Mouse Anti-Human CFTR Hybridoma [mAb 35-2]

Soluble secreted proteins and membrane proteins are subjected to protein quality control pathways during their synthesis in the endoplasmic reticulum (ER) and delivery to other destinations. Foremost among these quality control pathways is the selection of misfolded proteins for ER-associated degradation (ERAD). A growing number of diseases, including Cystic Fibrosis, are linked to the ERAD pathway. In most cases, a membrane protein known as the Cystic Fibrosis Transmembrane Conductance Regulator, or CFTR, is prematurely degraded by ERAD. Cell-based assays and in vitro studies have elucidated factors required for the recognition and degradation of CFTR, yet mechanistic details on how these factors target specific disease-causing variants is limited. Given the possibility that variants might exhibit unique susceptibilities to ubiquitin modification, which is required for proteasome-mediated degradation, we devised an assay that recapitulates this event. Here, we demonstrate that ER-enriched membranes from transfected human cells support CFTR ubiquitination when combined with radiolabeled ubiquitin and isolated enzymes in the ubiquitination cascade. We also show that select disease-causing variants are ubiquitinated more extensively than wild-type channels and to varying degrees. Our system provides a platform to examine how other purified factors impact CFTR ubiquitination and the ubiquitination of additional disease-associated membrane proteins.

Wearable sensors enable the monitoring of an individual's sweat composition in real time. In this work, we recorded real-time sweat chloride concentration for 12 healthy subjects in three different protocols involving step changes in exercise load and compared the results to laboratory-based analysis. The sensor results reflected the changes in exercise load in real time. On increasing the exercise load from 100W to 200W the sweat chloride concentration increased from 12.0 +/- 5.9 to 31.4 +/- 16mM (mean +/- SD). On decreasing the load from 200W to 100W, the sweat chloride concentration decreased from 27.7 +/- 10.5 to 14.8 +/- 8.1mM. The half-time associated with the change in sweat chloride, defined as the time at which the concentration reached half of the overall change, was about 6minutes. While the changes in sweat chloride were statistically significant, there was no correlation with changes in sweat rate or other physiological parameters, which we attribute to intra-individual variation (SD=1.6-8.1mM). The response to exercise-induced sweating was significantly different to chemically-induced sweating where the sweat chloride concentration was almost independent of sweat rate. We speculate that this difference is related to changes in the open probability of the CFTR channel during exercise, resulting in a decrease in reabsorption efficiency at higher sweat rates.