alerting other cells, with the release of cytokines IL-1β and IL-18 through the gasdermin-D-formed membrane pores. hGBP2 and mGBP2 primarily serve supporting roles in inflammasome assembly.

In the canonical pathway, activation is primarily mediated by PRRs, such as nucleotide-binding oligomerization domain-like receptors, such as NLRP3, or AIM2-like receptors, including AIM. PAMPs or DAMPs are recognized by PRRs, such as NLRP3 or AIM2. During this process, mGBP2 can facilitate the release of dsDNA by lysing pathogen-containing vacuoles or pathogens, enabling AIM2 recognition (7,34,35). Upon activation, NLRP3 or AIM2 interacts with the adaptor protein ASC through their pyrin domains (36). In this step, mGBP2 and mGBP5 can form a heterocomplex, where mGBP2 binds ASC and mGBP5 binds NLRP3. This mGBP2-mGBP5 complex brings NLRP3 and ASC together, thereby promoting the assembly of the NLRP3 inflammasome (7). ASC in turn recruits caspase-1 through its caspase recruitment domain, forming the inflammasome. Subsequently, caspase-1 within the inflammasome cleaves gasdermin-D, releasing its N-terminal fragment to form plasma membrane pores and induce pyroptosis. Caspase-1 also processes pro-IL-1β and pro-IL-18 into their active forms, which are then released through the membrane pores to alert neighboring cells (7).

In the non-canonical pathway, activation in humans is primarily mediated by hGBPs through PRRs while in mice, it is mediated by caspase-11 with the assistance of mGBP2. In humans, when bacteria enter cells, hGBP1 binds bacterial LPS and recruits hGBP2, hGBP3 and hGBP4 to the surface of bacteria, especially gram-negative bacteria, forming a coating (6,37). hGBP2 and hGBP4 expose the lipid moiety of LPS, recruiting caspase-4 to the bacterial surface for LPS binding, while hGBP3 regulates caspase-4 activation (6,37). Activated caspase-4 then cleaves pro-IL-1β, pro-IL-18 and gasdermin-D, leading to pyroptosis and cytokine release.

In mice, caspase-11 directly recognizes the mGBP2-LPS complex, oligomerizes and activates, gaining the ability to cleave gasdermin-D and induce pyroptosis (38). Additionally, the formation of plasma membrane pores causes potassium ion efflux due to the intracellular potassium gradient, further promoting NLRP3 inflammasome assembly and creating a positive-feedback amplification loop (39). In this process, mGBP2 assists caspase-11 in LPS recognition. A previous study has reported that mGBP2 can interact with gasdermin-D and mGBP3, potentially serving as a novel component of this pathway, although the precise mechanisms remain unclear and require further investigation (7).

The severe consequences of pyroptosis, such as further inflammation cascade caused by the release of IL-18 and IL-1β (40), may explain the need for the continuity and regulation of the GBP2/caspase pathway. This type of regulation allows for the existence of multiple regulatory checkpoints before caspase activation, increasing the difficulty of activation and preventing unnecessary triggering (6).

The expression of hGBP2 is increased in the kidney tissues of patients with lupus nephritis, particularly in the glomeruli and renal tubulointerstitium (41). In diabetic nephropathy, macrophages at the injury site are predominantly of the M1 subtype, where hGBP2 can promote the polarization of macrophages toward the pro-inflammatory M1 subtype through the Notch 1 pathway. Through the hGBP2-mediated pathway, macrophages can be induced into the M1 phenotype by LPS or IFN-γ (42).

In allergic rhinitis (AR), mGBP2 can alleviate oxidative stress and abnormal lipid metabolism by inhibiting the hypoxia-inducible factor-1 (HIF-1) pathway. It can also inhibit mitochondrial fission whilst maintaining mitochondrial fusion to mitigate oxidative stress-induced damage to cells (43). A previous study has demonstrated that the overexpression of mGBP2 can notably reduce inflammatory cell infiltration into the nasal mucosa and markedly decrease the levels of various factors, such as total cholesterol, low-density lipoprotein-cholesterol, TNF-α, IL-5, IFN-γ and trimethylamine N-oxide, in AR mouse models whilst increasing high-density lipoprotein-cholesterol levels (44). However, the underlying mechanisms require further investigation.

