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A yeast two-hybrid system reconstituting substrate recognition of the von Hippel-Lindau tumor suppressor protein

The von Hippel-Lindau tumor suppressor protein (pVHL) is inactivated in the hereditary cancer syndrome von Hippel-Lindau disease and in the majority of sporadic renal carcinomas. pVHL is the substrate-binding subunit of the CBC(VHL) ubiquitin ligase complex that negatively regulates cell growth by p...

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Autores principales: Bex, Claudia, Knauth, Katja, Dambacher, Silvia, Buchberger, Alexander
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2007
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2175351/
https://www.ncbi.nlm.nih.gov/pubmed/17986458
http://dx.doi.org/10.1093/nar/gkm932
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author Bex, Claudia
Knauth, Katja
Dambacher, Silvia
Buchberger, Alexander
author_facet Bex, Claudia
Knauth, Katja
Dambacher, Silvia
Buchberger, Alexander
author_sort Bex, Claudia
collection PubMed
description The von Hippel-Lindau tumor suppressor protein (pVHL) is inactivated in the hereditary cancer syndrome von Hippel-Lindau disease and in the majority of sporadic renal carcinomas. pVHL is the substrate-binding subunit of the CBC(VHL) ubiquitin ligase complex that negatively regulates cell growth by promoting the degradation of hypoxia-inducible transcription factor subunits (HIF1/2α). Proteomics-based identification of novel pVHL substrates is hampered by their short half-life and low abundancy in mammalian cells. The usefulness of yeast two-hybrid (Y2H) approaches, on the other hand, has been limited by the failure of pVHL to adopt its native structure and by the absence of prolylhydroxylase activity critical for pVHL substrate recognition. Therefore, we modified the Y2H system to faithfully reconstitute the physical interaction between pVHL and its substrates. Our approach relies on the coexpression of pVHL with the cofactors Elongin B and Elongin C and with HIF1/2α prolylhydroxylases. In a proof-of-principle Y2H screen, we identified the known substrates HIF1/2α and new candidate substrates including diacylglycerol kinase iota, demonstrating that our strategy allows detection of stable interactions between pVHL and otherwise elusive cellular targets. Additional future applications may include structure/function analyses of pVHL-HIF1/2α binding and screens for therapeutically relevant compounds that either stabilize or disrupt this interaction.
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spelling pubmed-21753512008-01-07 A yeast two-hybrid system reconstituting substrate recognition of the von Hippel-Lindau tumor suppressor protein Bex, Claudia Knauth, Katja Dambacher, Silvia Buchberger, Alexander Nucleic Acids Res Methods Online The von Hippel-Lindau tumor suppressor protein (pVHL) is inactivated in the hereditary cancer syndrome von Hippel-Lindau disease and in the majority of sporadic renal carcinomas. pVHL is the substrate-binding subunit of the CBC(VHL) ubiquitin ligase complex that negatively regulates cell growth by promoting the degradation of hypoxia-inducible transcription factor subunits (HIF1/2α). Proteomics-based identification of novel pVHL substrates is hampered by their short half-life and low abundancy in mammalian cells. The usefulness of yeast two-hybrid (Y2H) approaches, on the other hand, has been limited by the failure of pVHL to adopt its native structure and by the absence of prolylhydroxylase activity critical for pVHL substrate recognition. Therefore, we modified the Y2H system to faithfully reconstitute the physical interaction between pVHL and its substrates. Our approach relies on the coexpression of pVHL with the cofactors Elongin B and Elongin C and with HIF1/2α prolylhydroxylases. In a proof-of-principle Y2H screen, we identified the known substrates HIF1/2α and new candidate substrates including diacylglycerol kinase iota, demonstrating that our strategy allows detection of stable interactions between pVHL and otherwise elusive cellular targets. Additional future applications may include structure/function analyses of pVHL-HIF1/2α binding and screens for therapeutically relevant compounds that either stabilize or disrupt this interaction. Oxford University Press 2007-12 2007-11-05 /pmc/articles/PMC2175351/ /pubmed/17986458 http://dx.doi.org/10.1093/nar/gkm932 Text en © 2007 The Author(s) http://creativecommons.org/licenses/by-nc/2.0/uk/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Bex, Claudia
Knauth, Katja
Dambacher, Silvia
Buchberger, Alexander
A yeast two-hybrid system reconstituting substrate recognition of the von Hippel-Lindau tumor suppressor protein
title A yeast two-hybrid system reconstituting substrate recognition of the von Hippel-Lindau tumor suppressor protein
title_full A yeast two-hybrid system reconstituting substrate recognition of the von Hippel-Lindau tumor suppressor protein
title_fullStr A yeast two-hybrid system reconstituting substrate recognition of the von Hippel-Lindau tumor suppressor protein
title_full_unstemmed A yeast two-hybrid system reconstituting substrate recognition of the von Hippel-Lindau tumor suppressor protein
title_short A yeast two-hybrid system reconstituting substrate recognition of the von Hippel-Lindau tumor suppressor protein
title_sort yeast two-hybrid system reconstituting substrate recognition of the von hippel-lindau tumor suppressor protein
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2175351/
https://www.ncbi.nlm.nih.gov/pubmed/17986458
http://dx.doi.org/10.1093/nar/gkm932
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