Ephemeroptera (Mayfly)

One of the questions in antigen processing is what happens to peptides between the time they’re generated, and the time the they bind to MHC class I.

(The reason we care about peptides and MHC is that antiviral lymphocytes react with a complex of peptides and MHC class I, so this is a central point for antiviral immunity. Peptides are formed as a byproduct of normal protein degradation; an outline of the process, should you care, can be found here.)

In general, the peptides we’re interested in are produced by proteasomes. A protein (say, 500 amino acids long) enters the proteasome, the protein is chopped up, and peptides (between 3 and 30 amino acids long) come out. Almost all of those peptides are further chopped up, to produce amino acids – recycling and replenishing the amino acid pool for new protein synthesis. A small fraction of the peptides (perhaps between 0.01% and 1%), though, escape destruction and manage to bind to MHC class I. We would like to know more about that fraction of peptides, because they drive the lymphocyte attack on virus-infected cells. Why are they not destroyed — is it pure chance, or is there something special about the peptides that are not destroyed? How do they reach the MHC — is it chance again, just random diffusion, or is there some kind of specialized shuttle system that ferries the peptides to the proper subcellular location? Is there any active process modifying the peptides, to make them more (or less) suitable for binding MHC? And so on.

The problem is that it’s really hard to look at those peptides. Ideally, we’d like to grab samples of peptides at every point in the process: Exiting the proteasome, in transit, being degraded and processed, and so on. Then we could analyze what they’re like at each step, and develop a time course of modifications, interactions, and so on. But we can’t do that (yet), because it’s really difficult to measure peptides within a living cell.

A couple of years ago Jacques Neefjes (who always turns out cool papers) put some numbers on just how difficult is is.1

Blogging on Peer-Reviewed ResearchThere were a whole bunch of really cool things about this paper, but just focusing on one: Neefjes’ group came up with a way of measuring the rate of peptide destruction in living cells. They added a fluorescent tag to peptides in such a way that it would only fluoresce when the peptide was degraded; injected the tagged peptides into single cells; and measured (again in single cells) the rate at which the fluorescence appeared.

The injected peptides were destroyed with a half-life of 7 seconds. That is, a single cell can destroy hundreds of thousands, or millions, of peptides within a few seconds. (Most of this destruction, by the way, is performed by aminopeptidases, which are very abundant in cytosol.)

That’s not a long time, and it doesn’t give any individual peptide much chance to find its potential MHC binding partner. “A peptide will thus diffuse through the entire cell in 6 s and has to find TAP within this short period for translocation into the ER lumen.”

Why so fast? Why is the cell so worried about letting peptides hang about? Well, we presume this is because peptides are potentially very toxic. These peptides are generated, pretty much randomly, from active proteins. The peptides will therefore include short chunks of active protein domains, separated from any regulatory context; they could conceivably have biological activities by themselves. Also, you’d get hydrophobic chunks that could cluster into degradation-resistant clumps, if you let them accumulate, and it’s believed that such degradation-resistant complexes are themselves toxic. So you need to get rid of peptides fast, before they accumulate to form dangerous side-effects.

As a result, we antigen processing guys have to pretty much guess and use roundabout, indirect methods to measure peptides. Keeps us off the streets, I guess.

  1. Reits, E., Griekspoor, A., Neijssen, J., Groothuis, T., Jalink, K., van Veelen, P., Janssen, H., Calafat, J., Drijfhout, J. W., and Neefjes, J. (2003). Peptide diffusion, protection, and degradation in nuclear and cytoplasmic compartments before antigen presentation by MHC class I. Immunity 18, 97-108 .[]