LPS-induced macrophages-derived exosomes (L-Exo) can be transported from macrophages to lung epithelial cells, resulting in damage to the alveolar epithelial tissue. The mGBP2 content in L-Exo is higher compared with that in control Exo (45), leading to the hypothesis that the mGBP2 contained in L-Exo can trigger pyroptosis in the lung epithelial tissue, thereby causing injury. This may represent a potential therapeutic approach for sepsis-associated acute lung injury (46). In depression-like behaviors induced by neuroinflammation, reducing the expression of mGBP2 may also alleviate symptoms (47).

Previous studies have demonstrated that in glioblastoma (GBM) and low-grade glioma cells, hGBP2 can directly interact with kinesin family member 22 (KIF22) to post-transcriptionally regulate and elevate its levels, thereby enhancing KIF22-mediated EGFR internalization and signaling (Table II) (48–63). This in turn fosters cell proliferation (64). Unc-51 like autophagy activating kinase 1 (ULK1) is another critical regulator of autophagy (65), phosphorylating and inducing autophagy under low-nutrient conditions. hGBP2 can activate the ULK1 complex by suppressing PI3K/Akt/mTORC1 pathway (66), thereby enhancing cellular autophagy (66). Notably, the outcomes of this function may vary, where it may exert tumor-suppressive effects during early-stage cancer but potentially promote cancer progression at advanced stages (67).

hGBP2 can induce the assembly of inflammasomes, leading to a positive feedback loop of inflammatory responses. This forms the hGBP2/STAT3/fibronectin (FN1) pathway (49,53), where hGBP2 induces FN1, which is an extracellular glycoprotein involved in cell migration (68), on both transcriptional and translational levels to enhance glioblastoma migration and invasion without influencing proliferation in vitro (49). In addition, STAT3 contributes to maintaining the mesenchymal subtype of GBM (50) and is indispensable for hGBP2-induced FN1 elevation (49). FN1 can also promote MMP-9 secretion in a focal adhesion kinase (FAK)- and Ras-dependent manner (69) or through the FN1/MMP-2/MMP-9 pathway (70). However, hGBP2 can induce FN1 in vivo but lacks this capability in vitro (49). Furthermore, MMP-9 is an inducer of epithelial-mesenchymal transition (EMT) (71). Thus, hGBP2 should activate, rather than inhibit, MMP-9 through the STAT3/FN1/FAK or STAT3/FN1/MMP-2 signaling pathways. However, the regulatory effect of hGBP2 on migration has not been experimentally demonstrated.

Bak is a member of the Bcl-2 family that is crucial for the activation of apoptosis. It promotes MOMP, thereby triggering the apoptotic process (72). hGBP2 acts as a dual stimulator of Bak. It can not only release Bak but can also promote its expression (8). The LG domain of hGBP2 specifically binds to the BH3 domain of Mcl-1, a member of the Bcl-2 family, thereby preventing the interaction between Bak and Mcl-1. This then releases Bak, which oligomerizes to induce MOMP, ultimately promoting apoptosis (8). Simultaneously, hGBP2 upregulates the expression of Bak through its inhibitory effect on the PI3K/Akt pathway (8). In summary, hGBP2 can promote MOMP by activating Bak and enhancing its expression, thereby facilitating apoptosis. PTX, as an anticancer drug, occupies the upstream position in the hGBP2/Mcl-1/Bak pathway. Through this pathway, hGBP2 enhances cellular sensitivity to PTX (8,66). Notably, hGBP2 upregulation is a common feature of PTX treatment and other chemotherapeutic agents for hematologic malignancies (such as doxorubicin, cytarabine, vincristine, etoposide and IFN-γ). However, among these antineoplastic agents, only PTX possesses the unique ability to elevate Bak levels (8).

