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Cystic Fibrosis (CF) is present due to mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, the most frequent variant being p.phe508del. The CFTR protein is a chloride (Cl-) channel which is defective and almost absent of cell membranes when the p.Phe508del mutation is present. The p.Phe508del-CFTR protein is retained in the endoplasmic reticulum (ER) and together with inflammation and infection triggers the Unfolded Protein Response (UPR). During the UPR, the Activating Transcription Factor 6 (ATF6) is activated with cleavage and then decreases the expression of p.Phe508del-CFTR. We have previously shown that the inhibition of the activation of ATF6 alleviates the p.Phe508del-CFTR defects in cells overexpressing the mutated protein. In the present paper, our aim was to inhibit the cleavage of ATF6, and thus its activation in a human bronchial cell line with endogenous p.Phe508del-CFTR expression and in bronchial cells from patients, to be more relevant to CF. This was achieved by inhibiting the protease MBTP1 which is responsible for the cleavage of ATF6. We show here that this inhibition leads to increased mRNA and p.Phe508del-CFTR expression and, consequently, to increased Cl-efflux. We also explain the mechanisms linked to these increases with the modulation of genes when MBTP1 is inhibited. Indeed, RT-qPCR assays show that genes such as HSPA1B, CEBPB, VIMP, PFND2, MAPK8, XBP1, INSIG1, and CALR are modulated. In conclusion, we show that the inhibition of MBTP1 has a beneficial effect in relevant models to CF and that this is due to the modulation of genes involved in the disease.

The cleavage of viral surface proteins by furin is associated with some viruses' high virulence and infectivity. The human papillomavirus (HPV) requires the proteolytic processing of its capsid proteins for activation before entry. Variability in reactivity with furin and other proprotein convertases (PCs) among HPV types was investigated. HPV16, the most prevalent and carcinogenic HPV type, reacted with PCs with the broadest selectivity compared to other types in reactions of pseudoviral particles with the recombinant PCs, furin, PC4, PC5, PACE4, and PC7. Proteolytic preactivation was assessed using a well-established entry assay into PC-inhibited cells based on the green fluorescent protein as a reporter. The inhibition of the target cell PC activity with serpin-based PC-selective inhibitors also showed a diversity of PC selectivity among HPV types. HPV16 reacted with furin at the highest rate compared to the other types in time-dependent preactivation reactions and produced the highest entry values standardized to pseudoviral particle concentration. The predominant expression of furin in keratinocytes and the high reactivity of HPV16 with this enzyme highlight the importance of selectively targeting furin as a potential antiviral therapeutic approach.

Proprotein Convertases (PCs) are serine endoproteases that regulate the homeostasis of protein substrates in the cell. The PCs family counts 9 members-PC1/3, PC2, PC4, PACE4, PC5/6, PC7, Furin, SKI-1/S1P, and PCSK9. The first seven PCs are known as Basic Proprotein Convertases due to their propensity to cleave after polybasic clusters. SKI-1/S1P requires the additional presence of hydrophobic residues for processing, whereas PCSK9 is catalytically dead after autoactivation and exerts its functions using mechanisms alternative to direct cleavage. All PCs traffic through the canonical secretory pathway, reaching different compartments where the various substrates reside. Despite PCs members do not share the same subcellular localization, most of the cellular organelles count one or more Proprotein Convertases, including ER, Golgi stack, endosomes, secretory granules, and plasma membranes. The widespread expression of these enzymes at the systemic level speaks for their importance in the homeostasis of a large number of biological functions. Among others, PCs cleave precursors of hormones and growth factors and activate receptors and transcription factors. Notably, dysregulation of the enzymatic activity of Proprotein Convertases is associated to major human pathologies, such as cardiovascular diseases, cancer, diabetes, infections, inflammation, autoimmunity diseases, and Parkinson. In the current COVID-19 pandemic, Furin has further attracted the attention as a key player for conferring high pathogenicity to SARS-CoV-2. Here, we review the Proprotein Convertases family and their most important substrates along the secretory pathway. Knowledge about the complex functions of PCs is important to identify potential drug strategies targeting this class of enzymes., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Proprotein convertases (PCs) are involved in the pathogenesis of various diseases, making them promising drug targets. Most assays for PCs have been performed with few standard substrates, regardless of differences in cleavage efficiencies. Derived from studies on substrate-analogue inhibitors, 11 novel substrates were synthesized and characterized with five PCs. H-Arg-Arg-Tle-Lys-Arg-AMC is the most efficiently cleaved furin substrate based on its k cat /K M value. Due to its higher k cat value, acetyl-Arg-Arg-Tle-Arg-Arg-AMC was selected for further measurements to demonstrate the benefit of this improved substrate. Compared to our standard conditions, its use allowed a 10-fold reduction of the furin concentration, which enabled K i value determinations of previously described tight-binding inhibitors under classical conditions. Under these circumstances, a slow-binding behavior was observed for the first time with inhibitor MI-1148. In addition to furin, four additional PCs were used to characterize these substrates. The most efficiently cleaved PC1/3 substrate was acetyl-Arg-Arg-Arg-Tle-Lys-Arg-AMC. The highest k cat /K M values for PC2 and PC7 were found for the N-terminally unprotected analogue of this substrate, although other substrates possess higher k cat values. The highest efficiency for PC5/6A was observed for the substrate acetyl-Arg-Arg-Tle-Lys-Arg-AMC. In summary, we have identified new substrates for furin, PC1/3, PC2, and PC7 suitable for improved enzyme-kinetic measurements., Competing Interests: Declaration of competing interest None., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Serum lipids, including total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-c), increase during pregnancy. Serum Proprotein Convertase Subtilisin Kexin 9 (PCSK9) is a vital regulator in lipoprotein metabolism. Circulating PCSK9 downregulates the LDL receptor on the surface of liver cells inhibiting clearance of LDL-c., Objective: To determine the influence of weeks of pregnancy and obesity on circulating levels of essential lipid lipoproteins and PCSK9 in women with normal, uncomplicated pregnancies and deliveries., Methods: We performed a comprehensive lipid and lipoprotein profile during each trimester of pregnancy in 70 mostly Caucasian women with uncomplicated normal pregnancies and deliveries. Based on their first trimester BMI, we placed them into one of three categories: (<25 kg/m 2 n=23, 25-30 kg/m 2 n=25, or >30 n=22) kg/m 2 . Cholesterol, triglycerides, LDL cholesterol (LDL-c), non-HDL particles, and lipoprotein(a) were measured by spectrophotometry, ion mobility, and immunoturbidimetric assays. Elisa assay determined PCSK9 (active and total). Homeostatic Model Assessment (HOMA-IR) assessed insulin resistance in the second and third trimesters of pregnancy., Results: Total and active PCSK9, LDL-c, and nonHDL particle concentrations were higher than reported for non-pregnant normal values, increased after the first trimester of pregnancy, and were highest from mid-gestation to the last trimester of pregnancy in the overweight and the obese., Conclusion: PCSK9 levels rise as normal pregnancy progresses. Levels are higher in persons who are obese, even after adjustment for insulin resistance. Defining normal PCSK9 levels during pregnancy must adjust for gestational age and BMI., Competing Interests: Declaration of Competing Interest We confirm no known conflicts of interest associated with this publication, and there has been no financial support for this work that could have influenced the outcome. Dr Wren is Medical Director at Quest and Dr Lopez has a registered patent for the PCSK9 assay. Analysis and reporting was blind to the clinical data. We also confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that all have approved the order of our authors., (Copyright © 2022. Published by Elsevier Inc.)

