(653) Targeting CFTR ubiquitination to augment therapeutic strategies

Background: Trikafta revolutionized treatment for CF patients who express F508del-CFTR and some other disease alleles. Without treatment, F508del-CFTR is rapidly degraded in the endoplasmic reticulum (ER). However, it is likely that even with Trikafta, a fraction of F508del-CFTR is still degraded. Moreover, antagonistic effects with anti-fungal medications, adverse events, and treatment-resistant alleles necessitate the search for drugs that improve Trikafta efficacy. We showed that drug-mediated inhibition of CFTR ubiquitination improves the ability of correctors to repair F508del-CFTR folding, transport, and activity [1]. More recently, we identified tool compounds that inhibit a ubiquitin activating enzyme as well as a ubiquitin ligase, CHIP, which modifies and targets F508del-CFTR for degradation [2,3]. To map CHIP-dependent ubiquitin sites in CFTR that trigger degradation, we reconstituted CHIP-dependent ubiquitination of purified CFTR and membrane embedded CFTR [4].

Methods: Wild-type CFTR or a subset of disease-associated CFTR variants in ER-derived membranes were treated with ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and CHIP (E3). The acquisition of ¹²⁵I-ubiquitin in an ATP-dependent manner was quantified after immunoprecipitation. The reaction was then scaled-up using purified wild-type CFTR in solution to maximize reaction specificity. After resolution by SDS-PAGE, in-gel trypsin digested CFTR was analyzed by nano-flow liquid chromatography tandem mass spectrometry (LCMS) to detect ubiquitinated lysines.

Results: In ER membranes, F508del CFTR and several severe disease-causing alleles were ubiquitinated to a greater extent than wild-type CFTR or mutants with subtle differences in conformation compared to wild-type CFTR, suggesting that CFTR conformation is maintained after subcellular fractionation. When ubiquitination sites on purified CFTR were mapped by LCMS, we identified both known sites that had previously been detected in analyses of CFTR ubiquitination from whole cell lysates, as well as novel sites. Interestingly, two ubiquitinated sites in the R domain were suggested to regulate cell surface localization, and another site potentially alters gating.

Conclusions: Our in vitro assays capitalize on the ability of the ubiquitination machinery to detect subtle conformations between wild-type and CFTR mutant alleles. A better definition of the distinct conformers amongst CFTR alleles will refine our ability to correct discrete molecular defects amongst diverse CFTR variants.

Acknowledgements: Supported by CFF grants BRODSK21I0, KAPPES20xx0 and KAPPES21xx0

References: [1] Chung, et al. Increasing the Endoplasmic Reticulum Pool of the F508DEL Allele of the Cystic Fibrosis Transmembrane Conductance Regulator Leads to Greater Folding Correction by Small Molecule Therapeutics. PLoS ONE (2016) 11:e0163615

[2] Pabon, et al. Proteome-scale detection of small-molecule protein interactions using data from gene expression perturbations. PLoS Comp. Biol. (2018) 14:e1006651

[3] Goeckeler-Fried, et al. Improved correction of F508del-CFTR biogenesis with a folding facilitator and an inhibitor of protein ubiquitination. Bioorg. Med. Chem. Lettr. (2021) 48:128243 [4] Estabrooks, S.E. and J.L. Brodsky. Recapitulating disease-causing CFTR variant ubiquitination in isolated membranes. Analytic. Biochem. (2020) 604:113829

[4] Estabrooks, S.E. and J.L. Brodsky. Recapitulating disease-causing CFTR variant ubiquitination in isolated membranes. Analytic. Biochem. (2020) 604:113829