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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 high , CD83 high , CD86 high , MHC‐II high ) and functional stimulation (NO high , IL‐10 absent , IL‐1β high ) 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, eIF2α 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 p110α 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. Unravelling molecular mechanisms that trigger immunogenic apoptosis of cancer cells could improve therapeutic intervention. Here, photo‐oxidative ER stress increases presentation of ‘eat me’ (surface‐exposed calreticulin) and ‘find me’ (ATP secretion) signals via a novel, PERK‐dependent pathway.

Determination of the in vivo significance of LDL receptor-related protein 1 (LRP1) dysfunction on lipid metabolism and atherosclerosis development in absence of its main ligand apoE. LRP1 knock-in mice carrying an inactivating mutation in the NPxYxxL motif were crossed with apoE-deficient mice. In the absence of apoE, relative to LRP1 wild-type animals, LRP1 mutated mice showed an increased clearance of postprandial lipids despite a compromised LRP1 endocytosis rate and inefficient insulin-mediated translocation of the receptor to the plasma membrane, likely due to inefficient slow recycling of the mutated receptor. Postprandial lipoprotein improvement was explained by increased hepatic clearance of triglyceride-rich remnant lipoproteins and accompanied by a compensatory 1.6-fold upregulation of LDLR expression in hepatocytes. One year-old apoE-deficient mice having the dysfunctional LRP1 revealed a 3-fold decrease in spontaneous atherosclerosis development and a 2-fold reduction in LDL-cholesterol levels. These findings demonstrate that the NPxYxxL motif in LRP1 is important for insulin-mediated translocation and slow perinuclear endosomal recycling. These LRP1 impairments correlated with reduced atherogenesis and cholesterol levels in apoE-deficient mice, likely via compensatory LDLR upregulation.

The membrane protein low-density lipoprotein receptor related-protein 1 (LRP1) has been attributed a role in cancer. However, its presumably often indirect involvement is far from understood. LRP1 has both endocytic and signaling activities. As a matricellular receptor it is involved in regulation, mostly by clearing, of various extracellular matrix degrading enzymes including matrix metalloproteinases, serine proteases, protease inhibitor complexes, and the endoglycosidase heparanase. Furthermore, by binding extracellular ligands including growth factors and subsequent intracellular interaction with scaffolding and adaptor proteins it is involved in regulation of various signaling cascades. LRP1 expression levels are often downregulated in cancer and some studies consider low LRP1 levels a poor prognostic factor. On the contrary, upregulation in brain cancers has been noted and clinical trials explore the use of LRP1 as cargo receptor to deliver cytotoxic agents. This mini-review focuses on LRP1's role in tumor growth and metastasis especially by modulation of the extracellular tumor environment. In relation to this role its diagnostic, prognostic and therapeutic potential will be discussed.

The low-density lipoprotein receptor-related protein-1 (LRP1) has a dual role in the metabolism of the amyloid precursor protein (APP). In cellular models, LRP1 enhances amyloid-β (Aβ) generation via APP internalization and thus its amyloidogenic processing. However, conditional knock-out studies in mice define LRP1 as an important mediator for the clearance of extracellular Aβ from brain via cellular degradation or transcytosis across the blood-brain barrier (BBB). In order to analyze the net effect of LRP1 on production and clearance of Aβ in vivo, we crossed mice with impaired LRP1 function with a mouse model of Alzheimer’s disease (AD). Analysis of Aβ metabolism showed that, despite reduced Aβ clearance due to LRP1 inactivation in vivo, less Aβ was found in cerebrospinal fluid (CSF) and brain interstitial fluid (ISF). Further analysis of APP metabolism revealed that impairment of LRP1 in vivo shifted APP processing from the Aβ-generating amyloidogenic cleavage by beta-secretase to the non-amyloidogenic processing by alpha-secretase as shown by a decrease in extracellular Aβ and an increase of soluble APP-α (sAPP-α). This shift in APP processing resulted in overall lower Aβ levels and a reduction in plaque burden. Here, we present for the first time clear in vivo evidence that global impairment of LRP1’s endocytosis function favors non-amyloidogenic processing of APP due to its reduced internalization and subsequently, reduced amyloidogenic processing. By inactivation of LRP1, the inhibitory effect on Aβ generation overrules the simultaneous impaired Aβ clearance, resulting in less extracellular Aβ and reduced plaque deposition in a mouse model of AD.

