Emma Reeves

Naturally Occurring ERAP1 Haplotypes Encode Functionally Distinct Alleles with Fine Substrate Specificity

Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims peptides for MHC class I presentation, influencing the degree and specificity of CD8+ T cell responses. Single-nucleotide polymorphisms within the exons encoding ERAP1 are associated with autoimmune diseases and cervical carcinoma, but it is not known whether they act independently or as disease-associated haplotypes. We sequenced ERAP1 from 20 individuals and show that single-nucleotide polymorphisms occur as distinct haplotypes in the human population and that these haplotypes encode functionally distinct ERAP1 alleles. Using a wide range of substrates, we are able to demonstrate that for any given substrate distinct ERAP1 alleles can be “normal,” “hypofunctional,” or “hyperfunctional” and that each allele has a trend bias toward one of these three activities. Thus, the repertoire of peptides presented at the cell surface for recognition by CTL is likely to depend on the precise combination of both MHC class I and ERAP1 alleles expressed within an individual, and has important implications for predisposition to disease.

Functionally distinct ERAP1 allotype combinations distinguish individuals with Ankylosing Spondylitis

Significance The immune system performs surveillance to identify infected or cancerous cells through recognition of small protein fragments called antigenic peptides on their surface. To do this, the peptides must be cut to a specific length by an enzyme called endoplasmic reticulum aminopeptidase 1 (ERAP1). Variation in this enzyme has recently been linked to the inflammatory rheumatic disease Ankylosing Spondylitis (AS). We have found that ERAP1 is highly polymorphic in humans and that specific combinations of ERAP1 are found in people with AS. These disease-associated combinations have a reduced ability to generate peptides for presentation at the cell surface by MHC class I molecules, including HLA-B27. Understanding this finding may allow easier identification of individuals with AS and allow stratification into prognostic groups. For more than 40 y, expression of HLA-B27 has been strongly associated with the chronic inflammatory disease Ankylosing Spondylitis (AS); however, the mechanisms underlying this association are still unknown. Single nucleotide polymorphisms within the aminopeptidase endoplasmic reticulum aminopeptidase 1 (ERAP1), which is essential for trimming peptides before they are presented to T cells by major histocompatibility complex (MHC) class I molecules, have been linked with disease. We show that ERAP1 is a highly polymorphic molecule comprising allotypes of single nucleotide polymorphisms. The prevalence of specific ERAP1 allotypes is different between AS cases and controls. Both chromosomal copies of ERAP1 are codominantly expressed, and analysis of allotype pairs provided clear stratification of individuals with AS versus controls. Functional analyses demonstrated that ERAP1 allotype pairs seen in AS cases were poor at generating optimal peptide ligands for binding to murine H-2Kb and -Db and the AS-associated HLA-B*2705. We therefore provide strong evidence that polymorphic ERAP1 alters protein function predisposing an individual to AS via its influence on the antigen processing pathway.

Rationally designed inhibitor targeting antigen-trimming aminopeptidases enhances antigen presentation and cytotoxic T-cell responses

Significance The human immune system fights disease by eradicating sick cells after first recognizing that they are infected or cancerous. This is achieved by specialized cells that detect on the surface of other cells small molecules called antigenic peptides. Pathogens and cancer can evade the immune system by stopping the generation of antigenic peptides. We designed, synthesized and evaluated artificial small molecules that can effectively block a group of enzymes that are key for the production or destruction of antigenic peptides. We show that these compounds can enhance the generation of antigenic peptides in cells and enhance the immune system reaction toward cancer. Inhibitors of this kind may provide a new approach to coax the immune system into recognizing and eliminating cancer cells. Intracellular aminopeptidases endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2), and as well as insulin-regulated aminopeptidase (IRAP) process antigenic epitope precursors for loading onto MHC class I molecules and regulate the adaptive immune response. Their activity greatly affects the antigenic peptide repertoire presented to cytotoxic T lymphocytes and as a result can regulate cytotoxic cellular responses contributing to autoimmunity or immune evasion by viruses and cancer cells. Therefore, pharmacological regulation of their activity is a promising avenue for modulating the adaptive immune response with possible applications in controlling autoimmunity, in boosting immune responses to pathogens, and in cancer immunotherapy. In this study we exploited recent structural and biochemical analysis of ERAP1 and ERAP2 to design and develop phosphinic pseudopeptide transition state analogs that can inhibit this family of enzymes with nM affinity. X-ray crystallographic analysis of one such inhibitor in complex with ERAP2 validated our design, revealing a canonical mode of binding in the active site of the enzyme, and highlighted the importance of the S2’ pocket for achieving inhibitor potency. Antigen processing and presentation assays in HeLa and murine colon carcinoma (CT26) cells showed that these inhibitors induce increased cell-surface antigen presentation of transfected and endogenous antigens and enhance cytotoxic T-cell responses, indicating that these enzymes primarily destroy epitopes in those systems. This class of inhibitors constitutes a promising tool for controlling the cellular adaptive immune response in humans by modulating the antigen processing and presentation pathway.

