Abstract
Targeted protein degradation and stabilization are promising therapeutic modalities because of their potency, versatility and their potential to expand the druggable target space1,2. However, only a few of the hundreds of E3 ligases and deubiquitinases in the human proteome have been harnessed for this purpose, which substantially limits the potential of the approach. Moreover, there may be other protein classes that could be exploited for protein stabilization or degradation3,4,5, but there are currently no methods that can identify such effector proteins in a scalable and unbiased manner. Here we established a synthetic proteome-scale platform to functionally identify human proteins that can promote the degradation or stabilization of a target protein in a proximity-dependent manner. Our results reveal that the human proteome contains a large cache of effectors of protein stability. The approach further enabled us to comprehensively compare the activities of human E3 ligases and deubiquitinases, identify and characterize non-canonical protein degraders and stabilizers and establish that effectors have vastly different activities against diverse targets. Notably, the top degraders were more potent against multiple therapeutically relevant targets than the currently used E3 ligases cereblon and VHL. Our study provides a functional catalogue of stability effectors for targeted protein degradation and stabilization and highlights the potential of induced proximity screens for the discovery of new proximity-dependent protein modulators.
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Data availability
Mass spectrometry data have been deposited into the MassIVE repository with the accession number MSV000093202. The ProteomeXchange accession is PXD046426. Full versions of all blots are provided in Supplementary Fig. 3. Source data are provided with this paper.
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Acknowledgements
We would like to thank C. Mogg for his help with experiments and D. Durocher, F. Sicher and members of the Taipale Laboratory for their comments on the manuscript. This work was supported by the David Dime and Elisa Nuyten Catalyst Fund award to M.T., the Mark Foundation for Cancer Research ASPIRE Award to M.T. (joint principal investigators: F. Sicheri and D. Durocher), the Charles H. Best Postdoctoral Fellowship to J.P., and the CIHR Fellowship Award to H.C.
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Contributions
J.P., A.D., N.A. and M.T. conceptualized the study. J.P., H.C. and A.D. executed the majority of the experiments. S.H. conducted xenograft experiments. A.Z.I. conducted the CRISPR–Cas9 screen. M.H.Y.L., N.A. and L.M. helped with cloning and establishing and benchmarking the induced proximity assay. J.P. and C.W. conducted mass spectrometry experiments and J.L. helped with microscopy. J.P., H.C. and M.T. visualized the data and created figures. J.P. and M.T. wrote the original draft that was also reviewed and edited by H.C. and S.H. A.-C.G., D.S. and M.T. supervised the study and acquired funding.
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Competing interests
M.T. is a co-founder of Induxion Therapeutics. The University of Toronto has filed a patent application (pending; PCT/CA2023/050511) related to proteome-scale induced proximity screens. M.T., J.P., A.D., N.A. and L.M. are listed as co-inventors. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Establishing the induced proximity assay.
(a) Representative flow cytometry plots of the EGFP-ABI1-IRES-TagBFP dual-reporter 293T stable line transduced with C-terminal PYL1 fusions. SPOPΔMATH is lacking its native substrate-binding domain (residues 28–166). Cells were treated with either 0.5% DMSO or 100 µM abscisic acid (ABA) for 24 h. (b) Frequency of high EGFP population as a function of ABA concentration and treatment time in the reporter cell line transduced with the C-terminal SPOPΔMATH-PYL1 fusion. (c) Representative flow cytometry plots of the EGFP-ABI1-IRES-TagBFP dual-reporter 293T stable line transduced with C-terminal vhhGFP fusions of Nanoluc, CRBN, full-length SPOP, or SPOPΔMATH.
Extended Data Fig. 2 Features of effector proteins identified in pooled screens.
