To keep things simple, the scientists started their search in yeast cells, which have very similar secretory pathways to human cells but are easier to grow and study in the lab. They noticed that when they grew the yeast under nutrient-rich conditions the cells secreted a little bit of SOD1. But when the cells were starved of nutrients, they exported nine times the amount.
Next, Malhotra and his team used genetic engineering to change certain molecular building blocks (amino acids) in SOD1, focusing on a region that is the same in both the yeast and the human versions of the protein. They discovered that a sequence of just two amino acids was enough to act as a ‘stamp’ to send the protein to the CUPS pathway. And, crucially, the same two amino acids are also found in the other unconventionally secreted protein Acb1, suggesting that this might be a universal signal for CUPS.
Finally, to see whether this same pathway might be at work during the development of ALS, the researchers tested healthy and ALS versions of SOD1 in human cells and found that it also is exported through the CUPS pathway when the cells are starved of nutrients.
Putting this all together, Malhotra believes that this work proves that both healthy and faulty versions of SOD1 are secreted from starving cells through the CUPS pathway, and that the little two amino-acid ‘stamp’ is enough to send them there. But there is still a mystery that needs to be solved.
“Many proteins have the same two amino-acid motif – in fact, it is extremely common,” he says. “We still need to find out how SOD1 and proteins like it are specifically recognised and sent to the CUPS, while other proteins are not.”
Malhotra thinks that the two-piece ‘stamp’ is normally hidden in proteins like SOD1 and Acb1 under normal conditions. But when something changes – for example, the protein is faulty or the cell is starving, which affects the shape of proteins – then it becomes exposed. Molecular ‘chaperones’ then step in to prevent any further unravelling, and instead send the protein off to the CUPS to be secreted out of the cell.
The identities of these chaperones and the exact ways in which they shuttle proteins into the CUPS are currently unknown, but Malhotra and his team are busy tracking them down. They are particularly interested in finding out what triggers harmful SOD1 secretion in ALS patients and – more importantly – working out if they can stop it.
“The discovery of this unconventional ‘stamp’ directing the secretion of SOD1 is very exciting,” he says. “At the moment there are no effective treatments for ALS, so I hope that our findings might provide the basis for the development of much-needed therapies in the future.”