Beyond ISGlylation: Functions of Free Intracellular and Extracellular ISG15

Abstract

ISG15 is a ubiquitin-like type I IFN-stimulated protein of 15 kDa and is one of the most prominently expressed proteins in viral infections. ISG15 is widely known to be involved in a process called ISGylation, where it binds to over 150 targets from a variety of classes of proteins including central immune signaling pathways such as those mediated by NFκB, JNK, and IRF-3. However, ISG15 also exists in a free form that can act intra- or extracellularly. In vitro and in vivo evidences suggest that free ISG15 play different roles in several cellular processes, from cancer and defense against viral infections to activation of immune cells such as lymphocytes, monocytes, and NK cells. This review discusses the roles of free intracellular and secreted ISG15 approaching questions yet to be answered about the mechanism of action of this protein.

Introduction

In the context of viral infection, type I interferons (IFN-α and β) are secreted by infected cells and bind to their cognate receptors on the surface of neighboring cells where they initiate the JAK-STAT signaling pathway (Schreiber and Piehler 2015). This pathway leads to the induction of over 300 genes called interferon-stimulated genes (ISGs), which have different functions such as nucleic acid sensors, signal transducers, and signal activators (de Veer and others 2001) and together confer an antiviral state to the host cells.

The interferon-stimulated gene of 15 kDa (ISG15) is among the 10 most expressed ISGs (Der and others 1998). The first description of ISG15 was made in 1979 by Farrell and others (1979) who observed a polypeptide of ∼15 kDa accumulating in the cytoplasm of IFN-treated Erlich ascite tumor cells. After that, a series of studies published in the 80s and 90s allowed further characterization of this protein demonstrating that it is induced by IFN-α, IFN-β, and to a lesser extent, IFN-γ (Korant and others 1984; Haas and others 1987), it is expressed by human and other vertebrates immune and nonimmune cells (Haas and others 1987; Loeb and Haas 1992, 1994; D'Cunha and others 1996b) and, finally, that it is synthesized as a 17 kDa precursor that has its N-terminal methionine (Blomstrom and others 1986) and 8 C-terminal amino acids cleaved to produce the mature form of the protein (Knight and others 1988).

ISG15 has 2 main functions: a ubiquitin-like function where the protein is able to bind and regulate target proteins (Haas and others 1987; Loeb and Haas 1992, 1994) and a free form function, which can be secreted or act inside the cell and has been mainly associated with cytokine production and activation of immune cells (Recht and others 1991; D'Cunha and others 1996a). ISGylation has been the most studied function leaving the mechanism of action of the free form of ISG15 with several questions yet to be answered and which we will address in this review.

Intracellular Roles of ISGylation

ISG15 was the first ubiquitin-like protein to be described. It was so classified because it has 2 domains with around 50% similarity to the amino acid sequence of ubiquitin and a C-terminal sequence LRLRGG where the last glycine binds to lysine residues in target proteins (Haas and others 1987). For both ubiquitin and ISG15 this binding is mediated by 3 enzymes commonly referred to as E1 (activating enzyme), E2 (conjugating enzyme), and E3 (ligase) (Liu and others 2013). The binding of ISG15 is performed by the E1 UbE1 L (Yuan and Krug 2001), E2 UbcH8 (Zhao and others 2004), and E3 Herc5 (Dastur and others 2006; Wong and others 2006). This process is known as ISGylation and these enzymes and USP18, an enzyme able to remove ISG15 from target proteins (Malakhov and others 2002) are, as ISG15, interferon-stimulated genes (Yuan and Krug 2001; Zhao and others 2004; Wong and others 2006; Zou and others 2007).

Over 150 intracellular ISG15 targets have been identified (Giannakopoulos and others 2005; Zhao and others 2005; Wong and others 2006) but only a restricted number have had the biological function of this interaction elucidated. Most of these studies have shown that the binding of ISG15 can affect important signaling pathways involved in immune regulation such as IFN, NFκB, and JNK pathways. Of note, interferon regulatory transcription factor 3 (IRF3), a key transcriptional regulator of type I IFN-dependent responses, is one of ISG15 targets (Lu and others 2006). The binding of ISG15 to IRF3 inhibits its degradation by the proteasome leading to a more prominent IFN response in human cells (Shi and others 2010).

ISG15 also binds to protein kinase R (PKR). PKR is an IFN-inducible protein kinase that is activated by double-stranded RNA (Sadler and Williams 2007) and can be activated by ISG15 in the absence of viral RNA (Okumura and others 2013). Activated PKR is known to inhibit protein translation by phosphorylating eIF2α (eukaryotic Initiation Factor 2 alpha) and the activation of PKR by ISG15 leads to a reduction in protein translation in HEK293T cells (Okumura and others 2013).

