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PubMed2003

The mouse matrix metalloproteinase, epilysin (MMP-28), is alternatively spliced and processed by a furin-like proprotein convertase.

Epilysin (MMP-28) is a recently identified member of the matrix metalloproteinase (MMP) family. To explore the expression of epilysin in vivo and to gain insight into its biological functions, we have cloned the mouse epilysin cDNA and determined its expression. The amino acid sequence of the mouse protein is 85% identical with the human sequence and contains conserved features such as an RKKR furin-activation sequence following the prodomain. Unexpectedly, we found two alternatively spliced forms of the epilysin mRNA lacking 30 and 72 nt at the beginning of the seventh exon coding for part of the haemopexin domain. Expression of recombinant epilysin in HT-1080 fibrosarcoma cells indicated that epilysin was secreted as a major 48 kDa form and a minor 58 kDa form. Expression of the 58 kDa form was increased by a synthetic furin inhibitor at the expense of the 48 kDa form, suggesting that furin cleaves and activates epilysin. Epilysin mRNA was detected in a number of mouse tissues, with the highest expression in the lung, placenta, heart and uterus, and lower levels in the testis and gastrointestinal tract. The wide expression of epilysin in intact, healthy tissues suggests that this MMP functions in physiological tissue homoeostasis and turnover.

Alternative Splicing↗
PubMed1999

Mammalian subtilisin/kexin isozyme SKI-1: A widely expressed proprotein convertase with a unique cleavage specificity and cellular localization.

Using reverse transcriptase-PCR and degenerate oligonucleotides derived from the active-site residues of subtilisin/kexin-like serine proteinases, we have identified a highly conserved and phylogenetically ancestral human, rat, and mouse type I membrane-bound proteinase called subtilisin/kexin-isozyme-1 (SKI-1). Computer databank searches reveal that human SKI-1 was cloned previously but with no identified function. In situ hybridization demonstrates that SKI-1 mRNA is present in most tissues and cells. Cleavage specificity studies show that SKI-1 generates a 28-kDa product from the 32-kDa brain-derived neurotrophic factor precursor, cleaving at an RGLT downward arrowSL bond. In the endoplasmic reticulum of either LoVo or HK293 cells, proSKI-1 is processed into two membrane-bound forms of SKI-1 (120 and 106 kDa) differing by the nature of their N-glycosylation. Late along the secretory pathway some of the membrane-bound enzyme is shed into the medium as a 98-kDa form. Immunocytochemical analysis of stably transfected HK293 cells shows that SKI-1 is present in the Golgi apparatus and within small punctate structures reminiscent of endosomes. In vitro studies suggest that SKI-1 is a Ca2+-dependent serine proteinase exhibiting a wide pH optimum for cleavage of pro-brain-derived neurotrophic factor.

Amino Acid Sequence↗
PubMed2002

Structure-function analysis of the prosegment of the proprotein convertase PC5A.

To investigate if some residues within the prosegment of PC5A are important for its optimal proteolytic function, various PC5A mutants were cellularly expressed, and their processing activities were compared using pro-vascular endothelial growth factor C (pro-VEGF-C) as a substrate. Although wild type PC5A almost completely processes pro-VEGF-C, a prosegment deletion as well as both P1 mutants of the primary (R116A) and secondary (R84A) autocatalytic cleavage sites are inactive. The in vitro inhibitory potency of various decapeptides mimicking the C-terminal sequence of PC5 prosegment (pPC5) revealed that the native (107)QQVVKKRTKR(116) peptide is a nanomolar inhibitor, whereas its P6 mutant K111H is more selective toward PC5A than Furin. In vitro activity assays using the bacterially expressed pPC5 and its mutants revealed them to be very potent nanomolar inhibitors (IC(50)) and only approximately 6-fold more selective inhibitors of PC5A versus Furin. Expression of the preprosegment of PC5 (ppPC5) and its mutants in Chinese hamster ovary FD11 cells overexpressing pro-VEGF-C with either PC5A or Furin showed them to be as good inhibitors of PC5A as the serpin alpha1-antitrypsin Portland (alpha1-PDX), ppFurin, or ppPACE4 but less potent toward overexpressed Furin. In conclusion, cleavages of the prosegment of PC5A at both Arg(116) and Arg(84) are required for PC5A cellular activity, and ppPC5 is a very potent but modestly selective cellular inhibitor of PC5A.

