Endoplasmic Reticulum Aminopeptidase 1 beyond Antigenic Peptide-Processing Enzyme in the Endoplasmic Reticulum

Abstract

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is well known as a processing enzyme of antigenic peptides, which are presented to major histocompatibility complex (MHC) class I molecules in the lumen of endoplasmic reticulum. Besides antigen processing, ERAP1 performs multiple functions in various cells depending on its intracellular and extracellular localization. Of note is the secretion of ERAP1 into the extracellular milieu in response to inflammatory stimuli, which further activates immune cells including macrophages and natural killer cells. Furthermore, secreted ERAP1 enhances the expression of pro-inflammatory cytokines like tumor necrosis factor-α, interleukin-1β, and interleukin-6. Such findings indicate that ERAP1 plays a significant role in the field of innate and acquired immunity. This review summarizes the functional analyses of ERAP1 that support our current understanding of its role as more than an antigenic peptide-processing enzyme, specifically emphasizing on its secretory form.

1. INTRODUCTION

Aminopeptidases are exopeptidases that catalyze the hydrolysis of amino acid residues from N-terminal of peptide or protein substrates. They are known to play roles in protein maturation, activation, modulation, and degradation of bioactive peptides, as well as determining protein stability.¹⁾ Among them is endoplasmic reticulum aminopeptidase 1 (ERAP1) belonging to M1 family of aminopeptidases, which require Zn²⁺ for hydrolysis (gluzincin). Phylogenetic tree in Fig. 1 shows the 12 enzymes in human belonging to M1 family.²⁾ These enzymes share the consensus motifs, HEXXH(X)₁₈E and GXMEN. HEXXH(X)₁₈E gluzincin motif functions as a zinc ligand and is essential for the catalytic activity of the enzyme while GXMEN motif contributes to exopeptidase specificity of enzyme by interacting with N-terminal amino acid of the substrate.³⁾ It has become evident that M1 family-aminopeptidases play important pathophysiological roles in the regulation of blood pressure, angiogenesis, and immunological responses.³⁾ In this review, we will focus on the functional properties of ERAP1, which we cloned and initially, named it as adipocyte-derived leucine aminopeptidase (A-LAP).⁴⁾ It is now well recognized that ERAP1 is primarily an antigenic peptide-processing enzyme located in the lumen of endoplasmic reticulum (ER-lumen), and involved in several autoimmune and inflammatory diseases.^5–8) However, there are additional activities of ERAP1 that have not been well documented to date. Here, we summarize multiple functions of ERAP1 enzyme outside ER-lumen.

Fig. 1. Phylogenetic Analysis of M1 Family of Human Aminopeptidases

Among M1 family, ERAP1 belongs to the oxytocinase subfamily with ERAP2 and P-LAP/IRAP. (Color figure can be accessed in the online version.)

Human ERAP1 is named after its localization in the ER-lumen. Its murine orthologue is known as endoplasmic reticulum aminopeptidase associated with antigen presentation (ERAAP).^9,10) In this review, we have employed the term, ERAP1.

2. TWO FORMS OF ERAP1

Figure 2 shows the structure of human ERAP1 gene and its products (mRNAs and proteins). Initially, ERAP1 was cloned by us as a homologous protein together with placental leucine aminopeptidase (P-LAP)/insulin regulated aminopeptidase (IRAP) that is known to increase in the blood vessel during pregnancy.⁴⁾ Sequence alignment of M1 family of aminopeptidases reveals that ERAP1 is highly related to leukocyte-derived arginine aminopeptidase (L-RAP) or ERAP2.¹¹⁾ Presently, it is classified as part of the oxytocinase subfamily of M1 family alongside P-LAP/IRAP and ERAP2. Protein-coding genes of the human oxytocinase subfamily are clustered on chromosome 5q15.

