PTHrP(1-34)-mediated repression of the PHEX gene in osteoblastic cells involves the transcriptional repressor E4BP4.

PHosphate-regulating gene with homology to Endopeptidase on the X chromosome (PHEX) has been identified as the gene mutated in X-linked hypophosphatemia (XLH) syndrome, the most prevalent form of rickets in humans. The predominant expression of PHEX in bones and teeth, and the defective mineralization of these tissues in XLH patients indicate that PHEX is an important regulator of mineralization. Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are known to regulate the expression of numerous genes in osteoblastic cells through activation of the protein kinase A pathway, including repression of PHEX. PTH also activates the transcriptional repressor E4BP4 through the same pathway, suggesting that PTH or PTHrP-mediated repression of PHEX expression could involve E4BP4. To evaluate this possibility, we treated UMR-106 osteoblastic cells with PTHrP(1-34), and used RT-PCR and immunoblotting to analyze PHEX and E4BP4 expression. E4BP4 mRNA and protein levels were rapidly increased in cells treated with PTHrP(1-34), with a concomitant decrease in PHEX expression. This downregulation of PHEX could be reproduced by overexpression of E4BP4. Moreover, PTHrP(1-34)-mediated PHEX repression was blocked when cells were transfected with a siRNA targeting E4BP4 mRNA. Finally, DNA pull-down and luciferase assays showed that two E4BP4 response elements located in PHEX promoter were functional. These results underline the important role of E4BP4 in osteoblastic cells and further define the repression mechanism of PHEX gene by PTHrP(1-34).

Recombinant expression and affinity purification of a novel epididymal human sperm-binding protein, BSPH1.

Mammalian sperm undergo a series of maturation steps before acquiring fertilization competence. Our previous work demonstrated the importance of binder of sperm (BSP) proteins in bovine sperm capacitation. Recent studies identified a BSP-homologous DNA sequence in the human genome (BSPH1) and mRNA expression in the epididymis. The aim of this study was to develop an efficient method to express and purify recombinant human BSPH1. BSPH1 accumulates in inclusion bodies when expressed with an N-terminal hexahistidine tag in BL21 (DE3) Escherichia coli cells. Similar to other BSP proteins, BSPH1 contains two fibronectin type-II (Fn2) domains, each consisting of two disulfide bonds. Therefore, when expressed in Origami B (DE3)pLysS cells, a strain favouring disulfide bond formation, an improvement in soluble protein yield was observed. However, protein was aggregated, which complicated subsequent purification steps. Expression of glutathione-S-transferase-tagged BSPH1 in both cell types also led to accumulation in inclusion bodies. Finally, successful production of soluble and active protein was achieved when BSPH1 was expressed as a His(6)-thioredoxin-tagged protein. Recombinant protein bound phosphatidylcholine liposomes, low-density lipoproteins and human sperm, therefore displayed binding activities common to all BSP-family proteins, which may indicate similar biological function(s). This approach was also successful in producing the murine orthologue of BSPH1 in the soluble and active form. Thus, fusion to thioredoxin and expression in Origami B (DE3)pLysS cells may constitute a strategy applicable to all BSP-family proteins, and possibly to other proteins containing Fn2 domains. This work is important to elucidate the role of BSPH1 in human sperm functions and fertility.

The critical interaction of the metallopeptidase PHEX with heparan sulfate proteoglycans.

The PHEX gene (phosphate-regulating gene with homologies to endopeptidase on the X chromosome) identified as a mutated gene in patients with X-linked hypophosphatemia (XLH), encodes a protein (PHEX) that shows striking homologies to members of the M13 family of zinc metallopeptidases. In the present work the interaction of glycosaminoglycans with PHEX has been investigated by affinity chromatography, circular dichroism, protein intrinsic fluorescence analysis, hydrolysis of FRET substrates flow cytometry and confocal microscopy. PHEX was eluted from a heparin-Sepharose chromatography column at 0.8 M NaCl showing a strong interaction with heparin. Circular dichroism spectra and intrinsic fluorescence analysis showed that PHEX is protected by glycosaminoglycans against thermal denaturation. Heparin, heparan sulfate and chondroitin sulfate inhibited PHEX catalytic activity, however among them, heparin presented the highest inhibitory activity (Ki=2.5+/-0.2 nM). Flow cytometry analysis showed that PHEX conjugated to Alexa Fluor 488 binds to the cell surface of CHO-K1, but did not bind to glycosaminoglycans defective cells CHO-745. Endogenous PHEX was detected at the cell surface of CHO-K1 colocalized with heparan sulfate proteoglycans, but was not found at the cell surface of glycosaminoglycans defective cells CHO-745. In permeabilized cells, PHEX was detected in endoplasmic reticulum of both cells. In addition, we observed that PHEX colocalizes with heparan sulfate at the cell surface of osteoblasts. This is the first report that the metallopeptidase PHEX is a heparin binding protein and that the interaction with GAGs modulates its enzymatic activity, protein stability and cellular trafficking.

