In the coip experiment, substrate band can only be enriched by e3 ligase in the presence of MG132. So when there is no MG132, once the substrate binds to e3 ligase, ubiquitylation occurs at a very short time and dissociates. That's why the substrate can't be enriched. If MG132 presents, proteasome is inhibited and ubiquitin becomes less and less with ubiquitylation continues. Finally, ubiquitin is not enough for rapid ubiquitylation. And once substrate binds to e3 ligase, there is little e2-Ub. So the ubiquitylation is inhibited, the complex can be exist for a longer time and be detected by coip. Is the explanation above correct or appropriate? Is this possibility can be only achieved by a perfect enzyme? Or is this just a common and normal situation for coip for e3 ligase and its substrate?
Not sure if I got your experiment and result correctly. You did several hours MG132 treatment of cells, followed by IP for your E3 and detected a (potential?) substrate that co-IPed only when MG132 was present; no substrate detected in control IP without MG132 ?
In your explanation you would have to assume that affinity of substrate to E3 is very different depending on modification; the unmodified substrate binds strongly and the ubiquitinated susbtrate has much less affinity. I am not sure if something like this has been shown for other E3 ligases or if this would be the most likely explanation.
I find it much more likely that during the several hours of MG132 treatment something happens to the E3 ligase or your substrate that affects affinity. This could be a posttranslational modification of E3 or substrate, or with the MG132 treatment you stabilize a substrate adapter that brings together substrate and E3. This substrate adapter can be a very unstable protein that exists only very shortly in the cell, but is stabilized upon proteasome inhibition. A scenario like this would also perfectly match with your results and is to my opinion much more likely.
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