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Targeted protein relocalization via protein transport coupling

Nature volume 633, pages 941–951 (2024)Cite this article

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Abstract

Subcellular protein localization regulates protein function and can be corrupted in cancers1 and neurodegenerative diseases2,3. The rewiring of localization to address disease-driving phenotypes would be an attractive targeted therapeutic approach. Molecules that harness the trafficking of a shuttle protein to control the subcellular localization of a target protein could enforce targeted protein relocalization and rewire the interactome. Here we identify a collection of shuttle proteins with potent ligands amenable to incorporation into targeted relocalization-activating molecules (TRAMs), and use these to relocalize endogenous proteins. Using a custom imaging analysis pipeline, we show that protein steady-state localization can be modulated through molecular coupling to shuttle proteins containing sufficiently strong localization sequences and expressed in the necessary abundance. We analyse the TRAM-induced relocalization of different proteins and then use nuclear hormone receptors as shuttles to redistribute disease-driving mutant proteins such as SMARCB1Q318X, TDP43ΔNLS and FUSR495X. TRAM-mediated relocalization of FUSR495X to the nucleus from the cytoplasm correlated with a reduction in the number of stress granules in a model of cellular stress. With methionyl aminopeptidase 2 and poly(ADP-ribose) polymerase 1 as endogenous cytoplasmic and nuclear shuttles, respectively, we demonstrate relocalization of endogenous PRMT9, SOS1 and FKBP12. Small-molecule-mediated redistribution of nicotinamide nucleotide adenylyltransferase 1 from nuclei to axons in primary neurons was able to slow axonal degeneration and pharmacologically mimic the genetic WldS gain-of-function phenotype in mice resistant to certain types of neurodegeneration4. The concept of targeted protein relocalization could therefore inspire approaches for treating disease through interactome rewiring.

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Fig. 1: Development of a quantitative cell-by-cell analysis pipeline and application to targeted protein relocalization.
Fig. 2: Nuclear hormone receptors are shuttles for cytoplasmic and diffuse proteins and provide a basis for quantitative assessment of target relocalization susceptibility.
Fig. 3: Targeted relocalization can move mutant proteins into nuclei and out of stress granules.
Fig. 4: Endogenous targets are susceptible to relocalization with TRAMs that engage endogenous shuttles.
Fig. 5: Targeted relocalization of NMNAT1 can protect against axon injury.

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Data availability

All data supporting the findings of this study are available within the Article, Extended Data Figs. 1–15, Supplementary Information or associated data tables. All single-cell quantification data are available upon request.

Code availability

All new code generated for the data analysis pipeline is available at https://github.com/cngsc/RAFL.

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Acknowledgements

We thank M. Gray for experimental assistance. This work was supported by an A*STAR fellowship to C.S.C.N. This work was supported in part by grant no. DP2GM154016 from the NIH/NIGMS.

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Authors and Affiliations

  1. Department of Chemistry, Stanford University, Stanford, CA, USA

    Christine S. C. Ng, Aofei Liu, Bianxiao Cui & Steven M. Banik

  2. Sarafan ChEM-H, Stanford University, Stanford, CA, USA

    Steven M. Banik

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  1. Christine S. C. Ng
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Contributions

C.S.C.N. and S.M.B. conceived of the project. C.S.C.N. conducted all experiments and analysed all data. A.L. and B.C. provided essential expertise in neuron collection and biology. C.S.C.N and S.M.B. wrote the manuscript. S.M.B. provided supervision.

Corresponding author

Correspondence to Steven M. Banik.

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Competing interests

S.M.B. is a member of the scientific advisory board for Lycia Therapeutics. Stanford University has filed a provisional patent application covering aspects of this work listing S.M.B. and C.S.C.N. as authors. The remaining authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Examination of NMNAT1 export cell lines.

