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PCSK9: An enigmatic protease
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Biochimica et Biophysica Acta 1781 (2008) 184 – 191 www.elsevier.com/locate/bbalip
Review
PCSK9: An enigmatic protease Dayami Lopez ⁎ Department of Experimental Therapeutics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA Department of Molecular Medicine, School of Basic Biomedical Sciences, College of Medicine, University of South Florida, Tampa, FL 33612, USA Received 2 August 2007; received in revised form 11 December 2007; accepted 15 January 2008 Available online 2 February 2008
Abstract Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a critical role in cholesterol metabolism by controlling the levels of low density lipoprotein (LDL) particles that circulate in the bloodstream. Several gain-of-function and loss-of-function mutations in the PCSK9 gene, that occur naturally, have been identified and linked to hypercholesterolemia and hypocholesterolemia, respectively. PCSK9 expression has been shown to be regulated by sterol regulatory element binding proteins (SREBPs) and statins similar to other genes involved in cholesterol homeostasis. The most critical finding concerning PCSK9 is that this protease is able to influence the number of LDL receptor molecules expressed on the cell surface. Studies have demonstrated that PCSK9 acts mainly by enhancing degradation of LDL receptor protein in the liver. Inactivation of PCSK9 in mice reduces plasma cholesterol levels primarily by increasing hepatic expression of LDL receptor protein and thereby accelerating clearance of circulating LDL cholesterol. The objective of this review is to summarize the current information related to the regulation and function of PCSK9 and to identify gaps in our present knowledge. © 2008 Elsevier B.V. All rights reserved. Keywords: PCSK9; NARC-1; ADH; LDL; LDL receptor; Hypercholesterolemia; Hypocholesterolemia
1. Introduction
characterized in the LDL receptor gene, which have been grouped in a disorder called familial hypercholesterolemia (FH or FH1) [1,2]. The second gene was recognized when a group of patients presented with a clinical manifestation similar to FH and reduced rates of LDL catabolism, but their LDL receptor activity was normal [3,4]. The gene mutated in this case is apolipoprotein (apo) B-100, the ligand for the LDL receptor [3,4]. Five mutations of the apoB-100 gene have been reported to date [3,4], but the most common is a missense mutation (p. Arg3500Gln) in the LDL receptor-binding domain of apoB-100 [3,4]. Mutations in the apoB-100 gene have been grouped in a disorder called familial defective apoB-100 (FDB or FH2) [3,4]. The third gene recognized in connection with ADH codes for the recently identified proprotein convertase subtilisin/kexin type 9 (PCSK9) protein [5–7].
It is well known that high levels of low density lipoprotein (LDL) cholesterol in the bloodstream are directly correlated with the incidence of cardiovascular disease [1–8]. Variations in plasma LDL cholesterol levels between individuals generally results from genetic factors [1–8]. One of the most frequent inherited disorders shown to cause a selective increase in the serum concentration of LDL cholesterol is autosomal dominant hypercholesterolemia (ADH; MIN # 143890). Patients with ADH are characterized by having extremely elevated total and LDL cholesterol levels in their serum, cholesterol deposits in tissues, tendon and skin xanthomas, arcus cornea, and premature atherosclerosis [1–8]. Thus far, three genes have been connected with ADH [1–8]. The first gene identified in association with this disease was the LDL receptor [1]. Approximately 1000 mutations have been
2. PCSK9 and the proprotein convertase family
⁎ Department of Experimental Therapeutics, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, SRB2, Tampa, FL 33612, USA. Tel.: +1 813 745 6202; fax: +1 813 745 1984. E-mail address: [email protected].
PCSK9, or as also called, neural apoptosis-regulated convertase 1 (NARC-1), is the ninth member of the proprotein convertase (PC) family [9–11]. These proteases are involved in activation, inactivation, and regulation of cellular localization,
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of proteins that transit through the secretory pathway [12–15]. Among the types of proteins processed by PCs are zymogens, prohormones, growth factors, cytokines, adhesion molecules, and receptors [12–15]. Seven of these convertases (PC1/3, PC2, furin, PC4, PACE4, PC5/6, and PC7/LPS) belong to the bacterial/yeast kexin subfamily of subtilases and exhibit cleavage-specificity for basic sites in the consensus sequence (K,R)(Xn)(K,R)↓, where X is any amino acid except Cys and n = 0, 2, 4, or 6 [11–13,16]. The eighth member of this family is the subtilisin kexin isoenzyme-1 (SKI-1)/site-1-protease (S1P), whose protease activity activates sterol regulatory element binding proteins (SREBPs) and brainderived neutrophic factor [17,18]. SK1-1/S1P is very similar to the pyrolysin subfamily of bacterial subtilisin proteases and cleaves after nonbasic residues in the consensus sequence (K,R) (X2)(L,T)↓ [11,13,16,17]. PCSK9 shows highest similarity to the proteinase K subfamily of bacterial subtilisin proteases and cleaves after nonbasic residues as SKI-1/S1P [9–11]. 3. Structure and processing of PCSK9 The human PCSK9 gene is localized on chromosome 1p32.3 [19]. This gene is about 22-kb long and comprises 12 exons encoding a 692 amino acid glycoprotein [9,11,20]. In rodents, the PCSK9 gene encodes a protein of 691 amino acids [21]. This convertase is highly expressed in the liver, small intestine and kidney [9–11]. PCSK9 is synthesized as a 74 kDa soluble zymogen (proPCSK9) that undergoes autocatalytic processing in the endoplasmic reticulum to release the propeptide (14 kDa) from the N-terminal resulting in a processed enzyme of about 60 kDa [9,11,21,22]. This autocleavage is necessary both for activation of the convertase and to allow its departure from the endoplasmic reticulum [9–12]. The cleavage site for PCSK9 autoprocessing has been mapped to SVFAQ↓152, where Q indicates the P1 cleavage position [9,12,22]. The protein domains that comprise PCSK9 are a 30 amino acid signal peptide (SP), the propeptide or inhibitory prodo-
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main (amino acids 31–152), the subtilisin-like catalytic domain (amino acids 153–452), and a cysteine-rich, unique, C-terminal domain (CRD; amino acids 453–692) [23–26] (see Fig. 1). The central part of the catalytic domain consists of seven-stranded parallel β-strand sandwiches between sets of α-helices [24–26]. Three disulfide bonds are found within this region [26]. The N-terminal α-helix has undergone a substantial conformational change moving more than 25 Å from Gln152 and is found in a position similar to that observed in other mature subtilases [26]. It has been proposed that this change in conformation is required for secretion [26]. Like other proteases, the two α-helices form the interface for interaction with the inhibitory prodomain [26]. PCSK9's catalytic triad consists of Asp186, His226 and Ser386, as well as a highly conserved asparagine at position 317 (Asn317) [24–26]. Asn317 forms the oxyanion hole which has been proposed to be critical for catalysis [24–26]. The catalytic triad of PCSK9 is conserved and completely superimposable with other serine protease active sites, suggesting that PCSK9 could be an active protease [24]. One difference occurs in the substrate-binding groove which is mostly neutral in PCSK9 but negatively charged in other convertases [25]. This clearly explains the distinct substrate specificity of PCSK9 [25]. The CRD, which is situated next to the catalytic domain, is made up mainly of three tightly packed subdomains (SD1, SD2, and SD3) arranged in a pseudothreefold with no α-helices [24–26]. Each subdomain consists of three structurally conserved disulfide bonds between the first and six cysteines (457–527, 534–601, 608–679), the second and fifth cysteines (477–526, 552–600, 626–678) and the third and fourth cysteines (486–509, 562–588, 635–654) cross-linking β-sheets β1–β6, β2–β6, and β3–β5, respectively [24–26]. Except for the conserved cysteine residues, there is very little sequence identity between the three subdomains [25,26]. Strand β4 is not stabilized by disulfide bonds and is disordered in SD2, suggesting that there is considerable flexibility in this region [25,26].
