The Internal Sequence of the Peptide-Substrate Determines Its N-Terminus Trimming by ERAP1

Background: Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims N-terminally extended antigenic peptide precursors down to mature antigenic peptides for presentation by major histocompatibility complex (MHC) class I molecules. ERAP1 has unique properties for an aminopeptidase being able to trim peptides in vitro based on their length and the nature of their C-termini. Methodology/Principal Findings: In an effort to better understand the molecular mechanism that ERAP1 uses to trim peptides, we systematically analyzed the enzyme's substrate preferences using collections of peptide substrates. We discovered strong internal sequence preferences of peptide N-terminus trimming by ERAP1. Preferences were only found for positively charged or hydrophobic residues resulting to trimming rate changes by up to 100 fold for single residue substitutions and more than 40,000 fold for multiple residue substitutions for peptides with identical N-termini. Molecular modelling of ERAP1 revealed a large internal cavity that carries a strong negative electrostatic potential and is large enough to accommodate peptides adjacent to the enzyme's active site. This model can readily account for the strong preference for positively charged side chains. Conclusions/Significance: To our knowledge no other aminopeptidase has been described to have such strong preferences for internal residues so distal to the N-terminus. Overall, our findings indicate that the internal sequence of the peptide can affect its trimming by ERAP1 as much as the peptide's length and C-terminus. We therefore propose that ERAP1 recognizes the full length of its peptide-substrate and not just the N- and C- termini. It is possible that ERAP1 trimming preferences influence the rate of generation and the composition of antigenic peptides in vivo

The role of endoplasmic reticulum aminopeptidases in antigen presentation

Endoplasmic reticulum aminopeptidases ERAP1/2 have emerged in the last years as key enzymes for the production of antigenic peptides that are presented by MHC class I molecules at the cell surface as part of the adaptive immune response. ERAP1 has unusual enzymatic properties that make it particularly suitable for this biological function. Specifically, it efficiently degrades peptides longer than 9 residues and shows preferences for the whole substrate sequence and not only for the N-terminus. Recently, coding Single Nucleotide Polymorphisms (SNPs) in ERAP1/2 were associated with predisposition to autoimmune diseases, such as ankylosing spondylitis, with cancer and with resistance to HIV infection. The hypothesis of this thesis is that the particular molecular mechanism of action of ERAP1/2 and the way in which this mechanism is affected by coding polymorphisms is the underlying reason for the association of ERAP1/2 with predisposition to human disease. To verify this hypothesis we investigated the molecular mechanism of action of ERAP1/2 aminopeptidases in the context of their polymorphic variability. We first developed a novel fluorigenic assay to analyze the activity of these enzymes. Biochemical analysis in combination with the recently solved ERAP1 crystal structure allowed us to propose a molecular model of function that can account for both substrate length and sequence specificity. In parallel, we set the groundwork for the development of small molecular weight compounds that modulate ERAP1 activity, by scanning a chemical library of pharmaceuticals and discovering lead compounds that either act as inhibitors or as activators of the enzyme.In order to understand the association of ERAP1/2 polymorphicity with changes in antigen presentation, we expressed different ERAP1/2 alleles and studied their ability to degrade different antigenic epitope precursors. Michaelis-Menten analysis showed that specific SNPs in ERAP1 affect the ΚΜ and kcat parameters of the enzyme and that particular allele-substrate combinations demonstrate substrate inhibition kinetics. A common ERAP2 allele was found to have a stronger effect on enzyme function and, as ERAP1 alleles, not only affects enzymatic activity but also alters specificity. The above results support the hypothesis that genetic variability in ERAP1/2 aminopeptidases can affect the generation of antigenic epitopes and may represent a previously unrecognized facet of adaptive immune response variability

The role of endocytic trafficking in antigen T cell receptor activation

International audienceAntigen T cell receptors (TCR) recognize antigenic peptides displayed by the major histocompatibility complex (pMHC) and play a critical role in T cell activation. The levels of TCR complexes at the cell surface, where signaling is initiated, depend on the balance between TCR synthesis, recycling and degradation. Cell surface TCR interaction with pMHC leads to receptor clustering and formation of a tight T cell-APC contact, the immune synapse, from which the activated TCR is internalized. While TCR internalization from the immune synapse has been initially considered to arrest TCR signaling, recent evidence support the hypothesis that the internalized receptor continues to signal from specialized endosomes. Here, we review the molecular mechanisms of TCR endocytosis and recycling, both in steady state and after T cell activation. We then discuss the experimental evidence in favor of endosomal TCR signaling and its possible consequences on T cell activation

