Low temperature restoring effect on F508del-CFTR misprocessing: A proteomic approach

doi:10.1016/j.jprot.2009.09.001

Journal of Proteomics

Volume 73, Issue 2, 1 December 2009, Pages 218-230

https://doi.org/10.1016/j.jprot.2009.09.001 Get rights and content

Abstract

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 [³⁵S]-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.

Graphical abstract

The cold-shock-induced cellular environment that is favourable to the (partial) rescue of F508del-CFTR processing and trafficking to the cell surface is mainly characterised by cell-metabolism reduction and unfolded protein response (UPR) activation.

Introduction

Cystic fibrosis (CF) is an autossomal recessive disorder resulting from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (http://www.genet.sickkids.on.ca/cftr) coding for a membrane cAMP-regulated chloride (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 understanding of the mechanisms that some treatments correct protein folding and trafficking defect of F508del-CFTR could be crucial for the development of an effective therapeutic strategy for CF [5], [6], [7].

Low temperature apparently stabilizes the mutant protein during folding in the ER, allowing some CFTR molecules to escape ER quality control and traffic through the Golgi apparatus to the plasma membrane [4]. Very recently, Rennolds et al. [8] suggested that at low temperature F508del-CFTR may use a non-conventional trafficking pathway and by-passes the Golgi apparatus where CFTR becomes Endo-H resistant. Others concluded that at least one rate limiting step in CFTR maturation is its interaction with one or more members of the molecular chaperone family [9]. Although some explanations have been proposed, the mechanisms involved in the restoration of F508del-CFTR trafficking defect by low temperature remain to be elucidated.

We hypothesise that proteins differentially expressed in response to low temperature treatment may be involved in the trafficking rescue of F508del CFTR. To identify such proteins we have investigated and compared by a proteomics approach the protein expression profile of BHK cell lines stably expressing wt- or F508del-CFTR at normal physiologic growth conditions (37 °C) and/or under effect of low temperature (26 °C) incubation for 24 h or 48 h. Our ultimate goal was to propose new targets for development of novel therapeutic strategies for CF.

Section snippets

Cells and metabolic labelling

BHK cells stably expressing human wild type (wt)- or F508del-CFTR (kindly provided by M Roxo-Rosa, INSA, Lisboa) were cultivated as previously described [10]. Before analysis, 6 × 10⁵ cells were seeded per 35 mm culture dishes (Nunc), grown to about 80% of confluence and then incubated at 37 °C (control) or 26 °C for 24 h or 48 h in the same media without MTX. Cells were incubated 30 min in methionine-free α-minimal essential medium (MEM, Gibco, Invitrogen) prior to the metabolic labelling in the same

Data analysis

Gene Ontology (www.geneonthology.org) was used to search for the biological processes, molecular functions and cellular components of the identified proteins [16]. PIKE (Protein Information and Knowledge Extractor) software was also used to collect all the available information about each protein (http://proteo.cnb.uam.es:8080/pike/).

Three clustering techniques [17], [18], [19] were applied to the spot volume data. An agglomerative unsupervised hierarchical clustering (Euclidean distance) was

2DE proteome profiling

To identify proteins whose expression is modulated by low temperature that could be involved in the trafficking restoration process of F508del-CFTR, the total protein extracts from BHK cells stably expressing wt or F508del-CFTR grown at 37 °C or at 26 °C for 24 h or 48 h followed by 3 h of [³⁵S]-metabolic labelling were analysed by 2DE (for details see Materials and methods). A total of 26 2D-gel autoradiographic images were produced and the respective 2DE maps were computer-analysed. A total of 139

Discussion

To gain insight into the proteins potentially involved in the low temperature-induced F508del-CFTR rescue process, we explored in BHK-wt and BHK-F508del cells the global proteome modulation at 26 °C, with particular interest on the proteostasis environment [25] that may adequately support the processing of CFTR through the folding and trafficking pathways [1], [26].

Concluding remarks

By proteomics we showed that the cellular environment induced by low temperature that is favourable to (partial) rescue of F508del-CFTR processing and trafficking, is mainly characterised by cell-metabolism reduction and UPR activation. Numerous identified proteins have been described to be involved in protein folding and trafficking and many of them are located in the secretory pathway where mutated CFTR is retained.

At low temperature, several proteasome subunits presented different

Acknowledgements

We thank MS group of ITQB-Oeiras for MS technical assistance and all colleagues of our laboratory for their scientific and technical support, M Roxo-Rosa (INSA.I.P., Lisboa) for BHK cell lines and P Fanen (INSERM 841, Créteil, France) for CSN5/Cops5 antibody.