Critical role of endoplasmic reticulum aminopeptidase 1 in determining the length and sequence of peptides bound and presented by HLA-B27.

HLA–B27 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are the two strongest genetic factors predisposing to ankylosing spondylitis (AS). A key aminopeptidase in class I major histocompatibility complex presentation, ERAP1 potentially contributes to the pathogenesis of AS by altering HLA–B27 peptide presentation. The aim of this study was to analyze the effects of ERAP1 on the HLA–B27 peptide repertoire and peptide presentation to cytotoxic T lymphocytes (CTLs).

Antigen processing and immune regulation in the response to tumours

The MHC class I and II antigen processing and presentation pathways display peptides to circulating CD8+ cytotoxic and CD4+ helper T cells respectively to enable pathogens and transformed cells to be identified. Once detected, T cells become activated and either directly kill the infected / transformed cells (CD8+ cytotoxic T lymphocytes) or orchestrate the activation of the adaptive immune response (CD4+ T cells). The immune surveillance of transformed/tumour cells drives alteration of the antigen processing and presentation pathways to evade detection and hence the immune response. Evasion of the immune response is a significant event tumour development and considered one of the hallmarks of cancer. To avoid immune recognition, tumours employ a multitude of strategies with most resulting in a down‐regulation of the MHC class I expression at the cell surface, significantly impairing the ability of CD8+ cytotoxic T lymphocytes to recognize the tumour. Alteration of the expression of key players in antigen processing not only affects MHC class I expression but also significantly alters the repertoire of peptides being presented. These modified peptide repertoires may serve to further reduce the presentation of tumour‐specific/associated antigenic epitopes to aid immune evasion and tumour progression. Here we review the modifications to the antigen processing and presentation pathway in tumours and how it affects the anti‐tumour immune response, considering the role of tumour‐infiltrating cell populations and highlighting possible future therapeutic targets.

Both rare and common ERAP1 allotypes have distinct functionality defined by polymorphic context and are important in AS association.

We read with interest the study by Roberts et al. indicating that the association between endoplasmic reticulum aminopeptidase 1 (ERAP1) genotypes and ankylosing spondylitis (AS) is by common genotypes rather than rare haplotype combinations (1). The authors also suggest that the data presented by Reeves et al. (2) indicated that only “rare” allotypes/combinations are important in the association of ERAP1 with AS. However, our data (2) provide prima facie evidence that common allotypes are functionally relevant and may be important in disease. For example, we have shown that the two common haplotypes discussed in Roberts et al. (1) (I/*001 and IV/\*008/\*011) are both hypofunctional (3 … [↵][1]1To whom correspondence should be addressed. Email: eddjames{at}soton.ac.uk. [1]: #xref-corresp-1-1

The role of polymorphic ERAP1 in autoinflammatory disease

Autoimmune and autoinflammatory conditions represent a group of disorders characterized by self-directed tissue damage due to aberrant changes in innate and adaptive immune responses. These disorders possess widely varying clinical phenotypes and etiology; however, they share a number of similarities in genetic associations and environmental influences. Whilst the pathogenic mechanisms of disease remain poorly understood, genome wide association studies (GWAS) have implicated a number of genetic loci that are shared between several autoimmune and autoinflammatory conditions. Association of particular HLA alleles with disease susceptibility represents one of the strongest genetic associations. Furthermore, recent GWAS findings reveal strong associations with single nucleotide polymorphisms in the endoplasmic reticulum aminopeptidase 1 (ERAP1) gene and susceptibility to a number of these HLA-associated conditions. ERAP1 plays a major role in regulating the repertoire of peptides presented on HLA class I alleles at the cell surface, with the presence of single nucleotide polymorphisms in ERAP1 having a significant impact on peptide processing function and the repertoire of peptides presented. The impact of this dysfunctional peptide generation on CD8+ T-cell responses has been proposed as a mechanism of pathogenesis diseases where HLA and ERAP1 are associated. More recently, studies have highlighted a role for ERAP1 in innate immune-mediated pathways involved in inflammatory responses. Here, we discuss the role of polymorphic ERAP1 in various immune cell functions, and in the context of autoimmune and autoinflammatory disease pathogenesis.

