The Multifaceted Proprotein Convertases: Their Unique,Redundant, Complementary,and Opposite Functions

The secretory proprotein convertase (PC) family comprises nine members: PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, SKI-1/S1P, and PCSK9. The first seven PCs cleave their substrates at single or paired basic residues, and SKI-1/S1P cleaves its substrates at non-basic residues in the Golgi. PCSK9 cleaves itself once, and the secreted inactive protease escorts specific receptors for lysosomal degradation. It regulates the levels of circulating LDL cholesterol and is considered a major therapeutic target in phase III clinical trials. In vivo, PCs exhibit unique and often essential functions during development and/or in adulthood, but certain convertases also exhibit complementary, redundant, or opposite functions.     

Comparative study of the binding pockets of mammalian proprotein convertases and its implications for the design of specific small molecule inhibitors

Proprotein convertases are enzymes that proteolytically cleave protein precursors in the secretory pathway to yield functional proteins. Seven mammalian subtilisin/Kex2p-like proprotein convertases have been identified: furin, PC1, PC2, PC4, PACE4, PC5 and PC7. The binding pockets of all seven proprotein convertases are evolutionarily conserved and highly similar. Among the seven proprotein convertases, the furin cleavage site motif has recently been characterized as a 20-residue motif that includes one core region P6-P2´ inside the furin binding pocket. This study extended this information by examining the 3D structural environment of the furin binding pocket surrounding the core region P6-P2´ of furin substrates. The physical properties of mutations in the binding pockets of the other six mammalian proprotein convertases were compared. The results suggest that: 1) mutations at two positions, Glu230 and Glu257, change the overall density of the negative charge of the binding pockets, and govern the substrate specificities of mammalian proprotein convertases; 2) two proprotein convertases (PC1 and PC2) may have reduced sensitivity for positively charged residues at substrate position P5 or P6, whereas the substrate specificities of three proprotein convertases (furin, PACE4, and PC5) are similar to each other. This finding led to a novel design of a short peptide pattern for small molecule inhibitors: [K/R]-X-V-X-K-R. Compared with the widely used small molecule dec-RVKR-cmk that inhibits all seven proprotein convertases, a finely-tuned derivative of the short peptide pattern [K/R]-X-V-X-K-R may have the potential to more effectively inhibit five of the proprotein convertases (furin, PC4, PACE4, PC5 and PC7) compared to the remaining two (PC1 and PC2). The results not only provide insights into the molecular evolution of enzyme function in the proprotein convertase family, but will also aid the study of the functional redundancy of proprotein convertases and the development of therapeutic applications.     

In vivo evidence that furin from hepatocytes inactivates PCSK9

The proprotein convertase PCSK9 plays a key role in cholesterol homeostasis by binding the LDL receptor and targeting it toward degradation. PCSK9 is strongly expressed in the liver and is found in human and mouse plasma as mature (～ 62 kDa) and inactivated (～ 55 kDa) forms. Ex vivo data showed that human PCSK9 is inactivated by cleavage at Arg(218)↓ by the overexpressed convertases furin and PC5/6A. Analysis of the plasma of human heterozygotes for R218S and F216L mutations revealed a ～ 50% reduction in the levels of the ～ 55-kDa form. To identify the convertase(s) responsible for cleavage at Arg(218) in vivo, we inactivated the genes of furin and/or PC5/6 specifically in hepatocytes. The PCSK9-inactivated form was strongly reduced in mice lacking furin in hepatocytes (Fur-hKO) and only slightly reduced in PC5/6-hKO plasma. In agreement with a key role of furin in regulating PCSK9 activity in vivo, we observed an overall 26% drop in the LDL receptor protein levels of Fur-hKO livers, likely due to the compound effects of a 35% increase in PCSK9 mRNA levels and the loss of PCSK9 cleavage, suggesting a higher activity of PCSK9 in these mice. Overexpression of PCSK9 in primary hepatocytes obtained from these mice revealed that only full-length, membrane-bound, but not soluble, furin is the cognate convertase. We conclude that in hepatocytes furin regulates PCSK9 mRNA levels and is the key in vivo-inactivating protease of circulating PCSK9.     

