Chemical and Biological Folding Contribute to Temperature-Sensitive ΔF508 CFTR Trafficking

Proteostasis (Balch et al. (2008) Science 319: 916) refers to the biology that maintains the proteome in health and disease. Proteostasis is challenged by the most common mutation in cystic fibrosis, ΔF508, a chloride channel (the cystic fibrosis transmembrane conductance regulator (CFTR)) that exhibits a temperature-sensitive phenotype for coupling to the coatomer complex II (COPII) transport machine for exit from the endoplasmic reticulum (ER). Whether rescue of export of ΔF508-CFTR at reduced temperature simply reflects energetic stabilization of the chemical fold defined by its primary sequence, or requires a unique proteostasis environment is unknown. We now show that reduced temperature (30°C) export of ΔF508 does not occur in some cell types despite efficient export of wild-type CFTR. We find ΔF508 export requires a local biological folding environment that is sensitive to heat/stress inducible factors found in some cell types suggesting that the energetic stabilization by reduced temperature is necessary, but not sufficient for export of ΔF508. Thus, the cell may require a proteostasis environment that is in part distinct from the wild-type pathway to restore ΔF508 coupling to COPII. These results are discussed in the context of the energetics of the protein fold and the potential application of small molecules to achieve a proteostasis environment favoring export of a functional form of ΔF508

Using small molecules to facilitate exchange of bicarbonate and chloride anions across liposomal membranes

Bicarbonate is involved in a wide range of biological processes, which include respiration, regulation of intracellular pH and fertilization. In this study we use a combination of NMR spectroscopy and ion-selective electrode techniques to show that the natural product prodigiosin, a tripyrrolic molecule produced by microorganisms such as Streptomyces and Serratia, facilitates chloride/bicarbonate exchange (antiport) across liposomal membranes. Higher concentrations of simple synthetic molecules based on a 4,6-dihydroxyisophthalamide core are also shown to facilitate this antiport process. Although it is well known that proteins regulate Cl-/HCO3- exchange in cells, these results suggest that small molecules may also be able to regulate the concentration of these anions in biological systems

Efficient, non‐toxic anion transport by synthetic carriers in cells and epithelia

Transmembrane anion transporters (anionophores) have potential for new modes of biological activity, including therapeutic applications. In particular they might replace the activity of defective anion channels in conditions such as cystic fibrosis. However, data on the biological effects of anionophores are scarce, and it remains uncertain whether such molecules are fundamentally toxic. Here, we report a biological study of an extensive series of powerful anion carriers. Fifteen anionophores were assayed in single cells by monitoring anion transport in real time through fluorescence emission from halide-sensitive yellow fluorescent protein. A bis-(p-nitrophenyl)ureidodecalin shows especially promising activity, including deliverability, potency and persistence. Electrophysiological tests show strong effects in epithelia, close to those of natural anion channels. Toxicity assays yield negative results in three cell lines, suggesting that promotion of anion transport may not be deleterious to cells. We therefore conclude that synthetic anion carriers are realistic candidates for further investigation as treatments for cystic fibrosis.info:eu-repo/semantics/publishe

A Chaperone Trap Contributes to the Onset of Cystic Fibrosis

Protein folding is the primary role of proteostasis network (PN) where chaperone interactions with client proteins determine the success or failure of the folding reaction in the cell. We now address how the Phe508 deletion in the NBD1 domain of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein responsible for cystic fibrosis (CF) impacts the binding of CFTR with cellular chaperones. We applied single ion reaction monitoring mass spectrometry (SRM-MS) to quantitatively characterize the stoichiometry of the heat shock proteins (Hsps) in CFTR folding intermediates in vivo and mapped the sites of interaction of the NBD1 domain of CFTR with Hsp90 in vitro. Unlike folding of WT-CFTR, we now demonstrate the presence of ΔF508-CFTR in a stalled folding intermediate in stoichiometric association with the core Hsps 40, 70 and 90, referred to as a ‘chaperone trap’. Culturing cells at 30 C resulted in correction of ΔF508-CFTR trafficking and function, restoring the sub-stoichiometric association of core Hsps observed for WT-CFTR. These results support the interpretation that ΔF508-CFTR is restricted to a chaperone-bound folding intermediate, a state that may contribute to its loss of trafficking and increased targeting for degradation. We propose that stalled folding intermediates could define a critical proteostasis pathway branch-point(s) responsible for the loss of function in misfolding diseases as observed in CF

An interlaboratory comparison on the characterization of a sub-micrometer polydisperse particle dispersion

The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments. The received datasets were organized by instrument class to enable comparison of intralaboratory and interlaboratory performance. The main findings included high variability between datasets from different laboratories, with coefficients of variation from 13 % to 189 %. Intralaboratory variability was, on average, 37 % of the interlaboratory variability for an instrument class and particle sub-population. Drop-offs at either end of the size range and poor agreement on maximum counts of particle sub-populations were noted. The mean distributions from an instrument class, however, showed the size-coverage range for that class. The study shows that a poly-disperse sample can be used to assess performance capabilities of an instrument set-up (including hardware, software, and user settings) and provides guidance for the development of polydisperse reference materials.Drug Delivery Technolog

Minimal sequential ordering of intra- and inter-domain folding events responsible for CFTR folding and trafficking.

<p>Intra-domain folding of NBD1 is dictated by the Hsp90 system (step 1). A structural rearrangement occurs in response to the binding of cytoplasmic loop 4 (CL4) to the F508 containing hydrophobic pocket present WT NBD1 (step 2). The binding of CL4 provides a stabilizing effect on NBD1, releasing Hsp90 and promoting H8–H9 helix-coil transition. This H8–H9 transition would expose the NBD2-binding interface of NBD1 and allow NBD1 to ‘chaperone’ <i>in trans</i> the folding of NBD2 (step 3).</p

Quantification of CFTR, Hsc70 and Hsp90 in CFTR-containing complexes.

<p><b>A.</b> Absolute abundance (ng/µl) of Hsp90 calculated by ¹⁵N protein labeling, AQUA labeling and Western blotting (WB). <b>B.</b> Absolute abundance (ng/µl) of Hsc70 calculated by ¹⁵N protein labeling, AQUA labeling and Western blotting (WB). In all panels, data is shown as mean ± SD, n≥3.</p

Stoichiometry of the ΔF508 CFTR interaction with core chaperones at physiological and corrective temperatures.

<p>Table depicting the absolute amounts of CFTR, Hsp90, Hsc70 and Hsp40, expressed in pmol. Also shown are the molar ratios of chaperones to total ΔF508-CFTR at both 37°C and 30°C. The fold change in the absolute amounts of CFTR, Hsp90 and Hsc70, expressed in pmol, relative to ΔF508-CFTR at 37°C is shown in the final column.</p

Stoichiometry of the WT and ΔF508 CFTR interaction with core chaperones.

<p>Table depicting the absolute amounts of CFTR, Hsp90 and Hsc70, expressed in pmol. Also shown are the molar ratios of chaperones to total ΔF508- or WT-CFTR. The fold change in the absolute amounts of CFTR, Hsp90 and Hsc70, expressed in pmol, relative to ΔF508-CFTR is shown in the final column.</p