Amino Acid Sequence↗
PubMed2004

Proprotein convertase furin interacts with and cleaves pro-ADAMTS4 (Aggrecanase-1) in the trans-Golgi network.

A member of the A disintegrin and metalloproteinase domain with thrombospondin type-1 motifs (ADAMTS-4) protease family can efficiently cleave aggrecan at several sites detected in joints of osteoarthritic patients. Although recent studies have shown that removal of the prodomain of ADAMTS4 is critical for its ability to degrade aggrecan, the cellular mechanisms for its processing and trafficking remain unclear. In this study, by using both furin-specific inhibitor and RNA interference technique, we demonstrate that furin plays an important role in the intracellular removal of ADAMTS4 prodomain. Further, we demonstrate that proADAMTS4 can be processed by means of multiple furin recognition sites: (206)RPRR(209), (209)RAKR(212), or (211)KR(212). The processing of proADAMTS4 was completely blocked by brefeldin A treatment, suggesting that processing occurs in the trans-Golgi network. Indeed, ADAMTS4 is co-localized with furin in trans-Golgi network. Interestingly, the pro form of ADAMTS4, not its mature one, co-precipitates with furin, suggesting that furin physically interacts with the prodomain of ADAMTS-4. In addition, our evidence suggests that a furin-independent pathway may also contribute to the activation of ADAMTS4. These results indicate that the activation mechanism for ADAMTS4 can be targeted for therapeutical intervention against this enzyme.

ADAM Proteins↗
PubMed1999

The RGD motif and the C-terminal segment of proprotein convertase 1 are critical for its cellular trafficking but not for its intracellular binding to integrin alpha5beta1.

Cellular trafficking of subtilisin/kexin-like precursor convertases (PCs) may be regulated by a number of motifs, some of which are present within the P-domain and in the C-terminal sequence. Six of the seven known PCs contain a conserved RGD sequence within the P domain. In order to investigate the functional importance of this motif, we generated mutants of PC1 that contain a Myc tag epitope inserted between the prosegment and the catalytic subunit. Cellular expression of vaccinia virus recombinants revealed that this tag did not seem to influence the autocatalytic conversion of proPC1 into PC1 or its bioactivity. The two PC1 variants produced possess either the wild type RGD sequence or its RGE mutant. Stable transfectants of these variants in AtT20 cells revealed that similar to the wild type enzyme, PC1-RGD-Myc is sorted to secretory granules. In contrast, PC1-RGE-Myc exits the cell via the constitutive secretory pathway. In vitro, a 14-mer peptide spanning the RGD sequence of PC1, but not its RGE mutant, binds to cell surface vitronectin-binding integrins of Chinese hamster ovary cells. However, within the endoplasmic reticulum and in an RGD-independent fashion, integrin alpha5beta1 associates primarily with the zymogens proPC1, proPC1-DeltaC (missing the C-terminal 137 residues), as well as proPC2. Thus, the observed discrimination between the secretion routes of PC1-RGD and PC1-RGE does not implicate integrins such as alpha5beta1.

Amino Acid Sequence↗
PubMed2005

Proprotein convertase models based on the crystal structures of furin and kexin: explanation of their specificity.