Dual mechanisms for the fibrate-mediated repression of proprotein convertase subtilisin/kexin type 9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with familial autosomal dominant hypercholesterolemia and is a natural inhibitor of the LDL receptor (LDLr). PCSK9 is degraded by other proprotein convertases: PC5/6A and furin. Both PCSK9 and the LDLr are up-regulated by the hypocholesterolemic statins. Thus, inhibitors or repressors of PCSK9 should amplify their beneficial effects. In the present study, we showed that PPARalpha activation counteracts PCSK9 induction by statins by repressing PCSK9 promoter activity and by increasing PC5/6A and furin expression. Quantification of mRNA and protein levels showed that various fibrates decreased PCSK9 and increased PC5/6A and furin expression. Fenofibric acid (FA) reduced PCSK9 protein content in immortalized human hepatocytes (IHH) as well as its cellular secretion. FA suppressed PCSK9 induction by statins or by the liver X receptor agonist TO901317. PCSK9 repression is occurring at the promoter level. We showed that PC5/6A and furin fibrate-mediated up-regulation is PPARalpha-dependent. As a functional test, we observed that FA increased by 30% the effect of pravastatin on the LDLr activity in vitro. In conclusion, fibrates simultaneously decreased PCSK9 expression while increasing PC5/6A and furin expression, indicating a broad action of PPARalpha activation in proprotein convertase-mediated lipid homeostasis. Moreover, this study validates the functional relevance of a combined therapy associating PCSK9 repressors and statins.     

The proprotein convertase (PC) PCSK9 is inactivated by furin and/or PC5/6A: functional consequences of natural mutations and post-translational modifications

PCSK9 is the ninth member of the proprotein convertase (PC) family. Some of its natural mutations have been genetically associated with the development of a dominant form of familial hyper- or hypocholesterolemia. The exact mechanism of action of PCSK9 is not clear, although it is known to enhance the intracellular degradation of the low density lipoprotein (LDL) receptor in acidic compartments, likely the endosomes/lysosomes. We analyzed the post-translational modifications of PCSK9 and show that it is sulfated within its prosegment at Tyr38. We also examined the susceptibility of PCSK9 to proteolytic cleavage by the other members of the PC family. The data show that the natural gain-of-function mutations R218S, F216L, and D374Y associated with hypercholesterolemia result in total or partial loss of furin/PC5/6A processing at the motif RFHR218 downward arrow. In contrast, the loss-of-function mutations A443T and C679X lead either to the lack of trans-Golgi network/recycling endosome localization and an enhanced susceptibility to furin cleavage (A443T) or to the inability of PCSK9 to exit the endoplasmic reticulum (C679X). Furthermore, we report the presence of both native and furin-like cleaved forms of PCSK9 in circulating human plasma. Thus, we propose that PCSK9 levels are finely regulated by the basic amino acid convertases furin and PC5/6A. The latter may reduce the lifetime of this proteinase and its ability to degrade the cell-surface LDL receptor, thereby regulating the levels of circulating LDL cholesterol.     

The regulated cell surface zymogen activation of the proprotein convertase PC5A directs the processing of its secretory substrates

The proprotein convertases are synthesized as zymogens that acquire activity upon autocatalytic removal of their NH(2)-terminal prosegment. Based on the convertase furin, to fold properly and gain activity, the convertases PC5A, PACE4, and PC7 are presumed to undergo two sequential prosegment cleavages in the endoplasmic reticulum and then in the trans-Golgi network. However, biochemical and immunocytochemical experiments revealed that mouse PC5A is complexed to its prosegment at the plasma membrane. This labeling is lost upon treatment with heparin and is increased by overexpressing members of the syndecan family and CD44, suggesting attachment of secreted PC5A-prosegment complex to heparan sulfate proteoglycans. Following stimulation of Y1 cells with adrenocorticotropic hormone or 8-bromo-cyclic AMP, the cell surface labeling of the prosegment of PC5A is greatly diminished, whereas the signal for mature PC5A is increased. Moreover, after stimulation, the protease activity of PC5A is enhanced, as evidenced by the cleavage of the PC5A substrates Lefty, ADAMTS-4, endothelial lipase, and PCSK9. Our data suggest a novel mechanism for PC5A activation and substrate cleavage at the cell surface, through a regulated removal of its prosegment. A similar mechanism may also apply to the convertase PACE4, thereby extending our knowledge of the molecular details of the zymogen activation and functions of these heparan sulfate proteoglycan-bound convertases.     

