Gamma-Interferon-Inducible Lysosomal Thiol Reductase (GILT)
In 1988, the laboratory of Dr. Jeffrey Ravetch characterized a gene which is upregulated upon exposure to the cytokine, interferon-γ (IFN-γ). The protein is produced in a precursor form of 35 kDa and processed into a mature form of 30 kDa. The protein was originally named inducible protein 30 (IP30) and the gene named IFI30. IP30 is constitutively expressed in many antigen-presenting cells (APCs) and induced in other cell types including fibroblasts, endothelial cells, and keratinocytes (Maric et al. 2001; Lackman and Cresswell 2006; Phipps-Yonas et al. 2013a; Nguyen et al. 2016).
Homology to protein folding patterns of a cellular thiol reductase, thioredoxin, suggested that IP30 may be capable of breaking disulfide bonds. Indeed, a thioredoxin-like motif, CXXC, is found in the IP30 protein sequence and is conserved across species with IP30 homologs. IP30 reduces disulfide bonds, and the cysteines in the CXXC motif are required for this activity (Arunachalam et al. 2000; Phan et al. 2000). Therefore, IP30 was given a new descriptive name, gamma-interferon-inducible lysosomal thiol reductase (GILT). GILT is the only known thiol reductase in the endocytic pathway. GILT is found in late endosomes and lysosomes where it co-localizes with major histocompatibility complex (MHC) class II (Arunachalam et al. 2000).
The reductase function and localization to the endocytic pathway led Dr. Peter Cresswell’s group to investigate the role of GILT in antigen processing and presentation. MHC class II-restricted processing and presentation require that proteins in the endocytic pathway are broken down into antigenic peptides, or epitopes, which are then presented in the context of MHC class II to generate CD4 T cell responses. GILT’s thiol reductase function proved to be critical for MHC class II-restricted processing and presentation of certain epitopes from disulfide bond containing proteins important in the immune response to pathogens, cancer, and autoimmunity (Haque et al. 2002; Sealy et al. 2008; Rausch et al. 2010; Bergman et al. 2012). In addition, GILT’s reductase function facilitates MHC class I-restricted cross-presentation by dendritic cells by enabling the retrotranslocation of exogenous antigens from the phagosome to the cytosol (Singh and Cresswell 2010). Thus, GILT’s reductase activity helps to shape the peptide repertoire for eliciting immune responses.
Additionally, GILT can restrict infection, as in the case of human immunodeficiency virus (HIV) and dengue virus (Kubo et al. 2016), or enhance microbial pathogenesis and escape from immune recognition, as in Listeria (Singh et al. 2008). Less well-described roles for GILT include decreasing oxidative stress, autophagy, and proliferation (Maric 2009; Chiang and Maric 2011). GILT expression is also associated with improved survival in certain cancer types (Phipps-Yonas et al. 2013a; Xiang et al. 2014). Each of these roles for GILT will be discussed in greater detail throughout this chapter.
Regulation, Trafficking, and Enzymatic Activity
Expression and Regulation
IFN-γ binds to a specific cell surface receptor which activates Janus kinases 1 and 2 (JAK1/JAK2) and the transcription factor signal transducer and activator of transcription 1 (STAT1). While IFN-γ-inducible expression of MHC class II and other genes in the class II antigen presentation pathway is regulated by IFN-γ-inducible isoforms of class II transactivator (CIITA), neither basal nor IFN-γ-inducible GILT expression is mediated by CIITA (O’Donnell et al. 2004). Instead, STAT1 regulates both constitutive and IFN-γ-inducible expressions of GILT (O’Donnell et al. 2004; Srinivasan and Maric 2010).
Maturation and Trafficking
In the ER, precursor GILT either dimerizes (~20%) through interchain disulfide bond formation at Cys-222 and is either secreted through the Golgi apparatus or is posttranslationally modified with a mannose-6-phosphate at one of its three putative N-linked glycosylation sites (Fig. 4; Arunachalam et al. 2000). The M6PR recognizes this modification and results in trafficking of precursor GILT to the endocytic pathway, initially localizing to the early endosomes (Arunachalam et al. 2000). Here, precursor GILT is cleaved to form the mature 30 kDa form lacking N- and C-terminal propeptides. The N-terminus is cleaved at Asn-32 or Ala-33 by cathepsins D, L, and S, while the C-terminal propeptide is cleaved at Lys-206 by cathepsins B, L, and partially S in vitro (Phan et al. 2000). However, in vivo data suggests that there are other proteases besides cathepsins capable of processing precursor GILT to its mature form (Phan et al. 2000). Cathepsin S (CatS) is also responsible for turnover of mature GILT. The mature form of GILT primarily localizes in late endosomes and multilaminar lysosomes known as MIICs due to their association with MHC class II antigen presentation components (Arunachalam et al. 2000). Indeed, mature GILT co-localizes in MIICs with MHC class II (Arunachalam et al. 2000).
