Shedding of membrane type matrix metalloproteinase 5 by a furin-type convertase: a potential mechanism for down-regulation

The shedding of membrane-associated proteins has been recognized as a regulatory mechanism to either up-regulate or down-regulate cellular functions by releasing membrane-bound growth factors or removing ectodomains of adhesion molecules and receptors. We have reported previously that the ectoenzyme of membrane type matrix metalloproteinase 5 (MT5-MMP) is shed into extracellular milieu (Pei, D. (1999) J. Biol. Chem. 274, 8925-8932). Here we present evidence that MT5-MMP is shed by a furin-type convertase activity in the trans-Golgi network. Among proteinase inhibitors screened, only decanoyl-Arg-Val-Lys-Arg-chloromethylketone, a known inhibitor for furin-type convertases, blocked the shedding of MT5-MMP in a dose-dependent manner. As expected, decanoyl-Arg-Val-Lys-Arg-chloromethylketone also prevented the activation of MT5-MMP, raising the possibility that the observed shedding could be autolytic. However, an active site mutant devoid of any catalytic activity, is also shed efficiently, thus ruling out the autolytic pathway. The shedding cleavage was subsequently mapped to the stem region immediately upstream of the transmembrane domain, where a cryptic furin recognition site, (545)RRKERR, was recognized. Indeed, MT5-MMP and furin are co-localized in the trans-Golgi network and the shed species could be detected inside the cells. Furthermore, deletion mutations removing this cryptic site prevented MT5-MMP from shedding. The resulting mutants express a gain-of-function phenotype by mediating more robust activation of proMMP-2 than the wild type molecule. Thus, shedding provides a potential mechanism to regulate proteolytic activity of membrane-bound MMPs.     

Shedding of collagen XVII/BP180: structural motifs influence cleavage from cell surface

Collagen XVII/BP180, an epithelial adhesion molecule, belongs to the group of collagenous transmembrane proteins, which are characterized by ectodomain shedding. We recently showed that ADAMs can cleave collagen XVII, but also that furin participates in this process (Franzke, C. W., Tasanen, K., Sch?cke, H., Zhou, Z., Tryggvason, K., Mauch, C., Zigrino, P., Sunnarborg, S., Lee, D. C., Fahrenholz, F., and Bruckner-Tuderman, L. (2002) EMBO J. 21, 5026-5035). To define the cleavage region in the juxtamembranous NC16A linker domain and assess its structure and requirements for shedding, we constructed deletion mutants of the NC16A domain, expressed them in COS-7 cells, and analyzed their structural integrity and shedding behavior. A mutant lacking the furin consensus sequence was shed in a normal manner, demonstrating that furin does not cleave collagen XVII but rather activates ADAMs (a disintegrin and metalloproteinase). Large deletions of the NC16A domain prevented shedding, and analysis of defined smaller deletions pointed to the stretch of amino acid residues 528-547 as important for sheddase recognition and cleavage. Secondary protein structure predictions showed that deletion of this stretch resulted in an NC16A domain with a positive net charge and an amphipathic alpha-helix, which can cause conformational changes in the collagen XVII homotrimer. Assessment of triple-helix folding of the mutants revealed a lower thermal stability of all non-shed variants than of wild-type collagen XVII or the shed mutants. In contrast, deletion of the putative nucleation site for triple-helix folding of collagenous transmembrane proteins did not affect folding of collagen XVII. The data indicate that the conformation of the NC16A domain and steric availability of the cleavage site influence shedding and is important for folding of collagen XVII.     

Cloning and functional expression of Dfurin2, a subtilisin-like proprotein processing enzyme of Drosophila melanogaster with multiple repeats of a cysteine motif.

Production of a variety of regulatory eukaryotic proteins, such as growth factors and polypeptide hormones, often involves endoproteolytic processing of proproteins at cleavage sites consisting of paired basic residues. The first known mammalian proprotein processing enzyme with such specificity is the human fur gene product furin. Structurally and functionally, furin is related to the subtilisin-like serine endoprotease kexin (EC 3.4.21.61) of yeast Saccharomyces cerevisiae; unlike kexin, it contains a cysteine-rich region with an unknown function. Here, we describe cloning and sequencing of a 5.8-kbp cDNA of the Dfur2 gene, a fur-like gene of Drosophila melanogaster, which we found expressed during various stages of development. This Dfur2 cDNA has an open reading frame for a 1680-residue protein, called Dfurin2. Dfurin2 contains similar protein domains as mammalian furin, however, it has an extended amino-terminal region and its cysteine-rich region is much larger than that of mammalian furin. Because of this latter phenomenon, we were able to identify a particular cysteine motif that was repeated multiple times in Dfurin2 but present only twice in mammalian furin. Furthermore, we show that Dfur2 encodes an endoproteolytic enzyme with specificity for paired basic amino acid residues as, in cotransfection experiments, correct cleavage was demonstrated of the precursor of the von Willebrand factor but not of a cleavage mutant. Finally, Dfur2 was mapped to region 14C of the X chromosome of D. melanogaster.     

