Mystery Rays from Outer Space

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

August 27th, 2008

DRiPs, immunoribosomes, and immunostress

"Drips" -- Susan S. Roberts
“Drips” (Susan S. Roberts. 2007)

Fred Goldberg has just published a paper that may have interesting implications for Jon Yewdell’s DRiPs theory.

Over a decade ago,1 Yewdell proposed that, first, protein translation sucks in terms of accuracy, so that many defective proteins are produced; second, that these defective proteins are rapidly degraded, which was why they hadn’t been identified before; and third, that these defective proteins are the dominant source of T cell (MHC class I) epitopes. It had long been known, of course, that MHC class I epitopes are produced as a byproduct of protein degradation; Yewdell’s suggestion was that because defective proteins are the most abundant class of degraded proteins, they are also the dominant source of MHC class I epitopes.

Lots of people were uncomfortable with the concept of sloppy translation, since I think it was generally believed (perhaps without much evidence) that translation is a very accurate process. That particular issue never bothered me very much, for reasons I’ve discussed earlier.  (And there seems to be support for this concept, too; see the paper I quoted from a couple of weeks ago, that concluded that some 20% of newly-synthesized proteins are defective.)

However, it did bother me very much that there were quite a few examples of MHC class I epitope production that was clearly not linked to degradation of newly synthesized, defective proteins. As we interpreted Yewdell at the time, he wanted to propose that the vast majority of MHC class I epitopes came from DRiPs (“Defective Ribosomal Products“, a terrific acronym that has probably helped contribute to the success of the theory). Either he has subsequently softened his stance, or we overinterpreted his proposal, or — most likely — both; I think I’m fairly comfortable, now, with the current model that a significant fraction of epitopes come from DRiPs, and a significant fraction do not. What exactly a “significant fraction” is, remains to be determined.

One prediction Yewdell has made from his DRiPs theory is the presence of “immunoribosomes”. He reasons that if sloppy translation by ribosomes is a major source of MHC class I epitopes, then in the presence of inflammation (i.e. when there is the potential for infection, when you’d want to increase T cell surveillance) you would expect translation to become even sloppier, generating even more epitopes. (This, like the “immunoribosome” name, is an argument based on proteasomes and immunoproteasomes, which conceptually do something very similar.) This, I think, has been a much less successful prediction.

Fred Goldberg’s paper offers a bit of weak support to the concept,2 though it is far from vindication. The paper is

Medicherla, B., Goldberg, A.L. (2008). Heat shock and oxygen radicals stimulate ubiquitin-dependent degradation mainly of newly synthesized proteins. The Journal of Cell Biology, 182(4), 663-673. DOI: 10.1083/jcb.200803022
 

Proteasome
Proteasome

Because this work is done in yeast, which of course lack anything like MHC-based immunity, there’s no reason to expect immunoribosomes here. What Medicherla and Goldberg did find, though, is that cellular stress leads to differential degradation of proteins, with newly-synthesized proteins being particularly targeted for destruction. (There are a lot of other interesting things about this paper, including the evidence that these rapidly-degraded proteins require ubiquitination, which has been a little controversial.  But I’m just going to talk about the one aspect of the work here.)  

“Cellular stress” in this case was due to things like heat shock and toxins like paraquat, but mammalian cells undergo cell stress when they’re infected with viruses, among other things, so this might be something that could be adapted to immune responses. Their suggestion is that “many cytosolic proteins proceed through a prolonged “fragile period” during which they are sensitive to degradation induced by superoxide radicals or increased temperatures” and that this fragile period is because many proteins need to interact with binding partners or whatever before they become resistant to misfolding and degradation.

Because it is unlikely that the folding of many proteins would require 30-60 min, this “fragile period” presumably represents the time necessary for postsynthetic modifications, proper multimer formation, and localization, which together contribute to thermal resistance.

This fragile period apparently lasts an hour or so, longer than Yewdell had described for his DRiPs,3 and Goldberg seems to hint that such fragile, incompletely assembled proteins may be a more plausible source of rapidly-degraded proteins than DRiPs.

This fragile population, in Medicherla and Goldberg’s hands, represents a rather small fraction:

 In yeast growing at 20 or 30°C, such rapidly degraded components comprise 3-4% of newly synthesized proteins, but the short-lived fraction reached 13% after shift to 38 or 42°C, and 10-13% of the proteins synthesized in the presence of cadmium or paraquat at 30°C. These treatments accelerated the degradation of 10% of proteins that would otherwise be long lived so that they behaved like short-lived components.

(My emphasis.) Nevertheless, it represents a 4-fold increase in degradation, and potentially (if these findings can be extended to cells with an MHC class I system) a 4-fold increase in antigen presentation. What’s more, if the cellular stress was triggered by a viral infection, the targeted proteins would probably be disproportionately biased toward viral over cellular proteins. All in all, this sounds more like the concept Yewdell was pushing toward with his immunoribosomes: Shoud we call it “Immunostress”?


