PCSK9 binds to multiple receptors and can be functionally inhibited by an EGF-A peptide

doi:10.1016/j.bbrc.2008.07.106

Biochemical and Biophysical Research Communications

Volume 375, Issue 1, 10 October 2008, Pages 69-73

https://doi.org/10.1016/j.bbrc.2008.07.106 Get rights and content

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to low density lipoprotein receptor (LDLR) and induces its internalization and degradation. PCSK9 binding to LDLR is mediated through the LDLR epidermal growth factor-like repeat A (EGF-A) domain. We show for the first time that an EGF-A peptide inhibits PCSK9-mediated degradation of LDLR in HepG2 cells. In addition to LDLR, we show that PCSK9 also binds directly to ApoER2 and mouse VLDLR. Importantly, binding of PCSK9 to either LDLR or mouse VLDLR was effectively inhibited by EGF-A while binding to ApoER2 was less affected. In contrast, LDL receptor-associated protein (RAP), which interacts with LDL receptor repeat type A (LA) domains, inhibited PCSK9 binding to ApoER2 with greater efficacy than either LDLR or mVLDLR. These data demonstrate that while PCSK9 binds several receptors via its EGF-A binding domain, additional contacts with other receptor domains are also involved.

Section snippets

Materials and methods

Recombinant proteins and antibodies. Polyhistidine-tagged human LDLR, human ApoER2, mouse VLDLR and mouse reelin were purchased from R&D Systems as were antibodies to LDLR and mouse VLDLR. Polyhistidine-tagged human JNK2α2 and mouse endostatin were purchased from Calbiochem Inc. Recombinant rat RAP was obtained from PROSPEC Protein Specialists and human RAP from Molecular Innovations. Polyclonal rabbit anti-LDLR used for Western blotting and in-cell Western was obtained from PROGEN Biotechnik.

Results and discussion

Conflicting results have been reported in regard to the ability of PCSK9 to interact with proteins other than LDLR. Zhang et al. reported that no binding of PCSK9 to VLDLR could be detected in transfected COS-M cells [17]. In addition, these authors showed that binding to VLDLR could be conferred by introducing the EGF-A domain of LDLR into VLDLR. In contrast, Poirier et al. showed enhanced PCKS9 association with CHO-A7 cells expressing either ApoER2 or VLDLR after an overnight incubation [16].

Acknowledgments

The Schering Plough Research Institute is funded entirely by Schering-Plough Corporation. The authors would like to thank Drs Harry Davis and Diane Hollenbaugh for valuable discussion and suggestions.