Fig. 1. Protein domains of proprotein convertase subtilisin/kexin type 9 (PCSK9). The protein domains that comprise PCSK9 are a 30 amino acid signal peptide (SP), the propeptide, the subtilisin-like catalytic domain, and a cysteine-rich, unique, C-terminal domain. Important residues such as the triad of active site residues: aspartate 186 (D186), histidine 226 (H226) and serine 386 (S386), the highly conserved asparagine 317 (N317), and a N-glycosylation site (Asn533) have been indicated. The autocleavage site has been mapped to SVFAQ↓152.
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The first 30 residues of the prodomain, 31–60, are disordered, but residues 61–152 form a five-stranded antiparallel β-sheet that is covered by two α-helices on one side [25]. The β sheet forms the interface between the prodomain and the catalytic domain through an extensive network of both hydrophobic and electrostatic interactions [26]. The C-terminus of the prodomain, Gln152, forms hydrogen bonds with His226 and occupies the oxyanion hole located between the Ser386 backbone nitrogen and the Asn317 side chain amide [25]. This is consistent with PCSK9's ability to self-cleave at Gln152–Ser153 [25]. After self-cleavage, the prodomain remains in the catalytic groove and must obstruct the access of other proteins and peptides [25]. Then, the noncovalently bound PCSK9/prosegment complex departs from the endoplasmic reticulum and migrates through the secretory pathway until it is secreted into the bloodstream [27–29]. It has been found that normal PCSK9 levels in human plasma range from 50 to 600 ng/ml [30,31]. There are not significant differences in plasma PCSK9 levels between men (608 ng/ml) and women (646 ng/ml) [32]. It is currently unknown whether plasma PCSK9 is associated with LDL particles or exists in a free form. PCSK9 has been also detected in the serum of rats and PCSK9 transgenic mice, and in the culture media of different hepatoma cells lines [11,21,30,33]. In PCSK9, the orientation of the protein domains relative to one another appears to be independent of pH [24]. A calcium atom could be located in one of the known subtilisin calcium-binding sites coordinated by the side chains of Asp60 and Thr335 and the carbonyl oxygens of Ala328, Ala330, Val333, and Cys358 [24]. Since calcium is not a major determinant of PCSK9 activity, it has been suggested that PCSK9 can accommodate a calcium atom, but has little effect on its structure [24]. Asn533, the only glycosylation site in PCSK9, is on the surface of SD2 and is not essential to PCSK9 activity [25]. Interestingly, PCSK9 does not appear to contain a target loop typical of other convertases that serves as the site for a second cleavage that destabilizes the prodomain [25]. In other convertases, the second maturation step of the proteases is generally reached as a result of further proteolytic processing or a change in environmental stimulus, such as a change in pH or increase in calcium concentration [24]. However, in the case of PCSK9, no physiological event has been reported that causes the dissociation of the inhibitory prodomain from the protease [24]. It is important to mention that in many cell lines, PCSK9 is further cleaved to a 53 kDa protein [9,11]. The residue that appears to be involved in the production of this further cleaved form is Arg218 [28]. Not only is this Arg residue conserved between species, but it is also found within an RXXR or KXXXXR sequence which is a basic amino acid recognition site specific for furin and/or PC5/6-like enzymes [28,33]. In fact, studies have demonstrated that membrane bound furin and to a lesser extent the soluble PC5/6A are capable of cleaving PCSK9 through this RXXR motif [28,33]. The 53 kDa PCSK9 form is inactive and unable to bind the prosegment [28,33]. Whether this second cleavage of PCSK9 by furin and/or PC5/6 occurs or is significant in vivo requires further elucidation.
4. Regulation and function of PCSK9 This protease has been shown to be downregulated by cholesterol [21,34], and upregulated by SREBPs [34–37], cholesterol biosynthesis inhibitors [20,38,39], and cholestyramine [40], similar to other genes involved in cholesterol biosynthesis. Sterol regulatory element (SRE) and Sp1 sites have been identified in the promoter of the human, mouse, and rat PCSK9 genes [20,35]. In rat hepatocytes, PCSK9 expression is increased by the liver X receptor agonist T0901317 via SREBP1c, whereas in mice, the hepatic expression of PCSK9 is decreased by long term fasting and restored by re-feeding [35]. Furthermore, inducing diabetes in rats with streptozotocin causes a significant reduction in PCSK9 protein expression [41]. Another regulator appears to be fenofibrate which is able to downregulate PCSK9 expression in controls but not in PPARα-deficient mice [42]. Several findings connect PCSK9 to cholesterol metabolism. In fact, gain-of-function mutations, which are linked to hypercholesterolemia and ADH, as well as loss-of-function mutations, which are directly associated with hypocholesterolemia and a decrease in the risk of developing cardiovascular disease, have been described for this gene [21,25,26,29,30,43–53]. However, the most critical finding concerning PCSK9 is that this protease is able to influence the number of LDL receptor molecules expressed at the cell surface. It has been reported that gain-of-function mutations in PCSK9 have a 23% decreased level of cell surface expression of LDL receptors and a 38% decrease in internalization of LDL, while loss-of-function mutations are associated with a 16% increased level of cell surface LDL receptors and a 35% increased internalization of LDL [44]. Interestingly, two apparently healthy women with mutations affecting both alleles of the PCSK9 gene have been described with an extremely low level of LDL cholesterol (14 mg/dl) [29,54]. These findings not only demonstrate the critical role of PCSK9 in modulating LDL receptor protein expression and plasma LDL cholesterol levels, but also provide evidence that the loss of a functional PCSK9 in humans is not associated with apparent deleterious effects [29,54]. Table 1 summarizes the characteristics of several natural mutations and variations identified for the PCSK9 gene. 5. Structure and cycling of the LDL receptor protein The LDL receptor removes LDL from the circulation through a process that involves endocytosis of the LDL/LDL receptor complex within clathrin-coated regions [55–57]. The cytosolic domain of the LDL receptor contains a 823FDNPVY sequence that is necessary and sufficient for rapid clathrinmediated endocytosis [58,59]. Internalization of the LDL/LDL receptor complex in hepatic cells is controlled mainly by the LDLR adaptor protein 1 (LDLRAP1) [60,61]. The aminoterminal phosphotyrosine-binding (PTB) domain of LDLRAP1 interacts with the FDNPVY sequence of the LDL receptor [60,61], whereas LDLRAP1's carboxy-terminal region has been shown to interact with clathrin [60,61]. The extracellular region of the LDL receptor, on the other hand, is composed of a ligand-binding domain, an EGF
