Nuclear factor erythroid-derived 2-like-2 (Nrf2), a key antioxidant transcription factor, plays an important role in enhancing cellular resistance to toxic stimuli and oxidative stress damage (47). Under resting conditions, Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Within the E3 ubiquitin ligase complex based on the Cul3 scaffold, Keap1 forms homodimers through its BTB domain, which allows it to specifically recognize and expedite the ubiquitination and degradation of cytoplasmic Nrf2 (48). The oxidative modification of specific cysteine residues on Keap1 causes spatial conformational changes that cause Nrf2 to separate from ubiquitination (49). Furthermore, Nrf2 expression and nuclear translocation can be induced by oxidative stress, which eventually results in Nrf2 accumulation in the nucleus (50). Binding to small Maf proteins, nuclear Nrf2 activates antioxidant response elements (AREs) and triggers the expression of several genes related to detoxification and antioxidants (51).

HACE1 is an oxidative stress response factor that is essential for maintaining cellular antioxidant defense by mediating Nrf2 activation. Specifically, HACE1 prevents Nrf2 ubiquitination and proteasomal degradation by competing with Keap1, thereby stabilizing Nrf2 protein and enhancing its transcriptional activity in response to oxidative stress (52). This process requires HACE1 to have an intact domain, because the loss of either the HECT or ANK domain will lead to the disruption of Nrf2 protein homeostasis (52). Hence, the cytoprotective effect of HACE1 may be mainly attributed to the antioxidant activity of Nrf2. Rotblat et al. (45) found that HACE1 is closely associated with neurodegenerative diseases because its expression levels are reduced in the striatum of Huntington’s disease patients. Furthermore, knockout of HACE1 resulted in impaired antioxidant stress response in mice, and this sensitivity to oxidative stress could be restored by ectopic expression of Nrf2 (45). In addition to inhibiting neurodegeneration, HACE1 has been reported to activate the Nrf2/ARE signaling pathway, upregulate HO-1 and NQO1 antioxidant genes, and attenuate H/R-induced oxidative stress and inflammation, making it a promising therapeutic target and predictor of cardiac disease (42). Consistent with findings in the nervous system, the protective effect of HACE1 in the heart was also confirmed in Nrf2-mediated responses (53).

Ras-related C3 botulinum toxin substrate 1 (Rac1) is a small GTPase belonging to the Rho GTPase family and an essential intracellular signaling molecule (54). As a signal transduction molecular switch, Rac1 alternates between inactive and active states to transduce cellular signals. The activated form of Rac1 is an indispensable subunit of NOX, which is essential for the subsequent activation of NOX and ROS generation (55). NOX is a transmembrane protein composed of two catalytic subunits (gp91phox and p22phox) and four regulatory subunits (p47phox, p40phox, p67phox and Rac1) (56). In a resting cellular state, NOX is inactive because there are no cytoplasmic subunits bound to the cell membrane (57). When cells are stimulated by internal and external factors, the cytoplasmic subunit of NOX is modified by phosphorylation to undergo a conformational change and ultimately assembles with the membrane-bound subunit to form a catalytically active oxidase (58). In addition, previous study has reported that there is a mutual activation between Rac1 and NOX, which is instrumental in sustaining physiological levels of ROS required for axonal growth of hippocampal neurons (59).

The current study has found that the E3 ubiquitin ligases HACE1 and the IAPs are responsible for the ubiquitination and proteasome degradation of activated Rac1 (60). During the further identification process, Stéphanie et al. (61) have found that HACE1 is the E3 ubiquitin ligase with the greatest influence on the regulation of Rac1 turnover and activity. As shown in the Figure 3 , it has been shown that HACE1 controls the activity of the Rac1-dependent NOX complex by targeting Rac1 for degradation, which inhibits the production of superoxide and ROS (29, 62). The deficiency of HACE1 leads to the hyperactivation of Rac1, resulting in increased cellular malignancy and invasiveness, thereby promoting cancer progression (25, 55, 63). In addition to its therapeutic role in tumors, HACE1 is significant for embryonic development through the Rac1 signaling pathway (64). Lack of HACE1 causes the accumulation of Rac1 and NADPH oxidase-dependent ROS production, which ultimately results in vertebrate cardiac and neurological hypoplasia (65, 66). Zang et al. (67) have found that HACE1 expression decreased with age in mouse models of Parkinson’s disease. HACE1 knockdown exacerbates neuroinflammation and symptoms in PD in a Rac1-dependent NADPH oxidase manner (67). Therefore, targeting HACE1 to inhibit the Rac1 signaling pathway to alleviate cellular oxidative stress would be an effective therapeutic strategy in degenerative diseases.

