Proprotein convertase FURIN constrains Th2 differentiation and is critical for host resistance against Toxoplasma gondii.
ABSTRACT: The proprotein convertase subtilisin/kexin enzymes proteolytically convert immature proproteins into bioactive molecules, and thereby they serve as key regulators of cellular homeostasis. The archetype proprotein convertase subtilisin/kexin, FURIN, is a direct target gene of the IL-12/STAT4 pathway and it is upregulated in Th1 cells. We have previously demonstrated that FURIN expression in T cells critically regulates the maintenance of peripheral immune tolerance and the functional maturation of pro-TGF-?1 in vivo, but FURIN's role in cell-mediated immunity and Th polarization has remained elusive. In this article, we show that T cell-expressed FURIN is essential for host resistance against a prototypic Th1 pathogen, Toxoplasma gondii, and for the generation of pathogen-specific Th1 lymphocytes, including Th1-IL-10 cells. FURIN-deficient Th cells instead show elevated expression of IL-4R subunit ? on cell surface, sensitized IL-4/STAT6 signaling, and a propensity to polarize toward the Th2 phenotype. By exploring FURIN-interacting proteins in Jurkat T cells with Strep-Tag purification and mass spectrometry, we further identify an association with a cytoskeleton modifying Ras-related C3 botulinum toxin substrate/dedicator of cytokinesis 2 protein complex and unravel that FURIN promotes F-actin polymerization, which has previously been shown to downregulate IL-4R subunit ? cell surface expression and promote Th1 responses. In conclusion, our results demonstrate that in addition to peripheral immune tolerance, T cell-expressed FURIN is also a central regulator of cell-mediated immunity and Th1/2 cell balance.
Project description:Tuberculosis is a chronic bacterial disease with a complex pathogenesis. An effective immunity against Mycobacterium tuberculosis requires both the innate and adaptive immune responses, including proper T helper (Th) type 1 cell function. FURIN is a proprotein convertase subtilisin/kexin (PCSK) enzyme, which is highly expressed in Th1 type cells. FURIN expression in T cells is essential for maintaining peripheral immune tolerance, but its role in the innate immunity and infections has remained elusive. Here, we utilized Mycobacterium marinum infection models in zebrafish (Danio rerio) to investigate how furin regulates host responses against mycobacteria. In steady-state furinAtd204e/+ fish reduced furinA mRNA levels associated with low granulocyte counts and elevated Th cell transcription factor expressions. Silencing furin genes reduced the survival of M. marinum-infected zebrafish embryos. A mycobacterial infection upregulated furinA in adult zebrafish, and infected furinAtd204e/+ mutants exhibited a proinflammatory phenotype characterized by elevated tumor necrosis factor a (tnfa), lymphotoxin alpha (lta) and interleukin 17a/f3 (il17a/f3) expression levels. The enhanced innate immune response in the furinAtd204e/+ mutants correlated with a significantly decreased bacterial burden in a chronic M. marinum infection model. Our data show that upregulated furinA expression can serve as a marker for mycobacterial disease, since it inhibits early host responses and consequently promotes bacterial growth in a chronic infection.
Project description:Furin, also called proprotein convertase subtilisin/kexin 3 (PCSK3), is a calcium-dependent serine endoprotease that processes a wide variety of proproteins involved in cell function and homeostasis. Dysregulation of furin has been implicated in numerous disease states, including cancer and fibrosis. Mammalian cell expression of the furin ectodomain typically produces a highly glycosylated, heterogeneous protein, which can make crystallographic studies difficult. Here, the expression and purification of nonglycosylated human furin using the BacMam technology and site-directed mutagenesis of the glycosylation sites is reported. Nonglycosylated furin produced using this system retains full proteolytic activity indistinguishable from that of the glycosylated protein. Importantly, the nonglycosylated furin protein reliably forms extremely durable apo crystals that diffract to high resolution. These crystals can be soaked with a wide variety of inhibitors to enable a structure-guided drug-discovery campaign.
Project description:Furin represents a crucial member of secretory mammalian subtilase, the Proprotein Convertase (PC) or Proprotein Convertase Subtilisin/Kexin (PCSK) superfamily. It has been linked to cancer, tumorgenesis, viral and bacterial pathogenesis. As a result it is considered a major target for intervention of these diseases.Herein, we report, for the first time, the synthesis and biological evaluation of a newly designed potent furin inhibitor that contains a highly reactive beta-turn inducing and radical generating "enediynyl amino acid" (Eda) moiety. "Eda" was inserted between P1 and P1' residues of hfurin(98-112) peptide, derived from the primary cleavage site of furin's own prodomain. The resulting hexadecapeptide derivative inhibited furin in vitro with IC(50) approximately 40 nM when measured against the fluorogenic substrate Boc-RVRR-MCA. It also inhibited furin-mediated cleavage of a fluorogenic peptide derived from hSARS-CoV spike protein with IC(50) approximately 193 nM. Additionally it also blocked furin-processing of growth factors proPDGF-A, B and VEGF-C that are linked to tumor genesis and cancer. Circular dichroism study showed that this inhibitor displayed a predominantly beta-turn structure while western blots confirmed its ability to protect furin protein from self degradation.These findings imply its potential as a therapeutic agent for intervention of cancer and other furin-associated diseases.
