Mystery Rays from Outer Space

Meddling with things mankind is not meant to understand. Also, pictures of my kids

June 27th, 2007

More new tricks: Thymus-specific proteasomes

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.[]
June 21st, 2007

Non-lysine ubiquitination

In a new and dynamic field, everything-you-know-is-wrong papers appear regularly, and no one is too surprised. Usually, once a field of study has been around for a while (twenty years or more, say) most of the basics are settled in, and when an e-y-k-i-w paper comes along there’s either great skepticism or great angst or both. But there are also some long-established fields where paradigms seem to be shattered on a weekly basis. Ubiquitin is one of those. Another universal rule of ubiquitin was disproven recently, and I for one just nodded thoughtfully, unsurprised.

The paper is:

Ubiquitination of serine, threonine, or lysine residues on the cytoplasmic tail can induce ERAD of MHC-I by viral E3 ligase mK3

Xiaoli Wang, Roger A. Herr, Wei-Jen Chua, Lonnie Lybarger, Emmanuel J.H.J. Wiertz, and Ted H. Hansen

The Journal of Cell Biology, Vol. 177, No. 4, May 21, 2007 613-6241

It’s particularly interesting to me because it’s yet another example of the important insights into cell biology that arise from antigen presentation in general and viral immune evasion in particular.

The paradigm that was overthrown is that “poly-ubiquitination, the process in which a chain of at least four ubiquitin peptides are attached to a lysine on a substrate protein, most commonly results in the degradation of the substrate protein via the proteasome.” (That’s from Wikipedia– my first source for oversimplified summaries that miss important advances and misinterpret what they do find. But other articles on ubiquitin include similar statements.) It’s the “lysine” bit I’m taking issue with in this case.

Ubiquitin moleculeUbiquitin/proteasome pathwayUbiquitin was identified 30-odd years ago (picture on the left from the Nobel Prize web site). It’s a small, abundant protein that’s found in all eukaryotes, and it’s involved in protein destruction. That’s the last of the firm statements: For the rest of this paragraph, you should imagine every statement to be footnoted or qualified in some way, because throughout the past 30 years ubiquitin has made a habit of constantly revealing unexpected functions and new aspects. The simplest pattern is the one you’ll find in innumerable posters and illustrations (the one on the right is from Sigma-Aldrich, but there are scores of virtually-identical ones out there). In this pathway, ubiquitin is covalently attached to proteins, new ubiquitins are attached to the original one, a polyubiquitin chain forms, the proteasome recognizes the polyubiquitin chain, and the tagged protein is destroyed, releasing ubiquitin to kill again. It’s one way to put the regulation in your regulated proteolysis.

Poly-ubiquitin chain on Src

The canonical linkage for ubiquitin in this targeting to the proteasome is between a lysine on a substrate protein, and the terminal glycine on ubiquitin; followed by a chain of ubiquitins tagging onto the preceding ubiquitin’s lysine 48. The beautiful picture on the right is taken from the PDB’s Molecule of the Month from 2004, and shows “a string of ubiquitin molecules (colored pink and tan here, from PDB entry 1ubq) attached to old proteins, such as the src protein shown here (colored blue, from PDB entry 2src).”

There’s no room here to talk about all of the myriad other functions for ubiquitin that have been discovered over the years, but I want to highlight one in particular. In the mid 1990s2 it was discovered that ubiquitination of cell-surface molecules didn’t necessarily lead to destruction by the proteasome, but rather to internalization and in some cases destruction by the lysosome. What’s more, this receptor targeting mode of ubiquitin often involves polyubiquitin chains extending from ubiquitin’s lysine 63, not 48.3 So already there was precedent for flexibility in ubiquitin linkages.

A more recent observation came in the late 1990s and early 2000s, with the unexpected discovery4 that ubiquitin doesn’t even need lysines on its substrate protein; instead, ubiquitin can link up with the amino terminal residue of the protein and form a polyubiquitin chain there.

(I’m surprised that this doesn’t seem to be more widely known. I’ve talked to several people who have come to me, scratching their heads, because they’ve mutated all the lysines in their protein and still see it being ubiquitinated and destroyed — they were quite amazed when I pointed this phenomenon out to them.)

Wang et al, in the paper I’m highlighting here, take this one step further. They were looking at the way mK3 (a viral immune evasion molecule that causes class I major histocompatibility complexes to be rapidly degraded by the proteasome) causes degradation of MHC class I molecules. To make a long story short — hey, you should read the paper yourself! — they mutated all the lysines in the substrate protein and still saw polyubiquitination and degradation. But when they removed threonines and series — amino acids that are supposedly inaccessible to ubiquitin tagging — then the protein was no longer polyubiquitinated, and was no longer degraded. This seems to be a novel chemical process for ubiquitin, too, not involving the usual amide linkage but instead involving an ester bond.

What are the implications of this? On a purely technical basis, of course, it means that all the people who have decided ubiquitin can’t be important for their protein because there are no lysines available, have to go back and actually test directly. A more interesting question is whether non-lysine ubiquitination is a normal cellular process that the virus is just piggy-backing on, or whether this doesn’t occur normally and mK3 somehow forces the system in a new and bizarre direction. My guess is that this is in fact a normal cellular capability (there are hints in the paper and from previous literature that this may not be an abnormal event, but as yet they’re only hints). If so, the next question is whether this is a normal function that’s specific for the particular form of degradation here — that is, ER-associated degradation (ERAD), which is the process by which secreted or transmembrane proteins get destroyed during their maturation in the endoplasmic reticulum. ERAD is a fairly new and active field, and there’s a lot that’s not understood about it yet. If non-lysine ubiquitination is ERAD-specific, or especially if it’s actually a marker for ERAD, that would be really interesting and might offer a handle manipulating ERAD. Wang et al conclude:

It will be interesting to determine whether other ERAD pathways involving transmembrane protein substrates might also involve tail ubiquitination using non-K residues. Furthermore, the fact that mK3 has numerous viral (including MIR1) and cellular homologues makes it attractive to speculate that other ubiquitination-regulated processes use similar nonconventional methods of Ub conjugation.

  1. doi:10.1083/jcb.200611063[]
  2. I think the first papers were Hicke L and Riezman H (1996) Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Cell, 84, 277-287. and Strous GJ, Vankerkhof P, Govers R, Ciechanover A and Schwartz AL (1996) The ubiquitin conjugation system is required for ligand-induced endocytosis and degradation of the growth hormone receptor. EMBO J, 15, 3806-3812.[]
  3. Nice if now dated review: Dubiel W, Gordon C. Ubiquitin pathway: another link in the polyubiquitin chain? Curr Biol. 1999 Jul 29-Aug 12;9(15):R554-7.[]
  4. The EMBO Journal (1998) 17, 5964-5973. A novel site for ubiquitination: the N-terminal residue, and not internal lysines of MyoD, is essential for conjugation and degradation of the protein. Kristin Breitschopf, Eyal Bengal, Tamar Ziv, Arie Admon and Aaron Ciechanover. (doi:10.1093/emboj/17.20.5964); also see Ciechanover’s review in Trends Cell Biol. 2004 Mar;14(3):103-6 (doi:10.1016/j.tcb.2004.01.004) []
June 15th, 2007

Peptide splicing, proteasomes, and immunity

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[]