Integration of FRET and sequencing to engineer kinase biosensors from mammalian cell libraries.
Nat Commun
; 12(1): 5031, 2021 08 19.
Article
de En
| MEDLINE
| ID: mdl-34413312
ABSTRACT
The limited sensitivity of Förster Resonance Energy Transfer (FRET) biosensors hinders their broader applications. Here, we develop an approach integrating high-throughput FRET sorting and next-generation sequencing (FRET-Seq) to identify sensitive biosensors with varying substrate sequences from large-scale libraries directly in mammalian cells, utilizing the design of self-activating FRET (saFRET) biosensor. The resulting biosensors of Fyn and ZAP70 kinases exhibit enhanced performance and enable the dynamic imaging of T-cell activation mediated by T cell receptor (TCR) or chimeric antigen receptor (CAR), revealing a highly organized ZAP70 subcellular activity pattern upon TCR but not CAR engagement. The ZAP70 biosensor elucidates the role of immunoreceptor tyrosine-based activation motif (ITAM) in affecting ZAP70 activation to regulate CAR functions. A saFRET biosensor-based high-throughput drug screening (saFRET-HTDS) assay further enables the identification of an FDA-approved cancer drug, Sunitinib, that can be repurposed to inhibit ZAP70 activity and autoimmune-disease-related T-cell activation.
Texte intégral:
1
Collection:
01-internacional
Base de données:
MEDLINE
Sujet principal:
Phosphotransferases
/
Techniques de biocapteur
/
Transfert d'énergie par résonance de fluorescence
/
Séquençage nucléotidique à haut débit
Limites:
Humans
Langue:
En
Journal:
Nat Commun
Sujet du journal:
BIOLOGIA
/
CIENCIA
Année:
2021
Type de document:
Article
Pays d'affiliation:
États-Unis d'Amérique