Cardiac looping and trabeculation are key processes during cardiac chamber maturation. However, the underlying mechanisms remain incompletely understood. Here, we report the isolation, cloning and characterization of the proprotein convertase furina from the cardiovascular mutant loft in zebrafish. loft is an ethylnitrosourea-induced mutant and has evident defects in the cardiac outflow tract, heart looping and trabeculation, the craniofacial region and pharyngeal arch arteries. Positional cloning revealed that furina mRNA was barely detectable in loft mutants, and loft failed to complement the TALEN-induced furina mutant pku338, confirming that furina is responsible for the loft mutant phenotypes. Mechanistic studies demonstrated that Notch reporter Tg(tp1:mCherry) signals were largely eliminated in mutant hearts, and overexpression of the Notch intracellular domain partially rescued the mutant phenotypes, probably due to the lack of Furina-mediated cleavage processing of Notch1b proteins, the only Notch receptor expressed in the heart. Together, our data suggest a potential post-translational modification of Notch1b proteins via the proprotein convertase Furina in the heart, and unveil the function of the Furina-Notch1b axis in cardiac looping and trabeculation in zebrafish, and possibly in other organisms., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Inhibition of the binding of enveloped viruses surface glycoproteins to host cell receptor(s) is a major target of vaccines and constitutes an efficient strategy to block viral entry and infection of various host cells and tissues. Cellular entry usually requires the fusion of the viral envelope with host plasma membranes. Such entry mechanism is often preceded by "priming" and/or "activation" steps requiring limited proteolysis of the viral surface glycoprotein to expose a fusogenic domain for efficient membrane juxtapositions. The 9-membered family of Proprotein Convertases related to Subtilisin/Kexin (PCSK) serine proteases (PC1, PC2, Furin, PC4, PC5, PACE4, PC7, SKI-1/S1P, and PCSK9) participate in post-translational cleavages and/or regulation of multiple secretory proteins. The type-I membrane-bound Furin and SKI-1/S1P are the major convertases responsible for the processing of surface glycoproteins of enveloped viruses. Stefan Kunz has considerably contributed to define the role of SKI-1/S1P in the activation of arenaviruses causing hemorrhagic fever. Furin was recently implicated in the activation of the spike S-protein of SARS-CoV-2 and Furin-inhibitors are being tested as antivirals in COVID-19. Other members of the PCSK-family are also implicated in some viral infections, such as PCSK9 in Dengue. Herein, we summarize the various functions of the PCSKs and present arguments whereby their inhibition could represent a powerful arsenal to limit viral infections causing the present and future pandemics.

A wide variety of viruses exploit furin and other proprotein convertases (PCs) of the constitutive protein secretion pathway in order to regulate their cell entry mechanism and infectivity. Surface proteins of enveloped, as well as non-enveloped, viruses become processed by these proteases intracellularly during morphogenesis or extracellularly after egress and during entry in order to produce mature virions activated for infection. Although viruses also take advantage of other proteases, it is when some viruses become reactive with PCs that they may develop high pathogenicity. Besides reacting with furin, some viruses may also react with the PCs of the other specificity group constituted by PC4/PC5/PACE4/PC7. The targeting of PCs for inhibition may result in a useful strategy to treat infections with some highly pathogenic viruses. A wide variety of PC inhibitors have been developed and tested for their antiviral activity in cell-based assays.

