Hypolipidaemia among patients with PMM2-CDG is associated with low circulating PCSK9 levels: a case report followed by observational and experimental studies

International audienceBACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors are novel therapeutics for reducing low-density lipoprotein cholesterol (LDLc). While serious side-effects have not been observed in short-term clinical trials, there remain concerns that long-term PCSK9 inhibition may cause neurocognitive side-effects.METHODS AND RESULTS: An adult male with childhood-onset global developmental delay, cerebellar atrophy and severe hypolipidaemia underwent extensive biochemical and genetic investigations. Initial testing revealed low circulating PCSK9 levels and a common loss-of-function PCSK9 polymorphism, but these findings did not fully account for severe hypolipidaemia. Whole-exome sequencing was subsequently performed and identified two pathogenic phosphomannose mutase 2 (PMM2) variants (p.Arg141His and p.Pro69Ser) known to cause PMM2-associated congenital disorder of glycosylation (PMM2-CDG). A diagnosis of PMM2-CDG was consistent with the proband's neurological symptoms and severe hypolipidaemia. Given that PMM2-CDG is characterised by defective protein N-glycosylation and that PCSK9 is a negative regulator of LDLc, we postulated that loss of PCSK9 N-glycosylation mediates hypolipidaemia among patients with PMM2-CDG. First, in an independent cohort of patients with PMM2-CDG (N=8), we verified that circulating PCSK9 levels were significantly lower in patients than controls (p=0.0006). Second, we conducted in vitro experiments in hepatocyte-derived cells to evaluate the effects of PCSK9 N-glycosylation loss on LDL receptor (LDLR) activity. Experimental results suggest that defective PCSK9 N-glycosylation reduces the ability of circulating PCSK9 to degrade LDLR.CONCLUSION: Life-long exposure to genetically lower PCSK9 per se is unlikely to cause neurocognitive impairment. Both observational and experimental findings suggest that hypolipidaemia in PMM2-CDG may be partially mediated by loss of PCSK9 N-glycosylation and/or its regulators

SKI-1/S1P Facilitates SARS-CoV-2 Spike Induced Cell-to-Cell Fusion via Activation of SREBP-2 and Metalloproteases, Whereas PCSK9 Enhances the Degradation of ACE2

Proprotein convertases activate various envelope glycoproteins and participate in cellular entry of many viruses. We recently showed that the convertase furin is critical for the infectivity of SARS-CoV-2, which requires cleavage of its spike protein (S) at two sites: S1/S2 and S2′. This study investigates the implication of the two cholesterol-regulating convertases SKI-1 and PCSK9 in SARS-CoV-2 entry. The assays used were cell-to-cell fusion in HeLa cells and pseudoparticle entry into Calu-3 cells. SKI-1 increased cell-to-cell fusion by enhancing the activation of SREBP-2, whereas PCSK9 reduced cell-to-cell fusion by promoting the cellular degradation of ACE2. SKI-1 activity led to enhanced S2′ formation, which was attributed to increased metalloprotease activity as a response to enhanced cholesterol levels via activated SREBP-2. However, high metalloprotease activity resulted in the shedding of S2′ into a new C-terminal fragment (S2″), leading to reduced cell-to-cell fusion. Indeed, S-mutants that increase S2″ formation abolished S2′ and cell-to-cell fusion, as well as pseudoparticle entry, indicating that the formation of S2″ prevents SARS-CoV-2 cell-to-cell fusion and entry. We next demonstrated that PCSK9 enhanced the cellular degradation of ACE2, thereby reducing cell-to-cell fusion. However, different from the LDLR, a canonical target of PCSK9, the C-terminal CHRD domain of PCSK9 is dispensable for the PCSK9-induced degradation of ACE2. Molecular modeling suggested the binding of ACE2 to the Pro/Catalytic domains of mature PCSK9. Thus, both cholesterol-regulating convertases SKI-1 and PCSK9 can modulate SARS-CoV-2 entry via two independent mechanisms