D. Lopez / Biochimica et Biophysica Acta 1781 (2008) 184–191 Table 1 Summary of several natural mutations and variations identified for the PCSK9 gene Mutation/ Notes variation
References
p.S127R Associated with hypercholesterolemia and FH; residue highly conserved among species; undergoes autocatalytic cleavage poorly and is not secreted; has 5× higher affinity for the LDL receptor than the wild-type protein; is able to sharply decrease LDL receptor expression and activity p.D129G Associated with hypercholesterolemia and FH; residue highly conserved among species; undergoes autocatalytic cleavage poorly and is not secreted; is able to sharply decrease LDL receptor expression and activity p.D374Y Associated with hypercholesterolemia and FH; residue conserved in rats and mice; cleaved and secreted normally; has 10× higher affinity for the LDL receptor resulting in decreased receptor recycling and increased degradation p.H553R Associated with hypercholesterolemia; adds positive charges to the CRD that appears to affect its capacity to interact with the LDL receptor p.F216L Associated with hypercholesterolemia and FH; cleaved and secreted normally; located in a catalytic domain loop that is disordered (213–218); appears to prevent cleavage by furin that can inactivate PCSK9 resulting in increased stability or abundance of the mutant and thereby enhancing LDL receptor lowering p.R218S Associated with hypercholesterolemia; residue conserved in rats, mice and chicken; located in a catalytic domain loop that is disordered (213–218); appears to prevent cleavage by furin that can inactivate PCSK9 resulting in increased stability or abundance of the mutant and thereby enhancing LDL receptor lowering p.E670G Identified as the most important tagging polymorphism of the PCSK9 gene that acted as an independent determinant of plasma LDL cholesterol levels and coronary atherosclerosis severity; related to polygenic hypercholesterolemia in men; associated with large-vessel atherosclerosis stroke risk in the Belgian population p.R46L Associated with lower total cholesterol, apoB, and LDL-C levels, and decreased cardiovascular risk (the heterozygous have 15% reduction in LDL-C and 47% reduction in CHD); normally cleaved and secreted; affects an amino acid residue poorly conserved across 12 species of vertebrates; may alter the association between the prodomain and the protease after its activation by autocatalytic cleavage p.G106R Associated with hypocholesterolemia; found in the prodomain; results in a protein that is defective in autocatalysis and is not secreted; produces increased response to statin therapy p.N157K Residue conserved in rats and mice; introduces steric clashes which is probably a hindrance to folding; produces increased response to statin therapy
[21,25,30,33,43]
[25,43]
[25,30,44,45]
[46]
[25,33,44]
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Table 1 (continued ) Mutation/ Notes variation
References
p.Q554E Associated with hypocholesterolemia; is poorly [46] cleaved and poorly secreted; is capable of binding to the cell surface, but does not sort to endosomes p.L253F Associated with hypocholesterolemia; residue [25,26,29,33,46] conserved between species; is poorly cleaved and poorly secreted; is capable of binding to the cell surface, but does not sort to endosomes [25,26,29,33,46,51] p.C679X Associated with hypocholesterolemia; the heterozygous have 40% reduction in LDL-C and 88% reduction in CHD; cleaved normally but retained in the ER and not secreted; introduces a stop codon at residue 679 truncating the protein by 14 amino acids; results in disruption of the penultimate disulfide bond, suggesting a disruption in the folding pattern of the CRD and thus prevention of secretion p.Y142X Associated with hypocholesterolemia; the [51–53] heterozygous have 40% reduction in LDL-C and 88% reduction in CHD; produces no detectable protein; introduces a stop codon at residue 142; may result in a transcript that immediately undergoes nonsense-mediated mRNA decay p.A443T Associated with hypocholesterolemia; located [29,33] close to the S386 of the catalytic triad; normally cleaved and secreted; has higher susceptibility to furin cleavage
[25,33]
[47–49]
[29,33,50,51]
[25,26,46]
[25]
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precursor homology domain, and an O-linked sugar-rich domain [45]. The ligand-binding domain consists of seven LDL receptor type A modules (LA1–LA7), which use three conserved, calcium atom-binding acidic residues for protein– protein interactions [45]. The EGF precursor homology domain contains a six-bladed β-propeller region flanked by cysteinerich EGF repeats [45]. On the cell surface, the LDL receptor's extracellular domain is extended, exposing the ligand-binding domain to LDL [45]. After LDL binds to the receptor, the LDL receptor–ligand complex is internalized and delivered to endosomes [45]. Acidification of the endosome is carried out by the activity of V-type ATPases [57]. This low pH facilitates folding of the LDL receptor back upon itself, bringing the βpropeller region closer to the ligand-binding domain, which results in displacement of LDL [45,57,62]. LDL then moves to the lysozome, where cholesteryl esters are hydrolysed to form cholesterol and free fatty acids, whereas apoB-100 is degraded to free amino acids [57]. Most of the LDL receptor molecules are recycled back to the cell surface, where they can bind and internalize LDL again [45,62]. In each round of the cycle only a very small percentage of LDL receptor molecules are degraded [63]. It has been estimated that each LDL receptor molecule completes about 150 cycles before it is finally degraded [63]. One interesting structural feature is that LA7 forms a rigid structure with EGF-A, located in the EGF precursor homology domain, which is stabilized by calcium and hydrophobic interactions [64,65]. This rigid structure is not affected by a reduction in pH [64,65].
