Online Journal Club for November 2009

Our new format includes only relevant articles. If you have articles that you believe should be included in the online journal club, send them to me before the 20th of the month.

*1. SUMOylation of the mitochondrial fission protein Drp1 occurs at multiple nonconsensus sites within the B domain and is linked to its activity cycle. Claudia Figueroa-Romero, et al.

Presented at the Lab Meeting and summarized below by Leslie Foster:

Upon reading this paper, it appears to have given rise to more questions than answers. This is useful in that it gives ideas for possible future experiments when studying SUMOylation of a specific protein, yet also shows the complicated role of SUMOylation and the lingering uncertainty of what is really going on.

In this article, dynamin-related protein (Drp) 1 of HEK cells is discussed. Drp1, a cytoplasmic protein that binds the outer membrane of mitochondria, provides the driving force for mitochondrial membrane constriction and facilitates apoptosis by promoting this outer membrane’s permeabilization. It is a target for many post translational modifications, especially SUMOylation. It is made up of four domains: GTPase, Middle, B, and GTPase effector, N- to C- terminus respectively. These domains were mutated and/or deleted and analyzed to see how each variation affected SUMOylation. Specific lysine residues in the B domain were determined to be the main sites of SUMO interaction, which verifies previous papers stating SUMO binds to lysine. Upon sequence alteration, it was also shown that binding may occur at nonconsensus sites, allowing even more room for variation. This could also mean that SUMOylation, being unrestricted to a strictly conserved sequence, plays such an important role that even certain modifications or mutations will not prevent binding. The last point of this group was that Drp1’s activity was affected by SUMOylation upon substitution of K with A. There was an absence of the protein’s activity and an increase in its SUMOylation.

The relevant ideas to take from this paper include:

1. Importance of NEM in buffer. Before, we were not sure about NEM’s role in our SUMOylation experiments since some papers included it and others did not. Here, this lab performed an experiment with and without NEM (irreversibly inhibits SUMO proteases) and showed the presence and absence of a SUMOylation+protein band, respectively. Therefore to ensure SUMO will remain bound so that we may analyze it, NEM is necessary.

2. A method for determining binding sites of SUMO/Ubc9 on our protein of choice. This lab used cotransformations in yeast for determining interactions between Ubc9 and its target domain(s). This may be able to be applied to future studies in our lab once we have found a specific protein in the brain that is SUMOylated.

3. An alternate perspective on SUMOylation’s role. Although much of the discussion in this paper is non-conclusive, it is interesting to think that SUMOylation does not solely act as protein stabilizer or degrader, as stated previously in many other papers. Its interaction with the protein of interest may not directly effect that protein; rather, enable “functional consequences beyond SUMOylation of [protein of interest] itself”.

Link (PDF): http://www.fasebj.org/cgi/reprint/23/11/3917

*2. Update of the Stroke Therapy Academic Industry Roundtable Preclinical Recommendations. Marc Fischer et al.

As I warned you last month, this one is a bit depressing. It underscores the failure of the field to really move forward with clinically effective neuroprotectants, and responds with one more turn of the screw. You should take a look, but here’s a summary:

1. Sample size calculations need to be presented in detail. Parmetric data must be reported as 95% CI or std dev rather that std error.

2. Explicit exclusin and inclusion criteria, applied prospectively.

3. Randomization. Fortunately, we are ahead of the game here.

4. Allocation concealment. Again, we are ahead of the game.

5. Reporting of animals excluded from analysis. A significant change that we will incorporate into our standard approach.

6. Blinded assessment of outcome. Another area in which we have proactively caught the wave.

7. Reporting potential conflicts of interest and study funding. We should have more of these kinds of problems.

8. Studies in old, sick, female and gyrencephalic animals. We have discussed this at length. I won’t belabor the point.

9. Dose-response studies. It appears that the authors believe these should encompass both biochemical endpoints and outcome.

10. Window-of-opportunity.

11. Multiple outcome endpoints, including “long term” endpoints.

12. Physiological monitoring.

In summary: the bar is always getting higher. We have to keep up.

Link (PDF): http://stroke.ahajournals.org/cgi/reprint/40/6/2244