Stage-specific modulation of neprilysin and aminopeptidase N in the limbic system during kindling progression.

Aminopeptidase N (APN) and neprilysin (NEP) inactivate neuropeptides released into the brain extracellular fluid. We previously showed that the expression of pyroglutamyl peptidase II (PPII), the TRH degrading ecto-enzyme, is regulated in rat brain by amygdaline kindling, a paradigm that activates neuronal pathways in the limbic system increasing the expression of several neuropeptides including TRH and opioids. To understand the specificity of this phenomenon, we studied APN and NEP expression in brains of partially or fully kindled rats (stage II and V), sacrificed 6 h after last stimulus, compared with sham-operated animals. In situ hybridization analysis of NEP mRNA levels showed decreased expression at stage II in CA1, CA2, olfactory tubercle and medial mammillary nucleus, and increased at stage V in CA1 and CA2 cells. These changes were specific for the ipsilateral side. APN mRNA levels, semi-quantified by RT-PCR, were decreased at stage II and increased at stage V, in frontal cortex-olfactory tubercle, and hippocampus. NEP and APN enzymatic activities, determined by fluorometric assays, followed similar variations to their respective mRNA levels. The coordinated changes (in some regions) of NEP and APN expression were opposite to those previously observed for PPII mRNA and activity levels in limbic regions. These results demonstrate that expression of ectopeptidases can be regulated when peptide neurons are activated and, that regulation is enzyme-, region-, and stage-specific.

Reduced fertility in male mice deficient in the zinc metallopeptidase NL1.

Members of the M13 family of zinc metalloendopeptidases have been shown to play critical roles in the metabolism of various neuropeptides and peptide hormones, and they have been identified as important therapeutic targets. Recently, a mouse NL1 protein, a novel member of the family, was identified and shown to be expressed mainly in the testis as a secreted protein. To define its physiological role(s), we used a gene targeting strategy to disrupt the endogenous murine Nl1 gene by homologous recombination and generate Nl1 mutant mice. The Nl1(-/-) mice were viable and developed normally, suggesting that zygotic expression of Nl1 is not required for development. However, Nl1(-/-) males produced smaller litters than their wild-type siblings, indicating specific male fertility problems. Reduced fertility may be explained by two impaired processes, decreased egg fertilization and perturbed early development of fertilized eggs. These two phenotypes did not result from gross anatomical modifications of the testis or from impaired spermatogenesis. Basic sperm parameters were also normal. Thus, our findings suggest that one of the roles of NL1 in mice is related to sperm function and that NL1 modulates the processes of fertilization and early embryonic development in vivo.

Human recombinant membrane-bound aminopeptidase P: production of a soluble form and characterization using novel, internally quenched fluorescent substrates.

APP (aminopeptidase P) has the unique ability to cleave the N-terminal amino acid residue from peptides exhibiting a proline at P(1)'. Despite its putative involvement in the processing of bioactive peptides, among them the kinins, little is known about the physiological roles of both human forms of APP. The purpose of the present study is first to engineer and characterize a secreted form of hmAPP (human membrane-bound APP). Our biochemical analysis has shown that the expressed glycosylated protein is fully functional, and exhibits enzymic parameters similar to those described previously for mAPP purified from porcine or bovine lungs or expressed from a porcine clone. This soluble form of hmAPP cross-reacts with a polyclonal antiserum raised against a 469-amino-acid hmAPP fragment produced in Escherichia coli. Secondly, we synthesized three internally quenched fluorescent peptide substrates that exhibit a similar affinity for the enzyme than its natural substrates, the kinins, and a higher affinity compared with the tripeptide Arg-Pro-Pro used until now for the quantification of APP in biological samples. These new substrates represent a helpful analytical tool for rapid and reliable screening of patients susceptible to adverse reactions associated with angiotensin-converting enzyme inhibitors or novel vasopeptidase (mixed angiotensin-converting enzyme/neprilysin) inhibitors.

