Characterizing the beta-catenin interactome using inhibitor screens and novel interaction proteomics techniques
Characterizing the beta-catenin interactome using inhibitor screens and novel interaction proteomics techniques
Background
Wnt signalling is a critical developmental pathway and associated aberrant signaling drives oncogenesis including colorectal and bone cancer. β-catenin, a central protein forms a range of protein-protein interactions(PPIs), fundamental to driving the transcription of oncogenes inducing proliferation and tumour formation. To further elucidate these interactions, we have sought to i) identify inhibitors of β-catenin PPIs and ii) employ a proximity dependent labelling technique (MiniTurbo) to characterize novel PPIs.
Methods
MiniTurbo-Mutant Biotin ligase BirA is genetically fused to β-catenin and upon addition of Biotin, tagging is induced of proteins within 20nm in as quick as 10 minutes enabling streptavidin pull down and tagging to Mass spectrometry. CTNNB1 gene was successfully inserted into the Miniturbo plasmid construct using SLICE cloning. DH5alpha bacteria were transformed with plasmid and single colony selected with Ampicillin. Following plasmid purification, it was added with Retroviral components for viral transfection and production in HEK293 cells. U2OS adenocarcinoma cells transduced and selected using puromycin. Validated with western blot and immunofluorescence. 24-hour application of MSAB derivatives following induction of Miniturbo with Doxycycline. Streptavidin pulldown was carried out and samples were subjected to Mass Spectrometry following on-bead trypsin digestion. Proteomic analysis was carried out to elucidate novel PPI.
Results
-MSAB derivatives influence levels of β-catenin and Wnt target genes at protein and transcriptional level. -Successful introduction and selection of the miniturbo plasmid into U2OS cells. Western blots and immunofluorescence verification. -Novel Proteomic networks of Beta-catenin protein interactions in U2OS cells. The results illustrate the dynamic PPI network of β-catenin in a 2D cancer model and how those interactions are modulated in the presence of small molecule inhibitors for therapeutic indications.
Conclusions
Targeting β-catenin directly for Oncotherapy has been difficult with very little success. Further characterization and understanding of the dynamic β-catenin interactome may pave an avenue for addressing this challenge.
S1406-S1406
John, S.
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Ewing, R.
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Baud, M.
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Divecha, N.
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Skipp, P.
1ba7dcf6-9fe7-4b5c-a9d0-e32ed7f42aa5
John, S.
b5ffef11-a31e-4013-9bba-f492eb8b93c2
Ewing, R.
022c5b04-da20-4e55-8088-44d0dc9935ae
Baud, M.
8752d519-3d33-43b6-9a77-ab731d410c2e
Divecha, N.
5c2ad0f8-4ce7-405f-8a15-2fc4ab96d787
Skipp, P.
1ba7dcf6-9fe7-4b5c-a9d0-e32ed7f42aa5
John, S., Ewing, R., Baud, M., Divecha, N. and Skipp, P.
(2022)
Characterizing the beta-catenin interactome using inhibitor screens and novel interaction proteomics techniques.
Annals of Oncology, 33 (8), .
(doi:10.1016/j.annonc.2022.09.075).
Record type:
Meeting abstract
Abstract
Background
Wnt signalling is a critical developmental pathway and associated aberrant signaling drives oncogenesis including colorectal and bone cancer. β-catenin, a central protein forms a range of protein-protein interactions(PPIs), fundamental to driving the transcription of oncogenes inducing proliferation and tumour formation. To further elucidate these interactions, we have sought to i) identify inhibitors of β-catenin PPIs and ii) employ a proximity dependent labelling technique (MiniTurbo) to characterize novel PPIs.
Methods
MiniTurbo-Mutant Biotin ligase BirA is genetically fused to β-catenin and upon addition of Biotin, tagging is induced of proteins within 20nm in as quick as 10 minutes enabling streptavidin pull down and tagging to Mass spectrometry. CTNNB1 gene was successfully inserted into the Miniturbo plasmid construct using SLICE cloning. DH5alpha bacteria were transformed with plasmid and single colony selected with Ampicillin. Following plasmid purification, it was added with Retroviral components for viral transfection and production in HEK293 cells. U2OS adenocarcinoma cells transduced and selected using puromycin. Validated with western blot and immunofluorescence. 24-hour application of MSAB derivatives following induction of Miniturbo with Doxycycline. Streptavidin pulldown was carried out and samples were subjected to Mass Spectrometry following on-bead trypsin digestion. Proteomic analysis was carried out to elucidate novel PPI.
Results
-MSAB derivatives influence levels of β-catenin and Wnt target genes at protein and transcriptional level. -Successful introduction and selection of the miniturbo plasmid into U2OS cells. Western blots and immunofluorescence verification. -Novel Proteomic networks of Beta-catenin protein interactions in U2OS cells. The results illustrate the dynamic PPI network of β-catenin in a 2D cancer model and how those interactions are modulated in the presence of small molecule inhibitors for therapeutic indications.
Conclusions
Targeting β-catenin directly for Oncotherapy has been difficult with very little success. Further characterization and understanding of the dynamic β-catenin interactome may pave an avenue for addressing this challenge.
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e-pub ahead of print date: 17 October 2022
Identifiers
Local EPrints ID: 476166
URI: http://eprints.soton.ac.uk/id/eprint/476166
ISSN: 0923-7534
PURE UUID: 733da01b-c23a-45cf-b83e-fbd9ea7d28f6
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Date deposited: 12 Apr 2023 16:58
Last modified: 17 Mar 2024 03:41
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Author:
S. John
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