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Neutrophil myeloperoxidase/H2O2/chloride system is a key mechanism to control pathogen infection. This enzyme, myeloperoxidase, plays a pivotal role in the arsenal of azurophilic granules that are released through degranulation upon neutrophil activation, which trigger local hypochlorous acid production. Myeloperoxidase gene encodes a protein precursor named promyeloperoxidase that arbors a propeptide that gets cleaved later during secretory routing in post-endoplasmic reticulum compartments. Although evidence suggested that this processing event was performed by one or different enzymes from the proprotein convertases family, the identity of this enzyme was never investigated. In this work, the naturally producing myeloperoxidase promyelocytic cell line HL-60 was used to investigate promyeloperoxidase cleavage during granulocytic differentiation in response to proprotein convertase inhibitors decanoyl-RVKR-chloromethylketone and hexa-d-arginine. Stable PC knockdown of endogenously expressed proprotein convertases, furin and PC7, was achieved using lentiviral delivery of shRNAs. None of the knockdown cell line could reproduce the effect of the pan-proprotein convertases inhibitor decanoyl-RVKR-chloromethylketone that accumulated intracellular promyeloperoxidase stores in HL-60 cells, therefore illustrating that both furin and PC7 redundantly process this proprotein. [ABSTRACT FROM AUTHOR]

The locus of the human proprotein convertase subtilisin–kexin type‐7 (PC7) gene (PCSK7) is on chromosome 11q23.3 close to the gene cluster APOA5/APOA4/APOC3/APOA1, a region implicated in the regulation of lipoprotein metabolism. A GWAS reported the association of PCSK7 SNPs with plasma triglyceride (TG), and exome sequencing of African Americans revealed the association of a low‐frequency coding variant of PC7 (R504H; SNP rs142953140) with a ~ 30% TG reduction. Another PCSK7 SNP rs508487 is in linkage disequilibrium with a promoter variant of the liver‐derived apolipoprotein A‐V (apoA‐V), an indirect activator of the lipoprotein lipase (LpL), and is associated with elevated TG levels. We thus hypothesized that PC7 regulates the levels/activity of apoA‐V. Studies in the human hepatic cell line HuH7 revealed that wild‐type (WT) PC7 and its endoplasmic reticulum (ER)‐retained forms bind to and enhance the degradation of human apoA‐V in acidic lysosomes in a nonenzymatic fashion. PC7‐induced degradation of apoA‐V is inhibited by bafilomycin A1 and the alkalinizing agents: chloroquine and NH4Cl. Thus, the PC7‐induced apoA‐V degradation implicates an ER‐lysosomal communication inhibited by bafilomycin A1. In vitro, the natural R504H mutant enhances PC7 Ser505 phosphorylation at the structurally exposed Ser‐X‐Glu507 motif recognized by the secretory kinase Fam20C. Co‐expression of the phosphomimetic PC7‐S505E with apoA‐V resulted in lower degradation compared to WT, suggesting that Ser505 phosphorylation of PC7 lowers TG levels via reduced apoA‐V degradation. In agreement, in Pcsk7−/− mice fed high‐fat diet, plasma apoA‐V levels and adipocyte LpL activity are increased, providing an in vivo mechanistic link for a role of liver PC7 in enhanced TG storage in adipocytes. [ABSTRACT FROM AUTHOR]

Summary: Furin trafficking, and that of related proprotein convertases (PCs), may regulate which substrates are accessible for endoproteolysis, but tools to directly test this hypothesis have been lacking. Here, we develop targeted biosensors that indicate Furin activity in endosomes is 10-fold less inhibited by decanoyl-RVKR-chloromethylketone and enriched >3-fold in endosomes compared to the trans-Golgi network (TGN). Endogenous PC7, which resists this inhibitor, was active in distinct vesicles. Only overexpressed PC7 activity reached the cell surface, endosomes, and the TGN. A PLC motif in the cytosolic tail of PC7 was dispensable for endosomal activity, but it was specifically required for TGN recycling and to rescue proActivin-A cleavage in Furin-depleted B16F1 melanoma cells. In sharp contrast, PC7 complemented Furin in cleaving Notch1 independently of PLC-mediated TGN access. Our study provides a proof in principle that compartment-specific biosensors can be used to gain insight into the regulation of PC trafficking and to map the tropism of PC-specific inhibitors.

