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Differential expression of proteins in cell model systems

The most common mutation of the CFTR gene, the delta F508 prevents the protein from maturing correctly and reaching the apical membrane to exert its functions, which accelerates its intracellular degradation. A direct strategy is to investigate the differences in protein profiles between cells expressing the normal and the mutated channel. This is the rationale of a study performed within the context of the EUROPROCF project that scrutinizes the protein expression patterns in a cell system (HeLa) stably transfected with either the mutated or the wild-type CFTR. In the study, the 2-D gel screening showed five spots of greater intensity in the mutation-bearing line, identified as type II keratin 8 and type I keratin 18. It can be argued that interaction of mutated CFTR with these proteins could be one of the mechanisms responsible for proteasomal degradation and prevention of protein addressing to the apical membrane. This was reversed by low temperature, and confirmed by keratin 18 siRNA silencing. Because this study was conducted on undifferentiated HeLa cells, these exciting conclusions would be reinforced after re-investigation in a more physiological CF model.

Primary cultures of nasal polyps from normal individuals can be used as an alternative to cell lines of identical genetic back ground and compared with those of patients bearing the deltaF508 mutation. This cell model has been used in a preliminary study aiming to determine the proteins that are not changed in spite of differences in the genetic background of donors. 2-D gels in the pH range 5-8 revealed 24 unchanged proteins, including cytokeratins 6,7,8 and 19, HSP 27 and 70, and elongation factors ER-Tu and ER-2. In addition, 20 protein isoforms were found only in patients (i.e. keratins 4 and 18), and 13 only in controls (i.e., annexin-2, keratin 19). Some of them were identified in both CF and control cells (i.e. chaperonin GroEL precursor, keratins 4 and 17), but showing a migration displacement that could again be attributed to post-translational modifications. Despite the limitations of the study in terms of number of subjects and cell model, it seems to confirm the potential of cytoskeletal proteins as participants in the pathogenesis of CF.

Although multiple functions have been attributed to CFTR, the mature protein located at the apical membrane of epithelial cells is believed to act as a way out for chloride ions and water. The use of CFTR-null (cftr -/-) or CFTR-dysfunctinal mouse models is the most consequent procedure to analyze the effects of channel dysfunction. However, one of the limiting factors of proteomics is the difficulty to examine the membrane proteome, which is mainly composed of hydrophobic and basic proteins that cannot be properly resolved by the conventional IEF-based 2-D technology. A study performed within the scope of the EUROPROCF project successfully overcame this obstacle by a blue native-based electrophoresis approach (BN-PAGE). BN consists of a non-denaturing electrophoresis of protein samples solubilized in a non-ionic detergent that preserves native conformation and interactions, allowing the separation of membrane proteins and complexes. It can be combined with a classic SDS-PAGE to dissociated the separated complexes and resolve their constitutive subunits in a second dimension.

This way, not only the membrane proteome can be addressed and trans-membrane domain-containing proteins identified, but also protein-protein interactions may be unmasked. The study by Brouillard et al. (Mol. Cell. Proteomics, 4, 1762-1775, 2005) showed that in colonic crypts and lung epithelium from cftr -/- mice, the expression of the calcium (sensitive chloride channel ClCA3 is decreased. This channel, which has been suggested to compensate for the lack of functional CFTR in the human intestine, could account, when dysfunctional in the mouse, for the digestive pathology in this animal model. It is proposed that ClCA and most likely mClCA3, which is localized in goblet cells, is involved in this mucus secretory response. In turn, underexpression of mClCA3 protein in cftr -/- mice would participate in the defect of fluid and mucus transport in the colon. In addition, MS analysis per se of mClCA3 pointed to the existence of novel isoforms in mouse colonic epithelial cells that probably results from proteolytic cleavage. The following step is to investigate the expression and function of ClCA proteins in human patients.

One of the most intriguing pathological features of CF is the abnormal inflammatory response in several organs. In a study using a global proteomic approach on colonic crypts from the cftr -/- mouse model, the anti-inflammatory protein annexin-I was identified and found to be totally absent. A similar pattern was found in the pancreas and lung of the same model. Interestingly, the non-targeted organs showed a normal level of annexin-I expression.

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