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.     

Template-assisted rational design of peptide inhibitors of furin using the lysine fragment of the mung bean trypsin inhibitor

Highly active, small-molecule furin inhibitors are attractive drug candidates to fend off bacterial exotoxins and viral infection. Based on the 22-residue, active Lys fragment of the mung bean trypsin inhibitor, a series of furin inhibitors were designed and synthesized, and their inhibitory activity towards furin and kexin was evaluated using enzyme kinetic analysis. The most potent inhibitor, containing 16 amino acid residues with a Ki value of 2.45x10(-9) m for furin and of 5.60x10(-7) m for kexin, was designed with three incremental approaches. First, two nonessential Cys residues in the Lys fragment were deleted via a Cys-to-Ser mutation to minimize peptide misfolding. Second, residues in the reactive site of the inhibitor were replaced by the consensus substrate recognition sequence of furin, namely, Arg at P1, Lys at P2, Arg at P4 and Arg at P6. In addition, the P7 residue Asp was substituted with Ala to avoid possible electrostatic interference with furin inhibition. Finally, the extra N-terminal and C-terminal residues beyond the doubly conjugated disulfide loops were further truncated. However, all resultant synthetic peptides were found to be temporary inhibitors of furin and kexin during a prolonged incubation, with the scissile peptide bond between P1 and P1' being cleaved to different extents by the enzymes. To enhance proteolytic resistance, the P1' residue Ser was mutated to D-Ser or N-methyl-Ser. The N-methyl-Ser mutant gave rise to a Ki value of 4.70x10(-8) m for furin, and retained over 80% inhibitory activity even after a 3 h incubation with the enzyme. By contrast, the d-Ser mutant was resistant to cleavage, although its inhibitory activity against furin drastically decreased. Our findings identify a useful template for the design of potent, specific and stable peptide inhibitors of furin, shedding light on the molecular determinants that dictate the inhibition of furin and kexin.     

Shedding of collagen XXIII is mediated by furin and depends on the plasma membrane microenvironment

Collagen XXIII belongs to the class of type II orientated transmembrane collagens. A common feature of these proteins is the presence of two forms of the molecule: a membrane-bound form and a shed form. Here we demonstrate that, in mouse lung, collagen XXIII is found predominantly as the full-length form, whereas in brain, it is present mostly as the shed form, suggesting that shedding is tissue-specific and tissue-regulated. To analyze the shedding process of collagen XXIII, a cell culture model was established. Mutations introduced into two putative proprotein convertase cleavage sites showed that altering the second cleavage site inactivated much of the shedding. This supports the idea that furin, a major physiological protease, is predominantly responsible for shedding. Furthermore, our studies indicate that collagen XXIII is localized in lipid rafts in the plasma membrane and that ectodomain shedding is altered by a cholesterol-dependent mechanism. Moreover, newly synthesized collagen XXIII either is cleaved inside the Golgi/trans-Golgi network or reaches the cell surface, where it becomes protected from processing by being localized in lipid rafts. These mechanisms allow the cell to regulate the amounts of cell surface-bound and secreted collagen XXIII.     

Binding properties of chimeric insulin receptors containing the cysteine-rich domain of either the insulin-like growth factor I receptor or the insulin receptor related receptor.

We constructed and expressed chimeric receptor cDNAs with insulin receptor exon 3 (residues 191-297 of the cysteine-rich region) replaced with either the comparable region of the insulin-like growth factor I receptor (IGF-IR) or the insulin receptor related receptor (IRR). Both chimeric receptors still could bind insulin with as high affinity as the wild-type receptor. In addition, chimeric receptors containing exon 3 of the IGF-IR could also bind with high affinity both IGF-I and IGF-II. In contrast, chimeric receptors containing exon 3 of IRR did not bind either IGF-I, IGF-II, or relaxin. These results indicate that (1) the high affinity of binding of insulin to its receptor can occur in the absence of insulin receptor specific residues encoded by exon 3, the cysteine-rich region; (2) the cysteine-rich region of the IGF-I receptor can confer high-affinity binding to both IGF-I and IGF-II; and 3) the IRR is unlikely to be a receptor for either IGF-I, IGF-II, or relaxin.     

