Resource | Mollie's Blog No 14
Meet the Scientists
Over the past two weeks I have moved from cells to proteins – clearing away the clutter and amplifying my magnifying glass to focus solely on the possible proteins that comprise the part of the PINK1/Parkin pathway that I am studying.
I have carried out two types of in vitro assays – each studying opposite components of the PINK1/Parkin pathway. The first involved looking at the reverse components of the pathway, these proteins, such as PP1 and PP2A, are known as phosphatases and are able to remove phosphate tags from various substrates. The second assay involved monitoring the activity of PINK1.
Both assays require the use of recombinant proteins. We synthesize recombinant proteins are through a process in which we exploit the abilities of certain biological systems, often bacteria and yeast, to produce large amounts of a protein we need (often a human protein.) It is then possible to purify the protein from these systems, enabling it to be used for a variety of applications – including the study of their activity in assays such as mine. Another reason that the production proteins in this way is important is that it is possible to produce disease mutants of proteins and study how these mutations change the way the protein behaves. However, in my experiments I use the wild-type (or non-mutated) forms of the proteins, as we are just trying to unpick how the fully functioning proteins work in the PINK1/Parkin pathway.
My first assay essentially involves me mixing a phosphatase with a potential substrate and seeing if the substrate gets dephosphorylated. There are several ways of monitoring this presence/absence of phosphorylation, one of which is the use of PhosTag gels – a technique I struggled with for a while right at the start of my placement (see my 3rd, 4th and 5th blog). However, I am pleased to say that the technique has been working very nicely now and has given me some clear and exciting results. After my initial findings in cells that PP1 is able to dephosphorylate phospho-Ubiquitin, I was able to use this assay to show, without doubt, that PP1 is able to directly remove the phosphate tags from ubiquitin. This is a really exciting development (I am not sure I fully appreciated how significant it was at the time) and adds real validity to the idea that PP1 may be the phosphatase against phosphor-Ubiquitin. It has also given me a real boost of energy to try to uncover more details of the mechanism at play here before I finish my placement in July.
Despite PINK1 being the main focus of study in our lab, my project has been focused on the mechanisms that work in the reverse direction to PINK1 activity, hence, I have not introduced it in too much depth as of yet. PINK1 is classified as a kinase, a group of proteins with the ability to phosphorylate various substrates (the opposite function to the phosphatases I have been studying.) In the pathway that we study, we follow the PINK1-mediated phosphorylation of two substrates: Parkin and Ubiquitin. The current dogma is that, following its own activation upon mitochondrial damage or stress, PINK1 phosphorylates Parkin and that this phosphorylation in turn activates Parkin. It is Parkin that is then responsible for creating chains of Ubiquitin at the surface of the mitochondria. PINK1 is then also able to phosphorylate the Ubiquitin in these chains. These combined actions of PINK1 and Parkin lead to the accumulation of phosphorylated Ubiquitin at the mitochondrial surface, a signal that leads to the removal to the damaged mitochondria.
In my second assay, I was looking at whether PINK1 activity could have been affected by any of the chemical treatments I had used in my cell-based experiments. In a similar manner to my first experiment, I essentially mixed PINK1 with one of its substrates, in this case Parkin, and monitored changes to the levels of Parkin phosphorylation. The difference here being that I am hoping to see phosphorylation of the substrate rather than dephosphorylation, as in the previous assay.
Hopefully, the results of these experiments will help to give a clearer picture of the underlying mechanisms that lead to the previous results I have seen in my cell-based experiments and add to the validity of our findings.