Family Of Endoproteases Research Articles

Matrix metalloproteinases: expression and regulation in the endometrium during the estrous cycle and at the maternal-conceptus interface during pregnancy in pigs.

Matrix metalloproteinases (MMPs) are a family of endoproteases produced by various tissues and cells and play important roles in angiogenesis, tissue repair, immune response, and endometrial remodeling. However, the expression and function of MMPs in the pig endometrium during the estrous cycle and pregnancy have not been fully elucidated. Thus, we determined the expression, localization, and regulation of MMP2, MMP8, MMP9, MMP12, and MMP13 in the endometrium throughout the estrous cycle and at the maternal-conceptus interface during pregnancy in pigs. Endometrial tissues during the estrous cycle and pregnancy and conceptus and chorioallantoic tissues during pregnancy were obtained and the expression of MMPs was analyzed. The effects of steroid hormones and cytokines on the expression of MMPs were determined in endometrial explant cultures. Expression levels of MMP12 and MMP13 changed during the estrous cycle, while expression of MMP2, MMP9, MMP12, and MMP13 changed during pregnancy. Expression of MMP2, MMP8, and MMP13 mRNAs was cell type-specific at the maternal-conceptus interface. Gelatin zymography showed that enzymatically active MMP2 was present in endometrial tissues. In endometrial explant cultures, estradiol-17β induced the expression of MMP8 and MMP12, progesterone decreased the expression of MMP12, interleukin-1β increased the expression of MMP2, MMP8, MMP9, and MMP13, and interferon-γ increased the expression of MMP2. These results suggest that MMPs expressed in response to steroids and cytokines play an important role in the establishment and maintenance of pregnancy by regulating endometrial remodeling and processing bioactive molecules in pigs.

Caspase-11 promotes high-fat diet-induced NAFLD by increasing glycolysis, OXPHOS, and pyroptosis in macrophages.

Non-alcoholic fatty liver disease (NAFLD) has a global prevalence of 25% of the population and is a leading cause of cirrhosis and hepatocellular carcinoma. NAFLD ranges from simple steatosis (non-alcoholic fatty liver) to non-alcoholic steatohepatitis (NASH). Hepatic macrophages, specifically Kupffer cells (KCs) and monocyte-derived macrophages, act as key players in the progression of NAFLD. Caspases are a family of endoproteases that provide critical connections to cell regulatory networks that sense disease risk factors, control inflammation, and mediate inflammatory cell death (pyroptosis). Caspase-11 can cleave gasdermin D (GSDMD) to induce pyroptosis and specifically defends against bacterial pathogens that invade the cytosol. However, it's still unknown whether high fat diet (HFD)-facilitated gut microbiota-generated cytoplasmic lipopolysaccharides (LPS) activate caspase-11 and promote NAFLD. To examine this hypothesis, we performed liver pathological analysis, RNA-seq, FACS, Western blots, Seahorse mitochondrial stress analyses of macrophages and bone marrow transplantation on HFD-induced NAFLD in WT and Casp11-/- mice. Our results showed that 1) HFD increases body wight, liver wight, plasma cholesterol levels, liver fat deposition, and NAFLD activity score (NAS score) in wild-type (WT) mice; 2) HFD increases the expression of caspase-11, GSDMD, interleukin-1β, and guanylate-binding proteins in WT mice; 3) Caspase-11 deficiency decreases fat liver deposition and NAS score; 4) Caspase-11 deficiency decreases bone marrow monocyte-derived macrophage (MDM) pyroptosis (inflammatory cell death) and inflammatory monocyte (IM) surface GSDMD expression; 5) Caspase-11 deficiency re-programs liver transcriptomes and reduces HFD-induced NAFLD; 6) Caspase-11 deficiency decreases extracellular acidification rates (glycolysis) and oxidative phosphorylation (OXPHOS) in inflammatory fatty acid palmitic acid-stimulated macrophages, indicating that caspase-11 significantly contributes to maintain dual fuel bioenergetics-glycolysis and OXPHOS for promoting pyroptosis in macrophages. These results provide novel insights on the roles of the caspase-11-GSDMD pathway in promoting hepatic macrophage inflammation and pyroptosis and novel targets for future therapeutic interventions involving the transition of NAFLD to NASH, hyperlipidemia, type II diabetes, metabolic syndrome, metabolically healthy obesity, atherosclerotic cardiovascular diseases, autoimmune diseases, liver transplantation, and hepatic cancers.

