It seems like a theme here has been old dogs/new tricks, what with peptide splicing by proteasomes, new ubiquitin bonds, and new LCMV epitopes. Proteasomes, which have been studied intensively for well over 20 years, also showed a new trick recently.1

Proteasomes chop up proteins into peptides. Immunologists tend to think this is to make peptides that cytotoxic T lymphocytes (CTL) can recognize, but that’s a tiny, tiny fraction of a proteasome’s output. Mostly, proteasomes are there to destroy proteins that have reached the end of their useful life span. In fact, proteasomes are really designed to be (from the immunologists’ viewpoint) inefficient at producing CTL peptides.2 Almost all cells, almost all the time, are not infected by any viruses, and will never make use of the CTL recognition system, so any peptides that system draws away from the cell, are a strain on the natural recycling pathway.

Proteasome beta subunits

But what happens when a neighbouring cell is infected by a virus? That changes the whole equation. Now, local cells are much more likely to be infected, and it would behoove3 these neighbouring cells to divert more resources to antiviral defense systems. So, in the presence of interferon (which is produced in the presence of a viral infection), cells do many, many immune-related things, and one of those thing is to switch off one set of proteasomes and turn on a different set, evocatively known as “immunoproteasomes”.4 Immunoproteasomes differ from constitutive proteasomes in that they have a different set of catalytic subunits, so they have different cleavage preferences and they’re much more likely to make peptides that bind to MHC class I molecules and can therefore be examined by CTL. (Image on the left is of the three constitutive beta (catalytic) subunits; taken from: Chemistry & Biology 9:655-662 (May 2002). Probing Structural Determinants Distal to the Site of Hydrolysis that Control Substrate Specificity of the 20S Proteasome. Michael Groll, Tamim Nazif, Robert Huber and Matthew Bogyo)

This much has been known for 15 years or more. There are knockout mice, biochemical studies of the different kinds of proteasomes, calculations of their peptides’ lengths, and so on, and really the glitter has long since worn off proteasome subunits.

Except that Keiji Tanaka’s lab just turned up a new one.5 While rummaging through the genome, they noticed (right next door to one of the constitutive catalytic subunits of the proteasome) something that looked a lot like another, undescribed, proteasome catalytic subunit. On testing, sure enough, that’s exactly what it is. It incorporates into proteasomes, changes the catalytic activity of the proteasome, replaces its constitutive version … looks just like an interferon-inducible subunit. But it’s not interferon inducible. Instead, it’s tissue-specific; it’s only found in the thymus. (Image below is a small part of human chromosome 14, showing the new ß5 subunit, in blue, next door to its constitutive version, PSMB5, in red. Drawn with XPlasMap v.0.95, using the GenBank genomic sequence.)

Human chromosome 14

Why a thymus-specific proteasome subunit? The thymus is where T cells (including CTL) grow up; it’s where they learn how to recognize peptides, how not to recognize peptides that are part of the normal self, how to react only to abnormal (viral, tumour) peptides.6 So an obvious question was: Is this new subunit required for T cells to mature? And it is; quite dramatically so. Without this subunit, numbers of CTL exiting the thymus are reduced by maybe 75%. CD4 T cells, which are not believed to be dependent on proteasome-derived peptides for peptide recognition, weren’t affected. By comparison, knocking out the interferon-inducible catalytic subunits makes a small, often barely-detectable difference in CTL numbers and target recognition.7

Why is this one little subunit so important for CTL generation? Tanaka’s group proposed that this change in catalytic subunits makes thymic proteasomes generate a different set of peptides: peptides that are the opposite of immunoproteasome-generated peptides. Instead of being customized for MHC class I binding, these peptides are customized to be poor binders. By having low-affinity peptides, T cells would have their positive selection enhanced:

Considering that proteasomes are essential for the production of MHC class I ligands and that ß5t specifically attenuates the peptidase activities that cleave peptide bonds after hydrophobic amino acid residues, it is possible that thymoproteasomes predominantly produce low-affinity MHC class I ligands rather than high-affinity ligands in cTECs, as compared with constitutive- and immunoproteasomes, thereby supporting positive selection.

OK, if I’m interpreting this correctly, I’m not buying it. Clearly ß5t has a strong effect on CTL generation in the thymus; but I don’t see how simply generating low-affinity peptides can be the cause.