In eukaryotes, many secreted proteins and peptide hormones are excised from larger precursors by calcium-dependent serine proteinases, the proprotein/prohormone convertases (PCs). These PCs cleave their protein substrates very specifically following multiple basic residues. The seven mammalian PCs and their yeast orthologue kexin are multi-domain proteinases consisting of a subtilisin-related catalytic domain, a conserved P-domain and a variable, often cysteine-rich domain, which in some PCs is followed by an additional C-terminal trans-membrane domain and a short cytoplasmic domain. The recently published crystal structures of the soluble mouse furin and yeast kexin ectodomains have revealed the relative arrangement of catalytic and P domains, the exact domain fold and the detailed architecture of the substrate binding clefts. Based on these experimental structures, we now have modelled the structures of the other human/mouse PCs. According to topology and to structure-based sequence comparisons, these other PCs closely resemble furin, with PC4, PACE4 and PC5/6 being more similar, and PC1/3, PC2 and PC7 being less similar to furin. Except for PC1 and PC2, this order of similarity is valid for the catalytic as well as for the P domains, and is almost reversed using kexin as a reference molecule. A similar order results from the number and clustering of negative charges lining the non-prime subsites, explaining the gradually decreasing requirement for basic residues N-terminal to substrate cleavage sites. The preference of the different PCs for distinct substrates seems to be governed by overall charge compensation and matching of the detailed charge distribution pattern.

Amino Acid Sequence↗
PubMed2003

Novel insights into cadherin processing by subtilisin-like convertases.

Proprotein convertases (PCs) are known to activate many important molecules and their overexpression plays a significant role in tumor progression. Only little is known about the involvement of PCs in the processing of cadherin adhesion molecules, which are potent tumor suppressors. Here we show in a baculovirus overexpression system that the desmosomal cadherins Dsg1 and Dsg3 are substrates for the PC furin. Accordingly, inhibition of PCs in differentiating mouse keratinocytes by alpha 1-anti-trypsin Portland (alpha 1-PDX) negatively interfered with pro-epithelial (proE)-cadherin processing, but unexpectedly also resulted in a dramatic reduction of E-cadherin, Dsg1 and Dsg3 protein and Dsg1 mRNA. Because loss of intercellular adhesion is a rate-limiting step in the transition from benign to malignant tumors, these results have significant implications for the use of PC inhibitors as possible therapeutic tools.

Animals↗
PubMed2004

The proprotein convertase furin colocalizes with caveolin-1 in the Golgi apparatus and endosomes of hepatocytes.

The significance of furin in the maturation and activation of a wide array of proproteins in the secretory pathway has been well demonstrated. However, despite efforts made to characterize the subcellular locations where furin activates its substrates, doubts on the proprotein-processing compartments still persist. Using in vivo gene delivery, together with high-resolution immunogold electron microscopy, we were able to assign precise subcellular locations to furin. In rat hepatocyte, the enzyme was found concentrated in the Golgi apparatus, along the basolateral (sinusoidal) plasma membrane and in underlying endosomes. An asymmetry was detected in respect to amounts of furin between the basolateral domain and the apical (canalicular) one, favoring the former. The asymmetric recycling of furin through the basolateral domain may be of high importance for the polarized secretion of processed bioactive compounds. Double immunogold labelings indicate that furin colocalizes with the caveolae/raft-marker caveolin-1 in the Golgi apparatus and in the basolateral endosomes. Furthermore, co-immunoprecipitation experiments show the possible interaction of caveolin-1 and furin. Our results suggest that, in addition to the Golgi, furin-/caveolin-1-containing endosomes could represent a compartment where furin processes its substrates at the basolateral domain of the hepatocyte.

Animals↗
PubMed1994

Intracellular trafficking and activation of the furin proprotein convertase: localization to the TGN and recycling from the cell surface.

Furin is a membrane-associated endoprotease that efficiently cleaves precursor proteins on the C-terminal side of the consensus sequence, Arg-X-Lys/Arg-Arg1, and has been proposed to catalyze these reactions in both exocytic and endocytic compartments. To study its biosynthesis and routing, a furin construct (designated fur/f) containing the FLAG epitope tag inserted on the C-terminal side of the enzyme's autoproteolytic maturation site was used. Introduction of the epitope tag had no effect on the expression, proteolytic maturation or activity of furin. Analysis of the localization of fur/f by immunofluorescence microscopy showed that its staining pattern largely overlapped with those of several Golgi-associated markers. Treatment of cells with brefeldin A caused the fur/f distribution to collapse around the microtubule organizing center, indicating that furin is concentrated in the trans-Golgi network (TGN). Immunoelectron microscopy showed unequivocally that furin resides in the TGN where it colocalized with TGN38. In agreement with its proposed activity in multiple compartments, antibody uptake studies showed that fur/f cycles between the cell surface and TGN. Furthermore, targeting to the TGN requires sequences in the cytoplasmic tail of the enzyme. Pulse-chase and immunofluorescence analyses demonstrated that proregion removal occurs in the endoplasmic reticulum and that cleavage may be required for exist from this compartment. Finally, we show that proregion removal is necessary but not sufficient for enzyme activation.