Abstract Purpose To analyze the surgical outcomes and complication rates in a group of patients with refractory glaucoma who underwent simultaneous pars plana vitrectomy (PPV) and PC7 or PC8 Ahmed glaucoma valve (AGV) implantation. Methods Retrospective case series study of 10 eyes in 9 patients with secondary glaucoma, refractory to other treatment, who underwent 23G-PPV and implantation of PC7 or PC8 AGV between 2012 and 2014. Study variables were postoperative BCVA, IOP and the number of glaucoma medications, which were evaluated preoperatively and at 1 day, 1 week and 1, 3, 6, and 12 months after the surgical intervention. Absolute success was defined as IOP less than 21 mmHg in the absence of any medication and qualified success if medication was needed to control IOP under 21 mmHg. Results The average follow-up was 10.2 ± 2.89 months. Postoperative IOP levels decreased in all cases in comparison with preoperative values (p < 0.05). Absolute success rate was 60%, reaching 100% in terms of qualified success. Kaplan–Meier survival analysis showed 60% absolute success at 12 months. Changes in postoperative BCVA were not statistically significant in comparison with preoperative data. Early postoperative complications were athalamia, hyphema, and retinal detachment; late complications were pars plana clip extrusion and cystic bleb. Conclusions PC7 and PC8 Ahmed valve implantation via pars plana is a safe and useful option in patients with secondary refractory glaucoma who are either candidates for PPV or have been previously vitrectomized. [ABSTRACT FROM AUTHOR]

Proprotein Convertase 7 (PC7) is a Furin-like endoprotease that cleaves precursor proteins at basic amino acids. PC7 is concentrated in the trans -Golgi network (TGN) but it shuttles between the plasma membrane and the TGN depending on sequences in the cytoplasmic tail. A short region containing a three amino acids motif, P 724 -L 725 -C 726 , is essential and sufficient for internalization of PC7 but not for TGN localization, which requires the additional presence of the juxtamembrane region. In this study we have investigated the contribution of a cluster of basic amino acids and two reversibly palmitoylated cysteine residues to endocytic trafficking. Stable cell lines overexpressing chimeric proteins (CD25 and CD46) containing the cytoplasmic domain of PC7 in which the basic cluster alone or together with both palmitoylated cysteines are mutated showed enhanced surface expression as demonstrated by immunofluorescence experiments and surface biotinylation. The mutant proteins no longer recycled to the TGN in antibody uptake experiments and accumulated in an endosomal compartment. Recycling of wild type PC7 to the TGN is blocked by nocodazole, suggesting that PC7 shuttles to the TGN via late endosomes, similar to Furin. Unlike furin, however, PC7 was found to recycle to a region within the TGN, which is deficient in sialyltransferase, as shown by resialylation experiments. In conclusion, a novel motif, composed of a basic amino acid cluster and two palmitoylated cysteines are essential for TGN localization and endocytic trafficking. [ABSTRACT FROM AUTHOR]

