Here I’m picking up on a throwaway comment I made in a thread on Larry Moran’s “Sandwalk” blog. Larry wrote about protein turnover in the cell, a favourite topic of mine to start with, especially when proteasomes come into play, as they so often do.

In the comments, daedalus2u observed “Proteases only hydrolyze peptides when the equilibrium favors it. Under conditions of dehydration, the equilibrium favors the making of peptides.” He made this in the context of lysosomes (and frankly his train of thought seems to increasingly run off the rails as the comment progresses) but it prompted Ryan to say that “I doubt proteasomes could ever act in reverse. ”

Just about everyone else doubted it, too, until a few years ago, when some really cool evidence for just that happening came out of immunology. As it turns out, though, proteasomes almost certainly can act in reverse and splice peptides. For a while it even seemed possible that this could be a common event, but I think it’s becoming increasingly likely that it’s actually a very rare event, one that’s usually only detectable by the exquisitely-sensitive T cell recognition system.1

Ryan’s reasoning wasn’t bad. He argued that “dehydrating the proteasome would change it’s structure and probably eliminate any catalytic activity.” That makes sense, but it misses something unusual (though not unique) about the proteasome.

Proteasomes have been in the news quite a bit since they won the Nobel in 20042 and there are lots of friendly introductions to proteasome-mediated protein degradation around. The Nobel Foundation has a fairly friendly “Information for the public” thing, and a less friendly but more complete PDF . For the purpose of peptide splicing, though, you only need to know the basics.

Here’s the basics: Proteasomes are multi-catalytic proteases, and they’re very abundant throughout the cytoplasm and nucleus of most cells. From this, you can work out why peptide splicing works. Not that anyone actually did work it out, but in hindsight there’s a definite logic to it. Follow closely here:

Proteasomes are multicatalytic. That is, they can chop up many different peptide bonds. That’s in contrast to many proteases, that only cleave when a very precise sequence of amino acids line up. Proteasomes do have their preferences, sure; there are sequences they don’t like — but if you feed a protein to a purified proteasome you’ll find that virtually every possible amino acid pair has been cleaved (if only very rarely).

If they’re multicatalytic, and they’re abundant, then they’re a potential hazard to normal cell function. You can’t have a protease indiscriminately chewing up cellular proteins. So proteasomes are regulated proteases (the regulation part is what the Nobel was for). If they’re regulated, you have to have a way to shield the catalytic sites so they only attack what they’re supposed to. Proteasomes do this by hiding their active sites on the inside of a hollow cylinder.

Proteasome end viewProteasome side viewHere I get to throw in a couple of images of the proteasome, which is something I do at every opportunity anyway.3 There’s an end view and a side view.4 In fact in a real cell, you probably wouldn’t see the end view like this, because this is the central core of a larger particle that has caps over the open ends. But it makes the point that this is a hollow, barrel-shaped structure. The catalytic sites are on the inside, the caps normally prevent access to the inside, and the regulatory machinery ends up selecting proteins that feed into the open chamber for destruction.

A couple of other proteases follow this pattern, by the way — tricorn protease is a huge, hollow icosahedral particle, for example. Tripeptidyl peptidase II is also a gigantic particle, and I wonder if there’s some kind of regulatory aspect to its size, even though as far as I know from relatively crude evidence, the catalytic sites of TPPII are more or less exposed.

Anyway, the hollow barrel of a proteasome is probably the key to its ability to do peptide splicing. As daedalus2u pointed out, enzymes run both ways. Proteases in general act through hydrolysis, which requires, of course, water. If there’s no water, the reaction can run backwards. In the old days, I’m told, that was how you synthesized peptides: you took the appropriate enzyme and ran the reaction in a non-aqueous system. Normally, of course, there is water inside a proteasome, or it wouldn’t work. But it’s not hard to picture a scenario where peptides are being rapidly generated, and before they have a chance to diffuse out of the proteasome they’re squeezing away water molecules. There you have a high concentration of reactive peptide ends, crowded together in the absence of a water molecule and bumping up against a promiscuous active site. When that happens, you can get peptide splicing.

As I said, this was detected using T cells, which are very sensitive to peptides — recognizing fewer than ten per cell, perhaps. In 2004, Benoit van den Eynde showed that a peptide that was a T cell epitope was in fact generated by peptide splicing in the proteasome5 and later, he showed that you can even swap position, demonstrating this with a T cell epitope that was generated by splicing two peptides in the reverse order.6

How common is this? After the first paper or two, we really didn’t know. When you look at peptide epitopes associated with a cell, I’m told, there are often a significant number that can’t be identified by blasting through databases. Were all of these unidentified because they were peptide splices? That was Benoit’s original idea, I think, and I wouldn’t have been at all surprised to see a small flood of papers triumphantly identifying as spliced those pesky holdout peptides from previous work.

Hasn’t happened, though. It’s negative evidence, but for the most part peptide splicing doesn’t seem to have fixed the problem of the unidentified peptide.7 Perhaps there will still be a herd of peptide splicing examples popping up any day now, but for now I’m leaning to the idea that this really is a very rare event.

Too bad, because it’s pretty cool.


  1. But I’m not going to be dogmatic about it. It’s an open possibility that this is a common event that’s just very hard to detect[]
  2. At any event, they’ve been in the news more often, even if they haven’t caught up with Paris Hilton yet[]
  3. Just one of the many things that make me the life of any party. I wonder why I’m not invited to more?[]
  4. This is the mammalian 20S proteasome, ref. Unno et al., Structure 2002 May; 10(5):609-18. I made the images with iMol from the pdb files.[]
  5. Science. 2004 Apr 23;304(5670):587-90[]
  6. Science. 2006 Sep 8;313(5792):1444-7[]
  7. They’re probably allelic variants, or maybe sequencing errors, or something like that, is my guess now[]