[indot05]

I have to submit a research proposal for an assignment for my biochemistry seminar.  I'm proposing an experiment to monitor the activity of the ubiquitin proteasome degradation pathway in the presence of certain corrector therapeutics being tested in clinical trials for cystic fibrosis.  Essentially, I want to monitor whether these drugs upregulate or downregulate this degradation pathway. So, what method would some of you all suggest to monitor the activity of this degradation pathway? 

I don't need a full explanation, as I'll do the research myself.  However, it would be great if I could get a few names for some possible methods and maybe a quick summary of what they do/how they work.  Just looking for a good starting point.  Thanks!

Edit: As these proposals will not results in an actual experiment, we were given the guidelines that we have any instrumentation available to us that would be found in a major research instituted, research that can be completed in two or so years, all within a practical budget (no real number of dollar guideline.  Just can't be something like a $5,000,000 budget).

[Yggdrasil]

One option would be fluorescent reporter system like the one described in this paper (https://www.nature.com/articles/nmeth.1460).

[indot05]

Okay thanks!

This is my first time dealing with photoconversion.  So, what about the actual photoconversion in this experiment is indicating degradation of the UbG76V mutant? 

The way I'm understanding it is, the UbG76V-Dendra2 fusion protein is recognized by UPS, broken down by the 26S proteasome, then blasted with a particular wavelength of light (I think 405 and 488 nm in this case), which alters the emission wavelength.  This then changes the emission from green to red.  But, what about this process is used to quantify the actual activity of the UPS?  I'm assuming that only the proteins that are degraded are photoconverted, but what stops the proteins that haven't been degraded from being converted?  Thanks!

[Yggdrasil]

Photoconversion allows you to set a t = 0 timepoint when measuring protein degradation.  Dendra2 is a fluorescent protein that is normally green.  The cell is constantly producing Dendra2 as well as degrading it, so the levels of green fluorescence in the cell reach a constant, steady state level. 

The paper makes use of the fact that you can convert Dendra2 to a version that fluoresces red by hitting it with a UV laser.  Now, the fact that the cell is producing more Dendra2 does not matter because the newly synthesized Dendra2 will not be red.  Therefore, after photoconversion, you can measure decay of the red fluorescent signal which, if you correct for photobleaching, will reflect the rate of degradation of the protein.