Abstract
Dendritic 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.
Highlights
Dendritic cells (DCs) are immune myeloid cells that exert prime functions in the initiation of adaptive immune responses
Aiming to identify the peptidases involved in trimming the amino-terminus of MHC-I-presented antigenic peptides, we have previously reported that insulin-regulated aminopeptidase (IRAP) is rapidly recruited to newly formed phagosomes (Saveanu et al, 2009; Weimershaus et al, 2012, 2018)
In agreement with the results from the immunoblot analysis, fluorescence-activated cell sorting (FACS) staining for the late endosomal/lysosomal markers lysosome-associated membrane protein (LAMP)-1 and Rab7 showed higher fluorescence intensities on phagosomes isolated from IRAP−/− DCs, indicating an accelerated phagosome maturation in these cells compared to wt DCs (Figure 1B)
Summary
Dendritic cells (DCs) are immune myeloid cells that exert prime functions in the initiation of adaptive immune responses. While the C-terminus of most MHC-I-associated peptides is generated by the proteasome, different N-terminal trimming peptidases have been identified (reviewed in Weimershaus et al, 2013). In this context, we have discovered an unexpected role as trimming enzyme for insulin-regulated aminopeptidase (IRAP), a widely expressed enzyme best studied for its role in oxytocin and vasopressin cleavage and its colocalization with glucose transporter (GLUT) 4 to specific cytoplasmic insulin-responsive storage vesicles (GSVs; reviewed in Li et al, 2019)
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