GILT’s 11 conserved cysteines play important roles in protein stability, folding, and maturation. Mutation of Cys-106, Cys-122, Cys-136, or Cys-152 results in undetectable expression of GILT, suggesting a role for these residues in proper folding or stability of GILT. GILT mutants of cysteines at positions 91, 98, 200, and 211 are expressed, but fail to mature, resulting in an increased amount of precursor GILT that is secreted. Interestingly, Cys-211 is in the C-terminal propeptide, and mutation of Cys-200 or Cys-211 resulted in decreased stability of the secreted precursor form, suggesting these two cysteines likely form a disulfide bond that contributes to the dimer’s stability. Active sites Cys-46 and Cys-49 are also involved in GILT maturation, although the mechanism by which these cysteines participate in GILT processing is unknown (Hastings et al. 2006).
MHC Class II Presentation
The MHC class II-restricted antigen processing and presentation pathway is reviewed in Fig. 4. Newly synthesized MHC II α and β chains associate with invariant chain and form a trimer of trimers in the ER. Class II-invariant chain complexes can traffic to endosomes either via the cell surface or directly from the Golgi, directed by targeting motifs on the cytoplasmic tail of invariant chain. Cathepsin proteases are responsible for the sequential cleavage of invariant chain, proteolytic cleavage of antigenic proteins, and converting precursor GILT to its mature form. Proteolytic degradation of invariant chain ultimately leaves only the class II-associated invariant chain peptide (CLIP) in the peptide-binding groove of class II. CLIP prevents premature loading of peptides onto class II until the late endosome/lysosome (or MIIC) compartments. HLA-DM catalyzes the exchange of CLIP for a newly generated peptide and functions as a peptide editor. HLA-DO binds the same face of class II as HLA-DM and thus functions as a competitive inhibitor of class II interaction with HLA-DM. GILT-mediated reduction of protein disulfide bonds facilitates MHC class II-restricted presentation of certain epitopes from disulfide bond-containing proteins, presumably through exposing structurally constrained or buried regions of the protein for class II binding and protection from further proteolytic degradation (Hastings et al. 2006). The peptide/MHC class II complexes are directed to the plasma membrane for presentation to CD4 T cells.
APCs from GILT−/− mice have defects in MHC class II-restricted processing and presentation of particular epitopes (Maric et al. 2001). For instance, the model antigen hen egg lysozyme (HEL) contains four disulfide bonds. GILT is required for intracellular processing of HEL protein to generate the HEL peptide consisting of residues 74–88 (HEL74–88), which contains two cysteine residues that are each involved in disulfide bonds. Processing of HEL46–61, which does not contain cysteine residues, is partially diminished in the absence of GILT. The processing of two other HEL epitopes, HEL20–35 and HEL30–53, which share a cysteine that is involved a disulfide bond, is not affected by the absence of GILT. Thus, the mere presence of a cysteine involved in a disulfide bond in the epitope does not necessarily predict GILT dependence. A requirement for GILT may depend on the epitope’s location within the tertiary structure. A buried or disulfide bond constrained region is predicted to require GILT-mediated reduction for presentation, whereas a GILT-independent epitope may be surface exposed or, in a less structurally constrained site, accessible for class II binding without prior reduction. Despite not all HEL epitopes requiring GILT for efficient processing and presentation, the overall CD4 T cell response to HEL is decreased by 90% in GILT−/− mice compared to wild-type mice.
