Targeted deubiquitination rescues distinct trafficking-deficient ion channelopathies

Abstract

Impaired protein stability or trafficking underlies diverse ion channelopathies and represents an unexploited unifying principle for developing common treatments for otherwise dissimilar diseases. Ubiquitination limits ion channel surface density, but targeting this pathway for the purposes of basic study or therapy is challenging because of its prevalent role in proteostasis. We developed engineered deubiquitinases (enDUBs) that enable selective ubiquitin chain removal from target proteins to rescue the functional expression of disparate mutant ion channels that underlie long QT syndrome (LQT) and cystic fibrosis (CF). In an LQT type 1 (LQT1) cardiomyocyte model, enDUB treatment restored delayed rectifier potassium currents and normalized action potential duration. CF-targeted enDUBs synergistically rescued common (ΔF508) and pharmacotherapy-resistant (N1303K) CF mutations when combined with the US Food and Drug Administation (FDA)-approved drugs Orkambi (lumacaftor/ivacaftor) and Trikafta (elexacaftor/tezacaftor/ivacaftor and ivacaftor). Altogether, targeted deubiquitination via enDUBs provides a powerful protein stabilization method that not only corrects diverse diseases caused by impaired ion channel trafficking, but also introduces a new tool for deconstructing the ubiquitin code in situ.

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Fig. 1: enDUBs reverse NEDD4L-mediated ubiquitination of KCNQ1.
Fig. 2: enDUBs rescue trafficking-deficient mutant LQT1 channels in HEK293 cells.
Fig. 3: enDUBs restore action potential duration in an LQT1 cardiomyocyte model.
Fig. 4: enDUBs facilitate rescue of mutant CFTR channels in combination with Orkambi.
Fig. 5: CF-targeted enDUB combination therapy functionally rescues rare trafficking-deficient CFTR mutations.
Fig. 6: Dual-acting enDUBs synergize to improve the functional rescue and apical localization of the most common ΔF508 mutation.

Data availability

All data generated or analyzed during this study are included in this published article (and its Extended Data files). All supporting data are available from the corresponding author upon reasonable request. The CFTR structure was reproduced from PDB (PDB: 5UAK). The nanobody structure was reproduced from PDB (3K1K).

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Acknowledgements

We thank M. Chen for technical support, M.B. Johny (Columbia University) for help with the flow cytometry–FRET assay and helpful discussions and L. Pon (Columbia University) for guidance in initiating yeast cultures. This work was supported by grants RO1-HL121253 and 1RO1-HL122421 from the NIH (to H.M.C.). S.A.K. was supported by a Medical Scientist Training Program grant (T32 GM007367) and an NHLBI National Research Service Award (1F30-HL140878). This work was also supported by the TRx Accelerator of the CUMC Irving Institute, as supported by the NCATS, NIH (UL1TR001873). Flow cytometry experiments were performed in the CCTI Flow Cytometry Core, supported in part by the NIH (S10RR027050). Confocal images were collected in the HICCC Confocal and Specialized Microscopy Shared Resource, supported by the NIH (P30 CA013696).

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Authors

Contributions

S.A.K. and H.M.C. conceived of the study and designed experiments. Z.S. performed CFTR electrophysiological measurements and analyses. P.C. contributed to molecular biology and the isolation of cardiomyocytes. A.J. contributed to KCNQ1 flow cytometry experiments. S.A.K. and H.M.C. wrote the manuscript; H.M.C. supervised the project.

Corresponding author

Correspondence to Henry M. Colecraft.

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Competing interests

S.A.K. and H.M.C. have filed a patent application through Columbia University based on this work.