(a) Outline of the pooled ORFeome screens for protein stability regulators. (b) 5 days after completion of the selection, cells infected with the ORFeome-PYL1 library were treated with 100 µM abscisic acid (ABA) for 48 h and top 10% and bottom 10% of cells with EGFP/BFP ratio were sorted. About 1.5 million cells were sorted in duplicate for both subpopulations to reach a coverage of 100x. Biological replicates correlations were similar between unsorted (R2 = 0.74) and top 10% (R2 = 0.77) subpopulations, and a bit lower for the bottom 10% subpopulation (R2 = 0.60). (c) Similar FACS gates were used for the sorting of the ORFeome-vhhGFP infected cells 5 days after completion of the selection. 1.8 million cells were sorted in triplicate, with similar replicate correlations between unsorted (R2 = 0.75 to 0.80), top 10% (R2 = 0.88 to 0.91), and bottom 10% (R2 = 0.86 to 0.95) subpopulations. (d) Enrichment of ORFs in the low EGFP pool infected with the ORFeome-vhhGFP compared to unsorted cells. Significantly enriched ORFs are shown in blue. (e) Enrichment of ORFs in the high EGFP pool infected with the ORFeome-vhhGFP compared to unsorted cells. Significantly enriched ORFs are shown in red. (f-g) Enrichment of Gene Ontology (GO) categories in the ORFeome-PYL1 screen hits (f) and ORFeome-vhhGFP screen hits (g). (h) Enriched GO categories in the overlapping hits between ORFeome-vhhGFP and ORFeome-PYL1 stabilization screens. CC, cellular component; MF, molecular function; BP, biological process. (i) Enrichment of PFAM and Interpro domains in the degradation screen hits. (j) Enrichment of PFAM and Interpro domains in the stabilizer hits. (k-l) Comparison of protein half-lifes21 (k) and protein steady state levels22 (l) between hits and all tested proteins. Statistical significance was calculated with a two-tailed t-test. ns, non-significant; *, p≤0.0181; **, p≤0.0064; ****, p≤0.0001.
Extended Data Fig. 3 ORFeome-PYL1 and ORFeome-vhhGFP library characteristics.
(a) Distribution of sequencing reads across the 293T EGFP-ABI1 reporter cell line infected with the ORFeome-PYL1 library. (b) Distribution of sequencing reads across the 293T EGFP-ABI1 reporter cell line infected with the ORFeome-vhhGFP library (c) Distribution of ORF sizes (bp) in the 293T EGFP-ABI1 reporter cell line infected with the ORFeome-PYL1 library. (d) Distribution of ORF sizes (bp) in the 293T EGFP-ABI1 reporter cell line infected with the ORFeome-vhhGFP library. Median ORF length in the screen (black) and in the ORFeome libraries (blue) are indicated by vertical dotted lines.
Extended Data Fig. 4 Effect of induced-proximity dependency, tag location and linker size on effector activity.
(a) The EGFP-ABI1 293T reporter cell line was transfected with indicated effectors fused to vhhGFP or 3xFLAG. EGFP fluorescence was measured by flow cytometry and normalized to cells transfected with an unrelated construct (no effector). D, Degrader hit; S, Stabilizer hit. (b) The EGFP-ABI1 293T reporter cell line was transfected with indicated effectors fused in their C terminus or N terminus. EGFP fluorescence was measured and normalized as described in (a). (d) Indicated C-terminal vhGFP fusions cloned either with the original or a minimal linker were transfected in the EGFP-ABI1 293T reporter cell line. EGFP fluorescence was measured and normalized as described in (a). Of the two independent experiments performed, results from one representative experiment are shown.
Extended Data Fig. 5 Analysis of E3 activity in proximity-dependent degradation.
(a) Comparison of two independent replicates for the degradation assay with 290 individual vhhGFP-tagged E3s transfected into the EGFP-ABI1 293T reporter cell line. (b) Relative mRNA expression of EGFP in 293T EGFP-ABI1 reporter cell line transfected with indicated effectors fused to vhhGFP. GAPDH mRNA levels were used to normalize the EGFP mRNA values. (c) Comparison of degrader hits recovered from at least one of the pooled ORFeome screens (vhhGFP or PYL1) and non-hits in arrayed degradation assay. Statistical significance was calculated with unpaired two-tailed Mann Whitney test (**, p < 0.0036). (d) Effect of different E3 families on EGFP-ABI1 reporter stability in 293T cells. RING, Really Interesting New Gene; BTB (also known as POZ), BR-C, Ttk and Bab; CRL, Cullin E3 RING ubiquitin ligase; DCAF, DDB1 and Cul4 associated factor. (e) Phylogenetic clustering of FBXL family E3s and their activity in the degradation assay in the EGFP-ABI1 293T reporter cell line. (f) As in D, but for BTB-BACK-Kelch family E3s. (g) Comparison of the activity of full-length E3 constructs (FL) to their splice variants without the RING domain in the EGFP-ABI1 293T reporter cell line.
Extended Data Fig. 6 UBE2B mutations and truncations.