  1. Yewdell, J. W., Aton, L. C., and Benink, J. R. (1996). Defective ribosomal products (DRiPs): A major source of antigenic peptides for MHC class I molecules? J. Immunol. 157, 1823-1826[]
  2. Probably much to Fred’s dismay, since I think he is at best unenthusiastic about the whole DRiPs thing[]
  3. though the half-life of DRiPs as defined in this model seems to have increased over the years[]
May 28th, 2008

A little learning

Kopp et al 1995, proteasomeOne of the problems with genomics research is that the people who interpret it may not know much about the genes they identify. For example:

… single nucleotide polymorphisms (SNPs) in two genes critical for T-cell function are associated with susceptibility to MDD:1 PSMB4 (proteasome 4 subunit), important for antigen processing …2

Kopp et al 1995, HN3/PSMB4
PSMB4 location in proteasome3

OK, first of all, the proteasome is important in antigen processing, but antigen processing is probably about the least important of its myriad tasks. Suggesting that the function of the proteasome is antigen processing is like claiming the the function of my car is to hold coffee, because it happens to have cup-holders.4

Second, PSMB4 per se has never been implicated in antigen processing.5 Not only is it not one of the interferon-inducible trio of proteasome subunits that have been implicated in antigen processing, PSMB4 is not even catalytic.6

This looks like someone had a vague memory of seeing something in a textbook years ago, and didn’t try to look at the literature at all. Or who wanted to squeeze data into a preconceived theory.


  1. MDD: Major depressive disorder[]
  2. Wong ML, Dong C, Maestre-Mesa J, Licinio J.  Polymorphisms in inflammation-related genes are associated with susceptibility to major depression and antidepressant response. Mol Psychiatry. 2008 May 27. [Epub ahead of print] doi:10.1038/mp.2008.59 []
  3. Kopp F, Kristensen P, Hendil KB, Johnsen A, Sobek A, Dahlmann B. The human proteasome subunit HsN3 is located in the inner rings of the complex dimer. J Mol Biol. 1995 Apr 28;248:264-72 doi:10.1016/S0022-2836(95)80049-2 []
  4. Also, in my case, about 2000 little plastic dinosaurs, tucked into every cranny and hidden under the booster seats in the back.  I’m not sure what their function is either[]
  5. As far as I know; which is reasonably far[]
  6. Though it may be involved in proteolysis anyway.  But the point remains — not shown to have a role in antigen processing[]
February 17th, 2008

Classic paper: Presentation from ER proteins

Endoplasmic reticulum I have a very sporadic and idiosyncratic series in which I talk about “classic papers”, and in my idiosyncratic series Vic Engelhard’s paper on tyrosinase processing counts as a classic paper. It was one of the early indications that proteins in the ER must be degraded in the cytosol, and as such it’s one of a number of ways that antigen presentation has helped fundamental understanding of cell biology; but I think it hasn’t received as much recognition as it could have.

But perhaps I should begin at the beginning.

Proteolysis is a normal part of cell function. Proteins that are damaged, misfolded, or mis-translated, as well as proteins that have simply reached the end of their useful life, are degraded and converted to amino acids that can be recycled into new proteins. In the early- to mid-1990s, there were three general systems that were known to degrade proteins, depending on which subcellular compartment they were in:

  • In the cytosol and nucleus, proteins are predominately degraded by proteasomes.
  • Proteins taken up from the exterior of the cell can be degraded in acidic lysosomes
  • Mitochondrial proteins are degraded by a number of proteases within the mitochondrion1

Endoplasmic reticulum (pancreas cell)That leaves an obvious gap. What happens to proteins that are in the endoplasmic reticulum (ER)? This is particularly relevant because the ER is a ferociously active site of protein synthesis, folding, and assembly; when any of those steps goes awry, the protein is supposed to be degraded, a process known as “quality control”. It was clear in the 1980s that proteins that failed quality control in the ER were degraded; in human cells, a well-known example was the cystic fibrosis transmembrane conductance regulator (CFTR), which folds inefficiently and is rapidly degraded2. But it was not clear where the degradation happened (in the ER? The cytosol? Somewhere else?), and what proteases were responsible.

At first it was believed that “what happens in the ER stays in the ER” — ER proteins were degraded in the ER, by ill-defined proteases in that compartment. But I don’t think there was much enthusiasm for that belief, and basically, the field was a mess, as you can see from this introductory paragraph from the time:3

Other membrane proteins are also known to be degraded at the ER, but the process is poorly understood, and the responsible enzymes have not been identified. For example, some of these proteolytic events are ATP dependent, but some are not; some occur within the lumen while others take place on the cytoplasmic side; some exhibit inhibitor sensitivities characteristic of serine proteases, whereas others do not.

