[Show abstract][Hide abstract] ABSTRACT: Insulin-degrading enzyme (IDE) is a protease that cleaves insulin and other bioactive peptides such as amyloid-β. Knockout and genetic studies have linked IDE to Alzheimer's disease and type-2 diabetes. As the major insulin-degrading protease, IDE is a candidate drug target in diabetes. Here we have used kinetic target-guided synthesis to design the first catalytic site inhibitor of IDE suitable for in vivo studies (BDM44768). Crystallographic and small angle X-ray scattering analyses show that it locks IDE in a closed conformation. Among a panel of metalloproteases, BDM44768 selectively inhibits IDE. Acute treatment of mice with BDM44768 increases insulin signalling and surprisingly impairs glucose tolerance in an IDE-dependent manner. These results confirm that IDE is involved in pathways that modulate short-term glucose homeostasis, but casts doubt on the general usefulness of the inhibition of IDE catalytic activity to treat diabetes.
[Show abstract][Hide abstract] ABSTRACT: Members of the oxytocinase sub-family of M1 aminopeptidases (ERAP1, ERAP2 and IRAP) play important roles in both the adaptive and innate human immune responses. Their enzymatic activity can contribute to the pathogenesis of several major human diseases ranging from viral and parasitic infections to autoimmunity and cancer. We have previously demonstrated that diaminobenzoic acid derivatives show promise as selective inhibitors for this group of aminopeptidases. In this study we have thoroughly explored a series of 3,4-diaminobenzoic acid derivatives as inhibitors of this class of enzymes, achieving sub-micromolar inhibitors for ERAP2 (IC50=237 nM) and IRAP (IC50=105 nM). Cell-based analysis indicated that the lead compounds can be effective in down-regulating macrophage activation induced by lipopolysaccharide and interferon-?, as well as cross-presentation by bone marrow-derived dendritic cells. Our results indicate that this class of inhibitors may be useful for the targeted down-regulation of immune
[Show abstract][Hide abstract] ABSTRACT: The endoplasmic reticulum aminopeptidases (ERAP)1 and ERAP2 play a critical role in the production of final epitopes presented by MHC class I molecules. Formation of heterodimers by ERAP1 and ERAP2 has been proposed to facilitate trimming of epitope precursor peptides, but the effects of dimerization on ERAP function remain unknown. In this study, we produced stabilized ERAP1-ERAP2 heterodimers and found that they produced several mature epitopes more efficiently than a mix of the two enzymes unable to dimerize. Physical interaction with ERAP2 changes basic enzymatic parameters of ERAP1 and improves its substrate-binding affinity. Thus, by bringing the two enzymes in proximity and by producing allosteric effects on ERAP1, dimerization of ERAP1/2 creates complexes with superior peptide-trimming efficacy. Such complexes are likely to enhance Ag presentation by cells displaying coordinated expression of the two enzymes.
The Journal of Immunology 06/2014; 193(2). DOI:10.4049/jimmunol.1302855 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Peptide ligands presented by MHC class I (MHC-I) molecules are produced by degradation of cytosolic and nuclear, but also endoplasmic reticulum (ER)-resident, proteins by the proteasome. However, Ag processing of ER proteins remains little characterized. Studying processing and presentation of proinsulin, which plays a pivotal role in autoimmune diabetes, we found that targeting to the ER has profound effects not only on how proinsulin is degraded, but also on regulation of its cellular levels. While proteasome inhibition inhibited degradation and presentation of cytosolic proinsulin, as expected, it reduced the abundance of ER-targeted proinsulin. This targeting and protein modifications modifying protein half-life also had profound effects on MHC-I presentation and proteolytic processing of proinsulin. Thus, presentation of stable luminal forms was inefficient but enhanced by proteasome inhibition, whereas that of unstable luminal forms and of a cytosolic form were more efficient and compromised by proteasome inhibitors. Distinct stability of peptide MHC complexes produced from cytosolic and luminal proinsulin suggests that different proteolytic activities process the two Ag forms. Thus, both structural features and subcellular targeting of Ags can have strong effects on the processing pathways engaged by MHC-I-restricted Ags, and on the efficiency and regulation of their presentation.