IRAP Endosomes Control Phagosomal Maturation in Dendritic Cells

International audienceDendritic cells (DCs) contribute to the immune surveillance by sampling their environment through phagocytosis and endocytosis. We have previously reported that, rapidly following uptake of extracellular antigen into phagosomes or endosomes in DCs, a specialized population of storage endosomes marked by Rab14 and insulin-regulated aminopeptidase (IRAP) is recruited to the nascent antigen-containing compartment, thereby regulating its maturation and ultimately antigen cross-presentation to CD8 + T lymphocytes. Here, using IRAP –/– DCs, we explored how IRAP modulates phagosome maturation dynamics and cross-presentation. We find that in the absence of IRAP, phagosomes acquire more rapidly late endosomal markers, are more degradative, and show increased microbicidal activity. We also report evidence for a role of vesicle trafficking from the endoplasmic reticulum (ER)–Golgi intermediate compartment to endosomes for the formation or stability of the IRAP compartment. Moreover, we dissect the dual role of IRAP as a trimming peptidase and a critical constituent of endosome stability. Experiments using a protease-dead IRAP mutant and pharmacological IRAP inhibition suggest that IRAP expression but not proteolytic activity is required for the formation of storage endosomes and for DC-typical phagosome maturation, whereas proteolysis is required for fully efficient cross-presentation. These findings identify IRAP as a key factor in cross-presentation, trimming peptides to fit the major histocompatibility complex class-I binding site while preventing their destruction through premature phagosome maturation

The Role of Insulin Regulated Aminopeptidase in Endocytic Trafficking and Receptor Signaling in Immune Cells

International audienceInsulin regulated aminopeptidase (IRAP) is a type II transmembrane protein with broad tissue distribution initially identified as a major component of Glut4 storage vesicles (GSV) in adipocytes. Despite its almost ubiquitous expression, IRAP had been extensively studied mainly in insulin responsive cells, such as adipocytes and muscle cells. In these cells, the enzyme displays a complex intracellular trafficking pattern regulated by insulin. Early studies using fusion proteins joining the IRAP cytosolic domain to various reporter proteins, such as GFP or the transferrin receptor (TfR), showed that the complex and regulated trafficking of the protein depends on its cytosolic domain. This domain contains several motifs involved in IRAP trafficking, as demonstrated by mutagenesis studies. Also, proteomic studies and yeast two-hybrid experiments showed that the IRAP cytosolic domain engages in multiple protein interactions with cytoskeleton components and vesicular trafficking adaptors. These findings led to the hypothesis that IRAP is not only a cargo of GSV but might be a part of the sorting machinery that controls GSV dynamics. Recent work in adipocytes, immune cells, and neurons confirmed this hypothesis and demonstrated that IRAP has a dual function. Its carboxy-terminal domain located inside endosomes is responsible for the aminopeptidase activity of the enzyme, while its amino-terminal domain located in the cytosol functions as an endosomal trafficking adaptor. In this review, we recapitulate the published protein interactions of IRAP and summarize the increasing body of evidence indicating that IRAP plays a role in intracellular trafficking of several proteins. We describe the impact of IRAP deletion or depletion on endocytic trafficking and the consequences on immune cell functions. These include the ability of dendritic cells to cross-present antigens and prime adaptive immune responses, as well as the control of innate and adaptive immune receptor signaling and modulation of inflammatory responses

Activating FcγR function depends on endosomal-signaling platforms

Summary: Cell surface receptor internalization can either terminate signaling or activate alternative endosomal signaling pathways. We investigated here whether endosomal signaling is involved in the function of the human receptors for Fc immunoglobulin fragments (FcRs): FcαRI, FcγRIIA, and FcγRI. All these receptors were internalized after their cross-linking with receptor-specific antibodies, but their intracellular trafficking was different. FcαRI was targeted directly to lysosomes, while FcγRIIA and FcγRI were internalized in particular endosomal compartments described by the insulin responsive aminopeptidase (IRAP), where they recruited signaling molecules, such as the active form of the kinase Syk, PLCγ and the adaptor LAT. Destabilization of FcγR endosomal signaling in the absence of IRAP compromised cytokine secretion downstream FcγR activation and macrophage ability to kill tumor cells by antibody-dependent cell-mediated cytotoxicity (ADCC). Our results indicate that FcγR endosomal signaling is required for the FcγR-driven inflammatory reaction and possibly for the therapeutic action of monoclonal antibodies

Impact of the TAP-like transporter in antigen presentation and phagosome maturation

International audienc