Another ISG15 target is Retinoic Acid-Inducible Gene I (RIG-I), a RNA sensor that signal leads to the activation of IRF3 and NFκB. The binding of ISG15 to RIG-I reduces type I interferon promoter activity and NFκB response in mice cells (Kim and others 2008). Still on the NFκB pathway, ISG15 binds to Ubc13 (Takeuchi and Yokosawa 2005), a ubiquitin-conjugating enzyme that participates in the ubiquitination of transforming growth factor beta-activated kinase 1 (TAK1). The presence of ISG15 inhibits TAK1 ubiquitination leading to a negative regulation of the NFκB pathway (Minakawa and others 2008). Last, ISG15 binds to filamin B (Jeon and others 2009). Filamin B serves as a scaffold for RAC1, MEKK1, MKK4, and JNK facilitating IFN signaling (Jeon and others 2008) and binding of ISG15 to filamin B prevents the formation of that scaffold and, in consequence, diminishes IFN response (Jeon and others 2009).

Besides regulating specific signaling pathways, ISG15 also binds to viral proteins and it has been suggested that this protein has an important role in the response against viral infections in humans and mice. For instance, it has been demonstrated that ISG15 binds to HIV-1 Gag protein and to the Tsg101 protein from the endosomal sorting complexes required for transport (ESCRT) system compromising viral budding in 293T cells (Okumura and others 2006). Also, ISG15 binds to Nedd4, a ubiquitin ligase, inhibiting the ubiquitination of Ebola VP40 protein.

The lack of ubiquitination of VP40 avoids viral association with the ESCRT machinery reducing viral budding in 293T cells (Malakhova and Zhang 2008; Okumura and others 2008). In addition, binding of ISG15 to NS1 from Influenza A has also been reported and reduces viral propagation in human cell lines (Tang and others 2010). According to Zhao and others (2010), ISG15 is able to control influenza infection because it inhibits the conjugation of NS1 to importin-α and abrogates the transport of viral RNA to the nucleus where it should be replicated.

Although these and other studies have suggested that ISGylation is associated with protection against viral infections in humans in vitro (Morales and Lenschow 2013), works with individuals carrying a truncated and inactive form of ISG15 showed that they do not have increased susceptibility to several viruses (Bogunovic and others 2012; Zhang and others 2015; Speer and others 2016) when compared to normal individuals. These studies, which will be discussed in depth later on, showed that the absence of ISG15 increases a type I IFN response that would otherwise be regulated in normal individuals in a way dependent of ISG15 but independent of ISGylation. These results have driven the spotlight from ISGylation to free ISG15, giving opportunity to discuss how the free intracellular and extracellular forms of this protein participate in biological events.

The Roles of Free Intracellular and Extracellular ISG15

ISG15 is produced and secreted by several different types of cells including human primary monocytes, lymphocytes, neutrophils, plasmablasts, and immune (THP-1, Raji, Jurkart) and nonimmune cells lines (OVCAR-3 and A549) after type I IFN treatment (Knight and Cordova 1991; D'Cunha and others 1996b; Bogunovic and others 2012; Care and others 2016). Interestingly, ISG15 has been detected in the serum of patients after administration of IFNβ (D'Cunha and others 1996b) and of patients with hepatitis B virus (HBV) infection (Hoan and others 2016).

Only a few studies have contemplated potential ISG15 secretion pathways. Using inhibitors of cellular secretory processes, D'Cunha and others (1996b) suggested that ISG15 was not secreted by the Golgi complex, which is the classical protein transport pathway, or by the multidrug resistance glycoprotein, one of the nonclassical pathways. These data led the authors to associate ISG15 with the proinflammatory cytokine Interleukin-1β (IL-1β). Like IL-1β (Auron and others 1984; Rubartelli and others 1990), ISG15 is synthesized from a larger precursor (Knight and others 1988), lacks a secretory signal peptide (Knight and Cordova 1991). and is not secreted by the classical pathway (D'Cunha and others 1996b) (Fig. 1).

FIG. 1.

An overview of free-ISG15 secretion and function in known target cells: ISG15 is an interferon-stimulated gene produced in response to both IFN-α and β produced in viral or bacterial infections. Free ISG15 can be found in the extracellular milieu and carry out different functions. T cells, neutrophils, monocytes, and epithelial cells secrete this protein through nonclassical unknown mechanisms and it exerts cytokine-like responses in target cells. The main role of free extracellular ISG15 is the participation in IFN-γ production by T or NK cells. In the latter, ISG15 has been described to act in synergy with IL-12, a potent activator of IFN-γ production.