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Treating rats with cholesterol biosynthesis inhibitors significantly increases hepatic LDL receptor mRNA levels [66,67] and the rate of synthesis of LDL receptor protein [66]. However, the steady-state levels of the LDL receptor protein remain unchanged [67]. This apparent contradiction is explained by a corresponding increase in the rate of degradation of the hepatic LDL receptor protein [67]. Increases in the rate of synthesis and degradation of the LDL receptor could result from an increase in receptor cycling in response to treatment with cholesterol biosynthesis inhibitors. An increase in cycling of the LDL receptor is expected to enhance clearance of LDL particles from the circulation. In fact, zaragozic acid A, a cholesterol biosynthesis inhibitor shown to increase both the synthesis and degradation of the LDL receptor protein, causes a significant decrease in serum cholesterol levels [66,67]. 6. Degradation of the LDL receptor protein by PCSK9 The currently information indicates that PCSK9 autocatalysis triggers its progression through the secretory pathway, and that this protease directly interacts with the LDL receptor either within this pathway or at the cell surface [26,31]. It is important to mention that the catalytic activity of PCSK9 appears not to be required for LDL receptor degradation, but it is essential for activation and secretion of PCSK9 [25,43,68,69]. The region within the LDL receptor that is bound by PCSK9 corresponds to EGF-A located within the receptor EGF precursor homology domain [70]. Interestingly, mutations in EGF-A that inhibit PCSK9 binding also prevent PCSK9-induced degradation of the LDL receptor in cells [70]. There is a highly conserved leucine residue (Leu318) located between cysteine residues 4 (Cys317) and 5 (Cys319) of the LDL receptor EGF-A that contributes to the specificity of the PCSK9–LDL receptor interaction [70]. Furthermore, two calcium-binding sites located at the N terminus of EGF-A and at the interface with EGF-B have been shown to be necessary for PCSK9 to bind EGF-A [70]. Deletion of the receptor ligand-binding domain does not significantly affect PCSK9 binding, suggesting that PCSK9 can bind to the LDL receptor independently of lipoproteins [70]. However, it is currently unknown whether the LDL receptor is able to bind lipoproteins and PCSK9 simultaneously or whether PCSK9 binding to EGF-A affects lipoprotein binding to the receptor. [70] The interaction of PCSK9 with the LDL receptor occurs with 1:1 stoichiometry and a Kd of 170 nM at neutral pH [25]. Considering that the concentration of PCSK9 in the plasma is very low, it has been suggested that about 1%–5% of the LDL receptor molecules are bound by PCSK9 at neutral pH [25]. It may be possible that other plasma factors affect the affinity of this binding in vivo, but additional studies are required to determine this [25]. The PCSK9/LDL receptor complex subsequently enters the endosomal pathway [26,31]. In contrast to the binding of LDL to the LDL receptor, PCSK9–LDL receptor affinity is increased in the endosome [25,26,70]. Failure to release PCSK9 may prevent receptor recycling and direct the PCSK9/ LDL receptor complex to the lysozome, where degradation of
the LDL receptor occurs [25,26]. Findings also demonstrate that the LDL receptor plays a critical role in facilitating the trafficking of PCSK9 from the endoplasmic reticulum to downstream sites in the secretory and endocytic pathways [46]. In fact, it has been shown that in cells lacking the LDL receptor, PCSK9 is mostly found in the endoplasmic reticulum, whereas when both proteins are present, PCSK9 is always found colocalizing with the LDL receptor [46]. Furthermore, the endosomal immunoreactivity of these proteins could be enhanced in the presence of NH4Cl [46], which has been shown previously to prevent PCSK9dependent degradation of the LDL receptor [9]. Another critical finding is that LDL and VLDL affect the LDL receptor-binding affinity for PCSK9 suggesting that the contribution of secreted PCSK9 to LDL receptor lowering may be reduced by the plasma levels of LDL [71]. It is important to mention that overnight incubation of exogenous PCSK9 with COS-1 cells led to cellular association but did not result in the degradation of endogenous LDL receptor [72], suggesting that PCSK9 may require a tissue specific factor or factors to effectively degrade the LDL receptor protein. However, if a tissue specific factor is required, it does not appear to be secreted into the serum along with PCSK9 [73]. Instead, this tissue specific factor or factors could be bound at the cell surface or endogenously expressed inside responsive cells. Another possible explanation for the lack of PCSK9's effect on the LDL receptor protein in these cells could be that the receptor is internalized differently in unresponsive and responsive cells [73]. In fact, blocking the clathrin-coated pit route with hypertonic medium in HepG2 (responsive) cells did not prevent internalization and degradation of the LDL receptor protein by PCSK9 suggesting the presence of a clathrin-independent endocytotic pathway in these cells [73]. Interestingly, studies performed in fibroblasts [74] and in CHO cells [75] demonstrated that the LDL receptor is mainly located within clathrin-coated pits, whereas in hamster and rat liver, the LDL receptor is mainly associated with caveolae [76]. In addition to the LDL receptor, PCSK9 also appears to affect the levels of ApoER2 and VLDL receptor but with less affectivity [72]. 7. PCSK9 and levels of ApoB-100 In addition to its effects on the LDL receptor protein, PCSK9 seems to influence the levels of circulating apoB100 containing lipoproteins. This is supported by the finding that elimination of LDL receptor protein through overexpression of PCSK9 in mice results in a 9-fold increase in circulating LDL cholesterol, while the total lack of LDL receptor protein in LDL receptor KO mice only results in a 2-fold increase in LDL cholesterol [42]. Consistent with this view, cells expressing the pD374Y and pS127R PCSK9 mutants secrete more apoB-100 than control cells or cells expressing WT PCSK9 [42,77,78]. Similarly, patients having these mutations show an increase in apoB-100 production [77–79]. No other gain-of-function mutation seems to affect apoB-100 production [78]. Furthermore, the LDL receptor
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itself has been shown to directly affect apoB-100 levels by promoting intracellular degradation of this apolipoprotein and by enhancing apoB-100 recapture before it enters the circulation from the liver [54,80–82]. Overexpressing PCSK9 in LDL receptor KO mice does not further alter apoB-100 secretion [27]. Thus, further analysis will be required to determine whether PCSK9 affects apoB-100 levels directly or indirectly by regulating levels of LDL receptor protein.
[4]
[5]
[6]
8. Conclusion Even though tremendous advances in this area of research have been made, several key questions still need to be answered related to the convertase PCSK9. First, the substrate(s) of PCSK9, if any, need(s) to be identified. This is critical considering that not only the LDL receptor does not appear to be a substrate for this convertase, but also the enzymatic activity of PCSK9 does not seem to be required for degradation of the LDL receptor. Identification of PCSK9's substrate(s) will help to answer questions such as 1) how do natural mutations of PCSK9 enhance/reduce the activity of PCSK9 and 2) what other pathways are regulated by PCSK9 (i.e., liver regeneration). If PCSK9 is indeed an active protease, it will be necessary to determine the mechanism(s) of activation of the protease. Perhaps, PCSK9 interacts with an unidentified partner that facilitates the release of the prodomain activating PCSK9 as a protease. Another open question is whether PCSK9 acts primary as an intracellular or as a secreted factor. Furthermore, the specific interactions between PCSK9 and the LDL receptor need to be further elucidated in order to completely understand how PCSK9 regulates extracellular levels of the LDL receptor. An intriguing question is why are more LDL receptor protein produced by the liver when more PCSK9 are synthesized resulting in degradation of the LDL receptor protein. Apparently, PCSK9 serves as a control point to reduce uptake of cholesterol by degrading LDL receptors, thereby preventing excessive cholesterol accumulation within the cell. Acknowledgment This work was supported in part by Grant # 0555334B from the American Heart Association Florida-Affiliate and Bridge/ Departmental funding from the University of South Florida, College of Medicine and Department of Molecular Medicine.