Fig. 2. Structure of Human ERAP1 Gene and Its Products

ERAP1 gene is clustered with those of ERAP2 and P-LAP/IRAP on the 5q15 locus. Alternative splicing generates two forms of mRNAs with different C-terminal amino acid sequences (ERAP1-19E and ERAP1-20E). The C-terminal amino acid sequences of two isoforms (RM for ERAP-19E, HDPEADATG for ERAP1-20E) are shown in the figure. Both mRNAs have two polyadenylation sites to generate four different mRNAs. Full-size proteins translated from the mRNAs comprise of four domains. (Color figure can be accessed in the online version.)

Human ERAP1 gene contains 20 exons. Similarity has been reported among different members with respect to the exon/intron structures suggesting their origin through gene duplication. GXMEN and HEXXH motifs have been reported to be encoded by exon 6 while 19 amino acids downstream from the HEXXH motif encoded by exon 7 is the glutamic acid residue, which together with the two histidine residues in HEXXH motif binds the zinc ion.¹²⁾ Phylogenetic analysis of M1-aminopeptidase family also implies a divergence in ERAP from P-LAP/IRAP²⁾ (Fig. 1).

It is noteworthy that P-LAP/IRAP has been shown to lack a nucleotide sequence corresponding to exon 10 of ERAP1 and ERAP2.¹³⁾ This difference indicates that exon 10 sequences of ERAP1 and ERAP2 were inserted in the ancestral gene of the oxytocinase subfamily enzymes after diversion, and assist in encoding the functional properties, which are specifically assigned to ERAP1 and ERAP2 and typically retained in the ER-lumen. In addition, the coding sequence in exon 10 of ERAP2 has modest homology with the coding sequence of ERAP1 suggesting that exon 10 coding sequences were independently incorporated in the two genes.

Alternative splicing between exon19 and exon 20 generates at least two ERAP1 mRNAs that code for full-length proteins (Fig. 2). While we identified and initially designated these two isoforms as A-LAPa and A-LAPb,¹²⁾ another group termed them as ERAP1b and ERAP1a, respectively.¹⁴⁾ To avoid nomenclature confusion, the isoforms encoded by these mRNAs were re-designated as ERAP1–19E and ERAP1–20E, respectively.^15,16)

Exon 19 contains the coding sequence for the last 51 amino acids of ERAP1–19E, stop codon, and at least two consensus polyadenylation signals. On the other hand, exon 20 contains the coding sequence for the last nine amino acids of ERAP1–20E, stop codon, and at least two consensus polyadenylation signals. The two mRNAs for these isoforms are generated by alternative splicing that encodes different amino acids at the C-terminus (ERAP1–19E: RM, ERAP1–20E: HDPEADATG). A nucleotide sequence located on exon 19 functions as a splicing donor to generate ERAP1–20E mRNA. Thus, at least four cDNAs encoding full size ERAP1 proteins with potential absolute enzymatic activity have been identified¹²⁾ (Fig. 2). However, functional differences between ERAP1–19E and ERAP1–20E, if any, remain elusive.

On the other hand, murine ERAP1 gene seems to be composed of 19 exons. An exon corresponding to exon 20 of human gene has not been found. In addition, there is no consensus sequence of 5′-splicing site in exon 19 registered in the database (NCBI Reference Sequence: NM030711), suggesting a lack of alternative splicing. It is thus most plausible that in the protein level, only a single form of the murine enzyme is expressed.