Amino Acid Sequence↗
PubMed1995

Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin.

Furin, a subtilisin-like eukaryotic endoprotease, is responsible for proteolytic cleavage of cellular and viral proteins transported via the constitutive secretory pathway. Cleavage occurs at the C-terminus of basic amino acid sequences, such as R-X-K/R-R and R-X-X-R. Furin was found predominantly in the trans-Golgi network (TGN), but also in clathrin-coated vesicles dispatched from the TGN, on the plasma membrane as an integral membrane protein and in the medium as an anchorless enzyme. When furin was vectorially expressed in normal rat kidney (NRK) cells it accumulated in the TGN similarly to the endogenous glycoprotein TGN38, often used as a TGN marker protein. The signals determining TGN targeting of furin were investigated by mutational analysis of the cytoplasmic tail of furin and by using the hemagglutinin (HA) of fowl plague virus, a protein with cell surface destination, as a reporter molecule, in which membrane anchor and cytoplasmic tail were replaced by the respective domains of furin. The membrane-spanning domain of furin grafted to HA does not localize the chimeric molecule to the TGN, whereas the cytoplasmic domain does. Results obtained on furin mutants with substitutions and deletions of amino acids in the cytoplasmic tail indicate that wild-type furin is concentrated in the TGN by a mechanism involving two independent targeting signals, which consist of the acidic peptide CPSDSEEDEG783 and the tetrapeptide YKGL765. The acidic signal in the cytoplasmic domain of a HA-furin chimera is necessary and sufficient to localize the reporter molecule to the TGN, whereas YKGL is a determinant for targeting to the endosomes. The data support the concept that the acidic signal, which is the dominant one, retains furin in the TGN, whereas the YKGL motif acts as a retrieval signal for furin that has escaped to the cell surface.

Amino Acid Sequence↗
PubMed1998

The role of subtilisin-like proprotein convertases for cleavage of the measles virus fusion glycoprotein in different cell types.

The fusion (F) glycoprotein gene of measles virus (MV) encodes a nonfusogenic precursor protein (F0) that is activated by cleavage into the F1 and F2 subunits during transport to the cell surface. The F protein of both the Edmonston strain and a wild-type MV was found to be cleaved in the trans-Golgi cisternae and/or the trans-Golgi network (TGN). In HEp-2 cells, B lymphoblastoid cells, and PBMC, the cleavage process required calcium, and calcium deprivation prevented syncytium formation. The calcium dependence indicated the involvement of the pro-protein convertase (PC) endoprotease family. The expression of the presently recognized members of the PC family in human cell types known to be infected during measles was examined by RT-PCR. Among the PCs residing in the TGN, only furin was expressed in all cells. Soluble secreted human furin produced by a recombinant baculovirus cleaved MV F0 into proteins the exact size of F1 and F2 and increased the titer of MV particles released from calcium-deprived or endoprotease defective infected cells. These results strongly indicate that furin is the most important and maybe the only endoprotease involved in activation of the MV F protein.

Adenocarcinoma↗
PubMed2000

Mutational analysis of bli-4/kpc-4 reveals critical residues required for proprotein convertase function in C. elegans.