a, Unlinked warhead controls for 1. 2: trimethoprim (TMP), binds EcDHFR, 3: binds FKBP12F36V. 1a, TRAM which engages EcDHFR and FKBP12F36V containing a (PEG)3 linker between the warheads. b, Localization of either protein upon treatment of EL-A with unlinked warheads 2 and 3. c, Comparison of the relative mCherry/GFP median fluorescence intensity ratio between the three isolated clonal export lines (EL-A-C). d, Ability of three NMNAT1 export lines exhibiting different relative export shuttle (mCherry) to NMNAT1 (GFP) ratios to translocate NMNAT1 from the nucleus promoted by 1 or inhibited by leptomycin B (LMB) after 3-hour treatment. e, Representative live-cell images for NMNAT1 relocalization treated with 1 or control molecules, or 1 and leptomycin-B (LMB) in Export Line A (EL-A) after 3 h. f, Representative live-cell images for NMNAT1 relocalization treated with 1 or 1 and leptomycin-B (LMB) in Export Line B (EL-B) after 3 h. g, Representative live-cell images for NMNAT1 relocalization treated with 1, or 1 and leptomycin-B (LMB) in Export Line C (EL-C) after 3 h. Levels of mCherry h, and GFP i, after treatment with 1 for 3 h across four isolated export cell lines with varying nuclear and cytoplasmic protein expression levels. j, Representative live-cell images for NMNAT1 relocalization with export line EL-A treated with varying concentrations of 1a. k, Dose response curve of NMNAT1 and NES localization for EL-A in response to treatment with 1a. MFI: Median fluorescence intensity. Images in e, f, g, j are representative of three biological replicates. Data in b, c, d, h, i, k are mean ± s.d. of three independent experiments. Scale bars are 20 µm. P values in b were determined by unpaired two-tailed t-tests comparing treatment to the DMSO control. NMNAT1 = FKBP12F36V-GFP-NMNAT1, NES = mCherry-ecDHFR-NES(HIV-REV1) P values in d, k were determined by one-way ANOVA with Dunnett’s post hoc test comparing each condition to the DMSO control. P values in h, i were determined by two-way ANOVA with Šidák post hoc test comparing the treated condition to the DMSO control for each line. Hoechst stains the nucleus.

Extended Data Fig. 2 Illustration of gating and segmentation pictures automatically generated from analysis pipeline applied to three different cell types.

a, Imaging analysis pipeline applied on HeLa cells. Gating histograms generated with the set bounds indicated. Masks for nuclear and cytoplasm areas overlayed onto respective fluorophore images. Cytoplasm areas are circumferentially grown from nuclei masks retaining the relative size between the masks. b, Imaging analysis pipeline applied to HEK293T cells. Masks for nuclear and cytoplasm areas overlayed onto respective fluorophore images, with segmentation of highly confluent cells and gating out of dead cells (high nuclear stain over a small area) and debris. c, Representative live-cell images for NMNAT1 relocalization in EL-A treated with 1 or control molecules in cells seeded at high (0.04 × 106 cells) and low density (0.01 × 106 cells). d, Quantification of nuclear GFP and mCherry from c. Images in c are representative of three biological replicates. Data in d are mean ± s.d. of three independent experiments. Scale bars are 20 µm. P values in d were determined by one-way ANOVA with Dunnett’s post hoc test comparing each condition to the DMSO control. Hoechst stains the nucleus.

Extended Data Fig. 3 NMNAT1 can be utilized as a nuclear import shuttle.

a, Representative live-cell images of clonal import line A (IL-A) upon treatment with 1 or small molecule controls for 3 h. b, Quantification of localization upon treatment of the IL-A with unlinked warheads 2 and 3. c, Comparison of the relative mCherry/GFP median fluorescence intensity ratio between the three isolated clonal import lines (IL-A-C). d, Ability of three representative import cell lines (IL) with different relative target (mCherry) and shuttle (NMNAT1) ratios to redistribute a diffuse protein target after 3-hour treatment with 1. e, Representative live-cell images of clonal import line B (IL-B) upon treatment with 1 for 3 h. f, Representative live-cell images of clonal import line C (IL-C) upon treatment with 1 for 3 h. g, Quantification of NMNAT1 protein localization change upon treatment with 10 nM 1 in IL-A-C possessing different mCherry/GFP ratios. h, Levels of GFP after treatment with 1 for 3 h in four isolated import cell lines with varying nuclear protein expression levels. i, Levels of mCherry after treatment with 1 for 3 h across four isolated import cell lines with varying diffuse protein expression levels. Images in a, e, f, are representative of three biological replicates. Data in b, c, d, g, h, i are mean ± s.d. of three independent experiments. Scale bars are 20 µm. MFI = median fluorescence intensity. P values in h, i were determined by two-way ANOVA with Šidák post hoc test comparing the treated condition to the DMSO control for each line. P values in b, d were determined by unpaired two-tailed t-tests comparing treatment to the DMSO control.

