Finding a Solid Foundation

The key players in my PhD thesis: the lipid transfer protein, RdgBβ in lilac, ATRAP in black, right, spanning the lipid bilayer, and 14-3-3, an ‘m’ shape, black, left.

Don’t run before you can walk. Don’t get to the end before you’ve started.

Overwhelmed? Take a step back.

When I was in the lab and the results of my experiments started to get confusing, I cleared out my schedule, found a quiet spot, and went through things systematically. What’s consistent? What’s reproducible? What does make sense? Finding something solid gave me a chance to wind back to a safe point and move forward one step at a time, with clarity and purpose.

I’ve been working on a post about blood and getting myself in knots. Writing about therapies for blood disorders is a huge topic to tackle. With my target of two blogposts a month, I have put myself under so much pressure I’m not sure of what I’m doing anymore.

That, and my ideas for my book have hit a dead end. My tutor at art school told me once, “If it’s boring, it’s not worth doing.” I’m asking questions but the answers don’t excite me. The book is turning dull, cold, metallic.

So it’s time to wind things back. This book is about innovation - I need to speak to innovators. But first my own breakthrough moment. If I’m going to talk to other people about their breakthrough moments, I’m first going to tell you about mine.

I experienced two breakthrough moments during my PhD that became for me, my solid foundation.

The first breakthrough revealed the identity of the mystery lipid, a clear spot on the thin layer chromatography plate that we’d seen time and time again since my undergraduate project. But that mystery wasn’t mine alone to solve and required us to collaborate with a mass spec team in Southampton.

That mystery lipid spot was so clear, once we’d seen it, it wouldn’t go away. We couldn’t unsee it. The second breakthrough was like that too, only this was a blob on a western blot that wasn't there - until one day it was.

My undergraduate project centred around RdgBβ, a novel lipid transfer protein. Only two papers had been written on RdgBβ when I started work, and to look for clues as to its function, I began by scanning bioinformatics databases.

A potential interaction with the Angiotensin Receptor-Associated Protein, ATRAP, screamed hypotheses at us and led my supervisor, Prof Shamshad Cockcroft, to write to the British Heart Foundation to ask for a PhD studentship for me to work on it further.

ATRAP has no enzymatic activity yet has over 50 (and counting) potential interaction partners. At the time Shamshad submitted her application, one of these potential interactors was RdgBβ.

ATRAP gets its name from its interaction with the Angiotensin type 1 receptor, the receptor that causes the constriction of blood vessels, and through which repeated activation leads to chronic high blood pressure. In mice, ATRAP can protect heart muscle cells from enlarging due to continued Angiotensin II infusion. ATRAP can prevent blood pressure from rising.

ATRAP is a protective molecule.

So what’s it doing then, partnering with my lipid transfer protein?

Well, in order to find out, I first had to show the interaction was real. It turns out that was the hard part.

At the bottom of this post, there’s a photograph of the page in my lab book when I finally did this. Experiment number IP29. That means twenty-eight immunoprecipitation experiments came before this one, in 20 months. That’s 20 months of trying - and failing - to show this was a real interaction.

The results to IP29 came on a Friday afternoon. Crucial results so often arrive on a Friday afternoon, and let me tell you, they can completely make - or break - your weekend.

On this particular Friday afternoon, Shamshad was standing with post-doc Nico at Michelle, the technician’s desk. They were laughing and joking, no doubt talking about plans for the weekend. I don’t know what they were talking about, I was too busy finishing off my western blot.

We were a small research group. We’d been together through all 28 experiments before this one - and many other experiments besides. By this point, we’d started adding various activator or inhibitor molecules for different signalling pathways to see if the interaction needed some kind of signal before we could see it.

When the ATRAP band appeared on the developing western blot that afternoon, I couldn’t believe it. A year and 8 months and it had never been there before, not even once if you squinted. But here it was, clear as day. To say I was deeply suspicious is an understatement. I must have done something wrong, made a mistake. This can’t be real after so long. Things can’t actually be about to work out.

I printed the image and walked round the corner to my chattering lab mates. They stopped when they saw me. “Well?” Shamshad said.

“I’ve done it. I’ve got it. I’ve got the interaction,” I replied, cold with shock.

“Well why does your face look like that?” she said.

Once I’d seen this interaction - the innocent blob on the western blot - I couldn’t unsee it, and this became my solid foundation. It guided the experiments I needed to write my papers and to finish my thesis. It guided my choice of post-doc positions and my eternal quest for a cell line sensitive to Angiotensin II. It guided my fellowship application and eventually led to my own discovery of a therapeutic molecule.

What happened next is a story for another day.

Page 26 from PhD lab book 6 of 8 - almost two years in, I was now able to confirm the protein-protein interaction on which my whole PhD project rested.

Further Reading

Garner, K., Li, M., Ugwuanya, N., Cockcroft, S. (2011) The phosphatidylinositol transfer protein RdgBβ binds 14-3-3 via its unstructured C-terminus, whereas its lipid-binding domain interacts with the integral membrane protein ATRAP (angiotensin II type I receptor-associated protein) Biochem. J. 439(1):97–111.