Pairs Of Basic Amino Acids Research Articles

Circ_FURIN promotes trophoblast cell proliferation, migration and invasion in preeclampsia by regulating miR-34a-5p and TFAP2A.

Emerging evidence has shown that circular RNAs (circRNAs) play vital roles in the progression of diverse human diseases. However, the functions of circRNAs in preeclampsia (PE) are largely unknown. In this study, we aimed to explore the functions of the circRNA furin, paired basic amino acid cleaving enzyme (circ_FURIN) in PE development. qRT-PCR and western blot analyses were conducted to determine the levels of circ_FURIN, miR-34a-5p and transcription factor AP-2 alpha (TFAP2A). A Cell Counting Kit-8 (CCK-8) assay and a 5'-ethynyl-2'-deoxyuridine (EdU) incorporation assay were utilized to evaluate the cell proliferation ability. Transwell assays were adopted to estimate cell migration and invasion. A dual-luciferase reporter assay and an RNA pulldown assay were utilized to analyze the relationships among circ_FURIN, miR-34a-5p and TFAP2A. It was found that circ_FURIN was downregulated in PE placental tissues and hypoxia-treated placental trophoblast cells. Overexpression of circ_FURIN promoted trophoblast cell proliferation, migration and invasion under hypoxic conditions. Circ_FURIN functioned as the sponge for miR-34a-5p. MiR-34a-5p overexpression abrogated the effects of circ_FURIN on the proliferation, migration and invasion of trophoblast cells under hypoxic conditions. In addition, TFAP2A was demonstrated to be the target gene of miR-34a-5p. TFAP2A silencing ameliorated the promotive effects of miR-34a-5p inhibition on trophoblast cell proliferation, migration and invasion under hypoxic conditions. In conclusion, circ_FURIN enhanced trophoblast cell proliferation, migration and invasion under hypoxic conditions by elevating TFAP2A expression through sponging miR-34a-5p.

Single-cell analysis of SARS-CoV-2 receptor ACE2 and spike protein priming expression of proteases in the human heart

AimsSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly binds to ACE2 (angiotensin-converting enzyme 2) to facilitate cellular entry. Compared with the lung or respiratory tract, the human heart exhibits greater ACE2 expression. However, little substantial damage was found in the heart tissue, and no viral particles were observed in the cardiac myocytes. This study aims to analyse ACE2 and SARS-CoV-2 spike (S) protein proteases at the single-cell level, to explore the cardiac involvement in COVID-19 and improve our understanding of the potential cardiovascular implications of COVID-19.Methods and ResultsWith meta-analysis, the prevalence of cardiac injury in COVID-19 patients varies from 2% [95% confidence interval (CI) 0–5%, I2 = 0%] in non-ICU patients to 59% (95% CI 48–71%, I2 = 85%) in non-survivors. With public single-cell sequence data analysis, ACE2 expression in the adult human heart is higher than that in the lung (adjusted P < 0.0001). Inversely, the most important S protein cleavage protease TMPRSS2 (transmembrane protease serine protease-2) in the heart exhibits an extremely lower expression than that in the lung (adjusted P < 0.0001), which may restrict entry of SARS-CoV-2 into cardiac cells. Furthermore, we discovered that other S protein proteases, CTSL (cathepsin L) and FURIN (furin, paired basic amino acid cleaving enzyme), were expressed in the adult heart at a similar level to that in the lung, which may compensate for TMPRSS2, mediating cardiac involvement in COVID-19.ConclusionCompared with the lung, ACE2 is relatively more highly expressed in the human heart, while the key S protein priming protease, TMPRSS2, is rarely expressed. The low percentage of ACE2+/TMPRSS2+ cells reduced heart vulnerability to SARS-CoV-2 to some degree. CTSL and FURIN may compensate for S protein priming to mediate SARS-CoV-2 infection of the heart.

Structural variations and expression profiles of the SARS-CoV-2 host invasion genes in lung cancer.