It has been shown previously that IL-1β can reach the extracellular environment through secretory lysosomes (Rubartelli and others 1990), shedding of plasma membrane microvesicles (small membranous particles of 100–1,000 nm thought to provide a communication network between host cells) (MacKenzie and others 2001; Pizzirani and others 2007), exocytosis of exosomes (Qu and others 2007), and through apoptosis (Hogquist and others 1991). As for ISG15, neutrophil granules and microvesicles have been reported as potential secretion pathways. ISG15 colocalizes with gelatinase and secretory granules in neutrophils (Bogunovic and others 2012) suggesting the most likely ISG15 secretion pathway in granulocytes. Neutrophils are the first cells to arrive at the site of infections and are able to produce several cytokines and chemokines that are important for the initial inflammatory response (Lacy and Stow 2011).

IL-8, IL-6, and IL-1β are examples of cytokines that have also been detected inside gelatinase granules of neutrophils (Naegelen and others 2015). Besides gelatinase granules, ISG15 was found in microvesicles released from Mtb-infected macrophages. Interestingly, addition of ISG15-containing microparticles to uninfected macrophages increased the secretion of the proinflammatory cytokine and chemokines interleukin-8 (IL-8), macrophage inflammatory protein-1 α (MIP-1α), and IFN gamma-inducible protein 10 (IP-10) and induced the expression of more ISG15 along with other ISGs such as IFIT1, IFIT2, and IFIT3 (Interferon Induced Protein with Tetratricopeptide Repeats 1, 2, and 3) (Hare and others 2015).

ISG15 has also been found in exosomes released by TLR3-activated human brain microvascular endothelial cells. Primary macrophages are able to internalize these exosomes, an event that leads to an increase in the expression of ISGs mRNAs and proteins, including ISG15 itself (Sun and others 2016). It is important to note that not only neutrophils but also monocytes, T cells, cDC, pDC, and NK cells all had a constitutive production of ISG15 protein even in the absence of type I IFN (Bogunovic and others 2012) suggesting they produce and might be able to secret ISG15 in physiological circumstances (Fig. 1).

Once secreted, ISG15 is able to modulate the function of different immune cells. It has been demonstrated that this protein increased the cytotoxicity of LPS-stimulated primary monocytes (Recht and others 1991), induced the production of IFN-γ (Recht and others 1991), augmented NK cell proliferation in a B lymphocyte-depleted primary culture without inducing the production of IL-2 or IL-12 (D'Cunha and others 1996a) and had chemo-attractant properties to murine neutrophils (Owhashi and others 2003) (Fig. 1).

The production of IFN-γ induced by free extracellular ISG15 is an important function of this protein. Individuals carrying mutations in ISG15 can develop mendelian susceptibility to mycobacterium disease (Bogunovic and others 2012), a rare condition that makes the subject predisposed to severe clinical symptoms when infected even with less pathogenic species of mycobacteria including the Bacille Calmette-Guérin, used for Mycobacterium tuberculosis (Mtb) vaccination.

The lack of a functional ISG15 results in lower IFN-γ production, a cytokine known to promote macrophage activation, which in turn, is crucial for protection against Mtb (Flynn and others 1993). Bogunovic and others (2012) demonstrated that treatment with recombinant ISG15 rescued IFN-γ production by total leukocytes from ISG15-deficient subjects in vitro. In this system, NK cells were the major producers of IFN-γ among peripheral blood mononuclear cells treated with ISG15 and, in addition, this protein worked synergistically with IL-12 without increasing the expression of IL-12 receptor subunits in T or NK cells. It is important to note that the ability of ISG15 to induce IFN-γ is due to free ISG15 only and not due to ISGylation once a mutated form lacking the C-terminal glycines (ΔGG ISG15) displayed equivalent capability to induce IFN-γ production in NK cells (Bogunovic and others 2012) (Fig. 1).

In addition to tuberculosis, other malignancies have also been associated with free extracellular ISG15 such as lupus, cancer, and viral infections, but the presence of ISG15 does not necessarily have a protective phenotype.

