Dundee Research Interest Group

28/05/2021

The past two weeks have been very busy tying up lots of loose ends whilst also preparing for and setting up my final big experiments.

In previous blogs I have talked about carrying out in vitro dephosphorylation assays. These were controlled experiments in which I mixed phosphatases (proteins such as PP1 and PP2A, that remove phosphate groups from specific substrates) with various phosphorylated substrates (like phospho-Ubiquitin). From these experiments I found that PP1 was active against phospho-Ubiquitin (pUb). My next challenge is to validate this finding and also look for potential binding partners of PP1 that may direct it to the specific subcellular location of pUb in the cell (the mitochondria) in order to enable PP1 to dephosphorylate it.

This week I have undertaken a very similar experiment to the in vitro dephosphorylation assays with the exception being that rather than testing the purified PP1 protein for its activity against pUb, I have tested the whole cell extract. The whole cell extract is a solution that contains almost all the cellular contents, including proteins. We make this by lysing the cells – this essentially involves rupturing and removing the cell membranes, thereby releasing the cell contents into solution. Once we have the whole cell extract, it is possible to calculate its protein concentration, from which a calculated amount of the whole cell extract can be mixed with purified pUb in a similar in vitro assay. It was hoped we would observe the same dephosphorylation of pUb that occurred in the original in vitro assay.

The result was not as clear cut and conclusive as I had hoped, however, it has helped us to make some unexpected and interesting conclusions about the subcellular location of PP1. I was hoping to see complete removal of the phosphate groups from the phosphorylated Ubiquitin (pUb) but, unfortunately, this was not the case with the whole cell extract. Importantly, this does indicate that PP1 may be membrane-bound. This means that the protein is tethered to a particular membrane – usually at the site where its activity is required. For PP1 we could assume that this may be the mitochondrial membrane as that is where pUb is located, however, I need to investigate this experimentally.

Whilst these experiments didn’t produce the result we expected, it has brought about an interesting new line of enquiry. This year, I have seen how important it is in research to follow the science, even if it means going off on a complete tangent from your original project – researchers cannot be tunnel visioned and try to squash the results into the shape and direction they want. Hence, in light of these last results, it was back to the drawing board and I now have a new set of experiments to undertake and even more questions to answer. As ever, the more we learn, the more we realise how much we don’t know – such is the way of science.