A novel splicing variant of proprotein convertase subtilisin/kexin type 9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is the most recently identified member of the proprotein convertase family. Genetic and cell biology studies have suggested a critical role of PCSK9 in regulating low-density lipoprotein receptor (LDLR) protein levels and thus modulating plasma LDL cholesterol. Recent data on the molecular basis for PCSK9 action support the model in which PCSK9 is self-cleaved, secreted, and tightly bound to the EGF-A repeat of LDLR extracellular domain. PCSK9 binding to LDLR is essential for the ensuing receptor-mediated endocytosis and is speculated to lock LDLR in a specific conformation that favors degradation in lysosomal compartment instead of recycling back to plasma membrane. We report here a novel human PCSK9 splicing variant, which we named PCSK9sv. PCSK9sv had an in-frame deletion of the eighth exon of 58 amino acids and was expressed in multiple tissues, including liver, small intestine, prostate, uterus, brain, and adipose tissue. Unlike wild-type PCSK9, which is secreted, PCSK9sv expressed in human embryonic kidney HEK293 cells failed to process the prosegment intracellularly and thus was not secreted into the medium. Examination of potential functions revealed that PCSK9sv did not change the LDLR protein levels. Two mutations that have been reported in humans with the associated changes in plasma LDL cholesterol were within exon 8, and thus the expression and function of the two mutants were studied. Both N425S and A443T mutants were processed normally, secreted, and reduced LDLR levels. However, the physiological function of this novel splicing variant of PCSK9 has yet to be determined.     

Molecular characterization of proprotein convertase subtilisin/kexin type 9-mediated degradation of the LDLR

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that promotes degradation of cell surface LDL receptors (LDLRs) in selected cell types. Here we used genetic and pharmacological inhibitors to define the pathways involved in PCSK9-mediated LDLR degradation. Inactivating mutations in autosomal recessive hypercholesterolemia (ARH), an endocytic adaptor, blocked PCSK9-mediated LDLR degradation in lymphocytes but not in fibroblasts. Thus, ARH is not specifically required for PCSK9-mediated LDLR degradation. Knockdown of clathrin heavy chain with siRNAs prevented LDLR degradation. In contrast, prevention of ubiquitination of the LDLR cytoplasmic tail, inhibition of proteasomal activity, or disruption of proteins required for lysosomal targeting via macroautophagy (autophagy related 5 and 7) or the endosomal sorting complex required for trafficking (ESCRT) pathway (hepatocyte growth factor-regulated Tyr-kinase substrate and tumor suppressor gene 101) failed to block PCSK9-mediated LDLR degradation. These findings are consistent with a model in which the LDLR-PCSK9 complex is internalized via clathrin-mediated endocytosis and then routed to lysosomes via a mechanism that does not require ubiquitination and is distinct from the autophagy and proteosomal degradation pathways. Finally, the PCSK9-LDLR complex appears not to be transported by the canonical ESCRT pathway.     

Regulation of epithelial sodium channel trafficking by proprotein convertase subtilisin/kexin type 9 (PCSK9)

The epithelial Na(+) channel (ENaC) is critical for Na(+) homeostasis and blood pressure control. Defects in its regulation cause inherited forms of hypertension and hypotension. Previous work found that ENaC gating is regulated by proteases through cleavage of the extracellular domains of the α and γ subunits. Here we tested the hypothesis that ENaC is regulated by proprotein convertase subtilisin/kexin type 9 (PCSK9), a protease that modulates the risk of cardiovascular disease. PCSK9 reduced ENaC current in Xenopus oocytes and in epithelia. This occurred through a decrease in ENaC protein at the cell surface and in the total cellular pool, an effect that did not require the catalytic activity of PCSK9. PCSK9 interacted with all three ENaC subunits and decreased their trafficking to the cell surface by increasing proteasomal degradation. In contrast to its previously reported effects on the LDL receptor, PCSK9 did not alter ENaC endocytosis or degradation of the pool of ENaC at the cell surface. These results support a role for PCSK9 in the regulation of ENaC trafficking in the biosynthetic pathway, likely by increasing endoplasmic reticulum-associated degradation. By reducing ENaC channel number, PCSK9 could modulate epithelial Na(+) absorption, a major contributor to blood pressure control.     

