Structural requirements for PCSK9-mediated degradation of the low-density lipoprotein receptor

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that controls plasma LDL cholesterol levels by posttranslational regulation of the LDL receptor (LDLR). Previously, we showed that PCSK9 binds specifically to an EGF-like repeat (EGF-A) in LDLR and reroutes the receptor from endosomes to lysosomes rather than to the cell surface. Here, we defined the regions in LDLR and PCSK9 that are required for receptor degradation and examined the relationship between PCSK9 binding and LDLR conformation. Addition of PCSK9 to cultured hepatocytes promoted degradation of WT LDLR and of receptors lacking up to four ligand binding domains, EGF-B or the clustered O-linked sugar region. In contrast, LDLRs lacking the entire ligand binding domain or the ␤-propeller domain failed to be degraded, although they bound and internalized PCSK9. Using gel filtration chromatography, we assessed the effects of PCSK9 binding on an acid-dependent conformational change that happens in the extracellular domain of the LDLR. Although PCSK9 prevented the reduction in hydrodynamic radius of the receptor that occurs at a reduced pH, the effect was not sufficient for LDLR degradation. A truncated version of PCSK9 containing the prodomain and the catalytic domain, but not the C-terminal domain, bound the receptor but did not stimulate LDLR degradation. Thus, domains in both the LDLR and PCSK9 that are not required for binding (or internalization) are essential for PCSK9-mediated degradation of the LDLR.cholesterol ͉ proprotein convertase ͉ ␤-propeller ͉ endocytosis P lasma levels of LDL cholesterol are directly related to the risk of coronary atherosclerosis. The tight association between LDL levels and ischemic heart disease is illustrated by the fact that all monogenic forms of severe hypercholesterolemia are accompanied by premature coronary atherosclerosis. The major pathway for removal of LDL from the circulation is by LDL receptor (LDLR)-mediated endocytosis in the liver. Mutations in the LDLR (1), the ligand for the LDLR (apolipoprotein B-100) (2), or an LDLR adaptor protein (ARH/LDLRAP) (3) all cause hypercholesterolemia by reducing the clearance of circulating LDL. In 2003, a third form of autosomal-dominant hypercholesterolemia was identified that is caused by selected missense mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9) (4).PCSK9 is a 692-aa secreted glycoprotein composed of a 22-aa signal sequence, a prodomain, a catalytic domain that resembles the proteinase K family of subtilisin-like serine proteases, and a cysteine-and histidine-rich C-terminal domain that is unique to this member of the proprotein convertase family (5). In the endoplasmic reticulum, PCSK9 undergoes autocatalytic cleavage between residues 151 and 152 (5, 6), releasing the N-terminal prodomain, which remains noncovalently attached to the catalytic domain, physically shielding the catalytic triad as the protein transits through the secretory pathway (7,8). PCSK9 circulates in the blood (9) and binds the extracellular domain ...

Novel Domain Interaction Regulates Secretion of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Protein

“…Interestingly, the D70P mutant also did not impair PCSK9 self-processing and secretion. This suggests that 70 PPWRL 74 can preserve the 3 10 It is known that deletion of the entire CD protein produces a truncated protein that is cleaved and secreted, suggesting that CD is not required for PCSK9 secretion (39). However, it is also known that the C679X mutant, a natural loss-of-function mutant causing low cholesterol in humans (12,41), is not secreted by cells even though the autocleavage of PD is preserved (38).…”

Mapping the Binding Region on the Low Density Lipoprotein Receptor for Blood Coagulation Factor VIII

“…In LDLR, the RAP-binding region comprises the sites for FVIII (present study) and apoE (CR.4-5) (27), whereas a longer region involving CR.3-7 and an EGF-like repeat is required for apoB-100 (28,62). In contrast, proprotein convertase subtilisin/kexin type 9 (PCSK9) has an atypical site on the receptor, which is formed by the EGF-like domain (63). Thus, at least for LDLR and LRP, the binding sites for RAP typically comprise or overlap sites for other ligands.…”

Pcsk9

“…These data support the finding that aside from the EGF-A domain, ≥3 of the 7 ligand-binding repeats of the LDLR and the β-propeller domain are necessary for the LDLR to be degraded in the presence of PCSK9. 52 However, such a binding is likely to be weak because all reported crystal structures of PCSK9 at either neutral or acidic pH do not reveal any interaction of the CHRD with the N-terminal repeats (aa 25-313) of the LDLR. One exception is the predicted weak hydrophobic interaction of Leu 626 of the β-propeller domain of the LDLR with Leu 108 of the prosegment of PCSK9, 41 which was also deduced from a GOF L108R PCSK9 mutant that possibly strengthens this interaction by favoring the electrostatic binding of Glu 605 of the LDLR to the mutant Arg 108 of PCSK9.…”

“…51 Interestingly, both the intracellular and the extracellular LDLR degradation activities of PCSK9 require the presence of the CHRD because the [PCSK9-ΔCHRD≡LDLR] complex that lacks this domain, although still capable of internalization into endosomes, does not traffic to lysosomes and is likely recycled to the cell surface. 51,52 This has led to the search of other proteins that may interact with the CHRD and the LDLR and drive the [PCSK9≡LDLR] complex to lysosomes.…”