Extended Data Fig. 4 Validation of GR as import shuttle for FKBP12.

a, TRAM engaging GR and FKBP12. b, c Unlinked warhead controls for GR-based TRAMs. 8 binds to GR and 9 binds to FKBP12. d, Nuclear hormone receptor (ERα, GR) hijacking for import of target proteins. e, Nuclear localization of mCherry-FKBP12 after treatment with 6 or unlinked warheads 8 and 9 for 3 h. f, Nuclear localization of GR after treatment with 6 or unlinked warheads for 3 h. g, Genetic construct for stable incorporation. h, Representative live-cell images of GR-dependent FKBP12 import upon treatment with 6 or unlinked warheads 8 and 9 for 3 h. i, FKBP12 nuclear import dose response derived from the analysis pipeline after a 3-hour treatment with 6 engaging GR. j, Nuclear mCherry localization upon treatment with 6 in competition with either 1x or 10x of 8 and 9 for 3 h. k, Nuclear localization of GR upon treatment with 6 in competition with either 1x or 10x of 8 or 9 for 3 h. l, Representative live-cell images of the unlinked warhead competition assay for GR-dependent FKBP12 import after a 3-hour treatment. Images in h, l, are representative of three biological replicates. Data in e, f, i, j, k are mean ± s.d. of three independent experiments. Scale bars are 20 µm. P values in e, f, i, j, k were determined by one-way ANOVA with Dunnett’s post hoc test comparing each condition to the DMSO control. Hoechst stains the nucleus.

Extended Data Fig. 5 Validation of GR as import shuttle for FKBP12F36V.

a. TRAM engaging GR and FKBP12F36V. b, Representative live-cell images of GR dependent FKBP12F36V import upon treatment with 4 or unlinked warheads 8 and 3 for 3 h. c, FKBP12F36V nuclear import dose response derived from the analysis pipeline after a 3-hour treatment with 4 engaging GR. d, Nuclear localization of mCherry-FKBP12F36V after treatment with 4 or unlinked warheads 3 and 8 for 3 h. e, Nuclear localization of GR after treatment with 4 or unlinked warheads 3 and 8 for 3 h. f, Representative live-cell images of the unlinked warhead competition assay for GR-dependent FKBP12F36V import after 3-hour treatment. g, mCherry localization upon treatment with 4 in competition with either 1x or 10x of 8 or 3 for 3 h. h, Nuclear localization of GR upon treatment with 4 in competition with either 1x or 10x of 8 or 3 for 3 h. Images in b, f, are representative of three biological replicates. Data in c, d, e, g, h are mean ± s.d. of three independent experiments. Scale bars are 20 µm. P values in c, d, e, g, h were determined by one-way ANOVA with Dunnett’s post hoc test comparing each condition to the DMSO control. Hoechst stains the nucleus.

Extended Data Fig. 6 Validation of ERα as import shuttle for FKBP12.

a. TRAM engaging ERα and FKBP12, raloxifene (10) unlinked control binds to ERα. b, FKBP12 nuclear import dose response derived from the analysis pipeline after a 3-hour treatment with 7 engaging ERα. c, Representative live-cell images of ERα-dependent FKBP12 import upon treatment with 7 or unlinked warheads 10 and 9 for 3 h. d, Nuclear localization of mCherry-FKBP12 after treatment with 7 or unlinked warheads 10 and 9 for 3 h. e, Nuclear localization of ERα after treatment with TRAM 7 or unlinked warheads 10 and 9 for 3 h. f, Nuclear mCherry localization upon treatment with 7 in competition with either 1x or 10x of 10 or 9 for 3 h. g, Nuclear localization of ERα upon treatment with 7 in competition with either 1x or 10x of 10 or 9 for 3 h. h, Representative live-cell images of the unlinked warhead competition assay for ERα-dependent FKBP12 import after a 3-hour treatment. Images in c, h are representative of three biological replicates. Data in b, d, e, f, g are mean ± s.d. of three independent experiments. Scale bars are 20 µm. P values in b, d, e, f, g, were determined by one-way ANOVA with Dunnett’s post hoc test comparing each condition to the DMSO control. Hoechst stains the nucleus.