Purpose: Proprotein convertase subtilisin/kexin type 4 (PCSK4, a 54-kDa protease) is expressed in the plasma membrane of the acrosome in human spermatozoa. It plays a critical role in penetrating the zona pellucida. Synthesis of human anti-PCSK4 might be important for novel male immunocontraception., Materials and Methods: We used semen from adult males as the source of antigen (acrosomal PCSK4). Isolation and antigen characterization were done by immunohistochemistry followed by electrophoresis. Purification of PCSK4 was done by the electroelution method, followed by immunization with an animal model ( Oryctolagus cuniculus ). Antibody was collected, purified, and tested by using Western blot and dot blot. Antibody in vitro potential testing was performed on human spermatozoa by laser scanning microscopy with rhodamine stain under a light microscope and on rat spermatozoa., Results: Human anti-PCSK4 bound with PCSK4 on the head of human spermatozoa and could interfere with rat spermatozoa activity to penetrate the oocyte., Conclusions: The result of this study can be used as a basis to develop new immunocontraceptives targeted towards males. This study shows that antibodies from induction of PCSK4 from spermatozoa may hinder fertilization., Competing Interests: CONFLICTS OF INTEREST: The authors have nothing to disclose.

The activation of viral glycoproteins by the host protease furin is an essential step in the replication of numerous pathogenic viruses. Thus, effective inhibitors of furin could serve as broad-spectrum antiviral drugs. A crystal structure of an inhibitory hexapeptide derivative in complex with furin served as template for the rational design of various types of new cyclic inhibitors. Most of the prepared derivatives are relatively potent furin inhibitors with inhibition constants in the low nanomolar or even sub-nanomolar range. For seven derivatives the crystal structures in complex with furin could be determined. In three complexes, electron density was found for the entire inhibitor. In the other cases the structures could be determined only for the P6/P5-P1 segments, which directly interact with furin. The cyclic derivatives together with two non-cyclic reference compounds were tested as inhibitors of the proteolytic activation and replication of respiratory syncytial virus in cells. Significant antiviral activity was found for both linear reference inhibitors, whereas a negligible efficacy was determined for the cyclic derivatives., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Proprotein convertases are serine proteases responsible for the cleavage and subsequent activation of protein substrates, many of them relevant for the development of an ample variety of diseases. Seven of the PCs, including furin and PACE4, recognize and hydrolyze the C-terminal end of the general sequence RXRR/KXR, whereas PCSK-9 recognizes a series of non-basic amino acids. In some systems, PC-mediated substrate activation results in the development of pathological processes, such as cancer, endocrinopathies, and cardiovascular and infectious diseases. After establishing PCs as relevant contributors to disease processes, research efforts were directed towards the development of inhibition strategies, including small and large molecules, anti-sense therapies, and antibody-based therapies. Most of these inhibitors mimic the consensus sequence of PCs, blocking the active site in a competitive manner. The most promising inhibitors were designed as bioengineered proteins; however, some non-protein and peptidomimetic agents have also proved to be effective. These efforts led to the design of pre-clinical studies and clinical trials utilizing inhibitors to PCs. Although the initial studies were performed using non-selective PCs inhibitors, such as CMK, the search for more specific, and compartmentalized selective inhibitors resulted in specific activities ascribed to some, but not all of the PCs. For instance, PACE4 inhibitors were effective in decreasing prostate cancer cell proliferation, and neovascularization. Decreased metastatic ovarian cancer utilizing furin inhibitors represents one of the major endeavors, currently in a phase II trial stage. Antibodies targeting PCSK-9 decreased significantly the levels of HDL-cholesterol, in a phase III trial. The study of Proprotein convertases has reached a stage of maturity. New strategies based on the alteration of their activity at the cellular and clinical level represent a promising experimental pharmacology field. The development of allosteric inhibitors, or specific agents directed against individual PCs is one of the challenges to be unraveled in the future., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Chronic repetitive rounds of injury and repair in the airway lead to airway remodelling, including ciliated cell loss and mucous cell hyperplasia. Airway remodelling is mediated by many growth and differentiation factors including Notch1, which are proteolytically processed by proprotein convertases (PCs). The present study evaluated a novel approach for controlling basal cell-type determination based on the inhibition of PCs. It was found that decanoyl-RVKR-chloromethylketone (CMK), a PC inhibitor, promotes ciliated cell differentiation and has no effect on the ciliary beat frequency in air-liquid interface (ALI) cultures of human nasal epithelial cells (HNECs). Comparative microarray analysis revealed that CMK considerably increases ciliogenesis-related gene expression. Use of cell-permeable and cell-impermeable PC inhibitors suggests that intracellular PCs regulate basal cell-type determination in ALI culture. Furthermore, CMK effect on ciliated cell differentiation was reversed by a Notch inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). CMK inhibited the processing of Notch1, a key regulator of basal cell differentiation toward secretory cell lineages in the airway epithelium, and down-regulated the expression of Notch1 target genes together with furin, a PC. Specific lentiviral shRNA-mediated knockdown of furin resulted in reduced Notch1 processing and increased numbers of ciliated cells in HNECs. Moreover, CMK inhibited Notch1 processing and promoted regeneration and ciliogenesis of the mouse nasal respiratory epithelium after ZnSO 4 injury. These observations suggest that PC inhibition promotes airway ciliated cell differentiation, possibly through suppression of furin-mediated Notch1 processing. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd., (© 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.)