Endothelin-converting enzyme-like 1 (ECEL1) is present both in the plasma membrane and in the endoplasmic reticulum.

Enzymes of the M13 family of zinc-containing endopeptidases are recognized as important regulators of neuropeptide and peptide hormone activity. Peptidases of this family are type II integral-membrane proteins characterized by short cytosolic domains and large extracellular domains containing the active site. The M13 family has, at present, seven members, including ECEL1 (endothelin-converting enzyme-like 1), one of the newest members. ECEL1 is expressed predominantly in the central nervous system. It has been proposed that the enzyme has a role in the nervous regulation of the respiratory system. No physiological substrate has been identified yet. To better understand the function(s) of this enzyme, we have expressed human ECEL1 in cultured cells and monitored its biosynthesis and subcellular localization. Immunoblot and cell-surface biotinylation analysis of transfected cells expressing ECEL1 showed that only a fraction of the protein travelled to the cell surface, while most of the enzyme was present in an intracellular compartment identified by confocal immunofluorescence microscopy and cell fractionation as the ER (endoplasmic reticulum). Pulse-chase experiments showed that ER-localized ECEL1 was stable, with a half-life of more than 3 h. Endogenous ECEL1 from mouse pituitary gland had a similar distribution between the cell surface and the ER. Finally, using domain-swapping experiments with neprilysin, another member of the M13 family, we showed that localization of ECEL1 to the ER requires both the transmembrane and cytoplasmic domains. It thus appears that ECEL1 may have functions both at the cell surface and in the ER.

Cell viability and secretion of active proteins in Schizosaccharomyces pombe do not require the chaperone function of calnexin.

Folding of newly synthesized proteins within the ER (endoplasmic reticulum) is a rate-limiting step in protein secretion. Thus ER molecular chaperones and foldases have a major impact in determining the rate and yield of these crucial cellular processes. Calnexin is a key ER chaperone implicated in the folding, retention and targeting for degradation of proteins that go through the secretory pathway. Calnexin molecules contain a highly conserved central domain (hcd) that has been proposed to be involved in the interaction with folding substrates and other chaperones. To gain a better understanding of the roles played by calnexin in the secretory pathway, we examined the efficiency of fission yeast (Schizosaccharomyces pombe) strains expressing calnexin mutants to secrete different model proteins. Remarkably, calnexin hcd-deletion mutants, although devoid of detectable chaperone activity in vitro, confer viability and cause a considerable increase in the secretion of heterologous cellulase. Surprisingly the quality-control efficiency, measured as the activity/amount ratio of secreted model protein, was not severely reduced in these calnexin hcd-deletion mutant strains. Our results indicate that the essential function of calnexin does not reside in its role in the folding or in the retention of misfolded proteins. These observations suggest the existence of a highly stringent quality control mechanism in the ER of S. pombe that might reduce the secretion efficiency of endogenous proteins.

Declining expression of neprilysin in Alzheimer disease vasculature: possible involvement in cerebral amyloid angiopathy.

Molecular, genetic, and pharmacological studies have shown that neprilysin (also called NEP) catabolizes amyloid beta peptides (A beta) in healthy conditions. However, in Alzheimer disease (AD), A beta accumulates forming senile plaques in brain parenchyma and amyloid deposition around blood vessels. In this study, we tested at cellular level the relationship between neprilysin and A beta in human healthy and AD brain. Our results provided evidence for declining levels of neprilysin in AD brains as compared to healthy controls in parallel with increasing deposition of A beta. In hippocampus of AD individuals we observed a significant down-regulation of neprilysin expression in pyramidal neurons, consistent with the possibility that neprilysin controls the level of A beta accumulation and plaque formation in this area. In the cortex and leptomeninges, neprilysin was expressed in the smooth muscle cells of blood vessels. In sections from AD patients we observed a clear inverse relationship between neprilysin and A beta peptide levels in the vasculature, implicating neprilysin in cerebral amyloid angiopathy.

Parathyroid hormone-related protein(1-34) regulates Phex expression in osteoblasts through the protein kinase A pathway.