Extended Data Fig. 7 Validation of ERα as import shuttle for FKBP12F36V.

a. TRAM engaging ERα and FKBP12F36V. b, FKBP12F36V nuclear import dose response derived from the analysis pipeline after a 3-hour treatment with 5 engaging ERα. c, Representative live-cell images of ERα-dependent FKBP12F36V import upon treatment with 5 or unlinked warheads 3 and 10 for 3 h. d, Nuclear localization of mCherry-FKBP12F36V after treatment with 5 or unlinked warheads 3 and 10 for 3 h. e, Nuclear localization of ERα after treatment with 5 or unlinked warheads 3 and 10 for 3 h. f, Representative live-cell images of the unlinked warhead competition assay for ERα-dependent FKBP12F36V import for 3 h. g, mCherry localization upon treatment with 5 in competition with either 1x or 10x of 3 or 10 for 3 h. h, Nuclear localization of ERα upon treatment with 5 in competition with either 1x or 10x of 3 or 10 for 3 h. Images in c, f, are representative of three biological replicates. Data in b, d, e, g, h are mean ± s.d. of three independent experiments. Scale bars are 20 µm. P values in b, d, e, g, h were determined by one-way ANOVA with Dunnett’s post hoc test comparing each condition to the DMSO control. Hoechst stains the nucleus.

Extended Data Fig. 8 Nuclear import of mutant proteins with ERα.

a, Representative live-cell images of HeLa cells stably expressing target mutant protein SMARCB1Q318X (SMARCB1*) and ERα under an inducible promoter after a 3-hour treatment of 5. b, Representative live-cell images of HeLa cells stably expressing target mutant protein TDP43ΔNLS (TDP* = TDP43K82A/R83A/K84A) and ERα under an inducible promoter after a 3-hour treatment of 5. c, Representative live-cell images of HeLa cells stably expressing target mutant protein FUSR495X (FUS*) and ERα under an inducible promoter after a 3-hour treatment of 5. d, Violin plots of the distribution of respective target mutant protein and ERα in the three cell lines under different treatment conditions, across 3 repeats. e, Mean percent nuclear target protein and ERα upon treatment with 5 for 3 h in HeLa cells stably expressing the target mutant protein and ERα under an inducible promoter. f, Representative immunoblot of mutant proteins after a 3-hour treatment with 5. Images in a, b, c are representative of three biological replicates. Data in e, is mean ± s.d. of three independent experiments. Data in f is representative of two independent experiments. P values in e were determined by one-way ANOVA comparing each condition to the DMSO control. Scale bars are 20 µm. Hoechst stains the nucleus. Uncropped blots can be found in Supplementary Fig. 7.

Extended Data Fig. 9 TRAM-mediated protein relocalization from stress granules.

a, Timelapse imaging snapshots of FUSR495X when cells are treated with DMSO after stress granule formation. b, Timelapse imaging snapshots of FUSR495X extraction from granules when cells are treated with 5. c, Representative fixed-cell immunofluorescent images of stress granule marker G3BP1 and mCherry-FUSR495X after treatment with 5 or control compounds for 3 h. d, Quantification of stress granules in cells after treatment with varying concentrations of 5 or control compounds for 3 h. Images in c are representative of three biological replicates. Images in a, b are representative of two independent experiments. Data in d are mean ± s.d. of three independent experiments. Scale bars are 20 µm. P values in d were determined by two-way ANOVA with Dunnett’s post hoc test comparing each condition to their DMSO control.