Loss of endothelial furin leads to cardiac malformation and early postnatal death

In mammals, seven proprotein convertases (PCs) cleave secretory proteins after basic residues, and four of them are called furin-like PCs: furin, PC5, PACE4, and PC7. In vitro, they share many substrates. However, furin is essential during development since deficient embryos die at embryonic day 11 and exhibit multiple developmental defects, particularly defects related to the function of endothelial cells. To define the role of furin in endothelial cells, an endothelial cell-specific knockout (ecKO) of the Furin gene was generated. Newborns die shortly after birth, indicating that furin is essential in these cells. Magnetic resonance imaging revealed that ecKO embryos exhibit ventricular septal defects (VSD) and/or valve malformations. In addition, primary cultures of wild-type and ecKO lung endothelial cells revealed that ecKO cells are unable to grow. Growth was efficiently rescued by extracellular soluble furin. Analysis of the processing of precursors of endothelin-1 (ET-1), adrenomedullin (Adm), transforming growth factor β1 (TGF-β1), and bone morphogenetic protein 4 (BMP4) confirmed that ET-1, Adm, and TGF-β1 are in vivo substrates of endothelial furin. Mature ET-1 and BMP4 forms were reduced by ～90% in ecKO purified endothelial cells from lungs.     

Processing of secretogranin II by prohormone convertases: Importance ofPC1 in generation of secretoneurin

Secretoneurin is a recently characterized neuropeptidepresent in the primary amino acid sequence of secretogranin II. We investigated the proteolytic processing of secretogranin II by prohormone convertases in vivo in a cellular system using the vaccinia virus system. Both PC1 and PC2 can cleave the secretogranin II precursor at sites of pairs of basic amino acids to yield intermediate-sized fragments. Other convertases like PACE4, PC5 and furin were not active. For the formation of the free neuropeptide secretoneurin a different pattern was found. Only PC1 but none of the other convertases tested including PC2 were capable of generating secretoneurin. Our results demonstrate that the prohormone convertases PC1 and PC2 are involved in proteolytic processing of secretogranin II. The neuropeptide secretoneurin can only be generated by PC1 suggesting tissue-specific processing of secretogranin II in neurons expressing different subsets of the prohormone convertases.     

Mammalian subtilisin-related proteinases in cleavage activation of the paramyxovirus fusion glycoprotein: superiority of furin/PACE to PC2 or PC1/PC3.

The fusion glycoprotein precursor of Newcastle disease virus is ubiquitously cleaved in the constitutive secretory pathway if it possesses an oligobasic cleavage motif (RRQR/KR), whereas the precursor is refractory to cleavage if the motif is monobasic (GR/KQGR). We examined the cleavage activity of the mammalian subtilisin-related proteinases furin/PACE, PC2, and PC1/PC3, which are thought to be responsible for proprotein processing in either the constitutive (furin/PACE) or the regulated (PC2 and PC1/PC3) secretory pathway, for the viral precursors with different cleavage motifs. Only furin/PACE was fully capable of cleaving the precursors with the oligobasic motif. PC2 and PC1/PC3 were incapable or only partially capable of cleaving at this motif. None of the proteinases cleaved the monobasic motif. These results suggest involvement of furin/PACE in viral protein processing in the constitutive secretory pathway.     

