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PubMed2005

Human carcinoma cell growth and invasiveness is impaired by the propeptide of the ubiquitous proprotein convertase furin.

Furin, a potent proprotein convertase involved in activation of several cancer-related substrates, is synthesized as an inactive zymogen, thus minimizing the occurrence of premature enzymatic activity that would lead to inappropriate protein activation or degradation. This natural inhibitory mechanism is based on the presence of an inactivating prosegment at the NH2 terminal of the zymogen. After initial autocatalytic cleavage, the prosegment remains tightly associated with the convertase until it reaches the trans-Golgi network where the dissociation of the prosegment and activation of furin occurs. We hypothesized that the inhibitory properties of the preprosegment of furin (ppFur) could be beneficial if ectopically expressed in tumor cells. Transfection of four human head and neck squamous cell carcinoma cell lines with the complete ppFur cDNA sequence (pIRES-EGFP-ppFur) or with the empty expression vector (pIRES-EGFP) was done. The inhibitory effect was evaluated using in vivo tumorigenicity, invasion, anchorage-independent growth in soft agar, and proliferation assays, as well as by investigating impairment of furin substrates processing. Following transfection of ppFur, a significant reduction in cell proliferation, tumorigenicity, and invasiveness was observed in vitro and in vivo. These biological changes are directly related to the inhibition of furin-mediated activation of crucial cancer-related substrates, such as membrane type 1 matrix metalloproteinase, transforming growth factor-beta, insulin-like growth factor-1 receptor, and vascular endothelial growth factor-C. PpFur expression in head and neck squamous cell carcinoma cell lines showed a mechanistic link between furin inhibition, decreased substrate processing, cell proliferation, and invasive ability. These findings suggest that furin inhibition is a feasible approach to ameliorate and even abolish the malignant phenotype of various malignancies.

Carcinoma, Squamous Cell↗

PubMed2004

Inhibition of proprotein convertases enhances cell migration and metastases development of human colon carcinoma cells in a rat model.

Although proprotein convertases are involved in tumor development, nothing is known about their role in metastatic dissemination. To investigate the involvement of convertase inhibition, we used human colon carcinoma cells overexpressing alpha1-antitrypsin Portland (alpha1-PDX, PDX39P cells), a potent convertase inhibitor. We previously reported that these cells bear uncleaved integrin alpha subunits and display an altered attachment to vitronectin that is correlated with defects in the intracellular signaling pathways activated by alphavbeta5 integrin ligation. In this study, we demonstrate that the inhibition of proprotein convertase activity either by overexpression of alpha1-PDX or with the synthetic inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk) led to a significant increase in cell migration supported by the alphavbeta5 integrin. A collagen gel invasion assay showed that PDX39P cells also displayed an invasive ability, contrary to control cells. Moreover, when injected to immunosuppressed newborn rats, PDX39P cells were highly invasive, as they induce 10 times more metastases than mock-transfected cells. In addition, the aggressiveness of PDX39P cells can be greatly reduced by a function-blocking monoclonal antibody (mAb) against the alphav subunit. It thus seems that inhibition of proprotein convertases enhances the in vivo invasiveness of colon tumor cells likely due to an increase in cell migration mediated by alphav integrins.

Adenocarcinoma↗

PubMed2001

The EGL-3 proprotein convertase regulates mechanosensory responses of Caenorhabditis elegans.

Neuroactive peptides are packaged as proproteins into dense core vesicles or secretory granules, where they are cleaved at dibasic residues by copackaged proprotein convertases. We show here that the Caenorhabditis elegans egl-3 gene encodes a protein that is 57% identical to mouse proprotein convertase type 2 (PC2), and we provide evidence that this convertase regulates mechanosensory responses. Nose touch sensitivity (mediated by ASH sensory neurons) is defective in mutants lacking GLR-1 glutamate receptors (GluRs); however, mutations eliminating the egl-3 PC2 restored nose touch sensitivity to glr-1 GluR mutants. By contrast, body touch sensitivity (mediated by the touch cells) is greatly diminished in egl-3 PC2 mutants. Taken together, these results suggest that egl-3 PC2-processed peptides normally regulate the responsiveness of C. elegans to mechanical stimuli.