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Peer review information Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 KCNQ1 pulldown and ubiquitin analyses after cell lysis with modified RIPA buffer containing 1% SDS.

a, Left, KCNQ1 pulldowns probed with anti-KCNQ1 antibody from HEK293 cells expressing KCNQ1-YFP ± NEDD4L with nano alone or enDUB-O1. The four bands represent KCNQ1 monomer, dimer, trimer, and tetrameric species, respectively. Right, Anti-ubiquitin labeling of KCNQ1 pulldowns after stripping previous blot. b, Relative KCNQ1 ubiquitination computed by ratio of anti-ubiquitin to anti-KCNQ1 signal intensity (n = 3 independent experiments; mean). **p < 0.003, one-way ANOVA with Tukey’s multiple comparison test. Source data

Extended Data Fig. 2 Flow cytometry gating strategy for BTX-647 surface labeling experiments.

a, Flow cytometry pseudocolor dot plots displaying an initial selection gate (left) for cells (vs debris); and a second selection gate (right) for singlets (vs doublets). b, Single color fluorescent controls after applying the gating strategy in a. c, Analysis gate established with single color controls in b to quantify channel surface density (BTX-647) in YFP- and CFP-positive cells (left) with cumulative distribution histograms (right). Sample 1 is exemplified by BBS-Q1-YFP + CFP-P2a-nano. Sample 2 is exemplified by BBS-Q1-YFP + CFP-P2a-nano + NEDD4L.

Extended Data Fig. 3 enDUB-O1 requires catalytic activity and target specificity for ubiquitin-dependent rescue of KCNQ1 channels.

a, (Left) Schematic of experimental strategy; BBS-Q1-YFP was co-transfected with either nanobody alone (grey line), NEDD4L + nano (red line), or NEDD4L + enDUB-O1 (blue line). (Right) Cumulative distribution histograms of Alexa647 fluorescence from flow cytometry analyses (data adapted from Fig. 1). Plot generated from population of YFP- and CFP-positive cells (n ≥ 5000 cells per experiment; N = 4). b, Same experiment as in a, but using catalytically inactive enDUB-O1* with C320S (N = 2). c, Same experiment as in a, but with untagged BBS-Q1 co-expressed with enDUB-O1 as a control for target specificity (N = 2).

Extended Data Fig. 4 enDUB-U21 has greater efficacy than enDUB-O1 in surface rescue of N1303K CFTR mutant channels.

a, Cumulative distribution histograms of Alexa647 fluorescence from flow cytometry analyses for cells expressing WT BBS-CFTR-YFP + nano (dotted line) and N1303K mutation co-expressing nano alone (red line), enDUB-O1 (cyan line), enDUB-U21 (blue line). Plot generated from population of YFP- and CFP-positive cells (n ≥ 5000 cells per experiment; N = 2). b, Same experimental design as above but with 24 hour incubation of VX809 with nano (green line), enDUB-O1 (cyan line) and enDUB-U21 (blue line) (N = 2).

Extended Data Fig. 5 enDUB-U21 requires catalytic activity and target specificity for ubiquitin-dependent rescue of CFTR mutants.

a, (Left) Schematic of experimental strategy; WT BBS-CFTR-YFP + nano (dashed line) or N1303K mutants co-transfected with nano (red line) or enDUB-U21 (blue line). Cumulative distribution histograms (middle) and quantification (right) of Alexa647 fluorescence from flow cytometry analyses (data adapted from Fig. 4). Plots generated from population of YFP- and CFP-positive cells (n ≥ 5000 cells per experiment; N = 3; mean ± s.e.m). Data are normalized to values from the WT CFTR control group (dotted line). b, Same experiment as in a, but using catalytically inactive enDUB-U21* with C221S (N = 3). c, Same experiment as in a, but with an mCherry-targeted nanobody, m-enDUB-U21, as a control for target specificity (N = 4). Source data

Extended Data Fig. 6 enDUB-U21 increases functional rescue of 4326delTC CFTR mutant channels in combination with lumacaftor ± ivacaftor.

a, Exemplar family of basal (top, black), forskolin-activated (middle, red), and VX770-potentiated (bottom, green) currents for 4326delTC mutant channels after 24 hr VX809 treatment (3 µM) and co-expression with nano (left) or enDUB-U21 (right). b, Population I-V curves for basal (black squares), forskolin-activated (red circles), and VX770-potentiated (green triangles) currents from 4326delTC mutants co-expressing nano alone (left; n = 15) or enDUB-U21 (right; n = 14). c, Same format as a, but with N1303K mutants. d, Same format as b, but with N1303K mutants co-expressing nano alone (left; n = 9) or enDUB-U21 (right; n = 11). Data for VX770-potentiated currents adapted from Fig. 4. Source data

Extended Data Fig. 7 Development of NBD1 binders from a yeast surface display nanobody library.

a, On-yeast binding affinity measurements of 9 nanobody clones using serial dilutions of purified FLAG-NBD1. b, Flow cytometric surface labeling assay and cumulative distribution histograms of WT CFTR surface density alone (dotted line) or when co-expressed with nanobody clones.