PCSK9 Prosegment Chimera as Novel Inhibitors of LDLR Degradation

The proprotein convertase PCSK9, a target for the treatment of hypercholesterolemia, is a negative regulator of the LDL receptor (LDLR) leading to its degradation in endosomes/lysosomes and up-regulation of plasma LDL-cholesterol levels. The proprotein convertases, a family of nine secretory serine proteases, are first synthesized as inactive zymogens. Except for PCSK9, all other convertases are activated following the autocatalytic excision of their inhibitory N-terminal prosegment. PCSK9 is unique since the mature enzyme exhibits a cleaved prosegment complexed with the catalytic subunit and has no protease activity towards other substrates. Similar to other convertases, we hypothesized that the in trans presence of the PCSK9 prosegment would interfere with PCSK9''s activity on the LDLR. Since the prosegment cannot be secreted alone, we engineered a chimeric protein using the Fc-region of human IgG1 fused to the PCSK9 prosegment. The expression of such Fcpro-fusion protein in HEK293 and HepG2 cells resulted in a secreted protein that binds PCSK9 and markedly inhibits its activity on the LDLR. This was observed by either intracellular co-expression of PCSK9 and Fcpro or by an extracellular in vitro co-incubation of Fcpro with PCSK9. Structure-function studies revealed that the inhibitory function of Fcpro does not require the acidic N-terminal stretch (residues 31–58) nor the C-terminal Gln₁₅₂ of the prosegment. Fcpro likely interacts with the prosegment and/or catalytic subunit of the prosegment≡PCSK9 complex thereby allosterically modulating its function. Our data suggest a novel strategic approach for the design and isolation of PCSK9 inhibitors.     

Neuroinflammation-Induced Interactions between Protease-Activated Receptor 1 and Proprotein Convertases in HIV-Associated Neurocognitive Disorder

The proprotein convertases (PCs) furin, PC5, PACE4, and PC7 cleave secretory proteins after basic residues, including the HIV envelope glycoprotein (gp160) and Vpr. We evaluated the abundance of PC mRNAs in postmortem brains of individuals exhibiting HIV-associated neurocognitive disorder (HAND), likely driven by neuroinflammation and neurotoxic HIV proteins (e.g., envelope and Vpr). Concomitant with increased inflammation-related gene expression (interleukin-1β [IL-1β]), the mRNA levels of the above PCs are significantly increased, together with those of the proteinase-activated receptor 1 (PAR1), an inflammation-associated receptor that is cleaved by thrombin at ProArg₄₁↓ (where the down arrow indicates the cleavage location), and potentially by PCs at Arg₄₁XXXXArg₄₆↓. The latter motif in PAR1, but not its R46A mutant, drives its interactions with PCs. Indeed, PAR1 upregulation leads to the inhibition of membrane-bound furin, PC5B, and PC7 and inhibits gp160 processing and HIV infectivity. Additionally, a proximity ligation assay revealed that furin and PC7 interact with PAR1. Reciprocally, increased furin expression reduces the plasma membrane abundance of PAR1 by trapping it in the trans-Golgi network. Furthermore, soluble PC5A/PACE4 can target/disarm cell surface PAR1 through cleavage at Arg₄₆↓. PACE4/PC5A decreased calcium mobilization induced by thrombin stimulation. Our data reveal a new PC-PAR1-interaction pathway, which offsets the effects of HIV-induced neuroinflammation, viral infection, and potentially the development of HAND.     

The cysteine-rich domain of the secreted proprotein convertases PC5A and PACE4 functions as a cell surface anchor and interacts with tissue inhibitors of metalloproteinases

The proprotein convertases PC5, PACE4 and furin contain a C-terminal cysteine-rich domain (CRD) of unknown function. We demonstrate that the CRD confers to PC5A and PACE4 properties to bind tissue inhibitors of metalloproteinases (TIMPs) and the cell surface. Confocal microscopy and biochemical analyses revealed that the CRD is essential for cell surface tethering of PC5A and PACE4 and that it colocalizes and coimmunoprecipitates with the full-length and C-terminal domain of TIMP-2. Surface-bound PC5A in TIMP-2 null fibroblasts was only observed upon coexpression with TIMP-2. In COS-1 cells, plasma membrane-associated PC5A can be displaced by heparin, suramin, or heparinases I and III and by competition with excess exogenous TIMP-2. Furthermore, PC5A and TIMP-2 are shown to be colocalized over the surface of enterocytes in the mouse duodenum and jejunum, as well as in liver sinusoids. In conclusion, the CRD of PC5A and PACE4 functions as a cell surface anchor favoring the processing of their cognate surface-anchored substrates, including endothelial lipase.     

