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PCSK9-D374Y mediated LDL-R degradation can be functionally inhibited by EGF-A and truncated EGF-A peptides: An in vitro study

  • Author Footnotes
    1 These authors contributed equally to this work.
    Vincenza Valenti
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Dipartimento di Promozione della Salute, Materno Infantile, Medicina Interna e Specialistica Di Eccellenza “G. D'Alessandro” (PROMISE), Università degli Studi di Palermo, Via del Vespro 129, 90127, Palermo, Italy
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  • Author Footnotes
    1 These authors contributed equally to this work.
    Davide Noto
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Dipartimento di Promozione della Salute, Materno Infantile, Medicina Interna e Specialistica Di Eccellenza “G. D'Alessandro” (PROMISE), Università degli Studi di Palermo, Via del Vespro 129, 90127, Palermo, Italy
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  • Author Footnotes
    1 These authors contributed equally to this work.
    Antonina Giammanco
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Dipartimento di Promozione della Salute, Materno Infantile, Medicina Interna e Specialistica Di Eccellenza “G. D'Alessandro” (PROMISE), Università degli Studi di Palermo, Via del Vespro 129, 90127, Palermo, Italy
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  • Francesca Fayer
    Affiliations
    Dipartimento di Promozione della Salute, Materno Infantile, Medicina Interna e Specialistica Di Eccellenza “G. D'Alessandro” (PROMISE), Università degli Studi di Palermo, Via del Vespro 129, 90127, Palermo, Italy
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  • Rossella Spina
    Affiliations
    Dipartimento di Promozione della Salute, Materno Infantile, Medicina Interna e Specialistica Di Eccellenza “G. D'Alessandro” (PROMISE), Università degli Studi di Palermo, Via del Vespro 129, 90127, Palermo, Italy
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  • Grazia I. Altieri
    Affiliations
    Dipartimento di Promozione della Salute, Materno Infantile, Medicina Interna e Specialistica Di Eccellenza “G. D'Alessandro” (PROMISE), Università degli Studi di Palermo, Via del Vespro 129, 90127, Palermo, Italy
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  • Valeria Ingrassia
    Affiliations
    Dipartimento di Promozione della Salute, Materno Infantile, Medicina Interna e Specialistica Di Eccellenza “G. D'Alessandro” (PROMISE), Università degli Studi di Palermo, Via del Vespro 129, 90127, Palermo, Italy
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  • Chiara Scrimali
    Affiliations
    Dipartimento di Promozione della Salute, Materno Infantile, Medicina Interna e Specialistica Di Eccellenza “G. D'Alessandro” (PROMISE), Università degli Studi di Palermo, Via del Vespro 129, 90127, Palermo, Italy
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  • Carlo M. Barbagallo
    Affiliations
    Dipartimento di Promozione della Salute, Materno Infantile, Medicina Interna e Specialistica Di Eccellenza “G. D'Alessandro” (PROMISE), Università degli Studi di Palermo, Via del Vespro 129, 90127, Palermo, Italy
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  • Federica Brucato
    Affiliations
    Dipartimento di Promozione della Salute, Materno Infantile, Medicina Interna e Specialistica Di Eccellenza “G. D'Alessandro” (PROMISE), Università degli Studi di Palermo, Via del Vespro 129, 90127, Palermo, Italy
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  • Gabriella Misiano
    Affiliations
    Dipartimento di Promozione della Salute, Materno Infantile, Medicina Interna e Specialistica Di Eccellenza “G. D'Alessandro” (PROMISE), Università degli Studi di Palermo, Via del Vespro 129, 90127, Palermo, Italy
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  • Author Footnotes
    2 These authors share the senior authorship of this work.
    Angelo B. Cefalù
    Correspondence
    Corresponding author. Dipartimento di Promozione della Salute, Materno Infantile, Medicina Interna e Specialistica Di Eccellenza “G. D'Alessandro” (PROMISE)Policlinico “Paolo Giaccone”, Via del Vespro 129, 90127, Palermo, Italy.
    Footnotes
    2 These authors share the senior authorship of this work.
    Affiliations
    Dipartimento di Promozione della Salute, Materno Infantile, Medicina Interna e Specialistica Di Eccellenza “G. D'Alessandro” (PROMISE), Università degli Studi di Palermo, Via del Vespro 129, 90127, Palermo, Italy
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  • Author Footnotes
    2 These authors share the senior authorship of this work.
    Maurizio R. Averna
    Correspondence
    Corresponding author. Dipartimento di Promozione della Salute, Materno Infantile, Medicina Interna e Specialistica Di Eccellenza “G. D'Alessandro” (PROMISE), Università degli Studi Palermo, Via del Vespro 129, 90127 Palermo, Italy.
    Footnotes
    2 These authors share the senior authorship of this work.
    Affiliations
    Dipartimento di Promozione della Salute, Materno Infantile, Medicina Interna e Specialistica Di Eccellenza “G. D'Alessandro” (PROMISE), Università degli Studi di Palermo, Via del Vespro 129, 90127, Palermo, Italy
    Search for articles by this author
  • Author Footnotes
    1 These authors contributed equally to this work.
    2 These authors share the senior authorship of this work.