Furin is a proprotein convertase which activates a variety of regulatory proteins in the constitutive exocytic and endocytic pathway. The effect of genetic ablation of fur was studied in the endocrine pancreas to define its physiological function in the regulated secretory pathway. Pdx1-Cre/loxP furin KO mice show decreased secretion of insulin and impaired processing of known PC2 substrates like proPC2 and proinsulin II. Both secretion and PC2 activity depend on granule acidification, which was demonstrated to be significantly decreased in furin-deficient β cells by using the acidotrophic agent 3-(2,4-dinitroanilino)-3'amino-N-methyldipropylamine (DAMP). Ac45, an accessory subunit of the proton pump V-ATPase, was investigated as a candidate substrate. Ac45 is highly expressed in islets of Langerhans and furin was able to cleave Ac45 ex vivo. Furthermore, the exact cleavage site was determined. In addition, reduced regulated secretion and proinsulin II processing could be obtained in the insulinoma cell line βTC3 by downregulation of either furin or Ac45. Together, these data establish an important role for furin in regulated secretion, particularly in intragranular acidification most likely due to impaired processing of Ac45.

The proximal NPXY and distal NPXYXXL motifs in the intracellular domain of LRP1 play an important role in regulation of the function of the receptor. The impact of single and double inactivating knock-in mutations of these motifs on receptor maturation, cell surface expression, and ligand internalization was analyzed in mutant and control wild-type mice and MEFs. Single inactivation of the proximal NPXY or in combination with inactivation of the distal NPXYXXL motif are both shown to be associated with an impaired maturation and premature proteasomal degradation of full-length LRP1. Therefore, only a small mature LRP1 pool is able to reach the cell surface resulting indirectly in severe impairment of ligand internalization. Single inactivation of the NPXYXXL motif revealed normal maturation, but direct impairment of ligand internalization. In conclusion, the proximal NPXY motif proves to be essential for early steps in the LRP1 biosynthesis, whereas NPXYXXL appears rather relevant for internalization.

A U2 small nuclear RNA-associated protein, designated B′ ′, was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled us to isolate cDNA clone λ HB′ ′-1 from a phage λ gt11 expression library. This clone appeared to code for the B′ ′protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone λ HB′ ′-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone λ HB′ ′-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the λ HB′ ′-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B′ ′protein. These data demonstrate that clone λ HB′ ′-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens.

Furin's role in T cells The protease furin is induced on T cell activation and is a target of Stat-transcription factors in T cells. Studying furin has been hampered by the fact that germline deletion of this gene is embryonically lethal. Here furin's physiological role in T cells is studied using a T cell-specific furin knockout mice. The striking finding is that conditional deletion of furin in T cells results in loss of peripheral tolerance and systemic autoimmune disease. Furin deficiency compromises TGF-β release and Treg function, and is associated with inherently more aggressive effector T cells. This research suggests that inhibiting furin may promote the immune response but may also cause the loss of peripheral tolerance by reducing levels of bioavailable TGF-β-1. Conditional knockout of the protease furin in CD4 + T cells is shown to result in impaired peripheral tolerance and autoimmune disease. Furin deficiency compromises TGF-β release and T reg function, and is associated with inherently more aggressive effector T cells. Furin is one of seven proprotein convertase family members that promote proteolytic maturation of proproteins 1 . It is induced in activated T cells and is reported to process a variety of substrates including the anti-inflammatory cytokine transforming growth factor (TGF)-β1 (refs 2–4 ), but the non-redundant functions of furin versus other proprotein convertases in T cells are unclear. Here we show that conditional deletion of furin in T cells allowed for normal T-cell development but impaired the function of regulatory and effector T cells, which produced less TGF-β1. Furin-deficient T regulatory (T reg ) cells were less protective in a T-cell transfer colitis model and failed to induce Foxp3 in normal T cells. Additionally, furin-deficient effector cells were inherently over-active and were resistant to suppressive activity of wild-type T reg cells. Thus, our results indicate that furin is indispensable in maintaining peripheral tolerance, which is due, at least in part, to its non-redundant, essential function in regulating TGF-β1 production. Targeting furin has emerged as a strategy in malignant and infectious disease 5 , 6 . Our results suggest that inhibiting furin might activate immune responses, but may result in a breakdown in peripheral tolerance.