Anton Roebroek

A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death

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 (CD80high, CD83high, CD86high, MHC‐IIhigh) and functional stimulation (NOhigh, IL‐10absent, IL‐1βhigh) 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.

Limited redundancy of the proprotein convertase furin in mouse liver

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, α5 integrin, lipoprotein receptor-related protein, vitronectin and α1-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.

The functional role of the second NPXY motif of the LRP1 beta-chain in tissue-type plasminogen activator-mediated activation of N-methyl-D-aspartate receptors.

The low density lipoprotein receptor-related protein 1 (LRP1) emerges to play fundamental roles in cellular signaling pathways in the brain. One of its prominent ligands is the serine proteinase tissue-type plasminogen activator (tPA), which has been shown to act as a key activator of neuronal mitogen-activated protein kinase pathways via the N-methyl-d-aspartate (NMDA) receptor. However, here we set out to examine whether LRP1 and the NMDA receptor might eventually act in a combined fashion to mediate tPA downstream signaling. By blocking tPA from binding to LRP1 using the receptor-associated protein, we were able to completely inhibit NMDA receptor activation. Additionally, inhibition of NMDA receptor calcium influx with MK-801 resulted in dramatic reduction of tPA-mediated downstream signaling. This indicates a functional interaction between the two receptors, since both experimental approaches resulted in strongly reduced calcium influx and Erk1/2 phosphorylation. Additionally, we were able to inhibit Erk1/2 activation by competing for the LRP1 C-terminal binding motif with a truncated PSD95 construct resembling its PDZ III domain. Furthermore, we identified the distal NPXY amino acid motif in the C terminus of LRP1 as the crucial element for LRP1-NMDA receptor interaction via the adaptor protein PSD95. These results provide new insights into the mechanism of a tPA-induced, LRP1-mediated gating mechanism for NMDA receptors.

cDNA sequence of a Drosophila melanogaster gene, Dfur1, encoding a protein structurally related to the subtilisin-like proprotein processing enzyme furin

Screening a genomic library of Drosophila melanogaster DNA with a human fur cDNA probe resulted in the isolation of DNA clones that apparently belonged to two different DNA regions of the Drosophila genome. Subsequently, corresponding Drosophila cDNA clones were isolated. Nucleotide sequence analysis indicated that these cDNA clones originated from two different genes, which were called Dfur1 and Dfur2. From overlapping Dfur1 cDNA clones, a composite cDNA could be constructed and analysis of its nucleotide sequence revealed the coding sequence for a protein of 899 amino acid residues. This protein, designated Dfurin1, exhibited striking sequence homology to human furin and contained the same protein domains except for the cysteine‐rich region. Furthermore, unlike human furin. Dfurin1 possessed an extended amino‐terminal region in which a potential transmembrane anchor was present.

Mutant Lrp1 Knock-In Mice Generated by Recombinase-Mediated Cassette Exchange Reveal Differential Importance of the NPXY Motifs in the Intracellular Domain of LRP1 for Normal Fetal Development

ABSTRACT Lrp1 knock-in mice carrying either a wild-type allele or three different mutated alleles encoding the multifunctional endocytic receptor LRP1 were generated by recombinase-mediated cassette exchange (RMCE). Reinsertion by RMCE of a wild-type allele led to a normal pattern and level of gene expression and a completely normal phenotype, indicating that the RMCE procedure itself is neutral with respect to the function of the gene locus. In contrast, reinsertion of mutated LRP1 alleles carrying either inactivating mutations in the proximal NPXY motif (NPTY→AATA) of the cytoplasmic domain or in the furin cleavage site (RHRR→AHAA) caused distinctive liver phenotypes: respectively, either a late fetal destruction of the organ causing perinatal death or a selective enlargement of von-Kupffer cell lysosomes reminiscent of a mild lysosomal storage without an apparent negative effect on animal survival. Notably, mutation of the distal NPXY motif overlapping with an YXXL motif (NPVYATL→AAVAATL) did not cause any obvious pathological effect. The mutations showed no effect on the LRP1 expression level; however, as expected, the proteolytic maturation of LRP1 into its two subunits was significantly impaired, although not completely abolished, in the furin cleavage mutant. These data demonstrate that RMCE is a reliable and efficient approach to generate multiple mutant knock-in alleles for in vivo functional analysis of individual domains or motifs of large multidomain proteins. Its application in Lrp1 reveals dramatically variant phenotypes, of which further characterization will definitively contribute to our understanding of the biology of this multifunctional receptor.