Similar to other members of the major histocompatibility complex (MHC) class I molecule-loading machinery, transcriptional expression of ERAP1 is enhanced by interferon γ (IFN-γ).^9,10) It has been shown that non-stimulated cells express relatively higher levels of ERAP1–20E mRNA and protein compared to the mRNA and protein expression of ERAP1–19E.¹⁶⁾ On stimulation with IFN-γ, ERAP1–19E mRNA has been reported to be up-regulated, while the expression level of ERAP1–20E mRNA has been observed to be rather constant. Based on change in ERAP1–19E/ERAP1–20E ratio on IFN-γ stimulation, it is conceivable that ERAP1–19E and ERAP1–20E play different roles depending upon the pathophysiological conditions.¹⁶⁾

It is intriguing that human cytomegalovirus induces microRNAs (miRNAs) targeting ERAP1 as an immune evasion strategy. Two miRNAs that target ERAP1–19E mRNA (miR-US4-1 and miR-UL112-5p) have been identified.^14,17) In addition, it was reported that ERAP1–20E mRNA was not a target of miR-US4-1. It has been shown that ERAP1-induced trimming of virus-derived peptide is inhibited lowering the susceptibility of infected cells to virus-specific cytotoxic T lymphocytes. These results indicate that ERAP1 is an important host defense enzyme against viral infection. Considering that only miRNAs targeting ERAP1–19E mRNA for degradation or translational repression were detected so far, it is plausible that the contribution of ERAP1–19E in the generation of cytotoxic T cells against virus-infected cells is more significant than that of ERAP1–20E. These results support the idea that the two isoforms play different roles depending upon the pathophysiological conditions that are assigned to ERAP1, and thus, the enzyme exhibits multi-functional properties. However, it should be reminded that only a single form of the enzyme is expressed in murine.

3. MULTIPLE FUNCTIONS OF ERAP1

Mature ERAP1 comprises of four protein domains (Fig. 2), and its enzymatic properties are well characterized to date.^18–21) Synthetic substrate analogue shows that the enzyme preferentially cleaves neutral amino acids such as leucine (Leu) and methionine (Met), whereas charged amino acids are less efficiently hydrolyzed.⁴⁾ However, as an antigenic peptide-processing enzyme, it is capable of releasing broad range of amino acids from N-terminal end of peptide substrates excluding the residues that are followed by proline (Pro). Thus, the substrate specificity of ERAP1 for N-terminal residues of peptide substrates is rather obscure. In contrast, an evident preference has been observed for the peptide length and C-terminal hydrophobic amino acids of the substrates. Accordingly, a unique “molecular ruler model” for ERAP1 specificity related to peptide substrates has been proposed.¹⁹⁾ These properties make ERAP1 a suitable antigen-processing enzyme.

Following cloning of ERAP1, its diverse pathophysiological functions and relationships with autoimmune and inflammatory diseases have been proven by several independent groups.^11,22,23) Role of ERAP1 in trimming antigenic peptides that are presented to MHC class I molecules, and its involvement in autoimmune and inflammatory diseases has been documented extensively in many review articles to date.^24–27) Thus, we chose to focus on additional multiple functions of the enzyme for this review.

It is intriguing that depending on the function being studied, the subcellular localization of ERAP1 is observed to vary (Fig. 3).

Fig. 3. Multi-Functional Properties of ERAP1

Various pathophysiological functions that have been assigned to ERAP1 are dependent on its location in intracellular or extracellular milieu. (Color figure can be accessed in the online version.)

Studies have reported that on cloning ERAP1 as puromycin-insensitive leucyl-specific aminopeptidase (PILSAP), it was considered a cytosolic protein that promotes angiogenesis.^28–30) ERAP1/PILSAP expression was observed to be up-regulated during differentiation of murine stem cells into endothelial cells when induced by vascular endothelial growth factor (VEGF). Furthermore, treating ERAP1/PILSAP mRNA with an antisense oligodeoxynucleotide (AS-ODN) was reported to reduce VEGF-stimulated proliferation, migration, and network formation of endothelial cells in vitro. Moreover, study showed that AS-ODN treatment inhibited in vivo angiogenesis, which supported the notion of ERAP1/PILSAP -mediated promotion of angiogenesis.