Phex (a phosphate-regulating gene with homologies to endopeptidases on the X chromosome) is expressed predominantly in bone in which it has been implicated in the mineralization process. Multiple factors and hormones, including PTHrP, regulate formation, development, and/or homeostasis of bone. The purpose of the present study was to determine whether PTHrP(1-34) regulates Phex expression and identify the signaling pathway used. Phex mRNA and protein levels were analyzed by RT-PCR and immunoblotting, respectively. In UMR-106 cells, PTHrP(1-34) caused a time- and concentration-dependent decrease in Phex expression. Forskolin, an adenylate cyclase activator, had the same effect. Dibutiryl cAMP also decreased Phex expression, and its effect was blocked by H89, a protein kinase A (PKA) inhibitor. In contrast, 12-O-tetradecanoyl phorbol-13-acetate, a protein kinase C (PKC) activator, increased Phex expression in a time- and dose-dependent manner. This effect was reversed by bisindolylmaleimide Iota, a PKC inhibitor. Bovine PTH(3-34), which activates PKC but not PKA, had no effect. On the contrary, human PTH(1-31), which activates PKA but not PKC, decreased Phex expression. H89 but not bisindolylmaleimide Iota blocked the effect of PTHrP(1-34). PTHrP(1-34) also decreased Phex expression in cultures of fetal rat calvaria cells at d 7 of culture but not at later stages. These data demonstrate that PTHrP(1-34), through PKA, down-regulates Phex expression in osteoblasts.

Structure and function of disease-causing missense mutations in the PHEX gene.

The PHEX gene that is mutated in patients with X-linked hypophosphatemia (XLH) encodes a protein homologous to the M13 family of zinc metallopeptidases. The present study was undertaken to assess the impact of nine PHEX missense mutations on cellular trafficking, endopeptidase activity, and protein conformation. Secreted forms of wild-type and mutant PHEX proteins were generated by PCR mutagenesis; these included C85R, D237G, Y317F, G579R, G579V, S711R, A720T, and F731Y identified in XLH patients, and E581V, which in neutral endopeptidase 24.11 abolishes catalytic activity but not plasma membrane localization. The wild-type and D237G, Y317F, E581V, and F731Y proteins were terminally glycosylated and secreted into the medium, whereas the C85R, G579R, G579V, S711R, and A720T proteins were trapped inside the transfected cells. Growing the cells at 26 C permitted the secretion of G579V, S711R, and A720T proteins, although the yield of rescued G579V was insufficient for further analysis. Endopeptidase activity of secreted and rescued PHEX proteins, assessed using a novel internally quenched fluorogenic peptide substrate, revealed that E581V and S711R are completely inactive; D237G and Y317F exhibit 50-60% of wild-type activity; and A720T and F731Y retain full catalytic activity. Conformational analysis by limited proteolysis demonstrated that F731Y is more sensitive to trypsin and D237G is more resistant to endoproteinase Glu-c than the wild-type protein. Thus, defects in protein trafficking, endopeptidase activity, and protein conformation account for loss of PHEX function in XLH patients harboring these missense mutations.

Sorting of furin in polarized epithelial and endothelial cells: expression beyond the Golgi apparatus.

The conversion of proteins into their mature forms underlies the functionality of many fundamental cellular pathways. One posttranslational modification leading to maturation of precursor proteins consists of the cleavage of their prodomain at pairs of basic amino acids by enzymes of the subtilisin-like mammalian proprotein convertase family. One of these enzymes, furin, acts in the constitutive secretory pathway of almost every cell type. However, in spite of furin's major roles in many pathophysiological processes, the exact subcellular sites of processing and activation of its substrates remain elusive. In this study, furin antigenic sites were tracked in subcellular compartments of various tissues and corresponding cell lines by high-resolution immunogold electron microscopy, Western blotting, cell transfection, and in vivo gene delivery of the furin cDNA. In addition to the Golgi apparatus, furin was assigned to endosomes and plasma membranes of polarized intestinal and renal epithelial cells and endothelial cells of the continuous, fenestrated, and discontinuous capillaries. Roles of furin in endothelial permeability, basement membrane turnover, and shedding of transmembrane proteins are supported by our data.

Controlled secretion of beta-endorphin from human embryonic kidney cells carrying a Tet-on-beta-endorphin fusion gene.

To create a cell line with controlled and specific secretion of beta-endorphin, a new fusion gene was constructed by joining human beta-endorphin coding sequence to part of NL1 gene. HEK293 cells carrying Tet-on system transfected with this fusion gene secreted beta-endorphin in a dose-dependent manner upon doxycycline administration. These findings suggest that this system can direct the controlled secretion of any peptide hormones such as beta-endorphin.