(a) Western blot analysis of 293T cells transfected with wild-type UBE2B-vhhGFP or the catalytically inactive mutant C88A. (b) Expression of UBE2B mutants deficient in E3 binding transfected with the indicated vhhGFP fusions into 293T cells. RQRR, S25R/V39Q/N65R/T99R quadruple mutant. (c) Structure of UBE2B (PDB 2YB6). Fragments 1–18 (light orange), 124–134 (black), and 134–152 (grey) are indicated. (d) Western blot analysis of 293T transfected with wild-type or truncated UBE2B-vhhGFP. (e) Effect of UBE2B truncation on proximity-dependent degradation. The EGFP-ABI1 293T reporter cell line was transfected with indicated vhhGFP fusions and reporter stability was measured by flow cytometry. Of the two independent experiments performed for (a), (b) and (d), results from one representative experiment are shown.
Extended Data Fig. 7 Degrons can function in trans for proximity-dependent degradation.
(a) Full-length EID1 and PRR20A or their fragments were either directly fused to EGFP (left) to assess degradation in cis or to vhhGFP (right) for degradation in trans. EGFP fusions were transfected into 293T cells and protein stability measured as EGFP/DsRed ratio. VhhGFP fusions were transfected into the EGFP-ABI1 293T cell line. Fluorescence was measured with FACS and normalized to Renilla luciferase (RLuc) fused to EGFP (left) or to RLuc-vhhGFP (right). (b) Western blot analysis of FBXO21 protein expression in 293T EGFP-ABI1 reporter cells infected with sgRNA-Cas9 expression plasmids, targeting FBXO21 or the adeno-associated virus integration site 1 (AAVS1) locus. FBXO21-specific sgRNA1 and 2 show 80% and 90% reduction of target protein level, respectively, compared to AAVS1 sgRNA. Of the two independent experiments performed, results from one representative experiment are shown. (c) Impact of FBXO21 knockout on EID1 and PRR20A induced-proximity degradation. C-terminal vhhGFP fusions were transfected into the control (AAVS1 sgRNA) or FBXO21 knockout (FBXO21 sgRNA2) EGFP-ABI1 293T cells. Fluorescence was measured with flow cytometry and normalized to RLuc-vhhGFP.
Extended Data Fig. 8 GPI-anchored proteins can mediate proximity-induced degradation.
(a) Domain structures of FCGR3B, PRNP, and FCGR3A. GPI-anchored proteins contain an N-terminal signal peptide and a C-terminal GPI signal sequence. ω is the residue to which the GPI moiety is attached. FCGR3A is not GPI-anchored but contains a C-terminal transmembrane domain (TMD). (b) Effect of mutations and domain substitutions on GPI-anchored proteins proximity-induced degradation. The EGFP-ABI1 293T reporter cell line was transfected with indicated vhhGFP fusions. Reporter stability was measured by flow cytometry and normalized to reporter cells transfected with unrelated DNA (no effector). (c) C-terminal deletion constructs of FCGR3B were assayed as vhhGFP fusions in the EGFP-ABI1 293T reporter cells as in (b). (d) Cellular localization of the EGFP-ABI1 reporter upon expression of GPI-anchored effectors fused to vhhGFP. The EGFP-ABI1 293T reporter cell line was transfected with indicated effectors fused to vhhGFP. Microscopy images of EGFP-ABI1 293T reporter cell line expressing indicated GPI-anchored proteins or FCGR3B tail mutants. ConA, concanavaline A. Of the two independent experiments performed, results from one representative experiment are shown. (e) Alanine scanning of the C-terminal tail of FCGR3B. Indicated FCGR3B C-terminal tail mutants fused to vhhGFP were assessed in the EGFP-ABI1 293T reporter cell line. (f) Outline of the genome-wide CRISPR/Cas9 screen in the EGFP-ABI1 293T reporter cells stably expressing FCGR3B-vhhGFP. (g) Volcano plot comparing significance of sgRNA enrichment/depletion in the bottom 10% (left panel) or top10% (right panel) EGFP/TagBFP ratio sorted populations and log2 fold change (top 10% vs bottom 10%) for all sgRNAs in the CRISPR TKOv3 library. Data were analyzed with MAGeCK. (h) Effect of SIK inhibitor ARN-3236 on FCGR3B proximity-induced degradation. The assay was conducted as in (b). Cells were either treated with DMSO (control) or ARN-3236 (5 µM) for 24 h. (i) MAGeCK ranking of GPI-anchored biogenesis proteins in the genome-wide CRISPR/Cas9 screen in the EGFP-ABI1 293T reporter cells stably expressing FCGR3B-vhhGFP. Statistical significance of GPI-anchored genes enrichment among other genes was calculated with a two-tailed Mann-Whitney test. (j) Schematic of the addition of a GPI precursor to a protein. The GPI anchor is built up by sequential modifications of sugars and lipids, including the transfer of the first mannose to the GPI-precursor by PIGM, the catalytic component of the GPI mannosyltransferase 1 complex. The precursor protein is synthesized independently of the GPI precursor. The N-terminal signal sequence induces translocation of the nascent protein into the ER lumen, but the C-terminal hydrophobic sequence halts translocation and is inserted to the ER membrane. The protein precursor polypeptide chain is cleaved between the ω and ω + 1 residues by the GPI transamidase complex consisting of five proteins including PIGK and PIGT, followed by the attachment of the GPI anchor to the protein and release of the C-terminal hydrophobic peptide. (k) Cell surface expression of GPI-anchored protein CD55 in control (AAVS1) and indicated knockout EGFP-ABI1 293T reporter cells. CD55 surface expression was detected by flow cytometry, using a CD55 specific antibody coupled to APC. (l) Effect of knockouts on FCGR3B proximity-induced degradation. EGFP-ABI1 293T knockout reporter cell lines were transfected with indicated vhhGFP fusions and the reporter stability was measured by flow cytometry and normalized to reporter cells transfected with unrelated DNA (no effector).