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Biochimica et Biophysica Acta 1781 (2008) 184 – 191 www.elsevier.com/locate/bbalip
Review
PCSK9: An enigmatic protease Dayami Lopez ⁎ Department of Experimental Therapeutics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA Department of Molecular Medicine, School of Basic Biomedical Sciences, College of Medicine, University of South Florida, Tampa, FL 33612, USA Received 2 August 2007; received in revised form 11 December 2007; accepted 15 January 2008 Available online 2 February 2008
Abstract Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a critical role in cholesterol metabolism by controlling the levels of low density lipoprotein (LDL) particles that circulate in the bloodstream. Several gain-of-function and loss-of-function mutations in the PCSK9 gene, that occur naturally, have been identified and linked to hypercholesterolemia and hypocholesterolemia, respectively. PCSK9 expression has been shown to be regulated by sterol regulatory element binding proteins (SREBPs) and statins similar to other genes involved in cholesterol homeostasis. The most critical finding concerning PCSK9 is that this protease is able to influence the number of LDL receptor molecules expressed on the cell surface. Studies have demonstrated that PCSK9 acts mainly by enhancing degradation of LDL receptor protein in the liver. Inactivation of PCSK9 in mice reduces plasma cholesterol levels primarily by increasing hepatic expression of LDL receptor protein and thereby accelerating clearance of circulating LDL cholesterol. The objective of this review is to summarize the current information related to the regulation and function of PCSK9 and to identify gaps in our present knowledge. © 2008 Elsevier B.V. All rights reserved. Keywords: PCSK9; NARC-1; ADH; LDL; LDL receptor; Hypercholesterolemia; Hypocholesterolemia
1. Introduction
characterized in the LDL receptor gene, which have been grouped in a disorder called familial hypercholesterolemia (FH or FH1) [1,2]. The second gene was recognized when a group of patients presented with a clinical manifestation similar to FH and reduced rates of LDL catabolism, but their LDL receptor activity was normal [3,4]. The gene mutated in this case is apolipoprotein (apo) B-100, the ligand for the LDL receptor [3,4]. Five mutations of the apoB-100 gene have been reported to date [3,4], but the most common is a missense mutation (p. Arg3500Gln) in the LDL receptor-binding domain of apoB-100 [3,4]. Mutations in the apoB-100 gene have been grouped in a disorder called familial defective apoB-100 (FDB or FH2) [3,4]. The third gene recognized in connection with ADH codes for the recently identified proprotein convertase subtilisin/kexin type 9 (PCSK9) protein [5–7].
It is well known that high levels of low density lipoprotein (LDL) cholesterol in the bloodstream are directly correlated with the incidence of cardiovascular disease [1–8]. Variations in plasma LDL cholesterol levels between individuals generally results from genetic factors [1–8]. One of the most frequent inherited disorders shown to cause a selective increase in the serum concentration of LDL cholesterol is autosomal dominant hypercholesterolemia (ADH; MIN # 143890). Patients with ADH are characterized by having extremely elevated total and LDL cholesterol levels in their serum, cholesterol deposits in tissues, tendon and skin xanthomas, arcus cornea, and premature atherosclerosis [1–8]. Thus far, three genes have been connected with ADH [1–8]. The first gene identified in association with this disease was the LDL receptor [1]. Approximately 1000 mutations have been
2. PCSK9 and the proprotein convertase family
⁎ Department of Experimental Therapeutics, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, SRB2, Tampa, FL 33612, USA. Tel.: +1 813 745 6202; fax: +1 813 745 1984. E-mail address: [email protected].
PCSK9, or as also called, neural apoptosis-regulated convertase 1 (NARC-1), is the ninth member of the proprotein convertase (PC) family [9–11]. These proteases are involved in activation, inactivation, and regulation of cellular localization,
1388-1981/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2008.01.003
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of proteins that transit through the secretory pathway [12–15]. Among the types of proteins processed by PCs are zymogens, prohormones, growth factors, cytokines, adhesion molecules, and receptors [12–15]. Seven of these convertases (PC1/3, PC2, furin, PC4, PACE4, PC5/6, and PC7/LPS) belong to the bacterial/yeast kexin subfamily of subtilases and exhibit cleavage-specificity for basic sites in the consensus sequence (K,R)(Xn)(K,R)↓, where X is any amino acid except Cys and n = 0, 2, 4, or 6 [11–13,16]. The eighth member of this family is the subtilisin kexin isoenzyme-1 (SKI-1)/site-1-protease (S1P), whose protease activity activates sterol regulatory element binding proteins (SREBPs) and brainderived neutrophic factor [17,18]. SK1-1/S1P is very similar to the pyrolysin subfamily of bacterial subtilisin proteases and cleaves after nonbasic residues in the consensus sequence (K,R) (X2)(L,T)↓ [11,13,16,17]. PCSK9 shows highest similarity to the proteinase K subfamily of bacterial subtilisin proteases and cleaves after nonbasic residues as SKI-1/S1P [9–11]. 3. Structure and processing of PCSK9 The human PCSK9 gene is localized on chromosome 1p32.3 [19]. This gene is about 22-kb long and comprises 12 exons encoding a 692 amino acid glycoprotein [9,11,20]. In rodents, the PCSK9 gene encodes a protein of 691 amino acids [21]. This convertase is highly expressed in the liver, small intestine and kidney [9–11]. PCSK9 is synthesized as a 74 kDa soluble zymogen (proPCSK9) that undergoes autocatalytic processing in the endoplasmic reticulum to release the propeptide (14 kDa) from the N-terminal resulting in a processed enzyme of about 60 kDa [9,11,21,22]. This autocleavage is necessary both for activation of the convertase and to allow its departure from the endoplasmic reticulum [9–12]. The cleavage site for PCSK9 autoprocessing has been mapped to SVFAQ↓152, where Q indicates the P1 cleavage position [9,12,22]. The protein domains that comprise PCSK9 are a 30 amino acid signal peptide (SP), the propeptide or inhibitory prodo-
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main (amino acids 31–152), the subtilisin-like catalytic domain (amino acids 153–452), and a cysteine-rich, unique, C-terminal domain (CRD; amino acids 453–692) [23–26] (see Fig. 1). The central part of the catalytic domain consists of seven-stranded parallel β-strand sandwiches between sets of α-helices [24–26]. Three disulfide bonds are found within this region [26]. The N-terminal α-helix has undergone a substantial conformational change moving more than 25 Å from Gln152 and is found in a position similar to that observed in other mature subtilases [26]. It has been proposed that this change in conformation is required for secretion [26]. Like other proteases, the two α-helices form the interface for interaction with the inhibitory prodomain [26]. PCSK9's catalytic triad consists of Asp186, His226 and Ser386, as well as a highly conserved asparagine at position 317 (Asn317) [24–26]. Asn317 forms the oxyanion hole which has been proposed to be critical for catalysis [24–26]. The catalytic triad of PCSK9 is conserved and completely superimposable with other serine protease active sites, suggesting that PCSK9 could be an active protease [24]. One difference occurs in the substrate-binding groove which is mostly neutral in PCSK9 but negatively charged in other convertases [25]. This clearly explains the distinct substrate specificity of PCSK9 [25]. The CRD, which is situated next to the catalytic domain, is made up mainly of three tightly packed subdomains (SD1, SD2, and SD3) arranged in a pseudothreefold with no α-helices [24–26]. Each subdomain consists of three structurally conserved disulfide bonds between the first and six cysteines (457–527, 534–601, 608–679), the second and fifth cysteines (477–526, 552–600, 626–678) and the third and fourth cysteines (486–509, 562–588, 635–654) cross-linking β-sheets β1–β6, β2–β6, and β3–β5, respectively [24–26]. Except for the conserved cysteine residues, there is very little sequence identity between the three subdomains [25,26]. Strand β4 is not stabilized by disulfide bonds and is disordered in SD2, suggesting that there is considerable flexibility in this region [25,26].
Fig. 1. Protein domains of proprotein convertase subtilisin/kexin type 9 (PCSK9). The protein domains that comprise PCSK9 are a 30 amino acid signal peptide (SP), the propeptide, the subtilisin-like catalytic domain, and a cysteine-rich, unique, C-terminal domain. Important residues such as the triad of active site residues: aspartate 186 (D186), histidine 226 (H226) and serine 386 (S386), the highly conserved asparagine 317 (N317), and a N-glycosylation site (Asn533) have been indicated. The autocleavage site has been mapped to SVFAQ↓152.
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The first 30 residues of the prodomain, 31–60, are disordered, but residues 61–152 form a five-stranded antiparallel β-sheet that is covered by two α-helices on one side [25]. The β sheet forms the interface between the prodomain and the catalytic domain through an extensive network of both hydrophobic and electrostatic interactions [26]. The C-terminus of the prodomain, Gln152, forms hydrogen bonds with His226 and occupies the oxyanion hole located between the Ser386 backbone nitrogen and the Asn317 side chain amide [25]. This is consistent with PCSK9's ability to self-cleave at Gln152–Ser153 [25]. After self-cleavage, the prodomain remains in the catalytic groove and must obstruct the access of other proteins and peptides [25]. Then, the noncovalently bound PCSK9/prosegment complex departs from the endoplasmic reticulum and migrates through the secretory pathway until it is secreted into the bloodstream [27–29]. It has been found that normal PCSK9 levels in human plasma range from 50 to 600 ng/ml [30,31]. There are not significant differences in plasma PCSK9 levels between men (608 ng/ml) and women (646 ng/ml) [32]. It is currently unknown whether plasma PCSK9 is associated with LDL particles or exists in a free form. PCSK9 has been also detected in the serum of rats and PCSK9 transgenic mice, and in the culture media of different hepatoma cells lines [11,21,30,33]. In PCSK9, the orientation of the protein domains relative to one another appears to be independent of pH [24]. A calcium atom could be located in one of the known subtilisin calcium-binding sites coordinated by the side chains of Asp60 and Thr335 and the carbonyl oxygens of Ala328, Ala330, Val333, and Cys358 [24]. Since calcium is not a major determinant of PCSK9 activity, it has been suggested that PCSK9 can accommodate a calcium atom, but has little effect on its structure [24]. Asn533, the only glycosylation site in PCSK9, is on the surface of SD2 and is not essential to PCSK9 activity [25]. Interestingly, PCSK9 does not appear to contain a target loop typical of other convertases that serves as the site for a second cleavage that destabilizes the prodomain [25]. In other convertases, the second maturation step of the proteases is generally reached as a result of further proteolytic processing or a change in environmental stimulus, such as a change in pH or increase in calcium concentration [24]. However, in the case of PCSK9, no physiological event has been reported that causes the dissociation of the inhibitory prodomain from the protease [24]. It is important to mention that in many cell lines, PCSK9 is further cleaved to a 53 kDa protein [9,11]. The residue that appears to be involved in the production of this further cleaved form is Arg218 [28]. Not only is this Arg residue conserved between species, but it is also found within an RXXR or KXXXXR sequence which is a basic amino acid recognition site specific for furin and/or PC5/6-like enzymes [28,33]. In fact, studies have demonstrated that membrane bound furin and to a lesser extent the soluble PC5/6A are capable of cleaving PCSK9 through this RXXR motif [28,33]. The 53 kDa PCSK9 form is inactive and unable to bind the prosegment [28,33]. Whether this second cleavage of PCSK9 by furin and/or PC5/6 occurs or is significant in vivo requires further elucidation.
4. Regulation and function of PCSK9 This protease has been shown to be downregulated by cholesterol [21,34], and upregulated by SREBPs [34–37], cholesterol biosynthesis inhibitors [20,38,39], and cholestyramine [40], similar to other genes involved in cholesterol biosynthesis. Sterol regulatory element (SRE) and Sp1 sites have been identified in the promoter of the human, mouse, and rat PCSK9 genes [20,35]. In rat hepatocytes, PCSK9 expression is increased by the liver X receptor agonist T0901317 via SREBP1c, whereas in mice, the hepatic expression of PCSK9 is decreased by long term fasting and restored by re-feeding [35]. Furthermore, inducing diabetes in rats with streptozotocin causes a significant reduction in PCSK9 protein expression [41]. Another regulator appears to be fenofibrate which is able to downregulate PCSK9 expression in controls but not in PPARα-deficient mice [42]. Several findings connect PCSK9 to cholesterol metabolism. In fact, gain-of-function mutations, which are linked to hypercholesterolemia and ADH, as well as loss-of-function mutations, which are directly associated with hypocholesterolemia and a decrease in the risk of developing cardiovascular disease, have been described for this gene [21,25,26,29,30,43–53]. However, the most critical finding concerning PCSK9 is that this protease is able to influence the number of LDL receptor molecules expressed at the cell surface. It has been reported that gain-of-function mutations in PCSK9 have a 23% decreased level of cell surface expression of LDL receptors and a 38% decrease in internalization of LDL, while loss-of-function mutations are associated with a 16% increased level of cell surface LDL receptors and a 35% increased internalization of LDL [44]. Interestingly, two apparently healthy women with mutations affecting both alleles of the PCSK9 gene have been described with an extremely low level of LDL cholesterol (14 mg/dl) [29,54]. These findings not only demonstrate the critical role of PCSK9 in modulating LDL receptor protein expression and plasma LDL cholesterol levels, but also provide evidence that the loss of a functional PCSK9 in humans is not associated with apparent deleterious effects [29,54]. Table 1 summarizes the characteristics of several natural mutations and variations identified for the PCSK9 gene. 5. Structure and cycling of the LDL receptor protein The LDL receptor removes LDL from the circulation through a process that involves endocytosis of the LDL/LDL receptor complex within clathrin-coated regions [55–57]. The cytosolic domain of the LDL receptor contains a 823FDNPVY sequence that is necessary and sufficient for rapid clathrinmediated endocytosis [58,59]. Internalization of the LDL/LDL receptor complex in hepatic cells is controlled mainly by the LDLR adaptor protein 1 (LDLRAP1) [60,61]. The aminoterminal phosphotyrosine-binding (PTB) domain of LDLRAP1 interacts with the FDNPVY sequence of the LDL receptor [60,61], whereas LDLRAP1's carboxy-terminal region has been shown to interact with clathrin [60,61]. The extracellular region of the LDL receptor, on the other hand, is composed of a ligand-binding domain, an EGF
