The gene encoding the proprotein convertase subtilisin/kexin type 9 (PCSK9) is linked to familial hypercholesterolemia, as are those of the low‐density lipoprotein receptor (LDLR) and apolipoprotein ...B. PCSK9 enhances LDLR degradation, resulting in low‐density lipoprotein accumulation in plasma. To analyze the role of hepatic PCSK9, total and hepatocyte‐specific knockout mice were generated. They exhibit 42% and 27% less circulating cholesterol, respectively, showing that liver PCSK9 was responsible for two thirds of the phenotype. We also demonstrated that, in liver, PCSK9 is exclusively expressed in hepatocytes, representing the main source of circulating PCSK9. The data suggest that local but not circulating PCSK9 regulates cholesterol levels. Although transgenic mice overexpressing high levels of liver and circulating PCSK9 led to the almost complete disappearance of the hepatic LDLR, they did not recapitulate the plasma cholesterol levels observed in LDLR‐deficient mice. Single LDLR or double LDLR/PCSK9 knockout mice exhibited similar cholesterol profiles, indicating that PCSK9 regulates cholesterol homeostasis exclusively through the LDLR. Finally, the regenerating liver of PCSK9‐deficient mice exhibited necrotic lesions, which were prevented by a high‐cholesterol diet. However, lipid accumulation in hepatocytes of these mice was markedly reduced under both chow and high‐cholesterol diets, revealing that PCSK9 deficiency confers resistance to liver steatosis. Conclusion: Although PCSK9 is a target for controlling hypercholesterolemia, our data indicate that upon hepatic damage, patients lacking PCSK9 could be at risk. (HEPATOLOGY 2008;48:646–554.)
The first seven members of the proprotein convertase (PC) family activate protein precursors by cleavage after basic residues. While PC7 has no known specific substrates, it shows redundancy with ...other PCs. A genome‐wide association study suggested that circulating levels of shed human transferrin receptor 1 (hTfR1) are regulated by PC7. We thus examined whether hTfR1 constitutes a specific substrate for PC7. Coexpression of hTfR1 with PCs in several cell lines indicated that PC7 is the only convertase that sheds this receptor into the medium. Site‐directed mutagenesis showed that cleavage occurs at the unusual site KTECER100↓LA, in which the P1 Arg100 and P6 Lys95 are critical. Pharmacological treatments revealed that shedding of hTfR1 by PC7 requires endocytosis into acidic clathrin‐coated vesicles. A PC7 chimera, in which the transmembrane domain and the cytosolic tail of PC7 were replaced by that of the convertase furin, lost its ability to cleave the receptor, demonstrating the importance of these domains in the regulation of PC7 function. Analysis of primary hepatocytes from mice lacking furin, PC5, PACE4, or PC7 revealed that hepcidin, which limits iron availability in the circulation, is specifically generated by furin and not by PC7. Finally, depletion of iron in the medium of hepatoma cell lines incubated with the iron chelator desferrioxamine resulted in PC7 down‐regulation. Conclusion: Among the PC family members, only furin activates hepcidin in hepatocytes, and uniquely the full‐length membrane‐bound PC7 can directly shed hTfR1 by cleavage at Arg100↓. Our results support the notion that, when iron is limiting, hTfR1 levels increase at least in part by way of the down‐regulation of PC7 expression. (HEPATOLOGY 2013;)
The locus of the human proprotein convertase subtilisin–kexin type‐7 (PC7) gene (PCSK7) is on chromosome 11q23.3 close to the gene cluster APOA5/APOA4/APOC3/APOA1, a region implicated in the ...regulation of lipoprotein metabolism. A GWAS reported the association of PCSK7 SNPs with plasma triglyceride (TG), and exome sequencing of African Americans revealed the association of a low‐frequency coding variant of PC7 (R504H; SNP rs142953140) with a ~ 30% TG reduction. Another PCSK7 SNP rs508487 is in linkage disequilibrium with a promoter variant of the liver‐derived apolipoprotein A‐V (apoA‐V), an indirect activator of the lipoprotein lipase (LpL), and is associated with elevated TG levels. We thus hypothesized that PC7 regulates the levels/activity of apoA‐V. Studies in the human hepatic cell line HuH7 revealed that wild‐type (WT) PC7 and its endoplasmic reticulum (ER)‐retained forms bind to and enhance the degradation of human apoA‐V in acidic lysosomes in a nonenzymatic fashion. PC7‐induced degradation of apoA‐V is inhibited by bafilomycin A1 and the alkalinizing agents: chloroquine and NH4Cl. Thus, the PC7‐induced apoA‐V degradation implicates an ER‐lysosomal communication inhibited by bafilomycin A1. In vitro, the natural R504H mutant enhances PC7 Ser505 phosphorylation at the structurally exposed Ser‐X‐Glu507 motif recognized by the secretory kinase Fam20C. Co‐expression of the phosphomimetic PC7‐S505E with apoA‐V resulted in lower degradation compared to WT, suggesting that Ser505 phosphorylation of PC7 lowers TG levels via reduced apoA‐V degradation. In agreement, in Pcsk7−/− mice fed high‐fat diet, plasma apoA‐V levels and adipocyte LpL activity are increased, providing an in vivo mechanistic link for a role of liver PC7 in enhanced TG storage in adipocytes.