ADAM13 disintegrin and cysteine-rich domains bind to the second heparin-binding domain of fibronectin

ADAM13 is a member of the disintegrin and metalloprotease protein family that is expressed on cranial neural crest cells surface and is essential for their migration. ADAM13 is an active protease that can cleave fibronectin in vitro and remodel a fibronectin substrate in vivo. Using a recombinant secreted protein containing both disintegrin and cysteine-rich domains of ADAM13, we show that this "adhesive" region of the protein binds directly to fibronectin. Fibronectin fusion proteins corresponding to the various functional domains were used to define the second heparin-binding domain as the ADAM13 binding site. Mutation of the syndecan-binding site (PPRR --> PPTM) within this domain abolishes binding of the recombinant disintegrin and cysteine-rich domains of ADAM13. We further show that the adhesive disintegrin and cysteine-rich domain of ADAM13 can promote cell adhesion via beta(1) integrins. This adhesion requires integrin activation and can be prevented by antibodies to the cysteine-rich domain of ADAM13 and beta(1) integrin. Finally, wild type, but not the E/A mutant of ADAM13 metalloprotease domain, can be shed from the cell surface, releasing the metalloprotease domain associated with the disintegrin and cysteine-rich domains. This suggests that ADAM13 shedding may involve its own metalloprotease activity and that the released protease may interact with both integrins and extracellular matrix proteins.     

Furin is a subtilisin-like proprotein processing enzyme in higher eukaryotes

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 endo-proteolytic 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 prov WF. In contrast, cotransfection of fur cDNA with a recombinat 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.     

Metalloprotease-dependent protransforming growth factor-alpha ectodomain shedding in the absence of tumor necrosis factor-alpha-converting enzyme

Zinc-dependent metalloproteases can mediate the shedding of the extracellular domain of many unrelated transmembrane proteins from the cell surface. In most instances, this process, also known as ectodomain shedding, is regulated via protein kinase C (PKC). The tumor necrosis factor alpha-converting enzyme (TACE) was the first protease involved in regulated protein ectodomain shedding identified. Although TACE belongs to the family of metalloprotease-disintegrins, few members of this family have been shown to participate in regulated ectodomain shedding. In fact, the phenotype of tace-/- cells and that of Chinese hamster ovary cell mutants defective in ectodomain shedding points to the existence of a common PKC-activated ectodomain shedding system, whose proteolytic component is TACE, that acts on a variety of transmembrane proteins. Examples of these proteins include the Alzheimer's disease-related protein beta-amyloid precursor protein (betaAPP) and the transmembrane growth factors protransforming growth factor-alpha (pro-TGF-alpha) and, as shown in this report, proheparin-binding epidermal growth factor-like growth factor (pro-HB-EGF). Here we show that the mercurial compound 4-aminophenylmercuric acetate (APMA), frequently used to activate in vitro recombinant matrix metalloproteases, is an activator of the shedding of betaAPP, pro-HB-EGF, and pro-TGF-alpha. Treatment of tace-/- cells or Chinese hamster ovary shedding-defective mutants with APMA activates the cleavage of pro-TGF-alpha but not that of pro-HB-EGF or betaAPP, indicating that APMA activates TACE and also a previously unacknowledged proteolytic activity specific for pro-TGF-alpha. Characterization of this proteolytic activity indicates that it acts on pro-TGF-alpha located at the cell surface and that it is a metalloprotease active in cells defective in furin activity. In summary, treatment of shedding-defective cell lines with APMA unveils the existence of a metalloprotease activity alternative to TACE with the ability to specifically shed the ectodomain of pro-TGF-alpha.     

