Dancing dustbins: New insights into proteasome activation with implications for antimalarial drug action.
Capitalising on the structural cryo-EM revolution,our team has generated molecular movies of a sophisticated nano-machine, called the proteasome. We show that the barrel-shaped proteasome has a cap, called PA28, that “dances” back and forth, opening and closing a gap at the interface.
Every cell needs a garbage disposal unit to deal with its damaged and no longer needed proteins. A major component of this waste system is a fleet of barrel-shaped shredders called “proteasomes”. Unwanted proteins are fed into the central chamber of the barrel, where they are cut into tiny pieces by three different protease enzymes, then spat out the other end. However, if the proteasome destroyed proteins in an indiscriminate manner, mayhem would ensue; so “guard” proteins, called activators, control the entry and exit portals. To enter the shredder, proteins can be marked with a “kiss-of-death” signal, called ubiquitin. Proteins marked with ubiquitin are recognised by an activator complex called the regulatory particle, at one end of the barrel, and passed through to the central core. At the other end, a different activator, called PA28, helps release the resulting breakdown products. The PA28 activator can also let in unstructured proteins - even if they don’t have the ubiquitin signal. But exactly how the PA28 activator works is poorly understood.
Some cells such as cancerous blood cells make proteins at a gangbusters’ rate, creating so much waste they are particularly reliant on their proteasomes. As a consequence, proteasome inhibitors are used clinically as anti-cancer agents. Similarly, malaria parasites multiply rapidly inside human red blood cells and in doing so, generate a lot of waste protein. The parasites rely heavily on their proteasomes to clear this waste; and proteasome inhibitors are potential antimalarial drugs.
In this work, we studied the consequences of genetic deletion of PA28 in the malaria parasite, Plasmodium falciparum. We found that parasites lacking PA28 survive under normal conditions. However, they were not happy when we exposed them to a low concentration of artemisinin – an antimalarial drug that explodes like a cluster bomb inside the parasite, generating damaged proteins that need to cleared. It seems the activation of the proteasome by PA28 helps withstand the toxic damage initiated by artemisinin. We wanted to know more about how PA28 helps deal with damaged proteins.
To understand how living matter works, it helps to have detailed structural views of the molecular machinery. It is now possible to obtain that information, due to a revolution in structural biology, called cryo electron microscopy (cryoEM) – a revolution that was recognised by the 2017 Nobel Prize in Chemistry. Suddenly, we are able to directly image the internal mechanics of molecular machines.
We used cryoEM to probe the structure of the malaria parasite proteasome in complex with the PA28 activator. But first we needed to purify the proteasome. Malaria parasites are only one twentieth the diameter of a human hair, so we needed to culture parasites for many months to get enough pure protein for cryoEM studies. A heroic effort, but it was worth it!
Putting together many thousands of electron microscopy images, we could see that PA28 takes the form of conical cap that sits at one end (or both ends) of the proteasome barrel. At the top of the cap, flexible streamer-like loops form dynamic swirls. We could also see loops at the bottom of PA28 and we could see how they engage with some of the protein subunits at the surface of the barrel. This clasp mechanism opens up a pore in the top of the barrel, creating a channel that runs from the peak of the conical cap through to the central shredding enzymes in the barrel core.
CryoEM offers the exciting possibility of making molecular “movies” - views of protein machines in action. When we looked at the different conformations that the proteasome/PA28 complex adopts, we got a bit of a surprise. The PA28 cap is only loosely connected to the proteasome core. It is hinged on one side – a bit like a dustbin lid. But we were really taken aback when we saw that the cap undergoes a rocking motion on the barrel - a motion that opens and closes a gap at the interface. We refer to this as the “dancing proteasome” motion (see Poster Image).
We used computer-based simulations to show that the dancing motion could let small peptide products escape through the interface between the cap and the barrel – potentially providing short-cut access to and from the shredder.
Our cryoEM-based molecular movies provide new insights into the mechanism of action of this important waste disposal system. Importantly, the new high-resolution structure of the malaria parasite proteasome is also helping us to design inhibitors that specifically target the plasmodium proteasome. We are working to develop new drugs to prevent the more than 400,000 deaths caused each year by the malaria parasite.
Xie SC, Metcalfe RD, Hanssen E, Yang T, Gillett DL, Leis AP, et al. The structure of the PA28–20S proteasome complex from Plasmodium falciparum and implications for proteostasis. Nature Microbiology. 2019.