Secretory proprotein convertases PACE4 and PC6A are heparin-binding proteins which are localized in the extracellular matrix. Potential role of PACE4 in the activation of proproteins in the extracellular matrix

PACE4, PC6 and furin are potent subtilisin-like proprotein convertases (SPCs) which are responsible for the activation of transforming growth factor-beta (TGFbeta)-related factors such as bone morphogenetic proteins. Heparan sulfate proteoglycan within the extracellular matrix (ECM) is known to regulate the biological activity of various differentiation factors including TGFbeta-related molecules. PACE4 binds tightly to heparin and its heparin-binding region was found to be a cationic stretch of amino acids between residues 743 and 760. Furthermore, PACE4 was detected in the extracellular material fraction of the HEK293 cells, defined as the material remaining on the culture plate following the removal of the cells from the plate. PACE4 bound to the extracellular fraction was selectively dislodged by heparin into the culture medium. Heparin has no inhibitory activity against PACE4. Similarly, PC6A is also able to bind to heparin, whereas soluble furin does not. In human placenta, PACE4 is mainly present in syncytiotrophoblasts and can be released by heparin. These results suggest that PACE4 and PC6 are unique SPC family proteases that anchor heparan sulfate proteoglycans at the ECM. The interaction between PACE4 and heparan sulfate proteoglycans might play an important role in the delicate spatiotemporal regulation of TGFbeta-related factors' biological activity.     

Secretory proprotein convertases PACE4 and PC6A are heparin-binding proteins which are localized in the extracellular matrix: Potential role of PACE4 in the activation of proproteins in the extracellular matrix

PACE4, PC6 and furin are potent subtilisin-like proprotein convertases (SPCs) which are responsible for the activation of transforming growth factor-β (TGFβ)-related factors such as bone morphogenetic proteins. Heparan sulfate proteoglycan within the extracellular matrix (ECM) is known to regulate the biological activity of various differentiation factors including TGFβ-related molecules. PACE4 binds tightly to heparin and its heparin-binding region was found to be a cationic stretch of amino acids between residues 743 and 760. Furthermore, PACE4 was detected in the extracellular material fraction of the HEK293 cells, defined as the material remaining on the culture plate following the removal of the cells from the plate. PACE4 bound to the extracellular fraction was selectively dislodged by heparin into the culture medium. Heparin has no inhibitory activity against PACE4. Similarly, PC6A is also able to bind to heparin, whereas soluble furin does not. In human placenta, PACE4 is mainly present in syncytiotrophoblasts and can be released by heparin. These results suggest that PACE4 and PC6 are unique SPC family proteases that anchor heparan sulfate proteoglycans at the ECM. The interaction between PACE4 and heparan sulfate proteoglycans might play an important role in the delicate spatiotemporal regulation of TGFβ-related factors' biological activity.     

The PC6B cytoplasmic domain contains two acidic clusters that direct sorting to distinct trans-Golgi network/endosomal compartments

The mammalian proprotein convertases (PCs) are a family of secretory pathway enzymes that catalyze the endoproteolytic maturation of peptide hormones and many bioactive proteins. Two PCs, furin and PC6B, are broadly expressed and share very similar cleavage site specificities, suggesting that they may be functionally redundant. However, germline knockout studies show that they are not. Here we report the distinct subcellular localization of PC6B and identify the sorting information within its cytoplasmic domain (cd). We show that in neuroendocrine cells, PC6B is localized to a paranuclear, brefeldin A-dispersible, BaCl(2)-responsive post-Golgi network (TGN) compartment distinct from furin and TGN38. The 88-amino acid PC6B-cd contains sorting information sufficient to direct reporter proteins to the same compartment as full-length PC6B. Mutational analysis indicates that endocytosis is predominantly directed by a canonical tyrosine-based motif (Tyr(1802)GluLysLeu). Truncation and sufficiency studies reveal that two clusters of acidic amino acids (ACs) within the PC6B-cd contain differential sorting information. The membrane-proximal AC (AC1) directs TGN localization and interacts with the TGN sorting protein PACS-1. The membrane-distal AC (AC2) promotes a localization characteristic of the full-length PC6B-cd. Our results demonstrate that AC motifs can target proteins to distinct TGN/endosomal compartments and indicate that the AC-mediated localization of PC6B and furin contribute to their distinct roles in vivo.     

