Novel Approach to Monitor ER-Related Protein Quality Control

Protein synthesis in eukaryotic cells found in plants, animals, and fungi involves more than just the straightforward ribosome assembly of amino acids. During or soon after their synthesis, nearly one-third of all human proteins need to be transported to the Endoplasmic Reticulum (ER).

These proteins' structure and function depend on the critical folding and modifications that take place in the ER, such as the creation of disulfide (S–S) bonds.

Many diseases are caused by disruptions in the formation of disulfide bonds or the translocation of proteins to the ER, and biology and medicine must comprehend the mechanisms underlying these processes.

Unfortunately, few tools are available to study them, and they require very expensive equipment and meticulously repeated measurements.

To address these issues, a group of researchers led by specially appointed Associate Professor Hiroshi Kadokura and Professor Hideki Taguchi from the Institute of Science Tokyo in Japan created a novel “reporter” molecule that can identify ER-related issues during protein synthesis. The journal iScience published their findings.

The fusion protein MalF-LacZ, which is derived from the Escherichia coli bacteria, served as a model for this reporter's design. The protein's MalF portion aids in moving LacZ from the cytoplasm to the cell envelope in these microbes.

Once there, the LacZ enzyme is deactivated by oxidation caused by the formation of disulfide bonds. Therefore, an abnormally activated LacZ enzyme would result from issues with either transportation or the formation of disulfide bonds.

The research team was inspired by these sophisticated natural processes and created a reporter molecule based on firefly luciferase (FLuc) that functions similarly. When oxygen, adenosine triphosphate (ATP), and magnesium ions (Mg²⁺) are present, the firefly enzyme luciferase catalyzes the oxidation of D-luciferin, which results in light.

More precisely, they created a variant of FLuc that is inactive in the ER when disulfide bonds form but is active in the cytosol or in the absence of disulfide bonds. By making certain changes, they “targeted” this compound to the ER. They also made it more likely to misfold (and deactivate) inside the ER by purposefully substituting cysteine for amino acids in the FLuc sequence.

This reporter made it simple for the researchers to identify issues with disulfide bond formation and protein translocation to the ER. A different kind of bioluminescence-producing enzyme can be used as an internal control to guarantee accurate measurement.

Additionally, the reporter protein has a motif that is only modified (glycosylated) upon translocation into the ER. As a result, they were also able to identify which of the two scenarios was the root cause of the FLuc reporter activation.

The team conducted experiments in cells where the ER's redox environment was chemically changed, impairing the formation of disulfide bonds, to demonstrate the effectiveness of this technique. They also demonstrated that the suggested reporter can identify protein translocation abnormalities brought on by a possible anti-HIV medication, indicating that the virus has been successfully inhibited.

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.”

Hiroshi Kadokura, Associate Professor, Institute of Science Tokyo

Notably, this new reporter has several advantages over existing techniques, such as its high reproducibility, resilience to environmental changes, and ease of use.

Our reporter system will serve as a valuable tool across various fields related to secretory pathway proteins, extending beyond fundamental studies.”

 Hideki Taguchi, Professor, Institute of Science Tokyo

Hopefully, these endeavors will result in a deeper comprehension of life processes and illnesses, opening the door to novel medical discoveries and therapies.