Recent days have seen growing evidence of cancer's susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and of the effect of genomic differences on the virus' entrance genes in lung cancer. Genetic confirmation of the hypotheses regarding gene expression and mutation pattern of target genes, including angiotensin‐converting enzyme‐2 (ACE2), transmembrane serine protease 2 (TMPRSS2), basigin (CD147/BSG) and paired basic amino acid cleaving enzyme (FURIN/PCSK3), as well as correlation analysis, was done in relation to lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC) using in silico analysis. Not only were gene expression and mutation patterns detected, but also there were correlation and survival analysis between ACE2 and other target genes expression levels. The total genetic anomaly carrying rate of target genes, including ACE2, TMPRSS2, CD147/BSG, and FURIN/PCSK3, was determined as 8.1% and 21 mutations were detected, with 7 of these mutations having pathogenic features. p.H34N on the RBD binding residues for SARS‐CoV‐2 was determined in our LUAD patient group. According to gene expression analysis results, though the TMPRSS2 level was statistically significantly decreased in the LUSC patient group compared to healthy control, the ACE2 level was determined to be high in LUAD and LUSC groups. There were no meaningful differences in the expression of CD147 and FURIN genes. The challenge for today is building the assessment of genomic susceptibility to COVID‐19 in lung cancer, requiring detailed experimental laboratory studies, in addition to in silico analyses, as a way of assessing the mechanism of novel virus invasion that can be used in the development of effective SARS‐CoV‐2 therapy.

Paralogues of Mmp11 and Timp4 Interact during the Development of the Myotendinous Junction in the Zebrafish Embryo.

The extracellular matrix (ECM) of the myotendinous junction (MTJ) undergoes dramatic physical and biochemical remodeling during the first 48 h of development in zebrafish, transforming from a rectangular fibronectin-dominated somite boundary to a chevron-shaped laminin-dominated MTJ. Matrix metalloproteinase 11 (Mmp11, a.k.a. Stromelysin-3) is both necessary and sufficient for the removal of fibronectin at the MTJ, but whether this protease acts directly on fibronectin and how its activity is regulated remain unknown. Using immunofluorescence, we show that both paralogues of Mmp11 accumulate at the MTJ during this time period, but with Mmp11a present early and later replaced by Mmp11b. Moreover, Mmp11a also accumulates intracellularly, associated with the Z-discs of sarcomeres within skeletal muscle cells. Using the epitope-mediated MMP activation (EMMA) assay, we show that despite having a weaker paired basic amino acid motif in its propeptide than Mmp11b, Mmp11a is activated by furin, but may also be activated by other mechanisms intracellularly. One or both paralogues of tissue inhibitors of metalloproteinase-4 (Timp4) are also present at the MTJ throughout this process, and yeast two-hybrid assays reveal distinct and specific interactions between various domains of these proteins. We propose a model in which Mmp11a activity is modulated (but not inhibited) by Timp4 during early MTJ remodeling, followed by a phase in which Mmp11b activity is both inhibited and spatially constrained by Timp4 in order to maintain the structural integrity of the mature MTJ.

Deciphering the Role of EGL-3 for Neuropeptides Processing in Caenorhabditis elegans Using High-Resolution Quadrupole–Orbitrap Mass Spectrometry

Neuropeptides are derived from large and inactive proteins which require endoproteolytic processing for the biosynthesis of the bioactive peptides. The maturation of pro-neuropeptide to neuropeptide is believed to be performed by ortholog pro-protein convertase EGL-3 in Caenorhabditis elegans (C. elegans). Furthermore, ortholog of Cathepsin L, CPL-1 are found in C. elegans and can potentially cleave paired basic amino acids at the N-terminal suggesting the presence of both pathways. The objective of this study was to decipher the role of EGL-3 in the proteolysis of FMRF amide-related peptides (FLPs) or neuropeptide-like proteins (NLPs) using synthetic surrogate peptides based on a universal enzymatic cleavage pattern published by Schechter and Berger and used widely in enzymology. The results show evidence that proteolysis controls FLP-21 and NLP-8 related neuropeptide levels in C. elegans. Surrogate peptides were degraded rapidly when exposed to C. elegans S9 fractions leading to the formation of specific peptide fragments related to EGL-3 and CPL-1 pathway. The results suggest that CPL-1 pathway does not compensate for the loss of the EGL-3 pathway. Proteolysis of pro-neuropeptides associated to FLP-21 and NLP-8 in elg-3 mutants are severely hampered leading to a lack of mature bioactive neuropeptides.

Aeromonas sobria serine protease (ASP): a subtilisin family endopeptidase with multiple virulence activities.

Aeromonas sobria serine protease (ASP) is secreted from Aeromonas sobria, a pathogen causing gastroenteritis and sepsis. ASP resembles Saccharomyces cerevisiae Kex2, a member of the subtilisin family, and preferentially cleaves peptide bonds at the C-terminal side of paired basic amino acid residues; also accepting unpaired arginine at the P1 site. Unlike Kex2, however, ASP lacks an intramolecular chaperone N-terminal propeptide, instead utilizes the external chaperone ORF2 for proper folding, therefore, ASP and its homologues constitute a new subfamily in the subtilisin family. Through activation of the kallikrein/kinin system, ASP induces vascular leakage, and presumably causes edema and septic shock. ASP accelerates plasma clotting by α-thrombin generation from prothrombin, whereas it impairs plasma clottability by fibrinogen degradation, together bringing about blood coagulation disorder that occurs in disseminated intravascular coagulation, a major complication of sepsis. From complement C5 ASP liberates C5a that induces neutrophil recruitment and superoxide release, and mast cell degranulation, which are associated with pus formation, tissue injury and diarrhea, respectively. Nicked two-chain ASP also secreted from A. sobria is more resistant to inactivation by α2-macroglobulin than single-chain ASP, thereby raising virulence activities. Thus, ASP is a potent virulence factor and may participate in the pathogenesis of A. sobria infection.

Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A

Essentials Factor (F) VIII is an inefficiently expressed protein.Furin deletion FVIII variants were purified and characterized using in vitro and in vivo assays.These minimally modified novel FVIII variants have enhanced function.These variants provide a strategy for increasing FVIII expression in hemophilia A gene therapy. SummaryBackgroundThe major challenge for developing gene‐based therapies for hemophilia A is that human factor VIII (hFVIII) has intrinsic properties that result in inefficient biosynthesis. During intracellular processing, hFVIII is predominantly cleaved at a paired basic amino acid cleaving enzyme (PACE) or furin cleavage site to yield a heterodimer that is the major form of secreted protein. Previous studies with B‐domain‐deleted (BDD) canine FVIII and hFVIII‐R1645H, both differing from hFVIII by a single amino acid at this site, suggested that these proteins are secreted mainly in a single polypeptide chain (SC) form and exhibit enhanced function.ObjectiveWe hypothesized that deletion(s) of the furin site modulates FVIII biology and may enhance its function.MethodsA series of recombinant hFVIII‐furin deletion variants were introduced into hFVIII‐BDD [Δ1645, 1645‐46(Δ2), 1645‐47(Δ3), 1645‐48(Δ4), or Δ1648] and characterized.Results In vitro, recombinant purified Δ3 and Δ4 were primarily SC and, interestingly, had 2‐fold higher procoagulant activity compared with FVIII‐BDD. In vivo, the variants also have improved hemostatic function. After adeno‐associated viral (AAV) vector delivery, the expression of these variants is 2–4‐fold higher than hFVIII‐BDD. Protein challenges of each variant in mice tolerant to hFVIII‐BDD showed no anti‐FVIII immune response.ConclusionsThese data suggest that the furin deletion hFVIII variants are superior to hFVIII‐BDD without increased immunogenicity. In the setting of gene‐based therapeutics, these novel variants provide a unique strategy to increase FVIII expression, thus lowering the vector dose, a critical factor for hemophilia A gene therapy.

The Proprotein Convertases in Hypercholesterolemia and Cardiovascular Diseases: Emphasis on Proprotein Convertase Subtilisin/Kexin 9.

The secretory proprotein convertase (PC) family comprises nine members, as follows: PC1/3, PC2, furin, PC4, PC5/6, paired basic amino acid cleaving enzyme 4, PC7, subtilisin kexin isozyme 1/site 1 protease (SKI-1/S1P), and PC subtilisin/kexin type 9 (PCSK9). The first seven PCs cleave their substrates at single/paired basic residues and exhibit specific and often essential functions during development and/or in adulthood. The essential SKI-1/S1P cleaves membrane-bound transcription factors at nonbasic residues. In contrast, PCSK9 cleaves itself once, and the secreted inactive protease drags the low-density lipoprotein receptors (LDLR) and very LDLR (VLDLR) to endosomal/lysosomal degradation. Inhibitory PCSK9 monoclonal antibodies are now prescribed to treat hypercholesterolemia. This review focuses on the implication of PCs in cardiovascular functions and diseases, with a major emphasis on PCSK9. We present a phylogeny of the PCs and the analysis of PCSK9 haplotypes in modern and archaic human species. The absence of PCSK9 in mice led to the discovery of a sex- and tissue-specific subcellular distribution of the LDLR and VLDLR. PCSK9 inhibition may have other applications because it reduces inflammation and sepsis in a LDLR-dependent manner. Our present understanding of the cellular mechanism(s) that enables PCSK9 to induce the degradation of receptors is reviewed, as well as the consequences of its key natural mutations. The PCSK9 ongoing clinical trials are reviewed. Finally, how the other PCs may impact cardiovascular disease and the metabolic syndrome, and become relevant targets, is discussed.

