In vivo monitoring of the ubiquitination of newly synthesized proteins in living cells by combining a click reaction with fluorescence cross-correlation spectroscopy (FCCS).

Newly synthesized proteins are closely related to a series of biological processes, including cell growth, differentiation, and signaling. The post-translational modifications (PTMs) of newly synthesized proteins help maintain normal cellular functions. Ubiquitination is one of the PTMs and plays a prominent role in regulating cellular functions. Although great progress has been made in studying the ubiquitination of newly synthesized proteins, the in vivo monitoring of the ubiquitination of newly synthesized proteins in living cells still remains challenging. In this study, we propose a new method for measuring the ubiquitination of newly synthesized proteins in living cells by combining a click reaction with fluorescence cross-correlation spectroscopy (FCCS). In this study, a puromycin derivative (Puro-TCO) and a fluorescence probe (Bodipy-TR-Tz) were synthesized, and then, the newly synthesized proteins in living cells were labelled with Bodipy-TR via the click reaction between Puro-TCO and Tz. Ubiquitin (Ub) in living cells was labelled with the enhanced green fluorescence protein (EGFP) by fusion using a gene engineering technique. FCCS was used to quantify the newly synthesized proteins with two labels (EGFP and Bodipy-TR) in living cells. After measurements, the cross-correlation (CC) value was used to evaluate the ubiquitination degree of proteins. Herein, we established a method for monitoring the ubiquitination of newly synthesized proteins with EGFP-Ub in living cells and studied the effects of the ubiquitin E1 enzyme inhibitor on newly synthesized proteins. Our preliminary results document that the combination of FCCS with a click reaction is an efficient strategy for studying the ubiquitination of newly synthesized proteins in vivo in living cells. This new method can be applied to basic research in protein ubiquitination and drug screening at the living-cell level.