The Journal of Immunology 04/2014; 192(11). DOI:10.4049/jimmunol.1300631 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The recent HIV-1 vaccine failures highlight the need to better understand virus-host interactions. One key question is why CD8(+) T cell responses to two HIV-Gag regions are uniquely associated with delayed disease progression only in patients expressing a few rare HLA class I variants when these regions encode epitopes presented by ∼30 more common HLA variants. By combining epitope processing and computational analyses of the two HIV subtypes responsible for ∼60% of worldwide infections, we identified a hitherto unrecognized adaptation to the antigen-processing machinery through substitutions at subtype-specific motifs. Multiple HLA variants presenting epitopes situated next to a given subtype-specific motif drive selection at this subtype-specific position, and epitope abundances correlate inversely with the HLA frequency distribution in affected populations. This adaptation reflects the sum of intrapatient adaptations, is predictable, facilitates viral subtype diversification, and increases global HIV diversity. Because low epitope abundance is associated with infrequent and weak T cell responses, this most likely results in both population-level immune evasion and inadequate responses in most people vaccinated with natural HIV-1 sequence constructs. Our results suggest that artificial sequence modifications at subtype-specific positions in vitro could refocus and reverse the poor immunogenicity of HIV proteins.
[Show abstract][Hide abstract] ABSTRACT: Antigen presentation by MHC class I molecules requires degradation of epitope source proteins in the cytosol. Although the preeminent role of the proteasome is clearly established, evidence suggesting a significant role for proteasome-independent generation of class I ligands has been reported repeatedly. However, an enzyme responsible for such a role has not been identified. Recently insulin-degrading enzyme (IDE) was shown to produce an antigenic peptide derived from the tumor antigen MAGE-A3 in an entirely proteasome-independent manner, raising the question of the global impact of IDE in MHC class I antigen processing. Here we report that IDE knockdown in human cell lines, or knockout in two different mouse strains, has no effect on cell surface expression of various MHC class I molecules, including allomorphs such as HLA-A3 and HLA-B27 suggested to be loaded in an at least a partly proteasome-independent manner. Moreover, reduced or absent IDE expression does not affect presentation of five epitopes including epitopes derived from beta amyloid and proinsulin, two preferred IDE substrates. Thus, IDE does not play a major role in MHC class I antigen processing, confirming the dominant and almost exclusive role of the proteasome in cytosolic production of MHC class I ligands.
PLoS ONE 02/2014; 9(2):e88365. DOI:10.1371/journal.pone.0088365 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Insulin Degrading Enzyme (IDE) is a protease conserved through evolution with a role in diabetes and Alzheimer's disease. The reason underlying its ubiquitous expression including cells lacking identified IDE substrates remains unknown. Here we show that the fission yeast IDE homologue (Iph1) modulates cellular sensitivity to endoplasmic reticulum (ER) stress in a manner dependent on TORC1 (Target of Rapamycin Complex 1). Reduced sensitivity to tunicamycin was associated with a smaller number of cells undergoing apoptosis. Wild type levels of tunicamycin sensitivity were restored in iph1 null cells when the TORC1 complex was inhibited by rapamycin or by heat inactivation of the Tor2 kinase. Although Iph1 cleaved hallmark IDE substrates including insulin efficiently, its role in the ER stress response was independent of its catalytic activity since expression of inactive Iph1 restored normal sensitivity. Importantly, wild type as well as inactive human IDE complemented gene-invalidated yeast cells when expressed at the genomic locus under the control of iph1(+) promoter. These results suggest that IDE has a previously unknown function unrelated to substrate cleavage, which links sensitivity to ER stress to a pro-survival role of the TORC1 pathway.
PLoS ONE 06/2013; 8(6):e67705. DOI:10.1371/journal.pone.0067705 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The therapeutic efficacy of anthracyclines relies on antitumor immune responses elicited by dying cancer cells. How chemotherapy-induced cell death leads to efficient antigen presentation to T cells, however, remains a conundrum. We found that intratumoral CD11c(+)CD11b(+)Ly6C(hi) cells, which displayed some characteristics of inflammatory dendritic cells and included granulomonocytic precursors, were crucial for anthracycline-induced anticancer immune responses. ATP released by dying cancer cells recruited myeloid cells into tumors and stimulated the local differentiation of CD11c(+)CD11b(+)Ly6C(hi) cells. Such cells efficiently engulfed tumor antigens in situ and presented them to T lymphocytes, thus vaccinating mice, upon adoptive transfer, against a challenge with cancer cells. Manipulations preventing tumor infiltration by CD11c(+)CD11b(+)Ly6C(hi) cells, such as the local overexpression of ectonucleotidases, the blockade of purinergic receptors, or the neutralization of CD11b, abolished the immune system-dependent antitumor activity of anthracyclines. Our results identify a subset of tumor-infiltrating leukocytes as therapy-relevant antigen-presenting cells.