The neuropeptide-degrading enzyme NL1 is expressed in specific neurons of mouse brain.

Metalloendopeptidases of the M13 family were shown to play critical roles in normal physiological processes such as pain control, hypertension and phosphate metabolism, and in pathological states such as Alzheimer's disease. Recently, NL1, a novel member of the family, has been identified and shown to be expressed in several tissues both as a membrane-bound and a secreted protein. As a further step to understand the physiological role(s) of NL1 in mouse, we mapped NL1 mRNA expression pattern in embryos and in young animals at postnatal days p1 and p3, and in adult nervous tissue, using in situ hybridization at the cellular level. No expression could be detected in embryos and young animals. In contrast, NL1 expression was evident in adult brain, pituitary gland and spinal cord. In the central nervous system (CNS), NL1 mRNA was predominantly found in the ventro-posterior regions, which are mostly associated with vegetative functions. At the cellular level, NL1 mRNA was non-uniformly distributed within subpopulations of neurons. In the spinal cord, specific signal was observed in the gray matter. Then, in order to identify putative relevant substrates for NL1, we studied its enzymatic activity towards peptides known to be co-expressed in the NL1-positive domains. Our study showed that NL1 degrades several of these peptides in vitro, the most readily degraded peptides being Bradykinin and Substance P. These results suggest that NL1 is likely to play a critical role in the central nervous system.

Dose response of bone-targeted enzyme replacement for murine hypophosphatasia.

Hypophosphatasia (HPP) features rickets or osteomalacia from tissue-nonspecific alkaline phosphatase (TNSALP) deficiency due to deactivating mutations within the ALPL gene. Enzyme replacement therapy with a bone-targeted, recombinant TNSALP (sALP-FcD(10), renamed ENB-0040) prevents manifestations of HPP when initiated at birth in TNSALP knockout (Akp2(-/-)) mice. Here, we evaluated the dose-response relationship of ENB-0040 to various phenotypic traits of Akp2(-/-) mice receiving daily subcutaneous (SC) injections of ENB-0040 from birth at 0.5, 2.0, or 8.2mg/kg for 43days. Radiographs, µCT, and histomorphometric analyses documented better bone mineralization with increasing doses of ENB-0040. We found a clear, positive correlation between ENB-0040 dose and prevention of mineralization defects of the feet, rib cage, lower limbs, and jaw bones. According to a dose-response model, the ED(80) (the dose that prevents bone defects in 80% of mice) was 3.2, 2.8 and 2.9mg/kg/day for these sites, respectively. Long bones seemed to respond to lower daily doses of ENB-0040. There was also a positive relationship between ENB-0040 dose and survival. Median survival, body weight, and bone length all improved with increasing doses of ENB-0040. Urinary PP(i) concentrations remained elevated in all treatment groups, indicating that while this parameter is a good biochemical marker for diagnosing HPP in patients, it may not be a good follow up marker for evaluating response to treatment when administering bone-targeted TNSALP to mice. These dose-response relationships strongly support the pharmacological efficacy of ENB-0040 for HPP, and provide the experimental basis for the therapeutic range of ENB-0040 chosen for clinical trials.

Enzyme replacement therapy for murine hypophosphatasia.

INTRODUCTION: Hypophosphatasia (HPP) is the inborn error of metabolism that features rickets or osteomalacia caused by loss-of-function mutation(s) within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNALP). Consequently, natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5'-phosphate (PLP), a co-factor form of vitamin B6. Babies with the infantile form of HPP often die with severe rickets and sometimes hypercalcemia and vitamin B6-dependent seizures. There is no established medical treatment. MATERIALS AND METHODS: Human TNALP was bioengineered with the C terminus extended by the Fc region of human IgG for one-step purification and a deca-aspartate sequence (D10) for targeting to mineralizing tissue (sALP-FcD10). TNALP-null mice (Akp2-/-), an excellent model for infantile HPP, were treated from birth using sALP-FcD10. Short-term and long-term efficacy studies consisted of once daily subcutaneous injections of 1, 2, or 8.2 mg/kg sALP-FcD10 for 15, 19, and 15 or 52 days, respectively. We assessed survival and growth rates, circulating levels of sALP-FcD10 activity, calcium, PPi, and pyridoxal, as well as skeletal and dental manifestations using radiography, microCT, and histomorphometry. RESULTS: Akp2-/- mice receiving high-dose sALP-FcD10 grew normally and appeared well without skeletal or dental disease or epilepsy. Plasma calcium, PPi, and pyridoxal concentrations remained in their normal ranges. We found no evidence of significant skeletal or dental disease. CONCLUSIONS: Enzyme replacement using a bone-targeted, recombinant form of human TNALP prevents infantile HPP in Akp2-/- mice.