PACE4, a member of the proprotein convertases (PCs) family of serine proteases, is a validated target for prostate cancer. Our group has developed a potent and selective PACE4 inhibitor: Ac-LLLLRVKR-NH 2 . In seeking for modifications to increase the selectivity of this ligand toward PACE4, we replaced one of its P3 Val methyl groups with a basic group capable of forming a salt bridge with D160 of PACE4. The resulting inhibitor is eight times more potent than the P3 Val parent inhibitor and two times more selective over furin, because the equivalent salt bridge with furin E257 is not optimal. Moreover, the β-branched nature of the new P3 residue favors the extended β-sheet conformation usually associated with substrates of proteases. This work provides new insight for better understanding of β-sheet backbone-backbone interactions between serine proteases and their peptidic ligands., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

PACE4, one of the seven members of the proprotein convertase family, plays an important role in the progression of prostate cancer. Therefore, its inhibition has become an attractive target to develop new therapies against this disease. Recently, we have developed a highly potent and selective PACE4 inhibitor, known as the multi-Leu peptide with the following sequence Ac-LLLLRVKR-NH 2 . Herein, with the aim of improving the stability profile of this inhibitor for potential in vivo application, we investigated the impact of different cyclization strategies. The inhibitory activity of new peptides was tested and compared to their linear counterparts. The potent analogues were further selected for stability evaluation. Our results showed that the cyclization involving a C-terminal carboxylic acid (head-to-tail or side chain-to-tail) led to compounds with significantly diminished inhibitory potency towards PACE4, indicating that an appropriate balance between rigidity and flexibility of the structure is necessary to allow the optimal binding with the enzyme. On the other hand, the modification within a multi-Leu core in combination with the incorporation of a C-terminal 4-amidinobenzylamide (Amba) residue yielded potent cyclic analogues. The best compound derived from this group, (&)[Mpa]LLLC(&)RVK[Amba] (where & indicates cyclization, Mpa - 3-mercaptopropionic acid), exhibited promising overall profile comprising of potent inhibitory effect against PACE4 and prostate cancer cell lines as well as improved stability. We believe that this cyclic framework could be further used to design even more potent and stable PACE4 inhibitors., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Objective: To investigate the effects of apolipoprotein E deficiency (Apo E(-/-)) on plasma and lipoprotein distribution of sphingosine-1-phosphate (S1P) in mice. Methods: Five male or female Apo E(-/-) or wild type (WT) mice were fed with chow diet and sacrificed at 32-week-age and plasma was collected. The constituents of lipoprotein(very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL)) were separated by ultracentrifuge. The protein concentration of constituents was detected by BCA protein quantitative kit, and the S1P concentration in plasma and various lipoprotein constituents was detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Western blot was used to determine the plasma, liver, and kidney protein expression of apolipoprotein M(Apo M), which is considered as specific ligand of S1P.The S1P concentration in plasma and various constituents of lipoprotein in the Apo E(-/-) mice was compared to respective WT mice. Results: (1)Plasma S1P content was significantly higher in the Apo E(-/-) groups than that of WT groups (male: (535.7±78.5)nmol/L vs. (263.3±22.0)nmol/L; female: (601.1±64.0)nmol/L vs. (279.0±33.9)nmol/L; all P <0.01). (2) Compared with WT mice, S1P content in non-HDL(LDL+ VLDL) was significantly higher in Apo E(-/-) mice (male: (504.9±52.8)nmol/L vs. (28.7±9.0)nmol/L; female: (427.7±27.4) vs. (27.8±4.7)nmol/L; after standardization of protein concentration, male: (385.0±41.2)pmol/mg protein vs. (71.4±6.6)pmol/mg protein; female: (330.2±22.0)pmol/mg protein vs. (67.2±12.1)pmol/mg protein; all P <0.01). (3) The expression of Apo M in plasma, liver and kidney was significantly higher in Apo E(-/-) groups than that of WT groups(all P <0.05). Conclusion: The deficiency of Apo E could lead to upregulated S1P expression in the non-HDL, the underlying mechanism might be the increased transfer of HDL into the non-HDL by Apo M-S1P.

Proprotein convertases (PCs) are serine proteases with an active role in the post-translational processing of numerous inactive proteins to active proteins including many substrates of paramount importance in cancer development and progression. Furin (PCSKC3), a well-studied member of this family, is overexpressed in numerous human and experimental malignancies. In the present communication, we treated two furin-overexpressing non-small cell carcinoma (NSCLC) cell lines (Calu-6 and HOP-62) with the PC inhibitor CMK (Decanoyl-Arg-Val-Lys-Arg-chloromethylketone). This resulted in a diminished IGF-1R processing and a simultaneous decrease in cell proliferation of two NSCLC lines. Similarly, growth of subcutaneous xenografts of both cell lines, were partially inhibited by an in vivo treatment with the same drug. These observations point to a potential role of PC inhibitors in cancer therapy. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Pro-opiomelanocortin (POMC), is a polyprotein expressed in the pituitary and the brain where it is proteolytically processed into peptide hormones and neuropeptides with distinct biological activities. It is the prototype of multipotent prohormones. The prohormone theory was first suggested in 1967 when Chrétien and Li discovered γ-lipotropin and observed that (i) it was part of β-lipotropin (β-LPH), a larger polypeptide characterized 2 years earlier and (ii) its C-terminus was β-melanocyte-stimulating hormone (β-MSH). This discovery led them to propose that the lipotropins might be related biosynthetically to the biologically active β-MSH in a precursor to end product relationship. The theory was widely confirmed in subsequent years. As we celebrate the 50th anniversary of the sequencing of β-LPH, we reflect over the lessons learned from the sequencing of those proteins; we explain their extension to the larger POMC precursor; we examine how the theory of precursor endoproteolysis they inspired became relevant for vast fields in biology; and how it led, after a long and arduous search, to the novel proteolytic enzymes called proprotein convertases. This family of nine enzymes plays multifaceted functions in growth, development, metabolism, endocrine, and brain functions. Their genetics has provided many insights into health and disease. Their therapeutic targeting is foreseeable in the near future. Thus, what started five decades ago as a theory based on POMC fragments, has opened up novel and productive avenues of biological and medical research, including, for our own current interest, a highly intriguing hypocholesterolemic Gln152His PCSK9 mutation in French-Canadian families., (© 2016 Society for Endocrinology.)