PACE4 is an important driver of ZR-75-1 estrogen receptor-positive breast cancer proliferation and tumor progression

Breast cancer is the most frequent and deadly malignancy in women worldwide. Despite national screening programs combined with new treatments relapse rate remain high and new therapies are needed. From previous work, we identified PACE4, a member of the proprotein convertase (PCs) family of endoproteases, as a novel therapeutic target in prostate cancer. In the present study we asked the question if PACE4 could also be a potential target in breast cancer. In clinical samples of breast adenocarcinoma, we observed a specific overexpression of PACE4 in the estrogen-receptor (ER) positive subtype. We therefore looked for a breast cancer cell line model which would be representative and thus focused on the ZR-75-1 since it both expresses PACE4 and is estrogen-receptor positive. We compared stable knockdowns of furin, PACE4 and PC7 in the estrogen-receptor-positive cell line ZR-75-1 to evaluate their respective contribution to cell growth and tumor progression. PACE4 was the only PC displaying an impact on cell growth. A PACE4 peptide-based inhibitor (C23) was tested and shown to decrease proliferation of ZR-75-1 cells in cell based assays. C23 also had potent effects of tumor progression in vivo on xenografts of the ZR-75-1 cell line in athymic nude mice. Thus, PACE4-silencing and systemic administration of a PACE4 inhibitor resulted in hindered tumor progression with reduction in proliferative indices and increased cell quiescence assessed with biomarkers. Our results suggest that PACE4 is a promising target for estrogen-receptor-positive breast cancer.

Processing by Convertases Is Required for Glypican-3-induced Inhibition of Hedgehog Signaling

Glypican-3 (GPC3) is one of the six members of the mammalian glypican family. We have previously reported that GPC3 inhibits Hedgehog (Hh) signaling by competing with Patched (Ptc) for Hh binding. We also showed that GPC3 binds with high affinity to Hh through its core protein, but that it does not interact with Ptc. Several members of the glypican family, including GPC3, are subjected to an endoproteolytic cleavage by the furin-like convertase family of endoproteases. Surprisingly, however, we have found that a mutant GPC3 that cannot be processed by convertases is as potent as wild-type GPC3 in stimulating Wnt activity in hepatocellular carcinoma cell lines and 293T cells and in promoting hepatocellular carcinoma growth. In this study, we show that processing by convertases is essential for GPC3-induced inhibition of Hh signaling. Moreover, we show that a convertase-resistant GPC3 stimulates Hh signaling by increasing the binding of this growth factor to Ptc. Consistent with this, we show that the convertase-resistant mutant binds to both Hh and Ptc through its heparan sulfate (HS) chains. Unexpectedly, we found that the mutant core protein does not bind to Hh. We also report that the convertase-resistant mutant GPC3 carries HS chains with a significantly higher degree of sulfation than those of wild-type GPC3. We propose that the structural changes generated by the lack of cleavage determine a change in the sulfation of the HS chains and that these hypersulfated chains mediate the interaction of the mutant GPC3 with Ptc.

AB0267 Correlation of MMP3 Serum Levels with Disease Activity in Patients with Rheumatoid Arthritis

BackgroundThe cartilage destruction in Rheumatoid Arthritis (RA) is also influenced by the actions of matrix metalloproteinase enzymes (MMPs) released by synovial cells in response to IL-1, TNF-α, epidermal growth factor...

Caspase Functions in Cell Death and Disease

Caspases are a family of endoproteases that provide critical links in cell regulatory networks controlling inflammation and cell death. The activation of these enzymes is tightly controlled by their production as inactive zymogens that gain catalytic activity following signaling events promoting their aggregation into dimers or macromolecular complexes. Activation of apoptotic caspases results in inactivation or activation of substrates, and the generation of a cascade of signaling events permitting the controlled demolition of cellular components. Activation of inflammatory caspases results in the production of active proinflammatory cytokines and the promotion of innate immune responses to various internal and external insults. Dysregulation of caspases underlies human diseases including cancer and inflammatory disorders, and major efforts to design better therapies for these diseases seek to understand how these enzymes work and how they can be controlled.

