January 13, 2015
Bounce houses are a great way for kids to burn off their excess energy. They can bounce off the floor and walls and scream to their hearts’ content.
Of course, adults need to keep an eye on how many kids are in the house at any one time, to keep things safe. And if one child starts to push and kick the others, it might be easier to restore calm if the adults are careful about how many kids, and which ones, they allow inside.
The yeast mitochondrion is actually a lot like a bounce house. It’s full of energy, and it has multiple gatekeepers—protein complexes in the mitochondrial membrane that imported proteins must pass through on their way in.
And, just like a bounce house, things can go very wrong inside the mitochondrion if its proteins don’t behave properly. The end result isn’t just an upset child with a black eye, either. Genetic diseases that affect mitochondrial function are among the most severe and the hardest to treat.
Now, described in a new paper in Nature Communications, Aiyar and colleagues have used a yeast model of human mitochondrial disease to discover both a drug and a genetic means to regulate a mitochondrial import complex. Surprisingly, tweaking mitochondrial import slightly by either of these methods mitigated the disease symptoms in both yeast and human cells. They found a gatekeeper who can make sure there is the right number of kids in the bounce house and that they’re all behaving properly (at least, as well as they can!).
The researchers were interested in mitochondrial disorders that affected ATP synthase. This huge molecular machine in the mitochondrial inner membrane is responsible for generating most of the cell’s energy, so if it doesn’t work properly it can be a disaster for both yeast and human cells.
Aiyar and coworkers used a genetic trick to create a yeast model that had lower amounts of functional ATP synthase. This mimics many mitochondrial disorders.
They were able to reduce the amount of functional ATP synthase by using an fmc1 null mutant. Fmc1p is involved in assembly of the complex, so the fmc1 null mutant has lower amounts of functional ATP synthase and a reduced respiration rate.
First, they looked for a drug that would mitigate the effects of the fmc1 mutation. They tested the drugs in a collection that had already been FDA approved—a drug repurposing library—to see if any would improve the mutant’s respiratory growth.
The one candidate drug that emerged from the screen was sodium pyrithione (NaPT), which is used as an antiseptic. Not only did it improve the respiration of the yeast fmc1 mutant, it also improved the respiratory growth of a human cell line carrying the atp6-T8993G mutation found in patients with neuropathy, ataxia and retinitis pigmentosa (NARP, one type of ATP synthase disorder).
Aiyar and colleagues wondered exactly what was being affected by the NaPT. To figure this out, they used the S. cerevisiae genome-wide heterozygous deletion mutant collection. This is a set of diploid strains, each heterozygous for a null mutation of a different gene, that has been an incredibly useful resource for all kinds of studies in yeast.
They tested the effect of NaPT on each of the mutant strains and found that strains with mutations in the TIM17 and TIM23 genes were among the most sensitive. And, when they checked the data from previous chemogenomic screens, they saw that these two mutants were much more sensitive to NaPT than to any other drug, showing that the effect was specific.
TIM17 and TIM23 are both subunits of the Tim23 complex in the mitochondrial inner membrane that acts as a gate for many of the proteins that end up in mitochondria. The researchers found that NaPT specifically inhibited the function of this mitochondrial gatekeeper complex in an in vitro mitochondrial import assay, confirming its selectivity.
So, Aiyar and coworkers had found a drug that alleviates the effects of an ATP synthase disorder by modulating the function of a mitochondrial gatekeeper. This in itself was a huge advance: the discovery that a potentially useful, already-approved drug has a specific effect on this disease phenotype.
However, the scientists took things a step further by looking to see whether a genetic therapy could accomplish the same thing as the drug. It was already known that overexpressing Tim21p, a regulatory subunit of the Tim23 complex, could modulate the function of the complex similarly to the effects they had seen for NaPT.
So the researchers tested whether overexpressing Tim21p would improve respiratory growth of the fmc1 mutant. Sure enough, it did. Consistent with this, assembly of the respiratory enzyme complexes of the mitochondrial inner membrane was more efficient when Tim21p was overexpressed.
Most importantly, overexpression of Tim21p in the fmc1 mutant cells caused their total ATP synthesis to more than double. And even more exciting was the discovery that overexpressing TIMM21, the human ortholog of TIM21, in the NARP disease human cell line improved survival of those cells.
So, just like a parent deciding how many kids should be in a bounce house so that everyone has a good time, the Tim23 complex can be made to “decide” which proteins, or perhaps how many proteins, get into mitochondria, with the end result that ATP synthesis happens as efficiently as possible. The exact mechanism of this effect is still unclear, but it is clear that modulating import in this way can improve mitochondrial health even when disease mutant proteins are present.