In breast cancer (BC) cells, hGBP2 has been demonstrated to directly interact with dynamin-related protein 1 (Drp1) in a K51-dependent manner, preventing its translocation from the cytoplasm to the mitochondria. This interaction inhibits Drp1-dependent mitochondrial fission and elongation, thereby blocking mitochondrial division and cell metastasis in cancer cells (73). Notably, the activity of hGBP2 binding to Drp1 is influenced by all three major structural domains of hGBP2, where it is possible that hGBP2 and Drp1 bind to each other to form hetero-oligomers, although further investigations are required (73). hGBP2 also exerts its anticancer effects by inhibiting the Wnt/β-catenin/EMT pathway (55,71,74) and cancer cell metastasis and invasion, which has been demonstrated in cancers such as skin cutaneous melanoma (SKCM) and colon cancer, but the specific molecular mechanisms remain unclear. PTX primarily inhibits tumor metastasis and progression by blocking angiogenesis in tumor tissues, whilst hGBP2 enhances the sensitivity of colon cancer cells to PTX by inhibiting the Wnt/β-catenin pathway (75,76). Additionally, a previous study has indicated that inhibiting the Wnt/β-catenin pathway in BC and triple-negative BC (TNBC) can promote ferroptosis in BC cells and suppress cell cycle and growth regulatory proteins, such as cyclin D1 and c-Myc. Therefore, it can be speculated that this may be a potential function of hGBP2, warranting further investigation.

In mouse dendritic cells, mGBP2 has been reported to interact with the Akt-p110 complex, inhibiting the phosphorylation and activation of Akt (77,78). This in turn prevents the phosphorylation of TSC complex subunit 2, disrupting its ability to form a complex with tuberous sclerosis 1 that inhibits mTORC1 (77–80). VEGF promotes endothelial cell proliferation and migration by binding to VEGFRs on endothelial cells, thereby stimulating angiogenesis. Under basal conditions, the HIF-1α subunit is hydroxylated by prolyl hydroxylases and recognized by the von Hippel-Lindau protein complex, leading to its ubiquitination and degradation. Under hypoxic conditions, however, the HIF-1α subunit is stabilized, where it translocates to the nucleus and dimerizes with the HIF-1β subunit to form the HIF-1 transcription factor, which then translocates to the nucleus and binds to hypoxia-response element in the promoter of VEGF gene (81,82), regulating VEGF transcription (43). In oxygen-induced retinopathy mice model, mGBP2 has been shown to inhibit the HIF-1α/VEGF pathway by suppressing mTORC1 (78), thereby attenuating angiogenesis. This reduces blood supply to cancer lesions, maintaining hypoxic conditions and potentially curbing cancer progression.

mGBP2 can activate MMP-9 through distinct pathways, thereby influencing collagen degradation near the basement membrane (33), extracellular matrix remodeling, angiogenesis in cancer lesions and cancer cell invasion and metastasis (77). Previous studies in NIH 3T3 fibroblasts have shown that mGBP2 can inhibit Rac, disrupting its regulation of the cytoskeleton. This inhibition suppresses the TNF-α-mediated activation of the NF-κB pathway, thereby reducing NF-κB-induced MMP-9 transcription (83). mGBP2 has been demonstrated in NIH 3T3 fibroblasts to directly interact with the p65 (RelA) protein in the NF-κB pathway, preventing its binding to the MMP-9 promoter and subsequently lowering MMP-9 expression (83). In ovarian cancer, mGBP2 promotes the recruitment of the peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) protein, which activates the NF-κB pathway (62). Based on this, it can be hypothesized that mGBP2 may upregulate MMP-9 expression through the Pin1/NF-κB and Rac/NF-κB pathways, although further experimental validations are required.

hGBP2 exerts a dual influence on the TIME. It can promote immune activation, transforming the TIME into a ‘hot’ state, thereby addressing the immunologically ‘cold’ tumors, such as TNBC and proficient mismatch repair/microsatellite stable colorectal cancer (CRC) of the immune class (59). Simultaneously, it can also reduce immune cell infiltration through certain mechanisms, enhancing tumor immune evasion. Additionally, the expression of immunotherapy biomarkers is positively associated with hGBP2 expression (61). In CRC, hGBP2 expression is positively associated with CD8⁺ T cell infiltration, CD8, PD-L1, C-X-C motif chemokine ligand (CXCL) 9, CXCL10, CXCL11, CXCL13, HLA-I expression, antigen processing and presentation machinery and a variety of antitumor immunity steps, including the release of cancer cell antigens, cancer antigen presentation, priming and activation and trafficking of immune cells to tumors (59). By contrast, it is negatively associated with the cell count of cytokeratin (59). However, specific mechanisms of the regulation of hGBP2 in CRC require further study.