Animals↗

PubMed2005

Proprotein convertase subtilisin kexin 9: the third locus implicated in autosomal dominant hypercholesterolemia.

PURPOSE OF REVIEW: Autosomal dominant hypercholesterolemia is a genetic disease in which patients have elevated LDL cholesterol levels and premature atherosclerosis. Mutations in the LDL receptor and its ligand apolipoprotein B are causative for autosomal dominant hypercholesterolemia, and the study of this pathway has been crucial to understanding LDL metabolism and receptor-mediated endocytosis in general. Recently, families were identified with a clinical diagnosis of autosomal dominant hypercholesterolemia, but without linkage to the LDL receptor or apolipoprotein B genes. Identification and study of the causative genes in these families should provide additional insights into LDL metabolism. RECENT FINDINGS: Recent microarray studies and database searches identified a novel member of the proprotein convertase family called proprotein convertase subtilisin kexin 9 (PCSK9). A role for PCSK9 in cholesterol metabolism was proposed from the expression studies and confirmed by the discovery that PCSK9 missense mutations were associated with a form of autosomal dominant hypercholesterolemia, Hchola3. The cellular role for PCSK9 and the mechanism behind its mutations are under study, and a role for PCSK9 in regulating LDL receptor protein levels has been demonstrated. SUMMARY: PCSK9 is the third locus implicated in autosomal dominant hypercholesterolemia (Hchola3), and it appears to play an important role in cellular cholesterol metabolism. Understanding the function of PCSK9 will be important for broadening our knowledge of LDL metabolism and may aid in the development of novel hypocholesterolemic agents.

Apolipoproteins B↗

PubMed2004

Expression of proprotein convertase 2 mRNA in the ovarian follicles of the medaka, Oryzias latipes.

Proprotein convertases (PCs) are enzymes responsible for processing the precursors of many bioactive peptides in vertebrates and invertebrates. In the present study, a cDNA for proprotein convertase 2 (PC2) was cloned for the first time from a fish. The clone, which was isolated from the ovary of the medaka, Oryzias latipes, by a combination of RT-PCR cloning and 5'- and 3'-rapid amplification of cDNA ends, codes for a protein of 641 amino acid residues highly homologous to other vertebrate PC2. The medaka preproPC2 consists of a signal sequence, a propeptide with sites for autocatalytic activation, a Kex2-like catalytic domain, and a P-domain. The catalytic triad residues (Asp-169, His-210, and Ser-386) were all conserved. Northern blot analysis revealed that PC2 was expressed in the brain, ovary, and kidney of the fish. The size of PC2 mRNA expressed in the ovary was 2.3 kb, whereas those of the brain and kidney were 2.8 kb. This size difference was attributed to the lack of an approximately 300-bp nucleotide sequence just before the poly(A)+ tail of the ovarian PC2 mRNA. Ovarian expression of the PC2 gene was found in the medaka but not in the mouse, and therefore further analysis was conducted for the fish ovary. The greatest expression of PC2 mRNA in the oocytes of small growing follicles in the mature medaka was demonstrated by Northern blotting, RT-PCR and in situ hybridization analysis. These results suggest that PC2 may play a role in the processing of proproteins and/or pro-hormones expressed in the growing oocytes.

Amino Acid Sequence↗

PubMed1999

Subtilisin-like proprotein convertases, PACE4 and PC8, as well as furin, are endogenous proalbumin convertases in HepG2 cells.

Serum albumin is synthesized as a larger precursor form, proalbumin, which undergoes proteolytic processing at a dibasic site by a hepatic proprotein convertase within the secretory pathway to generate the mature form. Although furin, a member of the subtilisin-like proprotein convertase (SPC) family, was thought to be the only candidate hepatic convertase for proalbumin, SPC family members other than furin were recently suggested to also be involved in proalbumin processing. This study was designed to identify the endogenous proprotein convertases involved in proalbumin processing. Since human hepatoma HepG2 cells are highly differentiated and produce major plasma proteins, this cell line was used as a model for hepatocytes. Northern blot analysis revealed that PACE4, furin and PC8 of the SPC family were expressed in HepG2 cells as well as in the liver. Ribonuclease protection assay showed that PACE4A-II mRNA is the major transcript in HepG2 cells among the PACE4 isoforms. The coexpression studies showed that furin, PACE4A-II and PC8 were all able to convert proalbumin to albumin correctly. To elucidate the roles of these endogenous SPC family members in proalbumin processing, the antisense RNA for PACE4, furin and PC8 was stably expressed in HepG2 cells, respectively. The expression of each antisense RNA resulted in approximately 30% inhibition of endogenous proalbumin processing. We therefore concluded that PACE4 and PC8, as well as furin, are involved in the processing of proalbumin in HepG2 cells, and that these SPC family members are functionally redundant in this processing.