Post-translational processing of beta-secretase (beta-amyloid-converting enzyme) and its ectodomain shedding. The pro- and transmembrane/cytosolic domains affect its cellular activity and amyloid-beta production

Processing of the beta-amyloid precursor protein (betaAPP) by beta- and gamma-secretases generates the amyloidogenic peptide Abeta, a major factor in the etiology of Alzheimer's disease. Following the recent identification of the beta-secretase beta-amyloid-converting enzyme (BACE), we herein investigate its zymogen processing, molecular properties, and cellular trafficking. Our data show that among the proprotein convertase family members, furin is the major converting enzyme of pro-BACE into BACE within the trans-Golgi network of HK293 cells. While we demonstrate that the 24-amino acid prosegment is required for the efficient exit of pro-BACE from the endoplasmic reticulum, it may not play a strong inhibitory role since we observe that pro-BACE can produce significant quantities of the Swedish mutant betaAPP(sw) beta-secretase product C99. BACE is palmitoylated at three Cys residues within its transmembrane/cytosolic tail and is sulfated at mature N-glycosylated moieties. Data with three different antibodies show that a small fraction of membrane-bound BACE is shed into the medium and that the extent of ectodomain shedding is palmitoylation-dependent. Overexpression of full-length BACE causes a significant increase in the production of C99 and a decrease in the alpha-secretase product APPsalpha. Although there is little increase in the generation of Abeta by full-length BACE, overexpression of either a soluble form of BACE (equivalent to the shed form) or one lacking the prosegment leads to enhanced Abeta levels. These findings suggest that the shedding of BACE may play a role in the amyloidogenic processing of betaAPP.     

Evidence for a critical role of the tumor necrosis factor alpha convertase (TACE) in ectodomain shedding of the p75 neurotrophin receptor (p75NTR)

Protein ectodomain shedding, the proteolytic release of the extracellullar domain of membrane-tethered proteins, can dramatically affect the function of cell surface receptors, growth factors, cytokines, and other proteins. In this study, we evaluated the activities involved in ectodomain shedding of p75NTR, a neurotrophin receptor with critical roles in neuronal differentiation and survival. p75NTR is shed in a variety of cell types, including dorsal root ganglia cells and PC12 cells. In Chinese hamster ovary cells, inhibitors of the MEK/ERK and p38 MAP kinase pathways uncovered distinct signaling pathways required for the constitutive and stimulated shedding of p75NTR. Stimulated p75NTR shedding is abrogated in M2 mutant Chinese hamster ovary cells that lack functional tumor necrosis factor-alpha converting enzyme (TACE, also referred to as ADAM17) and in cells isolated from adam17-/- mice, but not in cells from adam9/12/15-/- or adam10-/- mice. Stimulated p75(NTR) shedding is strongly reduced by deletion of 15 amino acid residues in its extracellular membrane-proximal stalk domain. However, similar to other shed proteins, point mutations and overlapping shorter deletions within this region have little or no effect on shedding. Because ectodomain shedding of p75NTR releases a soluble ectodomain and could also be a prerequisite for its regulated intramembrane proteolysis, these findings may have important implications for the functional regulation of p75NTR.     

Coiled Coils Ensure the Physiological Ectodomain Shedding of Collagen XVII

α-Helical coiled coils, frequent protein oligomerization motifs, are commonly observed in vital proteins. Here, using collagen XVII as an example, we provide evidence for a novel function of coiled coils in the regulation of ectodomain shedding. Transmembrane collagen XVII, an epithelial cell surface receptor, mediates dermal-epidermal adhesion in the skin, and its dysfunction is linked to human skin blistering diseases. The ectodomain of this collagen is constitutively shed from the cell surface by proteinases of a disintegrin and metalloprotease family; however, the mechanisms regulating shedding remain elusive. Here, we used site-specific mutagenesis to target the coiled-coil heptad repeats within the juxtamembranous, extracellular noncollagenous 16th A (NC16A) domain of collagen XVII. This resulted in a substantial increase of ectodomain shedding, which was not mediated by disintegrin and metalloproteases. Instead, conformational changes induced by the mutation(s) unmasked a furin recognition sequence that was used for cleavage. This study shows that apart from their functions in protein oligomerization, coiled coils can also act as regulators of ectodomain shedding depending on the biological context.     

pH-induced conformational transitions of a molten-globule-like state of the inhibitory prodomain of furin: implications for zymogen activation

