Live-Cell Imaging of Protein Degradation Utilizing Designed Protein-Tag Mutant and Fluorescent Probe with Turn-Off Switch

Abstract

Protein degradation plays various roles in cellular homeostasis and signal transduction. Real-time monitoring of the degradation process not only contributes to the elucidation of relevant biological phenomena but also offers a powerful tool for drug discoveries targeting protein degradation. Fluorescent protein labeling with a protein tag and a synthetic fluorescent probe is a powerful technique that enables the direct visualization of proteins of interest in living cells. Although a variety of protein tags and their labeling probes have been reported, techniques for the visualization of protein degradation in living cells remain limited. In order to overcome this limitation, we herein employed a PYP-tag labeling probe with a fluorescence turn-off switch that enables the imaging of protein degradation. Furthermore, we performed a structure-based design of a PYP-tag to stabilize a complex formed by the probe and the protein tag for long-term live-cell imaging. We successfully applied this technique to live-cell imaging of the degradation process of Regnase-1 in response to immunostimulation.

An “OFF–ON–OFF” fluorescence protein-labeling probe for real-time visualization of the degradation of short-lived proteins in cellular systems

“…Reactive "OFF-ON-OFF" probes for in vitro uorescent labeling of PYP-tag proteins Guided by our previous PYP-tag probe-labeling studies, 32 we designed a new trifunctional 'contact quenching' "OFF-ON-OFF" uorescence probe (F3-DNB) for the rapid labeling of PYPtag proteins (Fig. 2).…”

“…Guided by our previous PYP-tag probe-labeling studies, 32 we designed a new trifunctional 'contact quenching' "OFF-ON-OFF" fluorescence probe (F3-DNB) for the rapid labeling of PYP-tag proteins (Figure 2). This probe contains a PYP-tag binding 7hydroxycoumarin ligand connected to a fluorescein fluorophore through a PEG linker (See Schemes SI1-5 for details of syntheses of all probes).…”

“…In order to address these issues, we have recently developed a fluorescent PYP-tag probe, CG2, that can be used for 'no-wash' monitoring of protein expression/degradation in living cells in real time (Figure 1a). 32 Unfortunately, the relatively poor fluorescence intensity of the coumarin fluorophore of the CG2 probe prevents it from being used for high-resolution cell imaging studies (Figure S1 and Table S1). This means that CG2 is also not suitable for imaging short-lived proteins and/or monitoring the degradation of proteins with low expression levels (Figures S1 & S2).…”

“…1a ). 32 Unfortunately, the relatively poor fluorescence intensity of the coumarin fluorophore of the CG2 probe prevents it from being used for high-resolution cell imaging studies (Fig. S1 and Table S1 † ).…”

Rapid no-wash labeling of PYP-tag proteins with reactive fluorogenic ligands affords stable fluorescent protein conjugates for long-term cell imaging studies

“…glutathione (GSH)], which resulted in the cleavage of the DMAC ligand and a corresponding decrease in protein fluorescence over time. 23 Attempts to address this problem using PYP-tag mutants that could bind DMAC ligands more effectively were only partially successful, 23 resulting in a gradual decrease in fluorescence in living cells over time ( ca. 10% per h).…”