Furin↗

PubMed2002

Proprotein convertases are important mediators of the adipocyte differentiation of mouse 3T3-L1 cells.

Mouse 3T3-L1 cells are widely used to study adipocyte differentiation in vitro. When treated with insulin, dexamethasone and isobutylmethylxanthine these fibroblastic cells differentiate into round triglyceride-rich adipocytes. Because several proteins implicated in adipocyte differentiation (e.g. type 1 IGF receptors) are proteolytically activated by endoproteinases of the proprotein convertase family, we sought to determine whether these endoproteinases are crucial for adipose conversion. In this study, we show that expression of the proprotein convertases PACE4, PC7 and furin increases when 3T3-L1 cells are induced to differentiate into adipocytes. The differentiation was blocked in transfected cells expressing alpha1-antitrypsin Portland or in normal cells pre-treated with the synthetic inhibitor decanoyl-RVKR-chloromethylketone. Both inhibitors are known to specifically inactivate proprotein convertases. The block was associated with impaired proteolytic activation of proIGF-1 receptor, absence of induction of the adipogenic transcriptional factor PPARgamma and marked reduction of the nuclear translocation of the C/EBPbeta factor. Taken together, these data constitute evidence that proprotein convertases are crucial mediators of adipogenesis.

3T3 Cells↗

PubMed1997

Comparative analysis of expression of the proprotein convertases furin, PACE4, PC1 and PC2 in human lung tumours.

Proprotein convertases mediate the production of a variety of peptidic mitogens by limited proteolysis of their precursors. These proteases may also participate in the autocrine production of such mitogens by cancer cells and thus contribute to the unchecked proliferation of these cells. As a step towards defining this contribution, we have examined the levels of four convertase mRNAs in human lung neoplasms using semiquantitative Northern blot analysis. Furin mRNA was expressed in all the tumours; its level in squamous cell carcinomas and adenocarcinomas was on average about threefold higher than in small-cell lung carcinomas (SCLCs). PACE4 transcripts were detected in eight of 14 adenocarcinomas and in seven of 17 squamous cell carcinomas; they were detectable in only two of seven SCLCs. PC1 mRNA was undetected in squamous cell carcinomas and in all but two adenocarcinomas; it was present in four of six SCLCs. PC2 mRNA was found in two adenocarcinomas, in one squamous cell carcinoma and in five of seven SCLCs. This preliminary survey indicates that SCLCs often carry more mRNA for the endocrine convertases PC1 and PC2 and less mRNA for the more ubiquitous furin and PACE4, suggesting inverse roles of these convertases in the development of this neoplasm.

Aspartic Acid Endopeptidases↗

PubMed2005

Proprotein convertase PC3 is not a transmembrane protein.

Proprotein convertase PC3 (also known as PC1) is an endopeptidase involved in proteolytic processing of peptide hormone precursors in granules of the regulated secretory pathway of endocrine cells. Lacking any extended hydrophobic segments, PC3 was considered to be a secretory protein only peripherally attached to the granule membrane. Recently, evidence has been presented that PC3 is a transmembrane protein with a 115-residue cytoplasmic domain and a membrane-spanning segment containing eight charged amino acids [Arnaoutova, I., et al. (2003) Biochemistry 42, 10445-10455]. Here, we analyzed the membrane topology of PC3 and of a PC3 construct containing a conventional transmembrane segment of 19 leucines. Alkaline extraction was performed to assess membrane integration. Exposure to the cytosol or to the ER lumen was tested by addition of C-terminal tags for phosphorylation or glycosylation, respectively. Protease sensitivity was assayed in permeabilized cells. The results show that the C-terminus of PC3 is translocated across the endoplasmic reticulum membrane. Furthermore, the proposed transmembrane segment of PC3 and a similar one of carboxypeptidase E did not stop polypeptide translocation when inserted into a stop-transfer tester construct. PC3 is thus not a transmembrane protein. These results have implications for the mechanism of granule sorting of PC3 as well as for the topology of PC2 and carboxypeptidase E, which have been reported to span the lipid membrane by homologous charged sequences.

