Integrating systems biology with clinical research

A report on the conference 'Systems Genomics 2008', Heidelberg, Germany, 2-3 May 2008

Plasma membrane-specific interactome analysis reveals calpain 1 as a druggable modulator of rescued Phe508del-CFTR cell surface stability

Cystic 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.This work was supported by a center grant UID/MULTI/04046/2019 to BioISI and project PTDC/BIA-CEL/28408/2017 and IF2012 to PM, both from FCT, Portugal. AMM was recipient of fellowship SFRH/BD/52490/2014 from BioSYS PhD programme PD65-2012, and PB of fellowship SFRH/BPD/94322/2013.N/

Inheritance of the mammalian Golgi apparatus during the cell cycle

AbstractThe creation and propagation of the intricate Golgi architecture during the cell cycle poses a fascinating problem for biologists. Similar to the inheritance process for nuclear DNA, the inheritance of the Golgi apparatus consists of biogenesis (replication) and partitioning (mitosis/meiosis) phases, in which Golgi components must double in unit mass, then be appropriately divided between nascent daughter cells during cytokinesis. In this article we focus discussion on the recent advances in the area of Golgi inheritance, first outlining our current understanding of the behaviour of the Golgi apparatus during cell division, then concluding with a more conceptual discussion of the Golgi biogenesis problem. Throughout, we attempt to integrate ultrastructural and biochemical findings with more recent information obtained using live cell microscopy and morphological techniques

La nanochirurgie laser en biologie cellulaire

La cellule est un univers dynamique et compartimenté où interagissent une multitude de sous composants à l’échelle nanométrique. Afin d’étudier l’organisation subcellulaire, il est devenu nécessaire de posséder des outils permettant une manipulation directe extrêmement précise et non invasive. L’avènement des lasers à impulsions, dès les années 60, a conduit à la naissance de la chirurgie au laser. Aujourd’hui, la réduction des impulsions laser en dessous de la nanoseconde permet de mieux comprendre leur interaction avec les tissus biologiques et de contrôler des interventions chirurgicales à une résolution de l’ordre de quelques centaines de nanomètres. Utilisant l’ionisation de la matière par la lumière, cette nanochirurgie laser permet d’effectuer des interventions chirurgicales intracellulaires telles que la découpe de microtubules ou de fibres de tension, sans endommager les structures environnantes ou compromettre la viabilité cellulaire. Ainsi, l’utilisation de lasers à impulsions ultra-courtes, plus précis et puissants, offre une nouvelle approche pour l’étude des forces en biologie ou pour la quantification de la dynamique du cytosquelette.Since their first use in the early 60’s, pulsed lasers have become increasingly popular for their ability to ablate biological tissue. Short laser pulses allow high precision surgery for biological and medical applications with minimal invasiveness. Performing highly targeted manipulation and ablation allows experiments impossible so far in development biology, cellular biology or even assisted reproductive technologies and laser surgery has been increasingly used over the last five years to answer key questions in Biology. Recently, picosecond UV and femtosecond IR laser pulses have been used to cleave microtubules and to severe actin stress fibers in vivo with a spatial precision in the submicrometer range to study their dynamics without affecting cell viability. We review recent findings on the underlying principles of pulsed laser nanosurgery mechanisms showing how the use of ultra short laser pulses increases precision and non-invasiveness of laser surgery. We show how the understanding of the surgical process allows one to distinguish between single cell ablation in living organisms or intracellular nanosurgery in living cells and we review recent applications to the study of forces and the quantification of cytoskeleton dynamics

Chemical Chaperones Improve Protein Secretion and Rescue Mutant Factor VIII in Mice with Hemophilia A.

nefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking