Biology subjects

Edelman, A.

Publications and source records attributed to Edelman, A..

3 recordsLinked to original sources

bioRxiv2022

Keratin 8 is a scaffolding and regulatory protein of ERAD complexes

Early recognition and enhanced degradation of misfolded proteins by the endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD) cause defective protein secretion and membrane targeting, as exemplified for Z-alpha 1 antitrypsin (Z-A1AT), responsible for alpha-1-antitrypsin deficiency (A1ATD) and F508del-CFTR (cystic fibrosis transmembrane conductance regulator) responsible for cystic fibrosis (CF). Prompted by our previous observation that decreasing Keratin 8 (K8) expression increased trafficking of F508del-CFTR to the plasma membrane, we investigated whether K8 impacts trafficking of soluble misfolded Z-A1AT protein. The subsequent goal of this study was to elucidate the mechanism underlying the K8-dependent regulation of protein trafficking, focusing on the ERAD pathway. The results show that diminishing K8 concentration in HeLa cells enhances secretion of both Z-A1AT and wild type (WT) A1AT with a 13-fold and 4-fold increase, respectively. K8 down-regulation triggers ER failure and cellular apoptosis when ER stress is jointly elicited by conditional expression of the s heavy chains, as previously shown for Hrd1 knock-out. Simultaneous K8 silencing and Hrd1 knock-out did not show any synergistic effect, consistent with K8 acting in the Hrd1-governed ERAD step. Fractionation experiments reveal that K8 is recruited to ERAD complexes containing Derlin2, Sel1 and Hrd1 proteins upon expression of Z/WT-A1AT and F508del-CFTR. Treatment of the cells with c407, a small molecule inhibiting K8 interaction, decreases K8 and Derlin2 recruitment to high-order ERAD complexes. This was associated with increased Z-A1AT secretion in both HeLa and Z-homozygous A1ATD patients respiratory cells. Overall, we provide evidence that K8 acts as an ERAD modulator. It may play a scaffolding protein role for early-stage ERAD complexes, regulating Hrd1-governed retrotranslocation initiation/ubiquitination processes. Targeting K8-containing ERAD complexes is an attractive strategy for the pharmacotherapy of A1ATD.

cell biology↗

bioRxiv2021

Elexacaftor/Tezacaftor/Ivacaftor alters branching morphogenesis of the mouse embryonic lung

IntroductionCFTR modulators triple combo-therapy Elexacaftor/Tezacaftor/Ivacaftor (ETI) has proven to clinically benefit homozygous and heterozygous F508del patients. As a result, an increasing number of pregnancies is expected. Studies of the potential impact of these modulators on the development of the foetus are mandatory. MaterialsWe used the early mouse embryonic lung organ culture model to analyse ex vivo the lung branching process and the relative expression of Fgf10, Fgfr2IIIb, Shh, and Hhip development regulator genes in different conditions: standard culture medium, treatment with ETI or with Forskolin {+/-} Inh172. Development of lung branching and distal bud caliber were evaluated in lung explants from heterozygous F508del Cftrtm1Eur/+ and control Cftrtm1Eur+/+ (WT) mouse embryos at E12.5 during pseudo-glandular stage. ResultsExposure to ETI of the Cftrtm1Eur/+ and WT lung explants induced a significant decrease in lung branching after 48h culture and the percentage of terminal bud dilations was significantly increased. These results were recapitulated by cAMP-dependent CFTR continuous activation by Forskolin and reversed by addition of Inh172. ETI induced a significant decrease in Fgf10, Fgfr2IIIb, Shh and Hhip expression in lung explants of both E12.5 Cftrtm1Eur/+ and WT embryos treated with ETI for 72h. ConclusionOur results provide evidence that the triple association Elexacaftor/Tezacaftor/Ivacaftor alters lung branching morphogenesis of WT and heterozygous F508del mouse embryos during the pseudo-glandular stage. Those results argue for a close monitoring of pregnancies in patients treated with these drugs. Plain LanguageO_ST_ABSIntroductionC_ST_ABSThe triple combo-therapy Elexacaftor/Tezacaftor/Ivacaftor (ETI) improves homozygous and heterozygous F508del patients. As a result, an increasing number of pregnancies is expected. Studies of this treatment on the development of the foetus are lacking. We incubated lungs of murine foetus not carrying CFTR mutation or F508del heterozygous. We show that ETI induces significant defect of lung development and the formation of cysts. These results are at least partly due to CFTR activation. Those results argue for a close monitoring of pregnancies in patients treated with these drugs.

pharmacology and toxicology↗

bioRxiv2021

CFTR corrector efficacy is associated with occupancy of distinct binding sites

CFTR misfolding due to cystic fibrosis causing mutations can be corrected with small molecules designated as correctors. VX-809, an investigational corrector compound, is believed to bind CFTR directly to either the first membrane-spanning domain (MSD1) and/or the first nucleotide-binding domain (NBD1). Blind docking onto the 3D structures of these domains, followed by molecular dynamics (MD) simulations, revealed the presence of two potential VX-809 binding sites which, when mutated, abrogated corrector rescue. Mutations altering protein maturation are also shown to be not equally sensitive to the occupancy of the two sites by VX-809, with the most frequent mutation F508del requiring integrity of both sites and allosteric coupling with the F508del region while L206W only requires the integrity of the MSD1 site. A network of charged amino acids in the lasso Lh2 helix and the intracellular loops ICL1 and ICL4 is involved in the allostery between MSD1 and NBD1. Corrector VX-445, which is used in combination in clinics with VX-661, a structurally close analog of VX-809, to fully correct F508del, is also shown to occupy two potential binding sites on MSD1 and NBD1, the latter being shared with VX-809. In conclusion, VX-809 and VX-445 appear to bind different CFTR domains to alleviate specific folding defects. These results provide new insights into therapeutics understanding and may help the development of efficient corrector combinations.

molecular biology↗