Identification of a functional proprotein convertase cleavage site in microfibril-associated glycoprotein 2

Microfibril-associated glycoprotein 2 (MAGP2) is a secreted protein associated with multiple cellular activities including the organization of elastic fibers in the extracellular matrix (ECM), angiogenesis, as well as regulating Notch and integrin signaling. Importantly, increases in MAGP2 positively correlate with poor prognosis for some ovarian cancers. It has been assumed that full-length MAGP2 is responsible for all reported effects; however, here we show MAGP2 is a substrate for the proprotein convertase (PC) family of endoproteases. Proteolytic processing of MAGP2 by PC cleavage could serve to regulate secretion and thus, activity and function as reported for other extracellular and cell-surface proteins. In support of this idea, MAGP2 contains an evolutionarily conserved PC consensus cleavage site, and amino acid sequencing of a newly identified MAGP2 C-terminal cleavage product confirmed functional PC cleavage. Additionally, mutagenesis of the MAGP2 PC consensus cleavage site or treatment with PC inhibitors prevented MAGP2 proteolytic processing. Finally, both cleaved and uncleaved MAGP2 were detected extracellularly and MAGP2 secretion appeared independent of PC cleavage, suggesting that PC processing occurs mainly outside the cell. Our characterization of alternative forms of MAGP2 present in the extracellular space not only enhances diversity of this ECM protein but also provides a previously unrecognized molecular mechanism for regulation of MAGP2 biological activity.

Non-peptide Inhibitors of Proprotein Convertase Subtilisin Kexins (PCSKs): An Overall Review of Existing and New Data

ABSTRACT The Ca+2-dependent mammalian Proprotein Convertase Subtilisin Kexins (PCSKs) or Proprotein/ Prohormone Convertases (PCs) are a family of endoproteases that play critical roles not only in normal development and metabolism but also in various physiological and pathological conditions. These were initiated by the proteolytic processing of large inactive proproteins into their shorter bioactive mature forms by the PCSK enzymes. These events take place in a highly selective, orchestrated, and stepwise manner. Among the various proprotein substrates of PCSK enzymes, particularly important are the precursor growth factors that include proPDGF-A, B, proIGF-1, 2 and proVEGF-C because of their strong implications in neoplasia initiation, progression, and metastasis. As a result of these findings, PCSK enzymes, particularly furin or PCSK3, became a major target for possible interventions of cancer via the use of their selective inhibitors. Significant progress has been accomplished in the development of pe...

Detection of in vivo matrix metalloproteinase activity using microdialysis sampling and liquid chromatography/mass spectrometry.

Matrix metalloproteinases (MMPs) are a family of endoproteases that break down extracellular matrix and whose upregulation contributes to several diseases. A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed to quantify MMP-1 and MMP-9 substrates and their N-terminal peptide products in samples obtained from implanted microdialysis sampling probes. In vitro studies with purified human MMP-1 and MMP-9 were used to optimize the assay and determine the effectiveness of the local delivery of a broad-spectrum MMP inhibitor, GM 6001. Localized delivery of GM 6001 at 10 microM was sufficient to completely inhibit product formation in vitro. In vivo studies in male Sprague-Dawley rats were performed with microdialysis probes implanted into the subcutaneous tissue. Directly after microdialysis probe implantation, infusions of the MMP-1 and MMP-9 substrates (50 microM each) resulted in recovered product concentrations of approximately 2 microM. During a 50 microM GM 6001 coinfusion with the substrates, a 30% and 25% reduction in product formation for the MMP-1 and MMP-9 substrates was obtained, respectively. Blank dialysates were negative for enzymatic activity that could cleave the MMP substrates. This method allowed for the activity of different MMPs surrounding the microdialysis probe to be observed during in vivo sampling.

A disruption of ctpA encoding carboxy-terminal protease attenuates Burkholderia mallei and induces partial protection in CD1 mice

Burkholderia mallei is the etiologic agent of glanders in solipeds (horses, mules and donkeys), and incidentally in carnivores and humans. Little is known about the molecular mechanisms of B. mallei pathogenesis. The putative carboxy-terminal processing protease (CtpA) of B. mallei is a member of a novel family of endoproteases involved in the maturation of proteins destined for the cell envelope. All species and isolates of Burkholderia carry a highly conserved copy of ctpA. We studied the involvement of CtpA on growth, cell morphology, persistence, and pathogenicity of B. mallei. A sucrose-resistant strain of B. mallei was constructed by deleting a major portion of the sacB gene of the wild type strain ATCC 23344 by gene replacement, and designated as strain 23344Δ sacB. A portion of the ctpA gene (encoding CtpA) of strain 23344Δ sacB was deleted by gene replacement to generate strain 23344Δ sacBΔ ctpA. In contrast to the wild type ATCC 23344 or the sacB mutant 23344Δ sacB, the ctpA mutant 23344Δ sacBΔ ctpA displayed altered cell morphologies with partially or fully disintegrated cell envelopes. Furthermore, relative to the wild type, the ctpA mutant displayed slower growth in vitro and less ability to survive in J774.2 murine macrophages. The expression of mRNA of adtA, the gene downstream of ctpA was similar among the three strains suggesting that disruption of ctpA did not induce any polar effects. As with the wild type or the sacB mutant, the ctpA mutant exhibited a dose-dependent lethality when inoculated intraperitoneally into CD1 mice. The CD1 mice inoculated with a non-lethal dose of the ctpA mutant produced specific serum immunoglobulins IgG1 and IgG2a and were partially protected against challenge with wild type B. mallei ATCC 23344. These findings suggest that CtpA regulates in vitro growth, cell morphology and intracellular survival of B. mallei, and a ctpA mutant protects CD1 mice against glanders.

