Shining Light on Protein Kinase Biomarkers with Fluorescent Peptide Biosensors

Abstract

Protein kinases (PKs) are established gameplayers in biological signalling pathways, and a large body of evidence points to their dysregulation in diseases, in particular cancer, where rewiring of PK networks occurs frequently. Fluorescent biosensors constitute attractive tools for probing biomolecules and monitoring dynamic processes in complex samples. A wide variety of genetically encoded and synthetic biosensors have been tailored to report on PK activities over the last decade, enabling interrogation of their function and insight into their behaviour in physiopathological settings. These optical tools can further be used to highlight enzymatic alterations associated with the disease, thereby providing precious functional information which cannot be obtained through conventional genetic, transcriptomic or proteomic approaches. This review focuses on fluorescent peptide biosensors, recent developments and strategies that make them attractive tools to profile PK activities for biomedical and diagnostic purposes, as well as insights into the challenges and opportunities brought by this unique toolbox of chemical probes.

Keywords: cancer; fluorescent biosensor; kinase activity; peptide.

Figure 1

Complexity of the protein kinase activity/function illustrated by the RAS/RAF/MEK/ERK/CDK4 pathway. (a) PK activity is the result of a number of factors: CDK4 expression level, its regulation by activating and inhibitory partners, such as cyclin D and structural inhibitor p16INK4, posttranslational modifications catalysed by upstream regulators, such as ERK, MEK, RAS and RAF. (b) In human cancers, CDK4 hyperactivity may occur through different mechanisms that affect CDK4, cyclin D or p16INK4.

Figure 2

Strategies to detect and quantify PK expression and function: (a) genetic profiling; (b) transcriptomic profiling; (c) proteomics and interaction networks based on mass spectrometry analyses; (d) antibody-based approaches (ELISA and Western blotting); (e) methods based on ATP labelling; (f) fluorescent biosensors—kinase activity reporters.

Figure 3

Toolbox and diversity of fluorescent biosensors. (a) Genetically encoded FRET kinase activity reporter; (b) synthetic peptide biosensors; (c) nanomaterial-based biosensors: single-wall and multiwall carbon nanotube biosensors (left and middle, respectively) and a graphene biosensor with antibodies and fluorescent enzymes (right).

Figure 4

Features characterizing performance and robustness of fluorescent biosensors.

Figure 5

Mechanisms of response of fluorescent peptide biosensors. (a) Phosphorylation of the peptide affects fluorescence emission of the fluorophore proximal to the phosphate group; (b) phosphorylation of the peptide substrate promotes binding of a phosphoamino acid-binding domain which alters the local environment; (c) a quencher proximal to the fluorophore reduces its basal fluorescence until phosphorylation disrupts quenching; (d) chelation-enhanced fluorescence of a fluorophore upon binding of a metal ion involved in the phosphorylation process; (e,f) aggregation-caused quenching of fluorophores is reversed upon phosphorylation which promotes disassembly of fluorescent biosensors, leading to fluorescence enhancement.

Figure 6

CDKACT fluorescent peptide biosensor technology. (a) Schematic representation of CDKACT biosensors: bipartite biosensors comprising a CDK-specific substrate moiety (orange) onto which an environmentally sensitive dye is conjugated, a short linker and a phosphoamino acid-binding domain (or PAABD, green) that folds onto the phosphorylated substrate, thereby altering the local environment of the fluorophore and promoting changes in fluorescence emission. (b) An RFP fusion of CDKACT expressed in E. coli. (c) CDKACTs or RFP-CDKACTs can be introduced into cultured cells for live imaging experiments through complexation with cell-penetrating peptides (Pep1) to form nanoparticles that cross cell membranes and release CDKACT into cells. (d) A self-cell-penetrating variant of CDKACT was generated through fusion of the N-terminal moiety of Pep1 to the PAABD of CDKACT5. (e) CDKACT1-multiwall carbon nanotube conjugates yield an ultrasensitive nano-biosensor for imaging the CDK1 activity in mice.

Figure 7

CDKACT fluorescent peptide biosensor technology. (a) CDKACT technology for quantifying the CDK activity in vitro by means of fluorescence spectroscopy and in living cells by means of fluorescence microscopy and live cell imaging following facilitated delivery into cultured cells. (b) Comparison of fluorescent and luminescent CDKACT4 biosensors. (c) Schematic representation of multiplex biosensing experiments performed by combining four different CDKACT biosensors to profile CDK1, CDK2, CDK4 and CDK6 in human cancer biopsies.