Chemical and Biological Strategies for Profiling Protein‐Protein Interactions in Living Cells

Abstract

Protein-protein interactions (PPIs) play critical roles in almost all cellular signal transduction events. Characterization of PPIs without interfering with the functions of intact cells is very important for basic biology study and drug developments. However, the ability to profile PPIs especially those weak/transient interactions in their native states remains quite challenging. To this end, many endeavors are being made in developing new methods with high efficiency and strong operability. By coupling with advanced fluorescent microscopy and mass spectroscopy techniques, these strategies not only allow us to visualize the subcellular locations and monitor the functions of protein of interest (POI) in real time, but also enable the profiling and identification of potential unknown interacting partners in high-throughput manner, which greatly facilitates the elucidation of molecular mechanisms underlying numerous pathophysiological processes. In this review, we will summarize the typical methods for PPIs identification in living cells and their principles, advantages and limitations will also be discussed in detail.

Comparison of two peroxidases with high potential for biotechnology applications – HRP vs. APEX2

“…Therefore, both enzymes, HRP/sHRP and APX/sAPEX2, are highly similar in terms of overall protein fold and catalytic site. Mentioned similarities are necessary because it is known that both proteins have highly similar mechanism of functioning [12] , [13] , [15] , [33] . However, the differences such as glycosylation, disulfide bridges and calcium ions present only in HRP, induce changes in protein structure rigidity and can influence the application of split variants of these enzymes.…”

“…However, the differences such as glycosylation, disulfide bridges and calcium ions present only in HRP, induce changes in protein structure rigidity and can influence the application of split variants of these enzymes. Since HRP/sHRP is glycosylated, it is bigger in size – 44 kDa compering to 28 kDa of non-glycosylated APX/sAPEX2 [12] . In our previously published data, glycosylation plays stabilizing role in HRP/sHRP structures [10] .…”

“…APX is especially popular for proximity labeling techniques in cell biology and cellular imaging. In general, peroxidase-catalyzed reactions have a high potential for biotechnology applications and have become a valuable tool to decipher in vivo protein-protein interactions by protein engineering [11] , [12] , [13] , [14] , [15] . A very interesting and highly specific approach to analyze protein-protein in vivo is based on the concept of split peroxidases.…”