Extended Data Fig. 10 Validation of PARP1 and METAP2 as protein shuttles in ectopic expression systems.

a, Unlinked warheads that engage METAP2 (14) and PARP1 (15). b, Co-immunoprecipitation of mCherry-FKBP12F36V upon GFP-METAP2 pulldown in the presence of TRAM. I = input, F = flowthrough, E = eluate. c, Representative live-cell images of mCherry-FKBP12F36V after treatment with 11 or with unlinked warheads, 14 or 3 for 3 h in the presence of METAP2. d, Quantification of FKBP12F36V relocalization upon treatment with 11 for 3 h. e, METAP2 localization upon TRAM 11 treatment for 3 h. 11. f, Representative live-cell-images of FKBP12F36V after treatment with 12 or the unlinked warheads, 15 and 3 or in the absence of PARP1 after 3 h. g, Quantification of mCherry-FKBP12F36V relocalization upon treatment with 12 for 3 h. h, Quantification of GFP-PARP1 localization upon TRAM upon treatment with 12 for 3 h. i, mCherry-FKBP12F36V localization upon treatment with 12 for 3 h in competition with either 1x or 10x of each individual unlinked warhead 15 and 3. j, Quantification of mCherry-FKBP12F36V localization from i. k, Quantification of GFP-PARP1 localization from i. Images in c, f, i, are representative of three biological replicates. Immunoblots in b are representative of two independent experiments. Data in d, e, g, h, j, k are mean ± s.d. of three independent experiments. Scale bars are 20 µm. P values in d, e, g, h, j, k were determined by one-way ANOVA with Dunnett’s post hoc test comparing each condition to the DMSO control. Hoechst stains the nucleus. Uncropped blots can be found in Supplementary Fig. 7.

Extended Data Fig. 11 Validation of Cas9-CRISPR mediated endogenously tagged lines.

a, Schematic for modified PITCh knock-in into target proteins. b, Sequencing of the regions near the N terminus of the endogenous PRMT9 gene after donor cassette insertion into endogenous PRMT9. c, Sequencing of the region downstream the N terminus of the endogenous SOS1 gene after donor cassette insertion into endogenous SOS1. d, Due to high GC content of the genomic region upstream of the N terminus of SOS1, immunoblotting was used to validate successful incorporation of donor cassette onto endogenous SOS1 after GFP pulldown, I = input, F = flowthrough, E = eluate. e, Sequencing of the regions near the N terminus of the endogenous FKBP12 gene after donor cassette insertion into endogenous FKBP12. f, Demonstration of GFP-FKBP12F36V tagging of endogenous PRMT9, SOS1 and GFP tagging of endogenous FKBP12 in HEK293T cells. Immunoblots in d,f are representative of two independent experiments. Uncropped blots can be found in Supplementary Fig. 7.

Extended Data Fig. 12 METAP2 as an endogenous nuclear export shuttle to relocalize endogenous PRMT9.

a, Representative live-cell images of endogenous PRMT9 localization upon treatment with different concentrations of TRAM 11 for 24 h. b, Violin plots illustrating the homogenous population shift in PRMT9 localization upon TRAM 11 treatment at different concentrations for 24 h. c, Representative live-cell images of endogenous PRMT9 localization upon treatment with unlinked warheads 14 and 3 for 24 h. d, Quantification of localization changes observed in c. e, Violin plots showcasing the lack of homogenous population shift in PRMT9 localization when treated with the unlinked warheads 14 and 3 versus TRAM 11 for 24 h. f, Quantification of the change in PRMT9 localization upon treatment with 11 after pre-treatment for 1 h with either 1x or 10x of 14 or competed with 1x, or 10x of 3 for 24 h. g, Representative images of the competition assay quantified in f. Images in a, c, g are representative of three biological replicates. Data in d, f are mean ± s.d. of three independent experiments. Scale bars are 20 µm. P values in b, d, e, f were determined by one-way ANOVA with Dunnett’s post hoc test comparing each condition to the DMSO control. Hoechst stains the nucleus.

