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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