Project description:Interleukin 12 (IL-12) is a major inducer of interferon gamma (IFN-gamma) and the principal mediator of T helper 1 (Th1) differentiation. To identify IL-12-regulated genes, which might contribute to Th1 differentiation and IFNG regulation, we employed microarray analysis. Surprisingly, a ubiquitously expressed proprotein convertase (PC), furin, was one of the most consistently IL-12-induced genes in T cells, and among PCs was the only one regulated by this cytokine. Furin was preferentially expressed in differentiated Th1 cells in a Stat4-dependent manner. Expression of furin enhanced IFN-gamma secretion, whereas inhibition of furin interfered with IFN-gamma production. Thus, we conclude that IL-12 induction of furin might represent a new aspect of IFN-gamma regulation and control of Th1 differentiation.
Project description:Proprotein convertase subtilisin/kexin 9 (PCSK9) regulates plasma LDL cholesterol levels by regulating the degradation of LDL receptors. Another proprotein convertase, furin, cleaves PCSK9 at Arg(218)-Gln(219) in the surface-exposed "218 loop." This cleaved form circulates in blood along with the intact form, albeit at lower concentrations. To gain a better understanding of how cleavage affects PCSK9 function, we produced recombinant furin-cleaved PCSK9 using antibody Ab-3D5, which binds the intact but not the cleaved 218 loop. Using Ab-3D5, we also produced highly purified hepsin-cleaved PCSK9. Hepsin cleaves PCSK9 at Arg(218)-Gln(219) more efficiently than furin but also cleaves at Arg(215)-Phe(216). Further analysis by size exclusion chromatography and mass spectrometry indicated that furin and hepsin produced an internal cleavage in the 218 loop without the loss of the N-terminal segment (Ser(153)-Arg(218)), which remained attached to the catalytic domain. Both furin- and hepsin-cleaved PCSK9 bound to LDL receptor with only 2-fold reduced affinity compared with intact PCSK9. Moreover, they reduced LDL receptor levels in HepG2 cells and in mouse liver with only moderately lower activity than intact PCSK9, consistent with the binding data. Single injection into mice of furin-cleaved PCSK9 resulted in significantly increased serum cholesterol levels, approaching the increase by intact PCSK9. These findings indicate that circulating furin-cleaved PCSK9 is able to regulate LDL receptor and serum cholesterol levels, although somewhat less efficiently than intact PCSK9. Therapeutic anti-PCSK9 approaches that neutralize both forms should be the most effective in preserving LDL receptors and in lowering plasma LDL cholesterol.
Project description:We previously reported that insulin-like growth factor 1 (IGF1) was involved in coregulating female sexual maturation and longevity. To understand the underlying genetic mechanisms, based on the strain survey assays of development and aging traits, we crossed two mouse strains, KK/HIJ and PL/J, and produced 307 female F2 mice. We observed the age of vaginal patency (AVP) and the life span of these females. We also measured circulating IGF1 level at 7, 16, 24, 52, and 76 weeks. IGF1 level at 7 weeks significantly correlated with AVP. IGF1 levels at ages of 52 and 76 weeks negatively correlated with longevity (p ? .05). A gene mapping study found 22, 4 ,and 3 quantitative trait loci for IGF1, AVP, and life span, respectively. Importantly, the colocalization of IGF1, AVP, and life span quantitative trait loci in the distal region of chromosome 2 suggests this locus carries gene(s) that could regulate IGF1, AVP, and life span. In this region, proprotein convertase subtilisin/kexin type 2 has been found to be associated with female sexual maturation in a human genome-wide association study. We verified the roles of proprotein convertase subtilisin/kexin type 2 in regulating IGF1 and AVP by showing that depletion of proprotein convertase subtilisin/kexin type 2 significantly reduced IGF1 and delayed AVP in mice, suggesting that it also might be involved in the regulation of aging.
Project description:The peptide hormone apelin is translated as a 77-residue preproprotein, truncated to the 55-residue proapelin and, subsequently, to 13-36-residue bioactive isoforms named apelin-13 to -36. Proapelin is hypothesized to be cleaved to apelin-36 and then to the shorter isoforms. However, neither the mechanism of proapelin processing nor the endoproteases involved have been determined. We show direct cleavage of proapelin to apelin-13 by proprotein convertase subtilisin/kexin 3 (PCSK3, or furin) in vitro, with no production of longer isoforms. Conversely, neither PCSK1 nor PCSK7 has appreciable proapelin cleavage activity. Furthermore, we show that both proapelin and PCSK3 transcript expression levels are increased in adipose tissue with obesity and during adipogenesis, suggesting that PCSK3 is responsible for proapelin processing in adipose tissue.
