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PreprintCited by 2Protein Degradation and InhibitorsUbiquitin and proteasome pathways14-3-3 protein interactions

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A ubiquitination-mediated degradation system to target phospho-14-3-3-binding-motif embedded proteins

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Li, Zhaokai, Huang, Xiaoqiang, Li, Mohan, Sabry, Ziad, Chen, Y. Eugene, Wang, Zhong, Liu, Liu·2022·Heliyon
DOI 10.1101/2022.11.22.517526PMC PMC10213371

Preprint — published as 10.1016/j.heliyon.2023.e16318 · Paper page

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Abstract

The phosphorylation of 14-3-3 binding motif is involved in many cellular processes. A strategy that enables targeted degradation of phospho-14-3-3-binding-motif embedded (P-14-3-3-BME) proteins for studying their functions is highly desirable for basic research. Here, we report a phosphorylation-induced, ubiquitin-proteasome-system-mediated targeted protein degradation (TPD) strategy that allows specific degradation of P-14-3-3-BME proteins. Specifically, by ligating a modified von Hippel-Lindau E3-ligase with an engineered 14-3-3 bait, we generated a protein chimera referred to as Targeted Degradation of P-14-3-3-BME Protein (TDPP). TDPP can serve as a universal degrader for P-14-3-3-BME proteins based on the specific recognition of the phosphorylation in 14-3-3 binding motifs. TDPP shows high efficiency and specificity to a difopein-EGFP reporter, general and specific P-14-3-3-BME proteins. TDPP can also be applied for the validation of P-14-3-3-BME proteins. These results strongly support TDPP as a powerful tool for 14-3-3 related research.

