Dataset Information

A genomic signature approach to rescue ?F508-cystic fibrosis transmembrane conductance regulator biosynthesis and function.

ABSTRACT: The most common cystic fibrosis (CF) mutation, ?F508, causes protein misfolding, leading to proteosomal degradation. We recently showed that expression of miR-138 enhances CF transmembrane conductance regulator (CFTR) biogenesis and partially rescues ?F508-CFTR function in CF airway epithelia. We hypothesized that a genomic signature approach can be used to identify new bioactive small molecules affecting ?F508-CFTR rescue. The Connectivity Map was used to identify 27 small molecules with potential to restore ?F508-CFTR function in airway epithelia. The molecules were screened in vitro for efficacy in improving ?F508-CFTR trafficking, maturation, and chloride current. We identified four small molecules that partially restore ?F508-CFTR function in primary CF airway epithelia. Of these, pyridostigmine showed cooperativity with corrector compound 18 in improving ?F508-CFTR function. There are few CF therapies based on new molecular insights. Querying the Connectivity Map with relevant genomic signatures offers a method to identify new candidates for rescuing ?F508-CFTR function.

SUBMITTER: Ramachandran S

PROVIDER: S-EPMC4189493 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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A genomic signature approach to rescue ΔF508-cystic fibrosis transmembrane conductance regulator biosynthesis and function.

Ramachandran Shyam S Osterhaus Samantha R SR Karp Philip H PH Welsh Michael J MJ McCray Paul B PB

American journal of respiratory cell and molecular biology 20140901 3

The most common cystic fibrosis (CF) mutation, ΔF508, causes protein misfolding, leading to proteosomal degradation. We recently showed that expression of miR-138 enhances CF transmembrane conductance regulator (CFTR) biogenesis and partially rescues ΔF508-CFTR function in CF airway epithelia. We hypothesized that a genomic signature approach can be used to identify new bioactive small molecules affecting ΔF508-CFTR rescue. The Connectivity Map was used to identify 27 small molecules with potent ...[more]

PMID: 24669817

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Project description:Background: Most cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that lead to protein misfolding and degradation by the ubiquitin–proteasome system. Previous studies demonstrated that PIAS4 facilitates the modification of wild-type (WT) and F508del CFTR by small ubiquitin-like modifier (SUMO)-1, enhancing CFTR biogenesis by slowing immature CFTR degradation and producing increased immature CFTR band B. Methods: We evaluated two correction strategies using misfolding mutants, including the common variant, F508del. We examined the effects on mutant expression of co-expression with PIAS4 (E3 SUMO ligase), and/or the corrector, C18. To study the impact of these correction conditions, we transfected CFBE410- cells, a bronchial epithelial cell line, with a CFTR mutant plus: (1) empty vector, (2) empty vector plus overnight 5 μM C18, (3) PIAS4, and (4) PIAS4 plus C18. We assessed expression at steady state by immunoblot of CFTR band B, and if present, band C, and the corresponding C:B band ratio. The large PIAS4-induced increase in band B expression allowed us to ask whether C18 could act on the now abundant immature protein to enhance correction above the control level, as reported by the C:B ratio. Results: The data fell into three mutant CFTR categories as follows: (1) intransigent: no observable band C under any condition (i.e., C:B = 0); (2) throughput responsive: a C:B ratio less than control, but suggesting that the increased band C resulted from PIAS4-induced increases in band B production; and (3) folding responsive: a C:B ratio greater than control, reflecting C18-induced folding greater than that expected from increased throughput due to the PIAS4-induced band B level. Conclusion: These results suggest that the immature forms of CFTR folding intermediates occupy different loci within the energetic/kinetic folding landscape of CFTR. The evaluation of their properties could assist in the development of correctors that can target the more difficult-to-fold mutant conformations that occupy different sites within the CFTR folding pathway.

Dataset Information

A genomic signature approach to rescue ?F508-cystic fibrosis transmembrane conductance regulator biosynthesis and function.

Publications

A genomic signature approach to rescue ΔF508-cystic fibrosis transmembrane conductance regulator biosynthesis and function.

Similar Datasets

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