The Cellular Trafficking of the Secretory Proprotein Convertase PCSK9 and Its Dependence on the LDLR

Nassoury, Nasha; Blasiole, Daniel A.; Oler, Angie T.; Benjannet, Suzanne; Hamelin, Josée; Poupon, Vivianne; McPherson, Peter S.; Attie, Alan D.; Prat, Annik; Seidah, Nabil G.

doi:10.1111/j.1600-0854.2007.00562.x

Cited by 228 publications

(186 citation statements)

References 48 publications

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“…An alternative hypothesis, and one consistent with current knowledge, would be the absence of proteolytic activity altogether, whereby binding of PCSK9 to LDLR (or other receptor) provides a signal to lower LDLR levels. A recent publication by Nassoury et al (32) has provided evidence that the CRD binds to the cell surface via LDLR. A nonproteolytic mechanism has been shown to exist in proteases of the trypsin family such as hepatocyte growth factor (HGF) (33), whereby the protease has lost catalytic activity but functions as a growth factor receptor.…”

Section: Discussionmentioning

confidence: 99%

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

Hampton

Knuth

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

138

102

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Mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9) are strongly associated with levels of low-density lipoprotein cholesterol in the blood plasma and, thereby, occurrence or resistance to atherosclerosis and coronary heart disease. Despite this importance, relatively little is known about the biology of PCSK9. Here, the crystal structure of a full-length construct of PCSK9 solved to 1.9-Å resolution is presented. The structure contains a fully folded C-terminal cysteine-rich domain (CRD), showing a distinct structural similarity to the resistin homotrimer, a small cytokine associated with obesity and diabetes. This structural relationship between the CRD of PCSK9 and the resistin family is not observed in primary sequence comparisons and strongly suggests a distant evolutionary link between the two molecules. This three-dimensional homology provides insight into the function of PCSK9 at the molecular level and will help to dissect the link between PCSK9 and CHD.hypercholesterolemia ͉ low-density lipoprotein receptor ͉ proprotein convertase ͉ x-ray crystallography ͉ adipocytokine P CSK9 (also known as neural apoptosis-regulated convertase, NARC-1) is a 692-residue extracellular protein expressed primarily in the kidneys, liver and intestines (1) representing the 9th member of the secretory subtilase family. Various genetic observations subsequently mapped PCSK9 as the third gene (along with LDLR and APOB) to cause autosomal dominant hypercholesterolemia (ADH). These studies suggested that gainof-function mutations increase plasma levels of LDL-c (2-6), whereas nonsense or missense (loss-of-function) mutations, which interfere with folding or secretion of PCSK9, lead to a reduction of plasma levels of LDL-c and an 88% decrease in the risk of coronary heart disease (CHD) (5). In mice, adenoviral overexpression of PCSK9 results in increased plasma LDL-c level in normal mice but not in LDLR-deficient mice (7). Deletion of PCSK9 causes an increase in level of LDLR protein but not mRNA (8). These findings lead to a hypothesis that PCSK9 exerts its role in cholesterol metabolism through posttranslational down-regulation of LDLR, the receptor responsible for clearing LDL-c from plasma. In support of this hypothesis, a recent mouse parabiosis study revealed a nearly complete loss of liver LDLR proteins in wild-type recipients of human PCSK9 parabiosed from a transgenic littermate (9).Like other secretory subtilisin-like serine proteases, PCSK9 is synthesized in the endoplasmic reticulum (ER) as a precursor that undergoes autocatalytic processing, intracellular trafficking, and, finally, release into the extracellular medium with the capability of decreasing the cellular LDLR level and LDL-c uptake (10). Despite the rapid progress in understanding the genetics and biology of PCSK9, little is known of its structure, substrate specificity, or mode of action of PCSK9 in the regulation of LDLR. The full-length, mature sequence consists of three domains, [the first 30 residues of the N terminus contain signal se...

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Section: Discussionmentioning

confidence: 99%

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

Hampton

Knuth

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

138

102

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“…The current studies provide direct experimental evidence that exogenously added PCSK9 reduces LDLRs in a manner that requires LDLR binding but not catalytic activity. Studies by Maxwell et al (25) showed that overexpression of PCSK9 in HepG2 cells induces degradation of LDLRs intracellularly in a post-ER compartment, and Nassoury et al (26) have suggested that the two proteins interact in the ER and co-localize in the Golgi apparatus in hepatocytes. The results of our studies do not preclude the possibility that PCSK9 can function intracellularly in a manner that requires catalytic activity.…”

