Repo-Man/protein phosphatase 1 SUMOylation mediates binding to lamin A and serine 22 dephosphorylation

© 2022 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License. http://creativecommons.org/licenses/by/4.0/Lamin A phosphorylation/de-phosphorylation is an important process during cells division as it allows for nuclear envelope (NE) disassembly at mitotic entry and its re-assembly during mitotic exit. Several kinases have been identified as responsible for these phosphorylations, but no protein phosphatase has been implicated in their reversal. One of the mitotic phosphosites in lamin A responsible for its dynamic behaviour is serine 22 (S22) which is de-phosphorylated during mitotic exit. Recent evidence has also linked the nuclear pool of lamin A S22ph in interphase to gene expression regulation. Previous work suggested that the phosphatase responsible for lamin A S22 de-phosphorylation is chromatin bound and interacts with lamin A via SUMO-SIM motives. We have previously reported that Repo-Man/protein phosphatase 1 (PP1) is a chromatin-associated phosphatase that regulates NE reformation. Here we propose that Repo-Man/PP1 phosphatase mediates lamin A S22 de-phosphorylation. We indeed show that depletion of Repo-Man leads to NE defects, causes hyperphosphorylation of lamin A S22 that can be rescued by a wild-type but not a SUMOylation-deficient mutant. Lamin A and Repo-Man interact in vivo and in vitro, and the interaction is mediated by SUMOylation. Moreover, the localization of Repo-Man/PP1 to the chromatin is essential for lamin A S22 de-phosphorylation.Peer reviewedFinal Published versio

Impact of TRPM7 and ATF6 modulation on cystic fibrosis transmembrane conductance regulator

La mucoviscidose est une maladie causée par des mutations du gène cftr entraînant des défauts importants de la protéine CFTR. La mutation la plus fréquente (F508del) est caractérisée par un repliement incorrect conduisant à la rétention de la protéine dans le RE.L’accumulation de CFTR-F508del dans le RE, l’inflammation et les infections vont déclencher un stress du RE dans les cellules épithéliales ainsi que l’UPR. Cette dernière est une réponse adaptative déclenchée par le stress du RE et permet de rétablir l’homéostasie de ce compartiment. L’UPR est constituée de trois voies majeures dont l’une d’entre elles est activée dans les cellules exprimant un CFTR-F508del. Il s’agit de la voie ATF6 qui est de plus responsable de la répression transcriptionnelle du CFTR, ce qui en fait une cible thérapeutique potentielle. Nous avons montré que son inhibition conduit à l’amélioration de la fonction duCFTR-F508del et à l’augmentation de sa présence à la membrane des cellules.Nous nous sommes également intéressés au Mg2+ et au TRPM7, le régulateur principal de la [Mg2+]i dans les cellules. Nous avons émis l’hypothèse que TRPM7 était en partie responsable de l’activation d’ATF6 dans les cellules exprimant un CFTR-F508del. Le but de cette seconde partie du projet était donc tout d’abord d’étudier la relation existante entre le Mg2+, TRPM7 et le CFTR. Nous avons montré qu’il existait des différences de [Mg2+]i selon le type de mutation du CFTR exprimé par les cellules. Ces différences sont en partie dues à un défaut d’activation de TRPM7, lui-même probablement lié à un défaut du CFTR. En augmentant l’activité de TRPM7 par du Naltriben, nous avons pu montrer un effet potentialisant sur leCFTR-G551D.Cystic fibrosis is caused by mutations in the cftr gene resulting in several defaults on the CFTR protein. The most frequent mutation is F508del which is characterized by an incorrect folding causing its retention within the ER. CFTR-F508del protein accumulation in the ER, inflammation and infections will trigger the ER stress in epithelial cells, as well as UPR. UPR constitutes an adaptive response of the ER in order to restore ER’s homeostasis. UPR consists in three major pathways. Among them, one is activated in cells expressing CFTR-F508del protein. The ATF6 pathway of UPR is responsible of the transcriptional repression of CFTR, which makes of it a potential therapeutic target. We showed that the inhibition of ATF6 leads to the improvement of CFTR-508del function, as well as its increased presence in the cellular membrane. We were also interested in Mg2+ and TRPM7, the main regulator of [Mg2+]i. We suspected that TRPM7 is, at least in part, responsible for the activation of ATF6 in cells expressing the mutant CFTR-F508del. Thus, the second part of my work was focused on the study of the relationship between Mg2+, TRPM7 and CFTR. We showed the existence of [Mg2+]I differences according to CFTR mutant expressed in cells. These differences are the result of an altered TRPM7 activation, probably in link with the mutated CFTR’s malfunction. We proved that increasing TRPM7 activity by Naltriben treatment potentiates CFTR-G551D