The endoprotease furin, which belongs to the family of mammalian proprotein convertase (PC), is synthesized as a zymogen with an N-terminal, 81-residue inhibitory prodomain. It has been shown that the proenzyme form of furin undergoes a multistep 'autocatalytic' removal of the prodomain at the C-terminal side of the two consensus sites, R(78)-T-K-R(81) approximately and R(44)-G-V-T-K-R(49) approximately. The furin-mediated cleavage at R(44)-G-V-T-K-R(49) approximately, in particular, is significantly accelerated in an 'acidic' environment. Here, we show that under neutral pH conditions, the inhibitory prodomain of furin is partially folded and undergoes conformational exchanges as indicated by extensive broadening of the NMR spectra. Presence of many ring-current shifted methyl resonances suggests that the partially folded state of the prodomain may still possess a 'semirigid' protein core with specific packing interactions among amino acid side chains. Measurements of the hydrodynamic radii and compaction factors indicate that this partially folded state is significantly more compact than a random chain. The conformational stability of the prodomain appears to be pH sensitive, in that the prodomain undergoes an unfolding transition towards acidic conditions. Our NMR analyses establish that the acid-induced unfolding is mainly experienced by the residues from the C-terminal half of the prodomain (residues R(44)-R(81)) that contains the two furin cleavage sites. A 38-residue peptide fragment derived from the entire pH-sensitive C-terminal region (residues R(44)-R(81)) does not exhibit any exchange-induced line broadening and adopts flexible conformations. We propose that at neutral pH, the cleavage site R(44)-G-V-T-K-R(49) approximately is buried within the protein core that is formed in part by residues from the N-terminal region, and that the cleavage site becomes exposed under acidic conditions, leading to a facile cleavage by the furin enzyme.     

Phylogenetic analysis of the teneurins: conserved features and premetazoan ancestry

Teneurins are type II transmembrane proteins expressed during pattern formation and neurogenesis with an intracellular domain that can be transported to the nucleus and an extracellular domain that can be shed into the extracellular milieu. In Drosophila melanogaster, Caenorhabditis elegans, and mouse the knockdown or knockout of teneurin expression can lead to abnormal patterning, defasciculation, and abnormal pathfinding of neurites, and the disruption of basement membranes. Here, we have identified and analyzed teneurins from a broad range of metazoan genomes for nuclear localization sequences, protein interaction domains, and furin cleavage sites and have cloned and sequenced the intracellular domains of human and avian teneurins to analyze alternative splicing. The basic organization of teneurins is highly conserved in Bilateria: all teneurins have epidermal growth factor (EGF) repeats, a cysteine-rich domain, and a large region identical in organization to the carboxy-half of prokaryotic YD-repeat proteins. Teneurins were not found in the genomes of sponges, cnidarians, or placozoa, but the choanoflagellate Monosiga brevicollis has a gene encoding a predicted teneurin with a transmembrane domain, EGF repeats, a cysteine-rich domain, and a region homologous to YD-repeat proteins. Further examination revealed that most of the extracellular domain of the M. brevicollis teneurin is encoded on a single huge 6,829-bp exon and that the cysteine-rich domain is similar to sequences found in an enzyme expressed by the diatom Phaeodactylum tricornutum. This leads us to suggest that teneurins are complex hybrid fusion proteins that evolved in a choanoflagellate via horizontal gene transfer from both a prokaryotic gene and a diatom or algal gene, perhaps to improve the capacity of the choanoflagellate to bind to its prokaryotic prey. As choanoflagellates are considered to be the closest living relatives of animals, the expression of a primitive teneurin by an ancestral choanoflagellate may have facilitated the evolution of multicellularity and complex histogenesis in metazoa.     

Effects of spontaneous deamidation on the cytotoxic activity of the Bacillus anthracis protective antigen

Protective antigen (PA) is a central virulence factor of Bacillus anthracis and a key component in anthrax vaccines. PA binds to target cell receptors, is cleaved by the furin protease, self-aggregates to heptamers, and finally internalizes as a complex with either lethal or edema factors. Under mild room temperature storage conditions, PA cytotoxicity decreased (t(1/2) approximately 7 days) concomitant with the generation of new acidic isoforms, probably through deamidation of Asn residues. Ranking all 68 Asn residues in PA based on their predicted deamidation rates revealed five residues with half-lives of <60 days, and these residues were further analyzed: Asn10 in the 20-kDa region, Asn162 at P6 vicinal to the furin cleavage site, Asn306 in the pro-pore translocation loop, and both Asn713 and Asn719 in the receptor-binding domain. We found that PA underwent spontaneous deamidation at Asn162 upon storage concomitant with decreased susceptibility to furin. A panel of model synthetic furin substrates was used to demonstrate that Asn162 deamidation led to a 20-fold decrease in the bimolecular rate constant (k(cat)/Km) of proteolysis due to the new negatively charged residue at P6 in the furin recognition sequence. Furthermore, reduced PA cytotoxicity correlated with a decrease in PA cell binding and also with deamidation of Asn713 and Asn719. On the other hand, neither deamidation of Asn10 or Asn306 nor impairment of heptamerization could be observed upon prolonged PA storage. We suggest that PA inactivation during storage is associated with susceptible deamidation sites, which are intimately involved in both mechanisms of PA cleavage by furin and PA-receptor binding.     