The PC7 gene (PCSK7) is on chromosome 11q23.3 close to the gene cluster APOA5/APOA4/APOC3/APOA1, a region implicated in the regulation of lipoprotein metabolism. We propose that PC7 binds apoA‐V directly in the endoplasmic reticulum (ER) and the complex is then sorted to autophagosomes. These autophagosomes then fuse with lysosomes for degradation by macro‐autophagy, which is sensitive to bafilomycin A1.
Proprotein convertase subtilisin kexin type 9 (PCSK9), the last member of the family of Proprotein Convertases related to Subtilisin and Kexin, regulates LDL-cholesterol by promoting the ...endosomal/lysosomal degradation of the LDL receptor (LDLR). Herein, we show that the LDLR cell surface levels dramatically increase in the liver and pancreatic islets of PCSK9 KO male but not female mice. In contrast, in KO female mice, the LDLR is more abundant at the cell surface enterocytes, as is the VLDL receptor (VLDLR) at the cell surface of adipocytes. Ovariectomy of KO female mice led to a typical KO male pattern, whereas 17β-estradiol (E2) treatment restored the female pattern without concomitant changes in LDLR adaptor protein 1 (also known as ARH), disabled-2, or inducible degrader of the LDLR expression levels. We also show that this E2-mediated regulation, which is observed only in the absence of PCSK9, is abolished upon feeding the mice a high-cholesterol diet. The latter dramatically represses PCSK9 expression and leads to high surface levels of the LDLR in the hepatocytes of all sexes and genotypes. In conclusion, the absence of PCSK9 results in a sex- and tissue-specific subcellular distribution of the LDLR and VLDLR, which is determined by E2 levels.
High circulating cholesterol is associated with hypercholesterolemia, atherosclerosis, and stroke. However, the relation between cholesterol and tumorigenesis/metastasis is controversial. The ...proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low-density lipoprotein cholesterol homeostasis by targeting the low-density lipoprotein receptor (LDLR) for degradation. PCSK9 is mostly expressed in liver, which is one of the most common sites for metastatic disease. To reveal the function of PCSK9 and also evaluate the impact of cholesterol in liver metastasis development, B16F1 melanoma cells were injected into wild-type (WT) and Pcsk9-/- mice to induce liver metastasis. On chow diet, Pcsk9-/- mice harbored two-fold less liver metastases than WT mice. This decrease is related to low cholesterol levels in Pcsk9-/- mice, as the protection was lost after normalizing Pcsk9-/- cholesterol levels by a 2-week high cholesterol diet. Furthermore, a prolongation of this diet strongly increased metastasis in both genotypes, suggesting that high cholesterol levels promote metastatic progression. The protective effect of the PCSK9 deficiency is also associated with increased apoptosis in liver stroma and metastases. Tumor necrosis factor.α (TNFα) mRNA and protein were, respectively, higher in liver stroma and plasma of injected mice, likely increasing the apoptotic TNFα signaling. Furthermore, the anti-apoptotic factor B-cell lymphoma 2 was downregulated. TNFα regulation is LDLR-independent, as its mRNA level was similarly upregulated in mice lacking both PCSK9 and LDLR. Our findings show that PCSK9 deficiency reduces liver metastasis by its ability to lower cholesterol levels and by possibly enhancing TNFα-mediated apoptosis.