      Highlights

      • PCSK9 acts by binding to the EGF-A domain of LDLR and promotes its degradation.
      • Gain-of-function D374Y PCSK9 mutation causes severe hypercholesterolemia in humans.
      • PCSK9-D374Y mediated LDLR degradation is inhibited by EGF-A analogs in vitro.

      Abstract

      Background and aims

      Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to low density lipoprotein receptor (LDLR) through the LDLR epidermal growth factor-like repeat A (EGF-A) domain and induces receptor internalization and degradation. PCSK9 has emerged as a novel therapeutic target for hypercholesterolemia. Clinical studies with PCSK9 inhibiting antibodies have demonstrated strong LDL-c lowering effects, but other therapeutic approaches using small molecule inhibitors for targeting PCSK9 functions may offer supplementary therapeutic options. The aim of our study was to evaluate the effect of synthetic EGF-A analogs on mutated (D374Y) PCSK9-D374Y mediated LDLR degradation in vitro.

      Methods

      Huh7 human hepatoma cells were transiently transfected to overexpress the gain-of-function D374Y PCSK9 mutation, which has been associated with severe hypercholesterolemia in humans.

      Results

      Transient transfection of cells with PCSK9-D374Y expression vector very effectively enhanced degradation of mature LDLR in Huh7. Treatment with both EGF-A and EGF-A truncated peptides inhibited this effect and showed increased LDLR protein in Huh7 cells transfected with PCSK9-D374Y in a clear concentration dependent manner. Huh7 transfected cells treated with increasing concentration of EGF-A analogs also showed an increase internalization of labeled Dil-LDL.

      Conclusions

      The result of our study shows that EGF-A analogs are able to effectively hamper the enhanced degradation of LDLR in liver cells expressing PCSK9-D374Y.

      Graphical abstract

      Keywords

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      Linked Article

      • EGF-A peptides: A promising strategy for PCSK9 inhibition
        AtherosclerosisVol. 292
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          PCSK9 inhibitors have recently emerged as a powerful strategy to decrease low-density lipoprotein cholesterol (LDL-C) on top of statin and/or ezetimibe therapy in patients at high cardiovascular (CV) risk [1]. PCSK9 was discovered in 2003 as the third gene responsible for autosomal dominant hypercholesterolemia (ADH) in two French families, in whom gain-of-function (GOF) variants were identified [2]. Shortly after, carriers of PCSK9 loss-of-function (LOF) variants were shown to be characterized by low levels of LDL-C and, as a consequence, by a drastically reduced risk of CV events compared to non-carriers [3].
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