Conformational analyses of a partially-folded bioactive prodomain of human furin

The 81-residue multifunctional prodomain of human furin adopts only a partially-folded conformational state under near physiological conditions. By use of NMR spectroscopy, we demonstrate that the N-terminal residues 1-46 of the prodomain in 50% trifluoroethanol (TFE) populates backbone conformations containing a short helix, a beta-strand and a helix-loop-helix super-secondary structure with elements of tertiary interactions. (15)N NMR relaxation measurements indicate that the helix-loop-helix region has similar motional characteristics in the fast picosecond to nanosecond timescales. On the other hand, the intervening segment (residues 47-65) is predominantly unstructured with a long and highly flexible region surrounding the protease 'activation loop' followed by a partially helical segment in the C-terminal end. Interestingly, the helix-loop-helix "fold" was found to be populated even when excised out of the full-length prodomain, since a peptide fragment derived from residues Pro16-Arg49 can also form the helix-loop-helix structure in aqueous solution in the absence of TFE. Structure analyses reveal that two helices orient in an antiparallel fashion directed by the sharing of hydrophobic residues involved in helix-capping interactions. Very importantly, a positively-charged Lys residue replacing His43 in the 16-49 fragment imparts stability to the super-secondary structure at both acidic and neutral pH, while a hydrophobic residue Leu at position 43 appears to destabilize the helical conformation in the 31-44 region. As such, this study provides valuable insights into the structural properties of the furin prodomain in relation to its role in the folding of the furin zymogen and its inhibitory action toward furin.     

Impaired trafficking and activation of tumor necrosis factor-alpha-converting enzyme in cell mutants defective in protein ectodomain shedding

Protein ectodomain shedding is a specialized type of regulated proteolysis that releases the extracellular domain of transmembrane proteins. The metalloprotease disintegrin tumor necrosis factor-alpha-converting enzyme (TACE) has been convincingly shown to play a central role in ectodomain shedding, but despite its broad interest, very little is known about the mechanisms that regulate its activity. An analysis of the biosynthesis of TACE in mutant cell lines that have a gross defect in ectodomain shedding (M1 and M2) shows a defective removal of the prodomain that keeps TACE in an inactive form. Using LoVo, a cell line that lacks of active furin, and alpha1-Antitrypsin Portland, a protein inhibitor of proprotein convertases, we show that TACE is normally processed by furin and other proprotein convertases. The defect in M1 and M2 cells is due to a blockade of the exit of TACE from the endoplasmic reticulum. The processing of other zinc-dependent metalloproteases, previously suggested to participate in activated ectodomain shedding is normal in the mutant cells, indicating that the component mutated is highly specific for TACE. In summary, the characterization of shedding-defective somatic cell mutants unveils the existence of a specific mechanism that directs the proteolytic activation of TACE through the control of its exit from the ER.     

Structure and expression of Furin mRNA in the ovary of the medaka, Oryzias latipes

A cDNA for furin was cloned from the ovary of the medaka, Oryzias latipes, by a combination of cDNA library screening, 5'-rapid amplification of cDNA ends (RACE), and 3'- RACE. The cDNA sequence codes for a protein of 814 amino acid residues highly homologous to other vertebrate furins, Ca(2+)-dependent serine proteases belonging to the subtilysin-like proprotein convertase family. The medaka preprofurin consists of a leader sequence, a propeptide with autoactivation sites, a Kex2-like catalytic domain, a P domain, a cysteine-rich domain, a putative transmembrane domain, and a cytoplasmic domain. The catalytic triad residues (Asp-164, His-205, and Ser-379) were all conserved. Furin mRNA was expressed in many tissues of this, including the ovary. In the ovary, the greatest expression of furin mRNA occurred in oocytes of small growing follicles, as demonstrated by Northern blotting, RT-PCR, and in situ hybridization analysis. Temporary and spatial expression patterns of the medaka fish furin were similar to those of stromelysin-3 and MT5-MMP during oocyte growth and postnatal development.