Expression in human lung tumor cells of the proprotein processing enzyme PC1/PC3 Cloning and primary sequence of a 5 kb cDNA

Northern blot analysis of human lung tumors indicated that the gene, which encodes the subtilisin‐like proprotein processing enzyme PC1/PC3, was highly expressed in almost all carcinoid tumors tested. In small cell lung carcinomas (SCLCs), expression varied. In non‐SCLCs and normal lung, no expression was found. Analysis of SCLC cell lines revealed that expression was restricted preferentially to cell lines of the classical type, to lung tumor cells expressing the PC1/PC3 gene, transcripts of 3 kb and 5 kb were detected, the 5 kb mRNA always being the most abundant species. We isolated a cDNA corresponding to the 5 kb human PC1/PC3 transcript, determined the nucleotide sequence of it and deduced the amino acid sequence of the corresponding protein. Furthermore, we conclude that the two PC1/PC3 transcripts have 3′ non‐coding regions of different size and encode the same protein.

Functional interaction of megalin with the megalinbinding protein (MegBP), a novel tetratrico peptide repeat-containing adaptor molecule

Megalin is a member of the LDL receptor gene family that plays an important role in forebrain development and in cellular vitamin D metabolism through endocytic uptake of vitamin D metabolites. Similar to other receptors in this gene family, megalin is believed to functionally interact with intracellular proteins through adaptors that bind to the receptor tail and regulate its endocytic and signal transducing activities. Using yeast two-hybrid screens, we identified a novel scaffold protein with tetratrico peptide repeats, the megalin-binding protein (MegBP) that associates with the receptor. The binding site of MegBP was mapped to an N-terminal region on the receptor tail harboring a proline-rich peptide element. MegBP binding did not block the endocytic activity of the receptor; however, overexpression resulted in cellular lethality. In further screens, we identified proteins that bound to MegBP and thus might be recruited to the megalin tail. MegBP-interacting partners included several transcriptional regulators such as the SKI-interacting protein (SKIP), a co-activator of the vitamin D receptor. These finding suggest a model whereby megalin directly participates in transcriptional regulation through controlled sequestration or release of transcription factors via MegBP.

Role of furin in granular acidification in the endocrine pancreas: Identification of the V-ATPase subunit Ac45 as a candidate substrate

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.

Neuronal differentiation is accompanied by NSP-C expression

Abstract Neuroendocrine-specific protein (NSP) reticulons are expressed in neural and neuroendocrine tissues and cell cultures derived therefrom, while most other cell types lack NSP-reticulons. Three major subtypes have been identified so far, designated NSP-A, NSP-B, and NSP-C. We have investigated the correlation between the degree of neuronal differentiation, determined by morphological and biochemical criteria, and NSP-reticulon subtype expression. For this purpose, several human neuroblastoma cell lines, exhibiting different degrees of neuronal differentiation, were examined immuno(cyto)chemically. It became obvious that the expression of NSP-C, as detected by immunofluorescence microscopy and Western blotting, is most prominent in cell lines with a high degree of neuronal differentiation, such as LA-N-5. Such highly differentiated cells also express other neural and neuroendocrine markers, such as neural cell adhesion molecule (NCAM), neurofilament proteins, synaptophysin, and chromogranin. NSP-A was observed in all cell lines to a different extent. However, no clear correlation was observed with the degree of neuronal differentiation as defined by other neuronal and neuroendocrine markers or morphology. NSP-B could not be detected. The induction of neuronal differentiation with nerve growth factor, dbcAMP, and retinoic acid in the rat pheochromocytoma cell line PC12 and the human teratocarcinoma cell line hNT2, respectively, induced the expression of NSP-A and NSP-C in these cell lines parallel to the induction of neurofilament protein expression. It is concluded that NSP-C expression, in particular, is strongly correlated with neuronal differentiation.

Proprotein processing activity and cleavage site selectivity of the Kex2-like endoprotease PACE4

Proprotein processing activity of the Kex2‐like mammalian endoprotease PACE4 and its cleavage selectivity for sites with basic amino acid residues were determined. Using a recombinant vaccinia virus‐based expression system, PACE4 was expressed in pig kidney PK(15) cells and, like two other Kex2‐like endoproteases furin and PC6A, shown to correctly process the precursor of von Willebrand factor (pro‐vWF). Furthermore, characteristics of the cleavage site selectivity of PACE4 were compared to those of furin and PC6A using the vWF cleavage site mutants vWFR‐lG, vWFK‐2A, and vWFR‐4A as substrates. Cleavage site selectivity of PACE4 and PC6A appeared to be similar but they differed from that of furin.