Neprilysin carboxydipeptidase specificity studies and improvement in its detection with fluorescence energy transfer peptides.

We examined the substrate specificity of the carboxydipeptidase activity of neprilysin (NEP) using fluorescence resonance energy transfer (FRET) peptides containing ortho-aminobenzoyl (Abz) and 2,4-dinitrophenyl (Dnp) as a donor/acceptor pair. Two peptide series with general sequences Abz-RXFK(Dnp)-OH and Abz-XRFK(Dnp)-OH (X denotes the position of the altered amino acid) were synthesized to study P1 (cleavage at the X-F bond) and P2 (cleavage at R-F bond) specificity, respectively. In these peptides a Phe residue was fixed in P1' to fulfill the well-known NEP S1' site requirement for a hydrophobic amino acid. In addition, we explored NEP capability to hydrolyze bradykinin (RPPGFSPFR) and its fluorescent derivative Abz-RPPGFSPFRQ-EDDnp (EDDnp=2,4-dinitrophenyl ethylenediamine). The enzyme acts upon bradykinin mainly as a carboxydipeptidase, preferentially cleaving Pro-Phe over the Gly-Phe bond in a 9:1 ratio, whereas Abz-RPPGFSPFRQ-EDDnp was hydrolyzed at the same bonds but at an inverted proportion of 1:9. The results show very efficient interaction of the substrates' C-terminal free carboxyl group with site S2' of NEP, confirming the enzyme's preference to act as carboxydipeptidase at substrates with a free carboxyl-terminus. Using data gathered from our study, we developed sensitive and selective NEP substrates that permit continuous measurement of the enzyme activity, even in crude tissue extracts.

trans-Complementation assay establishes the role of proregion hydrophobic amino acid residues in the biosynthesis of Saccharomyces cerevisiae Kex2p endoprotease.

The proregion of Saccharomyces cerevisiae endoprotease Kex2p is essential for the biosynthesis of an active enzyme. It has been suggested that the proregion acts in the endoplasmic reticulum to catalyse folding of the enzyme. To identify amino acid residues important for proregion function, we used an in vivo system in which the Kex2p proregion can act in trans to activate a Kex2p enzyme synthesized without its proregion. Activation of Kex2p by wild-type and mutated proregions revealed the essential role of hydrophobic residues F(37), V(39) and F(70) in enzyme activation. Further exploration of the role of these residues by in vitro inhibition of Kex2p activity by its proregion indicated that they are essential to form the proregion/enzyme bimolecular complex. In contrast, basic residues K(108) and R(109), located in the C-terminus of the proregion, are not involved in complex formation but are necessary for the biosynthesis of an active enzyme.

Furin interacts with proMT1-MMP and integrin alphaV at specialized domains of renal cell plasma membrane.

Matrix metalloproteinases (MMPs) and integrins are essential for cell and extracellular matrix homeostasis. Both membrane type-1 MMP (MT1-MMP) and the integrin alphaV subunit are fully activated upon cleavage at a furin recognition site. Furin is shuttled to the cell surface through the trans-Golgi network and endosomal system, and its only known role on plasma membrane consists in activation of opportunistic pathogenic entities. Here, we report findings about the interaction of furin with MT1-MMP and the integrin alphaV at the cell surface. By using in vivo gene delivery, western blotting and immunogold electron microscopy, we provide evidence of significant pools of furin and proMT1-MMP along the surface of cells lining basement membranes. Moreover, furin and integrin alphaV are frequently found associated with the slit diaphragm of renal podocytes and around endothelial fenestrations. ProMT1-MMP, by contrast, is concentrated at the slit diaphragm. Coimmunoprecipitations and double immunogold labelings indicate that furin interacts with proMT1-MMP and alphaV at points of insertion of the slit diaphragm. Our results suggest that these focalized complexes could trigger basement membrane proteolysis either directly by activation of proMT1-MMP or indirectly by promoting activation of proMMP2.

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.