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A Systematic Approach to Assess the Activity and Classification of PCSK9 Variants.

التفاصيل البيبلوغرافية
العنوان: A Systematic Approach to Assess the Activity and Classification of PCSK9 Variants.
المؤلفون: Uribe KB; Department of Molecular Biophysics, Biofisika Institute, University of Basque Country and Consejo Superior de Investigaciones Científicas (UPV/EHU, CSIC), 48940 Leioa, Spain.; Center for Cooperative Research in Biomaterials (CIC biomaGUNE), Basque Research and Technology Alliance (BRTA), 20014 Donostia San Sebastian, Spain., Chemello K; Inserm, UMR 1188 Diabète Athérothrombose Thérapies Réunion Océan Indien (DéTROI), Université de La Réunion, 97400 Saint-Denis de La Reunion, France., Larrea-Sebal A; Department of Molecular Biophysics, Biofisika Institute, University of Basque Country and Consejo Superior de Investigaciones Científicas (UPV/EHU, CSIC), 48940 Leioa, Spain.; Fundación Biofisika Bizkaia, 48940 Leioa, Spain., Benito-Vicente A; Department of Molecular Biophysics, Biofisika Institute, University of Basque Country and Consejo Superior de Investigaciones Científicas (UPV/EHU, CSIC), 48940 Leioa, Spain.; Department of Biochemistry and Molecular Biology, Universidad del País Vasco UPV/EHU, 48080 Bilbao, Spain., Galicia-Garcia U; Department of Molecular Biophysics, Biofisika Institute, University of Basque Country and Consejo Superior de Investigaciones Científicas (UPV/EHU, CSIC), 48940 Leioa, Spain.; Fundación Biofisika Bizkaia, 48940 Leioa, Spain., Bourane S; Inserm, UMR 1188 Diabète Athérothrombose Thérapies Réunion Océan Indien (DéTROI), Université de La Réunion, 97400 Saint-Denis de La Reunion, France., Jaafar AK; Inserm, UMR 1188 Diabète Athérothrombose Thérapies Réunion Océan Indien (DéTROI), Université de La Réunion, 97400 Saint-Denis de La Reunion, France., Lambert G; Inserm, UMR 1188 Diabète Athérothrombose Thérapies Réunion Océan Indien (DéTROI), Université de La Réunion, 97400 Saint-Denis de La Reunion, France., Martín C; Department of Molecular Biophysics, Biofisika Institute, University of Basque Country and Consejo Superior de Investigaciones Científicas (UPV/EHU, CSIC), 48940 Leioa, Spain.; Department of Biochemistry and Molecular Biology, Universidad del País Vasco UPV/EHU, 48080 Bilbao, Spain.
المصدر: International journal of molecular sciences [Int J Mol Sci] 2021 Dec 18; Vol. 22 (24). Date of Electronic Publication: 2021 Dec 18.
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: Publisher: MDPI Country of Publication: Switzerland NLM ID: 101092791 Publication Model: Electronic Cited Medium: Internet ISSN: 1422-0067 (Electronic) Linking ISSN: 14220067 NLM ISO Abbreviation: Int J Mol Sci Subsets: MEDLINE
أسماء مطبوعة: Original Publication: Basel, Switzerland : MDPI, [2000-
مواضيع طبية MeSH: Proprotein Convertase 9/*genetics, Gain of Function Mutation ; HEK293 Cells ; Hep G2 Cells ; Humans ; Hyperlipoproteinemia Type II/genetics ; Hyperlipoproteinemia Type II/metabolism ; Mutagenesis, Site-Directed ; Mutation ; Proprotein Convertase 9/metabolism ; Receptors, LDL/metabolism
مستخلص: Background: Gain of function (GOF) mutations of PCSK9 cause autosomal dominant familial hypercholesterolemia as they reduce the abundance of LDL receptor (LDLR) more efficiently than wild-type PCSK9. In contrast, PCSK9 loss of function (LOF) variants are associated with a hypocholesterolemic phenotype. Dozens of PCSK9 variants have been reported, but most remain of unknown significance since their characterization has not been conducted.
Objective: Our aim was to make the most comprehensive assessment of PCSK9 variants and to determine the simplest approach for the classification of these variants.
Methods: The expression, maturation, secretion, and activity of nine well-established PCSK9 variants were assessed in transiently transfected HEK293 cells by Western blot and flow cytometry. Their extracellular activities were determined in HepG2 cells incubated with the purified recombinant PCSK9 variants. Their binding affinities toward the LDLR were determined by solid-phase immunoassay.
Results: LDLR expression increased when cells were transfected with LOF variants and reduced when cells were transfected with GOF variants compared with wild-type PCSK9. Extracellular activities measurements yielded exactly similar results. GOF and LOF variants had increased, respectively reduced, affinities for the LDLR compared with wild-type PCSK9 with the exception of one GOF variant (R218S) that showed complete resistance to inactivation by furin. All variants were expressed at similar levels and underwent normal maturation and secretion patterns except for two LOF and two GOF mutants.
Conclusions: We propose that transient transfections of HEK293 cells with a plasmid encoding a PCSK9 variant followed by LDLR expression assessment by flow cytometry is sufficient to reliably determine its GOF or LOF status. More refined experiments should only be used to determine the underlying mechanism(s) at hand.
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فهرسة مساهمة: Keywords: LDL; PCSK9; cholesterol; dyslipidaemias; familial hypercholesterolemia; gain of function; in vitro characterization; lipoproteins; loss of function; receptors
المشرفين على المادة: 0 (LDLR protein, human)
0 (Receptors, LDL)
EC 3.4.21.- (PCSK9 protein, human)
EC 3.4.21.- (Proprotein Convertase 9)
تواريخ الأحداث: Date Created: 20211224 Date Completed: 20220124 Latest Revision: 20220124
رمز التحديث: 20231215
مُعرف محوري في PubMed: PMC8706470
DOI: 10.3390/ijms222413602
PMID: 34948399
قاعدة البيانات: MEDLINE
الوصف
تدمد:1422-0067
DOI:10.3390/ijms222413602