Class I and II epitopes altered by GILT expression. Class I epitopes (red) and class II epitopes (blue) are shown as GILT-dependent, GILT-independent, or GILT-prevented. Dependent epitopes require GILT expression for processing and presentation, while independent epitopes are presented regardless of GILT expression. GILT-prevented epitopes are either more abundantly or uniquely presented in the absence of GILT. Epitopes were determined by (1) reactivity of T cell hybridomas specific for a given peptide/MHC, (2) recall responses to specific peptides following immunization or infection of mice, and (3) peptide elution from MHC class II followed by mass spectrometry
Cross-Presentation on MHC Class I
Large protein antigens must be unfolded or undergo partial proteolysis for translocation from the phagosome to the cytosol. Thus, proteins constrained by disulfide bonds may require reduction for translocation. For herpes simplex virus 1 (HSV-1), cross-presentation of an immunodominant epitope from an envelope protein glycoprotein B (gB498–505), which contains five disulfide bonds, requires GILT’s reductase active site (Singh and Cresswell 2010). In contrast, direct presentation of gB498–505 in infected dendritic cells and cross-presentation of an epitope from an HSV-1 protein (ICP6822–829), which does not contain disulfide bonds, are GILT-independent. GILT is also required for cross-priming gB498–505-specific CD8 T cells in vivo. This role is not limited to HSV-1, as GILT is essential for cross-priming CD8 T cell responses following infection with influenza virus. GILT is required for efficient cross-priming of multiple epitopes from hemagglutinin (HA) and neuraminidase (NA) proteins, which contain disulfide bonds, while cross-priming of epitopes from polymerase (PA) and nucleoprotein (NP) proteins is GILT-independent (Table 1). Thus, GILT is not only involved in MHC class II-restricted processing in the endocytic pathway, but is also critical for facilitating cross-presentation on MHC class I.
T Cell Development and Activation
The effect of GILT in MHC class II-restricted antigen processing and presentation influences T cell development and activation. GILT is required for efficient MHC class II-restricted presentation of a self- and melanoma antigen, tyrosinase-related protein 1 (TRP1109–230; Rausch et al. 2010). Presentation of TRP1109–230 is not detected in GILT-deficient B cells, and presentation is ~100-fold more efficient in dendritic cells expressing GILT. Transfer of naïve TRP1-specific CD4 cells into recipient mice with and without GILT demonstrates that GILT accelerates the onset and severity of TRP1-specific T cell-mediated autoimmune vitiligo, which is associated with an increased frequency of TRP1-specific T cells with an effector memory phenotype. In addition to enhancing the T cell activation and function of mature T cells recognizing a GILT-dependent epitope, GILT also influences T cell development in the thymus. TRP1 is a self-antigen expressed in the thymus. In GILT-expressing mice, there is increased deletion of TRP1-specific T cells in the thymus (Rausch and Hastings 2012), demonstrating that expression of GILT enhances the thymic deletion of T cells recognizing a GILT-dependent epitope from an endogenously expressed self-protein. Thus, GILT’s role in antigen presentation modulates T cell development and peripheral activation.
GILT is the only known enzyme in the lysosomes and phagosomes to catalyze disulfide bond reduction with the potential to influence antigen processing and proteolysis within these compartments. The cysteine protease CatS is required for the cleavage of invariant chain and proteolysis of exogenous and endogenous proteins in these compartments for antigen presentation. However, excess proteolysis results in loss of epitopes and diminished antigen presentation. Thus, control of CatS activity can modulate proteolysis and antigen presentation. The proteolytic activity of CatS requires the active site cysteine to be in the reduced thiol state. An increasingly oxidizing environment, as in the case following respiratory burst, renders CatS less active. During respiratory burst, the phagocyte NADPH oxidase (NOX2) generates reactive oxygen species (ROS), namely, superoxide, which is then converted to H2O2. Macrophages alternately activated in response to IL-4 have decreased NOX2 expression and increased GILT expression and, thus, maintain CatS in the active state and display efficient proteolysis in phagosomes. In alternatively activated macrophages or in classically activated macrophages with NOX2 inhibition, GILT enhances bulk proteolysis, and in an entirely in vitro system, recombinant GILT enhances CatS activity, suggesting that GILT is responsible for maintaining the CatS cysteine active site in the reduced/active state in macrophages where proteolysis is favored (Balce et al. 2014).
In contrast, GILT expression in B cells inhibits proteolysis by CatS and may fine-tune the degree of proteolysis to favor antigen processing. In B cells, GILT’s reductase activity decreases the steady-state protein expression of CatS, and GILT expression decreases the half-life of CatS, suggesting that GILT-mediated reduction of protein disulfide bonds enhances CatS degradation (Phipps-Yonas et al. 2013b). Lysates from GILT-expressing B cells exhibit decreased proteolysis of a CatS selective substrate. Thus, in B cells, GILT inhibits proteolysis by CatS thereby regulating the degree of proteolysis to favor MHC class II-restricted antigen processing. Therefore, GILT may serve different functions regarding the regulation of cathepsins in different cell types and activation states and influence the bias toward proteolysis or antigen presentation.