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Figures

Fig. 1 Illustration and de novo structure modeling of a degrader of 14-3-3-BPPs. A. TDPP fused protein design. B. Sequence partition information of TDPP. C. Full-length structure model generated by ColabFold [26]. KR mutation sites are highlighted in black spheres. D. Comparison of predicted VHL 3 KR model and experimental VHL structure. Red: predicted VHL (extracted from full-length model); cyan: experimental VHL structure (PDB ID: 4WQO). E. Comparison of predicted 14-3-3ζ 20 KR model and experimental 14-3-3ζ structure. Blue: predicted 14-3-3ζ (extracted from full-length model); cyan: experimental 14-3-3ζ structure (PDB ID: 2O02). See also Fig. S1.
Fig. 1 Illustration and de novo structure modeling of a degrader of 14-3-3-BPPs. A. TDPP fused protein design. B. Sequence partition information of TDPP. C. Full-length structure model generated by ColabFold [26]. KR mutation sites are highlighted in black spheres. D. Comparison of predicted VHL 3 KR model and experimental VHL structure. Red: predicted VHL (extracted from full-length model); cyan: experimental VHL structure (PDB ID: 4WQO). E. Comparison of predicted 14-3-3ζ 20 KR model and experimental 14-3-3ζ structure. Blue: predicted 14-3-3ζ (extracted from full-length model); cyan: experimental 14-3-3ζ structure (PDB ID: 2O02). See also Fig. S1.CC BY-NC-ND · PubMed Central
Fig. 2 TDPP degrades difopein-EGFP. A. Working model of TDPP-induced degradation of difopein-EGFP. Undegraded difopein-EGFP protein chimera has a green fluorescent ability from the EGFP tag. Since the 14-3-3 bait in TDPP could specifically interact with the difopein tag in difopein-EGFP, TDPP will provide the proximity of UPS to difopein-EGFP and degrade it, in which condition the green fluorescent intensity will decrease. B, C. Western blot result (B) and quantification of band intensity (C) of difopein-EGFP in HEK293T/17 cells co-transfected with expression plasmids of 14-3-3ζ (1–230) 20 KR-HA (referred as ctrl) or VHL 3 KR-14-3-3ζ (1–230) 20 KR-HA (referred as TDPP). D, E. Fluorescence assay result (D) and fluorescence intensity quantification (E) of HEK293T/17 cells transfected with difopein-EGFP vector, along with control vectors (14-3-3ζ (1–230) 20 KR or VHL 3 KR) or TDPP vector. F. Immunoprecipitation result of interaction between 14-3-3ζ (1–230) 20 KR-HA/14-3-3ζ (1–230) 19 KR K49E-HA and difopein-EGFP. IP: 14-3-3ζ (1–230) 20 KR-HA/14-3-3ζ (1–230) 19 KR K49E-HA. IB: difopein-EGFP. G, H. Western blot result (G) and quantification of band intensity (H) of difopein-EGFP in HEK293T/17 cells co-transfected with expression plasmids of 14-3-3ζ (1–230) 20 KR-HA (referred as ctrl), 14-3-3ζ (1–230) 19 KR K49E-HA (referred as ctrl K49E), VHL 3 KR-14-3-3ζ (1–230) 20 KR-HA (referred as TDPP), or VHL 3 KR-14-3-3ζ (1–230) 19 KR K49E-HA (referred as TDPP K49E). Data are represented as mean ± SEM.
Fig. 2 TDPP degrades difopein-EGFP. A. Working model of TDPP-induced degradation of difopein-EGFP. Undegraded difopein-EGFP protein chimera has a green fluorescent ability from the EGFP tag. Since the 14-3-3 bait in TDPP could specifically interact with the difopein tag in difopein-EGFP, TDPP will provide the proximity of UPS to difopein-EGFP and degrade it, in which condition the green fluorescent intensity will decrease. B, C. Western blot result (B) and quantification of band intensity (C) of difopein-EGFP in HEK293T/17 cells co-transfected with expression plasmids of 14-3-3ζ (1–230) 20 KR-HA (referred as ctrl) or VHL 3 KR-14-3-3ζ (1–230) 20 KR-HA (referred as TDPP). D, E. Fluorescence assay result (D) and fluorescence intensity quantification (E) of HEK293T/17 cells transfected with difopein-EGFP vector, along with control vectors (14-3-3ζ (1–230) 20 KR or VHL 3 KR) or TDPP vector. F. Immunoprecipitation result of interaction between 14-3-3ζ (1–230) 20 KR-HA/14-3-3ζ (1–230) 19 KR K49E-HA and difopein-EGFP. IP: 14-3-3ζ (1–230) 20 KR-HA/14-3-3ζ (1–230) 19 KR K49E-HA. IB: difopein-EGFP. G, H. Western blot result (G) and quantification of band intensity (H) of difopein-EGFP in HEK293T/17 cells co-transfected with expression plasmids of 14-3-3ζ (1–230) 20 KR-HA (referred as ctrl), 14-3-3ζ (1–230) 19 KR K49E-HA (referred as ctrl K49E), VHL 3 KR-14-3-3ζ (1–230) 20 KR-HA (referred as TDPP), or VHL 3 KR-14-3-3ζ (1–230) 19 KR K49E-HA (referred as TDPP K49E). Data are represented as mean ± SEM.CC BY-NC-ND · PubMed Central