Extended Data Fig. 9 Deubiquitinases and KLHL40 as proximity-dependent stabilizers.
(a) Western blot analysis of deubiquitinase mutants fused to vhhGFP transfected into 293T cells. (b) Multiple sequence alignment of BTB domains from different BTB-BACK-Kelch domains proteins and SPOP (binds CUL3), colored by conservation. The φ-X-E motif residues (where φ represents a hydrophobic amino acid) that binds CUL3 is annotated with arrows. (c) Western blot analysis of KLHL40 constructs fused to vhhGFP transfected into 293T cells. Note that Hsp90 band could not be observed on the KLHL40 lane after stripping the blot; however, the same amount of total lysate was loaded. (d) Soluble/insoluble fraction western blots of EGFP-ABI1 (left). EGFP-ABI1 293T reporter cell line was transfected with indicated vhhGFP fusions. S and P indicate soluble and pellet fraction, respectively. HSP90 shows the soluble fraction and histone H3 shows the insoluble fraction. The relative proportion of EGFP-ABI1 present in each fraction is shown on the right. (e) KLHL40 stabilizes EGFP in C2C12 myoblast cell line. C2C12 were transduced with lentiviruses encoding EGFP-ABI1-IRES-TagBFP and Rluc-vhhGFP or KLHL40-vhhGFP. EGFP and TagBFP fluorescence was measured by flow cytometry in the vhhGFP positive population using flow cytometry. Two independent experiments were performed for (a), (c) and (d). Results from one representative experiment are shown.
Extended Data Fig. 10 Loss of EGFP signal is due to EGFP ubiquitin-mediated proteolysis.
293T cells were co-transfected with indicated effectors fused to vhhGFP and EGFP or EGFP13R fused to either ABI1 or to one of two lysine-less substrates, human p14ARF and oncoprotein E7 from human papillomavirus 58 (HPV58). EGFP13R is a GFP mutant where thirteen surface lysines are mutated to arginines. Of note, the vhhGFP epitope is not mutated in EGFP13R. EGFP fluorescence was measured 48 h after transfection by flow cytometry.
Extended Data Fig. 11 Cellular localization of EGFP-tagged targets.
Microscopy images of stable 293T cell lines expressing indicated doxycycline-inducible EGFP-tagged proteins. Cells were treated with 1 µg/ml doxycycline for 24 h prior imaging. Of the two independent experiments performed, results from one representative experiment are shown.
Extended Data Fig. 12 Targeting non-GFP tagged proteins with novel effectors.