PACE4 regulates apoptosis in human pancreatic cancer Panc-1 cells via the mitochondrial signaling pathway

Previous studies have demonstrated the overexpression of paired basic amino acid cleaving enzyme4 (PACE4) mRNA in prostate cancer tissues. This overexpression is correlated with higher circulating protein levels in certain patients, however, the role of PACE4 in apoptosis and the potential molecular mechanisms of pancreatic cancer remain to be elucidated. The aim of the present study was to investigate the effect and potential molecular mechanisms of PACE4 on apoptosis in the Panc‑1 pancreatic cancer cell line. Cell proliferation was assessed using a Cell Counting Kit‑8 assay. Apoptotic nuclear shrinkage was monitored using Hoechst33258 staining. Caspase‑3/7 activities were measured using a colorimetric caspase‑glo3/7 assay. Alterations in protein expression were monitored using Western blot analysis. The results indicated that PACE4 small interfering (si)RNA inhibited cell proliferation and activated caspase‑3/7 activities. In addition, PACE4 siRNA significantly increased apoptosis via the activation of caspase‑3 and the downregulation of anti‑apoptotic proteins, X‑linked inhibitor of apoptosis protein and phosphorylated‑Akt. In addition, the results showed deregulation of the Bcell lymphoma‑2 (Bcl‑2)-associated Xprotein/Bcl‑2 ratio which led to the release of cytochromec following PACE4 siRNA transfection. In conclusion, PACE4 siRNA may exert antitumor activity through the mitochondrial pathway and is expected to be a promising therapeutic strategy for the treatment of pancreatic cancer.

Cleavage of proglucagon by the prohormone converting enzymes PCSK1 and PCSK2 is not dictated by the amino acid sequence immediately adjacent to the cleavage site

The peptide hormones glucagon, glucagon‐like peptide‐1, and glucagon‐like peptide‐2 are all derived from proglucagon (PG), a 160 amino acid polypeptide containing multiple potential cleavage sites designated by either single or paired basic amino acids. Initial cleavage of PG takes place on the C‐terminal side of the K70‐R71 dibasic site, which liberates the intermediates glicentin and major proglucagon fragment (MPGF). My laboratory is interested in examining the specificity of the prohormone cleaving enzymes PCSK1 and PCSK2 using both PG and proinsulin (PI) as model substrates. PG is a useful model because it has one cleavage site (the glicentin/MPGF junction) recognized almost equally well by both enzymes and multiple potential cleavage sites that are not recognized by either enzyme. PI is a good model because it has one cleavage site (the B chain/C peptide junction) preferentially recognized by PCSK1 and a second cleavage site (the C peptide/A chain junction) preferentially recognized by PCSK2. My lab has previously described the expression and purification of these two model substrates. However, the purification of PG has presented a number of problems because of the presence of contaminants that obscure detection and participate in co‐precipitation during early stages of purification. I have now devised an improved, two‐step scheme using step elution from SP Sepharose as a capture step, followed by gradient elution from Q Sepharose as the major purification step. This method resolves the problems posed by the contaminants and generates PG samples suitable for analysis of the PCSK‐mediated cleavage reaction. An in vitro cleavage assay was then set up so that the PG substrate (~3 μM) incubated with either PCSK1 or PCSK2 (~100 nM at pH 6.0 or 5.4, respectively) results in ~30% cleavage at the glicentin/MPGF junction using either enzyme. As noted above, in contrast to the situation with PG, the normal maturation pathway for PI exhibits a marked preference for PCSK1‐mediated cleavage at the B chain/C peptide junction. To examine whether this cleavage selectivity is due to amino acids in the immediate vicinity of the cleavage site, the sequence EAED from the C‐terminal side of the B chain/C peptide cleavage site in PI was cloned into the C‐terminal side of the K70‐R71 cleavage site in PG. In contrast to expectations, this substitution resulted in a nearly 50% decrease in PG cleavage by PCSK1. Equally surprising was that this substitution had almost no effect on the efficiency of PCSK2‐mediated cleavage of the altered site, despite the fact that in the original context of the PI substrate, this sequence is recognized much less efficiently by PCSK2 than by PCSK1. Taken together, these results suggest that three‐dimensional features (secondary or tertiary structure) play a significant role in the cleavage site specificity exhibited by both PCSK1 and PCSK2.

