Effects of pH and Low Density Lipoprotein (LDL) on PCSK9-dependent LDL Receptor Regulation

Abstract

Mutations within PCSK9 (proprotein convertase subtilisin/ kexin type 9) are associated with dominant forms of familial hyper-and hypocholesterolemia. Although PCSK9 controls low density lipoprotein (LDL) receptor (LDLR) levels post-transcriptionally, several questions concerning its mode of action remain unanswered. We show that purified PCSK9 protein added to the medium of human endothelial kidney 293, HepG2, and Chinese hamster ovary cell lines decreases cellular LDL uptake in a dose-dependent manner. Using this cell-based assay of PCSK9 activity, we found that the relative potencies of several PCSK9 missense mutants (S127R and D374Y, associated with hypercholesterolemia, and R46L, associated with hypocholesterolemia) correlate with LDL cholesterol levels in humans carrying such mutations. Notably, we found that in vitro wild-type PCSK9 binds LDLR with an ϳ150-fold higher affinity at an acidic endosomal pH (K D ‫؍‬ 4.19 nM) compared with a neutral pH (K D ‫؍‬ 628 nM). We also demonstrate that wild-type PCSK9 and mutants S127R and R46L are internalized by cells to similar levels, whereas D374Y is more efficiently internalized, consistent with their affinities for LDLR at neutral pH. Finally, we show that LDL diminishes PCSK9 binding to LDLR in vitro and partially inhibits the effects of secreted PCSK9 on LDLR degradation in cell culture. Together, the results of our biochemical and cell-based experiments suggest a model in which secreted PCSK9 binds to LDLR and directs the trafficking of LDLR to the lysosomes for degradation.PCSK9 (proprotein convertase subtilisin/kexin type 9) encodes the ninth member of the mammalian proprotein convertase family of serine endoproteases. PCSK9 is translated as a 692-amino acid proprotein that includes several domains found in other proprotein convertases, including an N-terminal signal sequence, a prodomain, a catalytic domain, and a cysteine-rich C-terminal domain (1-3). The PCSK9 catalytic domain shares high sequence similarity with the proteinase K family of subtilases and contains a catalytic triad (Asp 186 , His 226 , and Ser 386 ) responsible for autoprocessing (1, 4). PCSK9 processing occurs in the secretory pathway, presumably in the endoplasmic reticulum, and results in proteolytic cleavage occurring after Gln 152 (FAQ2SIP). This cleavage generates a stable PCSK9 heterodimer composed of a 14-kDa prodomain fragment and a mature 57-kDa fragment containing the catalytic and C-terminal domains (4, 5). Following processing, the PCSK9 heterodimer exits the ER and is eventually secreted (1). The prodomain of PCSK9 remains strongly bound to the mature protein after secretion, presumably inhibiting the catalytic activity of PCSK9 (1, 5, 6). To date, there is no conclusive evidence that the processed secreted form of PCSK9 can cleave any substrates in a catalytic serine-dependent manner.The first evidence that PCSK9 plays a significant role in regulating plasma low density lipoprotein (LDL) 3 cholesterol (LDL-C) levels was the identification of several missense mutations in PCS...

Structural and Biochemical Characterization of the Wild Type PCSK9-EGF(AB) Complex and Natural Familial Hypercholesterolemia Mutants

“…Although an intracellular mechanism preceding secretion is not formally excluded, the introduction of PCSK9 into the circulation of mice through parabiosis reduces hepatic LDLR levels (14), as does the injection into mice of recombinant PCSK9 (15,16), suggesting that circulating PCSK9 can act by binding to LDLR on the cell surface. This in vivo study is further supported by in vitro experiments in which the addition of PCSK9 to cultured cell medium results in LDLR degradation (9,14) and decreases LDL uptake (17). Prior to its degradation, the PCSK9-LDLR complex is internalized by endocytosis in a manner dependent on clathrin (18) and on the association of a cytosolic region of the LDLR with the ARH protein (14).…”

“…Prior to its degradation, the PCSK9-LDLR complex is internalized by endocytosis in a manner dependent on clathrin (18) and on the association of a cytosolic region of the LDLR with the ARH protein (14). A plausible mechanism of action leading to LDLR degradation rather than its vesicular recycling has been proposed following binding studies demonstrating that PCSK9 binds the LDLR ectodomain with ϳ100-fold higher affinity at the acidic pH of late endosomes/lysosomes compared with the neutral pH of the cell surface (10,12,17,19). The enhanced affinity at acidic pH suggests that, differently from the LDLR-LDL complex, the LDLR-PCSK9 complex does not dissociate in the endosomes, thus preventing LDLR recycling and instead leading to shuttling of the complex to the lysosomes for degradation.…”

The self-inhibited structure of full-length PCSK9 at 1.9 Å reveals structural homology with resistin within the C-terminal domain

“…The Piper article (35) also details a cubic crystal form grown at pH 4.6. This, combined with the fact that our structure crystallized at pH 10.5, strongly suggests that the orientation of the domains relative to one another is independent of pH and suggests that recent studies of differential binding affinities of LDLR at different pHs (36) are due to conformational changes in LDLR or other physiochemical changes. In addition, the structures do not contain a Ca ϩ2 at the conserved calcium-binding site.…”

The Proprotein Convertase PCSK9 Induces the Degradation of Low Density Lipoprotein Receptor (LDLR) and Its Closest Family Members VLDLR and ApoER2

“…Thus, whereas quite active in enhancing the degradation of endogenous LDLR in HepG2 cells, it does not seem to work efficiently on LDLR in CHO cells (12), and PCSK9 degrades endogenous LDLR much more rapidly in HepG2 versus HEK293 cells (37). We thus examined whether PCSK9 and PCSK9-L1 could degrade the three receptors in a cell-specific fashion.…”