(a) HeLa cells were co-transfected with 3xFLAG-V5-KRAS and indicated effectors fused to an intracellular single domain antibody (iDab) targeting Ras or LMO2 (control). (b) 293T cells were co-transfected with IKBKE fused to ALFA-3xFLAG tag and indicated effectors fused to Nb(ALFA)-Myc, followed by western blotting for IKBKE (anti-FLAG), effector (anti-Myc), and Hsp90. (c) Indicated effectors fused to Nb(ALFA)-Myc were co-transfected with ALFA-3xFLAG-ARAF into 293T cells, followed by western blotting for ARAF (anti-FLAG), effector (anti-Myc), and Hsp90. (d) Stable HCT116 cell lines expressing doxycycline-inducible effectors fused to WDR5-targeting monobody Mb(WDR5) were treated with 1 µg/ml doxycycline for 48 h or left untreated. Top, Endogenous WDR5 levels and effector expression were assessed by western blotting. Bottom, Quantification of WDR5 levels after doxycycline induction. Statistical significance was calculated with an unpaired t-test with false discovery rate correction for multiple hypotheses. (e) Correlation between K562 cell proliferation and BCR-ABL levels in cells stably expressing effectors fused to Mb(ABL) treated with doxycycline for 6 days. (f) Volcano plots of the p-value versus the log2 fold change of proteins in K562 cells transduced with indicated doxycycline-inducible effector fused to Mb(ABL)-HA treated with doxycycline for 24 h versus control. (g) Correlation between FBXL15 and FBXL12 proteomics profiles in K562. (f and g) Each dot represents a protein. Points colored black, blue, and orange indicate the endogenous BCR-ABL fusion protein, FBXL12-Mb(ABL)-HA fusion protein, and FBXL15-Mb(ABL)-HA fusion protein, respectively. At least two independent experiments were performed for (a), (b), (c) and (d). Results from one representative experiment are shown. (h) Mouse xenograft model of K562 chronic myeloid leukemia cells expressing effectors fused to BCR-ABL targeting Mb(ABL) monobody. Doxycycline inducible K562 cell lines expressing the listed effectors were subcutaneously injected into NSG mice (10 mice per cell-line). 2 weeks post transplantation, mice were randomly divided into two groups: vehicle (no Dox) and doxycycline (+ Dox). Tumor growth was measured with a caliper. Data are presented as mean values +/− SD. N = 5 animals *, p≤0.0389; **, p≤0.0074; ***, p≤0.0006; ****, p≤0.0001 (two-way ANOVA with Geisser-Greenhouse correction and Šídák correction for multiple comparisons).
Extended Data Fig. 13 Doxycycline titration of effector expression and endogenous tagging of effectors.
(a) Top, relative mRNA expression of doxycycline-inducible effectors fused to ABL-targeting monobody Mb(ABL) in K562 treated for 48 h with different concentrations of doxycycline. Bottom, western blot analysis of endogenous BCR-ABL levels, effector expression (HA), and HSP90 or actin in the same K562 cells treated with increasing doses of doxycycline for 48 h. GAPDH mRNA levels were used to normalize the effector mRNA values. Of the two independent experiments performed, results from one representative experiment are shown. (b) Schematic of the CRISPR/Cas9 tagging system. The tagging cassette containing vhhGFP nanobody (wt or mutant), a P2A ribosomal skipping motif (P2A), TagRFP, an Internal Ribosome Entry Site (IRES), and EGFP, is flanked by homology arm sequences. Co-transfection of this tagging cassette with a CRISPR/Cas9 plasmid bearing the exon-specific single guide RNA (sgRNA) was used to add the tagging cassette at the C-terminus of the endogenous locus in 293T. (c) Diagnostic PCR of knock-ins compared to the 293T parental cell line. Expected DNA fragments are indicated as white arrows. Primer target sites are shown in (b). (d) EGFP and TagRFP fluorescence were measured by flow cytometry in 293T after endogenous tagging of FBXL12 (right) or FBXL15 (right) with the cloning cassette described in (b). The normalized histograms for wild type vhhGFP fusions are shown in blue and for vhhGFP mutant (R35A/Y37A/W47A/F102A/E103A) are displayed in orange.
Supplementary information
Supplementary Fig. 1
Gating strategy for induced proximity assays with the eGFP–ABI1 reporter cell line.
Supplementary Fig. 2
Gating strategy for induced proximity assays with the GNMT(H176N)–eGFP reporter construct.
Supplementary Fig. 3
Original blots and gels.
Supplementary Table 1
Results from the ORFeome-wide degradation and stabilization screens.
Supplementary Table 2
Results from arrayed E3, E2 and DUB screens.
Supplementary Table 3
CRISPR–Cas9 screen read counts and MAGeCK analysis.
Supplementary Table 4
Proteomics analysis of K562 cells expressing BCR–ABL monobody fusions.
Supplementary Table 5
Oligonucleotides and gRNAs used in the study.
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Poirson, J., Cho, H., Dhillon, A. et al. Proteome-scale discovery of protein degradation and stabilization effectors. Nature 628, 878–886 (2024). https://doi.org/10.1038/s41586-024-07224-3
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DOI: https://doi.org/10.1038/s41586-024-07224-3
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