Furin as proprotein convertase and its role in normaland pathological biological processes

Furin belongs to serine intracellular Ca2+-dependent endopeptidases of the subtilisin family, also known as proprotein convertase (PC). Human furin is synthesized as zymogen with a molecular weight of 104 kDà, which is then activated by autocatalytic in two stages. This process can occur when zymogen migrates from the endoplasmic reticulum to the Golgi apparatus, where a large part of furin is accumulated. The molecular weigh t of the active furin is 98 kDà. Furin relates to enzymes with a narrow substrate specificity: it hydrolyzes peptide bonds at the site of paired basic amino acids and furin activity exhibits in a wide pH range 5-8. Its main biological function is activation of the functionally important protein precursors. It is accompanied by the launch of a cascade of reactions, which lead to appearance of biologically active molecules involved in realization of specific biological functions both in normal and in some patologicheskih processes. Furin substrates are biologically important proteins such as enzymes, hormones, growth factors and differentiation, receptors, adhesion proteins, proteins of blood plasma. Furin plays an important role in the development of processes such as proliferation, invasion, cell migration, survival, maintenance of homeostasis, embryogenesis, as well as the development of a number of pathologies, including cardiovascular, oncologic and neurodegenerative diseases. Furin and furin-like proprotein convertases participate as key factors in the realization of the regulatory functions of proteolytic enzymes, the value of which is currently being evaluated as most important in comparison with the degradative function of proteases.

Characterization of endoproteolytic processing of dynorphins by proprotein convertases using mouse spinal cord S9 fractions and mass spectrometry

Dynorphins are important neuropeptides with a central role in nociception and pain alleviation. Many mechanisms regulate endogenous dynorphin concentrations, including proteolysis. Proprotein convertases (PCs) are widely expressed in the central nervous system and specifically cleave at C-terminal of either a pair of basic amino acids, or a single basic residue. The proteolysis control of endogenous big dynorphin (BDyn) and dynorphin A (Dyn A) levels has a profound impact on pain perception and the role of PCs remain unclear. The objective of this study was to decipher the role of PC1 and PC2 in the proteolysis control of BDyn and Dyn A levels using cellular fractions of spinal cords from wild-type (WT), PC1−/+ and PC2−/+ animals and mass spectrometry. Our results clearly demonstrate that both PC1 and PC2 are involved in the proteolysis regulation of BDyn and Dyn A with a more important role for PC1. C-terminal processing of BDyn generates specific peptide fragments dynorphin 1–19, dynorphin 1–13, dynorphin 1–11 and dynorphin 1–7, and C-terminal processing of Dyn A generates dynorphin 1–13, dynorphin 1–11 and dynorphin 1–7, all these peptide fragments are associated with PC1 or PC2 processing. Moreover, the proteolysis of BDyn leads to the formation of Dyn A and Leu-Enk, two important opioid peptides. The rate of formation of both is significantly reduced in cellular fractions of spinal cord mutant mice. As a consequence, even the partial inhibition of PC1 or PC2 may impair the endogenous opioid system.

Tachykinins Processing is Significantly Impaired in PC1 and PC2 Mutant Mouse Spinal Cord S9 Fractions.

Substance P (SP) play a central role in nociceptive transmission and it is an agonist of the Neurokinin-1 receptor located in the lamina I of the spinal cord. SP is a major proteolytic product of the protachykinin-1 primarily synthesized in neurons. Proprotein convertases (PCs) are extensively expressed in the central nervous system and specifically cleave at C-terminal of either a pair of basic amino acids, or a single basic residue. The proteolysis control of endogenous protachykinins has a profound impact on pain perception and the role of PCs remain unclear. The objective of this study was to decipher the role of PC1 and PC2 in the proteolysis surrogate protachykinins (i.e. Tachykinin 20-68 and Tachykinin 58-78) using cellular fractions of spinal cords from wild type (WT), PC1(-/+) and PC2(-/+) animals and mass spectrometry. Full-length Tachykinin 20-68 and Tachykinin 58-78 was incubated for 30min in WT, PC1(-/+) and PC2(-/+) mouse spinal cord S9 fractions and specific C-terminal peptide fragments were identified and quantified by mass spectrometry. The results clearly demonstrate that both PC1 and PC2 mediate the formation of SP and Tachykinin 58-71, an important SP precursor, with over 50% reduction of the rate of formation in mutant PC1 and PC2 mouse S9 spinal cord fractions. The results obtained revealed that PC1 and PC2 are involved in the C-terminal processing of protachykinin peptides and suggest a major role in the maturation of the protachykinin-1 protein.

