In eukaryotic cells — found in animals, plants, and fungi — protein synthesis involves more than the simple assembly of amino acids in ribosomes. Nearly one-third of all human proteins must be transported to the endoplasmic reticulum (ER) during or shortly after their synthesis. In the ER, these proteins undergo crucial folding and modifications, including the formation of disulfide (S-S) bonds, which are vital for their structure and function.
Disruptions in protein translocation to the ER or disulfide bond formation underlie several diseases, and understanding the mechanisms that govern these processes is essential in biology and medical science. Unfortunately, the tools available to study them are either quite limited in scope or require exceptionally expensive equipment and carefully repeated measurements.
In an effort to overcome these challenges, a research team including Specially-Appointed Associate Professor Hiroshi Kadokura and Professor Hideki Taguchi from Institute of Science Tokyo, Japan, developed an innovative ‘reporter’ molecule that can detect ER-related problems during protein synthesis. Their findings were published in the journal iScience.
While designing this reporter, the researchers took a page from a fusion protein called MalF-LacZ, derived from Escherichia coli bacteria. In these microorganisms, the MalF part of the protein helps with translocating LacZ from the cytoplasm to the cell envelope. Once transported there, the LacZ enzyme undergoes oxidation through disulfide bond formation, thus deactivating it. Therefore, problems in either transportation or disulfide bond formation would result in an abnormally activated LacZ enzyme.
Inspired by these elegant natural mechanisms, the research team developed a reporter molecule based on firefly luciferase (FLuc) that operates in a similar manner. Luciferase is a bioluminescence producing enzyme of firefly that produces light when it catalyzes the oxidation of D-luciferin in the presence of oxygen, adenosine triphosphate (ATP), and magnesium ions (Mg2+). More specifically, they engineered a FLuc variant that is rendered inactive by disulfide bond formation in the ER, but remains active in the cytosol or if disulfide bonds do not form. They ‘targeted’ this compound to the ER by introducing specific modifications, and made it more prone to misfolding (and deactivating) within the ER by strategically replacing amino acids in the FLuc sequence with Cysteine.
Using this reporter, the researchers could easily detect problems in protein translocation to the ER, as well as problems in disulfide bond formation. A bioluminescence producing enzyme of a different type can serve as an internal control and ensures precise measurement. Furthermore, the reporter protein is equipped with a motif that undergoes a modification (glycosylation) only when the protein is translocated into the ER. Thus, they could also determine which of the two possibilities were the underlying cause for the activation of the FLuc reporter.
To showcase the power of this method, the team ran experiments in cells where the redox environment of the ER was chemically altered, disrupting disulfide bond formation. Additionally, they showed that the proposed reporter can 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,” highlights Kadokura.
Notably, this novel reporter bears several advantages over other available methods, including its simplicity, robustness against environmental fluctuations, and high reproducibility. “Our reporter system will serve as a valuable tool across various fields related to secretory pathway proteins, extending beyond fundamental studies,” says Taguchi, hopeful for the future.