Extended Data Fig. 8 enDUB-U21CF.E3h deubiquitinates WT CFTR and N1303K.

a, Anti-ubiquitin labeling of WT CFTR and N1303K pulldowns. Lane 1 – untransfected cells; lane 2 – mCherry-CFTR + nanobody; lane 3 – mCherry-CFTR + enDUB-U21CF.E3h; lane 4 – mCherry-N1303K + nanobody; lane 5 – mCherry-N1303K + enDUB-U21CF.E3h. Proteins were pulled down with anti-mCherry and probed with anti-ubiquitin antibody (n = 2 independent experiments). b, Anti-CFTR labeling of WT CFTR and N1303K pulldowns. The top blot was stripped and probed with anti-CFTR antibody. Source data

Extended Data Fig. 9 enDUB-U21 restores functional currents of N1303K and F508del CFTR mutant channels in combination with Trikafta.

a, Population I-V curves for forskolin-activated currents from FRT cells stably expressing WT CFTR (black circles, n = 57) and N1303K + CFP (red squares, n = 8), and forskolin-activated, VX770-potentiated currents from N1303K + CFP treated with VX661 + VX445 (green triangles, n = 9); N1303K + enDUB-U21CF.E3h treated with VX661 + VX445 (blue triangles, n = 9); n cells examined over ≥3 independent experiments (mean ± s.e.m). Data for N1303K + CFP adapted from Fig. 5. b, Population I-V curves for forskolin-activated currents from FRT cells stably expressing WT CFTR (black circles, n = 57) and F508del + CFP (red squares, n = 12), and forskolin-activated, VX770-potentiated currents from F508del + CFP treated with VX661 + VX445 (green triangles, n = 10); F508del + enDUB-U21CF.T2a treated with VX661 + VX445 (blue triangles, n = 9); n cells examined over ≥3 independent experiments (mean ± s.e.m). Data for F508del + CFP adapted from Fig. 6. *p < 0.03, **p < 0.009,***p < 0.0001, two-way ANOVA with Tukey’s multiple comparison test. Source data

Extended Data Fig. 10 Mucociliary differentiation of human bronchial epithelial cells (hBECs) cultured at air-liquid interface (ALI).

a, H&E staining of immature (left; 5 day old) and mature hBEC ALI cultures (middle; 6 weeks), featuring a pseudostratified epithelium with mucin-containing goblet cells (*) (right). Apical (ap) and basal (bs) compartments labeled. b, Immunofluorescence staining of mature ALI cultures, featuring ciliated cells (green; acetylated-tubulin), mucin-containing goblet cells (pink; MUC5AC), and a basal cell layer (red; CK5), and merged image with DAPI staining (blue; nuclei). c, Immunostaining of mature WT hBEC cultures, with anti-CFTR signal (heat map) and anti-ezrin (green) labeling of the apical membrane. White box inset highlights the apical membrane (far right).

Supplementary information

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Data for bar charts and unprocessed western blots

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Source data for bar charts, IV curves and unprocessed western blots

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Source data for Ipeak and APD90 bar charts

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Source data for surface density bar charts and IV curves

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Source data for K-slope bar charts and IV curves

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Source data for surface density bar charts and IV curves

Source Data Extended Data Fig. 1

Source data for ubiquitin bar charts and unprocessed western blots

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Source data for surface density bar charts

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Source data for IV curves

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Unprocessed Western blots

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Source data for IV curves

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Kanner, S.A., Shuja, Z., Choudhury, P. et al. Targeted deubiquitination rescues distinct trafficking-deficient ion channelopathies. Nat Methods 17, 1245–1253 (2020). https://doi.org/10.1038/s41592-020-00992-6

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