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.

Screening a genomic library oT Drosophih rwhmgosrer DNA with a human_firrcDNA probe resulted in the isolation of DNA clones thaw apparently belonged to two different DNA regions of the Drosophikr genome. Subsequently. corresponding Drosophih cDNA ciones were isolated. Nucleotide sequence analysis indicated that these cDNA clones originated from two different genes. which were called Djitrl and Djirr2. From overlappihig Dfirl 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 Dfurinl. exhibited striking sequence homology to human furin and contained the same protein domains except for the cysteine-rich region. Furthermore. unlike human furin. Dfurinl possessed an extended amino-terminal region in which a potential transmembranc anchor was present.Subtilisin-like proprotein processing enzyme: Furin; DTurin I: Drosupllilcl rr~c/trr~ogcrs~er

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.

Lefty/Ebaf polypeptides, novel members of the TGF-beta superfamily, are involved in endometrial differentiation and embryo implantation. Recently, we showed that, during undisturbed estrous cycle, lefty is present in mouse uterine horn primarily in a precursor form. Here, we show that decidual differentiation of endometrial stroma leads to increased lefty (approximately 3.1- to 3.6-fold in vivo and 5- to 8-fold in vitro) and processing of its precursor primarily to its long form. This event occurs on d 5 of pregnancy, and is paralleled by proprotein convertase (PC)5/6 up-regulation (approximately 6-fold increase for PC5A and 3-fold increase for PC5B) in decidualized uterine horn, independent of embryo implantation. Among the known convertases, only PC5/6A processes lefty to its long form. Taken together, the findings show that decidualized differentiation of stroma, which is a prerequisite for embryo implantation, leads to processing of lefty by PC5/6A.