This was relevant to me as an immunologist, because viral glycoproteins (which, of course, are synthesized in the ER) are popular targets for cytotoxic T lymphocyte (CTL) recognition (a quick review of MHC class I antigen presentation is here). We knew in the early 1990s that most if not all CTL targets — even those derived from ER proteins — were generated in the cytosol. As I wrote in a 1996 review:4

Proteins targeted into the ER by signal sequences can also be presented on MHC I molecules. Since these molecules are cotranslationally transported into the endoplasmic reticulum, they might be expected to bypass hydrolysis in the cytosol. However, where analyzed, the presentation of most of these antigens is dependent on the TAP-transporter and on proteasome activity, and therefore the presented peptides are probably being generated in the cytosol.

The three obvious possible explanations were that either the putative glycoprotein never made it into the ER and was degraded as a mistargeted protein (Jon Yewdell would call that a “DRiP”; I can’t remember exactly when I heard him propose that first, but it was around that time); that the glycoprotein went in to the ER, got degraded there, and the peptides were first transfered to the cytosol; or that the glycoprotein was transfered from the ER to the cytosol and degraded there.

The simplest explanation, at the time, seemed to be the first one –  proteins never made it in to the ER, and were degraded in the cytosol. However, it wasn’t an explanation that we liked very much, for various reasons. Vic Engelhard’s paper (Remember Engelhard’s paper? This here’s a post about Engelhard’s paper) cleared that question up, at least for one epitope.

Skipper, J. C., Hendrickson, R. C., Gulden, P. H., Brichard, V., Van Pel, A., Chen, Y., Shabanowitz, J., Wolfel, T., Slingluff, C. L., Jr., Boon, T., Hunt, D. F., and Engelhard, V. H. (1996). An HLA-A2-restricted tyrosinase antigen on melanoma cells results from posttranslational modification and suggests a novel pathway for processing of membrane proteins. J. Exp. Med. 183, 527-534.

His finding is simple enough to describe, although it relied on a technically very difficult mass spec analysis:

  1. A peptide presented on MHC class I was derived from an ER protein (which they knew from its sequence);
  2. the protein had actually gone into the ER, because it had been N-glycosylated, which only happens in the ER;
  3. yet the peptide itself was probably generated in the cytosol, because enzymes that modified it are mainly found in the cytosol.

Mumps virus protein (turquoise) in endoplasmic reticulumThe most surprising and exciting part was the second point: Clear evidence that the protein had actually gone into the ER before the peptide was generated. 5 This wasn’t by any means definitive proof that ER proteins are degraded in the cytosol (a follow-up paper in 19986 took it a bit further) but it certainly was suggestive.

If Vic’s paper had come out a year or two earlier, it would probably have made much more of a splash than it did, but in February of 1996 it was only a nose ahead of several more focused papers. The field had started to clear up in the mid-1990s, with some observations in yeast in 1993 and 1994 7 and around 1995 moving into mammalian cells with (among others) the Jensen et al. paper I quoted above. 8  And later in 1996, the iceberg tipped over altogether, with a whole bunch of almost simultaneous papers that showed quite clearly that ER degradation wasn’t done by ER proteases at all, but was in fact performed by proteasomes. 9 All the papers demonstrated that there’s an export step before degradation: ER proteins that fail quality control are shunted out into the cytosol, where the proteasomes can grab onto them and chop them up. (The export step is still not all that well understood in molecular detail, though in 2007 it started to open up some, I think.)

Though Engelhard’s 1996 paper is reasonably widely cited (256 citations as I write this) it clearly didn’t have the impact on cell biology in general that it did on me, probably because it came out around the same time as a bunch of more specific papers. This blogpost is an attempt to give a bit more retroactive credit to a very nice example of logical reasoning from indirect evidence.