An increased production of type I IFNs and ISGs in active lupus have been reported in several studies (Baechler and others 2003; Bennett and others 2003; Kirou and others 2005; Feng and others 2006; Yao and others 2009). Feng and others (2006), for instance, have shown that ISG15 is among the genes overexpressed in patients with lupus nephritis, being suggested as a marker for the disease. Care and others (2016) identified a sustained production of ISG15 and components of the ISGylation pathway and ISG15 secretion in plasmablasts from patients with systemic lupus erymethematosus, a profile comparable with that of plasma cells from healthy donors treated with IFN-α. Although overexpression and secretion of ISG15 have been associated with active lupus, it is still elusive whether this protein plays a protective or detrimental role in the disease.

ISG15 is also associated with cancer. Many works have associated an increase in expression of ISG15 and/or of ISGylation with tumorigenesis (Padovan and others 2002; Andersen and Hassel 2006; Desai and others 2006, 2008; Bektas and others 2008; Kiessling and others 2009; Rajkumar and others 2011) but the literature is filled with conflicting results suggesting that ISG15 has both pro- and antitumor activity. Of note, it has been reported that ISG15 is more secreted by melanoma tumors that induced dendritic cell (DC) maturation than by tumors that did not and also that melanoma cells overexpressing ISG15 induced expression of E-cadherin in DCs in vitro (Padovan and others 2002). E-cadherin is an adhesion molecule and its expression can impair DC motility and work as a mechanism of tumor escape (Zeng and others 2007) implicating in a protumor role for free ISG15 in melanoma.

Protumor activity was also observed for free ISG15 in pancreatic ductal adenocarcinoma (PDAC). This protein is secreted by macrophages present in the tumor in response to IFN-β produced by PDAC cells and acts upon cancer stem cells enhancing their tumorigenicity. In addition, ISG15 secretion was shown to be increased in M2, or alternatively activated macrophages (Sainz and others 2014), which are often associated with promoting tumor growth directly and via angiogenesis as well as tissue remodeling and suppression of adaptive immunity (Mantovani and others 2002).

In human tissue, human cells lines and mouse models of breast cancer, although ISGylation is more prominent and related to an unfavorable prognosis (Bektas and others 2008; Desai and others 2012), free ISG15 appears to have an antitumor function (Wood and others 2012; Burks and others 2015). Injection in mice of a fusion of ISG15 gene downstream of a DNA sequence coding for truncated Listeriolysin O (tLLO), a hemolysin produced by the bacteria Listeria monocytogenes, resulted in secretion of the ISG15-tLLO protein, consequent induction of IFN-γ, and significant reductions in primary tumor burden, in metastatic spread and in tumor incidence for 3 separate models of mouse breast cancer (Wood and others 2012).

Supporting these data, breast cancer cells with a silenced ISG15 gene resulted in faster growing tumors in nude mice when compared to cells overexpressing ISG15 (Burks and others 2015). The same group has also shown that free ISG15 promoted intratumor infiltration of NK cells inhibiting tumor growth when added extracellularly and that intracellular free ISG15 enhanced antigen presentation and expression of MHC class I complexes on breast cancer cells.

The works of Wood and others (2012) and Burks and others (2015) have suggested that free ISG15 has an antitumor activity resulting from an induction of IFN-γ or an increase in cancer surveillance by NK cells, but the molecular mechanisms underlying these events are still elusive. Interestingly, a few antitumor mechanisms have been suggested for ISGylation. P53, a transcription factor that activates cell cycle arrest and apoptosis to prevent propagation of damaged cells (Bieging and others 2014) is a target for ISG15. The binding of ISG15 to misfolded p53 increases its degradation by the proteasome contributing to protein turnover (Huang and others 2014).

In another study, Park and others (2016) demonstrated that DNA damage induces ISG15 conjugation to p53 and this modification markedly enhances the binding of p53 to the promoters of its target genes leading to suppression of cell growth and of tumorigenesis. It is important to note that protumor mechanisms have also been associated with ISGylation (Park and others 2016). Desai and others (2006) have shown that different human tumor cell lines have an increased ISGylation and lower ubiquitination suggesting that ISG15 can interfere with the ubiquitin/26S proteasome pathway and, consequently, with the regulation of proteins involved in cell growth and death.

Last but not least, free intracellular ISG15 has also been associated with the regulation of immune response in viral infections. As mentioned previously in this review, individuals carrying a nonfunctional form of ISG15 have a normal response to viral infection and this response have a central participation of free intracellular ISG15. Individuals carrying a nonsense mutation in the isg15 gene had an enhanced type I IFN activity with high expression of ISGs and persistent phosphorylation of STAT1 and STAT2. This activity would lower to normal levels in cells transduced with the wild-type form of ISG15, what also applied to an ISG15 lacking the C-terminal glycines (ΔGG ISG15), a form incapable of ISGylating.

