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Fluorescent Biosensor of CDK5 Kinase Activity in Glioblastoma Cell Extracts and Living Cells
Marion Peyressatre1, Arthur Laure1, Morgan Pellerano1
1Institut des Biomolécules Max Mousseron, CNRS, UMR 5247, Université de Montpellier, Faculté de Pharmacie, 15, Av. Charles Flahault, Montpellier, 34093, France.
Abstract:
CDK5 plays a major role in neuronal functions, and is hyperactivated in neurodegenerative pathologies as well as in glioblastoma and neuroblastoma. Although this kinase constitutes an established biomarker and pharmacological target, there are few means of probing its activity in cell extracts or in living cells. To this aim a fluorescent peptide reporter of CDK5 kinase activity, derived from a library of CDK5-specific substrates, is engineered and its ability to respond to recombinant CDK5/p25 is established and CDK5 activity in glioblastoma cell extracts is reported on through sensitive changes in fluorescence intensity. A cell-penetrating variant of this biosensor which can be implemented to image CDK5 activation dynamics in space and in time is further implemented. This original biosensor constitutes a potent tool for quantifying differences in CDK5 activity following treatment with selective inhibitors and for monitoring CDK5 activation, following inhibition or stimulation, in a physiologically relevant environment. As such it offers attractive opportunities to develop a diagnostic assay for neuronal pathologies associated with hyperactivated CDK5, as well as a companion assay to evaluate response to new therapies targeting this kinase.
Insights
A novel fluorescent biosensor was developed to measure cyclin-dependent kinase 5 (CDK5) activity. This tool aids in diagnosing neurodegenerative diseases and evaluating therapies targeting CDK5.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- Cyclin-dependent kinase 5 (CDK5) is crucial for neuronal functions.
- Hyperactivated CDK5 is implicated in neurodegenerative diseases, glioblastoma, and neuroblastoma.
- Limited tools exist to probe CDK5 activity in cellular environments.
Purpose of the Study:
- To engineer a fluorescent peptide reporter for CDK5 kinase activity.
- To validate the biosensor's response to CDK5/p25 and in glioblastoma cell extracts.
- To develop a cell-penetrating variant for in vivo imaging of CDK5 dynamics.
Main Methods:
- Engineering a fluorescent peptide substrate library specific for CDK5.
- Testing biosensor response with recombinant CDK5/p25 and glioblastoma cell extracts.
- Developing and implementing a cell-penetrating version of the biosensor.
Main Results:
- Successful engineering of a fluorescent peptide reporter for CDK5 activity.
- Demonstrated CDK5 activity detection in cell extracts via fluorescence changes.
- Developed a cell-penetrating biosensor for spatiotemporal imaging of CDK5 activation.
Conclusions:
- The developed biosensor is a potent tool for quantifying CDK5 activity.
- It enables monitoring of CDK5 activation dynamics in relevant physiological settings.
- Offers potential for diagnostic assays in CDK5-related pathologies and companion assays for therapy evaluation.
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