Project description:Global inhibition of N-linked glycosylation broadly reduces glycan occupancy on glycoproteins, but identifying how this inhibition functionally impacts specific glycoproteins is challenging. This limits our understanding of pathogenesis in the congenital disorders of glycosylation (CDG). We used selective exo-enzymatic labeling of cells deficient in the two catalytic subunits of oligosaccharyltransferase - STT3A and STT3B - to monitor the presence and glycosylation status of cell surface glycoproteins. We show reduced abundance of two canonical tyrosine receptor kinases - the insulin receptor and insulin-like growth factor 1 receptor (IGF-1R) - at the cell surface in <i>STT3A</i>-null cells, due to decreased N-linked glycan site occupancy and proteolytic processing in combination with increased endoplasmic reticulum localization. Providing cDNA for Golgi-resident proprotein convertase subtilisin/kexin type 5a (PCSK5a) and furin cDNA to wild-type and mutant cells produced under-glycosylated forms of PCSK5a, but not furin, in cells lacking STT3A. Reduced glycosylation of PCSK5a in <i>STT3A</i>-null cells or cells treated with the oligosaccharyltransferase inhibitor NGI-1 corresponded with failure to rescue receptor processing, implying that alterations in the glycosylation of this convertase have functional consequences. Collectively, our findings show that STT3A-dependent inhibition of N-linked glycosylation on receptor tyrosine kinases and their convertases combines to impair receptor processing and surface localization. These results provide new insight into CDG pathogenesis and highlight how the surface abundance of some glycoproteins can be dually impacted by abnormal glycosylation.
Project description:Objective- Atherosclerotic coronary artery disease is the leading cause of death worldwide, and current treatment options are insufficient. Using systems-level network cluster analyses on a large coronary artery disease case-control cohort, we previously identified PCSK3 (proprotein convertase subtilisin/kexin family member 3; FURIN) as a member of several coronary artery disease-associated pathways. Thus, our objective is to determine the role of FURIN in atherosclerosis. Approach and Results- In vitro, FURIN inhibitor treatment resulted in reduced monocyte migration and reduced macrophage and vascular endothelial cell inflammatory and cytokine gene expression. In vivo, administration of an irreversible inhibitor of FURIN, ?-1-PDX (?1-antitrypsin Portland), to hyperlipidemic Ldlr-/- mice resulted in lower atherosclerotic lesion area and a specific reduction in severe lesions. Significantly lower lesional macrophage and collagen area, as well as systemic inflammatory markers, were observed. MMP2 (matrix metallopeptidase 2), an effector of endothelial function and atherosclerotic lesion progression, and a FURIN substrate was significantly reduced in the aorta of inhibitor-treated mice. To determine FURIN's role in vascular endothelial function, we administered ?-1-PDX to Apoe-/- mice harboring a wire injury in the common carotid artery. We observed significantly decreased carotid intimal thickness and lower plaque cellularity, smooth muscle cell, macrophage, and inflammatory marker content, suggesting protection against vascular remodeling. Overexpression of FURIN in this model resulted in a significant 67% increase in intimal plaque thickness, confirming that FURIN levels directly correlate with atherosclerosis. Conclusions- We show that systemic inhibition of FURIN in mice decreases vascular remodeling and atherosclerosis. FURIN-mediated modulation of MMP2 activity may contribute to the atheroprotection observed in these mice.
Project description:The low-density lipoprotein-cholesterol (LDL-C) level of a 60-year-old woman diagnosed with acute coronary syndrome (ACS) was 212 mg/dL. She was suspected of having familial hypercholesterolemia, therefore, administration of a proprotein convertase subtilisin-kexin type 9 (PCSK9) antibody in addition to atorvastatin plus ezetimibe was initiated, reducing her LDL-C level to 42 mg/dL. Nine months after initial ACS, the PCSK9 antibody was discontinued. Six months after the iturruption, she relapsed with ACS, and neoatherosclerosis progression was confirmed via intravascular ultrasound. Then, the PCSK9 antibody was reintroduced. Disruption of a PCSK9 may be associated with the progression and destabilization of neoatherosclerosis. <Learning objective: Administration of a proprotein convertase subtilisin-kexin type 9 (PCSK9) antibody in addition to statin decreases low-density lipoprotein-cholesterol level and is effective in suppressing cardiovascular events, the effect on neoatherosclerosis after coronary artery stent deployment is not clear. We experienced an interesting reccurent ACS case with familial hypercholesterolemia, and reported the possibility that PCSK9 antibody disruption might contribute to destabilization of neoatherosclerosis after coronary stenting utilizing intravascular ultrasound.>.