What the FLuc?! Revealing the pathways responsible for secretory protein biogenesis
A novel reporter protein has been developed to enable the investigation of protein biogenesis in the ER and its disruption in disease.
A recent research collaboration between multiple Japanese institutions has developed a technique to facilitate the study of protein biogenesis at the endoplasmic reticulum (ER). The team developed a reporter protein targeted to the ER that is inactivated by key functions of the ER, enabling the visualization and exploration of abnormalities in protein biogenesis.
After their initial translation, one third of human proteins (often secretory proteins) are transported to the ER, where they are folded further into more finalized forms, a process that involves the formation of disulfide bonds within the protein. The disruption of this process can lead to an array of diseases; however, our understanding of how it goes wrong, and the techniques available to explore this process, are severely lacking.
To address this shortfall, the team set out to develop a new technique with which to explore protein biogenesis at the ER. In preparation for the study, the team considered the E. coli fusion protein MalF-LacZ, which is transported to the ER through the MalF subunit, where the enzymatic LacZ component is oxidized by the formation of disulfide bonds and deactivated.
In the current study, the team selected firefly luciferase (FLuc), an enzyme that functions similarly to MalF-LacZ but also produces bioluminescence during catalysis. They engineered the protein to be directed to the ER and made it more prone to misfolding – and therefore deactivation – following the formation of disulfide bonds in the ER by substituting carefully selected residues for cysteine. The resulting reporter was a protein that fluoresced in the cytosol, would be transported to the ER and then stop fluorescing on successful processing. This would highlight any issues in translocation by continuing to fluoresce in the cytosol and any issues in disulfide bond formation by continuing to fluoresce in the ER.
Old World monkeys highlight new targets for Hepatitis B
Cryo-electron microscopy reveals the structural basis for the restriction of hepatitis B virus (HBV) entry in macaques, enhancing our understanding of viral dynamics.
To test the value of the reporter protein in the investigation of the ER and defects in biogenesis, the team used it to investigate the impact of inhibiting ER oxidoreductase 1 alpha (Ero1α)-dependent oxidation of protein disulfide isomerases (PDIs) on the ER’s redox environment. PDIs are the proteins responsible for the introduction of disulfide bonds into proteins transported to the ER, and PDI oxidases oxidize PDIs to facilitate their function.
Ero1α is assumed to play a key role in this process, but visualizing its activity with existing reporters has proven to be notoriously difficult. The team chemically inhibited Ero1α’s activity and observed a corresponding increase in FLuc activity. Further studies enabled the team to conclude that despite the existence of other pathways and enzymes to oxidize PDIs and enable disulfide bond formation, the inhibition of Ero1α activity alters the ER’s redox environment. This confirmed the vital importance of Ero1α in this pathway and FLuc’s ability to report the disruption of disulfide bond formation.
Introducing a version of the reporter fused to the signaling motif of a protein targeted by the anti-HIV drug CADA – which impairs protein translocation to the ER – into cells treated with the drug, the team again observed an increase in the activity of FLuc. This demonstrates its ability to report on protein translocation, as well as disulfide bond formation.
The team were highly enthused by this reporter, highlighting its simplicity and robustness against environmental fluctuations and the high reproducibility of the results produced using it. Senior author Hideki Taguchi (Institute of Science Tokyo, Japan) declared, “Our reporter system will serve as a valuable tool across various fields related to secretory pathway proteins, extending beyond fundamental studies.”