Angiopoietin-like Protein 3 Inhibits Lipoprotein Lipase Activity through Enhancing Its Cleavage by Proprotein Convertases

Lipoprotein lipase (LPL)-mediated lipolysis of triglycerides is the first and rate-limiting step in chylomicron/very low density lipoprotein clearance at the luminal surface of the capillaries. Angiopoietin-like protein 3 (ANGPTL3) is shown to inhibit LPL activity and plays important roles in modulating lipoprotein metabolism in vivo. However, the mechanism by which it inhibits LPL activity remains poorly understood. Using cell-based analysis of the interaction between ANGPTL3, furin, proprotein convertase subtilisin/kexin type 5 (PCSK5), paired amino acid converting enzyme-4 (PACE4), and LPL, we demonstrated that the cleavage of LPL by proprotein convertases is an inactivation process, similar to that seen for endothelial lipase cleavage. At physiological concentrations and in the presence of cells, ANGPTL3 is a potent inhibitor of LPL. This action is due to the fact that ANGPTL3 can enhance LPL cleavage by endogenous furin and PACE4 but not by PCSK5. This effect is specific to LPL but not endothelial lipase. Both N- and C-terminal domains of LPL are required for ANGPTL3-enhanced cleavage, and the N-terminal domain of ANGPTL3 is sufficient to exert its effect on LPL cleavage. Moreover, ANGPTL3 enhances LPL cleavage in the presence of either heparan sulfate proteoglycans or glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1). By enhancing LPL cleavage, ANGPTL3 dissociates LPL from the cell surface, inhibiting both the catalytic and noncatalytic functions of LPL. Taken together, our data provide a molecular connection between ANGPTL3, LPL, and proprotein convertases, which may represent a rapid signal communication among different metabolically active tissues to maintain energy homeostasis. These novel findings provide a new paradigm of specific protease-substrate interaction and further improve our knowledge of LPL biology.     

Proprotein convertases in post-menopausal endometrial cancer: distinctive regulation and non-invasive diagnosis

Proprotein convertases (PCs) play critical roles in cleaving precursor proteins (growth factors, hormones, receptors and adhesion molecules) for activation. PCs are implicated in a number of cellular functions, including oncogenesis. Endometrial cancer is the most common gynecological cancer in the developed world, but the involvement of PCs is unclear. To characterize the role of PCs in endometrial cancer, we assessed expression of seven PCs (PC1/3, PC2, PACE4, PC4, furin, PC5/6 and PC7) by RT-PCR in six well characterized endometrial cancer cell lines. Expression was variable in all lines, with furin being most consistently expressed in all cell lines tested. We next determined the cellular localization and expression levels of four ubiquitously expressed PCs (furin, PACE4, PC5/6 and PC7) in post-menopausal endometrial biopsies from control (n=7) and endometrial cancer patients (n=30) by immunohistochemistry. Furin increased in tumors, whereas PC5/6, PACE4 and PC7 expression was reduced with increasing cancer grades. Uterine lavage is a non-invasive source material for evaluating the endometrium. We thus assessed whether total PC activity was altered in uterine lavage of endometrial cancer patients (n=36) compared to controls (n=10). PC activity was detected in all uterine lavage samples, and significantly elevated in all grades of endometrial cancer. This study demonstrates a complex association between individual PCs and endometrial cancer. Importantly, we show that monitoring the total PC activity in uterine lavage may provide a rapid and non-invasive method for the diagnosis of endometrial cancer in postmenopausal women.