Surface-exposed calreticulin (ecto-CRT) and secreted ATP are crucial damage-associated molecular patterns (DAMPs) for immunogenic apoptosis. Inducers of immunogenic apoptosis rely on an endoplasmic reticulum (ER)-based (reactive oxygen species (ROS)-regulated) pathway for ecto-CRT induction, but the ATP secretion pathway is unknown. We found that after photodynamic therapy (PDT), which generates ROS-mediated ER stress, dying cancer cells undergo immunogenic apoptosis characterized by phenotypic maturation (CD80 ) of dendritic cells as well as induction of a protective antitumour immune response. Intriguingly, early after PDT the cancer cells displayed ecto-CRT and secreted ATP before exhibiting biochemical signatures of apoptosis, through overlapping PERK-orchestrated pathways that require a functional secretory pathway and phosphoinositide 3-kinase (PI3K)-mediated plasma membrane/extracellular trafficking. Interestingly, eIF2a phosphorylation and caspase-8 signalling are dispensable for this ecto-CRT exposure. We also identified LRP1/CD91 as the surface docking site for ecto-CRT and found that depletion of PERK, PI3K p110a and LRP1 but not caspase-8 reduced the immunogenicity of the cancer cells. These results unravel a novel PERK-dependent subroutine for the early and simultaneous emission of two critical DAMPs following ROS-mediated ER stress.

Proprotein convertases (PC) are a family of proteases that cleave target proproteins at basic aminoacids, generating mature, biologically active polypeptides. Furin, the founding member of this family, is reported to have a number of potential substrates. However, germline deletion of fur is embryonically lethal 1 , and therefore the cell-type specific functions of furin remain poorly understood. Although furin is one of the predominant PC family members expressed by T cells, is induced by T cell activation and is a direct target of Stat family transcription factors 2 , the function of furin in T cells is also not clear. Herein, we show that conditional deletion of furin in T cells results in loss of peripheral tolerance characterized by activated T cells that overproduce both Th1 and Th2 type cytokines, circulating autoantibodies and development of inflammatory bowel disease. PCs are reportedly involved in the processing of several key immunoregulatory cytokines and we found that

The human fur gene encodes a protein, designated furin, the C-terminal half of which contains a transmembrane and a cysteine-rich receptor-like domain. The N-terminal half of furin exhibits striking primary amino acid sequence similarity to the catalytic domains of members of the subtilisin family of serine proteases. We here report characteristics of the furin protein and propose a three-dimensional model for its presumptive catalytic domain with characteristics, that predict furin to exhibit an endoproteolytic cleavage selectivity at paired basic residues. This prediction is substantiated by transfection and cotransfection experiments, using COS-1 cells. Full length fur cDNA evokes the specific synthesis of two polypeptides of about 100 kDa and 90 kDa as appeared from Western blot analysis of transfected COS-1 cells using a polyclonal anti-furin antiserum. Functional analysis of furin was performed by cotransfection of fur cDNA with cDNA encoding the 'wild type' precursor of von Willebrand factor (pro-vWF) and revealed an increased proteolytic processing of provWF. In contrast, cotransfection of fur cDNA with a recombinant derivative (provWFgly763), having the arginine residue adjacent to the proteolytic cleavage site (arg-ser-lys-arg) replaced by glycine, revealed that provWFgly763 is not processed by the fur gene product. We conclude that in higher eukaryotes, furin is the prototype of a subtilisin-like class of proprotein processing enzymes with substrate specificity for paired basic residues.

Furin is an endoprotease of the family of mammalian proprotein convertases and is involved in the activation of a large variety of regulatory proteins by cleavage at basic motifs. A large number of substrates have been attributed to furin on the basis of in vitro and ex vivo data. However, no physiological substrates have been confirmed directly in a mammalian model system, and early embryonic lethality of a furin knock-out mouse model has precluded in vivo verification of most candidate substrates. Here, we report the generation and characterization of an interferon inducible Mx-Cre/loxP furin knock-out mouse model. Induction resulted in near-complete ablation of the floxed fur exon in liver.In sharp contrast with the general furin knock-out mouse model, no obvious adverse effects were observed in the transgenic mice after induction. Histological analysis of the liver did not reveal any overt deviations from normal morphology. Analysis of candidate substrates in liver revealed complete redundancy for the processing of the insulin receptor. Variable degrees of redundancy were observed for the processing of albumin, ␣ 5 integrin, lipoprotein receptor-related protein, vitronectin and ␣ 1 -microglobulin/bikunin. None of the tested substrates displayed a complete block of processing. The absence of a severe phenotype raises the possibility of using furin as a local therapeutic target in the treatment of pathologies like cancer and viral infections, although the observed redundancy may require combination therapy or the development of a more broad spectrum convertase inhibitor.