March 27, 2014
Most SGD users are probably too young to remember Saturday Night Live’s early years. One very funny commercial parody involved Gilda Radner and Dan Aykroyd arguing over a product called Shimmer. Gilda argues that it is a floor wax while Dan says it is a dessert topping. In comes Chevy Chase to tell them that it is both. Not quite as funny as Bassomatic, but still hilarious.
In a new study, Tsang and coworkers show something similar for the enzyme Sod1p. Most people know Sod1p as an enzyme that protects the cell and its DNA by directly deactivating harmful reactive oxygen species (ROS) like superoxide. Turns out that it may also be a transcription factor.
Now these two jobs aren’t quite as disconnected as a dessert topping and floor wax. When Sod1p acts as a transcription factor, it is regulating genes that affect a cell’s response to ROS. It is actually using its two functions to attack the same problem on multiple fronts.
Tsang and coworkers started out by looking at what happens to nuclear DNA under oxidative stress, using the Comet and TUNEL DNA damage assays. They found that endogenous and exogenous ROS caused DNA damage that was much worse in the sod1 null mutant – in other words, Sod1p protected the cells’ DNA. Using immunofluorescence, they also showed that Sod1p quickly went into the nucleus in the presence of ROS. But if they restricted Sod1p to the cytoplasm by adding a nuclear export signal, the protein no longer protected the DNA. In fact, it did no better than a strain deleted for SOD1.
In the course of these experiments one of the ways the researchers induced nuclear localization was with a burst of hydrogen peroxide. But since hydrogen peroxide isn’t a substrate of the enzyme Sod1p, Tsang and coworkers next wanted to figure out how Sod1p got its signal to go nuclear.
Previous work had shown that SOD1 genetically interacted with MEC1, a yeast homolog of ATM kinases which sense oxidative stress. They deleted MEC1 and found that Sod1p was trapped in the cytoplasm, unable to protect the cell’s DNA from damage. This result was confirmed in human cells by showing that Sod1p only went nuclear if the cell made ATM kinase.
Tsang and coworkers suspected that this interaction might happen through a protein kinase called Dun1p, whose human homolog is a Mec effector. They confirmed a previous mass spectrometry result that showed Sod1p interacted physically with Dun1p. And indeed, when DUN1 was deleted, Sod1p was again stranded in the cytoplasm. Further work showed that Dun1p does its job by phosphorylating Sod1p on two serine residues, S60 and S99. When both these serines are mutated to alanine, preventing phosphorylation, less of the mutant Sod1p makes it into the nucleus.
Using DNA microarrays, Tsang and coworkers next showed that SOD1 was required to activate 123 genes needed by the cell to respond to hydrogen peroxide. These genes fell into five categories: oxidative stress, replication stress, DNA damage response, general stress response and Cu/Fe homeostasis. The final experiment used chromosomal immunoprecipitation (ChIP) to show that in the presence of hydrogen peroxide more Sod1p was bound at the promoters of two of these genes, RNR3 and GRE2, but not the control gene ACT1.
Of course, the authors have only looked at two of the 123 genes and an obvious next step is to figure out how many of the 123 have more Sod1p bound to their promoters in the presence of hydrogen peroxide. Still, if these results can be confirmed and expanded they will suggest that Sod1p is able to combat oxidative damage in two completely different ways.
In the first it uses its enzymatic activity to directly inactivate the ROS superoxide, while in the second it helps the cell respond to other ROS apparently by acting as a transcription factor. While the jobs themselves are not as different as a floor wax and a dessert topping, how Sod1p goes about getting each job done is. “Calm down you two, Sod1p is an enzyme AND a transcription factor.”
In addition to these two roles, we’ve written before about yet another regulatory role for Sod1p: it regulates glucose repression by binding to two kinases and stabilizing them. This is truly an overachiever of a protein!
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight