Low temperature restoring effect on F508del-CFTR misprocessing: A proteomic approach Patricia Gomes-Alves a,b , Sofia Neves a , Ana V. Coelho b , Deborah Penque a, ⁎ a Laboratório de Proteómica, Departamento de Genética, Instituto Nacional de Saúde Dr Ricardo Jorge (INSA,I.P.), Lisboa, Portugal b Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal ARTICLE INFO ABSTRACT Article history: Received 7 August 2009 Accepted 2 September 2009 To gain insight into the proteins potentially involved in the low temperature-induced F508del-CFTR rescue process, we have explored by two-dimensional electrophoresis (2DE) the proteome of BHK cell lines expressing wt or F508del-CFTR, grown at 37 °C or 26 °C/24 h or 26 °C/48 h followed by 3 h of metabolic labelling with [ 35 S]-methionine. A set of 139 protein spots (yielding 125 mass spectrometry identifications) was identified as differentially expressed (p ANOVA < 0.05) among the six phenotypic groups analysed. The data analysis suggests that the unfolded protein response (UPR) induction and some cell-metabolism repression are the major cold-shock responses that may generate a favourable cellular environment to promote F508del-CFTR rescue. Down-regulation of proteasome regulatory PA28 and/or COP9 signalosome subunit, both involved in CFTR degradation, could also be a relevant cold-shock-induced condition for F508de-CFTR rescue. Moreover, cold-shock may promote the reestablishment of some proteostasis imbalance associated with over-expression of F508del-CFTR. In BHK-F508del cells, the deregulation of RACK1, a protein described to be important for stable expression of CFTR in the plasma membrane, is partially repaired after low temperature treatment. Together these findings give new insights about F508del-CFTR rescue by low temperature treatment and the proteins involved could ultimately constitute potential therapeutic targets in CF disease. © 2009 Elsevier B.V. All rights reserved. Keywords: F508del-CFTR Low temperature Protein trafficking Therapeutic targets 1. Introduction Cystic fibrosis (CF) is an autossomal recessive disorder resulting from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (http://www.genet.sick- kids.on.ca/cftr) coding for a membrane cAMP-regulated chlo- ride (Cl - ) channel that is functional in the apical surface of epithelial cells. F508del (deletion of a phenylalanine residue at position 508) is the most prevalent disease-causing mutation, found in ~ 70% of the CF chromosomes worldwide [1]. This mutation is the prototype example of class II mutations (defective intracellular trafficking) as the synthesised CFTR is unable to correctly fold and consequently is mostly retained in the ER via the action of molecular chaperones and is degraded [2], likely via ubiquitin/proteasome-dependent pathway [3]. As F508del-CFTR fails to mature and traffic to cell membrane cells expressing this mutant are unable to transport Cl - in response to increases in intracellular cAMP levels. The folding defect of F508del-CFTR mutant is temperature sensitive [4] and therefore, the most common strategy used to rescue F508del-CFTR to the cell surface is by incubating cells at sub-physiological temperatures (26–30 °C). Given that F508del-CFTR can function as a cAMP-regulated Cl - channel once it reaches the cell membrane, the JOURNAL OF PROTEOMICS 73 (2009) 218 – 230 ⁎ Corresponding author. Tel.: +351 217508137; fax: +351 217526410. E-mail address: deborah.penque@insa.min-saude.pt (D. Penque). 1874-3919/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2009.09.001 available at www.sciencedirect.com www.elsevier.com/locate/jprot