Novel domain interaction regulates secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) protein

PCSK9 (proprotein convertase subtilisin/kexin type 9) has emerged as a novel therapeutic target for hypercholesterolemia due to its LDL receptor (LDLR)-reducing activity. Although its structure has been solved, the lack of a detailed understanding of the structure-function relation hinders efforts to develop small molecule inhibitors. In this study, we used mutagenesis and transfection approaches to investigate the roles of the prodomain (PD) and the C-terminal domain (CD) and its modules (CM1-3) in the secretion and function of PCSK9. Deletion of PD residues 31-40, 41-50, or 51-60 did not affect the self-cleavage, secretion, or LDLR-degrading activity of PCSK9, whereas deletion of residues 61-70 abolished all of these functions. Deletion of the entire CD protein did not impair PCSK9 self-cleavage or secretion but completely abolished LDLR-degrading activity. Deletion of any one or two of the CD modules did not affect self-cleavage but influenced secretion and LDLR-reducing activity. Furthermore, in cotransfection experiments, a secretion-defective PD deletion mutant (ΔPD) was efficiently secreted in the presence of CD deletion mutants. This was due to the transfer of PD from the cotransfected CD mutants to the ΔPD mutant. Finally, we found that a discrete CD protein fragment competed with full-length PCSK9 for binding to LDLR in vitro and attenuated PCSK9-mediated hypercholesterolemia in mice. These results show a previously unrecognized domain interaction as a critical determinant in PCSK9 secretion and function. This knowledge should fuel efforts to develop novel approaches to PCSK9 inhibition.     

Characterization of a quantitative method to measure free proprotein convertase subtilisin/kexin type 9 in human serum

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that plays an important role in the regulation of serum low-density lipoprotein (LDL) cholesterol by downregulation of LDL receptor, and as such is considered a novel target in cholesterol lowering therapy. In support of the drug development program for Evolocumab, a fully human IgG₂ antibody that targets PCSK9, a quantitative ELISA to measure free PCSK9 in human serum was developed. PCSK9 serves as a biomarker of pharmacological response during treatment, and measuring levels of the free ligand post-dosing was of interest as an aid to establishing the pharmacokinetic and pharmacodynamic properties of the therapeutic. Given the complexities associated with the measurement of free ligand in the presence of high concentrations of circulating drug, it was important to challenge the method with experiments designed to assess ex vivo conditions that have the potential to affect the binding equilibrium of drug and ligand within test samples during routine sampling handling and assay conditions. Herein, we report results of experiments that were conducted to characterize the assay in alignment with regulatory guidance and industry standards, and to establish evidence that the method is measuring the free ligand in circulation at the time serum was collected. A robust supporting data package was generated that demonstrates the method specifically and reproducibly measures the free ligand, and is suitable for its intended use.     

Characterization of a quantitative method to measure free proprotein convertase subtilisin/kexin type 9 in human serum

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that plays an important role in the regulation of serum low-density lipoprotein (LDL) cholesterol by downregulation of LDL receptor, and as such is considered a novel target in cholesterol lowering therapy. In support of the drug development program for Evolocumab, a fully human IgG₂ antibody that targets PCSK9, a quantitative ELISA to measure free PCSK9 in human serum was developed. PCSK9 serves as a biomarker of pharmacological response during treatment, and measuring levels of the free ligand post-dosing was of interest as an aid to establishing the pharmacokinetic and pharmacodynamic properties of the therapeutic. Given the complexities associated with the measurement of free ligand in the presence of high concentrations of circulating drug, it was important to challenge the method with experiments designed to assess ex vivo conditions that have the potential to affect the binding equilibrium of drug and ligand within test samples during routine sampling handling and assay conditions. Herein, we report results of experiments that were conducted to characterize the assay in alignment with regulatory guidance and industry standards, and to establish evidence that the method is measuring the free ligand in circulation at the time serum was collected. A robust supporting data package was generated that demonstrates the method specifically and reproducibly measures the free ligand, and is suitable for its intended use.     

Secreted proprotein convertase subtilisin/kexin type 9 reduces both hepatic and extrahepatic low-density lipoprotein receptors in vivo

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that is known to reduce hepatic low-density lipoprotein receptor (LDLR) levels and increase plasma LDL cholesterol. It is not clear, however, whether secreted PCSK9 degrades extrahepatic LDLRs. We present evidence that recombinant PCSK9, either injected intravenously into or expressed in the liver of C57BL/6 mice, significantly reduced LDLR levels in multiple extrahepatic tissues. During the initial characterization, we found that injected human recombinant PCSK9 at 30 μg/mouse had a half-life of 15 min in serum in mice. Hepatic LDLR levels were reduced within 30 min and the degradation of hepatic LDLR reached the maximum 2 h after the initial protein injection. Endocytosis of PCSK9 in liver occurred within 5 min of protein injection and internalized PCSK9 was only barely detectable within 1 h. When extrahepatic LDLRs were examined by Western blotting analysis, we found significant reductions of LDLRs in multiple extrahepatic tissues including lung, adipose and kidney along with the more dramatic reduction of LDLRs in liver. These studies were further extended using adenoviral expression of human PCSK9 in C57BL/6 mice to demonstrate that PCSK9 produced in liver impacted extrahepatic tissue LDLR levels as well. Taken together, our studies indicate that secreted PCSK9 can potentially impact extrahepatic tissue cholesterol homeostasis by regulating extrahepatic tissue LDLR levels.     

Affinity and kinetics of proprotein convertase subtilisin/kexin type 9 binding to low-density lipoprotein receptors on HepG2 cells

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that regulates the number of cell surface low-density lipoprotein receptors (LDLRs) and the levels of low-density lipoprotein cholesterol in plasma. Intact cells have not previously been used to determine the characteristics of binding of PCSK9 to LDLR. Using PCSK9 iodinated by the tyramine cellobiose (TC) method ([(125)I]TC-PCSK9), we measured the affinity and kinetics of binding of PCSK9 to LDLR on HepG2 cells at 4 °C. The extent of [(125)I]TC-PCSK9 binding increased as cell surface LDLR density increased. Unlabeled wild-type and two gain-of-function mutants of PCSK9 reduced binding of [(125)I]TC-PCSK9. The Scatchard plot of the binding-inhibition curve was curvilinear, indicative of high-affinity and low-affinity sites for PCSK9 binding on HepG2 cells. Nonlinear regression analysis of the binding data also indicated that a two-site model better fitted the data. The time course of [(125)I]TC-PCSK9 binding showed two phases in the association kinetics. Dissociation of [(125)I]TC-PCSK9 also occurred in two phases. Unlabeled PCSK9 accelerated the dissociation of [(125)I]TC-PCSK9. At low pH, only one phase of dissociation was apparent. Furthermore, the dissociation of [(125)I]TC-PCSK9 under pre-equilibrium conditions was faster than under equilibrium conditions. Overall, the data suggest that PCSK9 binding to cell surface LDLR cannot be described by a simple bimolecular reaction. Possible interpretations that can account for these observations are discussed.     

Proprotein convertase subtilisin/kexin type 9: A new target molecule for gene therapy

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a novel target for controlling plasma levels of low-density lipoprotein cholesterol (LDL-C) and decreasing the risk of cardiovascular diseases. At present it is clear that the major classes of commonly prescribed lipid-lowering medications increase serum PCSK9 levels and fail to protect a significant percentage of patients from cardiovascular events. Therefore development of new LDL-C lowering medications that either do not increase circulating PCSK9 levels or work through inhibition of PCSK9 expression and protease activity is a highly desirable approach to overcome hypercholesterolemia. Since there are several agents which are being evaluated in human preclinical and clinical trials, this review summarizes current therapeutic strategies targeting PCSK9, including specific antibodies, antisense oligonucleotides, small interfering RNAs (siRNAs) and other small-molecule inhibitors.     

Immunization against proprotein convertase subtilisin-like/kexin type 9 lowers plasma LDL-cholesterol levels in mice

Successful development of drugs against novel targets crucially depends on reliable identification of the activity of the target gene product in vivo and a clear demonstration of its specific functional role for disease development. Here, we describe an immunological knockdown (IKD) method, a novel approach for the in vivo validation and functional study of endogenous gene products. This method relies on the ability to elicit a transient humoral response against the selected endogenous target protein. Anti-target antibodies specifically bind to the target protein and a fraction of them effectively neutralize its activity. We applied the IKD method to the in vivo validation of plasma PCSK9 as a potential target for the treatment of elevated levels of plasma LDL-cholesterol. We show that immunization with human-PCSK9 in mice is able to raise antibodies that cross-react and neutralize circulating mouse-PCSK9 protein thus resulting in increased liver LDL receptor levels and plasma cholesterol uptake. These findings closely resemble those described in PCSK9 knockout mice or in mice treated with antibodies that inhibit PCSK9 by preventing the PCSK9/LDLR interaction. Our data support the IKD approach as an effective method to the rapid validation of new target proteins.     