Carboxyl-Terminal Protease RegulatesBrucella suisMorphology in Culture and Persistence in Macrophages and Mice

The putative carboxyl-terminal processing protease (CtpA) of Brucella suis 1330 is a member of a novel family of endoproteases involved in the maturation of proteins destined for the cell envelope. The B. suis CtpA protein shared up to 77% homology with CtpA proteins of other bacteria. A CtpA-deficient Brucella strain (1330DeltactpA), generated by allelic exchange, produced smaller colonies on enriched agar plates and exhibited a 50% decrease in growth rate in enriched liquid medium and no growth in salt-free enriched medium compared to the wild-type strain 1330 or the ctpA-complemented strain 1330DeltactpA[pBBctpA]. Electron microscopy revealed that in contrast to the native coccobacillus shape of wild-type strain 1330, strain 1330DeltactpA possessed a spherical shape, an increased cell diameter, and cell membranes partially dissociated from the cell envelope. In the J774 mouse macrophage cell line, 24 h after infection, the CFU of the strain 1330DeltactpA declined by approximately 3 log(10) CFU relative to wild-type strain 1330. Nine weeks after intraperitoneal inoculation of BALB/c mice, strain 1330DeltactpA had cleared from spleens but strain 1330 was still present. These observations suggest that the CtpA activity is necessary for the intracellular survival of B. suis. Relative to the saline-injected mice, strain 1330DeltactpA-vaccinated mice exhibited 4 to 5 log(10) CFU of protection against challenge with virulent B. abortus strain 2308 or B. suis strain 1330 but no protection against B. melitensis strain 16 M. This is the first report correlating a CtpA deficiency with cell morphology and attenuation of B. suis.

Transcriptional regulation and structural modelling of the Synechocystis sp. PCC 6803 carboxyl-terminal endoprotease family

The Synechocystis sp. PCC 6803 ctp gene family members ctpA (slr0008), ctpB (slr0257) and ctpC (slr1751), encoding carboxyl-terminal endoproteases (Ctps), were studied at levels of gene transcription and protein structure. Northern blot analysis revealed differential activation and accumulation of the ctp transcripts upon induction of various environmental conditions, including light, temperature, salinity and growth mode, supporting the view of distinct roles of Ctps in Synechocystis sp. PCC 6803 cellular processes. Amino acid sequence comparison of 16 ctp gene products showed that they fall into three distinct groups: the eukaryotic CtpA-like proteins, the prokaryotic CtpA-like proteins and the prokaryotic CtpB/C-like proteins. Structural models of the Synechocystis sp. PCC 6803 Ctps, constructed based on the amino acid sequence alignment and the crystal structure of the Scenedesmus obliquus D1 processing protease, revealed that although the overall structure of the Synechocystis sp. PCC 6803 Ctps is very similar, differences exist in the putative membrane contact regions and in the active site environment.

Profibrillin-1 maturation by human dermal fibroblasts: proteolytic processing and molecular chaperones.

Fibrillin-1 is synthesized as a proprotein that undergoes proteolytic processing in the unique C-terminal domain by a member of the PACE/furin family of endoproteases. This family of endoproteases is active in the trans-Golgi network (TGN), but metabolic labeling studies have been controversial as to whether profibrillin-1 is processed intracellularly or after secretion. This report provides evidence that profibrillin-1 processing is not an intracellular event. Bafilomycin A(1) and incubation of dermal fibroblasts at 22 degrees C were used to block secretion in the TGN to confirm that profibrillin-1 processing did not occur in this compartment. Profibrillin-1 immunoprecipitation studies revealed that two endoplasmic reticulum-resident molecular chaperones, BiP and GRP94, interacted with profibrillin-1. To determine the proprotein convertase responsible for processing profibrillin-1, a specific inhibitor of furin, alpha-1-antitrypsin, Portland variant, was both expressed in the cells and added to cells exogenously. In both cases, the inhibitor blocked the processing of profibrillin-1, providing evidence that furin is the enzyme responsible for profibrillin-1 processing. These studies delineate the secretion and proteolytic processing of profibrillin-1, and identify the proteins that interact with profibrillin-1 in the secretory pathway.

