I am trying to identify the substrates of a lysine deacetylase by proteomics. I treated the cells with a presumed activator of the protein deacetylase, lysed the cells and performed immunoprecipitation with anti-acetyl-lysine antibody. Surprisingly, a strong band showed up in the treated cells, which means accumulation of an acetylated protein. This was confirmed by Western blot. One possibility is that the presumed activator is indeed an inhibitor of the enzyme. Another possibility is that activation of this enzyme causes inhibition of another lysine deacetylase, the substrate of which accumulates. Is there a good example of the second possibility?
Hi!
You should also work with the possibility that the activation of your deacetylase is inducing hyperactivation or increased transcription/translation of an acetyltransferase, as a compensatory response. If you know a protein that counteracts your protein of interest you could investigate, e.g. if its protein level changes with your treatment.
Very good suggestion. I indeed performed RNA-seq to look at the transcriptome changes. I will look through the data.
you said you treated the cells with a presumed activator so why are you now thinking that perhaps it is an inhibitor? In any case, the easy thing to do is to measure the specific activity of your enzyme and thus determine if there is an activator or inhibitor at work. In addition, you could recover the immunoprecipitate complex and pull it down with either protein A or protein G and see what type of protein/enzyme is being ppt with the inhibitor. Though I am not familiar with the enzyme, as far as doing research with it, there is always a possibility of isozymes that will also be activated/inhibited by the same molecule. Lastly, you can run a protein gel embedded with the enzyme substrate (zymogram)to determine if your enzyme is more/less active after treatment with activator.
good luck.
If it were an activator, the acetylated protein should be reduced not increased compared to the untreated one. I saw the opposite. Yes I am planning on doing the enzyme activity assay. The protein is a nuclear protein, and the extraction condition (high-salt) for nuclear proteins may not be optimal for the activity assay. I am still trying to optimize it.
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