This work was supported by FCT/FEDER research grants POCTI/MGI/40878/2001 and POCI/SAU-MMO/56163/2004, FCT/Poly-Annual Funding Program and FEDER/Saúde XXI Program (Portugal). PGA is a recipient of FCT-PhD fellowship SFRH/BD/17744/2004.

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Our approach has permitted to pay attention to the molecular mechanisms which regulate pathways directly or indirectly involved with CFTR defects: heat shock response, cross-talk between cytoskeleton and signal transduction, chronic inflammation and alteration of CFTR gating. Our data could open new perspectives into the understanding of CF, identifying potential targets for drug treatments in order to alleviate Δ508CFTR membrane instability and consequently increase life expectancy for CF patients.
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Lowering the temperature improves CFTR conformation not only by thermodynamic stabilization but also by changes in cellular factors, because correction is not possible in every cell type [32]. Cellular proteostasis as well as the F508del CFTR interactome are different at low temperature [33••,34]. This includes differences in chaperone and co-chaperone associations and their kinetics.
Pharmacological intervention to treat the lethal genetic disease cystic fibrosis has become reality, even for the severe, most common folding mutant F508del CFTR. CFTR defects range from absence of the protein, misfolding that leads to degradation rather than cell-surface localization (such as F508del), to functional chloride-channel defects on the cell surface. Corrector and potentiator drugs improve cell-surface location and channel activity, respectively, and combination therapy of two correctors and a potentiator have shown synergy. Several combinations are in the drug-development pipeline and although the primary defect is not repaired, rescue levels are reaching those resembling a cure for CF. Combination therapy with correctors may also improve functional CFTR mutants and benefit patients on potentiator therapy.
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Cystic 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 (Ca²⁺) homeostasis. In this study, we discovered that the Ca²⁺ binding protein Calumenin (CALU) is a key regulator in the maintenance of ER-Ca²⁺ calcium homeostasis in both wild type and F508del-CFTR expressing cells. Calumenin modulates SERCA pump activity without drastically affecting ER-Ca²⁺ 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.
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In addition, a “repaired” Phe508del-CFTR, in which RXR ER retention motifs were mutated to rescue its maturation, induced apart from Grp78 also other ER stress markers, including Grp94 and Grp75, to higher extent than Phe508del-CFTR (Gomes-Alves et al., 2010). It has been known that several other approaches that rescue Phe508del-CFTR from ERAD and transport it to the cell surface, such as low temperature (Gomes-Alves et al., 2009) and chemical chaperone treatments, cause UPR activation in cells (Egan et al., 2002). Analysis of the primary CF airway epithelial cells, however, did not give coherent results.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride (Cl⁻) channel. Mutations of its gene lead to the disease of cystis fibrosis (CF) among which the most common is the deletion of phenylalanine at position 508 (Phe508del). CFTR is a multi-domain glycoprotein whose biosynthesis, maturation and functioning as an anion channel involve multi-level post-translational modifications of CFTR molecules and complex folding processes to reach its native, tertiary conformation. Only 20–40% of the nascent chains achieve folded conformation, while the remaining molecules are targeted for degradation by endoplasmic reticulum, lysosomes, or autophagy. A large number of mutations causing CF impair processing of CFTR. Growing knowledge of CFTR biosynthesis has enabled understanding the cellular basis of CF and has brought to light various potential targets for novel, promising therapies.
This article is part of a Directed Issue entitled: Cystic fibrosis: From o-mics to cell biology, physiology, and therapeutic advances.

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Low temperature restoring effect on F508del-CFTR misprocessing: A proteomic approach

Abstract

Graphical abstract

Introduction

Section snippets

Cells and metabolic labelling

Data analysis

2DE proteome profiling

Discussion

Concluding remarks

Acknowledgements

Cell

J Biol Chem

Biochem Biophys Res Commun

J Biol Chem

J Cyst Fibros

Biochem Biophys Res Commun

Cell

Mol Cell Proteomics

J Biol Chem

Biochim Biophys Acta

Trends Biochem Sci

Blood

Biochem Biophys Res Commun

Biochim Biophys Acta

Trends Cell Biol

Cell

Cystic fibrosis in the 21st century

Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive

Nature

Chloride conductance expressed by delta F508 and other mutant CFTRs in Xenopus oocytes

Science

Altered chloride ion channel kinetics associated with the delta F508 cystic fibrosis mutation

Nature

The cystic fibrosis mutation (delta F508) does not influence the chloride channel activity of CFTR

Nat Genet

Chemical chaperones correct the mutant phenotype of the delta F508 cystic fibrosis transmembrane conductance regulator protein

Cell Stress Chaperones

Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis

Electrophoresis