Human Factor VIII Variants with Diminished PACE-Furin Cleavage Exhibit Increased Biological Activity and Therapeutic Efficacy

During intracellular processing, factor VIII (FVIII) undergoes proteolysis at multiple sites with the most predominant cleavage at a Paired basic Amino acid Cleaving Enzyme (PACE)-furin cleavage site at the carboxy-terminus of the B-domain at residue 1648 to give rise to two polypeptide chains, the heavy chain and the light chain. Through a metal ion dependent association, these chains form a heterodimer that is the secreted form of the protein. Previously, we observed that canine B-domain deleted FVIII (cFVIII-BDD) is secreted primarily as a single chain (SC) molecule (170kD) while human FVIII-BDD (hFVIII-BDD) is secreted predominantly as a heterodimer. In addition, cFVIII-BDD is more stable and has higher biological activity. Amino acid sequence analysis revealed a single amino acid difference between cFVIII (1645-HHQR-1648) and hFVIII (1645-RHQR-1648) at the PACE-furin cleavage recognition site. Characterization of hFVIII-R1645H demonstrated that this residue is responsible for the secretion of cFVIII predominantly as a single polypeptide chain and its inherent increased stability and specific activity. Furthermore, hFVIII-R1645H has enhanced hemostatic effects upon vascular injury and in the setting of AAV delivery was associated with increased circulating levels compared to wild type hFVIII-BDD. These data suggest that there is suboptimal cleavage of cFVIII and hFVIII-R1645H by PACE-furin. Here we tested whether deletion of part or all of the PACE-furin recognition sequence may further decrease the efficiency of cleavage at the site resulting in a larger portion of single SC molecules and thus may confer increased stability compared to hFVIII-R1645H. A series of hFVIII-BDD deletion variants (n=5) of the PACE-furin cleavage recognition site (1645-1648) were generated: del1645, del1645-46, del1645-47, del1645-48 and del1648. Stable BHK cell lines were established for each variant and protein was purified using ion exchange chromatography. On an SDS-PAGE gel under reducing conditions the deletion variants migrate in a similar manner, however, the proportion of the hFVIII in the single chain form compared to hFVIII-BDD (15% SC) was 3.5-fold higher for del1645-46 (57% SC), del1645-47 (53% SC) and del1645-48 (48% SC) which is similar to R1645H (52% SC). The single deletion variants on SDS-PAGE were 2-fold higher (del1645, 34% SC) or identical (del1648, 15% SC) to hFVIII-BDD. In a one-stage aPTT assay, all variants had activity similar to hFVIII-BDD. While in the two-stage aPTT assay, three variants (del1645-46, del1645-47, del1645-48) had 2-fold higher activity than hFVIII-BDD. Variants del1645 and del1648 had similar activity to hFVIII-BDD. Using an A2 domain dissociation clotting-based assay, we found that del1645-46, del1645-47, del1645-48 and del1648 were more stable following thrombin (IIa) activation compared to hFVIII-BDD suggesting that the A2 domain of these variants may dissociate more slowly than the wild type human A2 domain following IIa cleavage. These findings explain, at least in part, the superior clotting activity determined in multiple assays. The hFVIII PACE-furin variants were also introduced into an adeno-associated viral vector serotype 8 (AAV8-hAAT-hFVIII-BDD) for liver targeted delivery. Each AAV8-hAAT-hFVIII-P/F variant was delivered to hemophilia A mice (5x1011vector genomes/mouse)(n=6/group). At 4 weeks post vector administration, the hFVIII expression of del1645-47 was 3-fold higher than hFVIII-BDD followed by del1645, del1645-48, del1645-46 while del1648 was similar to hFVIII-BDD. In summary, these in vitro data demonstrate that deletion of residues 1645-46, 1645-47 or 1645-48 of the PACE-furin cleavage recognition site confers 2-3-fold higher biological activity than wild type hFVIII-BDD. In vivo these variants result in 2-3 fold higher levels of hFVIII expression after AAV delivery compared to wild type hFVIII-BDD. Together these data suggest that the hFVIII-P/F deletion variants have enhanced hemostatic function and are superior to wild type hFVIII. In the setting of gene-based therapeutics for hemophilia A, these novel variants provide a unique strategy for increasing factor VIII expression which will allow the use of a lower vector dose which is a critical improvement for translation of this approach. DisclosuresNguyen:Pfizer, Inc.: Research Funding. Camire:Pfizer: Patents & Royalties, Research Funding. Sabatino:Spark Therapeutics: Research Funding; Pfizer, Inc.: Research Funding.

Unexpected Role of PACE/Furin Cleavage Site in FVIII Biology: Implications for Hemophilia a Therapy

The paired basic amino acid cleaving enzyme (PACE)/Furin is a protein convertase system that plays a vital role in several biological processes, including coagulation. The propeptide processing of human FIX by PACE/Furin is a critical posttranslational modification, so cells co-expressing PACE/Furin and FIX are used for production of clinical recombinant protein. In the development of recombinant B-domain deleted (BDD) FVIII for hemophilia A (HA), a 14 amino acid B-domain sequence containing a putative cleavage site for PACE/Furin was retained because it was believed to be critical for intracellular processing and secretion. In contrast to FIX, we report here a surprising detrimental effect of PACE/Furin in FVIII activity and intracellular processing and secretion.We engineered a human FVIII variant where the PACE/Furin site at residues 1645-1648 was deleted from FVIII-BDD (FVIII-ΔP/F). Notably, FVIII-ΔP/F exhibits a 3-fold increased activity over FVIII-BDD (p=0.0004) in a 2-stage APTT assay. Moreover, the A2-domain dissociation of activated FVIII-ΔP/F was also 3-fold longer compared to FVIII-BDD, suggesting a more stable activated FVIII molecule. The amount of FVIII secreted from stably transduced BHK cells was about 3-fold higher for FVIII-ΔP/F than for FVIII-BDD. Conversely, the amount of intracellular FVIII antigen was lower for FVIII-ΔP/F than for FVIII-BDD.To confirm that PACE/Furin was implicated in the underlying mechanisms for the observed differences in FVIII secretion, we inhibited PACE/Furin in FVIII-BDD producing BHKs by transducing them with vectors expressing an engineered α1-antitrypsin variant (haat-PDX) that specifically inhibits PACE/Furin. This resulted in a 40% increase in FVIII secretion (p=0.017) and a decrease in intracellular FVIII-BDD, whereas transduction with the haat-wild type control, which does not inhibit PACE/Furin, did not significantly change the amount of FVIII secreted (p=0.32). Importantly, the secretion and intracellular levels of FVIII-ΔP/F were not affected by the inhibition of PACE/Furin by haat-PDX, indicating that the secretion of this FVIII variant does not benefit from further inhibition of PACE/Furin cleavage. Together these data suggest that the increased secretion of FVIII-ΔP/F compared to FVIII-BDD is due to the former circumventing PACE/Furin.Furin is ubiquitously expressed in mammal tissues. In a stringent cellular model, we used LoVo, a unique human cell line that lacks functional Furin to determine whether expression of FVIII-ΔP/F and FVIII-BDD would differ, as we have observed in cells expressing Furin. Interestingly, the secretion of FVIII-ΔP/F and FVIII-BDD were comparable. This result confirms that FVIII-BDD is secreted better in the absence of Furin. In summary, our novel variant FVIII-ΔP/F exhibits enhanced secretion primarily by bypassing PACE/Furin cleavage; inhibiting this cellular process also enhances the secretion of FVIII. Futhermore in vivo experiments also demonstrated a beneficial effect of FVIII-ΔP/F: HA mice (n=4-7/dose) given adeno-associated viral 8 (AAV8) vectors for liver gene expression of FVIII-ΔP/F resulted in a 3-fold higher circulating FVIII levels than FVIII-BDD-expressing mice (p=0.025).These exciting results from human FVIII-ΔP/F prompt us to test this variant HA canine model. First we found that recombinant canine FVIII with the entire PACE/Furin site deleted (cFVIII-ΔP/F) had increased activity in a 2-stage aPTT assay compared to wild-type cFVIII-BDD. Injection of cFVIII-ΔP/F effectively corrects the hemophilia coagulopathy in two HA dogs. Further, AAV8 liver gene therapy with cFVIII-ΔP/F in additional two HA dogs at doses of ~6 x 1012 vg/kg, a log lower than previously used for canine FVIII-BDD AAV8 gene therapy, resulted in therapeutic levels of cFVIII and shortening of clotting times. Preliminary data on injection of cFVIII-BDD protein was well tolerated in cFVIII-ΔP/F-expressing dogs.In conclusion, these data suggest that PACE/Furin cleavage of FVIII hampers protein biological activity. FVIII variants lacking PACE/Furin recognition sequences are secreted more efficiently and exhibit improved hemostatic effects in both protein- and gene-based strategies. Inhibition of PACE/Furin in manufacturing systems for recombinant human FVIII may increase the yields of protein production. Thus these strategies have a strong rationale for translation to HA therapy. DisclosuresNo relevant conflicts of interest to declare.

PACE4 regulates proliferation, migration and invasion in human breast cancer MDA-MB-231 cells.

PACE4 is one of the proprotein convertases (PC) that participate in the post-translational activation of inactive proteins, leading to mature, biologically active proteins. The processing reactions occur in pairs of basic amino acids. PACE4 is an extracellular PC that binds to growth factors and several components of the extracellular matrix contributing to tumor progression. In the present study, the PACE4 gene was silenced by small interfering RNA (siRNA), and the knockdown human breast cancer MDA-MB-231 cells showed significantly reduced proliferation, migration and invasion rates. Flow cytometry analysis indicated that downregulation of PACE4 increases the percentage of cells arrested at the G0/G1 phase. Moreover, the expression of genes involved in cell growth, invasion and adhesion, i.e., IGF-2, MMP9 and MPZL2 was significantly decreased following siRNA-mediated silencing of PACE4. Taken together, these results indicate that PACE4 plays an important role in human breast cancer, and that it might represent a novel target for breast cancer therapy.