Single domain antibodies (sdAbs) correspond to the antigen-binding domains of camelid antibodies. They have the same antigen-binding properties and specificity as monoclonal antibodies (mAbs) but are ...easier and cheaper to produce. We report here the development of sdAbs targeting human PCSK9 (proprotein convertase subtilisin/kexin type 9) as an alternative to anti-PCSK9 mAbs. After immunizing a llama with human PCSK9, we selected four sdAbs that bind PCSK9 with a high affinity and produced them as fusion proteins with a mouse Fc. All four sdAb-Fcs recognize the C-terminal Cys-His-rich domain of PCSK9. We performed multiple cellular assays and demonstrated that the selected sdAbs efficiently blocked PCSK9-mediated low density lipoprotein receptor (LDLR) degradation in cell lines, in human hepatocytes, and in mouse primary hepatocytes. We further showed that the sdAb-Fcs do not affect binding of PCSK9 to the LDLR but rather block its induced cellular LDLR degradation. Pcsk9 knock-out mice expressing a human bacterial artificial chromosome (BAC) transgene were generated, resulting in plasma levels of ∼300 ng/ml human PCSK9. Mice were singly or doubly injected with the best sdAb-Fc and analyzed at day 4 or 11, respectively. After 4 days, mice exhibited a 32 and 44% decrease in the levels of total cholesterol and apolipoprotein B and ∼1.8-fold higher liver LDLR protein levels. At 11 days, the equivalent values were 24 and 46% and ∼2.3-fold higher LDLR proteins. These data constitute a proof-of-principle for the future usage of sdAbs as PCSK9-targeting drugs that can efficiently reduce LDL-cholesterol, and as tools to study the Cys-His-rich domain-dependent sorting the PCSK9-LDLR complex to lysosomes.
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that binds and escorts the low density lipoprotein receptor (LDLR) into the lysosomal degradation pathway. Prescribed ...monoclonal antibodies (mAbs) against PCSK9 prevent its binding to the LDLR, and result in ~60% lower LDL cholesterol (LDLc) levels. Although efficient, mAbs are expensive. Hence other PCSK9 inhibitors are needed. For screening purpose, we developed C57BL/6J mice expressing the human PCSK9 gene under the control of its own promoter, but lacking endogenous mouse PCSK9. All lines recapitulate the endogenous PCSK9 expression pattern. The Tg2 line that expresses physiological levels of human PCSK9 (hPCSK9) was selected to characterize the inhibitory properties of a previously reported single domain antibody (sdAb), PKF8-mFc, which binds the C-terminal domain of PCSK9. Upon intraveinous injection of 10 mg/kg, PKF8-mFc and the mAb evolocumab neutralized ~50% and 100% of the hPCSK9 impact on total cholesterol (TC) levels, respectively, but PKF8-mFc had a more sustained effect. PKF8-mFc barely affected hPCSK9 levels, whereas evolocumab promoted a 4-fold increase 3 days post-injection, suggesting very different inhibitory mechanisms. The present study also shows that the new transgenic mice are well suited to screen a variety of hPCSK9 inhibitors.
PC7 belongs to the proprotein convertase family, whose members are implicated in the cleavage of secretory precursors. The in vivo function of PC7 is unknown. Herein, we find that the precursor ...proBDNF is processed into mature BDNF in COS-1 cells coexpressing proBDNF with either PC7 or Furin. Conversely, the processing of proBDNF into BDNF is markedly reduced in the absence of either Furin or PC7 in mouse primary hepatocytes. In vivo we observe that BDNF and PC7 mRNAs are colocalized in mouse hippocampus and amygdala and that mature BDNF protein levels are reduced in these brain areas in PC7 KO mice but not in the hippocampus of PC1/3 KO mice. Various behavioral tests reveal that in PC7 KO mice spatial memory is intact and plasticity of responding is mildly abnormal. Episodic and emotional memories are severely impaired, but both are rescued with the tyrosine receptor kinase B agonist 7,8-dihydroxyflavone. Altogether, these results support an in vivo role for PC7 in the regulation of certain types of cognitive performance, in part via proBDNF processing. Because polymorphic variants of human PC7 are being characterized, it will be important in future studies to determine their effects on additional physiological and behavioral processes.
Toll-like receptor 7 (TLR7) triggers antiviral immune responses by recognizing viral single-stranded RNA in endosomes, but the biosynthetic pathway of human TLR7 (hTLR7) remains unclear. Here, we ...show that hTLR7 is proteolytically processed and that the C-terminal fragment selectively accumulates in endocytic compartments. hTLR7 processing occurred at neutral pH and was dependent on furin-like proprotein convertases (PCs). Furthermore, TLR7 processing was required for its functional response to TLR7 agonists such as R837 or influenza virus. Notably, proinflammatory and differentiation stimuli increased the expression of furin-like PCs in immune cells, suggesting a positive feedback mechanism for TLR7 processing during infection. Because self-RNA can under certain conditions activate TLR7 and trigger autoimmunity, our results identify furin-like PCs as a possible target to attenuate TLR7-dependent autoimmunity and other immune pathologies.
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•Human TLR7 is cleaved and accumulates in endosomes independently of low pH•Calcium-dependent furin-like proprotein convertases (PCs) process TLR7•Inhibition or knockdown of furin-like PCs reduces responsiveness to TLR7 agonists•Mutating a furin-like PC recognition site in TLR7 reduces receptor processing
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.
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