Proteasome Catalysed Peptide Splicing: How studying proteasome arts and crafts can increase our understanding of epitope generation

Abstract

In order to fight off pathogens and tumours, the body makes a lot of use of little pieces of information called peptides, which can be derived from proteins in a cell. By using the proteasome, a molecular scissor inside the cell, the body can cut up these proteins and present them on a silver platter on the cell surface, also known as the MHC molecules. From there, it can be recognized by immune cells in the body and a fitting reaction can take place, such as the production of antibodies or the killing of tumour cells. This creates a system where there is continuous sampling of the waste products of the cells and allows the body to respond to unknown peptides in a quick and specific matter. Interestingly, it has been discovered that peptides exist that are not made from simply cutting up existing proteins, called non-canonical peptides. So far it has been unclear what the actual mechanism behind the generation of these peptides is, although possible origins have been proposed. Most theories propose that these peptides are cut from proteins made from faulty blueprints, which normally should not be present. In this report, one such origin will be explored, the so-called Proteasome Catalysed Peptide Splicing (PCPS). According to this theory, the proteasome does not simply cut all the different proteins, but is also able to paste distant regions of the protein together to create new peptides. To achieve this, we took a protein that was known to produce non-canonical peptides and replaced that part with a widely used reporter peptide (SIINFEKL, derived from egg white proteins), that was either complete or split into two parts (split SIIN/FEKL). These proteins could then be produced inside bacteria and could be purified after, allowing us to use them in further experiments. Subsequently, we transferred this protein into a cell by giving it electric shocks. After giving the cells time to recover and process the proteins, we then used a reporter cell that could recognize (non-split) SIINFEKL. Afterwards, we could then compare the cells that we gave SIINFEKL-containing proteins, split SIINFEKL-containing proteins or proteins containing neither. Using this method, we were able to efficiently bring proteins that were produced in bacteria into mammalian cells. Additionally, we saw a presence of non-split SIINFEKL in cells that we only electroporated with split SIINFEKL. From this, we concluded that the proteasome could indeed paste distant parts of proteins together through PCPS. By modifying parts of the model protein, we can further study the mechanism behind PCPS in future experiments. By better understanding the way in which these non-canonical peptides are produced, we can more accurately predict in the future which peptides can be generated from a given protein. In turn, this will allow us to make better vaccines against pathogens and even tumour cells.