On the other hand, it was reported that ERAP1 overexpression in endometrial carcinoma cells suppresses angiotensin II (Ang II)-induced VEGF expression and did not induce VEGF-mediated angiogenesis in vivo.³¹⁾ The study further explained that overexpressed ERAP1 is probably secreted into blood vessels to inactivate Ang II. Overall, it is conceivable that the enzyme exhibits dual effects in angiogenesis depending on the condition of ERAP1. While ERAP1 associated with endothelial cells is reported to promote angiogenesis presumably via activation of integrins,²⁹⁾ the secreted enzyme acts as a negative regulator for angiogenesis via Ang II inactivation.

ERAP1 is also known as aminopeptidase regulator of TNFR1 shedding 1 (ARTS-1). Specifically, ERAP1/ARTS-1 has been shown to bind and facilitate ectodomain shedding of the cytokine receptors such as tumor necrosis factor R1 (TNFR1), interleukin 6 receptor α (IL-6Rα), and IL-1RII.^32–34) As the translated product of ERAP1/ARTS-1 cDNA was found to contain N-terminal hydrophobic region capable as a membrane-spanning domain, ERAP1/ARTS-1 was proposed to be a transmembrane protein that bound to cytokine receptors in the plasma membrane.³²⁾ Cells overexpressing ERAP1/ARTS-1 demonstrated an increase in receptor shedding whereas cells expressing AS-ODN to ERAP1/ARTS-1 mRNA were found to have decreased membrane-associated ERAP1/ARTS-1 level and receptor shedding. These results supported the role of ERAP1/ARTS-1 in receptor shedding. In addition, it was reported that while catalytic activity of ERAP1/ARTS-1 was required to exhibit receptor shedding of IL-6Rα and IL-1RII, TNFR1 shedding did not require such activity. Even if the enzymatic activity of ERAP1/ARTS-1 is required for shedding of cytokine receptors, it is unlikely that ERAP1/ARTS-1 itself acts as an endo-protease to liberate the receptors. This is because the enzyme is an aminopeptidase that efficiently cleaves N-terminal amino acid of peptide substrates having approximately 9 to 16 amino acids in length.^18,19) It is likely that unidentified proteases are involved in receptor shedding. Thus, it is most plausible that ERAP1/ARTS-1 primarily acts as a binding partner to these receptors forming a scaffold for receptor shedding.

Several proteins have been identified such as phosphatidylinositol-dependent kinase 1 (PDK1), nucleobindin 2 (NUCB2), and X-linked RNA-binding motif protein that bind to ERAP1 and modulate its diverse functional activities.^35–37) It is speculative that ERAP1 changes its binding partners depending upon its function and location in the cells.

4. SECRETION OF ERAP1

It is possible that the N-terminal hydrophobic region of ERAP1 functions not only to align the protein in the plasma membrane, but also to act as a signal sequence to load enzymes in the secretory pathway. Indeed, ERAP1 is frequently found in the ER-lumen and observed to function as a final processing enzyme for antigenic peptides that are presented to MHC class I molecules.^24–26) However, ERAP1 has no ER-retention signal in its primary amino acid sequence. Moreover, we discovered that when ERAP1 is overexpressed in COS-7 cells, it was secreted into the culture medium.³⁸⁾ Overall, we postulated that ERAP1 is primarily secreted into the extracellular milieu; however, can be retained in the ER-lumen in the presence of putative binding proteins.^6,11) Further, we depleted the exon 10 coding sequence, and observed that the mutant enzyme was no longer retained in the ER-lumen and was constitutively secreted into the culture medium; thereby, supporting our hypothesis.¹³⁾ It is plausible that saturation with putative binding protein resulted in free residual ERAP1 in the ER-lumen, which was then secreted into the extracellular milieu. Indeed, it was recently reported that ERp44 in the ER-lumen can bind to Cys487 (encoded on exon 10) in a redox-dependent manner.³⁹⁾ Knockout of ERp44 has been found to cause constitutive secretion of the enzyme. The catalytic activity of the mutant enzyme lacking exon 10 coding sequence was found to be fully retained. This indicated that the sequence is indispensable for solely retaining the enzyme in the ER-lumen; however, not for its enzymatic activity.