As research progressed, it has been discovered that activation of Rac1 is necessary for NADPH oxidase-dependent ROS production (62). Thus, one might wonder whether HACE1 regulates oxidative stress through degrading Rac1. To confirm this conjecture, we summarize the functional mechanisms and actions of HACE1 in regulating Rac1 in Table 2 . The expression of HACE1 protein is influenced by various factors, including gene editing, HACE1 mutations, and the regulation of HACE1-PTMs. HIF inhibitors (FIH) have been found to inhibit the ability of HACE1 to ubiquitinate Rac1 by hydroxylating HACE1 at residue N191 of the ANK domain (68). Neuroepithelial transforming gene 1 (NET1) protects Rac1 from degradation by HACE1 and is essential for actin dynamics and meiotic spindle formation (69). Furthermore, HACE1-driven Rac1 ubiquitination degradation also requires another E3 ubiquitin ligase, TRIP12. Silencing TRIP12 blocks the inhibitory effect of HACE1 on Rac1 protein degradation and esophageal cancer (ESCA) tumor growth (70). Rac1 has been considered a central signaling hub essential for many oncogene-induced transformations, playing a crucial role in cancer cell proliferation, migration, and invasion (71). HACE1 inhibits the progression of a variety of tumors, including breast cancer, lung cancer, prostate cancer, and osteosarcoma, by regulating the ubiquitination and proteasomal degradation of Rac1. Specifically, HACE1 protects cells from oxidative stress-induced DNA damage and cycle D1-driven hyperproliferation by blocking ROS production through Rac1-dependent NADPH oxidase. In addition, HACE1 plays a crucial role in the growth and development of the organism, especially the heart and nervous system. Taken together, HACE1 regulates cellular oxidative stress in a Rac1-dependent manner and is essential in organ development and tumor suppression.

Optineurin (OPTN) is a multifunctional protein with multiple domains and is broadly expressed in many organs and tissues in the human body (81). OPTN consists of two coiled-coil (CC) domains, a leucine zipper (LZ) domain, an LC3-interaction region (LIR), a HACE1-interaction region (HIR), a ubiquitin-binding (UBAN) domain, and a C-terminal Npl4-type zinc finger (NZF) domain (82). The N-terminal CC domain of OPTN binds directly to the C-terminal domain of TBK1, forming stable OPTN-TBK1 heterotetrameric complexes that are recruited into ubiquitinated aggregates (83). The LZ domain of OPTN interacts with ATG9A, which facilitates the recruitment of ATG9A vesicles for autophagosome formation (84). The LZ and LIR domains of OPTN bind to the N-terminal death effector domains (DEDs) of caspase 8 and regulate TNF-α-induced apoptosis (82). In addition, Wauters et al. have discovered that OPTN binds to ubiquitinated mitochondria through its UBAN domain and recruits LC3 to wrap damaged mitochondria to form autophagosomes through its LIR domain (85). Finally, Liu et al. have found that HACE1 can interact with OPTN through its N-terminal ANK structural domain, and the region on OPTN that interacts with HACE1 is defined as the HACE1 interaction region (HIR) (32).

Previous studies have established that TANK-binding kinase 1 (TBK1) induces autophagy through phosphorylation of the autophagy receptors p62 and OPTN (86). TBK1 phosphorylates directly OPTN and promotes its interaction with LC3 to accelerate autophagy efficiency (86). In turn, OPTN enhances the phosphorylation ability of TBK1 by promoting TBK1 activation (87). This process of autophagy activation forms a positive feedback loop to increase autophagic flux. It has been shown that HACE1 is upstream of TBK1 and can inhibit virus-triggered type I IFN signaling by disrupting the formation of the MAVS-TRAF3 complex (88). Therefore, in addition to focusing on the direct interaction of HACE1 with OPTN, HACE1 may also be involved in regulating autophagy efficiency by affecting TBK1 activity.

