This month’s paper comes once again from Science, as recommended to me by my old college roommate Eric. It was a pertinent recommendation for me, as the primary investigator on the paper is Uri Alon, a relatively famous scientist in the field of systems biology and author of the great introductory book An Introduction to Systems Biology: Design Principles of Biological Circuits , a book I happened to cite in my thesis :-).
In a previous paper-a-month post, I mentioned that scientists tend to assume proteins follow a basic first-order degradation relationship, where the higher the level of protein, the faster the proteins are cleared out. This gives a relationship which is not unlike the relationship you get with radioactive isotopes: they have half-lives where after a certain amount of time, half of the previous quantity is consumed. Within a cell, there are two ways for proteins to be cleared away in this fashion: either the cell grows/splits (so the same amount of protein has to be “shared” by more space – i.e. dilution) or the cell’s internal protein “recycling” machinery actively destroys the proteins. (i.e. degradation)
This paper tried to study this by developing a fairly ingenious experimental method, as described in Figure 1B (below). The basic idea is to use well-understood genetic techniques to introduce the proteins of interest tagged with fluorescent markers (like YFP = Yellow Fluorescent Protein) which will glow if subject to the right frequency of light. The researchers would then separate a sample of cells with the tagged proteins into two groups. One group would be the control (duh, what else do scientists do when they have two groups), and one would be subject to photobleaching – where fluorescent proteins lose their ability to glow over time if they are continuously excited. The result, hopefully, is one group of cells where the level of fluorescence is a balance between protein creation and destruction (the control) and one group of cells where the level of fluorescence stems from the creation of new fluorescently tagged proteins. Subtract the two, and you should get a decent indicator of the rate of protein destruction within a cell.
But, how do you figure out whether or not the degradation is caused by dilution or degradation? Simple, if you know the rate at which the cells divide (or can control the cells to divide at a certain rate), then you effectively know the rate of dilution. Subtract that from the total and you have the rate of degradation! The results for a broad swatch of proteins is shown below in Figure 2, panels E & F, which show the ratio of the rate of dilution to rate of degradation for a number of proteins and classifies them by which is the biggest factor (those in brown are where degradation is much higher and hence they have a shorter half-life, those in blue are where dilution is much higher and hence they have a longer half-life, and those in gray are somewhere in between).
I was definitely very impressed with the creativity of the assay method and their ability to get data which matched up relatively closely (~10-20% error) with the “gold standard” method (which requires radioactivity and a complex set of antibodies), I was frankly disappointed by the main thrust of the paper. Cool assays don’t mean much if you’re not answering interesting questions. To me, the most interesting questions would be more functional: why do some proteins have longer or shorter half-lives? Why are some of their half-lives more dependent on one thing than the other? Do these half-lives change? If so, what causes them to change? What functionally determines whether a protein will be degraded easily versus not?
Instead, the study’s authors seemed to lose their creative spark shortly after creating their assay. They wound up trying to rationalize that which was already pretty self-evident to me:
- If you a stress a cell, you make it divide more slowly
- Proteins which have slow degradation rates tend to have longer half-lives
- Proteins which have slow degradation rates will have even longer half-lives when you get cells to stop dividing (because you eliminate the dilution)
- Therefore, if you stress a cell, proteins which have the longest half-lives will have even longer half-lives
Now, this is a worthy finding – but given the high esteem of a journal like Science and the very cool assay they developed, it seemed a bit anti-climactic. Regardless, I hope this was just the first in a long line of papers using this particular assay to understand biological phenomena.
(Figures from paper)
Paper: Eden et al. “Proteome Half-Life Dynamics in Living Human Cells.” Science 331 (Feb 2011) – doi: 10.1126/science.1199784