Kex2/subtilisin-like proteinase activity is required for the production of the adult cuticle in the nematode Caenorhabditis elegans. Deletion of the carboxy termini of four of the bli-4/kpc-4 convertase isoforms results in blistering of the adult cuticle. The blisters vary in severity (expressivity) and are not evident in all individuals (reduced penetrance). We have isolated 13 bli-4/kpc-4 mutants that arrest development in late embryogenesis. Using a PCR-based heteroduplex technique, we have identified nucleotide changes responsible for eight of these lethal mutations. The lesions reside within the first 12 exons that are shared by all of the bli-4/kpc-4 gene products, with the majority of mutations clustered within the protease domain. This finding suggests that the protease domain represents a large mutable target. Among these mutations, allele h384 represents a molecular null mutant in which the catalytically essential serine residue (Ser415) is replaced by phenylalanine. Novel missense mutations that change the identity of amino acids evolutionary conserved in all kex2/subtilisin-convertases highlight critical residues essential for activity. We examined the functional activity of BLI-4/KPC-4 products expressed from several lethal mutants by testing their effect on the variable penetrance of blistering exhibited by the e937 allele. We found that the combination of a bli-4/kpc-4 lethal mutation in trans to the bli-4(e937) mutation was sufficient to cause severe blistering in heteroallelic progeny, even in the presence of a known dominant suppressor.

Alleles↗
PubMed1998

Bone morphogenetic protein-1 processes the NH2-terminal propeptide, and a furin-like proprotein convertase processes the COOH-terminal propeptide of pro-alpha1(V) collagen.

Bone morphogenetic protein-1 (BMP-1) plays key roles in regulating the deposition of vertebrate extracellular matrix; it is the procollagen C-proteinase that processes the major fibrillar collagen types I-III, and it may process prolysyl oxidase to the mature enzyme necessary to the formation of covalent cross-links in collagen and elastic fibers. Type V collagen is a fibrillar collagen of low abundance that is incorporated into and helps regulate the shape and diameter of type I collagen fibrils. Here we show that, in contrast to its action on procollagens I-III, BMP-1 does not cleave the C-propeptide of pro-alpha1(V) homotrimers. Instead, the single BMP-1-specific cleavage site within pro-alpha1(V) chains, lies within the large globular N-propeptide. This cleavage site is immediately upstream of a glutamine, thus redefining the specificity of cleavage for BMP-1-like enzymes. It also produces an NH2 terminus that corresponds to an equivalent NH2 terminus on the processed matrix form of the similar alpha1(XI) chain, thus suggesting physiological significance. Cleavage of the C-propeptide occurs efficiently in recombinant pro-alpha1(V) homotrimers produced in 293-EBNA human embryonic kidney cells, and this cleavage is shown to occur immediately downstream of the sequence RTRR. This is similar to sites cleaved by subtilisin-like proprotein/prohormone convertases and is shown to be specifically cleaved by the recombinant subtilisin-like proprotein/prohormone convertase furin.

Amino Acid Sequence↗
PubMed1998

Processing of human intestinal prolactase to an intermediate form by furin or by a furin-like proprotein convertase.

Human lactase-phlorizin hydrolase (human-LPH) is synthesized as a large precursor (prepro-LPH), then cleaved to a pro-LPH of 220 kDa which is further cut to a "mature-like LPH" of a size close to that of mature LPH, i.e. about 150 kDa (in the processing of rabbit pro-LPH the intermediate has a mass of approximately 180 kDa). By coexpression of human prepro-LPH with furin in COS-7 cells we show that furin generates a mature-like LPH. Radioactive amino acid sequence analysis reveals that furin recognizes the motif R-T-P-R832, a protein convertase consensus, to generate a NH2 terminus located 36 amino acids upstream of the NH2 terminal found in vivo at Ala869. This intermediate is ultimately cleaved to the mature LPH form by other proteases including the pancreatic ones. These data demonstrate that human pro-LPH, like the rabbit enzyme, is processed to the mature enzyme by furin or furin-like enzymes through at least an intermediate form that has, however, an apparent mass close to that of the mature enzyme.

Amino Acid Sequence↗
PubMed2000

Processing of beta-secretase by furin and other members of the proprotein convertase family.