First of all, the number of CTL coming out are reduced to 25% of normal. But they show in their Supplemental Figures that only about 20% of proteasomes in the thymic cells have the thymus-specific subunit. The vast majority of proteasomes in these cells are either constitutive or immunoproteasomes, not thymoproteasomes. Five times as many high-affinity peptides will beat out low affinity peptides every time.

Thymic subunitsSecond, it’s well known that when MHC class I is associated with low-affinity peptides, the peptide falls off more readily, and the MHC class I is no longer recognized by most antibodies. Therefore, if ß5t forces MHC class I to have low-affinity peptides, eliminating ß5t should increase detectable surface levels of MHC class I. But Murata actually measure cell-surface MHC class I levels in wild-type and knockout mice, and there’s no difference (again this is in their Supplemental Figures). So there’s no indication that there is a significant amount of low affinity peptide involved. (Image on the right is from Murata et al’s paper, showing the distribution of ß5t in the thymic cortex vs. medulla)

So I don’t think that thymic epithelial cells are normally coated with low-affinity peptides that are important for CTL positive selection. Michael Bevan has a commentary8 on Murata et al, and he has a more plausible spin on the same question:

However, if the thymus cortical epithelium expresses a unique range of self peptides as Murata et al. suggest, this raises the possibility that positive selection may be mediated by self antigens that are not seen outside the thymus. Such sequestration of the positively selecting peptide may provide a greater safety window between high and low affinity to better guard against activated T cells cross-reacting on self antigens and causing autoimmunity.

I think this is a subtly but critically different suggestion. (Or, I could be misunderstanding either Bevan’s or Tanaka’s argument.) Tanaka’s group seem to be proposing that because of ß5t, the peptides associated with thymic epithelial cells are low-affinity. Bevan seems to be suggesting that there is a specific low-affinity peptide (or a small number of them) that are thymus-specific, and that are especially designed for T cell selection. This starts to sound a little reminiscent of CLIP for the MHC class II system,9 though not identical. I find this a more attractive model, and it’s testable (as is Tanaka’s model, of course). It makes me wonder — if it is CLIP-like — what the CLIP analogue peptide is. It would have to bind to a wide range of MHC class I alleles that have very different binding motifs … It occurs to me that it wouldn’t have to be a low-affinity binding at all, nor for that matter would it even have to bind entirely to the peptide-binding groove of MHC class I (maybe do something analogous to superantigens). That would get around the motif problem.

Should be interesting to see what comes up in the next year or two.

  1. Well, they’ve had the trick for the past 400 million years, but we just found out about it.[]
  2. Of course, it’s the other way around — CTL are designed to be inefficient at using proteasomes’ peptides, because proteasomes are phylogenetically much older than CTL; proteasomes are present in Archaebacteria, plants, yeast — things that have never even come close to a CTL. When CTL and the class I MHC system arose, they were optimized to not suck too many peptides out of the protein recycling pathway.[]
  3. This footnote exists solely to give me another chance to say “behoove”[]
  4. A term coined by Keiji Tanaka, from whose lab today’s paper comes: J Leukoc Biol. 1994 Nov;56(5):571-5. []
  5. Regulation of CD8+ T Cell Development by Thymus-Specific Proteasomes. Shigeo Murata, Katsuhiro Sasaki, Toshihiko Kishimoto, Shin-ichiro Niwa, Hidemi Hayashi, Yousuke Takahama, Keiji Tanaka. Science 316:1349-1353 (DOI: 10.1126/science.1141915 ) []
  6. Less delicately, they never actually learn, but they’re killed if they fail the test.[]
  7. Immunity. 1994 Oct;1(7):533-41; Science. 1994 Aug 26;265(5176):1234-7; and J Immunol. 2006 Jun 1;176(11):6665-72.[]
  8. Science 316:1291-1292 (June 2007) []
  9. Immunity. 1999 Jan;10(1):83-92. Thymic selection by a single MHC/peptide ligand: autoreactive T cells are low-affinity cells. Lee DS, Ahn C, Ernst B, Sprent J, Surh CD. And Eur J Immunol. 2000 Dec;30(12):3542-51. CLIP-derived self peptides bound to MHC class II molecules of medullary thymic epithelial cells differ from those of cortical thymic epithelial cells in their diversity, length, and C-terminal processing. Kasai M, Kropshofer H, Vogt AB, Kominami E, Mizuochi T.[]