OPTN is a recognized selective autophagy receptor that mediates interactions between ubiquitinated proteins and autophagy machinery, and lack of OPTN leads to impairment of the autophagy pathway and accumulation of oxidized proteins (32). Furthermore, the autophagy activity of OPTN is affected by its expression level as well as post-translational modifications (89). HACE1 was found to enhance the interaction between OPTN and p62 by promoting ubiquitination of OPTN, and promote the formation of autophagy receptor complex to accelerate autophagy flux (32). Here, E3 ubiquitin ligase activity of HACE1 and subsequent ubiquitination of OPTN (specifically ubiquitination at Lys193) are essential for activation of autophagy. Disruption of the polyubiquitin chain on OPTN leads to impaired formation of the p62-OPTN complex, resulting in disruption of the HACE1-OPTN-p62 autophagy axis. More and more studies show that that HACE1 activates selective autophagy mediated by the HACE1-OPTN-P62 axis through ubiquitination of OPTN, thereby inhibiting tumor cell growth and progression (20, 90, 91). Thus, HACE1 bridges the gap between the ubiquitination and autophagy pathways by ubiquitinating the selective autophagy receptor OPTN. Targeting HACE1 to induce the ubiquitination of OPTN, thereby activating cellular autophagy, holds promise for the treatment of diseases characterized by autophagy deficiency.

p62 (also known as sequestosome-1, SQSTM1) is an adapter protein with multiple functional domains, including N-terminal PB1 domain, ZZ domain, TB domain, nuclear localization signal, nuclear export signal, PEST domain, LIR domain, KIR domain and C-terminal UBA domain (92). The LIR and UBA domains are key regulatory elements of p62 as a selective autophagy adaptor protein. p62 directly binds to LC3-II via the LIR domain to mediate autophagic degradation of specifically ubiquitylated cargoes bound to the UBA domain of p62 (93). The efficiency of p62-mediated autophagy is not only influenced by its UBA domain and ubiquitinated cargo but also by oligomerization mediated by the PB1 domain (94). p62 regulates the recruitment of target cargo and the formation of autophagosomes through the oligomerization mediated by the PB1 domain (95). The KIR domain of p62 participates in the regulation of cellular oxidative stress by binding to Keap1 and promoting the autophagic degradation of it (96). Besides, the linker region between the PB1 and ZZ domains is known as the regulatory linker (RL) region, which acts as a regulatory switch for p62 activity by interacting with the ZZ domain to regulate the binding ability of the ZZ domain (97). Taken together, these findings indicate that p62 interacts with various cellular signaling molecules through its multiple domains, thereby executing diverse cellular functions.

Autophagy is a dynamic process including the formation of autophagosomes, fusion of lysosomes and autophagosomes, and degradation of autophagosomes by lysosomes (98). Prior research has demonstrated that the autophagic activity of HACE1 is dependent on p62. The ablation of p62 disrupted the HACE1-OPTN autophagy axis, which could be reactivated by reintroducing p62 (32). Furthermore, this study also found that the HACE1-OPTN-p62 axis activates autophagy by promoting LC3II lipidation and the formation of LC3 puncta, thereby clearing p62 and carbonylated proteins to reduce ROS production (32). This avoidance of the production and accumulation of oxidative damage may be the underlying mechanism of the HACE1-OPTN-p62 axis inhibiting tumor growth and tumorigenicity. When the heart experiences hemodynamic stress, HACE1 exerts cardioprotective effects by activating p62-dependent selective autophagy to clear ubiquitinated protein aggregates. In contrast, mice lacking HACE1 have impaired autophagic flow and are less able to resist pressure stress, leading to accelerated heart failure and increased mortality (27). Mechanistically, HACE1 directly interacts with p62 to affect the cellular level of p62 independently of its E3 ubiquitin ligase activity (27). These findings have identified HACE1 as a regulator of autophagy, which activates cellular autophagy by promoting the activation of p62, thereby mitigating cellular stress injury.

Cyclin D1 (CCND1) is a protein encoded by CCND1 gene located on chromosome 11q13 and involved in regulating cell cycle progression (102). During the process of cell proliferation, cyclin D1 activates and binds to cyclin-dependent kinase 4 and 6 (CDK4/6) to form the cyclin D1-CDK4/6 complex (103). This complex phosphorylates retinoblastoma (Rb) protein, leading to the release of the E2F transcription factor, which promotes gene expression associated with G1/S phase transition (104). However, overexpression of cyclin D1 leads to cell cycle checkpoint failure and CDK dysregulation, which promotes tumor initiation and progression (105). Furthermore, the current studies demonstrate that cyclin D1 can also recruit methyltransferase G9a independently of CDK kinase activity to induce H3K9 demethylation, thereby regulating gene expression and signaling (106).

