Last Wednesday, two new papers (1,2) were published that describe the function of Rpn13 as a new proteasomal ubiquitin receptor. Since I happen to be a coauthor on one of the papers, I thought it might be a good idea for a blog entry. Actually, it was Ian’s suggestion, but it is not his fault if the text is boring. In any case, it is going to be too long – a complex as interesting as the proteasome deserves more than a few lines.
If there is anything the non-expert knows about ubiquitin, it is the fact that ubiquitin becomes attached to other proteins and earmarks them for destruction by the proteasome. In order to do this job, the proteasome has to recognize if a protein carries a ubiquitin degradation signal. The complete 26S proteasome consists of at least 32 different stoichiometric subunits and some of them are thought to act as ubiquitin recognition components, or ‘ubiquitin receptors’. There is no shortage of contenders for this job, though. At least three different ubiquitin recognition systems had been described previously:
Rpn10 or S5a, depending on if you are a yeast or mammalian person. Or PSMD4, if you are a nomenclature person. The ubiquitin recognition properties of this 19S subunit, positioned at the interface between base and lid of the 19S cap, have been known for more than 10 years. The ubiquitin binding is done by a short helical motif, which was later shown to occur in lots of other ubiquitin binding proteins and has been called ubiquitin interaction motif or UIM.
Rpn1 or S2 (PSMD2) is another subunit of the 19S base complex involved in the recognition of ubiquitinated targets. This recognition, however, is not direct. Instead of recognizing ubiquitin itself, Rpn1 recognizes ubiquitin-like domains that are present in a class of ‘adaptor proteins’. These adaptors contain a ubiquitin-like domain at their N-terminus, while the C-terminus carries a UBA domain, another widespread class of ubiquitin recognizing modules. It is this UBA domain (in proteins like Rad23, Dsk1, Ddi1) that is responsible for the actual recognition of the degradation target.
Rpt5 or S8 (PSMC3) is one of the six AAA ATPases that form the base complex of the 19S proteasome cap. All six ATPases are quite similar to each other, but Rpt5 has ben singled out as ‘the’ receptor for polyubiquitin chains, which constitute the degradation signal. I don’t have an opinion on this myself, but I have noticed that most people in the area don’t believe in Rpt5 being a real ubiquitin receptor under physiological conditions.
This was the situation before the Rpn13 paper appeared. I will summarize the contents of the paper further down, but you can also read the associated news & views article to get a better idea. Let me briefly recount the story behind this paper and my (admittedly very minor) contribution. In 2000, Rati Verma in Ray Deshaies group performed a proteomics screen in yeast, looking for new proteasome-associated proteins. One of the proteins they focused on was Rpn13, which they described to be a new stoichiometic proteasome subunit. When I heard Rati talk about their results on one of the early Zomes conferences, we began looking for a human version of Rpn13. To our surprise, we came up with a protein known as ADRM1, described as a cell adhesion protein. Not what you would expect for a proteasome subunit. Nevertheless, I was sure that we were right, and expected the cell adhesion phenotype to be an epiphenomenon, if not an artifact.
Some years later, in 2004, Grzegorz Zapart in the lab of Ivan Dikic in Frankfurt performed a Y2H screen for novel ubiquitin interactors. Among the long list of putative interactors, many of them very interesting proteins, was ADRM1 – the adhesion molecule. Now, this is my only real contribution to the paper: when I saw the list of interacting proteins, I remembered our identification of ADRM1 as the human Rpn13 ortholog and thus a likely proteasome subunit. I felt that a new proteasome subunit binding to ubiquitin would be something to write home about, and recommended to Ivan that he should focus on ADRM1 rather than the other interactors. It (kind of) worked, and Koraljka Husnjak did the further characterization of the protein, showed that it really binds to ubiquitin, mapped the interaction region to the N-terminus, and found lots of other interesting details about the protein. Next thing, the collaboration was joined by the group of Dan Finley (Harvard), who had shown that a simultaneous knockout of all known proteasomal ubiquitin receptors in yeast still supported ubiquitin-dependent protein degradation. Thus, it was an obvious idea to test if Rpn13 is responsible for this residual activity.
In the meantime, several other groups (1,2,3,4) demonstrated that ADRM1 really is a proteasome subunit. With these publications, my own contribution to the current project became virtually obsolete, except maybe giving Ivan’s and Dan’s groups two years head start. It was also shown that one important function of ADRM1 is to anchor the deubiquitinating enzyme Uch37 to the proteasome. This could not be the whole story, though, as yeast has an Rpn13 but no Uch37. Later on, the collaboration was further extended by adding two structure groups: Kylie Walters (Minneapolis) solved the NMR structure and Michael Groll (Munich) did the X-ray structure of ADRM1 (or hRpn13, as it is called now) in isolation and bound to ubiquitin. The majority of the structure work is published in a second paper, although some of the NMR work features also in the main article.
The ubiquitin binding mode of Rpn13 is somewhat unusual. Most other ubiquitin binding domains are shared by many proteins, use a contiguous binding surface and use one or more alpha-helices for contacting the Ile-44 patch of ubiquitin. The N-terminal ubiquitin binding domain of Rpn13 has a beta-sheet fold, very similar to the PH domain despite the absence of sequence relationship. Contact to ubiquitin is made through multiple non-contiguous loop regions, and no homolog outside of the Rpn13 family could be detected. The above figure shows yeast Rpn13 (botton) in contact with ubiquitin (top). Highlighted in red are three Rpn13 residues, which – when mutated – no longer support ubiquitin binding. Two other noteworthy features of ubiquitin recognition by Rpn13 are the unusually high affinity (Kd of 300 nM for monoubiquitin, 90 nM for diubiquitin, compared to the uM Kd valued measured for other ubiquitin receptors) and the ability to also recognize ubiquitin-like domains besides ubiquitin itself.
What is the significance of the finding? Rpn13 is certainly one of the more interesting ubiquitin recognition modes by the proteasome. Is it a ‘New Target for Cancer Drugs’, as the press releases ask (see e.g. here) ? Not very likely. Is it true that ‘A discovery of this kind happens only once in a researcher’s lifetime’, as Ivan Dikic is cited in the release? I hope not. As usual, the press releases on this publication (there are several ones) consist mainly of hot air. At least they agree with me on the insignificance of my contribution, illustrated by dropping me from the list of groups involved. That’s fine by me, though.
One final question: It this the end of the line for proteasome ubiquitin receptors? Chances are that there is still more to come. Apparently, yeast cells deleted for Rpn10, all UBL-UBA adaptors, and Rpn13 are still viable (while deletions of the catalytic proteasome subunits are lethal). Either there is still one more ubiquitin receptor around that would support some basal level of recognition and degradation, or ubiquitin independet proteasomal degradation is more important than expected.
P.S. As usual, other bloggers have beaten me in reporting on Rpn13. See e.g. Think Gene.