New & Noteworthy

A Radical Discovery About a Well-Known Enzyme

May 22, 2013

Living your life puts a lot of wear and tear on you. A big reason is that as your cells go about their business, they churn out lots of damaging chemicals. 

This radical wanted us to rethink our role in Vietnam. The radical superoxide is making us rethink what the enzyme Sod1p does in a cell.

One of the worst offenders is the free radical superoxide, O2. Cells can’t help producing this powerful oxidant during normal metabolism, but it’s so toxic that it can destroy proteins and damage DNA.

Cells have come up with a two-step process to deal with this toxic waste. In the first step, they use the enzyme superoxide dismutase (Sod1p is the cytosolic form in yeast) to convert superoxide into the less harmful hydrogen peroxide (H2O2) and water. The cells then use catalases to take care of the H2O2, converting it to water and molecular oxygen. 

We’ve known about the first enzyme, superoxide dismutase, for decades. It has always been thought to have a simple role, sitting in the cytoplasm and detoxifying O2. But new research shows that its job is considerably more interesting than that: it also has a role in a regulatory process known as the Crabtree effect.

The Crabtree effect is named after the scientist who first described it way back in 1929. Some types of cells are able to produce energy by either fermentation or respiration in the presence of oxygen. Since these two processes have different metabolic costs and consequences, which one to use is a critically important choice.

If lots of glucose is around, yeast cells choose fermentation. They prevent respiration by repressing production of the necessary enzymes, and this glucose-dependent repression is the Crabtree effect. It happens not only in yeast, but also in some types of proliferating cancer cells.

A new study by Reddi and Culotta shows that Sod1p is actually a key player in the Crabtree effect. In response to oxygen, glucose, and superoxide levels, it stabilizes two key kinases that are involved in glucose repression.

It was recently found that the sod1 null mutant can’t repress respiration when glucose is around.  This is different from the wild type, which is subject to the Crabtree effect. 

Reddi and Culotta started by investigating this observation and found that SOD1 is part of the glucose repression pathway that also involves the two homologous protein kinases Yck1p and Yck2p. They found that Sod1p binds to Yck1p, which wasn’t totally unexpected since this interaction had been seen before in a large-scale screen. The unexpected part was that Sod1p binding actually stabilizes Yck1p and Yck2p.  These stabilized kinases can now phosphorylate targets that propagate the glucose signal down the pathway and ultimately repress respiration.

Now the question is why does Sod1p binding stabilize the kinases? It turns out that its enzymatic activity is crucial for stabilization. One idea is that the hydrogen peroxide that Sod1p makes in the neighborhood of the kinases could inactivate ubiquitin ligases that would target them for degradation. Ubiquitin ligases are rich in cysteine residues, and so could be especially sensitive to oxidation by H2O2.

This regulation might also feed into other pathways: these kinases are also involved in response to amino acid levels, and the sod1 null mutant was seen to affect the amino acid sensing pathway in this study.

Most excitingly, this mechanism is not just a peculiarity of yeast Sod1p. The authors mixed and matched yeast, worm, and mammalian superoxide dismutases and casein kinase gamma (the mammalian equivalent of Yck1p/Yck2p), and found that binding and stabilization works in the same way across all these species.

Superoxide dismutases may have been drafted into this regulatory role during evolution because they are the only molecules that sense superoxide, whose levels reflect both glucose and oxygen conditions. A radical idea indeed!

by Maria Costanzo, Ph.D., Senior Biocurator, SGD

Categories: Research Spotlight

Tags: regulation , Saccharomyces cerevisiae , respiration , fermentation

More Going on in the Ribosome Than Expected

January 20, 2012

More ORFs than previously thought are interacting with ribosomes.

As scientists peer ever more deeply into a cell, the picture of how things work becomes more and more complicated.  This was true when scientists took a hard look at transcription and gene regulation and found lots of little RNAs scurrying around the cell, regulating genes.  And it now appears to be true for what is being translated and how translation is regulated.

In a new study, Brar and coworkers used ribosome profiling to explore what happens in yeast cells during meiosis at the level of translation.  What they found was that a whole lot more was being translated (or at the very least gumming up the translation machinery) than anyone expected. They also found that translation is as finely regulated as is transcription. 

And this doesn’t just happen in yeast.  The same group has also generated similar findings in mice embryos as well.  Results with human cells should be right around the corner…

Ribosome Profiling

In ribosome profiling, scientists determine what RNAs are contained in a ribosome at a given time point.  The basic idea is that they isolate ribosomes, treat them with nucleases and then harvest the associated 30-35 nucleotide long mRNAs.  They then sequence all of the isolated RNAs and identify where they came from.

Like lots of biology these days, this technique has only become possible with the advent of cheap, robust sequencing.  In fact, the size of these sequences is ideal for modern sequencing techniques.

Researchers in the Weissman lab are finding all sorts of interesting things using this new tool.  For example, in meiosis they were better able to determine which proteins are involved at various stages of meiosis, to see how involved “untranslated” mRNA leaders are in translation, and to identify smaller, previously ignored transcripts associated with ribosomes.  In this post we’ll just focus on the last point but encourage the reader to learn about the study’s other findings here.

Of Shorter ORFs

Ribosome profiling has revealed that a lot more is being translated in yeast than the standard set of genes identified in the Saccharomyces Genome Database (SGD).  For example, Brar and coworkers found that the mRNA of many open reading frames (ORFs) shorter than the usual 250 or so base pairs were associated with the ribosomes.  Shorter ORFs like these aren’t routinely thought of as genes and so have not been extensively studied.

However, given how many of these ORFs were associated with ribosomes, scientists probably should start paying more attention now.  Even before meiosis, 5% of the ribosomes tested in yeast contained RNAs from these shorter ORFs.  Once meiosis kicked in, the number went up to an astonishing 30%.

Since scientists have only just started to focus on them, it isn’t surprising they don’t know how many of these smaller ORFs are translated into smaller peptides.  Or what any of these peptides that do get translated might be doing in a cell.

In a recent study, Kondo and coworkers have shown that one of these ORFs is translated into a peptide and proposed it affects how the transcription regulator Shavenbaby works in Drosophila.  Work similar to this will need to get underway before we have a good handle on what exactly is going on with these shorter ORFs.

Whatever they turn out to do, these small ORFs will probably change what we consider to be a gene.  Again.  The cell just keeps getting more and more complicated! 

Lengthy but informative lecture on ribosome profiling.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: ribosome profiling , ribosome , UTR , regulation , translation , untranslated leader