Curbing activation: proprotein convertases in homeostasis and pathology.

The proprotein convertases (PCs) are a seven-member family of endoproteases that activate proproteins by cleavage at basic motifs. Expression patterns for individual PCs vary widely, and all cells express several members. The list of substrates activated by PCs has grown to include neuropeptides, peptide hormones, growth and differentiation factors, receptors, enzymes, adhesion molecules, blood coagulation factors, plasma proteins, viral coat proteins, and bacterial toxins. It has become clear that the PC family plays a crucial role in a variety of physiological processes and is involved in the pathology of diseases such as cancer, viral infection, and Alzheimer's disease. Recent studies using PC inhibitors have demonstrated their potential as therapeutic targets. Despite the avalanche of in vitro data, the physiological role of individual PCs has remained largely elusive. Recently, however, knockout mouse models have been developed for furin, PC1, PC2, PC4, PC6B, LPC, and PACE4, and human patients with PC1 deficiency have been identified. The phenotypes range from undetectable to early embryonic lethality. The major lesson learned from these studies is that specific PC-substrate pairs do exist, but that there is substantial redundancy for the majority of substrates. To some extent, redundancy may be cell type and even species dependent.

Amino Acid Residues That Are Critical for in VivoCatalytic Activity of CtpA, the Carboxyl-terminal Processing Protease for the D1 Protein of Photosystem II

CtpA, a carboxyl-terminal processing protease, is a member of a novel family of endoproteases that includes a tail-specific protease from Escherichia coli. In oxygenic photosynthetic organisms, CtpA catalyzes C-terminal processing of the D1 protein of photosystem II, an essential event for the assembly of a manganese cluster and consequent light-mediated water oxidation. We introduced site-specific mutations at 14 conserved residues of CtpA in the cyanobacterium Synechocystis sp. PCC 6803 to examine their functional roles. Analysis of the photoautotrophic growth capabilities of these mutants, their ability to process precursor D1 protein and hence evolve oxygen, along with an estimation of the protease content in the mutants revealed that five of these residues are critical for in vivo activity of CtpA. Recent x-ray crystal structure analysis of CtpA from the eukaryotic alga Scenedesmus obliquus (Liao, D.-I., Qian, J., Chisholm, D. A., Jordan, D. B. and Diner, B. A. (2000) Nat. Struct. Biol. 7, 749-753) has shown that the residues equivalent to Ser-313 and Lys-338, two of the five residues mentioned above, form the catalytic center of this enzyme. Our in vivo analysis demonstrates that the three other residues, Asp-253, Arg-255, and Glu-316, are also important determinants of the catalytic activity of CtpA.

Role of Endoproteolytic Processing in the Adhesive and Signaling Functions of αvβ5 Integrin

Some integrin alpha subunits undergo a post-translational cleavage in their extracellular domain. However, the role of this cleavage in integrin function is unclear. Enzymes involved in this maturation belong to the subtilisin-like endoprotease family (convertases). To understand the role of the alpha subunit cleavage in integrin function, we have designed stable transfectants (PDX39P cells) expressing alpha(1)-PDX, a convertase inhibitor. Immunoprecipitation of cell surface proteins from PDX39P showed that alpha(3), alpha(6) and alpha(v) integrins lack endoproteolytic cleavage. We have compared adhesion between PDX39P cells and mock-transfected cells on different extracellular matrix proteins. No difference in adhesion could be observed on laminin-1 and type I collagen, while attachment of PDX39P cells to vitronectin (ligand of the alpha(v)beta(5) integrin) was dramatically reduced. The reduced adhesion of PDX39P cells was not due to changes in integrin affinity as determined by solid-phase receptor assay in a cell-free environment. Intracellular signaling pathways activated by alpha(v) integrin ligation were also affected in PDX39P cells. It thus seems that the absence of endoproteolytic cleavage of alpha(v) integrins has important consequences on signal transduction pathways leading to alterations in integrin function such as cell adhesion.

