Recent findings

ERAP2 Inhibition Induces Cell-Surface Presentation by MOLT-4 Leukemia Cancer Cells of Many Novel and Potentially Antigenic Peptides

Recent studies have linked the activity of ER aminopeptidase 2 (ERAP2) to increased efficacy of immune-checkpoint inhibitor cancer immunotherapy, suggesting that pharmacological inhibition of ERAP2 could have important therapeutic implications. To explore the effects of ERAP2 inhibition on the immunopeptidome of cancer cells, we treated MOLT-4 T lymphoblast leukemia cells with a recently developed selective ERAP2 inhibitor, isolated Major Histocompatibility class I molecules (MHCI), and sequenced bound peptides by liquid chromatography tandem mass spectrometry. Inhibitor treatment induced significant shifts on the immunopeptidome so that more than 20% of detected peptides were either novel or significantly upregulated. Most of the inhibitor-induced peptides were 9mers and had sequence motifs and predicted affinity consistent with being optimal ligands for at least one of the MHCI alleles carried by MOLT-4 cells. Such inhibitor-induced peptides could serve as triggers for novel cytotoxic responses against cancer cells and synergize with the therapeutic effect of immune-checkpoint inhibitors. (Int J Mol Sci. 2022)

The endoplasmic-reticulum aminopeptidase ERAP1 processes antigenic peptides for loading on MHC-I proteins and recognition by CD8 T cells as they survey the body for infection and malignancy. Crystal structures have revealed ERAP1 in either open or closed conformations, but whether these occur in solution and are involved in catalysis is not clear. Here, we assess ERAP1 conformational states in solution in the presence of substrates, allosteric activators, and inhibitors by small-angle X-ray scattering. We also characterize changes in protein conformation by X-ray crystallography, and we localize alternate C-terminal binding sites by chemical crosslinking. Structural and enzymatic data suggest that the structural reconfigurations of ERAP1 active site are physically linked to domain closure and are promoted by binding of long peptide substrates. These results clarify steps required for ERAP1 catalysis, demonstrate the importance of conformational dynamics within the catalytic cycle, and provide a mechanism for the observed allosteric regulation and Lys/Arg528 polymorphism disease association. (Nature Communications 2021).

Presentation of antigenic peptides by MHCI is central to cellular immune responses against viral pathogens. While adaptive immune responses versus SARS-CoV-2 can be of critical importance to both recovery and vaccine efficacy, how protein antigens from this pathogen are processed to generate antigenic peptides is largely unknown. Here, we analyzed the proteolytic processing of overlapping precursor peptides spanning the entire sequence of the S1 spike glycoprotein of SARS-CoV-2, by three key enzymes that generate antigenic peptides, aminopeptidases ERAP1, ERAP2 and IRAP. All enzymes generated shorter peptides with sequences suitable for binding onto HLA alleles, but with distinct specificity fingerprints. ERAP1 was the most efficient in generating peptides 8-11 residues long, the optimal length for HLA binding, while IRAP was the least efficient. The combination of ERAP1 with ERAP2 greatly limited the variability of peptide sequences produced. Less than 7% of computationally predicted epitopes were found to be produced experimentally, suggesting that aminopeptidase processing may constitute a significant filter to epitope presentation. These experimentally generated putative epitopes could be prioritized for SARS-CoV-2 immunogenicity studies and vaccine design. We furthermore propose that this in vitro trimming approach could constitute a general filtering method to enhance the prediction robustness for viral antigenic epitopes (J Proteome Res).

Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims antigenic peptide precursors to generate mature antigenic peptides for presentation by major histocompatibility complex class I (MHCI) molecules and regulates adaptive immune responses. ERAP1 has been proposed to trim peptide precursors both in solution and in pre-formed MHCI-peptide complexes, but which mode is more relevant to its biological function remains controversial. Here, we compared ERAP1-mediated trimming of antigenic peptide precursors in solution or when bound to three MHCI alleles, HLA-B*58, HLA-B*08 and HLA-A*02. For all MHCI-peptide combinations, peptide binding onto MHCI protected against ERAP1-mediated trimming. In only a single MHCI-peptide combination, trimming of an HLA-B*08-bound 12mer progressed at a considerable rate, albeit still slower than in solution. Results from thermodynamic, kinetic and computational analyses suggested that this 12mer is highly labile and that apparent on-MHC trimming rates are always slower than that of MHCI-peptide dissociation. Both ERAP2 and leucine aminopeptidase, an enzyme unrelated to antigen processing, could trim this labile peptide from pre-formed MHCI complexes as efficiently as ERAP1. A pseudopeptide analogue with high affinity for both HLA-B*08 and the ERAP1 active site could not promote the formation of a ternary ERAP1-MHCI-peptide complex. Similarly, no interactions between ERAP1 and purified peptide loading complex (PLC) were detected in the absence or presence of a pseudopeptide trap. We conclude that MHCI binding protects peptides from ERAP1 degradation and that trimming in solution, along with the dynamic nature of peptide binding to MHCI, are sufficient to explain ERAP1 processing of antigenic peptide precursors. (JBC 2020)

ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that generates antigenic peptides and is an emerging target for cancer immunotherapy and the control of autoimmunity. ERAP1 inhibitors described previously target the active site, and are limited in selectivity, minimizing their clinical potential. To address this, we targeted the regulatory site of ERAP1 using a high-throughput screen and discovered a small molecule hit that is highly selective for ERAP1. (4aR,5S,6R,8S,8aR)-5-(2-(Furan-3-yl)ethyl)-8-hydroxy-5,6,8a-trimethyl-3,4,4a,5,6,7,8,8a-octahydronaphthalene-1-carboxylic acid is a natural product found in Dodonaea viscosa, that constitutes a sub-microM, highly-selective and cell-active modulator of ERAP1. Although the compound activates hydrolysis of small model substrates, it is a competitive inhibitor for physiologically-relevant longer peptides. Crystallographic analysis confirmed that the compound targets the regulatory site of the enzyme that normally binds the C-terminus of the peptide substrate. Our findings constitute a novel starting point for the development of selective ERAP1 modulators that have potential for further clinical development (Journal of Medicinal Chemistry, 2020).

Endoplasmic reticulum aminopeptidase 1 (ERAP1), an enzyme important for regulating adaptive immune responses, is an emerging target for immunotherapy applications. One of the hallmarks of ERAP1 is that it must be able to select substrates out of hundreds of thousands of possible ones. An incomplete understanding of how it performs this selection has been a major barrier to predicting and manipulating its effects on the immune response. We solved high-resolution crystal structures of ERAP1 with substrate analogs, which, along with biochemical and computational analysis, shed light on the mechanism that ERAP1 uses to select its substrates. Our structures constitute an important framework for better predicting epitope selection and immunodominance (PNAS 2019).

The efficacy of cancer immunotherapy, including treatment with immune-checkpoint inhibitors, often is limited by ineffective presentation of antigenic peptides that elicit T-cell-mediated anti-tumor cytotoxic responses. Manipulation of antigen presentation pathways is an emerging approach for enhancing the immunogenicity of tumors in immunotherapy settings. ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that trims peptides as part of the system that generates peptides for binding to MHC class I molecules (MHC-I). We hypothesized that pharmacological inhibition of ERAP1 in cells could regulate the cellular immunopeptidome. To test this hypothesis, we treated A375 melanoma cells with a recently developed potent ERAP1 inhibitor and analyzed the presented MHC-I peptide repertoire by isolating MHC-I, eluting bound peptides, and identifying them using capillary chromatography and tandem mass spectrometry (LC-MS/MS). Although the inhibitor did not reduce cell-surface MHC-I expression, it induced qualitative and quantitative changes in the presented peptidomes. Specifically, inhibitor treatment altered presentation of about half of the total 3204 identified peptides, including about one third of the peptides predicted to bind tightly to MHC-I. Inhibitor treatment altered the length distribution of eluted peptides without change in the basic binding motifs. Surprisingly, inhibitor treatment enhanced the average predicted MHC-I binding affinity, by reducing presentation of sub-optimal long peptides and increasing presentation of many high-affinity 9–12mers, suggesting that baseline ERAP1 activity in this cell line is destructive for many potential epitopes. Our results suggest that chemical inhibition of ERAP1 may be a viable approach for manipulating the immunopeptidome of cancer (Cancer Immunology Immunotherapy, 2019).

Edited by Piotr Kusnierczyk, Efstratios Stratikos Volume 80, Issue 5,Pages 277-348 (May 2019)

The vertebrate organisms (as well as all others) coexist not only with other members of their own species, but also with a great multiplicity of other species. Some of these are symbiotic or commensal microorganisms, others are infectious and nosogenic microbes and parasites, still others are providing food ingredients. All of them differ from us biochemically, and these differences are recognized by our immune system, both innate and adaptive. Infectious microorganisms and parasites try to escape this recognition by variation and molecular mimicry, and this drives the evolution of components of the immune system according to Red Queen Paradigm (RQP), i.e., an evolutionary race between infectious agents and molecules of the immune system. As Jack points out, “Host–pathogen interactions are classical RQPs—arms races in which the pathogens are forever evolving new virulence strategies while the host responds with ever more effective resistance mechanisms.”