Proprotein convertase furin regulates osteocalcin and bone endocrine function

Osteocalcin (OCN) is an osteoblast-derived hormone that increases energy expenditure, insulin sensitivity, insulin secretion, and glucose tolerance. The cDNA sequence of OCN predicts that, like many other peptide hormones, OCN is first synthesized as a prohormone (pro-OCN). The importance of pro-OCN maturation in regulating OCN and the identity of the endopeptidase responsible for pro-OCN cleavage in osteoblasts are still unknown. Here, we show that the proprotein convertase furin is responsible for pro-OCN maturation in vitro and in vivo. Using pharmacological and genetic experiments, we also determined that furin-mediated pro-OCN cleavage occurred independently of its γ-carboxylation, a posttranslational modification that is known to hamper OCN endocrine action. However, because pro-OCN is not efficiently decarboxylated and activated during bone resorption, inactivation of furin in osteoblasts in mice resulted in decreased circulating levels of undercarboxylated OCN, impaired glucose tolerance, and reduced energy expenditure. Furthermore, we show that Furin deletion in osteoblasts reduced appetite, a function not modulated by OCN, thus suggesting that osteoblasts may secrete additional hormones that regulate different aspects of energy metabolism. Accordingly, the metabolic defects of the mice lacking furin in osteoblasts became more apparent under pair-feeding conditions. These findings identify furin as an important regulator of bone endocrine function.status: publishe

Furin Is the Primary in Vivo Convertase of Angiopoietin-like 3 and Endothelial Lipase in Hepatocytes

The proprotein convertases (PCs) furin, PC5/6, and PACE4 exhibit unique and/or complementary functions. Their knockout (KO) in mice resulted in strong and specific phenotypes demonstrating that, in vivo, these PCs are unique and essential during development. However, they also exhibit redundant functions. Liver angiopoietin-like 3 (ANGPTL3) inhibits lipolysis by binding to lipoprotein lipases. It is found in the plasma as full length and truncated forms. The latter is more active and generated by cleavage at a furin-like site. Endothelial lipase (EL) binds heparin sulfate proteoglycans on cell surfaces and catalyzes the hydrolysis of HDL phospholipids. EL activity is regulated by two endogenous inhibitors, ANGPTL3 and ANGPTL4, and by PCs that inactivate EL through cleavage releasing the N-terminal catalytic and C-terminal lipid-binding domains. Herein, because furin and PC5/6 complete KOs are lethal, we used mice lacking furin or PC5/6 specifically in hepatocytes (hKO) or mice completely lacking PACE4. In primary hepatocytes, ANGPTL3 was processed into a shorter form of ANGPTL3 intracellularly by furin only, and extracellularly mainly by PACE4. In vivo, the absence of furin in hepatocytes reduced by similar to 50% the circulating levels of cleaved ANGPTL3, while the lack of PACE4 had only a minor effect. Analysis of the EL processing in primary hepatocytes and in vivo revealed that it is mostly cleaved by furin. However, the lack of furin or PC5/6 in hepatocytes and complete PACE4 KO did not appreciably modify plasma HDL levels or EL activity. Thus, inhibition of furin in liver would not be expected to modify the plasma lipid profiles

SKI-1/S1P Facilitates SARS-CoV-2 Spike Induced Cell-to-Cell Fusion via Activation of SREBP-2 and Metalloproteases, Whereas PCSK9 Enhances the Degradation of ACE2