Reply to Robinson and Brown: It is the combination of ERAP1 allotypes that identifies individuals with Ankylosing Spondylitis.

We would like to thank Robinson and Brown (1) for their interest in our work (2). In their comments, the authors suggest that the comparison of endoplasmic reticulum aminopeptidase 1 (ERAP1) allotypes in Ankylosing Spondylitis (AS) cases and controls are not statistically different, and therefore do not support the conclusions of the work presented (1). We do not claim an association between specific allotypes (haplotypes) and disease, but rather that the inheritance of particular combinations of allotypes (haplotypes) allows identification of individuals with disease from controls (2). This is a robust observation, which is further validated at the functional level.

Functionally distinct ERAP1 and ERAP2 are a hallmark of HLA-A29-(Birdshot) Uveitis

&NA; Birdshot Uveitis (Birdshot) is a rare eye condition that affects HLA‐A29‐positive individuals and could be considered a prototypic member of the recently proposed ‘MHC‐I (major histocompatibility complex class I)‐opathy’ family. Genetic studies have pinpointed the endoplasmic reticulum aminopeptidase (ERAP1) and (ERAP2) genes as shared associations across MHC‐I‐opathies, which suggests ERAP dysfunction may be a root cause for MHC‐I‐opathies. We mapped the ERAP1 and ERAP2 haplotypes in 84 Dutch cases and 890 controls. We identified association at variant rs10044354, which mediated a marked increase in ERAP2 expression. We also identified and cloned an independently associated ERAP1 haplotype (tagged by rs2287987) present in more than half of the cases; this ERAP1 haplotype is also the primary risk and protective haplotype for other MHC‐I‐opathies. We show that the risk ERAP1 haplotype conferred significantly altered expression of ERAP1 isoforms in transcriptomic data (n = 360), resulting in lowered protein expression and distinct enzymatic activity. Both the association for rs10044354 (meta‐analysis: odds ratio (OR) [95% CI]=2.07[1.58‐2.71], P = 1.24 × 10(−7)) and rs2287987 (OR[95% CI]: =2.01[1.51‐2.67], P = 1.41 × 10(−6)) replicated and showed consistent direction of effect in an independent Spanish cohort of 46 cases and 2103 controls. In both cohorts, the combined rs2287987‐rs10044354 haplotype associated with Birdshot more strongly than either variant alone [meta‐analysis: P=3.9 × 10(−9)]. Finally, we observed that ERAP2 protein expression is dependent on the ERAP1 background across three European populations (n = 3353). In conclusion, a functionally distinct combination of ERAP1 and ERAP2 are a hallmark of Birdshot and provide rationale for strategies designed to correct ERAP function for treatment of Birdshot and MHC‐I‐opathies more broadly.

Tumour and placenta establishment: The importance of antigen processing and presentation

Classical and non-classical MHC class I (MHC I) molecules displayed at the cell surface are essential for the induction of innate and adaptive immune responses. Classical MHC I present endogenously derived peptides to CD8+ T cells for immunosurveillance of infected or malignant cells. By contrast, non-classical MHC I, in particular HLA-G, also display peptides, but primarily act as immunomodulatory ligands for the innate immune response and are an important component for extravillous trophoblast invasion to form the placenta in pregnancy. Endoplasmic Reticulum AminoPeptidase 1 (ERAP1), which trims peptides in the ER to generate ligands for MHC I loading, is a key regulator of the peptide repertoire and has a significant impact on the formation of stable MHC I at the cell surface. ERAP1 also plays a role in angiogenesis, cell cycle progression and migration, events that are shared between tumour cells and placenta formation. Here we discuss the similarities between tumour and extravillous trophoblast cells in their immune modulatory, invasion, migration and proliferation properties in the context of ERAP1 and its role in establishment of solid tumours and placenta formation.