[Show abstract][Hide abstract] ABSTRACT: Development of CD8(+) T cell (CTL) immunity or tolerance is linked to the conditions during T cell priming. Dendritic cells (DCs) matured during inflammation generate effector/memory T cells, whereas immature DCs cause T cell deletion/anergy. We identify a third outcome of T cell priming in absence of inflammation enabled by cross-presenting liver sinusoidal endothelial cells. Such priming generated memory T cells that were spared from deletion by immature DCs. Similar to central memory T cells, liver-primed T cells differentiated into effector CTLs upon antigen re-encounter on matured DCs even after prolonged absence of antigen. Their reactivation required combinatorial signaling through the TCR, CD28, and IL-12R and controlled bacterial and viral infections. Gene expression profiling identified liver-primed T cells as a distinct Neuropilin-1(+) memory population. Generation of liver-primed memory T cells may prevent pathogens that avoid DC maturation by innate immune escape from also escaping adaptive immunity through attrition of the T cell repertoire.
[Show abstract][Hide abstract] ABSTRACT: Cross-presentation is defined as the ability of certain professional antigen-presenting cells to take up, process and present extracellular antigens on major histocompatibility class I (MHC-I) molecules to CD8+ T cells. The stimulation of naive cytotoxic CD8+ T cells by this process, termed cross-priming, is involved in many different responses, including those to tumors, pathogens, graft tissues, and self-antigens. Dendritic cells (DCs), a heterogeneous cell population, are endowed with the highest cross-priming capacity. Investigation of their cross-presentation capacities, important both for vaccination and for the induction of immune tolerance can be performed by in vivo and in vitro assays. In this chapter we describe the preparation of antigens that can be used to test cross-presentation via pinocytosis, receptor-mediated endocytosis, and phagocytosis.
[Show abstract][Hide abstract] ABSTRACT: In vitro cultures of bone marrow-derived precursors are a convenient method for generating dendritic cells (DC). This method additionally overcomes the problem of low availability of certain DC types, DC heterogeneity, and laborious procedures encountered using ex vivo isolation protocols.Here we describe two standard protocols for in vitro differentiation of steady-state DC equivalents with Fms-like tyrosine kinase 3 ligand (Flt3L) and inflammatory-like DC using granulocyte-macrophages-colony-stimulating factor (GM-CSF). These protocols allow for obtaining up to 2 × 10(8) CD11c(high) inflammatory-like DC and up to 5 × 10(6) equivalents of each CD8+ and CD8- conventional DC and plasmacytoid DC.
[Show abstract][Hide abstract] ABSTRACT: Peptide epitopes presented by MHC class I molecules are produced through sequential proteolysis, frequently terminating with an aminoterminal trimming step. While the trimming enzymes processing endogenous MHC class I ligands in the endoplasmic reticulum have by now been characterized extensively, we have only recently identified an endosomal enzyme, insulin-regulated aminopeptidase (IRAP) that can trim cross-presented peptides derived from proteins internalized by dendritic cells. Here we summarize the essential features of IRAP as a trimming enzyme, propose an updated model of cellular cross-presentation pathways, and discuss potential additional functions of IRAP and its compartment in dendritic cell biology.
[Show abstract][Hide abstract] ABSTRACT: Peptides presented by MHC class I molecules are typically produced through antigen degradation by the proteasome followed by trimming by exopeptidases. According to recent results, these include both aminopeptidases and carboxypeptidases in the cytosol and the endoplasmic reticulum. While cytosolic peptidases have a net neutral or destructive effect on MHC ligands, endoplasmic reticulum aminopeptidases are required for efficient class I loading and have a strong effect on the repertoire of peptide/MHC complexes. Cells lacking these enzymes can be eliminated both by NK cells and by CD8+ T cells recognizing complexes formed between an MHC class Ib molecule and a conserved peptide. Cross-presented peptides derived from internalized antigens can be processed by insulin-regulated aminopeptidase, the only endosomal trimming peptidase.
Current opinion in immunology 10/2012; 25(1). DOI:10.1016/j.coi.2012.10.001 · 7.48 Impact Factor