Endoproteases in the secretory pathway process pro-cholecystokinin (CCK) into the biologically active forms found in the tissues that express CCK mRNA. Thus far, the endoproteases involved in CCK processing include cathepsin L and the prohormone convertases (PC) 1, 2, and 5. This study finds that PC7 is also critical for normal production of CCK in specific areas of the brain. Loss of PC7 results in decreased levels of CCK in more brain regions than any other endoprotease studied to date. Substantial decreases in brain levels of CCK are found in the prefrontal, frontal, parietal-insular-pyriform, and temporal cortex, caudate-putamen, basal forebrain, thalamus, hippocampus, septum, and medulla of PC7 knock-out (KO) mice. A tissue-specific sexual dimorphism of PC7 activity was also identified. This is the first report that loss of PC7 alters levels of a neuropeptide in the brain. This loss of PC7 and CCK may independently contribute to the decrease in Brain Derived Neurotrophic Factor production and be partially responsible for the learning and memory defects observed in mice that lack PC7. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor (VEGF)-D is a secreted glycoprotein that induces anglogenesis and lymphangiogenesis. It consists of a central domain, containing binding sites for VEGF receptor-2 (VEGFR-2) and VEGFR-3, and N- and C-terminal propeptides. It is secreted from the cell as homodimers of the full-length form that can be proteolytically processed to remove the propeptides. It was recently shown, using adenoviral gene delivery, that fully processed VEGF-D induces angiogenesis in vivo, whereas full-length VEGF-D does not. To better understand these observations, we monitored the effect of VEGF-D processing on receptor binding using a full-length VEGF-D mutant that cannot be processed. This mutant binds VEGFR-2, the receptor signaling for angiogenesis, with ∼17,000-fold lower affinity than mature VEGF-D, indicating the importance of processing for interaction with this receptor. Further, we show that members of the proprotein convertase (PC) family of proteases promote VEGF-D processing, which facilitates the VEGF-D/VEGFR-2 interaction. The PCs furin and PC5 promote cleavage of both propeptides, whereas PC7 promotes cleavage of the C-terminal propeptide only. The finding that PCs promote activation of VEGF-D and other proteins with roles in cancer such as matrix metalloproteinases, emphasizes the importance of these enzymes as potential regulators of tumor progress. ion and metastasis. [ABSTRACT FROM AUTHOR]

Abstract: Processing of precursor proteins by the proprotein convertases is thought to occur mainly in the trans-Golgi network or post-Golgi compartments. Such cleavage is inhibited by the prosegment of the convertases. During our studies of the use of the inhibitory prosegment of PC1, we noticed that a construct containing the prosegment fused to the C-terminal secretory granule sorting domain was cleaved in the endoplasmic reticulum (ER) at a pair of basic residues, best recognized by furin and PC7. This was further confirmed when this construct was fused at the C-terminus with a KDEL ER-retention signal. This suggests that the convertases could cleave some substrates within the ER, possibly by displacing the inhibitory prosegment associated with them. [Copyright &y& Elsevier]

Tumor necrosis factor-α converting enzyme (TACE or ADAM17) is a member of the ADAM (a disintegrin and metalloproteinase) family of type I membrane proteins and mediates the ectodomain shedding of various membrane-anchored signaling and adhesion proteins. TACE is synthesized as an inactive zymogen, which is subsequently proteolytically processed to the catalytically active form. We have identified the proprotein-convertases PC7 and furin to be involved in maturation of TACE. This maturation is negatively influenced by the phorbol ester phorbol-12-myristate-13-acetate (PMA), which decreases the cellular amount of the mature form of TACE in PMA-treated HEK293 and SH-SY5Y cells. Furthermore, we found that stimulation of protein kinase C or protein kinase A signaling pathways did not influence long-term degradation of mature TACE. Interestingly, PMA treatment of furin-deficient LoVo cells did not affect the degradation of mature TACE. By examination of furin reconstituted LoVo cells we were able to exclude the possibility that PMA modulates furin activity. Moreover, the PMA dependent decrease of the mature enzyme form is specific for TACE, as the amount of mature ADAM10 was unaffected in PMA-treated HEK293 and SH-SY5Y cells. Our results indicate that the activation of TACE by the proprotein-convertases PC7 and furin is very similar to the maturation of ADAM10 although there is a significant difference in the cellular stability of the mature enzyme forms after phorbol ester treatment. [ABSTRACT FROM AUTHOR]

