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Fluorescent Peptide Biosensor for Probing the Relative Abundance of Cyclin-Dependent Kinases in Living Cells

Cyclin-dependant kinases play a central role in coordinating cell growth and division, and in sustaining proliferation of cancer cells, thereby constituting attractive pharmacological targets. However, there are no direct means of assessing their relative abundance in living cells, current approache...

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Autores principales: Kurzawa, Laetitia, Pellerano, Morgan, Coppolani, J. B., Morris, May C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3196589/
https://www.ncbi.nlm.nih.gov/pubmed/22028905
http://dx.doi.org/10.1371/journal.pone.0026555
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author Kurzawa, Laetitia
Pellerano, Morgan
Coppolani, J. B.
Morris, May C.
author_facet Kurzawa, Laetitia
Pellerano, Morgan
Coppolani, J. B.
Morris, May C.
author_sort Kurzawa, Laetitia
collection PubMed
description Cyclin-dependant kinases play a central role in coordinating cell growth and division, and in sustaining proliferation of cancer cells, thereby constituting attractive pharmacological targets. However, there are no direct means of assessing their relative abundance in living cells, current approaches being limited to antigenic and proteomic analysis of fixed cells. In order to probe the relative abundance of these kinases directly in living cells, we have developed a fluorescent peptide biosensor with biligand affinity for CDKs and cyclins in vitro, that retains endogenous CDK/cyclin complexes from cell extracts, and that bears an environmentally-sensitive probe, whose fluorescence increases in a sensitive fashion upon recognition of its targets. CDKSENS was introduced into living cells, through complexation with the cell-penetrating carrier CADY2 and applied to assess the relative abundance of CDK/Cyclins through fluorescence imaging and ratiometric quantification. This peptide biosensor technology affords direct and sensitive readout of CDK/cyclin complex levels, and reports on differences in complex formation when tampering with a single CDK or cyclin. CDKSENS further allows for detection of differences between different healthy and cancer cell lines, thereby enabling to distinguish cells that express high levels of these heterodimeric kinases, from cells that present decreased or defective assemblies. This fluorescent biosensor technology provides information on the overall status of CDK/Cyclin complexes which cannot be obtained through antigenic detection of individual subunits, in a non-invasive fashion which does not require cell fixation or extraction procedures. As such it provides promising perspectives for monitoring the response to therapeutics that affect CDK/Cyclin abundance, for cell-based drug discovery strategies and fluorescence-based cancer diagnostics.
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spelling pubmed-31965892011-10-25 Fluorescent Peptide Biosensor for Probing the Relative Abundance of Cyclin-Dependent Kinases in Living Cells Kurzawa, Laetitia Pellerano, Morgan Coppolani, J. B. Morris, May C. PLoS One Research Article Cyclin-dependant kinases play a central role in coordinating cell growth and division, and in sustaining proliferation of cancer cells, thereby constituting attractive pharmacological targets. However, there are no direct means of assessing their relative abundance in living cells, current approaches being limited to antigenic and proteomic analysis of fixed cells. In order to probe the relative abundance of these kinases directly in living cells, we have developed a fluorescent peptide biosensor with biligand affinity for CDKs and cyclins in vitro, that retains endogenous CDK/cyclin complexes from cell extracts, and that bears an environmentally-sensitive probe, whose fluorescence increases in a sensitive fashion upon recognition of its targets. CDKSENS was introduced into living cells, through complexation with the cell-penetrating carrier CADY2 and applied to assess the relative abundance of CDK/Cyclins through fluorescence imaging and ratiometric quantification. This peptide biosensor technology affords direct and sensitive readout of CDK/cyclin complex levels, and reports on differences in complex formation when tampering with a single CDK or cyclin. CDKSENS further allows for detection of differences between different healthy and cancer cell lines, thereby enabling to distinguish cells that express high levels of these heterodimeric kinases, from cells that present decreased or defective assemblies. This fluorescent biosensor technology provides information on the overall status of CDK/Cyclin complexes which cannot be obtained through antigenic detection of individual subunits, in a non-invasive fashion which does not require cell fixation or extraction procedures. As such it provides promising perspectives for monitoring the response to therapeutics that affect CDK/Cyclin abundance, for cell-based drug discovery strategies and fluorescence-based cancer diagnostics. Public Library of Science 2011-10-18 /pmc/articles/PMC3196589/ /pubmed/22028905 http://dx.doi.org/10.1371/journal.pone.0026555 Text en Kurzawa et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Kurzawa, Laetitia
Pellerano, Morgan
Coppolani, J. B.
Morris, May C.
Fluorescent Peptide Biosensor for Probing the Relative Abundance of Cyclin-Dependent Kinases in Living Cells
title Fluorescent Peptide Biosensor for Probing the Relative Abundance of Cyclin-Dependent Kinases in Living Cells
title_full Fluorescent Peptide Biosensor for Probing the Relative Abundance of Cyclin-Dependent Kinases in Living Cells
title_fullStr Fluorescent Peptide Biosensor for Probing the Relative Abundance of Cyclin-Dependent Kinases in Living Cells
title_full_unstemmed Fluorescent Peptide Biosensor for Probing the Relative Abundance of Cyclin-Dependent Kinases in Living Cells
title_short Fluorescent Peptide Biosensor for Probing the Relative Abundance of Cyclin-Dependent Kinases in Living Cells
title_sort fluorescent peptide biosensor for probing the relative abundance of cyclin-dependent kinases in living cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3196589/
https://www.ncbi.nlm.nih.gov/pubmed/22028905
http://dx.doi.org/10.1371/journal.pone.0026555
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