Cyclin D1 acts as a signal transduction factor in response to various intracellular and extracellular signaling factors. For example, extracellular growth factors induce Cyclin D1 expression by activating the Ras/Raf/MAPK signaling pathway (107). Recent studies have shown that the degradation of cyclin D1 can be regulated by proteasomal and autophagic lysosomes, and GSK3β-induced T286 phosphorylation of cyclin D1 is considered to be the main mechanism of cyclin D1 proteasomal degradation (108). Activation of the PI3K/AKT signaling pathway promotes Ser9 phosphorylation of GSK3β, which leads to GSK3β inactivation and cyclin D1 stabilization (109). Additionally, cytoplasmic membrane-associated cyclin D1 augmented the phosphorylation of AKT1 at Ser473 (110). This creates the positive feedback loop required to maintain intracellular cyclin D1 levels. AMBRA1 (autophagy and beclin 1 regulator 1) is a major regulator of cyclin D1 and mediates ubiquitination and proteasomal degradation of cyclin D1 (111). It has been reported that ubiquitinated cyclin D1 can be degraded via p62-mediated selective autophagy (101).

As a well-known tumor suppressor, HACE1 can inhibit the growth, invasion, and metastasis of multiple cancer types. Penninger and colleagues have found that the tumor suppressor effect of HACE1 may be attributed to cyclin D1, and HACE1 prevents cellular stress-induced tumor cell proliferation and adhesion-dependent growth through the degradation of cyclin D1 (30). Meanwhile, they have also shown that HACE1 inhibits ROS de novo synthesis and cyclin D1 expression by blocking Rac1-dependent NOX activity (29). Although there are no studies to show that HACE1 directly targets cyclin D1 for degradation, cyclin D1 transcription requires Rac1 and NOX activities to complete (112). Lack of HACE1 results in increased Rac1-dependent cyclin D1 transcription (29). Moreover, the expression level of cyclin D1 is inhibited by OPTN, a substrate protein of HACE1, which did this by limiting Rac1 activation (113). Taken together, these studies support the notion that HACE1 exerts a tumor suppressor function by regulating cyclin D1.

Cyclin C (CCNC) is an important cell cycle protein that regulates transcription and forms the kinase module of the mediator complex with the partner kinase CDK8 and the two auxiliary subunits Med12 and Med13 (114). The mediator complex stimulates the assembly of a pre-initiation complex (PIC) and recruitment of RNA Polymerase II, which is responsible for the transcription of all protein-coding genes and most non-coding RNA genes (115, 116). Apart from transcriptional function, cyclin C is involved in the regulation of oxidative stress by activating the mitochondria-dependent cell death pathway (117). Loss of cyclin C blocks the stress-induced mitochondrial apoptosis pathway, rendering malignant tumor cells insensitive to chemotherapy (118). Recent studies report that subcellular localization of cyclin C is critical for regulating cell fate (119). Cyclin C is primarily located in the nucleus during the resting state, interacting with the Med13 to regulate transcription (120). Under oxidative stress, cyclin C translocates from the nucleus to the cytoplasm, leading to activation of the mitochondrial apoptotic pathway (121). These findings suggest that subcellular localization of cyclin C is essential for oxidative stress-induced apoptosis.

The subcellular localization of cyclin C is closely related to its protein post-translational modification. Research suggests that the phosphorylation of cyclin C is essential for its translocation from the cytoplasm to the nucleus. Oxidative stress activates Slt2p to phosphorylate cyclin C protein at Ser266 triggering the mitochondrial intrinsic pathway of apoptosis (122). HACE1 mediates the non-proteolytic ubiquitination of cyclin C in the cytoplasm, thereby promoting nuclear–mitochondrial translocation of cyclin C and maintaining the chemosensitivity of gastric cancer cells to cisplatin (118). However, it has also been shown that cyclin C relocalizes for cytoplasmic degradation in response to oxidative stress, thereby inducing normal stress-responsive gene expression (123). This apparent contradiction could be explained by the differential stages of oxidative stress processes in different cell types. However, further research is needed to explore these speculative mechanisms. In conclusion, these studies suggest that cyclin C is an important regulator of cell fate determination by regulating oxidative stress-induced mitochondrial apoptosis.

HACE1 is an indispensable tumor suppressor gene, and hypermethylation of its gene region has been found in a variety of human malignancies, including gastric cancer, colorectal cancer, liver cancer, Wilms tumor, B-cell lymphomagenesis (124). DNA methylation of CpG islands in the promoter is an important mechanism for silencing gene expression. Under normal physiological circumstances, most of the genome is methylated, and CpG islands commonly near promoters remain typically unmethylated (125). During the process of tumorigenesis, human cancer cells undergo CpG island promoter hypermethylation and loss of non-CpG island promoter CpG methylation, resulting in cancer-specific methylation patterns (126). This epigenetic alteration affects tumor suppressor genes and oncogene expression, which may lead to the malignant progression of tumors. Previous research has shown that inactivation of HACE1 renders mice more susceptible to spontaneous and carcinogen-induced tumors (30). The inactivation of HACE1 may be attributed to gene silencing due to hypermethylation of CpG islands on its promoter region (124). Analysis of the TCGA database data further has confirmed that hypermethylation of CpG islands in the HACE1 promoter region in tumors is associated with low HACE1 expression (124). Taken together, the epigenetic phenomenon of CpG island hypermethylation in HACE1 gene promoters, leading to repression and silencing of expression, is an important contributor to oncogenesis.