The higher IFN-α/β response in ISG15-deficient patients is due to an inability to sustain proper levels of USP18 (Zhang and others 2015). USP18 is responsible for downregulating type I IFN response (Malakhova and others 2006; Francois-Newton and others 2012). In normal individuals, ISG15 regulates USP18 in an ISGylation-independent way, controlling the expression of ISGs while in ISG15-deficient patients, the absence of this protein leads to lower levels of USP18 and an increased expression of ISGs (Zhang and others 2015).

In another study, fibroblasts from ISG15-deficient individuals were infected with human immunodeficiency virus type 1 (HIV-1), human cytomegalovirus (HCMV), influenza A virus (IAV), sendai virus (SeV), rift valey fever virus (RVFV), and nipah virus (NiV). With the exception of NiV, levels of viral replication were significantly lower when compared to the controls (Speer and others 2016). As demonstrated previously by Zhang and others (2015), ISG15-deficient patients displayed an increased IFN-α/β response and increased expression of ISGs that enhanced their antiviral protection. Interestingly, when they transduced ISG15-deficient cells with wild-type or ΔGG ISG15, viral susceptibility was rescued showing that not ISGylation but free intracellular ISG15 is able to regulate IFN response. It is worth noting that none of what was observed in ISG15-deficient human cells applied to ISG15-silenced murine cells (Speer and others 2016).

Previous studies have shown an important role for ISG15 in the antiviral response in vivo in mice (Lenschow and others 2005, 2007; Guerra and others 2008; Giannakopoulos and others 2009; Lai and others 2009), however, the works of Zhang and others (2015) and Speer and others (2016) suggest this might not be the most important role of ISG15 in humans. One explanation for this difference between species could be that ISG15-silenced murine cells have normal levels of USP18 and decreased levels of ISGs (Speer and others 2016), a different phenotype than that observed for ISG15-deficient humans.

Potential Mechanisms of ISG15 Signaling

The mechanism through which secreted ISG15 induces immune cell activation and IFN-γ production is still unknown. As the known cytokines, it is possible that ISG15 signals through a receptor, triggering signaling pathways responsible for its functions. In another scenario, it is possible that ISG15 acts through the IL-12 receptor (IL-12R) since it acts synergistically with IL-12 in the IFN-γ production (Bogunovic and others 2012).

A third hypothesis is that ISG15 binds to a surface molecule, which is not a receptor but a transporter. CD36, for example, works as a facilitator for the endocytosis of soluble ligands such as amiloyd-β, oxidized low-density lipoprotein, and islet amyloid polypeptides, which induce NLRP3-inflammasome activation once inside the cell (Sheedy and others 2013). Another possibility to be considered is that ISG15 is delivered to the cell through microvesicles and granules that are released from macrophages and neutrophils, as mentioned previously. These structures could be captured by other cells where ISG15 would exert its function (Hare and others 2015). Similar to what is suggested by this hypothesis is the new mechanism of action recently described for OAS1 (2′-5′-Oligoadenylate Synthetase 1).

OAS1 is an ISG and an UBL with antiviral properties (Eskildsen and others 2003) that, like ISG15, is secreted despite the absence of a signal peptide and accumulates in the serum of patients with HCV infection or undergoing IFN-α treatment (Yang and others 1997; Merritt and others 2010). OAS1 is captured by cells from the medium, potentially through vesicles such as lysosomes or endosomes, executing inside the cell an alternative antiviral mechanism different from the RNA degradation together with RNAse L (Kristiansen and others 2010; Thavachelvam and others 2015).

Lastly, in case ISG15 is present in apoptotic cells, it could be released after cell lysis and phagocytized by other cells. This is seen for ASC, an adaptor protein central for the inflammasome activation (Srinivasula and others 2002). Franklin and others (2014) showed that ASC specks released by pyroptotic cells are phagocytized by macrophages where they induce lysossomal damage being released into the cell cytoplasm leading to IL-1β production. Nonetheless, it has been reported previously that ISG15 secretion is not due to cell death (D'Cunha and others 1996b).

Conclusion

ISG15 is a recent evolutionary acquisition present only in vertebrates and there is still a long way before we can conclude the full spectrum of its actions. There are several evidences that it participates in a wide range of cellular processes and malignancies but the molecular mechanisms underlying many of these events, especially those regarding free ISG15, are still elusive. Also, ISG15 is able to trigger IFN-γ production and other signaling pathways suggesting it is a potential cytokine that could interact with still unknown receptors. Therefore, more studies on the molecular mechanisms of action of free ISG15 and its abilities to modulate other cells could contribute to our understanding of immune responses.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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