Japanese Researchers Develop Innovative Reporter Molecule to Detect Protein Synthesis Issues in Cells

Tokyo, December 6: Protein synthesis in eukaryotic cells, found in plants, animals, and fungi, involves more than just the ribosome’s assembly of amino acids. During or shortly after their synthesis, over one-third of all human proteins need to be transported to the endoplasmic reticulum (ER), where their structure and function are determined through critical folding processes, such as the formation of disulfide (S-S) bonds.

To address challenges in understanding this process, a research team led by Associate Professor Hiroshi Kadokura and Professor Hideki Taguchi from the Institute of Science in Tokyo has developed an innovative ‘reporter’ molecule that can detect ER-related problems during protein synthesis. This breakthrough aims to shed light on the disruptions in protein translocation to the ER or disulfide bond formation, which can lead to several diseases.

Currently, studying these mechanisms is hindered by limited tools or the need for expensive equipment and meticulous measurements. The researchers overcame these challenges by designing a reporter molecule based on firefly luciferase (FLuc), a bioluminescent enzyme that produces light when it catalyzes the oxidation of D-luciferin in the presence of oxygen, ATP, and magnesium ions (Mg2+).

The researchers adapted the FLuc enzyme to be inactive when disulfide bonds form in the ER, but active if disulfide bonds do not form or if the enzyme remains in the cytosol. By introducing specific modifications to target the compound to the ER, and making it more prone to misfolding, they developed a system that could easily detect problems in protein translocation and disulfide bond formation.

The FLuc-based reporter system can also detect which of the two issues (translocation or bond formation) is responsible for any observed activation. The researchers demonstrated its effectiveness by running experiments where the redox environment of the ER was chemically altered, disrupting disulfide bond formation. The reporter was also able to detect defects in protein translocation induced by a potential anti-HIV drug, signaling the successful inhibition of the virus.

“Given that luciferase-based assays are well-suited for high-throughput platforms, we suggest that this approach will facilitate large-scale screening of small molecules that specifically block the biosynthesis of harmful secretory pathway proteins,” said Kadokura.

The team’s reporter system offers several advantages over existing methods, including simplicity, robustness against environmental fluctuations, and high reproducibility. Taguchi added, “Our reporter system will serve as a valuable tool across various fields related to secretory pathway proteins, extending beyond fundamental studies.”

This breakthrough in understanding protein synthesis and its related diseases could pave the way for new medical advances and treatments, offering hope for better diagnosis and intervention in the future.