8] Use of expression of antisense mRNA for proprotein convertases 1 and 2 in prohormone processing

Studies using antisense inhibition have been criticized because of the possible nonspecific cellular effects complicating interpretation of the results. In light of these concerns, several controls have been performed to ensure that the effects of antisense expression on the processing of pro-cholecystokinin (CCK) are specific. The two most important controls performed were to measure the effects of antisense expression on target messenger ribonucleic acid (mRNA) and target protein. Another control that was performed was to measure the effect of polycystin 2 (PC2) message and protein level in antisense PC1-transfected cells and vice versa. Although PC1 and PC2 are homologous genes within the same family of endoproteases, the effect of antisense is specific for each. These observations suggest that the effect of antisense on the RNA level is complex but specific and may occur at multiple levels to produce inhibition of the protein product. Knowledge of the actual enzymes responsible for prohormone cleavages in specific tissues lags behind progress in other areas of the peptide hormone field owing to the technical difficulty of identification, isolation, characterization, and inhibition of these enzymes. As important as the PC enzymes appear to be, it is possible that proteases other than the PCs are involved in prohormone processing.

Endoprotease PACE4 is Ca2+-dependent and temperature-sensitive and can partly rescue the phenotype of a furin-deficient cell strain

PACE4 is a member of the eukaryotic subtilisin-like endoprotease family. The expression of human PACE4 in RPE.40 cells (furin-null mutants derived from Chinese hamster ovary K1 cells) resulted in the rescue of a number of wild-type characteristics, including sensitivity to Sindbis virus and the ability to process the low-density-lipoprotein receptor-related protein. Expression of PACE4 in these cells failed to restore wild-type sensitivity to Pseudomonas exotoxin A. Co-expression of human PACE4 in these cells with either a secreted form of the human insulin pro-receptor or the precursor form of von Willebrand factor resulted in both proproteins being processed; RPE.40 cells were unable to process either precursor protein in the absence of co-expressed PACE4. Northern analysis demonstrated that untransfected RPE.40 cells express mRNA species for four PACE4 isoforms, suggesting that any endogenous PACE4 proteins produced by these cells are either non-functional or sequestered in a compartment outside of the secretory pathway. In experiments in vitro, PACE4 processed diphtheria toxin and anthrax toxin protective antigen, but not Pseudomonas exotoxin A. The activity of PACE4 in vitro was Ca2+-dependent and, unlike furin, was sensitive to temperature changes between 22 and 37 degrees C. RPE.40 cells stably expressing human PACE4 secreted an endoprotease with the same Ca2+ dependence and temperature sensitivity as that observed in membrane fractions of these cells assayed in vitro. These results, in conjunction with other published work, demonstrate that PACE4 is an endoprotease with more stringent substrate specificity and more limited operating parameters than furin.

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 F1and F2subunits 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 thetrans-Golgi cisternae and/or thetrans-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 F0into proteins the exact size of F1and F2and 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.

Proprotein Convertase PC1/3-related Peptides Are Potent Slow Tight-binding Inhibitors of Murine PC1/3 and Hfurin

The proprotein convertase PC1/3 belongs to the subtilisin/kexin-like endoprotease family and is synthesized as a preproenzyme. To investigate the function of its propeptide, murine proPC1/3 and preproPC1/3 were isolated from the inclusion bodies of recombinant preproPC1/3 baculovirus-infected insect cells, rendered soluble with 6 M guanidine HCl and 20 mM dithiothreitol, and purified by gel filtration and metal-binding affinity chromatography. Two NH2-terminal fragments containing the complete propeptide 1-84 region were obtained after CNBr cleavage, purified, and chemically characterized. Progress curve kinetic analysis with enzymatically active murine 71-kDa PC1/3 or 50-kDa human furin demonstrated that both fragments were potent slow tight-binding inhibitors of either enzyme with Ki in the low nanomolar range. Additional cleavages at Trp residues yielded fragment9-71, which no longer represents a potent inhibitor. Upon incubation at pH 5.5 in the presence of excess 71-kDa murine PC1/3, NH2-terminal fragment1-98 is cleaved at two sites, as revealed through Western blotting using NH2-terminal-directed PC1/3 antibodies. Finally, murine PC2 is inhibited by the proPC1/31-98 peptide, albeit at a much lesser extent with a micromolar Ki and in a strictly competitive manner. These results suggest that the proregion of PC1/3 is an important feature in regulating its activity.