112 research outputs found

    The Role of Conformational Dynamics in Antigen Trimming by Intracellular Aminopeptidases

    Get PDF
    Antigenic peptides presented by the major histocompatibility complex class I (MHC-I) molecules for recognition by cytotoxic T-lymphocytes are processed by members of the oxytocinase sub-family of M1 aminopeptidases ERAP1, ERAP2, and IRAP. These three homologous zinc metallopeptidases trim N-terminally extended precursor antigenic peptides down to the correct length for loading onto the MHC-I but can also destroy some antigenic peptides by over-trimming, therefore, influencing the antigenic peptide repertoire and immunodominance hierarchy. Polymorphic variation has been found to affect their trimming function and predispose to human disease in complex and poorly understood patterns. Structural and biochemical analysis have pointed toward a complicated trimming mechanism that involves a major conformational transition during each catalytic cycle. Here, we provide an overview of current knowledge on the structure and mechanism of action of those enzymes with a focus on the proposed key role of conformational dynamics in their function

    Identification of the Lateral Interaction Surfaces of Human Histocompatibility Leukocyte Antigen (HLA)-DM with HLA-DR1 by Formation of Tethered Complexes That Present Enhanced HLA-DM Catalysis

    Get PDF
    Human histocompatibility leukocyte antigen (HLA)-DM is a major histocompatibility complex (MHC)-like protein that catalyzes exchange of antigenic peptides from MHC class II molecules. To investigate the molecular details of this catalysis we created four covalent complexes between HLA-DM and the MHC class II allele DR1. We introduced a disulfide bond between the naturally occurring cysteine β46 on HLA-DM and an engineered cysteine on the end of a linker attached to either the NH2- or the COOH terminus of an antigenic peptide that is tightly bound on DR1. We find that when DM is attached to the NH2 terminus of the peptide, it can, for all linker lengths tested, catalyze exchange of the peptide with a half-life a few minutes (compared with uncatalyzed t1/2 > 100 h). This rate, which is several orders of magnitude greater than the one we obtain in solution assays using micromolar concentrations of HLA-DM, is dominated by a concentration independent factor, indicating an intramolecular catalytic interaction within the complex. A similar complex formed at the COOH terminus of the peptide shows no sign of DM-specific intramolecular catalysis. Restrictions on the possible interaction sites imposed by the length of the linkers indicate that the face of DR1 that accommodates the NH2 terminus of the antigenic peptide interacts with the lateral face of HLA-DM that contains cysteine β46

    A systematic re-examination of processing of MHCI-bound antigenic peptide precursors by ER aminopeptidase 1

    Get PDF
    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

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

    Get PDF
    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 Antigen Processing and Presentation in Cancer and the Efficacy of Immune Checkpoint Inhibitor Immunotherapy

    No full text
    Recent clinical successes of cancer immunotherapy using immune checkpoint inhibitors (ICIs) are rapidly changing the landscape of cancer treatment. Regardless of initial impressive clinical results though, the therapeutic benefit of ICIs appears to be limited to a subset of patients and tumor types. Recent analyses have revealed that the potency of ICI therapies depends on the efficient presentation of tumor-specific antigens by cancer cells and professional antigen presenting cells. Here, we review current knowledge on the role of antigen presentation in cancer. We focus on intracellular antigen processing and presentation by Major Histocompatibility class I (MHCI) molecules and how it can affect cancer immune evasion. Finally, we discuss the pharmacological tractability of manipulating intracellular antigen processing as a complementary approach to enhance tumor immunogenicity and the effectiveness of ICI immunotherapy

    Antigenic peptide trimming by ER aminopeptidases--insights from structural studies

    No full text
    Generation and destruction of antigenic peptides by ER resident aminopeptidases ERAP1 and ERAP2 have been shown in the last few years to be important for the correct functioning and regulation of the adaptive immune response. These two highly homologous aminopeptidases appear to have evolved complex mechanisms well suited for their biological role in antigen presentation. Furthermore, polymorphic variability in these enzymes appears to affect their function and predispose individuals to disease. This review discusses our current understanding of the molecular mechanisms behind ERAP1/2 function as suggested by several recently determined crystallographic structures of these enzymes
    corecore