Lipoprotein glomerulopathy (LPG) is a rare renal disease, characterized by lipoprotein thrombi in glomerular capillaries. A series of apoE mutations have been associated with LPG development. We previously showed that three mutants based on apoE3 sequence, in which an arginine was substituted by proline, are thermodynamically destabilized and aggregation-prone. To examine whether other LPG-associated apoE3 mutations induce similar effects, we characterized three nonproline LPG-associated apoE3 mutations, namely, R25C (apoEKyoto), R114C (apoETsukuba), and A152D (apoELasVegas). All three apoE3 variants are found to have significantly reduced helical content and to be thermodynamically destabilized, both in lipid-free and lipoprotein-associated form, and to expose a larger portion of hydrophobic surface to the solvent compared with WT apoE3. Furthermore, all three apoE3 variants are aggregation-prone, as shown by dynamic light-scattering measurements and by their enhanced capacity to bind the amyloid probe thioflavin T. Overall, our data suggest that the LPG-associated apoE3 mutations R25C, R114C, and A152D induce protein misfolding, which may contribute to protein aggregation in glomerular capillaries. The similar effects of both LPG-associated proline and nonproline mutations on apoE3 structure suggest that the thermodynamic destabilization and enhanced aggregation of apoE3 may constitute a common underlying mechanism behind the pathogenesis of LPG (read here).

Insulin-regulated aminopeptidase (IRAP) is an enzyme with several important biological functions that is known to process a large variety of different peptidic substrates although the mechanism behind this wide specificity is not clearly understood. We describe a crystal structure of IRAP in complex with a recently developed bioactive and selective inhibitor at 2.53 Å resolution. In the presence of this inhibitor the enzyme adopts a novel conformation in which domains II and IV are juxtaposed, forming a hollow structure that excludes external solvent access to the catalytic center. A loop adjacent to the enzyme's GAMEN motif undergoes structural reconfiguration, allowing the accommodation of bulky inhibitor side-chains. Atomic interactions between the inhibitor and IRAP that are unique to this conformation can explain the strong selectivity compared to homologous aminopeptidases ERAP1 and ERAP2. This conformation provides insight on IRAP's catalytic cycle and reveals significant active site plasticity that may underlie its substrate permissiveness.

Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) is an intracellular enzyme important for the generation of antigenic epitopes and class I MHC-restricted adaptive immune responses. ERAP1 processes a vast variety of different peptides but still shows length and sequence selectivity although the mechanism behind these properties is poorly understood. X-ray crystallographic analysis has revealed that ERAP1 can assume at least two distinct conformations in which the C-terminal domain IV is either proximal or distal to the active site domain II. To help understand the role of this conformational change in the catalytic mechanism of ERAP1 we used site-directed mutagenesis to perturb key salt-bridges between domains II and IV. Enzymatic analysis revealed that these mutations, although located away from the catalytic site, greatly reduce catalytic efficiency and shift allosteric kinetic behavior. The variants were more efficiently activated by small peptides, and bound a competitive inhibitor with weaker affinity and faster dissociation kinetics. Molecular dynamics analysis suggested that the mutations affect the conformational distribution of ERAP1, reducing the population of closed states. Small-angle x-ray scattering indicated that both wild-type and the ERAP1 variants are predominantly in an open conformational state in solution. Overall, our findings suggest that electrostatic interactions between domains II and IV in ERAP1 are crucial for driving a conformational change that regulates the structural integrity of the catalytic site. The extent of domain opening in ERAP1 probably underlies its specialization for antigenic peptide precursors and should be taken into account for inhibitor development efforts (Biochemistry 2017).

The oxytocinase subfamily of M1 aminopeptidases, consisting of ER aminopeptidase 1 (ERAP1), ER aminopeptidase 2 (ERAP2) and insulin-regulated aminopeptidase (IRAP), plays critical roles in the generation of antigenic peptides and indirectly regulates human adaptive immune responses. We have previously demonstrated that phosphinic pseudotripeptides can constitute potent inhibitors of this group of enzymes. In this study, we used synthetic methodologies able to furnish a series of stereochemically defined phosphinic pseudotripeptides and demonstrate that side-chains at P1' and P2' positions are critical determinants in driving potency and selectivity. We identified low nanomolar inhibitors of ERAP2 and IRAP that display selectivity of more than 2 and 3 orders of magnitude, respectively. Cellular analysis demonstrated that one of the compounds that is a selective IRAP inhibitor can reduce IRAP-dependent but not ERAP1-dependent cross-presentation by dendritic cells with nanomolar efficacy. Our results encourage further pre-clinical development of phosphinic pseudotripeptides as regulators of adaptive immune responses. (J Med Chem 2016)

