Cargando…

Imaging of hypoxia-inducible factor 1α and septin 9 interaction by bimolecular fluorescence complementation in live cancer cells

Hypoxia-inducible factor 1 (HIF-1) is a major mediator of the hypoxic response involved in tumor progression. We had earlier described the interaction between septin 9 isoform 1 (SEPT9_i1) protein and the oxygen-regulated subunit, HIF-1α. SEPT9_i1 is a member of the conserved family of GTP-binding c...

Descripción completa

Detalles Bibliográficos
Autores principales: Golan, Maya, Mabjeesh, Nicola J
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Impact Journals LLC 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5458251/
https://www.ncbi.nlm.nih.gov/pubmed/28380438
http://dx.doi.org/10.18632/oncotarget.16527
_version_ 1783241722409517056
author Golan, Maya
Mabjeesh, Nicola J
author_facet Golan, Maya
Mabjeesh, Nicola J
author_sort Golan, Maya
collection PubMed
description Hypoxia-inducible factor 1 (HIF-1) is a major mediator of the hypoxic response involved in tumor progression. We had earlier described the interaction between septin 9 isoform 1 (SEPT9_i1) protein and the oxygen-regulated subunit, HIF-1α. SEPT9_i1 is a member of the conserved family of GTP-binding cytoskeleton septins. SEPT9_i1 stabilizes HIF-1α and facilitates its cytoplasmic-nuclear translocation. We utilized split yellow fluorescent protein (YFP) bimolecular fluorescence complementation (BiFC) methodology to monitor the interaction between HIF-1α and SEPT9_i1 in live cells. N-terminal (YN) and C-terminal (YC) split YFP chimeras with HIF-1α and SEPT9_i1 on both their amino and carboxyl termini were generated. HIF-1α and SEPT9_i1 chimeras were expressed in cancer cells and screened for functional complementation. SEPT9_i1-YN and YC-HIF-1α formed a long-lived highly stable complex upon interaction. The BiFC signal was increased in the presence of hypoxia-mimicking agents. In contrast, YC-ΔHLH-HIF-1α chimera, which lacked the helix-loop-helix domain that is essential for the interaction with SEPT9_i1 as well as the expression of SEPT9_i1 252-379 amino acids fragment required for the interaction with HIF-1α, significantly reduced the BiFC signal. The signal was also reduced when cells were treated with 17-N-allylamino-17-demethoxygeldanamycin, an HSP90 inhibitor that inhibits HIF-1α. It was increased with fourchlorfenuron, a small molecule that increases the interaction between HIF-1α and SEPT9_i1. These results reconfirmed the interaction between HIF-1α and SEPT9_i1 that was imaged in live cells. This BiFC system represents a novel approach for studying the real-time interaction between these two proteins and will allow high-throughput drug screening to identity compounds that disrupt this interaction.
format Online
Article
Text
id pubmed-5458251
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher Impact Journals LLC
record_format MEDLINE/PubMed
spelling pubmed-54582512017-06-08 Imaging of hypoxia-inducible factor 1α and septin 9 interaction by bimolecular fluorescence complementation in live cancer cells Golan, Maya Mabjeesh, Nicola J Oncotarget Research Paper Hypoxia-inducible factor 1 (HIF-1) is a major mediator of the hypoxic response involved in tumor progression. We had earlier described the interaction between septin 9 isoform 1 (SEPT9_i1) protein and the oxygen-regulated subunit, HIF-1α. SEPT9_i1 is a member of the conserved family of GTP-binding cytoskeleton septins. SEPT9_i1 stabilizes HIF-1α and facilitates its cytoplasmic-nuclear translocation. We utilized split yellow fluorescent protein (YFP) bimolecular fluorescence complementation (BiFC) methodology to monitor the interaction between HIF-1α and SEPT9_i1 in live cells. N-terminal (YN) and C-terminal (YC) split YFP chimeras with HIF-1α and SEPT9_i1 on both their amino and carboxyl termini were generated. HIF-1α and SEPT9_i1 chimeras were expressed in cancer cells and screened for functional complementation. SEPT9_i1-YN and YC-HIF-1α formed a long-lived highly stable complex upon interaction. The BiFC signal was increased in the presence of hypoxia-mimicking agents. In contrast, YC-ΔHLH-HIF-1α chimera, which lacked the helix-loop-helix domain that is essential for the interaction with SEPT9_i1 as well as the expression of SEPT9_i1 252-379 amino acids fragment required for the interaction with HIF-1α, significantly reduced the BiFC signal. The signal was also reduced when cells were treated with 17-N-allylamino-17-demethoxygeldanamycin, an HSP90 inhibitor that inhibits HIF-1α. It was increased with fourchlorfenuron, a small molecule that increases the interaction between HIF-1α and SEPT9_i1. These results reconfirmed the interaction between HIF-1α and SEPT9_i1 that was imaged in live cells. This BiFC system represents a novel approach for studying the real-time interaction between these two proteins and will allow high-throughput drug screening to identity compounds that disrupt this interaction. Impact Journals LLC 2017-03-23 /pmc/articles/PMC5458251/ /pubmed/28380438 http://dx.doi.org/10.18632/oncotarget.16527 Text en Copyright: © 2017 Golan and Mabjeesh http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Paper
Golan, Maya
Mabjeesh, Nicola J
Imaging of hypoxia-inducible factor 1α and septin 9 interaction by bimolecular fluorescence complementation in live cancer cells
title Imaging of hypoxia-inducible factor 1α and septin 9 interaction by bimolecular fluorescence complementation in live cancer cells
title_full Imaging of hypoxia-inducible factor 1α and septin 9 interaction by bimolecular fluorescence complementation in live cancer cells
title_fullStr Imaging of hypoxia-inducible factor 1α and septin 9 interaction by bimolecular fluorescence complementation in live cancer cells
title_full_unstemmed Imaging of hypoxia-inducible factor 1α and septin 9 interaction by bimolecular fluorescence complementation in live cancer cells
title_short Imaging of hypoxia-inducible factor 1α and septin 9 interaction by bimolecular fluorescence complementation in live cancer cells
title_sort imaging of hypoxia-inducible factor 1α and septin 9 interaction by bimolecular fluorescence complementation in live cancer cells
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5458251/
https://www.ncbi.nlm.nih.gov/pubmed/28380438
http://dx.doi.org/10.18632/oncotarget.16527
work_keys_str_mv AT golanmaya imagingofhypoxiainduciblefactor1aandseptin9interactionbybimolecularfluorescencecomplementationinlivecancercells
AT mabjeeshnicolaj imagingofhypoxiainduciblefactor1aandseptin9interactionbybimolecularfluorescencecomplementationinlivecancercells