PACE4 plays important roles in prostate cancer cell proliferation. The inhibition of this enzyme has been shown to slow prostate cancer progression and is emerging as a promising therapeutic strategy. In previous work, we developed a highly potent and selective PACE4 inhibitor, the multi-Leu (ML) peptide, an octapeptide with the sequence Ac-LLLLRVKR-NH2 . Here, with the objective of developing a useful compound for in vivo administration, we investigate the effect of N-terminal modifications. The inhibitory activity, toxicity, stability, and cell penetration properties of the resulting analogues were studied and compared to the unmodified inhibitor. Our results show that the incorporation of a polyethylene glycol (PEG) moiety leads to a loss of antiproliferative activity, whereas the attachment of a lipid chain preserves or improves it. However, the lipidated peptides are significantly more toxic when compared with their unmodified counterparts. Therefore, the best results were achieved not by the N-terminal extension but by the protection of both ends with the d-Leu residue and 4-amidinobenzylamide, which yielded the most stable inhibitor, with an excellent activity and toxicity profile., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A large group of secretory proteins involved in proper functioning of living organisms, is synthesized as inactive precursor molecules. Their biologically active forms are obtained as a result of numerous post-translational modifications. Some of these processes occur irreversibly, permanently changing the initial compound structure. An example of such modifications is catalytic treatment of proteins performed by proteolytic enzymes. Among five separate classes of these enzymes, the most numerous are serine proteases. Mammalian proprotein convertases (PCs), which include: furin, PC1/3, PC2, PACE4, PC4, PC5/6, PC7, PCSK9, SKI-1, represent serine endoproteases family. PCs play a key role in the activation of a number of precursor proteins causing formation of biologically active forms of enzymes, hormones, signaling molecules, transcription and growth factors. This article summarizes current state of knowledge on biosynthesis, structure and substrate specificity of PCs, identifies the relationship between location and intracellular activity of these enzymes, and their physiological functions in mammals.

PACE4 (PCSK6) is a proprotein convertase (PC) capable of processing numerous substrates involved in tumor growth, invasion, and metastasis. Because of the human relevancy of the tobacco-associated carcinogen benzo[a]pyrene (B(a)P) we investigated whether transgenic mice in which this PC is targeted to the epidermis (K5-PACE4) may be more susceptible to B(a)P complete carcinogenesis than wild type (WT) mice. In an in vitro experiment, using cell lines derived from skin tumors obtained after B(a)P treatment, we observed that PACE4 overexpression and activity accounts for an increased proliferation rate, exaggerated sensitivity to the PC inhibitor CMK, and interference with IGF-1R autophosphorylation. Squamous cell carcinomas, obtained from K5-PACE4 mice subjected to complete chemical carcinogenesis, were characterized by a 50% increase in cell proliferation, when compared with similar tumors from WT mice. In addition, tumors from K5-PACE4 mice showed deeper invasion into the underlying dermis. Thus, mice overexpressing PACE4 exhibited tumors of increased growth rate and invasive potential when exposed to the human carcinogen B(a)P, further supporting the significance of PCs in tumor growth and progression., (© 2014 Wiley Periodicals, Inc.)

Background: Proprotein convertase subtilisin/kexin-type 9 (PCSK9) downregulates clearance of plasma cholesterol by liver. Its inactivation increases this clearance, reducing cardiovascular risk. However, a lack of PCSK9 could also lead to cholesterol accumulation in pancreatic islet beta cells, impairing insulin secretion. We reported earlier that 4-month-old male PCSK9-deficient (KO) C57BL/6 mice were hyperglycemic and insulin-insufficient relative to their wild-type (WT) counterparts. Here, we examined how gender and diet affect lipid and glucose homeostasis in these mice at 8 months of age., Methods: After being fed a normal diet or a Western diet for over 6 months, KO mice were compared with same-gender WT mice for fasting plasma levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), glucose, and insulin; for glucose disposal and glucose-stimulated insulin secretion (GSIS); and for pancreatic islet morphology., Results: A. Females: On normal diet, KO mice showed lower plasma TC, HDL-C, and LDL-C, higher plasma glucose, and normal glucose disposal despite impaired GSIS. On Western diet, they showed comparable plasma TC and HDL-C, but lower LDL-C, higher plasma glucose, and normal glucose disposal despite impaired GSIS. B. Males: On normal and Western diets, KO mice showed lower plasma TC, HDL-C, and LDL-C, similarly elevated plasma glucose, glucose intolerance, and impaired GSIS. C. Both: KO mice on either diet showed pancreatic islet dysmorphism, with larger, possibly immature secretory granules., Conclusions: Lower LDL-C and impaired GSIS are two major phenotypes in aged PCSK9-deficient C57BL/6 mice. These phenotypes are modulated by gender and diet., (© 2014 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.)

The glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) serves as virus attachment protein to its receptor on host cells and is a key determinant for cell tropism, pathogenesis, and epidemiology of the virus. The GP of LCMV is posttranslationally cleaved by the subtilase SKI-1/S1P into two subunits, the peripheral GP1, which is implicated in receptor binding, and the transmembrane GP2 that is structurally similar to the fusion active membrane proximal portions of the glycoproteins of other enveloped viruses. The present study shows that cleavage by SKI-1/S1P is not required for cell surface expression of LCMVGP on infected cells but is essential for its incorporation into virions and for the production of infectious virus particles. In absence of SKI-1/S1P cleavage, cell-to-cell propagation of the virus was markedly reduced. Further, proteolytic processing of LCMVGP depends on the presence of a cluster of basic amino acids at the C-terminus of the cytoplasmic domain of GP2, a structural motif that is conserved in Old World arenaviruses. The effect of the truncation of the cytoplasmic tail on cleavage suggests a structural interdependence between the cytoplasmic domain and the ectodomains of LCMVGP.

Mammalian cells express several transcription factors embedded in the endoplasmic reticulum (ER) as transmembrane proteins that are activated by proteolysis, and two types of these proteins have been extensively investigated. One type comprises the sterol regulatory element-binding proteins (SREBP-1 and SREBP-2). The other type comprises the activating transcription factors 6 (ATF6alpha and ATF6beta), which are activated in response to ER stress. It was shown previously that both SREBP and ATF6 are cleaved sequentially first by the Site-1 protease (serine protease) and then by the Site-2 protease (metalloprotease) (Ye, J., Rawson, R. B., Komuro, R., Chen, X., Dave, U. P., Prywes, R., Brown, M. S., and Goldstein, J. L. (2000) Mol. Cell 6, 1355-1364). In this study, we examined various protease inhibitors and found that 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor, prevented ER stress-induced cleavage of ATF6alpha and ATF6beta, resulting in inhibition of transcriptional induction of ATF6-target genes. AEBSF also inhibited production of the mature form of SREBP-2 that was induced in response to sterol depletion, and appeared to directly prevent cleavage of ATF6alpha and ATF6beta by inhibiting Site-1 protease. As the Site-1 protease is localized in the Golgi apparatus, both SREBP and ATF6 must relocate to the Golgi apparatus to be cleaved. We showed here that AEBSF treatment had little effect on ER stress-induced translocation of ATF6 from the ER to the Golgi apparatus, but blocked nuclear localization of ATF6. These results indicate that the transport of ATF6 from the ER to the Golgi apparatus and that from the Golgi apparatus to the nucleus are distinct steps that can be distinguished by treatment with AEBSF.

Crimean-Congo hemorrhagic fever (CCHF) virus is a tick-borne member of the genus Nairovirus, family Bunyaviridae. The mature virus glycoproteins, Gn and Gc (previously referred to as G2 and G1), are generated by proteolytic cleavage from precursor proteins. The amino termini of Gn and Gc are immediately preceded by tetrapeptides RRLL and RKPL, respectively, leading to the hypothesis that SKI-1 or related proteases may be involved (A. J. Sanchez, M. J. Vincent, and S. T. Nichol, J. Virol. 76:7263-7275, 2002). In vitro peptide cleavage data show that an RRLL peptide representing the Gn processing site is efficiently cleaved by SKI-1 protease, whereas an RKPL peptide representing the Gc processing site is cleaved at negligible levels. The efficient cleavage of RRLL peptide is consistent with the known recognition sequences of SKI-1, including the sequence determinants involved in the cleavage of the Lassa virus (family Arenaviridae) glycoprotein precursor. These in vitro findings were confirmed by expression of wild-type or mutant CCHF virus glycoproteins in CHO cells engineered to express functional or nonfunctional SKI-1. Gn processing was found to be dependent on functional SKI-1, whereas Gc processing was not. Gn processing occurred in the endoplasmic reticulum-cis Golgi compartments and was dependent on an R at the -4 position within the RRLL recognition motif, consistent with the known cleavage properties of SKI-1. Comparison of SKI-1 cleavage efficiency between peptides representing Lassa virus GP2 and CCHF virus Gn cleavage sites suggests that amino acids flanking the RRLL may modulate the efficiency. The apparent lack of SKI-1 cleavage at the CCHF virus Gc RKPL site indicates that related proteases, other than SKI-1, are likely to be involved in the processing at this site and identical or similar sites utilized in several New World arenaviruses.

Polypeptide protease inhibitors are often found to inhibit targets with which they did not coevolve, as in the case of high-affinity inhibition of bacterial subtilisin by the leech inhibitor eglin c. Two kinds of contacts exist in such complexes: (i) reactive site loop-active site contacts and (ii) interactions outside of these that form the broader enzyme-inhibitor interface. We hypothesized that the second class of "adventitious" contacts could be optimized to generate significant increases in affinity for a target enzyme or discrimination of an inhibitor for closely related target proteases. We began with a modified eglin c, Arg-42-Arg-45-eglin, in which the reactive site loop had been optimized for subtilisin-related processing proteases of the Kex2/furin family. We randomized 10 potential adventitious contact residues and screened for inhibition of soluble human furin. Substitutions at one of these sites, Y49, were also screened against yeast Kex2 and human PC7. These screens identified not only variants that exhibited increased affinity (up to 20-fold), but also species that exhibited enhanced selectivity, that is, increased discrimination between the target enzymes (up to 41-fold for furin versus PC7 and 20-fold for PC7 versus furin). One variant, Asp-49-Arg-42-Arg-45-eglin, exhibited a Ki of 310 pM for furin and blocked furin-dependent processing of von Willebrand factor in COS-1 cells when added to the culture medium of the cells. The exploitation of adventitious contact sites may provide a versatile technique for developing potent, selective inhibitors for newly discovered proteases and could in principle be applied to optimize numerous protein-protein interactions.

