Slowing ribosome velocity restores folding and function of mutant CFTR
Kathryn E. Oliver, … , Zoya Ignatova, Eric J. Sorscher
Kathryn E. Oliver, … , Zoya Ignatova, Eric J. Sorscher
Published October 28, 2019
Citation Information: J Clin Invest. 2019;129(12):5236-5253. https://doi.org/10.1172/JCI124282.
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Research Article Cell biology Genetics

Slowing ribosome velocity restores folding and function of mutant CFTR

Abstract

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), with approximately 90% of patients harboring at least one copy of the disease-associated variant F508del. We utilized a yeast phenomic system to identify genetic modifiers of F508del-CFTR biogenesis, from which ribosomal protein L12 (RPL12/uL11) emerged as a molecular target. In the present study, we investigated mechanism(s) by which suppression of RPL12 rescues F508del protein synthesis and activity. Using ribosome profiling, we found that rates of translation initiation and elongation were markedly slowed by RPL12 silencing. However, proteolytic stability and patch-clamp assays revealed RPL12 depletion significantly increased F508del-CFTR steady-state expression, interdomain assembly, and baseline open-channel probability. We next evaluated whether Rpl12-corrected F508del-CFTR could be further enhanced with concomitant pharmacologic repair (e.g., using clinically approved modulators lumacaftor and tezacaftor) and demonstrated additivity of these treatments. Rpl12 knockdown also partially restored maturation of specific CFTR variants in addition to F508del, and WT Cftr biogenesis was enhanced in the pancreas, colon, and ileum of Rpl12 haplosufficient mice. Modulation of ribosome velocity therefore represents a robust method for understanding both CF pathogenesis and therapeutic response.

Authors

Kathryn E. Oliver, Robert Rauscher, Marjolein Mijnders, Wei Wang, Matthew J. Wolpert, Jessica Maya, Carleen M. Sabusap, Robert A. Kesterson, Kevin L. Kirk, Andras Rab, Ineke Braakman, Jeong S. Hong, John L. Hartman IV, Zoya Ignatova, Eric J. Sorscher

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Figure 4

Surface function and baseline PO of F508del-CFTR are enhanced following repression of RPL12.

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Surface function and baseline PO of F508del-CFTR are enhanced following ...
(A, left panel) Ussing chamber analysis in CFBE shows RPL12 knockdown at low temperature (27°C). Following a temperature ramp to approximately 35–37°C, F508del-CFTR channels retained significantly more residual function with RPL12 repressed compared with that in NS control (n = 3–4). Asterisks represent statistical comparison between siRPL12 and NS siRNA. *P < 0.025, unequal variance t test with post-hoc Bonferroni’s correction; α = 0.025. (A, right panel) Silencing RPL12 at 37°C led to an approximately 7-fold higher F508del-CFTR ion transport activity than the NS control and appeared thermally stable for at least 15 minutes at 37°C (n = 3–4). Asterisks represent statistical comparison between siRPL12 and NS siRNA. **P < 0.01; ***P < 0.001; ****P < 0.0001, unequal variance t test with post-hoc Bonferroni correction; α = 0.025. (B, left panel) HEK-293T cells, amenable to membrane-patch analysis, were transfected with F508del-CFTR cDNA (without RPL12 knockdown) and cultured at 27°C for 24 hours to promote maturation (n = 9). In the setting of 27°C rescue, F508del-CFTR PO is significantly blunted compared with WT, yet can be stimulated by VX-770 (49, 58). (B, right panel) HEK-293T cells transfected with F508del-CFTR and siRPL12 (at 37°C) were analyzed by excised inside-out patch (n = 12). (C) Mean PO comparison of 27°C-corrected and siRPL12-rescued (at 37°C) F508del-CFTR prior to VX-770 addition. All channels were activated with PKA catalytic subunit (110 U/mL) and MgATP (1.5 mM). VX-770 (200 nM) was added with holding potential of 60 mV. Data are shown as mean ± SEM. **P < 0.01, 2-sample t test. siRPL12, RPL12-targeting siRNA;forskolin, 5 μM; Inh172, 10 μM.