D. Lopez / Biochimica et Biophysica Acta 1781 (2008) 184–191 Table 1 Summary of several natural mutations and variations identified for the PCSK9 gene Mutation/ Notes variation
References
p.S127R Associated with hypercholesterolemia and FH; residue highly conserved among species; undergoes autocatalytic cleavage poorly and is not secreted; has 5× higher affinity for the LDL receptor than the wild-type protein; is able to sharply decrease LDL receptor expression and activity p.D129G Associated with hypercholesterolemia and FH; residue highly conserved among species; undergoes autocatalytic cleavage poorly and is not secreted; is able to sharply decrease LDL receptor expression and activity p.D374Y Associated with hypercholesterolemia and FH; residue conserved in rats and mice; cleaved and secreted normally; has 10× higher affinity for the LDL receptor resulting in decreased receptor recycling and increased degradation p.H553R Associated with hypercholesterolemia; adds positive charges to the CRD that appears to affect its capacity to interact with the LDL receptor p.F216L Associated with hypercholesterolemia and FH; cleaved and secreted normally; located in a catalytic domain loop that is disordered (213–218); appears to prevent cleavage by furin that can inactivate PCSK9 resulting in increased stability or abundance of the mutant and thereby enhancing LDL receptor lowering p.R218S Associated with hypercholesterolemia; residue conserved in rats, mice and chicken; located in a catalytic domain loop that is disordered (213–218); appears to prevent cleavage by furin that can inactivate PCSK9 resulting in increased stability or abundance of the mutant and thereby enhancing LDL receptor lowering p.E670G Identified as the most important tagging polymorphism of the PCSK9 gene that acted as an independent determinant of plasma LDL cholesterol levels and coronary atherosclerosis severity; related to polygenic hypercholesterolemia in men; associated with large-vessel atherosclerosis stroke risk in the Belgian population p.R46L Associated with lower total cholesterol, apoB, and LDL-C levels, and decreased cardiovascular risk (the heterozygous have 15% reduction in LDL-C and 47% reduction in CHD); normally cleaved and secreted; affects an amino acid residue poorly conserved across 12 species of vertebrates; may alter the association between the prodomain and the protease after its activation by autocatalytic cleavage p.G106R Associated with hypocholesterolemia; found in the prodomain; results in a protein that is defective in autocatalysis and is not secreted; produces increased response to statin therapy p.N157K Residue conserved in rats and mice; introduces steric clashes which is probably a hindrance to folding; produces increased response to statin therapy
[21,25,30,33,43]
[25,43]
[25,30,44,45]
[46]
[25,33,44]
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Table 1 (continued ) Mutation/ Notes variation
References
p.Q554E Associated with hypocholesterolemia; is poorly [46] cleaved and poorly secreted; is capable of binding to the cell surface, but does not sort to endosomes p.L253F Associated with hypocholesterolemia; residue [25,26,29,33,46] conserved between species; is poorly cleaved and poorly secreted; is capable of binding to the cell surface, but does not sort to endosomes [25,26,29,33,46,51] p.C679X Associated with hypocholesterolemia; the heterozygous have 40% reduction in LDL-C and 88% reduction in CHD; cleaved normally but retained in the ER and not secreted; introduces a stop codon at residue 679 truncating the protein by 14 amino acids; results in disruption of the penultimate disulfide bond, suggesting a disruption in the folding pattern of the CRD and thus prevention of secretion p.Y142X Associated with hypocholesterolemia; the [51–53] heterozygous have 40% reduction in LDL-C and 88% reduction in CHD; produces no detectable protein; introduces a stop codon at residue 142; may result in a transcript that immediately undergoes nonsense-mediated mRNA decay p.A443T Associated with hypocholesterolemia; located [29,33] close to the S386 of the catalytic triad; normally cleaved and secreted; has higher susceptibility to furin cleavage
[25,33]
[47–49]
[29,33,50,51]
[25,26,46]
[25]
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precursor homology domain, and an O-linked sugar-rich domain [45]. The ligand-binding domain consists of seven LDL receptor type A modules (LA1–LA7), which use three conserved, calcium atom-binding acidic residues for protein– protein interactions [45]. The EGF precursor homology domain contains a six-bladed β-propeller region flanked by cysteinerich EGF repeats [45]. On the cell surface, the LDL receptor's extracellular domain is extended, exposing the ligand-binding domain to LDL [45]. After LDL binds to the receptor, the LDL receptor–ligand complex is internalized and delivered to endosomes [45]. Acidification of the endosome is carried out by the activity of V-type ATPases [57]. This low pH facilitates folding of the LDL receptor back upon itself, bringing the βpropeller region closer to the ligand-binding domain, which results in displacement of LDL [45,57,62]. LDL then moves to the lysozome, where cholesteryl esters are hydrolysed to form cholesterol and free fatty acids, whereas apoB-100 is degraded to free amino acids [57]. Most of the LDL receptor molecules are recycled back to the cell surface, where they can bind and internalize LDL again [45,62]. In each round of the cycle only a very small percentage of LDL receptor molecules are degraded [63]. It has been estimated that each LDL receptor molecule completes about 150 cycles before it is finally degraded [63]. One interesting structural feature is that LA7 forms a rigid structure with EGF-A, located in the EGF precursor homology domain, which is stabilized by calcium and hydrophobic interactions [64,65]. This rigid structure is not affected by a reduction in pH [64,65].