The GAS1-related genes of fungi encode GPI-anchored proteins with beta-1,3-glucanosyltransferase activity. Loss of this activity results in defects in the assembly of the cell wall. We isolated mutants that show a synthetic defect when combined with a gas1Delta allele in Saccharomyces cerevisiae, and identified nine wild-type genes that rescue this defect. The indispensability of BIG1 and KRE6 for the viability of gas1Delta cells confirmed the important role of beta-1,6-glucan in cells that are defective in the processing of beta-1,3-glucan. The identification of the Wsc1p hypo-osmotic stress sensor and components of the PKC signal transduction pathway in our screen also confirmed that the cell wall integrity response attenuates the otherwise lethal gas1Delta defect. Unexpectedly, we found that the KEX2 gene is also required for the viability of the gas1Delta mutant. Kex2p is a Golgi/endosome-membrane-anchored protease that processes secretory preproteins. A cell wall defect was also found in the kex2Delta mutant, which was suppressible by multiple copies of the MKC7 or YAP3 gene, both of which encode other GPI-anchored proteases. Therefore, normal cell wall assembly requires proteolytic processing of secretory preproteins. Furthermore, the genes CSG2 and IPT1 were found to be required for normal growth of gas1Delta cells in the presence of 1 M sorbitol. This finding suggests that complex sphingolipids play a role in the hyper-osmotic response.

This paper reports the first structure of a member of the Kex2/furin family of eukaryotic pro-protein processing proteases, which cleave sites consisting of pairs or clusters of basic residues. Reported is the 2.4 A resolution crystal structure of the two-domain protein ssKex2 in complex with an Ac-Ala-Lys-boroArg inhibitor (R = 20.9%, R(free) = 24.5%). The Kex2 proteolytic domain is similar in its global fold to the subtilisin-like superfamily of degradative proteases. Analysis of the complex provides a structural basis for the extreme selectivity of this enzyme family that has evolved from a nonspecific subtilisin-like ancestor. The P-domain of ssKex2 has a novel jelly roll like fold consisting of nine beta strands and may potentially be involved, along with the buried Ca(2+) ion, in creating the highly determined binding site for P(1) arginine.

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., (Copyright 2003 John Wiley & Sons, Ltd.)

The envelope glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) is posttranslationally cleaved into two subunits. We show here that this endoproteolytic processing is not required for transport to the cell surface but is essential for LCMV GP to mediate infectivity of pseudotyped retroviral vectors. By systematic mutational analysis of the LCMV GP cleavage site, we determined that the consensus motif R-(R/K/H)-L-(A/L/S/T/F)(265) is essential for the endoproteolytic processing. In agreement with the identified consensus motif, we show that the cellular subtilase SKI-1/S1P cleaves LCMV GP.

Deletion of the kexin gene (KEX2) in Candida albicans has a pleiotropic effect on phenotype and virulence due partly to a defect in the expression of two major virulence factors: the secretion of active aspartyl proteinases and the formation of hyphae. kex2/kex2 mutants are highly attenuated in a mouse systemic infection model and persist within cultured macrophages for at least 24 h without causing damage. Pathology is modest, with little disruption of kidney matrix. The infecting mutant cells are largely confined to glomeruli, and are aberrant in morphology. The complex phenotype of the deletion mutants reflects a role for kexin in a wide range of cellular processes. Taking advantage of the specificity of Kex2p cleavage, an algorithm we developed to scan the 9168 open reading frames in Assembly 6 of the C. albicans genome identified 147 potential substrates of Kex2p. These include all previously identified substrates, including eight secreted aspartyl proteinases, the exoglucanase Xog1p, the immunodominant antigen Mp65, and the adhesin Hwp1p. Other putative Kex2p substrates identified include several adhesins, cell wall proteins, and hydrolases previously not implicated in pathogenesis. Kexins also process fungal mating pheromones; a modification of the algorithm identified a putative mating pheromone with structural similarities to Saccharomyces cerevisiae alpha-factor.

We have cloned and characterized the kex1 gene of Pneumocystis jiroveci. Unlike the case for Pneumocystis carinii, in which the homologous PRT-1 genes are multicopy, kex1 is a single-copy gene encoding a protein homologous to fungal serine endoproteases, which localize to the Golgi apparatus. Thus, substantial biological differences can be seen among Pneumocystis species.