Fig. 3 TDPP degrades 14-3-3-BPPs. A. Western blot of phospho-(Ser)-14-3-3-binding-motif treated with control/TDPP vector with/without 10 nM OA. Different molecular weight ranges were grouped as different regions and marked as different colors, which were roughly grouped by similar band intensity and reflect the difference among treatments. The grey value of bands and regions were quantified and further analyzed in panel B and C, respectively. B. Illustration of the grey value distribution of phospho-(Ser)-14-3-3-binding-motif in each treatment in panel A. X axis represents the distance to the top edge of the blot results. Upper panel: lane 1 (black) vs. lane 3 (green). Lower panel: lane 2 (red) vs. lane 4 (blue). C. Quantification of phospho-(Ser)-14-3-3-binding-motifembedded proteins with different molecular weight in panel A. D and E. Western blot results (D) and the quantifications (E) of several different phospho-14-3-3-binding-motif embedded proteins (HDAC4, HDAC5, FoxO1) in HEK293T/17 cells treated with control/TDPP vectors. F and G. Western blot result (F) and the quantification (G) of HDAC4 phospho-S632 in a HDAC4 wildtype vectors transfected HEK293T/17 cells treated with control/TDPP vector. Data are represented as mean ± SEM. See also Fig. S2.
Fig. 3 TDPP degrades 14-3-3-BPPs. A. Western blot of phospho-(Ser)-14-3-3-binding-motif treated with control/TDPP vector with/without 10 nM OA. Different molecular weight ranges were grouped as different regions and marked as different colors, which were roughly grouped by similar band intensity and reflect the difference among treatments. The grey value of bands and regions were quantified and further analyzed in panel B and C, respectively. B. Illustration of the grey value distribution of phospho-(Ser)-14-3-3-binding-motif in each treatment in panel A. X axis represents the distance to the top edge of the blot results. Upper panel: lane 1 (black) vs. lane 3 (green). Lower panel: lane 2 (red) vs. lane 4 (blue). C. Quantification of phospho-(Ser)-14-3-3-binding-motifembedded proteins with different molecular weight in panel A. D and E. Western blot results (D) and the quantifications (E) of several different phospho-14-3-3-binding-motif embedded proteins (HDAC4, HDAC5, FoxO1) in HEK293T/17 cells treated with control/TDPP vectors. F and G. Western blot result (F) and the quantification (G) of HDAC4 phospho-S632 in a HDAC4 wildtype vectors transfected HEK293T/17 cells treated with control/TDPP vector. Data are represented as mean ± SEM. See also Fig. S2.CC BY-NC-ND · PubMed Central
Fig. 4 TDPP validates BICRA as a new 14-3-3 interaction target protein. A. Western blot of BICRA-HA level in its expression HEK293T/17 cells transfected with control/TDPP vectors. B. Relative mean band intensity of BICRA-HA Western blot. Data are represented as mean ± SEM.
Fig. 4 TDPP validates BICRA as a new 14-3-3 interaction target protein. A. Western blot of BICRA-HA level in its expression HEK293T/17 cells transfected with control/TDPP vectors. B. Relative mean band intensity of BICRA-HA Western blot. Data are represented as mean ± SEM.CC BY-NC-ND · PubMed Central
Fig. 5 Working model of TDPP, a ubiquitination-mediated degradation system to target 14-3-3-binding phosphoproteins.
Fig. 5 Working model of TDPP, a ubiquitination-mediated degradation system to target 14-3-3-binding phosphoproteins.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Li, Z., Huang, X., Li, M., Sabry, Z., Chen, Y. E., Wang, Z., & Liu, L.
Affiliations
  • University of Michigan (US)
  • Chinese Academy of Medical Sciences & Peking Union Medical College (CN)
Corresponding author
  • Wang, Zhong
  • Liu, Liu
ORCID
Venue
Heliyon
Publisher
openRxiv
Published
2022-11-23
Volume
9
Issue
5
Pages
e16318-e16318
ISSN
2405-8440 (ISSN-L)
DOI
10.1101/2022.11.22.517526
PMC
PMC10213371
OpenAlex
W4377086858
Cited by
2
References
1
Citations by year
2024: 2
FWCI
0.18
Type
journal article
Language
en
Open access
Licence
cc-by-nc-nd
Locations
  • Heliyon (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Institutes of Health
  • National Heart, Lung, and Blood Institute (R01HL139735, R01HL163672)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › 14-3-3 protein interactions
Keywords
targeted protein degradation, ubiquitination, ubiquitin-proteasome system, 14-3-3 binding motif, protein chimeras

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Li, Z., Huang, X., Li, M., Sabry, Z., Chen, Y. E., Wang, Z., & Liu, L. (2022). A ubiquitination-mediated degradation system to target phospho-14-3-3-binding-motif embedded proteins. Heliyon. https://doi.org/10.1101/2022.11.22.517526