Proteomic and ionomic profiling reveals significant alterations of protein expression and calcium homeostasis in cystic fibrosis cells

Abstract

Cystic fibrosis (CF) is an autosomal recessive disorder associated with mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and defective chloride transport across the epithelial cell membranes. Abnormal epithelial ion transport is the primary cause of persistent airway infections and chronic inflammation in CF patients. In order to gain further insight into the mechanisms of epithelial dysfunctions linked to CFTR mutations, we performed and integrated proteomic and ionomic analysis of human bronchial epithelial IB3-1 cells and compared them with a CFTR-complemented isogenic cell line (C38). Aside from changes that were consistent with known effects related to CFTR mutations, such as differences in glycolytic and gluconeogenic pathways and unfolded protein responses, differential proteomics highlighted significant alteration of protein expression and, in particular, of the 14-3-3 signalling pathway that is known to be involved in cellular calcium (Ca) homeostasis. Of note, restoring chloride efflux by acting on Ca cellular homeostasis has been shown to be a promising therapeutic intervention for CF. Ionomic analysis showed significant changes in the IB3-1 element profile compared with C38 cells and in particular we observed an increase of intracellular Ca that significantly correlates with intracellular zinc (Zn) levels, suggesting a synergistic role of Ca and Zn influx. This finding is particularly intriguing because Zn has been reported to be effective in CF treatment increasing Ca influx. Taken together, our proteomic and ionomic data reveal that CFTR mutation sets in motion endogenous mechanisms counteracting impaired chloride transport mainly acting on epithelial ion transport and increasing intracellular Ca, suggesting potential links between protein expression and this response

Stenotrophomonas maltophilia Virulence and Specific Variations in Trace Elements during Acute Lung Infection: Implications in Cystic Fibrosis

“…In particular, specific mutations of CF transmembrane conductance regulator (CFTR) significantly affected metal homeostasis in vitro [58]. Furthermore, dyshomeostasis of K, Zn and NO 3 − was observed in exhaled air condensate in CF patients [33], while increased levels of Cu, total Fe, and Fe-binding proteins were also found in CF sputum [19], [20].…”

Breast cancer stem cells rely on fermentative glycolysis and are sensitive to 2-deoxyglucose treatment

“…52, 53, 54 A total of 100  μ g of protein extracts from the different conditions in separate experiments were precipitated with a mix of ethanol, methanol, and acetone (ratio 2 : 1 :  1, V/V) and then dissolved in 100 mmol/l Tris/HCl pH 7.9 containing 6 M urea and 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and sonicated. The reduction of proteins was performed by adding 100 mmol/l dithiothreitol (DTT) (1 h at 36 °C) and 200 mmol/l iodoacetamide (1 h at room temperature).…”

The capability of Pseudomonas aeruginosa to recruit zinc under conditions of limited metal availability is affected by inactivation of the ZnuABC transporter

“…Pellets corresponding to the whole cells were freeze dried by using a Modulyo freeze-dryer (ThermoSavant, USA) and weighted by an AX26 DeltaRange™ balance (Mettler-Toledo, Greifensee, Switzerland). Acid digestion of samples and ICP-MS analysis was carried out essentially as previously described 38, 39 Method sensitivity, repeatability, and trueness were assessed by calculating the limits of detection, limits of quantification, coefficients of variation, and the recovery functions, respectively, for each analysed elements 38 . The periplasmic fractions were diluted tenfold with 1% HNO 3 and analyzed by applying the standard addition method as described in a previous study 38 .…”