The filamentous fungus Aspergillus fumigatus is one of the causes of invasive lung disease in immunocompromised individuals. It is classified as asexual because no direct observation of mating or meiosis has been reported. Sequencing of the complete genome by an international collaboration, including the Wellcome Trust Sanger Institute (UK) and The Institute for Genomic Research (TIGR, USA), has made most of the genomic sequence information from A. fumigatus publicly available. By searching the incomplete genome sequence of A. fumigatus, I have identified the coding capacity for a set of proteins that could be involved in mating and the pheromone response pathway. These include one putative mating-type gene, one gene encoding a pheromone and two pheromone-receptor genes. The mating-type gene encodes a high-mobility group domain protein exhibiting significant similarity with mating-type proteins from sexually reproducing filamentous ascomycetes. The pheromone gene is predicted to encode a precursor pheromone that is processed by a KEX2-like protease to yield a pheromone that is structurally similar to the alpha-factor of the yeast Saccharomyces cerevisiae. In addition, the deduced gene products of the receptor genes are putative seven-transmembrane proteins, which display a high-level amino acid identity with the a-factor receptor Ste3p and the alpha-receptor Ste2p of S. cerevisiae, respectively. The identification of these homologues suggests the existence of a sexual cycle in A. fumigatus.

We have cloned and expressed genes encoding the allergenic brazil nut 2S albumin (Ber e 1) and the sunflower albumin 8 (SFA8) in the methylotrophic yeast Pichia pastoris. We show that both proteins were secreted at high levels and that the purified proteins were properly folded. We also showed that Ber e 1 is glycosylated during secretion and that the glycan does not interfere with the folding or immunoreactivity. The disulphide map of the Ber e 1 protein was experimentally established and is in agreement with the conserved disulphide structure of other members of the 2S albumin family. A model three-dimensional structure of the allergen was generated. During the expression studies and through mutation we have also shown that alteration of the sequences around the Kex2 endoproteolytic processing site in the expressed fusion protein can compromise the secretion by targeting part of the protein for possible degradation. The secreted production of these properly folded sulphur-rich plant albumins presents an opportunity to delineate the attributes that make an allergen and to facilitate the diagnosis and therapy of type I allergy.

Zygocin, a monomeric protein toxin secreted by a virus-infected killer strain of the osmotolerant spoilage yeast Zygosaccharomyces bailii, kills a broad spectrum of human and phytopathogenic yeasts and filamentous fungi by disrupting cytoplasmic membrane function. The toxin is encoded by a double-stranded (ds)RNA killer virus (ZbV-M, for Z. bailii virus M) that stably persists within the yeast cell cytosol. In this study, the protein toxin was purified, its N-terminal amino acid sequence was determined, and a full-length cDNA copy of the 2.1 kb viral dsRNA genome was cloned and successfully expressed in a heterologous fungal system. Sequence analysis as well as zygocin expression in Schizosaccharomyces pombe indicated that the toxin is in vivo expressed as a 238-amino-acid preprotoxin precursor (pptox) consisting of a hydrophobic N-terminal secretion signal, followed by a potentially N-glycosylated pro-region and terminating in a classical Kex2p endopeptidase cleavage site that generates the N-terminus of the mature and biologically active protein toxin in a late Golgi compartment. Matrix-assisted laser desorption mass spectrometry further indicated that the secreted toxin is a monomeric 10.4 kDa protein lacking detectable post-translational modifications. Furthermore, we present additional evidence that in contrast with other viral antifungal toxins, zygocin immunity is not mediated by the toxin precursor itself and, therefore, heterologous pptox expression in a zygocin-sensitive host results in a suicidal phenotype. Final sequence comparisons emphasize the conserved pattern of functional elements present in dsRNA killer viruses that naturally infect phylogenetically distant hosts (Saccharomyces cerevisiae and Z. bailii) and reinforce models for the sequence elements that are in vivo required for viral RNA packaging and replication.

Kexin-like protein is a component of the subtilase family of proteinases involved in the processing of proproteins to their active forms. Kexin-like proteins are also synthesized as a propeptide and this is involved in (auto)inhibition, correct folding and subcellular sorting of proteins. The kexin-like protein was described as the product of the kex2 gene for Aspergillus niger, Candida albicans, Saccharomyces cerevisiae, Yarrowia lipolytica and other fungi. Disruption of the kex2 gene in C. albicans and Y. lipolytica affects hyphae production and induces morphological cell defects, strongly suggesting a possible role of kexin-like proteins in dimorphism of human pathogenic fungi. In this work, we report the nucleotide sequence of the kex2 gene cloned from the dimorphic and human pathogenic fungus Paracoccidioides brasiliensis (Pbkex2). An open reading frame (ORF) of 2622 bp was identified in the complete sequence, interrupted by only one intron of 93 bp. The 5' non-coding region contains consensus sequences such as canonical TATA, CAAT boxes and putative motifs for transcriptional factors binding sites, such as HSE-like regulating genes involved in thermo-dependent processes; Xbp1, reported as a transcriptional factor that may control genes involved in cell morphology; and StuAp, which may regulate spore differentiation and pseudohyphal growth in fungi. In the 3' non-coding region were observed the canonical motifs necessary for correct mRNA processing and polyadenylation. The deduced protein sequence consists of 842 amino acid residues, showing identity to kexin-like proteinases from A. niger (55%), Emericella nidulans (53%) and C. albicans (48%). Comparative sequence analysis of P. brasiliensis kexin-like protein reveals the presence of homologous regions related to a signal peptide, a propeptide, a subtilisin-like catalytic domain, a P domain, a S/T rich region and a transmembrane domain. A putative Golgi retrieval signal (YEFEMI) has also been found in the cytoplasmic tail. The complete nucleotide sequence of Pbkex2 and its flanking regions have been submitted to GenBank database under Accession No. AF486805., (Copyright 2002 John Wiley & Sons, Ltd.)