Impact de la modulation de TRPM7 et ATF6 sur le cystic fibrosis transmembrane conductance regulator

Cystic fibrosis is caused by mutations in the cftr gene resulting in several defaults on the CFTR protein. The most frequent mutation is F508del which is characterized by an incorrect folding causing its retention within the ER. CFTR-F508del protein accumulation in the ER, inflammation and infections will trigger the ER stress in epithelial cells, as well as UPR. UPR constitutes an adaptive response of the ER in order to restore ER’s homeostasis. UPR consists in three major pathways. Among them, one is activated in cells expressing CFTR-F508del protein. The ATF6 pathway of UPR is responsible of the transcriptional repression of CFTR, which makes of it a potential therapeutic target. We showed that the inhibition of ATF6 leads to the improvement of CFTR-508del function, as well as its increased presence in the cellular membrane. We were also interested in Mg2+ and TRPM7, the main regulator of [Mg2+]i. We suspected that TRPM7 is, at least in part, responsible for the activation of ATF6 in cells expressing the mutant CFTR-F508del. Thus, the second part of my work was focused on the study of the relationship between Mg2+, TRPM7 and CFTR. We showed the existence of [Mg2+]I differences according to CFTR mutant expressed in cells. These differences are the result of an altered TRPM7 activation, probably in link with the mutated CFTR’s malfunction. We proved that increasing TRPM7 activity by Naltriben treatment potentiates CFTR-G551D.La mucoviscidose est une maladie causée par des mutations du gène cftr entraînant des défauts importants de la protéine CFTR. La mutation la plus fréquente (F508del) est caractérisée par un repliement incorrect conduisant à la rétention de la protéine dans le RE.L’accumulation de CFTR-F508del dans le RE, l’inflammation et les infections vont déclencher un stress du RE dans les cellules épithéliales ainsi que l’UPR. Cette dernière est une réponse adaptative déclenchée par le stress du RE et permet de rétablir l’homéostasie de ce compartiment. L’UPR est constituée de trois voies majeures dont l’une d’entre elles est activée dans les cellules exprimant un CFTR-F508del. Il s’agit de la voie ATF6 qui est de plus responsable de la répression transcriptionnelle du CFTR, ce qui en fait une cible thérapeutique potentielle. Nous avons montré que son inhibition conduit à l’amélioration de la fonction duCFTR-F508del et à l’augmentation de sa présence à la membrane des cellules.Nous nous sommes également intéressés au Mg2+ et au TRPM7, le régulateur principal de la [Mg2+]i dans les cellules. Nous avons émis l’hypothèse que TRPM7 était en partie responsable de l’activation d’ATF6 dans les cellules exprimant un CFTR-F508del. Le but de cette seconde partie du projet était donc tout d’abord d’étudier la relation existante entre le Mg2+, TRPM7 et le CFTR. Nous avons montré qu’il existait des différences de [Mg2+]i selon le type de mutation du CFTR exprimé par les cellules. Ces différences sont en partie dues à un défaut d’activation de TRPM7, lui-même probablement lié à un défaut du CFTR. En augmentant l’activité de TRPM7 par du Naltriben, nous avons pu montrer un effet potentialisant sur leCFTR-G551D

Evaluation of aminopyrrolidine amide to improve chloride transport in CFTR-defective cells

International audienc

Rapid detection of the mature form of cystic fibrosis transmembrane regulator by surface plasmon resonance

International audiencetractCystic fibrosis is the most common lethal autosomal recessive disease in theCaucasian population, and is due to mutations in the cystic fibrosis transmembraneconductance regulator (CFTR) gene. The CFTR protein functions as a chloride channel.Electrophoretic analysis shows that normal CFTR exists as three different molecular weightforms of 127, 131 and 170 kDa (band A, B and C, respectively) representing differentglycosylation forms. Band A is the non glycosylated form of CFTR, band B is the coreglycosylated CFTR and band C is the mature form of CFTR with complex glycosylation. Theglycosylation state of CFTR is representative of its maturation and is an important marker ofthe protein processing and function. The most common mutation in CF is a missingphenylalanine at position 508 (F508del-CFTR). The misfolded F508del-CFTR protein whichexhibits an altered glycosylation, is observed as bands A and B only and does not trafficcorrectly to the plasma membrane. Laboratory experiments (SDS-PAGE, immunoblotting,glycosidase digestions, mobility shift of deglycosylated CFTR) aimed to assess the expressionof CFTR and to depict which band is observed have been developed. Nevertheless, theseexperimental procedures are time consuming and poorly specific. The aim of the presentstudy was to provide an easy, rapid and reproducible methodology to assess whether theCFTR protein within a protein extract is expressed and matured. We show here that surfaceplasmon resonance (SPR) permits a direct detection of the mature form of CFTR in crude celllysates, providing a new tool to characterize CFTR in cells without any labelling or pre-treatment before cell lysis. The study of the effects of correctors for F508del-CFTR is themain task of many laboratories. Therefore, the proposed method is likely a useful tool for arapid detection of mature CFTR in complex samples. We also show here that our methodpermits the characterization of CFTR in patients cell extracts in a minimum tim

Additional file 1 of Ki-67 is necessary during DNA replication for fork protection and genome stability

Additional file 1: Supplementary Figures. Fig. S1. Generation of AID:mclover endogenously tagged HCT116 cell line. Fig. S2. Ki-67 degradation alters gene expression when cells exit mitosis in its absence. Fig. S3. Ki-67 is in proximity of the replication machinery. Fig. S4. Ki-67 degradation at G1/S leads to CDK2 inactivation. Fig. S5. Correlation between Ki-67 and the interferon pathway

Calumenin contributes to ER-Ca2+ homeostasis in bronchial epithelial cells expressing WT and F508del mutated CFTR and to F508del-CFTR retention

International audienceCystic Fibrosis (CF) is the most frequent fatal genetic disease in Caucasian populations. Mutations in the chloride channel CF Transmembrane Conductance Regulator (CFTR) gene are responsible for functional defects of the protein and multiple associated dysregulations. The most common mutation in patients with CF, F508del-CFTR, causes defective CFTR protein folding. Thus minimal levels of the receptor are expressed at the cell surface as the mutated CFTR is retained in the endoplasmic reticulum (ER) where it correlates with defective calcium (Ca(2+)) homeostasis. In this study, we discovered that the Ca(2+) binding protein Calumenin (CALU) is a key regulator in the maintenance of ER-Ca(2+) calcium homeostasis in both wild type and F508del-CFTR expressing cells. Calumenin modulates SERCA pump activity without drastically affecting ER-Ca(2+) concentration. In addition, reducing Calumenin expression in CF cells results in a partial restoration of CFTR activity, highlighting a potential function of Calumenin in CFTR maturation. These findings demonstrate a pivotal role for Calumenin in CF cells, providing insights into how modulation of Calumenin expression or activity may be used as a potential therapeutic tool to correct defects in F508del-CFTR

cAMP measurement in control and CF HNEC, with or without Buserelin treatment (10⁻¹² M, 2h).

<p>cAMP was assessed in control (n = 2, in triplicate) and in CF HNEC (n = 2, in triplicate). Results are expressed in pmol cAMP/mg of total proteins. Beside an observed variability between patients, cAMP was increased in CF cells after buserelin treatment. Data are mean ± S.E.M. Student’s t test was used to evaluate the significance (*: p<0.05).</p

Patch-clamp analysis of the chloride channel function of CFTR in CF HNEC in the presence of buserelin.

<p><b>A.</b> Representative current traces were recorded in non-treated CF HNEC at the basal level (a), in the presence of CFTR activators (Fsk/Gst) (b), in the presence of CFTR inhibitor (CFTR_inh172) (c). Current traces were recorded in buserelin treated CF HNEC at the basal level (d), in the presence of CFTR activators (Fsk/Gst) (e), in the presence of CFTR inhibitor (CFTR_inh172) (f). <b>B.</b> I/V curves with normalized currents by cell capacitance (pA/pF) are presented for CFTR basal current in CF HNEC, with and without buserelin treatment (1, 2 or 4 hours). <b>C.</b> Mean CFTR-related normalized current amplitudes were recorded at +80 mV and are presented as bar graphs. The statistical analysis indicated that CFTR-related currents were highly increased in the presence of buserelin (2 and 4 hours). Data are presented as mean ± S.E.M. for n = 4. Student’s t test was used to evaluate significant differences (***: p<0.001).</p

Localization and expression of GnRH-R in HBEC.

<p><b>A.</b> Representative confocal images of HBEC co-stained by cell markers CK13 (mucous cells, left panel, red labelling) and Muc5AC (ciliated cells, middle panel, red labelling) and GnRH-R (green labelling). Co-distribution of GnRH-R (green labelling) and CFTR (red labelling) was observed in ciliated cells, in apical cell membranes. <b>B.</b> Representative immunoblot performed to assess GnRH-R expression in 6 different HBEC cultures.</p