Cellular Redox State
A series of studies have revealed that GILT regulates the cellular redox state. GILT expression in fibroblasts has been shown to decrease oxidative stress. Glutathione is the major low molecular weight redox buffer for controlling the cellular redox state, with an excess of the oxidized form of glutathione (GSH) over the reduced form of glutathione (GSSG) available to remove ROS, specifically H2O2. GILT expression diminishes oxidative stress by increasing GSH, thereby increasing the GSH/GSSG ratio, and altering the glutathione redox potential to maintain a more reduced state (Chiang and Maric 2011). Increased oxidative stress results in inactivation of multiple protein tyrosine phosphatases leading to increased phosphorylation of growth factor receptors and increased phosphorylation of mitogen-activated protein kinase cascades, including ERK1/ERK2. In the presence of GILT, there is decreased ERK1/ERK2 phosphorylation/activation and cell proliferation (Maric et al. 2009; Chiang and Maric 2011). GILT expression is also associated with increased levels of superoxide dismutase 2 (SOD2), a mitochondrial enzyme involved in removal of superoxide (Maric et al. 2009). This effect is likely indirect as GILT and SOD2 are localized to different cellular compartments (i.e., the late endosomes/lysosomes and the mitochondria, respectively). Oxidative stress and ERK1/ERK2 activation induce autophagy resulting in removal of dysfunctional mitochondria damaged by ROS. GILT-expressing fibroblasts exhibit decreased autophagy, which may in part explain increased SOD2 levels (Chiang and Maric 2011). Thus, GILT has been implicated in decreasing oxidative stress and impacting redox-sensitive cellular processes, proliferation, and autophagy.
T Cell Sensitivity
GILT expression in T cells results in a similar decrease in ROS, ERK1/ERK2 phosphorylation, and proliferation following T cell activating stimuli (Maric et al. 2009). These results suggest that GILT plays an inhibitory role in T cell activation by dampening the response to activating stimuli. In further support of this, GILT expression levels increase with T cell development from double-positive thymocytes to single-positive thymocytes or peripheral T cells and inversely correlates with induction of the early T cell activation marker CD69. Therefore, GILT expression may serve to control TCR sensitivity as T cells mature. In support of this, GILT−/− mice developed earlier onset and more severe hyperglycemia in streptozocin-induced diabetes, a CD8 T cell-mediated autoimmunity. Thus, GILT expression in T cells is critical for regulating T cell activation, which, when absent, can lead to aberrant responses and autoimmunity.
GILT’s role in autoimmunity has also been studied in experimental autoimmune encephalitis (EAE), a mouse model of multiple sclerosis. GILT expression alters the MHC class II epitopes recognized and the pathogenic mechanism in EAE (Bergman et al. 2012). Wild-type mice immunized with myelin oligodendrocyte glycoprotein (MOG) develop EAE that is mediated by CD4 T cells and depends on the presentation of the GILT-dependent epitope MOG35–55. MOG35–55 is the sole encephalitogenic epitope in MOG protein-induced EAE in wild-type mice. In contrast, T cells from GILT−/− mice immunized with MOG protein proliferate in response to several overlapping MOG peptides, which did not elicit responses in wild-type mice (Table 1). Consistent with the GILT dependence of the MOG35–55 epitope, GILT−/− mice do not generate T cell responses to MOG35–55 and are resistant to EAE induced by MOG35–55 peptide. Surprisingly, GILT−/− mice develop more severe EAE following immunization with MOG protein compared to wild-type mice. This unanticipated finding is due to a switch in the pathogenic mechanism from T cell-mediated autoimmunity in wild-type mice to antibody-mediated disease in GILT−/− mice.
Modulating antigen presentation is one way in which GILT may alter the host response to infection. As discussed above, several viruses have GILT-dependent MHC class I and II epitopes for generation of CD8 or CD4 T cell responses, respectively (Table 1). Some of these antigens include HSV gB and influenza HA and NA (Singh and Cresswell 2010). However, both viruses also have GILT-independent epitopes, including one in HA of influenza that is reduction-dependent, but GILT-independent. A GILT-dependent MHC class II epitope was identified for HIV in the envelope glycoprotein (Env/gp120; Sealy et al. 2008). This epitope lies at the base of the V1/V2 loop in an antiparallel beta sheet, with three disulfide bonds in the immediate vicinity. However, a second epitope in this region and overlapping with the GILT-dependent epitope is less affected by GILT.