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Treating rats with cholesterol biosynthesis inhibitors significantly increases hepatic LDL receptor mRNA levels [66,67] and the rate of synthesis of LDL receptor protein [66]. However, the steady-state levels of the LDL receptor protein remain unchanged [67]. This apparent contradiction is explained by a corresponding increase in the rate of degradation of the hepatic LDL receptor protein [67]. Increases in the rate of synthesis and degradation of the LDL receptor could result from an increase in receptor cycling in response to treatment with cholesterol biosynthesis inhibitors. An increase in cycling of the LDL receptor is expected to enhance clearance of LDL particles from the circulation. In fact, zaragozic acid A, a cholesterol biosynthesis inhibitor shown to increase both the synthesis and degradation of the LDL receptor protein, causes a significant decrease in serum cholesterol levels [66,67]. 6. Degradation of the LDL receptor protein by PCSK9 The currently information indicates that PCSK9 autocatalysis triggers its progression through the secretory pathway, and that this protease directly interacts with the LDL receptor either within this pathway or at the cell surface [26,31]. It is important to mention that the catalytic activity of PCSK9 appears not to be required for LDL receptor degradation, but it is essential for activation and secretion of PCSK9 [25,43,68,69]. The region within the LDL receptor that is bound by PCSK9 corresponds to EGF-A located within the receptor EGF precursor homology domain [70]. Interestingly, mutations in EGF-A that inhibit PCSK9 binding also prevent PCSK9-induced degradation of the LDL receptor in cells [70]. There is a highly conserved leucine residue (Leu318) located between cysteine residues 4 (Cys317) and 5 (Cys319) of the LDL receptor EGF-A that contributes to the specificity of the PCSK9–LDL receptor interaction [70]. Furthermore, two calcium-binding sites located at the N terminus of EGF-A and at the interface with EGF-B have been shown to be necessary for PCSK9 to bind EGF-A [70]. Deletion of the receptor ligand-binding domain does not significantly affect PCSK9 binding, suggesting that PCSK9 can bind to the LDL receptor independently of lipoproteins [70]. However, it is currently unknown whether the LDL receptor is able to bind lipoproteins and PCSK9 simultaneously or whether PCSK9 binding to EGF-A affects lipoprotein binding to the receptor. [70] The interaction of PCSK9 with the LDL receptor occurs with 1:1 stoichiometry and a Kd of 170 nM at neutral pH [25]. Considering that the concentration of PCSK9 in the plasma is very low, it has been suggested that about 1%–5% of the LDL receptor molecules are bound by PCSK9 at neutral pH [25]. It may be possible that other plasma factors affect the affinity of this binding in vivo, but additional studies are required to determine this [25]. The PCSK9/LDL receptor complex subsequently enters the endosomal pathway [26,31]. In contrast to the binding of LDL to the LDL receptor, PCSK9–LDL receptor affinity is increased in the endosome [25,26,70]. Failure to release PCSK9 may prevent receptor recycling and direct the PCSK9/ LDL receptor complex to the lysozome, where degradation of
the LDL receptor occurs [25,26]. Findings also demonstrate that the LDL receptor plays a critical role in facilitating the trafficking of PCSK9 from the endoplasmic reticulum to downstream sites in the secretory and endocytic pathways [46]. In fact, it has been shown that in cells lacking the LDL receptor, PCSK9 is mostly found in the endoplasmic reticulum, whereas when both proteins are present, PCSK9 is always found colocalizing with the LDL receptor [46]. Furthermore, the endosomal immunoreactivity of these proteins could be enhanced in the presence of NH4Cl [46], which has been shown previously to prevent PCSK9dependent degradation of the LDL receptor [9]. Another critical finding is that LDL and VLDL affect the LDL receptor-binding affinity for PCSK9 suggesting that the contribution of secreted PCSK9 to LDL receptor lowering may be reduced by the plasma levels of LDL [71]. It is important to mention that overnight incubation of exogenous PCSK9 with COS-1 cells led to cellular association but did not result in the degradation of endogenous LDL receptor [72], suggesting that PCSK9 may require a tissue specific factor or factors to effectively degrade the LDL receptor protein. However, if a tissue specific factor is required, it does not appear to be secreted into the serum along with PCSK9 [73]. Instead, this tissue specific factor or factors could be bound at the cell surface or endogenously expressed inside responsive cells. Another possible explanation for the lack of PCSK9's effect on the LDL receptor protein in these cells could be that the receptor is internalized differently in unresponsive and responsive cells [73]. In fact, blocking the clathrin-coated pit route with hypertonic medium in HepG2 (responsive) cells did not prevent internalization and degradation of the LDL receptor protein by PCSK9 suggesting the presence of a clathrin-independent endocytotic pathway in these cells [73]. Interestingly, studies performed in fibroblasts [74] and in CHO cells [75] demonstrated that the LDL receptor is mainly located within clathrin-coated pits, whereas in hamster and rat liver, the LDL receptor is mainly associated with caveolae [76]. In addition to the LDL receptor, PCSK9 also appears to affect the levels of ApoER2 and VLDL receptor but with less affectivity [72]. 7. PCSK9 and levels of ApoB-100 In addition to its effects on the LDL receptor protein, PCSK9 seems to influence the levels of circulating apoB100 containing lipoproteins. This is supported by the finding that elimination of LDL receptor protein through overexpression of PCSK9 in mice results in a 9-fold increase in circulating LDL cholesterol, while the total lack of LDL receptor protein in LDL receptor KO mice only results in a 2-fold increase in LDL cholesterol [42]. Consistent with this view, cells expressing the pD374Y and pS127R PCSK9 mutants secrete more apoB-100 than control cells or cells expressing WT PCSK9 [42,77,78]. Similarly, patients having these mutations show an increase in apoB-100 production [77–79]. No other gain-of-function mutation seems to affect apoB-100 production [78]. Furthermore, the LDL receptor
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itself has been shown to directly affect apoB-100 levels by promoting intracellular degradation of this apolipoprotein and by enhancing apoB-100 recapture before it enters the circulation from the liver [54,80–82]. Overexpressing PCSK9 in LDL receptor KO mice does not further alter apoB-100 secretion [27]. Thus, further analysis will be required to determine whether PCSK9 affects apoB-100 levels directly or indirectly by regulating levels of LDL receptor protein.
[4]
[5]
[6]
8. Conclusion Even though tremendous advances in this area of research have been made, several key questions still need to be answered related to the convertase PCSK9. First, the substrate(s) of PCSK9, if any, need(s) to be identified. This is critical considering that not only the LDL receptor does not appear to be a substrate for this convertase, but also the enzymatic activity of PCSK9 does not seem to be required for degradation of the LDL receptor. Identification of PCSK9's substrate(s) will help to answer questions such as 1) how do natural mutations of PCSK9 enhance/reduce the activity of PCSK9 and 2) what other pathways are regulated by PCSK9 (i.e., liver regeneration). If PCSK9 is indeed an active protease, it will be necessary to determine the mechanism(s) of activation of the protease. Perhaps, PCSK9 interacts with an unidentified partner that facilitates the release of the prodomain activating PCSK9 as a protease. Another open question is whether PCSK9 acts primary as an intracellular or as a secreted factor. Furthermore, the specific interactions between PCSK9 and the LDL receptor need to be further elucidated in order to completely understand how PCSK9 regulates extracellular levels of the LDL receptor. An intriguing question is why are more LDL receptor protein produced by the liver when more PCSK9 are synthesized resulting in degradation of the LDL receptor protein. Apparently, PCSK9 serves as a control point to reduce uptake of cholesterol by degrading LDL receptors, thereby preventing excessive cholesterol accumulation within the cell. Acknowledgment This work was supported in part by Grant # 0555334B from the American Heart Association Florida-Affiliate and Bridge/ Departmental funding from the University of South Florida, College of Medicine and Department of Molecular Medicine.
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