Tag Archives: Ubiquitin

Ubiquitination isn’t just for proteins

Time to look up from the plow biochemists.  Everyone knows that ubiquitin is added to proteins to destroy them.  The carboxy terminal amino acid of ubiquitin (glycine) forms an amide with the epsilon amino acid of a lysine called an isopeptide bond, and off  the protein goes to the proteasome for destruction.  This is simplistic and ubiquitination has many other other roles in the cell, but there isn’t time for it here.

I couldn’t resist putting in two interesting facts about ubiquitin.

#1. Like sharks,  evolution hasn’t changed ubiquitin much — only 3/71 amino acids differ between yeast and us.

#2 Ubiquitin is so stable that boiling water doesn’t denature it < Science vol. 365 pp. 502 – 505 ’19 >.

We have over 600 E3 enzymes (ubiquitin ligases), 40 E2 enzymes, and 8 E1 enzymes, and all 3 types are required to add ubiquitin to proteins.

Once a bacterium gets inside a cell, one of the ways the innate immune system attacks it is by ubiquitinating its proteins.  Nothing out of the ordinary there.

Salmonella (the organism responsible for most cases of food poisoning) is one such.  Our cells ubiquitinate the hell out of it.  However Nature vol. 594 pp. 28 – 29, 111 – 116 ’21 shows that, not just Salmonella proteins are the only sites of ubiquitination.  We also ubiquitinate endotoxin (lipopolysaccharide) which is a combination of sugars and lipids, with nary an amino acid in sight.  Endotoxin is a component of the outer membrane of every Gram negative bacterium, so the effect is likely not confined to Salmonella.

Even more spectacular is the enzyme adding ubiquitin.  It is called RNF213 (aka Mysterin), which looks like nothing the classic E3 enzymes we know and love.  For one thing in addition to E3 activity, it has a motor domain, a zinc binding domain and other domains of unknown function.  It’s a real monster with 5,184 amino acids and a molecular mass of 584 kiloDaltons.

There is a lot of interesting molecular biology to RNF213 — mutations cause Moya moya disease.

But the papers are particularly interesting because they show a lot of work of a new type needs to be done.

What else does Mysterin ubiquitinate?  Are there other enzymes in the cell adding ubiquitin, and if so, what do they ubiquitinate?

Definitely time to expand the well plowed field of ubiquitin.

The ubiquitin wars

Ubiquitin used to be simple.  All it had to do was form an amide between its carboxy terminal glycine and the epsilon amino group of lysine of a target protein, and bingo — the protein was targeted for degradation by the proteasome.

Before proceeding, it’s worth thinking why this sort of thing doesn’t happen more often, by which I mean amide formation between carboxyl groups on aspartic and glutamic acid on one protein and lysines on the surface of another.  That’s where the 3 amino acids are likely to be found, because they are charged at physiological pH, meaning they cost energy (and probably entropy) to put into the relatively hydrophobic interior of a protein where there isn’t a lot of water around to hide their charges.   Also, every noncyclic protein (which is just about all of them) has a carboxy terminal amino acid — why don’t they link up spontaneously to the lysines on the surface of other proteins?

Well, ubiquitin does NOT link up spontaneously.  It has a suite of enzymes to do so.  Like a double play in baseball, 3 enzymes are involved, which move ubiquitin to E1 (the shortstop) to E2 (the second baseman) to E3 (the first baseman).  We have over 600 E3 enzymes, 40 E2s and 9 E1s.  650/20,000 protein coding genes is a significant number — and the 600 E3s are likely there to provide specificity to just what protein gets linked to.

Addendum 21 Feb — Silly me, I should have added in the nearly 100 genes coding for proteins that remove attached ubiquitins (e.g. the deubiquitinases).

A few more fun facts and then down to business.  First ubiquitin is so stable that boiling water doesn’t denature it [ Science vol. 365 pp. 502 – 505 ’19 ].  Second ubiquitin can link to itself, as it contains 7 lysines at amino acids 6, 11, 27, 28, 33, 48 and 63 of the 72 amino acids contained in the protein.

Polyubiquitin chains are often made up of multiple ubiquitin monomers with lengths up to 10 [ Nature. vol. 462 pp. 615 – 619 ’09  2009 ] meaning that there could be a lot of different ones ( 7^10 = 282,475,249.  However chains found in nature seem to use just one type of link, e.g. linking the carboxyl group of one ubiquitin to just one of the 7 lysines over and over, forming a rather monotonous polymer.

On to the interesting paper, namely the ubiquitin wars inside a macrophage invaded by TB [ Nature vol. 577 pp. 682 – 688 ’20 ]  Ubiquitin initially was thought to be a tag marking a protein for destruction.  It’s much more complicated than that.  A host E3 ubiquitin ligase (ANAPC2, a core subunit of the anaphase promoting complex/cyclosome) promotes the attachment of lysine #11 linked ubiquitin chains to lysine #76 of the TB protein Rv0222.  In some way this helps Rv022 to suppress the expression of proinflammatory cytokines.

We do know that the ubiquitination of Rv022 facilitates in some way the recruitment of the protein tyrosine phosphatase SHP1 to the adaptor protein TRAF6 (Tumor necrosis factor Receptor Associated Family member 6) preventing the its ubiquitination and activation.  Of interest is the fact that TRAF6 itself is an E3 ubiquitin ligase which acts on many proteins.

Now to continue and show the further complexity of what’s going on inside our cells.  Autophosphorylated IRAK leaves the TLR (Toll Like Receptor) signaling complex forming a complex with TRAF6 resulting in the oligomerization of TRAF6.  Somehow this activates TAK1, a member of the MAP3 kinase family and this leads to the activation of the family of IKappaB kinases which phosphorylate IKappaB leading to its proteolysis.  Once IKappaB is removed from NFKappaB, translation of NFKappaB to the nucleus occurs where it turns on transcription of cytokines and other proinflammatory genes.

It is really amazing when you think of all the checks and balances going on down there.  How crude our weapons against inflammation are now, compared to what we might have when we know all the mechanisms behind it.

In which we find the new tricks an old dog can do

We all know that the carboxy terminal glycine of ubiquitin forms an amide with with the epsilon amino group of lysine.  Like a double play in baseball, 3 enzymes are involved, which move ubiquitin to E1 (the shortstop) to E2 (the second baseman) to E3 (the first baseman).  We have over 600 E3 enzymes, 40 E2s and 9 E1s.

A new paper [ Nature vol. 556 pp. 381 – 385 ’18 ] describes an E3 enzyme (called MYCBP2 aka PHR1) with a different specificity — it forms esters between the carboxy terminal glycine of ubiquitin and the hydroxyl group of serine or threonine.  The authors speculate a bit, noting that there are a lot of hydroxyl groups around in the cell that aren’t on proteins — sugars and lipids come to mind.   Just how widespread this is and whether any of the other 600 E3’s have similar activity isn’t known.

So now we have yet another new (to us) player in the metabolic life of the cell. It is yet another post-translational protein modification.   The enzyme is found in neurons, making understanding the workings of the brain even harder.