Additionally, we found that ERAP1 was secreted from the macrophage cell line (RAW264.7 cells) in response to activation by IFN-γ and lipopolysaccharide (LPS), which enhanced the phagocytic activity of the cells.⁴⁰⁾ IFN-γ/LPS-induced enhancement of phagocytosis was partially inhibited by amastatin, a potent inhibitor of M1 aminopeptidases. Adding wild-type ERAP1, but not the inactive mutant, to the culture medium showed enhanced phagocytosis. These results suggested that ERAP1 secreted by IFN-γ/LPS-stimulated macrophages promoted their phagocytic activity through generation of active peptides processed by the enzyme. We also reported that secreted ERAP1 can contribute to up-regulating nitric oxide (NO) synthesis in a substrate-dependent manner.⁴¹⁾ In the absence of free arginine (Arg) in the culture medium, Ang III (RVYIHPF)-dependent, and not Ang IV (VYIHPF)-dependent NO synthesis was observed in RAW264.7 cells, which indicated that cells could utilize extracellular Arg derived from ERAP1-cleaved Ang III for NO synthesis. Overall, these results indicate that stimulus-dependent secretion of ERAP1 from activated macrophages mediates (at least in part) their biological defense responses through its enzymatic activity.

Subsequently, we characterized the secretion of ERAP1 in vivo by injecting mice with LPS.⁴²⁾ Mice that were intraperitoneally injected with LPS were found to secrete ERAP1 into blood vessel in a time-dependent manner. When NO synthesis was compared between the wild-type and ERAP1 knockout mice, the wild-type mice was found to accumulate much more NO in the serum. This indicated that secreted ERAP1 could indeed play roles in inflammatory conditions in vivo.

Aldhamen et al. also analyzed the functional properties of secreted ERAP1.⁴³⁾ Using ERAP1 specific inhibitor, DG03A, they confirmed the IFN-γ/LPS-induced secretion of ERAP1 from RAW264.7 cells and increase in their phagocytic activity. In addition, their treatment of peripheral blood mononuclear cells with catalytically active ERAP1 showed activation of natural killer (NK) cells, dendritic cells (DC), and T cells. ERAP1-mediated production of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 were also found to be up-regulated by pathways involving NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome. These results indicated that secreted ERAP1 can regulate the activities of several types of immune cells; thereby, modulating innate and acquired immune as well as inflammatory responses in addition to its final antigenic peptide-processing role in the ER-lumen. It is noteworthy here that ERAP1 exhibits dual effects on functioning of NK cells similar to that found in angiogenesis. While ERAP1 down-regulates NK cell functions as an antigenic peptide-processing enzyme in the ER-lumen,⁴⁴⁾ the enzyme activates NK cells when secreted into the extracellular milieu.⁴³⁾