Cancer cells release anaphylatoxin C5a from C5 by serine protease to enhance invasiveness.

Anaphylatoxin C5a indirectly fosters cancer cells through recruitment of myeloid-derived suppressor cells (MDS) for inhibiting antitumor CD8+ T cells and induction of neovascularization. We recently found activation of cancer cells by C5a directly via the C5a-receptor (C5aR; CD88) to enhance invasiveness. Thus, C5a possibly contributes to cancer progression rather than elimination. C5a generation in cancer tissues has been reported; however, the mechanism is not fully elucidated. Cancer cell expression of complement regulatory molecules suggests inefficient C5a generation through activation of the complement system in response to cancer cells. To explore another C5a generation mechanism in cancer tissues, we examined cancer cells for C5a-releasing activity from C5. C5a was present in C5-supplemented culture media of cancer cells including C5aR-expressing cells, and the media enhanced C5aR-expressing cancer cell invasion, which was abolished by anti-C5a antibody. The C5a-releasing activity was absent in the supernatants of the media and was inhibited by aprotinin, a serine protease inhibitor, and decanoyl-Arg-Val-Lys-Arg-chloromethylketone but not by inhibitors specific for cysteine, acid, or metal proteases. These results indicated C5a release from C5 by a cancer cell membrane-bound serine protease that can cleave peptide bonds at the carboxy-terminal site of paired basic amino acid residues. Cancer cell C5a release from the complement-immobilized plasma supported feasibility of this cancer cell protease-dependent C5a generation in cancer tissues. The new mechanism of C5a generation suggests self-activation of C5aR-expressing cancer cells to enhance invasiveness and induction of MDS recruitment and neovascularization to create a microenvironment favorable for cancer progression.

Paired basic amino acid cleaving enzyme 4 expression in prostate cancers and its clinicopathologic significance

Objective To investigate the expression of paired basic amino acid cleaving enzyme 4 (PACE4) in prostate cancers and its clinical significance.Methods Expression of PACE4 was examined by immunohistochemistry in 72 cases of prostate cancer (PCa),20 cases of prostatic intraepithelial neoplasia(PIN) and 40 cases of benign prostatic hyperplasia (BPH).Results The positive rates of PACE4 in PCa,PIN and BPH were 92％,80％ and 20％ respectively,with a significant difference among them (P ＜ 0.05).Its accuracy rate and Youden index(95％ confidence interval) were 82％ and 60％ (45％-74％) respectively when diagnosing PCa and Non-PCa.A positive linear trend was found between PACE4 expression and clinical stage,preoperative serum prostate specific antigen (PSA) level,Gleason score and estimated-risk.Conclusion PACE4 expression in prostate tissue could well differentiate PCa from PIN & BPH,and could represent the prognostic risk appropriately. Key words: Paired basic amino acid cleaving enzyme 4; Prostate cancer; Diagnosis; Risk grade

Exploring a role for the serine protease PACE4 in pain associated with experimental OA

Exploring a role for the serine protease PACE4 in pain associated with experimental OA

Improved Expression of Recombinant Human Factor IX by Co-expression of GGCX, VKOR and Furin

Recombinant human FIX concentrates (rhFIX) are essential in the treatment and prevention of bleeding in the bleeding disorder haemophilia B. However, due to the complex nature of FIX production yields are low which leads to high treatment costs. Here we report the production of rhFIX with substantially higher yield by co-expressing human FIX with GGCX (γ-glutamyl carboxylase), VKOR (vitamin K epoxide reductase) and furin (paired basic amino acid cleaving enzyme) in Chinese hamster ovary (CHO) cells. Our results show that controlled co-expression of GGCX with FIX is critical to obtain high rhFIX titre, and, that co-expression of VKOR further increased the yield of active rhFIX. Furin co-expression improved processing of the leader peptide of rhFIX but had a minor effect on yield of active rhFIX. The optimal expression level of GGCX was surprisingly low and required unusual engineering of expression vector elements. For VKOR and furin the control of expression was less critical and could be achieved by standard vector element. Using our expression vectors an rhFIX-producing clone with an expression level of up to 30 mg/L of active rhFIX was obtained. In addition an efficient single step purification method was developed to obtain pure and active rhFIX with up to 94 % yield.