Browsing by Author "Pepperkok, Rainer"
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- Functional profiling of LDLR variants: Important evidence for variant classification: Functional profiling of LDLR variantsPublication . Graça, Rafael; Alves, Ana Catarina; Zimon, Magdalena; Pepperkok, Rainer; Bourbon, MafaldaBackground: Familial Hypercholesterolemia (FH) is a semidominant disorder of the lipid metabolism associated with premature atherosclerosis and coronary heart disease. So far, about 3,000 unique LDLR variants have been described, most of which lack functional evidence proving their effect on LDLR function, despite the important role that functional studies play in variant classification. Objective: In this work, we aimed to functionally characterize 13 rare missense variants, identified worldwide and in Portugal, in clinical FH patients. Methods: LDLR-deficient CHO-ldlA7 cells were transfected with plasmids carrying different LDLR variants generated by site-directed mutagenesis. LDLR activity and expression were assessed by FACS. Results: 11/13 variants affect LDLR function (p.Cys109Phe; p.Cys143Arg; p.Glu267Lys; p.Cys352Ser; p.Ile451Thr; p.His485Gln; p.Asp492Asn; p.Val500Ala; p.Gly529Arg; p.Phe614Ile; p.Glu626Lys) and 2/13 are inconclusive (p.Arg81Cys; p.Gly98Arg;). Conclusion: Of the 13 variants studied, 8 were classified as VUS by ACMG criteria, but for 7 of these 8, our functional studies were able to reassign them as Likely pathogenic or Pathogenic. For an accurate diagnosis, an effort must be made to improve functional characterization of putative disease-causing variants.
- Inhibition of calpain 1 restores plasma membrane stability to pharmacologically rescued Phe508del-CFTR variantPublication . Matos, Ana M.; Pinto, Francisco R.; Barros, Patrícia; Amaral, Margarida D.; Pepperkok, Rainer; Matos, PauloCystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a chloride channel normally expressed at the surface of epithelial cells. The most frequent mutation, resulting in Phe-508 deletion, causes CFTR misfolding and its premature degradation. Low temperature or pharmacological correctors can partly rescue the Phe508del-CFTR processing defect and enhance trafficking of this channel variant to the plasma membrane (PM). Nevertheless, the rescued channels have an increased endocytosis rate, being quickly removed from the PM by the peripheral protein quality-control pathway. We previously reported that rescued Phe508del-CFTR (rPhe508del) can be retained at the cell surface by stimulating signaling pathways that coax the adaptor molecule ezrin (EZR) to tether rPhe508del-Na+/H+-exchange regulatory factor-1 complexes to the actin cytoskeleton, thereby averting the rapid internalization of this channel variant. However, the molecular basis for why rPhe508del fails to recruit active EZR to the PM remains elusive. Here, using a proteomics approach, we characterized and compared the core components of wt-CFTR- or rPhe508del-containing macromolecular complexes at the surface of human bronchial epithelial cells. We identified calpain 1 (CAPN1) as an exclusive rPhe508del interactor that prevents active EZR recruitment, impairs rPhe508del anchoring to actin, and reduces its stability in the PM. We show that either CAPN1 down-regulation or its chemical inhibition dramatically improves the functional rescue of Phe508del-CFTR in airway cells. These observations suggest that CAPN1 constitutes an appealing target for pharmacological intervention, as part of CF combination therapies restoring Phe508del-CFTR function.
- Plasma membrane-specific interactome analysis reveals calpain 1 as a druggable modulator of rescued Phe508del-CFTR cell surface stabilityPublication . Matos, Ana Margarida; Pinto, Francisco R.; Barros, Patrícia; Amaral, Margarida D.; Pepperkok, Rainer; Matos, PauloCystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a chloride channel normally expressed at the surface of epithelial cells. The most frequent mutation, resulting in Phe-508 deletion, causes CFTR misfolding and its premature degradation. Low temperature or pharmacological correctors can partly rescue the Phe508del-CFTR processing defect and enhance trafficking of this channel variant to the plasma membrane (PM). Nevertheless, the rescued channels have an increased endocytosis rate, being quickly removed from the PM by the peripheral protein quality-control pathway. We previously reported that rescued Phe508del-CFTR (rPhe508del) can be retained at the cell surface by stimulating signaling pathways that coax the adaptor molecule ezrin (EZR) to tether rPhe508del–Na+/H+-exchange regulatory factor-1 (NHERF1) complexes to the actin cytoskeleton, thereby averting the rapid internalization of this channel variant. However, the molecular basis for why rPhe508del fails to recruit active EZR to the PM remains elusive. Here, using a proteomics approach, we characterized and compared the core components of wt-CFTR– or rPhe508del–containing macromolecular complexes at the surface of human bronchial epithelial cells. We identified calpain 1 (CAPN1) as an exclusive rPhe508del interactor that prevents active EZR recruitment, impairs rPhe508del anchoring to actin, and reduces its stability in the PM. We show that either CAPN1 downregulation or its chemical inhibition dramatically improves the functional rescue of Phe508del-CFTR in airway cells. These observations suggest that CAPN1 constitutes an attractive target for pharmacological intervention, as part of CF combination therapies restoring Phe508del-CFTR function.