Proprotein convertases activate various envelope glycoproteins and participate in cellular entry of many viruses. We recently showed that the convertase furin is critical for the infectivity of SARS-CoV-2, which requires cleavage of its spike protein (S) at two sites: S1/S2 and S2′. This study investigates the implication of the two cholesterol-regulating convertases SKI-1 and PCSK9 in SARS-CoV-2 entry. The assays used were cell-to-cell fusion in HeLa cells and pseudoparticle entry into Calu-3 cells. SKI-1 increased cell-to-cell fusion by enhancing the activation of SREBP-2, whereas PCSK9 reduced cell-to-cell fusion by promoting the cellular degradation of ACE2. SKI-1 activity led to enhanced S2′ formation, which was attributed to increased metalloprotease activity as a response to enhanced cholesterol levels via activated SREBP-2. However, high metalloprotease activity resulted in the shedding of S2′ into a new C-terminal fragment (S2”), leading to reduced cell-to-cell fusion. Indeed, S-mutants that increase S2′’ formation abolished S2′ and cell-to-cell fusion, as well as pseudoparticle entry, indicating that the formation of S2′’ prevents SARS-CoV-2 cell-to-cell fusion and entry. We next demonstrated that PCSK9 enhanced the cellular degradation of ACE2, thereby reducing cell-to-cell fusion. However, different from the LDLR, a canonical target of PCSK9, the C-terminal CHRD domain of PCSK9 is dispensable for the PCSK9-induced degradation of ACE2. Molecular modeling suggested the binding of ACE2 to the Pro/Catalytic domains of mature PCSK9. Thus, both cholesterol-regulating convertases SKI-1 and PCSK9 can modulate SARS-CoV-2 entry via two independent mechanisms

Modulation of protease activated receptor 1 influences human metapneumovirus disease severity in a mouse model

Human metapneumovirus (hMPV) infection causes acute respiratory tract infections (RTI) which can result in hospitalization of both children and adults. To date, no antiviral or vaccine is available for this common viral infection. Immunomodulators could represent an interesting strategy for the treatment of severe viral infection. Recently, the role of protease-activated receptors (PAR) in inflammation, coagulation and infection processes has been of growing interest. Herein, the effects of a PAR1 agonist and a PAR1 antagonist on hMPV infection were investigated in BALB/c mice. Intranasal administration of the PAR1 agonist resulted in increased weight loss and mortality of infected mice. Conversely, the PAR1 antagonist was beneficial to hMPV infection by decreasing weight loss and clinical signs and by significantly reducing pulmonary inflammation, pro-inflammatory cytokine levels (including IL-6, KC and MCP-1) and recruitment of immune cells to the lungs. In addition, a significant reduction in pulmonary viral titers was also observed in the lungs of PAR1 antagonist-treated mice. Despite no apparent direct effect on virus replication during in vitro experiments, an important role for PAR1 in the regulation of furin expression in the lungs was shown for the first time. Further experiments indicated that the hMPV fusion protein can be cleaved by furin thus suggesting that PAR1 could have an effect on viral infectivity in addition to its immunomodulatory properties. Thus, inhibition of PAR1 by selected antagonists could represent an interesting strategy for decreasing the severity of paramyxovirus infections

Neuroinflammation-induced interactions between protease-activated receptor 1 and proprotein convertases in HIV-associated neurocognitive disorder

© 2015, American Society for Microbiology. 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-1ß [IL-1ß]), 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

New Sequencing technologies help revealing unexpected mutations in Autosomal Dominant Hypercholesterolemia

International audienceAutosomal dominant hypercholesterolemia (ADH) is characterized by elevated LDL-C levels leading to coronary heart disease. Four genes are implicated in ADH: LDLR, APOB, PCSK9 and APOE. Our aim was to identify new mutations in known genes, or in new genes implicated in ADH. Thirteen French families with ADH were recruited and studied by exome sequencing after exclusion, in their probands, of mutations in the LDLR, PCSK9 and APOE genes and fragments of exons 26 and 29 of APOB gene. We identified in one family a p.Arg50Gln mutation in the APOB gene, which occurs in a region not usually associated with ADH. Segregation and in-silico analysis suggested that this mutation is disease causing in the family. We identified in another family with the p.Ala3396Thr mutation of APOB, one patient with a severe phenotype carrying also a mutation in PCSK9: p.Arg96Cys. This is the first compound heterozygote reported with a mutation in APOB and PCSK9. Functional studies proved that the p.Arg96Cys mutation leads to increased LDL receptor degradation. This work shows that Next-Generation Sequencing (exome, genome or targeted sequencing) are powerful tools to find new mutations and identify compound heterozygotes, which will lead to better diagnosis and treatment of ADH