The amyloid peptide is the main constituent of the amyloid plaques in brain of Alzheimer's disease patients. This peptide is generated from the amyloid precursor protein by two consecutive cleavages. Cleavage at the N terminus is performed by the recently discovered beta-secretase (Bace). This aspartyl protease contains a propeptide that has to be removed to obtain mature Bace. Furin and other members of the furin family of prohormone convertases are involved in this process. Surprisingly, beta-secretase activity, neither at the classical Asp(1) position nor at the Glu(11) position of amyloid precursor protein, seems to be controlled by this maturation step. Furthermore, we show that Glu(11) cleavage is a function of the expression level of Bace, that it depends on the membrane anchorage of Bace, and that Asp(1) cleavage can be followed by Glu(11) cleavage. Our data suggest that pro-Bace could be active as a beta-secretase in the early biosynthetic compartments of the cell and could be involved in the generation of the intracellular pool of the amyloid peptide. We conclude that modulation of the conversion of pro-Bace to mature Bace is not a relevant drug target to treat Alzheimer's disease.

Amyloid beta-Protein Precursor↗
PubMed2001

Inhibitory specificity and potency of proSAAS-derived peptides toward proprotein convertase 1.

Prohormone convertase 1 (PC1), mediating the proteolytic processing of neural and endocrine precursors, is thought to be regulated by the neuroendocrine protein proSAAS. The PC1 inhibitory sequence is mostly confined within a 10-12-amino acid segment near the C terminus of the conserved human proSAAS and contains the critical KR(244) dibasic motif. Our results show that the decapeptide proSAAS-(235-244)( 235)VLGALLRVKR(244) is the most potent reversible competitive PC1-inhibitor (K(i) approximately 9 nm). The C-terminally extended proSAAS-(235-246) exhibits a 5-6-fold higher K(i) ( approximately 51 nm). The additional LE sequence at P1'-P2', resulted in a competitive substrate cleaved by PC1 at KR(244) downward arrowLE(246). Systematic alanine scanning and in some cases lysine scanning tested the contribution of each residue within proSAAS-(235-246) toward the PC1-inhibition's specificity and potency. The amino acids P1 Arg, P2 Lys, and P4 Arg are all critical for inhibition. Moreover, the aliphatic P3 Val and P5, P6, and P1' Leu significantly affect the degree of enzyme inactivation and PC1 specificity. Interestingly, a much longer N- and C-terminally extended endogenous rat proSAAS-(221-254) called little PenLen, was found to be a 3-fold less potent PC1 inhibitor with reduced selectivity but a much better substrate than proSAAS-(235-246). Molecular modeling studies and circular dichroism analysis indicate an extended and poly-l-proline II type structural conformation for proSAAS-(235-244), the most potent PC1 inhibitor, a feature not present in poor PC1 inhibitors.

Amino Acid Sequence↗
PubMed1998

Failure of ventral closure and axial rotation in embryos lacking the proprotein convertase Furin.

We have examined the role of Furin in postimplantation-stage mouse embryos by analyzing both the expression pattern of fur mRNA and the developmental consequences of a loss-of-function mutation at the fur locus. At early stages (day 7.5), fur mRNA is abundant in extraembryonic endoderm and mesoderm, anterior visceral endoderm, and in precardiac mesoderm. 1 day later fur is expressed throughout the heart tube and in the lateral plate mesoderm, notochordal plate and definitive gut endoderm. Embryos lacking Furin die between days 10.5 and 11.5, presumably due to hemodynamic insufficiency associated with severe ventral closure defects and the failure of the heart tube to fuse and undergo looping morphogenesis. Morphogenesis of the yolk sac vasculature is also abnormal, although blood islands and endothelial precursors form. Analysis of cardiac and endodermal marker genes shows that while both myocardial precursors and definitive endoderm cells are specified, their numbers and migratory properties are compromised. Notably, mutant embryos fail to undergo axial rotation, even though Nodal and eHand, two molecular markers of left-right asymmetry, are appropriately expressed. Overall, the present data identify Furin as an important activator of signals responsible for ventral closure and embryonic turning.

Animals↗