Section: Discussionmentioning

confidence: 99%

Catalytic Activity Is Not Required for Secreted PCSK9 to Reduce Low Density Lipoprotein Receptors in HepG2 Cells

McNutt

Lagace

Horton

2007

Journal of Biological Chemistry

259

209

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Proprotein convertase subtilisin/kexin type 9 (PCSK9), a member of the proteinase K subfamily of subtilases, promotes internalization and degradation of low density lipoprotein receptors (LDLRs) after binding the receptor on the surface of hepatocytes. PCSK9 has autocatalytic activity that releases the prodomain at the N terminus of the protein. The prodomain remains tightly associated with the catalytic domain as the complex transits the secretory pathway. It is not known whether enzymatic activity is required for the LDLR-reducing effects of PCSK9. Here we expressed the prodomain together with a catalytically inactive protease domain in cells and purified the protein from the medium. The ability of the catalytically inactive PCSK9 to bind and degrade LDLRs when added to culture medium of human hepatoma HepG2 cells at physiological concentrations was similar to that seen using wild-type protein. Similarly, a catalytic-dead version of a gain-of-function mutant, PCSK9(D374Y), showed no loss of activity compared with a catalytically active counterpart; both proteins displayed ϳ10-fold increased activity in degradation of cell surface LDLRs compared with wild-type PCSK9. We conclude that the ability of PCSK9 to degrade LDLRs is independent of catalytic activity and suggest that PCSK9 functions as a chaperone to prevent LDLR recycling and/or to target LDLRs for lysosomal degradation.

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“…More recently, the proprotein convertases subtilisin kexin 9 (PCSK9) gene (4), which is highly expressed in liver and small intestine (5), was identified as the third locus associated with familial hypercholesterolemia (6). It is now clear that PCSK9 binds the LDLR and triggers its intracellular degradation in acidic compartments, resulting in increased circulating plasma cholesterol (7)(8)(9)(10).…”

mentioning

confidence: 99%

“…Overexpression studies in liver suggested that both intraand extracellular PCSK9 target the LDLR (9,15,16) toward degradation in late endosomes/lysosomes (LE/L) (7)(8)(9)(10). It was shown that the adaptor protein ARH, which interacts with the cytosolic tail of the LDLR, is essential for the endocytosis and degradation of the cell surface PCSK9⅐LDLR complex in vivo (16).…”

mentioning

confidence: 99%

Dissection of the Endogenous Cellular Pathways of PCSK9-induced Low Density Lipoprotein Receptor Degradation

Poirier

Mayer

Poupon

et al. 2009

Journal of Biological Chemistry

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245

185

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Elevated levels of plasma low density lipoprotein (LDL)-cholesterol, leading to familial hypercholesterolemia, are enhanced by mutations in at least three major genes, the LDL receptor (LDLR), its ligand apolipoprotein B, and the proprotein convertase PCSK9. Single point mutations in PCSK9 are associated with either hyperor hypocholesterolemia. Accordingly, PCSK9 is an attractive target for treatment of dyslipidemia. PCSK9 binds the epidermal growth factor domain A (EGF-A) of the LDLR and directs it to endosomes/ lysosomes for destruction. Although the mechanism by which PCSK9 regulates LDLR degradation is not fully resolved, it seems to involve both intracellular and extracellular pathways. Here, we show that clathrin light chain small interfering RNAs that block intracellular trafficking from the trans-Golgi network to lysosomes rapidly increased LDLR levels within HepG2 cells in a PCSK9-dependent fashion without affecting the ability of exogenous PCSK9 to enhance LDLR degradation. In contrast, blocking the extracellular LDLR endocytosis/degradation pathway by a 4-, 6-, or 24-h incubation of cells with Dynasore or an EGF-AB peptide or by knockdown of endogenous autosomal recessive hypercholesterolemia did not significantly affect LDLR levels. The present data from HepG2 cells and mouse primary hepatocytes favor a model whereby depending on the dose and/or incubation period, endogenous PCSK9 enhances the degradation of the LDLR both extraand intracellularly. Therefore, targeting either pathway, or both, would be an effective method to reduce PCSK9 activity in the treatment of hypercholesterolemia and coronary heart disease.

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The Cellular Trafficking of the Secretory Proprotein Convertase PCSK9 and Its Dependence on the LDLR

Cited by 228 publications

References 48 publications

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

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

Catalytic Activity Is Not Required for Secreted PCSK9 to Reduce Low Density Lipoprotein Receptors in HepG2 Cells

Dissection of the Endogenous Cellular Pathways of PCSK9-induced Low Density Lipoprotein Receptor Degradation

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