GILT also alters microbial pathogenesis independent of antigen processing. GILT increases the pathogenesis of certain bacteria strains which utilize hemolysins. For instance, during infection with Listeria monocytogenes, the bacterium is phagocytosed by macrophages and evades immune destruction using the pore-forming listeriolysin O to escape into the cytosol. GILT−/− mice are resistant to Listeria monocytogenes infection (Singh et al. 2008). GILT-mediated reduction activates listeriolysin O (LLO), thus permitting bacterial escape to the cytosol. In GILT−/− mice, bacterial escape from the phagosome is delayed, accounting for the diminished infectivity of Listeria in GILT−/− mice. Additionally, secreted precursor GILT can activate streptolysin O, a virulence factor of Streptococcus pyogenes, suggesting that secreted GILT may enhance hemolysin-mediated tissue damage.
GILT expression can also restrict virus infection at different stages of the viral life cycle. For example, GILT expression confers resistance to dengue virus by reducing translation and genome replication efficiency, strongly correlating with decreased autophagy. GILT can also limit virion production posttranslation. GILT appears to deter HIV virion production by reducing disulfide bonds in CD63, a member of the tetraspanin family localized to the endosome/lysosome, thus preventing proper Gag-CD63 complex formation required for assembly (Kubo et al. 2016). Enzymatically active, but not mutant, GILT also diminishes infectivity of viral vectors displaying envelope proteins from HIV, murine leukemia virus (MLV), and vesicular stomatitis virus (Kubo et al. 2016). Thus, GILT is presumably reducing disulfide bonds within the envelope proteins. Consistent with this, in the presence of GILT, MLV’s SU protein is specifically depleted from extracellular virions when SU is normally tethered to the virion through a disulfide bond with TM (Kubo et al. 2016). This is an intriguing role for GILT as a restriction factor for viral replication and infection by its apparent action on viral envelope proteins and host factors, although the proteins mentioned here have not been directly verified as GILT substrates.
GILT expression is associated with improved patient survival in multiple cancers. GILT expression in diffuse large B-cell lymphoma (DLBCL) is associated with improved overall survival, and the association of GILT expression with survival is independent of established prognostic factors (Phipps-Yonas et al. 2013a). Similarly, in breast cancer patients, GILT expression in tumors correlates with less adverse tumor characteristics and improved disease-free survival (Xiang et al. 2014). Consistent with findings that GILT expression in T cells and fibroblasts decreases proliferation, GILT expression in breast cancer inversely correlates with staining of the proliferation marker Ki67. Another cancer type with the potential for GILT expression to be associated with improved prognosis is melanoma. MHC class II-restricted epitopes from two melanoma antigens, tyrosinase and TRP1, are GILT-dependent (Table 1; Haque et al. 2002; Rausch et al. 2010). In patient specimens, GILT expression is upregulated in melanoma cells compared with benign melanocytes, and GILT expression in melanoma cells is heterogeneous between patients (Nguyen et al. 2016). Multiple known functions of GILT have the potential to improve cancer survival, including enhancing antigen presentation and immune recognition and diminishing oxidative stress, proliferation, and autophagy. However, whether GILT is directly responsible for improved cancer survival and the mechanism of GILT’s effect remains to be determined.
Overall, GILT has a diverse set of cellular functions. The most well-characterized function is GILT’s role in antigen presentation via disulfide bond reduction. GILT also alters the activity or stability of lysosomal proteases in different antigen-presenting cells. GILT influences the peptide repertoire presented and the subsequent T cell responses in autoimmunity, infection, and cancer. GILT can even alter the immune-mediated mechanism of autoimmune pathogenesis. A further understanding of the role of lysosomal reduction in proteolysis and antigen presentation in different cells types, such as antigen presentation in the thymus for T cell selection as well as different dendritic cells populations, is warranted. Outside of antigen presentation, GILT’s reductase activity is important in microbial pathogenesis as a host factor used by bacterial hemolysins for activity and restricting viral infection. GILT also is associated with decreased oxidative stress, proliferation, and autophagy and improved patient survival in cancer; however, the mechanisms of these effects remain to be defined.
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