  1. I believe that some, though not all, of the mitochondrial proteases were identified in the late 1980s/early 1990s, and that the broad outline of mitochondrial proteolysis was understood in the early 1980s (Desautels, M. and Goldberg, A. L. (1982) Liver mitochondria contain an ATP-dependent, vanadate-sensitive pathway for the degradation of proteins. Proc Natl Acad Sci USA 79 , pp. 1869-1873.). I don’t know all that much about mitochondrial proteolysis, though, so if someone wants to correct me, please do so.[]
  2. For example, Ward C, Kopito R (October 14, 1994) Intracellular turnover of cystic fibrosis transmembrane conductance regulator. Inefficient processing and rapid degradation of wild-type and mutant proteins. J. Biol. Chem. 269.:25710-25718[]
  3. Taken from Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, et al. (October 6, 1995) Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83:129-35. with references removed[]
  4. York, I. A., and Rock, K. L. (1996). Antigen processing and presentation by the class I major histocompatibility complex. Annual review of immunology Annu Rev Immunol 14, 369-396.[]
  5. Technical explanation: The mass spec analysis showed that the asparagine that is encoded in the DNA was actually an aspartic acid in the presented peptide; deglycosylating enzymes that were believed to only be present in the cytosol remove carbohydrates from Asn to generate Asp.[]
  6. Mosse, C. A., Meadows, L., Luckey, C. J., Kittlesen, D. J., Huczko, E. L., Slingluff, C. L., Shabanowitz, J., Hunt, D. F., and Engelhard, V. H. (1998). The class I antigen-processing pathway for the membrane protein tyrosinase involves translation in the endoplasmic reticulum and processing in the cytosol. J Exp Med 187, 37-48.[]
  7. (Sommer T, Jentsch S (September 9, 1993) A protein translocation defect linked to ubiquitin conjugation at the endoplasmic reticulum. Nature 365.:176-9.
    and
    Kölling R, Hollenberg CP (July 15, 1994) The ABC-transporter Ste6 accumulates in the plasma membrane in a ubiquitinated form in endocytosis mutants. EMBO J 13.:3261-71.[]
  8. Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, et al. (October 6, 1995) Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83:129-35. []
  9. I may be missing some:
    Hampton RY, Gardner RG, Rine J (December 1996) Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein. Mol Biol Cell 7.:2029-44.
    Werner ED, Brodsky JL, McCracken AA (November 26, 1996) Proteasome-dependent endoplasmic reticulum-associated protein degradation: an unconventional route to a familiar fate. Proc Natl Acad Sci U S A 93.:13797-801.
    Hiller MM, Finger A, Schweiger M, Wolf DH (September 20, 1996) ER degradation of a misfolded luminal protein by the cytosolic ubiquitin-proteasome pathway. Science 273.:1725-8.
    Qu D, Teckman JH, Omura S, Perlmutter DH (September 13, 1996) Degradation of a mutant secretory protein, alpha1-antitrypsin Z, in the endoplasmic reticulum requires proteasome activity. J Biol Chem 271.:22791-5.
    Wiertz EJ, Jones TR, Sun L, Bogyo M, Geuze HJ, et al. (March 8, 1996) The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell 84.:769-79.[]
January 23rd, 2008

Proteasome subunit-dependent signaling in T cells?

Mammalian proteasomeWe all know, now, that the proteasome is the most important factor in generating peptides for MHC class I. (See the antigen processing overview here if you want a quick review.) There are lots of lines of evidence pointing to this conclusion, but one of the early clues was the presence of interferon-inducible proteasome subunits in the MHC region of the genome.

It turns out (to make a long story short) that proteasomes have at least two different options for their catalytic subunits, and one of these options uses three interferon-inducible subunits. Two of the three are MHC-encoded (LMP2 and LMP7); the third is encoded outside the MHC (MECL1). The twin red flags of genomic organization and response to inflammation suggested that they might have a role in MHC class I antigen presentation; biochemical analysis suggested that the proteasome that incorporated these subunits turned out peptides that are better suited for MHC class I antigen presentation.1 (MECL1 identification trailed the other two by a few years2 and by the time it was shown to be interferon-regulated, some of the excitement was over.) The next step was to make knockout mice lacking each of the subunits.

Mammalian proteasomeThe LMP2 and LMP7 knockout mice were duly made3 and sure enough both showed (rather modest) phenotypic changes that could be ascribed to changes in MHC class I antigen presentation. MECL1 knockout mice weren’t made until quite recently (as I say, the excitement had sort of passed on to other things by then); and these knockout mice, too, showed T cell anomalies that could be ascribed to antigen presentation changes.4

Blogging on Peer-Reviewed ResearchA simple straightforward story (in hindsight), right? 5 Not so fast. Double knockout mice, lacking both MECL1 and LMP7, are dysfunctional in a completely unexpected way: the double knockout T cells (but not either of the single knockout LMP7 -/- or MECL1 -/- cells) are hyperproliferative, overresponding to some signals. 6 What’s more, this applies to not only CD8 T cells (which one imagines might be affected by MHC class I antigen oddities) but also to CD4 T cells, which shouldn’t much care about MHC class I antigen presentation.

An even more recent paper, still in press,7 analyzes this further with chimeric mice. It turns out that even when the T cells mature in the same environment (in other words, when MHC class I antigen presentation is held constant) the MECL1-knockout T cells are still different:

On the other hand, we provide evidence that the effects of MECL-1 on CD4 or CD8 T cell expansion are entirely unrelated to its role in antigen processing. Our findings suggest that MECL-1 influences the homeostatic regulatory processes that maintain the relative proportions of both T cell subsets, through a T cell-intrinsic mechanism independent from thymic or lymphoid interaction partners.

