ESR 13 - Ana Madalina Ion
Characterization of the translation release factors that operate in human mitochondrial protein synthesis
Principal Investigator and Sending Institution:
Prof. Zofia Chrzanowska-Lightowlers, Newcastle University, UK
Principal Investigator and Host Institution:
Prof. Hans Spelbrink, Radboud University, Nijmegen, the Netherlands
04 November - 06 December 2019
Report - My secondment in Nijmegen was part of the C12orf65 study...
My secondment in Nijmegen was part of the C12orf65 study.
Like mtRF1 and ICT1, C12orf65 is a nuclear-encoded protein that putatively acts as mitochondria translation release factor, but whose function has not been proved yet. To investigate its function I am using the BioID2 technique, which has been previously used and optimized by Maria Falkenberg’s group in Sweden and Hans Spelbrink’s group in Netherlands.
BioID2 involves a bacterial biotin-ligase (from A.aeolicus), which has been made promiscuous due to an artificially-induced mutation in the target recognition domain. Fused with a protein of interest, this biotin-ligase adds biotin residues to any protein that the protein of interest encounters within a 10nm radius. Those interactor proteins are then isolated by pull down with streptavidin beads, trypsinized, then analyzed and identified by mass-spectrometry.
I used the BioID2 technique in an attempt to understand the function of C12orf65 by determining its interacting partners. I used HEK cells overexpressing different variants of BioID2 from which I isolated mitochondria (part done in Newcastle) that I shipped to Nijmegen, where I performed the pull-down, sample preparation and analysis by mass-spectrometry.
The cells used were:
- C12orf65-BioID2-HA HEK 293
- C12orf65-Linker-BioID2-HA HEK 293 (the Linker is made of 13*GSSG repeats that widen the biotinylation radius with 10nm and reduce steric hindrance between C12orf65 and BioID2)
- Mitochondria targetting sequence (from COX8)-BioID2-HA (the control for the endogenous biotynylation).
For each cell line, I had one sample induced (tetracycline, 1ug/ml) and another sample induced and fed with biotin (5mM) for 16h. I isolated mitochondria, then snap-frozen them and sent them on dry ice to Nijmegen.
The part performed in Nijmegen consisted of:
1) pull-down of biotinylated proteins with magnetic beads coated with streptavidin
3) sample concentration (STAGE-tips) and purification (detergent removal)
4) injection of the sample, measuring and protein identification by mass-spectrometry-HPLC
As each sample was done in triplicate, I analyzed 18 samples in total. The final results from mass-spectrometry were not as I would have hoped. I obtained a low amount of total peptides and the ‘hits’ which stand out were mostly endogenous biotinylated proteins (carboxylases) from mitochondria.
However, in terms of personal development, I consider my secondment a success. There have been five weeks of intensive learning and troubleshooting. I am grateful for all the help that I have received from Alisa Potter, the Marie-Curie ITN fellow from Nijmegen, and from Prof. Spelbrink. I learned what happens ‘behind the curtains’ of mass-spectrometry. I did with my own hands all the washes, the sample concentration and the sample purification. I understood how the Mass-spectrometer is calibrated. From now on, whenever I see written in an article that ‘samples were sent to the mass-spectrometry facility’, I will know what happens in there. It will not be just a black box with a stamp on it that says ‘mass-spectrometry’. I learned to appreciate more the work that the mass-spectrometer specialist puts into. I understand why it takes so long to receive the data, where things can go wrong, how manual the process can be (STAGE-tips) and, most importantly, why we have to do all the long washes to have the sample clean. I see now why everything must be as pure and clean as possible-reagents, bench, water. It takes a lot of effort to clean a mass-spectrometer and it delays everyone’s experiments.
I received valuable input from Hans Spelbrink, Alisa and the other people from the lab. Hans Spelbrink gave me the opportunity to present my work during their weekly seminars, which I did. I received several interesting questions which will be helpful while writing the ‘Discussion’ part of my thesis.
Also, it was a pleasure to work in Hans Spelbrink’s lab.
People were kind and helpful, there was always someone to whom to turn for help if I couldn’t find something, for example. And I felt welcomed by Hans Spelbrink’s team. I felt I belonged there the moment I arrived. I was invited to every social gathering they have organized, they invited me for lunch together, they celebrated with me their achievements, I got to say goodbye to people who were leaving. I was one of them, even if only for five weeks.
As a fortunate chain of events, Petra Palenikova, the REMIX fellow from Michal Minczuk’s lab, came to Nijmegen for a workshop in November. So, me, her and Xin had a small non-formal REMIX gathering shortly before she left.
A challenging part of my secondment was biking. I never learned biking as a kid, I had to learn it by necessity as an adult during my masters, then I stopped doing it. Now in Nijmegen, I had to start biking again. I am grateful for Alisa’s patience and guidance to improve my biking skills. I am happy to say that by the end of the five weeks I did much better compared to when I have started. Suffering a harsh fall only increased my resilience and determination. The bruises I made that day became the proof of how strong my body can be. So, by the end of the secondment, me and my bike almost made friends.
All in all, my secondment was a great experience and I am happy and grateful I had the chance to do it. I salute the Marie Curie initiative of asking the fellows to move to a different lab, because a change of environment is enlightening. The benefits outnumber by far the small difficulties of finding accommodation and moving to another place.