J. W. M. Creemers

Insulin crystallization depends on zinc transporter ZnT8 expression, but is not required for normal glucose homeostasis in mice

Zinc co-crystallizes with insulin in dense core secretory granules, but its role in insulin biosynthesis, storage and secretion is unknown. In this study we assessed the role of the zinc transporter ZnT8 using ZnT8-knockout (ZnT8−/−) mice. Absence of ZnT8 expression caused loss of zinc release upon stimulation of exocytosis, but normal rates of insulin biosynthesis, normal insulin content and preserved glucose-induced insulin release. Ultrastructurally, mature dense core insulin granules were rare in ZnT8−/− beta cells and were replaced by immature, pale insulin “progranules,” which were larger than in ZnT8+/+ islets. When mice were fed a control diet, glucose tolerance and insulin sensitivity were normal. However, after high-fat diet feeding, the ZnT8−/− mice became glucose intolerant or diabetic, and islets became less responsive to glucose. Our data show that the ZnT8 transporter is essential for the formation of insulin crystals in beta cells, contributing to the packaging efficiency of stored insulin. Interaction between the ZnT8−/− genotype and diet to induce diabetes is a model for further studies of the mechanism of disease of human ZNT8 gene mutations.

Generation of structural and functional diversity in furin-like proteins in Drosophila melanogaster by alternative splicing of the Dfur1 gene.

To investigate whether or not alternative splicing might be a mechanism by which in Drosophila melanogaster diversity is generated in endoproteases of the novel eukaryotic family of subtilisin‐like proprotein processing enzymes, we determined structural and functional characteristics of the Dfur1 gene. Northern blot analysis revealed Dfur1 transcripts of 7.6, 6.5, 4.5 and 4.0 kb. By comparative nucleotide sequence analysis of Dfur1 genomic and cDNA clones, 10 coding exons were identified and, together with Northern blot analysis using exon‐specific probes, evidence was obtained that the four transcripts are generated by alternative splicing and polyadenylation. The apparently complete open reading frames of three Dfur1 cDNAs revealed that these coded for three furin‐like proteins, Dfurin1 (892 residues), Dfurin1‐CRR (1101 residues) and Dfurin1‐X (1269 residues), which possessed common but also unique structural domains. These various isoforms of furin in Drosophila were characterized in gene transfer studies using immunoprecipitation analysis. Differential expression of Dfur1 transcripts was found in Northern blot analysis of RNA from various developmental stages of Drosophila. RNA in situ hybridization experiments revealed that the Dfurin1‐X and Dfurin1‐CRR isoforms are expressed in non‐overlapping sets of tissues during Drosophila embryogenesis. In gene transfer experiments in which the Dfurin1, Dfurin1‐CRR and Dfurin1‐X proteins were expressed at high levels together with the precursor of the beta A‐chain of activin‐A, a member of the transforming growth factor beta (TGF beta) superfamily, or the precursor of von Willebrand factor, all three proteins appeared capable of processing these substrates. Our studies indicate that the Dfur1 gene encodes structurally different subtilisin‐like proprotein processing enzymes with distinct physiological functions in Drosophila.

PREPL deficiency: Delineation of the phenotype and development of a functional blood assay

PurposePREPL deficiency causes neonatal hypotonia, ptosis, neonatal feeding difficulties, childhood obesity, xerostomia, and growth hormone deficiency. Different recessive contiguous gene deletion syndromes involving PREPL and a variable combination of SLC3A1 (hypotonia–cystinuria syndrome), CAMKMT (atypical hypotonia–cystinuria syndrome), and PPM1B (2p21 deletion syndrome) have been described. In isolated PREPL deficiency, previously described only once, the absence of cystinuria complicates the diagnosis. Therefore, we developed a PREPL blood assay and further delineated the phenotype.MethodsClinical features of new subjects with PREPL deficiency were recorded. The presence of PREPL in lymphocytes and its reactivity with an activity-based probe were evaluated by western blot.ResultsFive subjects with isolated PREPL deficiency, three with hypotonia-cystinuria syndrome, and two with atypical hypotonia-cystinuria syndrome had nine novel alleles. Their IQs ranged from 64 to 112. Adult neuromuscular signs included ptosis, nasal dysarthria, facial weakness, and variable proximal and neck flexor weakness. Autonomic features are prevalent. PREPL protein and reactivity were absent in lymphocytes from subjects with PREPL deficiency, but normal in the clinically similar Prader–Willi syndrome.ConclusionPREPL deficiency causes neuromuscular, autonomic, cognitive, endocrine, and dysmorphic clinical features. PREPL is not deficient in Prader–Willi syndrome. The novel blood test should facilitate the confirmation of PREPL deficiency.

Therapeutic Potential of Furin Inhibition: An Evaluation Using a Conditional Furin Knockout Mouse Model

Abstract The proprotein convertase Furin is a serine endoprotease which cleaves protein precursors carboxyterminal of basic residues in motifs such as Arg–X–X–Arg and Lys/Arg–Arg. Cleavage usually results in activation of the proprotein but can also inactivate or modify the activity. Therefore, it is not surprising that it plays a major role in many physiological processes and pathologies, including cancer. The other proprotein convertases belonging to the same family, PC1/3, PC2, PACE4, PC4, PC5/6, and PC7, cleave at similar cleavage sites and provide partial redundancy. To unravel the specific role of Furin in vivo, knockout mouse models have been generated. Furin null mice die between e10.5 and e11.5 due to severe ventral closure defects and the failure of the heart tube to fuse and undergo looping morphogenesis. Therefore, a conditional Furin knockout mouse was generated to investigate the role of Furin in specific organs, such as pancreas, liver, T-cells, endothelial cells, and salivary glands, resul...

Abstract 4627: Generation and characterization of non-competitive furin-inhibiting nanobodies

Furin belongs to a family of seven closely related subtilisin-like serine endoproteases, known as proprotein convertases (PCs), PC1/3, PC2, PC4, PC5/6, PACE4 and PC7. The physiological role of furin is to cleave and hence activate a large variety of proproteins. Therefore, it is not surprising that it plays a major role in many pathologies such as cancer and infectious diseases. Furin inhibition might be a good strategy for therapeutic intervention, and several furin inhibitors have been generated, although thus far none are entirely furin-specific. To reduce potential side effects caused by cross-reactivity with other proteases, dromedary heavy chain antibodies against catalytically active furin were developed as specific furin inhibitors. Nanobodies derived from these antibodies bind only to mouse and human furin but not to other PCs. In addition, upon overexpression in cell lines, these nanobodies can inhibit the cleavage of three different furin substrates, TGFβ, BAFF and GPC3. The purified nanobodies can also inhibit the cleavage of diphtheria toxin into its enzymatically active A fragment, but do not inhibit cleavage of a small synthetic peptide-based substrate, suggesting a mode of action based on steric hindrance. The inhibitory constant of the purified nanobodies was shown to be in the micromolar range and the nanobodies are non-competitive inhibitors as demonstrated by Dixon plot. Furthermore, anti-furin nanobodies can protect HEK293T cells from diphtheria toxin-induced cytotoxicity as efficiently as the well characterized PC inhibitor nona-D-arginine. In conclusion, these antibody-based single-chain nanobodies represent the first generation of highly specific, non-competitive furin inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4627. doi:1538-7445.AM2012-4627