The apolipoprotein (apo) E4 isoform is the strongest risk factor for late-onset Alzheimer's disease (AD). ApoE4 is more susceptibleto proteolysis than apoE2 and apoE3 isoforms and carboxyl-terminal truncated apoE4 forms have been found in AD patients' brain. We have previously shown that a specific apoE4 fragment, apoE4-165, promotes amyloid-peptide beta 42 (Aβ42) accumulation in humanneuroblastoma SK-N-SH cells and increased intracellular reactive oxygen species formation, two events considered to occur early in AD pathogenesis. Here, we show that these effects are allele-dependent and absolutely require the apoE4 background. Furthermore, the exact length of the fragment is critical since longer or shorter length carboxyl-terminal truncated apoE4 forms do not elicit the same effects. Structural and thermodynamic analyses showed that apoE4-165 has a compact structure, in contrast to other carboxyl-terminal truncated apoE4 forms that are instead destabilized. Compared however to other allelic backgrounds, apoE4-165 is structurally distinct and less thermodynamically stable suggesting that the combination of a well-folded structure with structural plasticity is a unique characteristic of this fragment. Overall, our findings suggest that the ability of apoE fragments to promote Aβ42 intraneuronal accumulation is specific for both the apoE4 isoform and the particular structural and thermodynamic properties of the fragment (Sci Rep 2016)

Endoplasmic reticulum aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences although the structural basis of this mechanism is unclear. To address this issue, we solved crystal structures of ER aminopeptidase 2 (ERAP2) in complex with peptides. Biochemical and structural analysis suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias(JBC, 2015)

Aminopeptidases that generate antigenic peptides influence immunodominance and adaptive cytotoxic immune responses. The mechanisms that allow these enzymes to efficiently process a vast number of differentlong peptide substrates are poorly understood. In this work, we report the structure of insulin-regulated aminopeptidase, an enzyme that prepares antigenic epitopes for cross-presentation in dendritic cells, in complex with an antigenic peptide precursor analog. Insulin-regulated aminopeptidase is found in a semiclosed conformation with an extended internal cavity with limited access to the solvent. The N-terminal moiety of the peptide is located at the active site, positioned optimally for catalysis, whereas the C-terminal moiety of the peptide is stabilized along the extended internal cavity lodged between domains II and IV. Hydrophobic interactions and shape complementarity enhance peptide affinity beyond the catalytic site and support a limited selectivity model for antigenic peptide selection that may underlie the generation of complex immunopeptidomes. (J Immunol, 2015)

Apolipoprotein (apo) E4 isoform has consistently emerged as a susceptibility factor for late-onset Alzheimer’s disease (AD) although the exact mechanism is not clear. A rare apoE4 mutant, apoE4[L28P] Pittsburgh, burdens carriers with added risk for late-onset AD and may be a useful tool for gaining insight on the role of apoE4 in disease pathogenesis. Towards this end, we evaluated the effect of the L28P mutation on the structural and functional properties of apoE4. ApoE4[L28P] was found to have significantly perturbed thermodynamic properties, to have reduced helical content and to expose a larger portion of hydrophobic surface to the solvent. Furthermore, this mutant is thermodynamically destabilized and more prone to proteolysis. When interacting with lipids, apoE4[L28P] formed populations of lipoprotein particles with structural defects. The structural perturbations brought about by the mutation were accompanied by aberrant functions associated with the pathogenesis of AD. Specifically, apoE4[L28P] promoted the cellular uptake of extracellular amyloid-beta peptide 42 (Aβ42) by human neuroblastoma SK-N-SH cells as well as by primary mouse neuronal cells and led to increased formation of intracellular reactive oxygen species (ROS) that persisted for at least 24hrs. Furthermore, lipoprotein particles containing apoE4[L28P] induced intracellular ROS formation and reduced SK-N-SH cell viability. Overall, our findings suggest that the L28P mutation leads to significant structural and conformational perturbations in apoE4, and can induce functional defects associated with neuronal Aβ42 accumulation and oxidative stress. We propose that these structural and functional changes underlie the observed added risk for AD development in carriers of apoE4[L28P]. (J Biol Chem, 2014)

The human immune system fights disease by eradicating sick cells after first recognising that they are infected or cancerous. This is achieved by specialised 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 recognising and eliminating cancer cells. (Proc Natl Acad Sci USA, 2013).