Amino Acid Sequence↗

PubMed2006

The proprotein convertases and their implication in sterol and/or lipid metabolism.

The proprotein convertases represent a family of nine proteinases, comprising seven basic amino acid-specific subtilisin-like serine proteinases related to yeast kexin, known as PC1/3, PC2, furin, PC4, PC5/6, PACE4 and PC7, and two other subtilases that cleave at non-basic residues, called SKI-1/S1P and NARC-1/PCSK9. The present review concentrates on the regulatory role played by some of these convertases in cholesterol and lipid metabolism. Thus, PC5/6, PACE4 and Furin upregulate high-density lipoprotein (HDL) levels via the inactivation of endothelial and lipoprotein lipases. The SKI-1/S1P-directed cleavage of membrane-bound transcription factors known as sterol regulatory element binding proteins (SREBP-1 and SREBP-2) results in upregulation of the synthesis of sterols, lipids and the LDL receptor (LDLR). Finally, PCSK9 downregulates the protein levels of the LDLR by enhancement of its intracellular metabolic pathway in subcellular acidic compartments.

Amino Acid Sequence↗

PubMed2005

Lipopolysaccharide mediated regulation of neuroendocrine associated proprotein convertases and neuropeptide precursor processing in the rat spleen.

Within the secretory pathway, the family of proprotein convertases cleave inactive precursors at paired basic residues to generate a myriad of biologically active peptides. Within the PC family, PC1/3 and PC2 are well known for their preferential expression within neuroendocrine cells. However, various data now indicate their potential expression in immune cells. The aim of our study was two fold: (1) survey PC expression in immune tissues, with emphasis on PC1/3 and PC2 and (2) examine PC expression under conditions that mimic an infectious state using lipopolysaccharide, known to activate immune cells via toll-like receptors. Spatial and temporal analyses of tissues from control and lipopolysaccharide treated rats were carried out using in situ hybridization histochemistry, Northern blot, mass spectrometry and antibacterial assays. Our tissue survey showed the basal expression of all PCs in the lymph nodes, thymus and spleen including PC1/3 and PC2. Focusing on the spleen, basal expression of PC1/3 was seen in the red pulp/marginal zone areas, suggesting expression within macrophages. Lipopolysaccharide treatment produced significant changes in PC1/3 expression and notably an induction in B lymphocytes within germinal centers. Similarly, PC2, which was undetectable in control spleens, was induced in germinal centers following lipopolysaccharide treatment. The PC1/3 and PC2 substrate proenkephalin was also induced following lipopolysaccharide treatment in the marginal zone, where PC1/3 expression was also found. Mass spectrometry analysis of spleen extracts demonstrated the presence of the antibacterial peptide enkelytin. Our studies confirmed that PC1/3 and PC2 expression was not restricted to neurons and endocrine cells, but was also found under basal conditions in both macrophage and lymphocytes. Additionally, plasticity of PC expression in immune cells was observed under conditions that mimic pathogen-like infections, suggesting a mechanistic link through Toll-like receptors. Collectively, these data clearly implicate PCs in immune responses, both innate and acquired.

Animals↗

PubMed2001

Single-nucleotide polymorphisms of the proprotein convertase subtilisin/ kexin type 5 (PCSK5) gene.

The proprotein convertase. subtilisin/kexin type 5, or PCSK5, mediates post-translational endoproteolytic processing for several integrin alpha subunits. We identified two silent single-nucleotide polymorphisms (SNPs) in PCSK5, which were found to vary in frequency across ethnic groups. The identification of these amplification primers and SNPs provides tools to investigate PCSK5 for association with inflammatory or vascular phenotypes.

Alleles↗

PubMed2000

A predicted alpha -helix mediates targeting of the proprotein convertase PC1 to the regulated secretory pathway.