It seems likely that MECL1 somehow affects T cell regulation through affecting some signaling pathways.  This isn’t completely out of the blue — the proteasome has effects on signaling in several systems (in fact the proteasome affects just about every aspect of cell biology, one way or another). And it’s somewhat reassuring that so far there’s no indication that LMP2 or LMP7 might have caused their effects through anything other than antigen presentation. Still, it’s a warning signal not to take the easy explanation every time.

As a side note, it also makes me wonder about the thymus-specific subunit that was recently identified. When that came out I found it hard to imagine that the very large effects associated with the knockout of this subunit could be explained by antigen presentation. Now I wonder even more, because signaling is something that could easily explain the size of the effects they saw, though I’m not quite sure if it could explain the nature of the effects.


  1. There are a bunch of references for this, but I’m going to be lazy and just include a couple that I don’t have to think about: Gaczynska, M., Rock, K. L., and Goldberg, A. L. (1993). Gamma-interferon and expression of MHC genes regulate peptide hydrolysis by proteasomes. Nature 365, 264-267.
    and
    Gaczynska, M., Rock, K. L., Spies, T., and Goldberg, A. L. (1994). Peptidase activities of proteasomes are differentially regulated by the major histocompatibility complex-encoded genes for LMP2 and LMP7. Proc Natl Acad Sci U S A 91, 9213-9217.[]
  2. Groettrup, M., Kraft, R., Kostka, S., Standera, S., Stohwasser, R., and Kloetzel, P. M. (1996). A third interferon-gamma-induced subunit exchange in the 20S proteasome. Eur J Immunol 26, 863-869.
    and
    Hisamatsu, H., Shimbara, N., Saito, Y., Kristensen, P., Hendil, K. B., Fujiwara, T., Takahashi, E., Tanahashi, N., Tamura, T., Ichihara, A., and Tanaka, K. (1996). Newly identified pair of proteasomal subunits regulated reciprocally by interferon gamma. J Exp Med 183, 1807-1816.[]
  3. Van Kaer, L., Ashton-Rickardt, P. G., Eichelberger, M., Gaczynska, M., Nagashima, K., Rock, K. L., Goldberg, A. L., Doherty, P. C., and Tonegawa, S. (1994). Altered peptidase and viral-specific T cell response in LMP2 mutant mice. Immunity 1, 533-541.
    and
    Fehling, H. J., Swat, W., Laplace, C., Kuhn, R., Rajewsky, K., Muller, U., and von Boehmer, H. (1994). MHC class I expression in mice lacking the proteasome subunit LMP-7. Science 265, 1234-1237.[]
  4. Basler, M., Moebius, J., Elenich, L., Groettrup, M., and Monaco, J. J. (2006). An altered T cell repertoire in MECL-1-deficient mice. J Immunol 176, 6665-6672.[]
  5. Especially because I am carefully avoiding mention of some of the blind alleys and red herrings that were puzzling at the time.[]
  6. Caudill, C. M., Jayarapu, K., Elenich, L., Monaco, J. J., Colbert, R. A., and Griffin, T. A. (2006). T cells lacking immunoproteasome subunits MECL-1 and LMP7 hyperproliferate in response to polyclonal mitogens. J Immunol 176, 4075-4082.[]
  7. Zaiss, D.M.W., de Graaf, N., & Sijts, A.J.A.M., 2007. The proteasome immunosubunit MECL-1 is a T cell intrinsic factor influencing homeostatic expansion. Infect Immun. doi:10.1128/IAI.01134-07 []
November 9th, 2007

Ephemera

Ephemeroptera (Mayfly)
Ephemeroptera

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 .[]
October 29th, 2007

RNA, protein, and information

ENCODE logo Not long ago there was some keruffle over the ENCODE data,1 and the unrelated but almost simultaneous Cell paper,2 that demonstrated widespread transcription even from apparently-inactive genes — for example, this discussion and this one at Ars Technica’s Nobel Intent , and this one at Sandwalk. The observations were considered surprising because RNA was (and is) usually considered to be fairly tightly regulated. The ENCODE data, in particular, were used as arguments pro and con “junk” RNA — non-functional transcription.

The presence of non-functional RNA, though, didn’t strike me as very surprising at all, and part of the reason for that was that I had been primed to think about efficiency in cellular processes by Jon Yewdell’s “DRiPs” hypothesis.3

Briefly (I want to talk about DRiPs in detail some other time) Yewdell suggests that peptides that are presented on MHC class I to cytotoxic T lymphocytes are usually derived, not from full-length, functional proteins, but from “defective ribosomal products” — proteins that began translation and got screwed up partway through, or that completed translation and failed to fold properly. Proteins, in other words, that were defective from the get-go, that never had a chance to contribute to the whole happy economy of the cell. This contrasts to the traditional view, that antigenic peptides are derived from proteins during their normal turnover, often over a period of many hours.