Spatiotemporal expression of proprotein convertase subtilisin/kexin type 5 in the development of anorectal malformations in fetal rats

This study examined the expression patterns of proprotein convertase subtilisin/kexin type 5 (Pcsk5) during anorectal development in normal and anorectal malformations (ARM) rat embryos, determine the possible role of Pcsk5 in the pathogenesis of ARM. An ARM rat model was developed by the administration of ethylenethiourea gestational day 10 (GD10). Embryos were harvested by surgical excision from GD13 to GD16, and the spatiotemporal expression of Pcsk5 was evaluated, using immunohistochemistry staining, Western blotting and real time RT-PCR. Immunohistochemistry staining in normal embryos revealed that Pcsk5 was abundantly expressed on the epithelium of the cloaca (CL) on GD13. On GD14 and GD15, positive cells were noted on the urorectal septum and the thin anal membrane. However, the epithelium of the CL of ARM embryos only faintly expressed Pcsk5 from GD13 to GD15. Western blotting and real time RT-PCR showed time-dependent increase of Pcsk5 expression in the developing hindgut. Pcsk5 expression levels were lower in the ARM group from GD14 to GD16 (p?≤?0.05). These results indicate that downregulation of Pcsk5 during cloaca development into the rectum and urethra might be related to the formation of ARMs.     

Binding of proprotein convertase subtilisin/kexin type 9 to epidermal growth factor-like repeat A of low density lipoprotein receptor decreases receptor recycling and increases degradation

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes degradation of hepatic low density lipoprotein receptors (LDLR), the major route of clearance of circulating cholesterol. Gain-of-function mutations in PCSK9 cause hypercholesterolemia and premature atherosclerosis, whereas loss-of-function mutations result in hypocholesterolemia and protection from heart disease. Recombinant human PCSK9 binds the LDLR on the surface of cultured hepatocytes and promotes degradation of the receptor after internalization. Here we localized the site of binding of PCSK9 within the extracellular domain of the LDLR and determined the fate of the receptor after PCSK9 binding. Recombinant human PCSK9 interacted in a sequence-specific manner with the first epidermal growth factor-like repeat (EGF-A) in the EGF homology domain of the human LDLR. Similar binding specificity was observed between PCSK9 and purified EGF-A. Binding to EGF-A was calcium-dependent and increased dramatically with reduction in pH from 7 to 5.2. The addition of PCSK9, but not heat-inactivated PCSK9, to the medium of cultured hepatocytes resulted in redistribution of the receptor from the plasma membrane to lysosomes. These data are consistent with a model in which PCSK9 binding to EGF-A interferes with an acid-dependent conformational change required for receptor recycling. As a consequence, the LDLR is rerouted from the endosome to the lysosome where it is degraded.     

Proprotein convertase subtilisin kexin9 (PCSK9): a novel target for cholesterol regulation

Proprotein Convertase Subtilisin Kexin9 (PCSK9), originally called Neural Apoptosis-Regulated Convertase1 (NARC1), is the latest member of mammalian subtilase super-family. Since its discovery in 2003, it has drawn significant attention because of its function in the degradation of Low Density Lipoprotein Receptor (LDL-R). LDL-R removes circulating LDL-cholesterol (LDL-C) in the blood. Increased level of PCSK9 functional activity will lead to an accumulation of cholesterol in the blood - a high risk factor for cardiovascular disease. This is confirmed by PCSK9 knock out and transgenic animals, various biochemical and clinical studies involving "gain and loss of function" genetic mutations of PCSK9 found in various subset of populations. Owing to this finding, development of strategies for inhibition of PCSK9 function has drawn significant research interest for therapeutic intervention of hypercholesterolemia. Thus PCSK9 is a target for the development of new cholesterol lowering drugs.     

Proprotein convertase subtilisin kexin 9: the third locus implicated in autosomal dominant hypercholesterolemia

PURPOSE OF REVIEW: Autosomal dominant hypercholesterolemia is a genetic disease in which patients have elevated LDL cholesterol levels and premature atherosclerosis. Mutations in the LDL receptor and its ligand apolipoprotein B are causative for autosomal dominant hypercholesterolemia, and the study of this pathway has been crucial to understanding LDL metabolism and receptor-mediated endocytosis in general. Recently, families were identified with a clinical diagnosis of autosomal dominant hypercholesterolemia, but without linkage to the LDL receptor or apolipoprotein B genes. Identification and study of the causative genes in these families should provide additional insights into LDL metabolism. RECENT FINDINGS: Recent microarray studies and database searches identified a novel member of the proprotein convertase family called proprotein convertase subtilisin kexin 9 (PCSK9). A role for PCSK9 in cholesterol metabolism was proposed from the expression studies and confirmed by the discovery that PCSK9 missense mutations were associated with a form of autosomal dominant hypercholesterolemia, Hchola3. The cellular role for PCSK9 and the mechanism behind its mutations are under study, and a role for PCSK9 in regulating LDL receptor protein levels has been demonstrated. SUMMARY: PCSK9 is the third locus implicated in autosomal dominant hypercholesterolemia (Hchola3), and it appears to play an important role in cellular cholesterol metabolism. Understanding the function of PCSK9 will be important for broadening our knowledge of LDL metabolism and may aid in the development of novel hypocholesterolemic agents.