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Proteinuria is a prominent feature of chronic kidney disease. Interventions that reduce proteinuria slow the progression of chronic kidney disease and the associated risk of cardiovascular disease. Here, we propose a mechanistic coupling between proteinuria and proprotein convertase subtilisin/kexin type 9 (PCSK9), a regulator of cholesterol and a therapeutic target in cardiovascular disease. PCSK9 undergoes glomerular filtration and is captured by megalin, the receptor responsible for driving protein reabsorption in the proximal tubule. Accordingly, megalin-deficient mice and patients carrying megalin pathogenic variants (Donnai Barrow syndrome) were characterized by elevated urinary PCSK9 excretion. Interestingly, PCSK9 knockout mice displayed increased kidney megalin while PCSK9 overexpression resulted in its reduction. Furthermore, PCSK9 promoted trafficking of megalin to lysosomes in cultured proximal tubule cells, suggesting that PCSK9 is a negative regulator of megalin. This effect can be accelerated under disease conditions since either genetic destruction of the glomerular filtration barrier in podocin knockout mice or minimal change disease (a common cause of nephrotic syndrome) in patients resulted in enhanced tubular PCSK9 uptake and urinary PCSK9 excretion. Pharmacological PCSK9 inhibition increased kidney megalin while reducing urinary albumin excretion in nephrotic mice. Thus, glomerular damage increases filtration of PCSK9 and concomitantly megalin degradation, resulting in escalated proteinuria.
Identification of a PCSK9-LDLR disruptor peptide with in vivo function
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Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma low-density lipoprotein cholesterol (LDL-C) levels by promoting hepatic LDL receptor (LDLR) degradation. Therapeutic antibodies that disrupt PCSK9-LDLR binding reduce LDL-C concentrations and cardiovascular disease risk. The epidermal growth factor precursor homology domain A (EGF-A) of the LDLR serves as a primary contact with PCSK9 via a flat interface, presenting a challenge for identifying small molecule PCSK9-LDLR disruptors. We employ an affinity-based screen of 10¹³ in vitro-translated macrocyclic peptides to identify high-affinity PCSK9 ligands that utilize a unique, induced-fit pocket and partially disrupt the PCSK9-LDLR interaction. Structure-based design led to molecules with enhanced function and pharmacokinetic properties (e.g., ₁₃PCSK9i). In mice, ₁₃PCSK9i reduces plasma cholesterol levels and increases hepatic LDLR density in a dose-dependent manner. ₁₃PCSK9i functions by a unique, allosteric mechanism and is the smallest molecule identified to date with in vivo PCSK9-LDLR disruptor function.
The emerging landscape of peptide-based inhibitors of PCSK9
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Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a clinically validated target for treating cardiovascular disease (CVD) due to its involvement in cholesterol metabolism. Although approved monoclonal antibodies (alirocumab and evolocumab) that inhibit PCSK9 function are very effective in lowering cholesterol, their limitations, including high treatment costs, have so far prohibited widespread use. Accordingly, there is great interest in alternative drug modalities to antibodies. Like antibodies, peptides are valuable therapeutics due to their high target potency and specificity. Furthermore, being smaller than antibodies means they have access to more drug administration options, are less likely to induce adverse immunogenic responses, and are better suited to affordable production. This review surveys the current peptide-based landscape aimed towards PCSK9 inhibition, covering pre-clinical to patented drug candidates and comparing them to current cholesterol lowering therapeutics. Classes of peptides reported to be inhibitors include nature-inspired disulfide-rich peptides, combinatorially derived cyclic peptides, and peptidomimetics. Their functional activities have been validated in biophysical and cellular assays, and in some cases pre-clinical mouse models. Recent efforts report peptides with potent sub-nanomolar binding affinities to PCSK9, which highlights their potential to achieve antibody-like potency. Studies are beginning to address pharmacokinetic properties of PCSK9-targeting peptides in more detail. We conclude by highlighting opportunities to investigate their biological effects in pre-clinical models of cardiovascular disease. The anticipation concerning the PCSK9-targeting peptide landscape is accelerating and it seems likely that a peptide-based therapeutic for treating PCSK9-mediated hypercholesterolemia may be clinically available in the near future.
PCSK9 and cancer: Rethinking the link
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Cancer is emerging as a major problem globally, as it accounts for the second cause of death despite medical advances. According to epidemiological and basic studies, cholesterol is involved in cancer progression and there are abnormalities in cholesterol metabolism of cancer cells including prostate, breast, and colorectal carcinomas. However, the importance of cholesterol in carcinogenesis and thereby the role of cholesterol homeostasis as a therapeutic target is still a debated area in cancer therapy. Proprotein convertase subtilisin/kexin type-9 (PCSK9), a serine protease, modulates cholesterol metabolism by attachment to the LDL receptor (LDLR) and reducing its recycling by targeting the receptor for lysosomal destruction. Published research has shown that PCSK9 is also involved in degradation of other LDLR family members namely very-low-density-lipoprotein receptor (VLDLR), lipoprotein receptor-related protein 1 (LRP-1), and apolipoprotein E receptor 2 (ApoER2). As a result, this protein represents an interesting therapeutic target for the treatment of hypercholesterolemia. Interestingly, clinical trials on PCSK9-specific monoclonal antibodies have reported promising results with high efficacy in lowering LDL-C and in turn reducing cardiovascular complications. It is important to note that PCSK9 mediates several other pathways apart from its role in lipid homeostasis, including antiviral activity, hepatic regeneration, neuronal apoptosis, and modulation of various signaling pathways. Furthermore, recent literature has illustrated that PCSK9 is closely associated with incidence and progression of several cancers. In a number of studies, PCSK9 siRNA was shown to effectively suppress the proliferation and invasion of the several studied tumor cells. Hence, a novel application of PCSK9 inhibitors/silencers in cancer/metastasis could be considered. However, due to poor data on effectiveness and safety of PCSK9 inhibitors in cancer, the impact of PCSK9 inhibition in these pathological conditions is still unknown.
A vast literature search was conducted to find intended studies from 1956 up to 2020, and inclusion criteria were original peer-reviewed publications.
To date, PCSK9 has been scantly investigated in cancer. The question that needs to be discussed is “How does PCSK9 act in cancer pathophysiology and what are the risks or benefits associated to its inhibition?”. We reviewed the available publications highlighting the contribution of this proprotein convertase in pathways related to cancer, with focus on the potential implications of its long-term pharmacological inhibition in cancer therapy.
In vitro selection generates RNA aptamer that antagonizes PCSK9–LDLR interaction and recovers cellular LDL uptake
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Proprotein convertase subtilisin/kexin type 9 (PCSK9) induces low-density lipoprotein (LDL)-receptor (LDLR) degradation, increasing plasma LDL-cholesterol levels and causing hypercholesterolemia. Therefore, inhibition of PCSK9–LDLR interaction is an attractive therapeutic target for hypercholesterolemia treatment. In this study, we have identified a novel RNA aptamer that binds specifically to PCSK9 by in vitro selection, also known as systematic evolution of ligands by exponential enrichment (SELEX). The binding kinetics of the PCSK9-binding RNA aptamer was measured by biolayer interferometry assay, showing that the aptamer has higher affinity compared to PCSK9–LDLR interaction. Competitive inhibition assay using chemiluminescence detection revealed that the RNA aptamer inhibits PCSK9–LDLR interaction. In cellular LDL-uptake assays with HepG2 cells, the RNA aptamer recovered LDL uptake in the PCSK9-treated cells, demonstrating its anti-PCSK9 antagonistic activity. These results indicated that the PCSK9-binding RNA aptamer has the potential to be an affinity reagent for PCSK9 protein analysis and a therapeutic reagent for hypercholesterolemia treatment.
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¹: These authors contributed equally to this work.

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PCSK9 binds to multiple receptors and can be functionally inhibited by an EGF-A peptide

Abstract

Section snippets

Materials and methods

Results and discussion

Acknowledgments

J. Am. Coll. Cardiol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

Anal. Biochem.

Mol. Cell

Mol. Cell

The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation

Proc. Natl. Acad. Sci. USA

Implication of the proprotein convertase NARC-1/PCSK9 in the development of the nervous system

J. Neurochem.

Proapoptotic effects of NARC 1 (= PCSK9), the gene encoding a novel serine proteinase

Cytometry A

Mutations in PCSK9 cause autosomal dominant hypercholesterolemia

Nat. Genet.