Pollock Untitled (Green Silver)
Pollock’s drips: “Untitled (Green Silver)”

Yewdell argued that in fact a large percentage of translation (he’s offered various percentages, but let’s say 30% of translation) ends up in this defective pool, and because it’s defective it’s destroyed very rapidly by the proteasome — again, he’s offered various numbers, but let’s say for the sake of argument that it’s destroyed within a handful of minutes.

I don’t mind saying that I was extremely skeptical when I read his initial paper, and I am still quite skeptical about the overall contribution; but over the years I have (reluctantly) come a long way to accepting the general principle. But — unlike many of the people who disagreed with the DRiP hypothesis — I didn’t find the principle of DRiPs per se implausible.

In fact, it was one of those things that I had never thought of, but that made immediate sense to me as soon as I read the idea. I think of it as an information theory thing: Preventing errors in translation must take a certain amount of energy; at some point the incremental energy needed to reduce the error rate from N to N-1 would be greater than the amount of energy needed to degrade a defective product. And as soon as you consider it as that equation, it becomes a slider, and the set-point could be almost anywhere. It’s quite plausible (to me, anyway) that the amount of energy used in error prevention is relatively high, whereas the energy loss in protein degradation is relatively low — and so it’s cheaper, energetically, to simply make error-riddled protein, and let the proteasome sort it out after the fact.

(I’m simplifying all the arguments here, pro and con. I’ll probably take them up in bits and pieces later on.)

Anyway, exactly the same reasoning applies to transcription. The amount of energy that it would take to clamp down and make absolutely perfect identification of proper transcriptional start sites, must at some point be greater than the amount of energy involved in destruction of aberrant RNA. So this is why I thought it was quite predictable that there would be widespread, low-level, transcription of non-functional RNA that would then run into the next level of information processing.

Blogging on Peer-Reviewed ResearchThe reason I thought about this today, months after the fuss has pretty much died down, is a paper from Nilabh Shastri’s group4 that demonstrates another instance of what, I suspect, is another example of aberrance that’s tolerated by cells. (There’s also a commentary on the paper,5 by Yewdell.) Today’s post was in fact supposed to be entirely about that paper, but what with all the time I’ve spent blathering about the background, I’ll finish up with a new post later this week. In any case it’s time for me to go read “Green Eggs and Ham” to my kids.

(I’m trying out including the BPR3 icon here. I’m not entirely convinced by the BPR3 rationale, but I’m willing to see what happens for a while, anyway.)


  1. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 2007 447, 799-816. []
  2. Guenther, M. G., Levine, S. S., Boyer, L. A., Jaenisch, R., and Young, R. A. (2007). A chromatin landmark and transcription initiation at most promoters in human cells. Cell 130, 77-88.[]
  3. Yewdell, J. W., Aton, L. C., and Benink, J. R. (1996). Defective ribosomal products (DRiPs): A major source of antigenic peptides for MHC class I molecules? J. Immunol. 157, 1823-1826.[]
  4. Maness, N. J., Valentine, L. E., May, G. E., Reed, J., Piaskowski, S. M., Soma, T., Furlott, J., Rakasz, E. G., Friedrich, T. C., Price, D. A., Gostick, E., Hughes, A. L., Sidney, J., Sette, A., Wilson, N. A., and Watkins, D. I. (2007). AIDS virus specific CD8+ T lymphocytes against an immunodominant cryptic epitope select for viral escape. J Exp Med 204:2505-2512 []
  5. Yewdell, J. W., and Hickman, H. D. (2007). New lane in the information highway: alternative reading frame peptides elicit T cells with potent antiretrovirus activity. J Exp Med 204:2501-2504 []
September 21st, 2007

An embarassment of riches: The new E1, yet again

E1 conformational changesYeah, so this is getting a little repetitious. The new E1 I noted back in June, and then again in August, has been reported one more time.

Whining aside,1 the new paper2 is a nice addition to the pack, because it offers a quick look at a knockout mouse, and a new binding partner.

The significance of a new E1, if you don’t want to look at my previous posts, is that E1s are the upstream-most enzyme involved in the ubiquitin cascade,3 which is critical to all kinds of cellular function starting from (but not limited to) regulated proteolysis . It had been believed that there was only a single E1 enzyme in most mammalian genomes, although the downstream enzyme families E2 and E3 are much more variable (a couple dozen genes, and close to a thousand, respectively). Jin et al4 and Pelzer et al5 both identified a new E1 (Jin et al called it UBA6; Pelzer et al stuck to an established name and called it UBE1L2) and showed that it is capable of charging ubiquitin but not any of several other ubiquitin-like substrates. Jin et al took it a little further, showing, for example, that the E1 did have a specific E2.