There are three CpG islands in HACE1 gene regions, including CpG-88, CpG-177, and CpG-29. The hypermethylation of CpG29 and CpG177 islands upstream of the HACE1 transcription start site (TSS) is associated with its low expression level was reported in human Wilms tumor (22). Increased methylation of CpG177 is associated with decreased HACE1 expression in tumor tissue compared to normal tissue (30). This has also been shown in other tumors, such as aggressive natural killer cell leukemia (ANKL) (127) and natural killer-cell neoplasms (128). These findings indicate that the expression of HACE1 through demethylation may be a promising therapeutic strategy for malignant tumors.

Previous studies have provided evidence that HACE1 inactivation in multiple cancers is due to promoter methylation (63). Enhancing HACE1 expression by regulating methylation leads to extensive research on its regulatory mechanism and its tumor-suppressive effect. For example, effective restoration of HACE1 expression in nephroblastoma by the addition of the specific DNA methylation inhibitor 5-azacytidine (5-Aza) (22). NKX6.3 attenuates Helicobacter pylori CagA-induced cellular oxidative stress by inhibiting DNA methyltransferase 1 (DNMT1) and promoting HACE1 expression. The antioxidant activity of NKX6.3 could be eliminated by HACE1 silencing (129). The histone methyltransferase inhibitor 3-deazaneplanocin A (DZNep) inhibits lymphoma cell growth by promoting HACE1 expression by reducing histone methylation modifications (130). Demethylation of the HACE1 gene promoter has increased HACE1 expression, which inhibited liver cancer cell proliferation by activating OPTN-dependent selective autophagy (90). LINC00161 is a long non-coding RNA closely related to the occurrence and development of hepatocellular carcinoma (HCC), and its expression is upregulated in HCC cells. Inhibition of HACE1 expression by inducing HACE1 promoter methylation can promote the growth and migration of HCC cells. Knockdown of LINC00161 can reduce the methylation level and increase the expression of HACE1, thereby inhibiting the progression of HCC (131). The expression of MBD3, a demethylation molecule, induced by propofol in a dose-dependent manner, promotes HACE1 protein expression and inhibits lung cancer cell proliferation by activating HACE1-OPTN axis autophagy. However, these therapeutic effects of propofol are eliminated by MBD3 knockout (91). DNA methylation is an epigenetic modification that regulates gene expression that regulates gene expression. It is an important direction for future development and has research value and exploratory significance.

Gene expression is regulated by multiple pathways, including epigenetic regulation through DNA methylation and histone acetylation (132). We have also discussed in the previous section that HACE1 expression is regulated by the DNA methylation level of the promoter regions. However, HACE1 protein expression is regulated at multiple levels, including protein degradation and gene expression. Previous studies have reported that dimethyl fumarate (DMF), an Nrf2 activator, induces the protein expression of both Nrf2 and HACE1 in a dose-dependent manner, thereby inhibiting the proliferation of T-cell acute lymphoblastic leukemia cells (19). Additionally, Huang and colleagues have found that Magnolol, extracted from the renowned traditional Chinese medicine Magnolia officinalis, exerts its antitumor effects by activating HACE1-OPTN axis-mediated autophagy (20). Finally, the supplementation of the blueberry anthocyanin malvidin 3-glucoside (MG) in colon tissues has been found to increase HACE1 expression and improve the gut microbiota composition (21). The prevalence of degenerative diseases in recent years has triggered extensive research into their pathomechanisms and treatment. At the same time, growing evidence suggests that HACE1 also plays a crucial role in degenerative diseases. HACE1 is an important regulator of cell survival, it maintains cellular homeostasis by activating cellular autophagy and the antioxidant defense system, and its biological mechanism is shown in Figure 4 . Although the mechanism of action of HACE1 has been well studied, unfortunately, there are no recognized activators of targeted therapy. Therefore, a promising research focus to develop specific and potent activators of HACE1 is an urgent research problem that needs to be addressed.