The proprotein convertase PC1 is a protease whose activity is largely confined to the dense core secretory granules of neuroendocrine cells. Efficient processing of PC1 substrates in granules requires a mechanism that will both limit the activity of the enzyme to these organelles and promote its targeting to the nascent secretory granules. In the current study, we provide evidence that targeting of PC1 to secretory granules is mediated by alpha-helical structures in its C-terminal tail and, at least in part, is dependent on interactions with specific components of the secretory granule membrane.

Amino Acid Sequence↗

PubMed2000

Dynamic palmitoylation of lymphoma proprotein convertase prolongs its half-life, but is not essential for trans-Golgi network localization.

Proprotein convertases are responsible for the endoproteolytic activation of proproteins in the secretory pathway. The most recently discovered member of this family, lymphoma proprotein convertase (LPC), is a type-I transmembrane protein. Previously, we have demonstrated that its cytoplasmic tail is palmitoylated. In this study, we have identified the two most proximal cysteine residues in the cytoplasmic tail as palmitoylation sites. Substitution of either cysteine residue by alanine interfered with palmitoylation of the other. Palmitoylation of LPC was found to be sensitive to the protein palmitoyltransferase inhibitor tunicamycin but not cerulenin. It was also insensitive to the drugs brefeldin A, monensin and cycloheximide, indicating that the modification occurs in a late exocytic or endocytic compartment. Turnover of palmitoylated LPC is significantly faster (t(1/2) approximately 50 min) than that of the LPC polypeptide backbone (t(1/2) approximately 3 h), suggesting that palmitoylation is reversible. Abrogation of palmitoylation reduced the half-life of the LPC protein, but did not affect steady-state localization of LPC in the trans-Golgi network. Finally, LPC could not be detected in detergent-resistant membrane rafts. Taken together, these results suggest that dynamic palmitoylation of LPC is important for stability, but does not function as a dominant trafficking signal.

Amino Acid Substitution↗

PubMed2000

The proprotein convertases furin and PACE4 play a significant role in tumor progression.

Processing of latent precursor proteins by proprotein convertases (PCs) into their biologically active products is a common mechanism required for many important biologic functions. This process is tightly regulated, leading to the generation of active peptides and proteins including neuropeptides and polypeptide hormones, protein tyrosine phosphatases, growth factors and their receptors, and enzymes including matrix metalloproteases (MMPs). These processing reactions occurs at pairs of basic amino acids. Within the past several years, a novel family of Ca(2+)-dependent serine proteases has been identified, all of which possess homology to the endoproteases subtilisin (bacteria) and kexin (yeast). This family of PCs is currently comprised of fewer than a dozen members, known as furin/paired basic amino-acid-cleaving enzyme (PACE), PC1/PC3, PC2, PC4, PACE4, PC5/PC6, and PC7/PC8/lymphoma proprotein convertase. They share a high degree of amino-acid identity of 50-75% within their catalytic domains. Despite the relatively high degree of homology in the PC family, only PACE4 and furin localize to the same chromosome: mouse chromosome 7 and human chromosome 15. Recent reports have supported a possible functional role for PCs in tumorigenesis. For instance, convertases have been shown to be expressed in various tumor lines and human primary tumors. Furin and PACE4 process stromelysin 3 (MMP-11 or Str-3), an MMP involved in tumor invasion, into its mature, active form. Similarly, a growing family of MMPs, known as membrane-type metalloproteinases (MT-MMPs), and growth factors and adhesion molecules such as E-cadherin show similar amino-acid motifs and thus could be activated by furin and PACE4. These data, taken together with the high expression levels of PACE4 in 50% of murine chemically induced spindle cell tumors, confer to PACE4 and possibly other PCs a possible functional role in the activation of MMPs and consequently in tumor cell invasion and tumor progression. This was further supported by the remarkable enhancement in the invasive ability of the PACE4-transfected murine tumor cell lines. Mol. Carcinog. 28:63-69, 2000.

Animals↗

PubMed2003

Epigenetic regulation of proprotein convertase PACE4 gene expression in human ovarian cancer cells.