The next step will be to translate this discovery into therapies that will help mitochondrial disease patients. People with various mitochondrial disorders may finally be able to turn their mitochondria into safe, fun places.
by Maria Costanzo, Ph.D., Senior Biocurator, SGD
March 06, 2014
Imagine the heater at your house is run by a homemade copper-zinc battery. You are counting on a delivery of a copper solution that will keep the thing going. Unfortunately it fails to come, which means the battery doesn’t work and you are left out in the cold.
Turns out that something similar can happen in cells too. The respiratory chain that makes most of our energy needs copper to work. In a recent study, Ghosh and coworkers showed that if Coa6p doesn’t do its job delivering copper to the respiratory chain, the cell can’t make enough energy.
This isn’t just interesting biology. In this same study, the researchers showed that mutations in the COA6 gene cause devastating disease in humans and zebrafish. And their discovery that added copper can cure the “disease” in yeast just might have therapeutic applications for humans.
The respiratory chain is a group of large enzyme complexes that sit in the mitochondrial inner membrane and pass electrons from one to another during cellular respiration. This process generates most of the energy that a cell needs. Hundreds of genes, in both the nuclear and mitochondrial genomes, are involved in keeping this respiratory chain working.
Yeast has been the ideal experimental organism for studying these genes, because it can survive just fine without respiration. If it can’t respire for any reason, yeast simply switches over to fermentation, generating the alcohol and CO2 byproducts that we know and love.
Human cells aren’t as versatile though. Genes involved in respiration can cause mitochondrial respiratory chain disease (MRCD) when mutated. This is one of the most common kinds of genetic defect, with over 100 different genes known so far that can cause this phenotype.
Ghosh and colleagues wondered whether there were as-yet-unidentified human genes involved in maintaining the respiratory chain. They reasoned that any such genes would be highly conserved across species, because they are so important to life, and that the proteins they encoded would localize to mitochondria.
One of the candidates, C1orf31, caught their eye for a couple of reasons. First, some variations in this gene had been found in the DNA of a MRCD patient. And second, the yeast homolog, COA6, encoded a mitochondrial protein that had been implicated in assembly of one of the respiratory complexes, Complex IV or cytochrome c oxidase.
They first did some more detailed characterization of COA6 in yeast. They were able to verify that the coa6 null mutant had reduced respiratory growth because it had lower levels of fully assembled Complex IV.
They also looked to see what happens in human cell culture. When they knocked down expression of the human homolog, they also saw less assembly of Complex IV. This suggested that the function of this protein is conserved across species.
Next they turned to a sequencing study of an MRCD patient who had, sadly, died of a heart defect (hypertrophic cardiomyopathy) before reaching his first birthday. The sequence showed a mutation in a conserved cysteine-containing motif of COA6. To see whether this might be the cause of the defect, they created the analogous mutation in yeast COA6. The mutant protein was completely nonfunctional in yeast.
To nail down the physiological role of COA6 in a multicellular organism, they turned to zebrafish. The embryos of these fish are transparent, so it’s easy to follow organ development. Given the phenotype, the fact that they can live without a functional cardiovascular system for a few days after fertilization was important too.
When the researchers knocked down expression of COA6 in zebrafish, they found that the embryos’ hearts failed to develop normally and they eventually died. The abnormal development of the fish hearts paralleled that seen in the human MRCD patient carrying the C1orf31/COA6 mutation. And reduced levels of Complex IV were present in the fish embryos.
Going back to yeast for one more experiment, Ghosh and colleagues decided to see whether Coa6p might be involved in delivering copper to Complex IV. They knew that Complex IV uses copper ions as a cofactor, and furthermore Coa6p had similarities to several other yeast proteins that are known to be involved in the copper delivery.
They tested this by supplying the coa6 null mutant with large amounts of copper. Sure enough, its respiratory growth defect and Complex IV assembly problems were reversed. The delivery of copper kept the energy flowing in these cells. And this result showed that Coa6p is involved in getting copper to Complex IV.
These experiments showcase the need for model organism research even in the face of ever more sophisticated techniques applied to human cells. The mutation in human C1orf31/COA6 was discovered in a next-generation sequencing study, but yeast genetics established the relationship between the mutation and its phenotype. The zebrafish system allowed the researchers to follow the effects of the mutation in an embryo from the earliest moments after fertilization. And the rescue of the yeast mutant by copper supplementation offers an intriguing therapeutic possibility for some types of MRCD. Just another testament to the awesome power of model organism research!
YeastMine now lets you explore human homologs and disease phenotypes. Enter “COA6” into the template Yeast Gene -> OMIM Human Homolog(s) -> OMIM Disease Phenotype(s) to link to the Gene page for human COA6 (the connection between COA6 and disease is too new to be represented in OMIM). To browse some diseases related to mitochondrial function, enter “mitochondrial” into the template OMIM Disease Phenotype(s) -> Human Gene(s) -> Yeast Homolog(s).
by Maria Costanzo, Ph.D., Senior Biocurator, SGD
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.
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