Depending on your system, ubiquitin mutants can be somewhat inviable. A more specific way to do this would be to mutate your protein so that it does not contain any lysine residues on to which ubiquitin can be conjugated, though this may not qualify as "simple". It wouldn't eliminate addition of ubiquitin to the N-terminal, but it would likely abrogate much ubiquitin-mediated degradation. Unfortunately, I do not have any hands-on experience with such constructs.

You can use ubiquitin mutants. Since K48 polyubiquitin chain is responsible to mediate most the proteasomal-dependent degradation, if you transfect cells with a mutated lysin in the ubiquitin protein (a mutation a lysine substitution to arginine), you should prevent the formation of polyubiquitin chains through this lysine residue and therefore prevent ubiquitination in your protein of interest.

You asked for a simple way... Well, there is an inhibitor for the ubiquitin E1 available. But the so called PYR41 is very toxic to the cells. That is why its not that easy to handle... Maybe its worth a try.

But even better and maybe simpler than mutating your protein might be a temperature sensitive E1 mutant cell line.

Hi David,

Depending on your measurement system......? - Ubiquitination needs ATP (E1/ activation of ubi). With depletion of ATP you could distinguish between UPS-dependend and other (non-ATP dep, e.g. lysosomal) degradation. - Ubiquitin-dependent protein function is a matter of modification typ (aa modified, mono/oligomeric, linear/branched) and for example could be determined by mass spec analysis. nice day np

Of course you can use ubiquitin mutants ( and also you can mutate all lysines involved in the ubiquitination of your interest protein). If you transfect your cell line with a mutated lysine in the ubiquitin protein (lysine substitution to arginine), you should prevent the formation of polyubiquitin chains (K48 implicate in proteasomal mediated degradation) through this lysine residue and therefore prevent ubiquitination in your protein of interest. The assays with inhibitors, deubiquitinases, ATP, etc. .... are recommend in ubiquitination in vitro assays.

Thanks for all the suggestions. Lysine mutant is technically plausible, but I didn't necessarily want to go there. PYR41 seems interesting, might be useful in the short term if toxicity isn't too high. I'd much rather block lysosomal and proteasomal ubiquitin mediated degradation so I think K48 is out.

The lysine to arginine mutant of the site of ubiquitin modification on your protein is the most straight forward method. This method is well established, has a high degree of specificity (unlike global small-molecule inhibitors of ubiquitin-modification), and is accepted by the field. If you don't know the site of ubiquitination, it should be relatively easy to locate using mass spectrometry.

Lysine-less mutant is a good way to go. Nowadays, companies such as eurofins will provide a synthesis service, so you don't need to do that yourself. The downside is the price, though.

If you want to block proteasomal degradation, then you can use proteasomal inhibitors, such as MG132 or lactacystin. However, as noted above, this isn't a specific method and you run the risk of having pleiotropic effects which might render the interpretation of your results difficult. That is, if you look at phenotypes. Besides, if your protein is ubiquitinated but not degraded, then the treatment with a proteasome inhibitor will have no effect.

You also can use various ubiquitin mutants; they will tell you which type of ubiquitin chain, if any, is anchored to your favorite protein this might also give you a hint as to the consequences of the ubiquitination. Briefly:

- Mono or multi-monoubiquitination: modification of the localisation of the protein, its activity or its interactions with other proteins or with nucleic acid

- K48, K11, K29: proteasomal degradation

- K63 lysosomal degradation, membrane trafficking, modification of activity (at least of PCNA)

That paper, among others, might give you an idea: Ye, Y., & Rape, M. (2009). Building ubiquitin chains: E2 enzymes at work. Nat Rev Mol Cell Biol, 10(11), 755–764. doi:10.1038/nrm2780

Probably the answer is...there is no easy answer. Mutating your protein (lysins) is not an easy way as Gregory mentioned. Needless to say, it might cause unwanted changes in your protein.

If you don´t want to use the mutant ones you can express the wild-type ubiquitin. It should answear your question in another manner. Even so, all methods have its own pitfalls. Try also using an E1 inhibitor.

Targeting E1-E2-E3 cascade is a viable approach although you will block ubiquitylation en mass. Craig Crews of Yale has interesting collection of inhibitors. Visit his website and check recent publication.Recently, GSK announced collaboration with Crew’s lab to explore PROTAC technology.

Http://crewslab.yale.edu/

fuse your protein to a hyperactive deubiquitinase (DUB). I would recommend USP21, USP2 or a viral OTU. that will keep it clear of Ub.

best

david

Hi James, if I understand you correctly, you want to know whether your protein is ubiquitinated or not, and if so for proteolytic or other function. When working with tissue or cell culture, it would be possible to use an in vitro ubiquitination assay to determine ubiquitinated proteins (with or without inhibition of proteasome activity). The specific representation of your protein was then analyzed by Western blotting and MS.

Nice day norbert

Hi,

If you wish to follow the K to R mutation strategy, they are proteomics data on the phosphosite.org database that might give you the lysine(s) to mutate. 

I guess the degradation aspect is easier to address, by getting smear signal enrichment by blocking the proteasome, measure half-life with co expression of Ub 0 Lysines mutant and so on.

For the function as mentioned in the discussion thread, there are difficult to control parameters,

However in any case, mutating lysines will also alter ubiquitin like modifiers (e.g. SUMO, NEDD8) that may have potent effect on your protein's function, the same keeps true for the use of DUBs which can often cleave off Ubl in addition to Ub. Overexpression of Ub dominant negative will probably yield a protein of interest with lots of target lysines decorated with mono-Ub that are unable to form chains and potentially create artifactual functional changes.

Anyway, in these kinds of situation where ideal control does not really exist I guess that converging results with different approaches helps confirm.

Good luck!

I noticed this old post is getting quite some reads and thought it deserves an update regarding the Ubiquitin E1 Inhibitor TAK243/MLN7243. It’s another Millennium compound (like the Nedd8 Inhibitor MLN4924), now Takeda.

It was finally published in February and works really well! I attach one of my results, it works for shorter treatments as well and tends to hit neddylation of CRLs at high conc. or long treatments.

https://www.ncbi.nlm.nih.gov/pubmed/29334375

https://www.nature.com/articles/nm.4474

Best of luck,

Kurt