Our knowledge of prohormone and proneuropeptide processing and its relationship to the secretory pathway has advanced significantly in the last five years. The recent discovery of the prohormone convertase family of proteolytic enzymes has provided new candidates for the prohormone and proneuropeptide convertases. The increasing appreciation of the role of proteolysis in diverse cellular processes has also brought the prohormone processing field closer to the fields of growth factor processing, the role of host proteases in viral and bacterial pathogenesis and toxicity, control of the cell cycle, inflammation, and apoptosis. The last five years have been very productive, but the most interesting questions are still unanswered. Which enzymes are actually responsible for prohormone cleavages in specific tissues? What structural features of the prohormones determine where it will be processed or how it is recognized as secretory material by the sorting machinery? How is tissue-specific processing determined and regulated? The availability of protease knockout mice and a more detailed understanding of the complex biosynthetic activation of these enzymes will provide at least some of the answers. [ABSTRACT FROM AUTHOR]

Proprotein convertases (PC), a family of serine proteases, process cancer-related substrates such as growth factors, growth factor receptors, cell adhesion molecules, metalloproteinases, etc. HIF-1α is a major transcription factor involved in tumorigenesis by sensing intratumoral hypoxia. Furin (PCSK3) is one of the numerous target genes regulated by HIF-1α transactivation and its distribution into endosomal compartments and onto the cell surface can be triggered by hypoxia via HIF-1α. siRNAs to knockdown PCs were transfected into cells alone or in combination with different drug treatments. Protein and RNA expression levels were analyzed by Western blotting or RT-PCR, respectively. PC7 (PCSK7) and furin siRNAs upregulated HIF-1α protein under normoxic condition to a level similar to that obtained by cobalt chloride treatment, eventually leading to activation of VEGF-A synthesis in two human head and neck squamous cell carcinoma cell lines. The unchanged levels of HIF-1α mRNA expression under siRNA treatment and the additive HIF-1α induction of PC siRNAs and either cobalt chloride or the 26S ribosome inhibitor, MG-132, suggested a post-transcriptional PC-mediated regulation. Furthermore, cycloheximide chase showed that PC7/furin siRNA regulation occurred at the level of HIF-1α translation. A specific IGF-1R signaling inhibitor was able to attenuate the PC siRNA induction of HIF-1α, suggesting the involvement of the IGF-1R pathway. Thus, the data show that PCs regulate HIF-1α. Furin and PC7 siRNAs induced HIF-1α protein by increasing its translation, resulting in upregulation of VEGF-A. This finding may provide insight into intricate PC functions that seem to be independent from their substrate-processing activity., (© 2014 Wiley Periodicals, Inc.)

Proprotein convertase subtilisin/kexin (PCSK) enzymes convert proproteins into bioactive end products. Although other PCSK enzymes are known to be essential for biological processes ranging from cholesterol metabolism to host defense, the in vivo importance of the evolutionarily ancient PCSK7 has remained enigmatic. Here, we quantified the expressions of all pcsk genes during the 1st week of fish development and in several tissues. pcsk7 expression was ubiquitous and evident already during the early development. To compare mammalian and zebrafish PCSK7, we prepared homology models, which demonstrated remarkable structural conservation. When the PCSK7 function in developing larvae was inhibited, we found that PCSK7-deficient fish have defects in various organs, including the brain, eye, and otic vesicle, and these result in mortality within 7 days postfertilization. A genome-wide analysis of PCSK7-dependent gene expression showed that, in addition to developmental processes, several immune system-related pathways are also regulated by PCSK7. Specifically, the PCSK7 contributed to the mRNA expression and proteolytic cleavage of the cytokine TGFβ1a. Consequently, tgfβ1a morphant fish displayed phenotypical similarities with pcsk7 morphants, underscoring the importance of this cytokine in the zebrafish development. Targeting PCSK activity has emerged as a strategy for treating human diseases. Our results suggest that inhibiting PCSK7 might interfere with normal vertebrate development.