I wish to perform a cell viability assay using Ubiquitin activating enzyme inhibitor (UBE1). PYR41, the first generation is toxic to the cells of my experiments (human primary foreskin fibroblasts). I heard about MLN7243 as a substrate analogue of UBE1 but haven't tried it. Is there any tricks to use this inhibitor?
Dear Debanjan,
In my hands, MLN7243 is far superior to PYR-41. There are no specific tricks to using this inhibitor, however, it will also be toxic over time to any cell type, since abolishing ubiquitylation will have a multitude of effects on signalling, protein degradation and cell cycle regulation/progression. I would suggest that you try concentrations ranging from 1 - 10 μM for 1h (if this is compatible with your experimental/biological set-up), while monitoring any signs of toxicity as well cellular poly-ubiquitin levels (via western blot). You should expect to see the typical ubiquitin smear seen on western blot completely disappearing (this will depend on time of treatment, as well as concentration of inhibitor).
Depending on your research question, you may also want to consider MLN4924, which inhibits the Nedd8 activating enzyme. This in turn will inhibit the activity of a group of ubiquitin-ligases called the Cullin-RING ligases (whose activity required Nedd8 modification), and if your substrate/biological readout is dependent on ubiquitylation mediated by this group of ligases, the MLN4924 inhibitor would be a more specific tool.
I hope this was of some help - best of luck with your research!
Cheers,
Roland.
As mentioned by Roland, I assume there is no ubiquitinylation inhibitor which would be non-toxic, since this system definitively is fundamental to many vital functions.
As mentioned by Roland, I assume there is no ubiquitinylation inhibitor which would be non-toxic, since this system definitively is fundamental to many vital functions.
As mentioned by Roland, I assume there is no ubiquitinylation inhibitor which would be non-toxic, since this system definitively is fundamental to many vital functions.
- Badges
- Science topic