In gastric cancer, hGBP2 is reported to be positively associated with an inflamed TIME, immunophenoscore (IPS), abundance of PD-1⁺ cells and the expression of immunotherapy biomarkers, such as PD-L1, PD-L2, IFN-γ, CD8A, secreted and transmembrane protein 1 and IFN-induced transmembrane protein 3 (61). In GBM, it has been demonstrated that urokinase, secreted protein acidic and rich in cysteine, TGFB1, FN1 and colony-stimulating factor 1 are induced by elevated hGBP2 expression (49). In SKCM, previous studies have demonstrated that hGBP2 expression is positively associated with the infiltration of B cells, CD8⁺ T cells, CD4⁺ T cells, macrophages, neutrophils and dendritic cells (11,12,57,61), This may serve a role in therapies that block PD-1/PD-L1 interactions to prevent tumor immune evasion, since CD8⁺ T cells are the primary effector cells in tumor killing (84). In BC, hGBP2 is positively associated with T cell infiltration levels and can serve as a marker for T cell infiltration (53). In clear cell renal cell carcinoma (ccRCC), hGBP2 promotes the infiltration of CD8⁺ T cells, regulator T cells and both M1 and M2 macrophages (57).

From the perspective of reducing immune infiltration and promoting tumor immune evasion, hGBP2 can exert oncogenic effects by increasing the phosphorylation of STAT2 and STAT3, modulating the JAK/STAT signaling pathway and reducing tumor immune infiltration (51). hGBP2 can also induce immune checkpoints, such as PD-1/PD-L1, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T cell immunoreceptor with Ig and ITIM domains, LAG3, indoleamine 2,3-dioxygenase 2 and V-domain Ig suppressor of T cell activation (VISTA) (11), which are inhibitory immune checkpoints. Additionally, hGBP2 can promote PD-L1 expression on the transcriptional level by binding to and phosphorylating STAT1, thereby reducing tumor immune infiltration and facilitating immune evasion (85). hGBP2 can also induce PD-L2; however, the relevant mechanisms remain unstudied (61). Based on these functions, hGBP2 will likely be a crucial target for restoring the TIME for the treatment of immunologically ‘cold’ tumors, such as TNBC.

mGBP2 has been found to upregulate the secretion of IL-6, IL-12 and TNF-α (77). Furthermore, mGBP2 promotes the maturation of dendritic cells and enhances their antigen-presenting capacity, thereby boosting T cell activation (77). mGBP2 also promotes the polarization of macrophages into M1 and M2 subtypes by promoting STAT3 pathway and by activating the NF-κB signaling pathway (62), while hGBP2 promotes polarization by stimulating the secretion of IL-18 (56). M2 macrophages, in turn, can enhance the migration and invasion of tumor cells by secreting IL-10 and TGF-β, which upregulate the hGBP2/STAT3 and ERK axes (part of the MAPK signaling pathway) as demonstrated in ccRCC (56).

Notably, a number of downstream factors promoted by hGBP2 can exhibit both pro-tumor and antitumor effects. CD80 in pancreatic adenocarcinoma can bind to either CD28 (activates T cells) or CTLA-4 (inhibits T cells), thereby bidirectionally modulating immune responses (11). Notably, hGBP2 is reported to promote the polarization of M0 to M2 in ccRCC by activating the secretion of IL-18 (56) and to M1 in ovarian cancer (62) and in diabetic nephropathy by activating the Notch 1 signaling pathway (42). By contrast, mGBP2 has only been reported to promote the polarization of M0 to M1 under the activation of the PLGA-CpG@ID8-M nano vaccine (62). The underlying mechanisms of these outcomes and whether mGBP2 can also promote the polarization of M0 to M2 require further investigation.

In primary CRC, hGBP2 has been found to exhibit a high mutation rate alongside genes such as G protein subunit β1 and GATA zinc finger domain containing 2A. However, only hGBP2 showed an even higher mutation rate in CRC with liver metastasis (60). Additionally, in CRC with liver metastasis, the hGBP2 gene undergoes methylation at four specific sites (m1A, m5C, m6A and m7G) (60). Peroxisome proliferator-activated receptor α, an anticancer factor, can inhibit hGBP2 expression (58). In BC (52) and SKCM (12), the methylation level of hGBP2 increases, leading to a decrease in hGBP2 expression.