Chiu et al call the gene E1-L2, which is near as dammit to the authentic name but just different enough that I had to compare sequences to make sure it was the same.6 As well as ubiquitin, they do find a different substrate for E1-L2: the ubiquitin-like molecule FAT10, which had been specifically ruled out by the other groups (Chiu et al explain it as a techinical problem with the system,7 which I don’t know well enough to comment on).

The knockout mouse is embryo-lethal, which was already known though not, I think, published (thanks to Jianping Jin for sending me some unpublished info a while back). This is in contrast to FAT10 knockout mice, which are viable and not grossly abnormal,8 so E1-L2/UBE1L2/UBA6 does something else — consistent with a role for this thing in authentic ubiquitination pathways.

The connection with FAT10 makes this thing even more interesting to me, because FAT10 is encoded in the major histocompatibility complex — a region that contains many genes important in immunity — and FAT10 has been linked to some aspects of the immune response.


  1. But before I stop whining, doesn’t Molecular Cell use DOIs? I can’t find one associated with the paper. Get with the new millennium, guys![]
  2. E1-L2 Activates Both Ubiquitin and FAT10. Yu-Hsin Chiu, Qinmiao Sun, and Zhijian J. Chen. Molecular Cell, Vol 27, 1014-1023, 21 September 2007 []
  3. (The figure at top, taken from VanDemark, A. P., and Hill, C. P. (2005). E1 on the move. Mol Cell 17, 474-475. , shows a model of E1′s mechanism of action.[]
  4. Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging. Jianping Jin, Xue Li, Steven P. Gygi & J. Wade Harper. Nature 447, 1135-1138 (28 June 2007) []
  5. UBE1L2, a Novel E1 Enzyme Specific for Ubiquitin. Christiane Pelzer, Ingrid Kassner, Konstantin Matentzoglu, Rajesh K. Singh, Hans-Peter Wollscheid, Martin Scheffner, Gunter Schmidtke, and Marcus Groettrup. J. Biol. Chem., Vol. 282, Issue 32, 23010-23014, August 10, 2007 []
  6. They make it clear in their discussion, so I should have read the whole paper before grumbling.[]
  7. “We believe it is important to remove the GST tag or replace it with a smaller tag such as His6 in order to observe the activation of FAT10 by E1-L2.”[]
  8. FAT10/diubiquitin-like protein-deficient mice exhibit minimal phenotypic differences. Canaan, A., Yu, X., Booth, C. J., Lian, J., Lazar, I., Gamfi, S. L., Castille, K., Kohya, N., Nakayama, Y., Liu, Y. C., Eynon, E., Flavell, R., and Weissman, S. M. (2006). Mol Cell Biol 26, 5180-5189. []
August 17th, 2007

Pat poove

 

C'est ne pas un proteosomeThey’ve done it again! The online table of contents for today’s issue of Science has an article on a ubiquitin ligase 1 and the editors have added this helpful blurb:

In developing worms, the pruning of excess synapses requires proteosome-mediated protein degradation and is selectively prevented by a neural adhesion molecule.

No, no, no, no, no! There’s no such word as “proteosome“! It’s “proteasome“. It’s a horribly common mistake (PubMed has 7280 cites for the misspelled version, and 11813 for the correct spelling — a ratio that’s actually nearly ten times worse than the generic web’s)  2 but it’s a mistake nonetheless.

This is far from the first time Science has done this3 and I wrote to them the last time they did it, which was in June for the teaser “Selective Proteosomes”. I wrote to them again today, but I’m not optimistic; the misspelled version from June is still there.

Some editor at Science needs a sharp smack upside the head.

 


  1. Spatial Regulation of an E3 Ubiquitin Ligase Directs Selective Synapse Elimination. Mei Ding, Dan Chao, George Wang, and Kang Shen. Science 17 August 2007: 947-951. []
  2. Google claims “about 240,000″ hits for -o- and 2,990,000 for -a-, a 12:1 ratio compared to the presumably more technical literature’s 1.6:1 ratio.[]
  3. 82 hits in Pubmed for “Proteosome AND science[Journal]“![]
August 3rd, 2007

Quick E1 update

A few weeks ago, when I posted about the identification of a new E1 enzyme,1 suicyte drew my attention to another paper on the same enzyme, in J Biol Chem, from Groettrup’s group. At the time that paper was online but not officially published; as of now it’s officially out.2Just as a couple of notes:3

  • suicyte implied that the Groettrup group beat Jin et al to publication — in fact the Jin et al paper was accepted first (Received 27 February 2007; Accepted 1 May 2007; Pelzer et al was Received June 4, 2007, accepted June 18, 2007).
  • Suicyte also commented that “what I like about the JBC paper is that they keep the original name UBE1L2 for the gene instead of inventing a new one”. The author of the Nature paper (where they call it Uba6) explained to me that they had already talked about the gene at conferences before the name “UBE1L2″ was assigned, and so they decided to keep the name they had already talked about and (I believe) published in conference proceedings. The whole priority thing gets kind of messy under these circumstances.


