By substituting a hydrogen atom of the protein with an iodine atom, IISc researchers have been successful in directly delivering proteins into mammalian cells. As a result, there was nearly six-fold increase in green fluorescent protein uptake by the cells. Supplementing the cellular protein becomes important during diseased conditions.
In a breakthrough which might have huge medical implications, researchers at Bengaluru’s Indian Institute of Science (IISc) have used a novel strategy to directly deliver proteins into mammalian cells. Proteins are big molecules and so cannot enter the cells on their own. So a team led by Prof. Govindasamy Mugesh from the institute’s Department of Inorganic and Physical Chemistry substituted a hydrogen atom of the protein with an iodine atom to achieve a nearly six-fold increase in protein uptake by cells. The increased protein uptake was seen even when the molecular weight of the protein was 28,000 dalton, meaning the protein was much bigger in size than most of the therapeutic small molecules.
The researchers also tried replacing a hydrogen atom with an atom of bromine and chlorine but the uptake was way lower than when iodine was used. In the case of bromine, the uptake of proteins increased by only about two times, while the uptake increased only marginally when chlorine was used. The results were published in the journal Angewandte Chemie.
Other researchers have tried tagging the protein with cell-penetrating peptides, supercharged proteins and even used virus-like particles to ferry the proteins into cells. But these approaches have severe limitations including altering the protein function inside the cell. For this reason, most of the applications involving proteins are directed to extracellular targets.
Proteins inside the cells get impaired during diseased conditions such as neurodegenerative and cardiovascular disease. Supplementing the cellular protein in such cases becomes important and this is where the method used by the IISc team will come in handy.
The team had to first synthesise a green fluorescent protein with one hydrogen atom being replaced with an iodine atom. “To introduce iodine at a specific site on the protein, we had to use an iodinated amino acid. Since the iodinated amino acid used is unnatural (not genetically coded), protein synthesis machinery does not accept it. So we had to expand the genetic code of the organism to accept and incorporate the iodinated amino acid into the proteins during the biosynthesis in the cells,” says Prof. Mugesh.
Since the iodinated amino acid is introduced on the surface of the protein, the secondary structure is not altered and so the protein remains functionally intact.
Protein uptake by cells
Iodine forms a halogen bond with a specific receptor (caveolin) that transports the protein from the cell membrane surface to inside the cells. “Compared with bromine and chlorine, iodine is heavier and so it forms a stronger halogen bond with the receptor. This might be responsible for more proteins getting into the cells when we substitute a hydrogen with an iodine atom,” says Surendar R. Jakka from IISc’s Department of Inorganic and Physical Chemistry and first author of the paper.
To be functionally useful, the proteins must enter the cytoplasm of the cell. However, the moment proteins are ferried into the cell by the receptor they are trapped inside the endosomes and transported to lysosomes, where the proteins are degraded. Significant decrease in protein concentration as measured by the fluorescence intensity was seen by the researchers after 24 hours.
To overcome the problem of protein degradation, the team treated the cells with a peptide (ppTG21). “The peptide also gets into the endosomes along with the protein and changes the pH of the endosomes. The endosome gets ruptured due to pH change leading to release of the proteins into the cytoplasm. In this case, there was no decrease in the protein concentration even after 24 hours,” says Prof. Mugesh.
“We are substituting only one hydrogen atom with an iodine atom in the entire protein. So the toxicity is similar to native protein,” says Prof. Mugesh. “We tested the cell viability by treating the cells with different concentrations of the proteins for 90 minutes. The cells were healthy after taking up the protein. The morphology of the cells that had taken up the proteins did not change even at the end of 24 hours,” says Jakka.
“We want to use this strategy to deliver more functional proteins which can regulate other biological processes such as redoc regulation, which goes awry in neurodegenerative diseases, ” says Prof. Mugesh. The researchers intend testing it on animal models.