Extended Data Fig. 13 PARP1 as an endogenous nuclear import shuttle to relocalize endogenous SOS1.

a, Representative live-cell images of endogenous SOS1 localization upon treatment with different concentrations of TRAM 12 for 24 h. b, Violin plots illustrating the homogenous population shift in SOS1 localization upon TRAM 12 treatment at different concentrations for 24 h. c, Representative live-cell images of endogenous SOS1 localization after treatment with unlinked warheads 15 and 3 for 24 h. d, Quantification of localization changes observed in c. e, Violin plots showcasing the lack of homogenous population shift in SOS1 localization when treated with the unlinked warheads 15 and 3 versus TRAM 12 for 24 h. f, Quantitation of change in SOS1 localization upon treatment with 12 in competition with either 1x or 10x of 15 or 3 for 24 h. g, Representative images of SOS1 localization in the competition assay in f. Images in a, c, g are representative of three biological replicates. Data in d, f are mean ± s.d. of three independent experiments. Scale bars are 20 µm. P values in b, d, e, f were determined by one-way ANOVA with Dunnett’s post hoc test comparing each condition to the DMSO control. Hoechst stains the nucleus.

Extended Data Fig. 14 PARP1 as an endogenous nuclear import shuttle to relocalize endogenous FKBP12.

a, Representative live-cell images of partial endogenous FKBP12 localization upon treatment with different concentrations of TRAM 13 for 24 h. b, Violin plots illustrating a homogenous population shift in FKBP12 localization upon TRAM 13 treatment at different concentrations for 24 h. c, Representative live-cell images of endogenous FKBP12 localization after treatment with unlinked warheads 15 and 9 for 24 h. d, Quantification of localization changes observed in c. e, Violin plots showcasing the lack of homogenous population shift in FKBP12 localization when treated with the unlinked warheads 15 and 9 versus TRAM 13 for 24 h. f, Quantification of the change in FKBP12 localization upon treatment with 13 in competition with either 1x or 10x of 15 or 9 for 24 h. g, Representative images of FKBP12 localization in the competition assay in f. Images in a, c, g are representative of three biological replicates. Data in d, f are mean ± s.d. of three independent experiments. Scale bars are 20 µm. P values in b, d, e, f were determined by one-way ANOVA with Dunnett’s post hoc test comparing each condition to the DMSO control. Hoechst stains the nucleus.

Extended Data Fig. 15 Nuclear export of NMNAT1 in neurons.

a, Representative live-cell images of NMNAT1 upon treatment with TRAM 1 or unlinked warheads 2 and 3 at 10 nM for 24 h. b, Representative images of before and after axotomy on explants. Axotomies were performed using a biopsy punch, the mRuby3-Axontag protein serves as an axonal marker, Axontag = GAP43at-mRuby3-ecDHFR. c, Representative images of axons and axon termini post-axotomy, that have been pre-treated with unlinked controls for 24 h prior. d, Representative images of mNMNAT1 in axons after exposure to 1 for 24 h followed by axotomy. mRuby3 signal was used to monitor degeneration across treated neurons. d, Quantification of axon degeneration over time. e, Quantification of axon degeneration over time in axon termini. f, Comparison of the degeneration index in axona at different time points with different treatment conditions. g, Comparison of the degeneration index (DI) in the axon termini at different time points with different treatment conditions. DI was calculated as the ratio of fragmented axon area over total axon area. For plots in d, f, a total of 12 replicates derived from 4 different embryos were combined in the plots for all conditions except 1 nM 2 + 1 nM 3. A total of 6 replicates derived from 2 different embryos were combined in the plots for the 1 nM 2 + 1 nM 3 condition. For plots in e, g, a total of 6 replicates derived from 4 different embryos were combined in the plots for all conditions except 1 nM 2 + 1 nM 3. A total of 4 replicates derived from 2 different embryos were combined in the plots for the 1 nM 2 + 1 nM 3 condition. Data in d, e is shown as the mean ± s.e.m. values. Data in f, g, is shown as the mean ± s.d. values. P values in d, e, were determined by two-way ANOVA with Tukey’s post hoc test comparing the 1 nM treatment of 1 with the DMSO (purple) or the unlinked warhead control (green) at each time point. P values in f, g, were determined by one-way ANOVA with Dunnett’s post hoc test comparing each treatment to the DMSO control at each time point. Images in a, b, c are representative of neurons harvested from 4 different embryos. Scale bars are 20 µm.

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Ng, C.S.C., Liu, A., Cui, B. et al. Targeted protein relocalization via protein transport coupling. Nature 633, 941–951 (2024). https://doi.org/10.1038/s41586-024-07950-8

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