In addition to free ERAP1 in the culture medium, a subset of secreted ERAP1 molecules was found to bind to exosomes that are released from IFN-γ/LPS-treated RAW264.7 cells.⁴⁵⁾ While ERAP1 is secreted via the conventional secretion pathway through Golgi apparatus,⁴⁰⁾ exosomes are known to be derived from endosomal pathway and formed within multivesicular bodies (MVB).⁴⁶⁾ It is most conceivable that on fusion of MVB with plasma membrane, exosomes are secreted into the extracellular milieu and then, bind to ERAP1 (Fig. 4). Exon 10 coding sequence of the enzyme has been shown responsible for association with exosomes, suggesting that the sequence provides multiple binding sites. Although untreated cells were also observed to release exosomes, their association with ERAP1 was not found. ERAP1-bound exosomes were observed to enhance the phagocytic and NO-synthetic activities of macrophages more efficiently than that by free ERAP1 and exosomes derived from untreated cells. On comparing the activities of exosomes derived from the wild-type and ERAP1-deficient RAW264.7 cells, it has been shown that ERAP1 certainly contributes, at least partially, to exosome-dependent phagocytosis and NO synthesis. Furthermore, IFN-γ/LPS-induced stimulation of RAW264.7 cells revealed that several cytokines and chemokines including TNF-α, IFN-γ, and C–C motif chemokine ligand 3 (CCL3) were also associated with exosomes. It was found that while CCL3 in the exosomes are crucial for the phagocytic activity of RAW264.7 cells, TNF-α and IFN-γ primarily contributes to the up-regulation of NO synthesis. However, ERAP1 is indispensable for these cells to reach the maximum levels of both macrophage activities. Consequently, inflammatory stimuli may be altering the properties of exosomes derived from macrophages to facilitate the association between ERAP1 and several cytokines/chemokines. The concurrent packaging and condensation of several effector molecules into exosomes might be advantageous to facilitate their actions on macrophages.

Fig. 4. Binding of ERAP1 to Exosomes in the Extracellular Milieu

ERAP1 is secreted from ER through the conventional secretion pathway. Exosomes are formed within MVB and released into the extracellular milieu on fusion of MVB with the plasma membrane to associate with the enzyme. (Color figure can be accessed in the online version.)

When thioglycollatem-elicited murine peritoneal macrophages were employed, only LPS was required for ERAP1 secretion⁴⁰⁾ suggesting that LPS is primarily an inducer of ERAP1 secretion while IFN-γ acts as a primer for macrophage activation. To evaluate this hypothesis, the effect of toll-like receptor (TLR) agonists on ERAP1 secretion was investigated. Several agonists including Pam3CSK4, FSL-1, and ODN1826 were found to induce ERAP1 secretion from RAW264.7 cells only in the presence of IFN-γ.⁴⁷⁾ On using cytokine antagonists, it was found that TNF-α and IFN-β are involved in ERAP1 secretion. In addition, ERAP1 secretion was found to be induced by A23187 and thapsigargin while inhibited by BAPTA-AM and calmodulin inhibitor, W-7. Overall, IFN-γ/LPS-induced secretion of ERAP1 from macrophages is mediated by TLRs via induction of intermediate cytokines, which further enhances cytosolic calcium levels and activates calmodulin. Since treating macrophages with ERAP1 enhanced the production of TNF-α and IL-1β, it can be speculative that the inflammatory stimuli provided by IFN-γ/LPS induces the initial secretion of ERAP1 and pro-inflammatory cytokines, which further enhances ERAP1 secretion. We propose here that the process of ERAP1 secretion may be involved in the positive network system comprising ERAP1 and cytokines.

Recently, ERAP2 was also shown to be secreted from macrophages in response to IFN-γ and LPS.⁴⁸⁾ It was speculated that similar to ERAP1, ERAP2 could act as an antigen processing enzyme in the ER-lumen and regulate immune cells when secreted into the extracellular milieu on inflammatory stimuli. However, we can only speculate whether or not deletion of exon 10 coding sequence of ERAP2 would generate a secreted protein as exon 10 sequences of ERAP2 and ERAP1 share little homology.

While probing for naturally occurring peptide substrates, we found that Ang II (DRVIHPF) and kallidin (KRPPGFSPFR), both of which have charged amino acids at their N-terminus, are cleaved most efficiently among the peptides investigated so far.³⁸⁾ As ERAP1 inactivates Ang II and generates bradykinin efficiently, we speculated that ERAP1 could regulate blood pressure. Subsequently, single nucleotide polymorphism (SNP) analysis identified the rs30187 variant form, an array with low enzymatic activity (ERAP1R528) as a risk factor for high blood pressure.^49,50) These results were later confirmed by genome-wide association studies (GWAS).⁵¹⁾ Considering that ERAP1 is primarily retained in the ER-lumen where the enzyme meets and processes peptides that have been localized out of the cells, is a mystery. Investigating that the enzyme is secreted into the extracellular milieu by inflammatory stimuli might be the answer.