  1. Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging. Jianping Jin, Xue Li, Steven P. Gygi & J. Wade Harper. Nature 447, 1135-1138 (28 June 2007) []
  2. UBE1L2, a Novel E1 Enzyme Specific for Ubiquitin. Christiane Pelzer, Ingrid Kassner, Konstantin Matentzoglu, Rajesh K. Singh, Hans-Peter Wollscheid, Martin Scheffner, Gunter Schmidtke, and Marcus Groettrup. J. Biol. Chem., Vol. 282, Issue 32, 23010-23014, August 10, 2007[]
  3. Minor points, perhaps, but since they’re already on the blog it’s worth clarification[]
June 29th, 2007

Yet another new trick: A new E1

The new tricks for the ubiquitin/proteasome system are coming thick and fast these days. Hot on the heels of the discovery of non-lysine ubiquitination and thymus-specific proteasome subunits, today’s issue of Nature reports that there’s a second E1! That may not be as startling to everyone as it is to me, but it’s yet another example of well-established observations being overturned.

Ubiquitin is attached to its substrate proteins through a multi-enzyme cascade. First, ubiquitin is “activated” by a ubiquitin-activating enzyme; then the ubiquitin is transferred to a ubiquitin-conjugating enzyme which, in combination with a ubiquitin ligase, transfers the ubiquitin to a specific substrate. The ubiquitinated substrate then does whatever its supposed to do when it’s ubiquitinated — gets degraded by the proteasome, perhaps, or trundles off to a new spot in the cell.

The ubiquitin ligase (an “E3″ enzyme) is mainly responsible for the specificity of the reaction; there are thousands of ubiquitin ligases in the human genome, and probably each interacts with a small number of specific substrates.

Ubiquitin conjugating enzymes, the second in the chain (“E2″ enzymes) are less abundant and less specific; there are a couple dozen of them. Each UBC interacts with a number of ubiquitin ligases, though the relationships here are not well understood in general.

The first link in the chain is the ubiquitin activating enzyme, the E1 (the gene in humans is “Ube1″). There’s only one of them — say all the reviews.1 If you knock out the single E1, as in some temperature-sensitive cell lines, then the cells die in a hurry.

The new discovery is described in:

Nature 447, 1135-1138 (28 June 2007) (doi:10.1038/nature05902)

Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging

Jianping Jin, Xue Li, Steven P. Gygi & J. Wade Harper

RelationshipsThis is a gene called “Uba6″, it has the three domains that Ube1 does, it’s about 40% identical to Ube1, and it’s found in vertebrates and sea urchins but not invertebrates or fungi (both of which do, of course, have their own versons of Ube1). (Figure on the right is from the paper, showing the relationships between some E1-related genes of various species; note that the Uba6 genes are most closely related to Ube1 genes even from different species).

Uba6 is found throughout the body (so unlike the mouse thestis-specific version of E1, it’s not tissue-specific), though at much lower levels than E1 Classic. It acts like an authentic E1, in that it charges ubiquitin, but (and this is a cool and critical point) it seems to be specific for different ubiquitin-conjugating enzymes; two of the three UBCs they tested were strictly dependent on Ube1, while the third was strictly dependent on Uba6. It’s not merely a redundant, backup E1.

What are the implications? Once again there’s a technical point: The authors point out that “it is conceivable that certain pathways that were previously thought to be independent of ubiquitin on this basis may nevertheless require ubiquitin by means of a Uba6-dependent pathway.” But the bigger question is why vertebrates need two E1s, where invertebrates get along fine with just one.2 The authors propose (a little feebly, I think) that this may allow differential regulation: “One possibility is that Uba6 and Ube1 are differentially regulated by upstream signalling pathways to enhance flux through a specific conjugating pathway under particular circumstances.” That’s a pretty vague suggestion that covers a host of possibilities.

Still, being able to tease apart different pathways should be a very useful way of tracking down their function, and also may help actually understand what some of the different UBCs are doing. Might this be a way to organize some of the myriad ubiquitin functions? Could Uba6 lead to a different set of ubiquitin reactions — say, trafficking instead of degradation?


  1. Mice, but not most other species (including humans) express a second E1 in their testes. Also, there are a couple of transcriptional variants of human E1, but there doesn’t seem to be much functional significance of that, as far as I know.[]
  2. Unless, of course, there’s another E1 hiding in an invertebrate genome that we haven’t found yet[]