Proprotein convertases (PC) are a family of serine endoproteases that play important roles in regulating cell function by converting proproteins to biologically active molecules. Several lines of evidence suggest that overexpression of PCs contributes to tumor formation and progression in various types of cancer. In this study, we examined PC expression in six normal ovarian surface epithelium (OSE) cultures, nine primary ovarian cancer (OC) cultures, and three established OC cell lines (Hey, HeyC2, and OCC-1). Our results show that furin and PC7 expression in OC cells was comparable to that in normal OSE. However, PACE4 expression was greatly reduced in all OC samples studied. PACE4 promoter activity was measured in HeyC2 and OCC-1 cells using transiently transfected luciferase reporter plasmids. Both cell lines supported PACE4 promoter activity, showing that the transcription factors critical for PACE4 expression are present in OC cells. The observation that established OC cell lines have reduced PACE4 expression, but maintained the ability to support PACE4 promoter activity, led to the hypothesis that reduced expression may be due to epigenetic modification of the PACE4 gene, such as DNA methylation and histone deacetylation. Methylation analysis of 79 CpG dinucleotides within the PACE4 promoter and exon I (-196/+340) revealed that the percentage of methylated cytosine nucleotides was 8-9% in normal OSE, but 58-93% in OC cells. Treatment with the demethylating agent 5-aza-2'-deoxycytidine and/or the histone deacetylase inhibitor trichostatin A greatly increased PACE4 expression in OC cells. These data suggest that the reduction of PACE4 expression in OC cells is caused, in part, by DNA hypermethylation and histone deacetylation.

DNA Methylation↗

PubMed2003

Functional regulation of semaphorin receptors by proprotein convertases.

PLEXIN genes encode receptors for secreted and membrane-bound semaphorins. It was proposed that the extracellular domain of plexins acts as an inhibitory moiety, preventing receptor activation. Here we show that plexin-B1 and plexin-B2 undergo proteolytic processing in their extracellular portion, thereby converting single-chain precursors into non-disulfide-linked, heterodimeric receptors. We demonstrate that plexin processing is mediated by subtilisin-like proprotein convertases, by inhibition with alpha1-antitrypsin Portland, and by mutagenesis of the substrate-cleavage sites. We provide evidence indicating that proprotein convertases cleave plexins in a post-Golgi compartment and, likely, at the cell surface. In addition, we find that both cell surface targeting and proteolytic processing of plexin-B1 depend on protein-protein interaction motifs in the cytoplasmic domain of the receptor. We then show that proteolytic conversion of plexin-B1 into a heterodimeric receptor greatly increases the binding and the functional response to its specific ligand semaphorin 4D/CD100. Thus, we conclude that cleavage by proprotein convertases is a novel regulatory step for semaphorin receptors localized at the cell surface.

3T3 Cells↗

PubMed2004

Statins upregulate PCSK9, the gene encoding the proprotein convertase neural apoptosis-regulated convertase-1 implicated in familial hypercholesterolemia.

OBJECTIVE: Neural apoptosis-regulated convertase (NARC)-1 is the newest member of the proprotein convertase family implicated in the cleavage of a variety of protein precursors. The NARC-1 gene, PCSK9, has been identified recently as the third locus implicated in autosomal dominant hypercholesterolemia (ADH). The 2 other known genes implicated in ADH encode the low-density lipoprotein receptor and apolipoprotein B. As an approach toward the elucidation of the physiological role(s) of NARC-1, we studied its transcriptional regulation. METHODS AND RESULTS: Using quantitative RT-PCR, we assessed NARC-1 regulation under conditions known to regulate genes involved in cholesterol metabolism in HepG2 cells and in human primary hepatocytes. We found that NARC-1 expression was strongly induced by statins in a dose-dependent manner and that this induction was efficiently reversed by mevalonate. NARC-1 mRNA level was increased by cholesterol depletion but insensitive to liver X receptor activation. Human, mouse, and rat PCSK9 promoters contain 2 typical conserved motifs for cholesterol regulation: a sterol regulatory element (SRE) and an Sp1 site. CONCLUSIONS: PCSK9 regulation is typical of that of the genes implicated in lipoprotein metabolism. In vivo, PCSK9 is probably a target of SRE-binding protein (SREBP)-2.

Alitretinoin↗