As mentioned above, Hisatsune et al. showed that ERp44 binds to ERAP1 via disulfide bond formation to retain the enzyme in the ER-lumen.³⁹⁾ In embryonic fibroblasts derived from ERp44 knockout mice, it has been seen that in the absence of its binding partner, ERAP1 levels are significantly elevated in the culture medium. Concurrent with the elevated levels of ERAP1 in the plasma, reduced Ang II stability was observed in ERp44 knockout mice. During sepsis, the interaction between ERAP1 and ERp44 was observed to be intensified to keep the enzyme retained in the ER-lumen. The authors speculated that ER-lumen-retained ERAP1 cannot access Ang II in the blood vessels to prevent hypotension during sepsis. Thus, the identification of naturally occurring peptides/hormones that are processed by secreted ERAP1 will be critical to completely elucidate its pathophysiological functions, which remains elusive to date.

In our unpublished work (Hattori, A. and Tsujimoto, M.), we have identified another ERAP1-binding protein termed anchor protein of endoplasmic reticulum aminopeptidase (AERA), which has an ER-retention HDEL sequence at its C-terminus. Unlike ERp44, we could not observe ERAP1 secretion even on knockdown of AERA. However, in the presence of a HDEL-deletion mutant of AERA, ERAP1 was observed to be co-transported into the culture medium, suggesting that AERA can indeed bind to ERAP1 in the ER-lumen. Thus, it is conceivable that multiple proteins contribute to retaining ERAP1 in the ER-lumen and AERA is one of the proteins, which acts in cooperation with ERp44. Considering the contribution of cytokine network and multiple binding proteins in the process of ERAP1 secretion, it is tempting to speculate that its retention in the ER-lumen is tightly regulated to prevent the aberrant action in the absence of inflammatory stimuli.

When the differential effects of IFN-γ on the expression of ERAP1–19E and ERAP1–20E mRNAs are taken into account, it is speculative that while the latter is the superior population that plays roles as an antigenic peptide-processing enzyme in the ER-lumen, the former primarily acts in the extracellular milieu on IFN-γ/LPS stimulation. It will be interesting to determine C-terminal amino acid sequence of secretory ERAP1 to reveal which form is secreted preferentially.

5. CONCLUSION

It has been two decades since we first reported the molecular cloning of ERAP1 in 1999. Subsequently, evidences have developed in support of ERAP1 as a multi-functional enzyme with roles in both, innate and acquired immune system as well as inflammatory responses, and thus, crucial in the biological defense systems. Presumably acting as an antigenic peptide-processing enzyme, ERAP1 was recently reported to regulate the efficiency of anti-PD-1/PD-L1 immunotherapy. Specifically, deletion of ERAP1 in mouse tumors was reported to increase the efficiency of anti-PD-1 immunotherapy.^52,53) Furthermore, low levels of ERAP2 were found to be associated with improved response to anti-PD-L1 in human patients with bladder cancer.⁵⁴⁾ On the other hand, besides acting as a processing enzyme, ERAP1 secreted into the extracellular milieu activates immune cells such as macrophages and NK cells, and stimulates the production of pro-inflammatory cytokines. Additionally, ERAP1 modulates angiogenesis and promotes receptor shedding of cytokines, both of which apparently depend on its localization in the cells. Therefore, we propose here that ERAP1 can be regarded as a moonlighting protein, which acts not only as an aminopeptidase processing peptide substrates, but also as a scaffold protein binding to several regulatory proteins involved in angiogenesis and receptor shedding.⁵⁵⁾ Further research is required to elucidate the processes and conditions that regulate the intracellular and extracellular location of the enzyme.

Acknowledgments

This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of Interest

The authors declare no conflict of interest.

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