Overlapping synthetic peptides as a tool to map protein-protein interactions ̶ FSH as a model system of nonadditive interactions.

In earlier work, we used partially overlapped synthetic peptides as a tool to find regions of interaction between the human FSH hormone and its receptor, aiming to find possible antagonists or agonists. Years later, the FSH and FSH receptor 3D structures were reported by other laboratories. The 3D results were in close agreement with the interacting regions predicted by using synthetic peptides. These earlier studies are reviewed here, and the predicted regions of interaction compared to the FSH and FSH receptor 3D structures to illustrate the usefulness of the synthetic peptide strategy to find binding regions. Different contact regions contribute multiplicatively to the high affinity of the entire ligand; thus, peptides covering a fraction of the anchor sites and with low free energy density cannot reach the affinity of the entire molecule. The earlier use of multiple linear regression to find the relevant predictors for effective binding, and a new way to estimate ΔG° and nonadditive interactions for the synthetic peptides in solution, by using the buried surface area (BSA), will be discussed.

Extracellular pH and lung infections in cystic fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease caused by CFTR mutations. It is characterized by high NaCl concentration in sweat and the production of a thick and sticky mucus, occluding secretory ducts, intestine and airways, accompanied by chronic inflammation and infections of the lungs. This causes a progressive and lethal decline in lung function. Therefore, finding the mechanisms driving the high susceptibility to lung infections has been a key issue. For decades the prevalent hypothesis was that a reduced airway surface liquid (ASL) volume and composition, and the consequent increased mucus concentration (dehydration), create an environment favoring infections. However, a few years ago, in a pig model of CF, the Na+/K+ concentrations and the ASL volume were found intact. Immediately a different hypothesis arose, postulating a reduced ASL pH as the cause for the increased susceptibility to infections, due to a diminished bicarbonate secretion through CFTR. Noteworthy, a recent report found normal ASL pH values in CF children and in cultured primary airway cells, challenging the ASL pH hypothesis. On the other hand, recent evidences revitalized the hypothesis of a reduced ASL secretion. Thus, the role of the ASL pH in the CF is still a controversial matter. In this review we discuss the basis that sustain the role of CFTR in modulating the extracellular pH, and the recent results sustaining the different points of view. Finding the mechanisms of CFTR signaling that determine the susceptibility to infections is crucial to understand the pathophysiology of CF and related lung diseases.

CFTR impairment upregulates c-Src activity through IL-1ß autocrine signaling.

Cystic Fibrosis (CF) is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Previously, we found several genes showing a differential expression in CFDE cells (epithelial cells derived from a CF patient). One corresponded to c-Src; its expression and activity was found increased in CFDE cells, acting as a signaling molecule between the CFTR activity and MUC1 overexpression. Here we report that bronchial IB3-1 cells (CF cells) also showed increased c-Src activity compared to 'CFTR-corrected' S9 cells. In addition, three different Caco-2 cell lines, each stably transfected with a different CFTR-specific shRNAs, displayed increased c-Src activity. The IL-1ß receptor antagonist IL1RN reduced the c-Src activity of Caco-2/pRS26 cells (expressing a CFTR-specific shRNA). In addition, increased mitochondrial and cellular ROS levels were detected in Caco-2/pRS26 cells. ROS levels were partially reduced by incubation with PP2 (c-Src inhibitor) or IL1RN, and further reduced by using the NOX1/4 inhibitor GKT137831. Thus, IL-1ß→c-Src and IL-1ß→NOX signaling pathways appear to be responsible for the production of cellular and mitochondrial ROS in CFTR-KD cells. In conclusion, IL-1ß constitutes a new step in the CFTR signaling pathway, located upstream of c-Src, which is stimulated in cells with impaired CFTR activity.

c- Src and its role in cystic fibrosis.

Cystic fibrosis (CF) is a lethal inherited disease produced by mutations in the gene encoding the CFTR chloride channel. Loss of function in the CFTR gene is associated with a not much noticed increased expression and activity of the non-receptor protein-tyrosine kinase c-Src. CF is therefore the result from the loss of CFTR chloride transport function and its consequences, including a chronic and excessive c-Src signaling. On the other hand, c-Src, encoded by the SRC gene, is involved in diverse signaling mechanisms that regulate key cellular functions such as cell proliferation, apoptosis, oxidative stress, inflammation, and innate immunity. These c-Src-regulated cellular functions are also affected in CF; however, studies exploring a direct role of c-Src in the regulation of these cellular functions in CF are yet scarce and often controversial. Here we describe the c-Src regulation and functions, with emphasis in those altered in CF, and describe the role of CFTR as a "signaling molecule" that negatively modulates c-Src expression and activity. It is also discussed the emerging role of intracellular Cl- and IL-1ß as intermediate signaling effectors between CFTR and c-Src.