New & Noteworthy

Turning to the Prion Expert: Yeast

February 15, 2017


You need an expert (not this guy!) for your plumbing problem. Just like you need yeast, and not some other organism, to study prions Image from flickr.

If you have a legal problem, you get a lawyer. A medical problem, a doctor. A leaky faucet, a plumber.

And if you are trying to find and figure out if a protein is a prion, you turn to the model organism where it is best understood. Yes, that is our old friend, the yeast Saccharomyces cerevisiae.

Prions became famous in the 1980’s when mad cow disease started to pop up in Britain. These are fascinating proteins that can cause an inheritable disease without affecting a cell’s DNA.

What happens is that prions can undergo a spontaneous conformational change. Now of course, this isn’t all that special. Lots of proteins can exist in different conformations.

But what makes a prion special is that the spontaneous change sets off a chain reaction where pretty much every copy of that prion protein is converted to that second conformation. And every translated prion protein thereafter both in the cell and any cells derived from that cell have that conformation too. So the new conformation along with the new traits it confers is passed on stably.

In mad cow disease, this spontaneous change causes neurological problems eventually resulting in death. But not every prion is so dangerous. Sometimes, as is the case in yeast, they can give new properties that allow survival in a new environment. Now these yeast have a new advantage in the absence of changed DNA.

Up until now prions have pretty much been confined to eukaryotes. That looks to change if a new study in Science by Yuan and Hochschild holds up.

They found that some bacteria have proteins that can and do behave as prions in a laboratory setting. The next step is to determine if they ever do so in the wild. If they do, then this would tell us that functioning prions may have evolved before the Bacteria/Eukaryota split and so be older than scientists previously thought.

The first step in finding a bacterial prion involved combing through a few bacterial genomes. Did I say a few? I meant something like 60,000 of them!

Of course you need the right tool for your search. This is where yeast’s incredibly well characterized set of prions comes in handy.

Yuan and Hochschild used an algorithm trained on known yeast prions to search through the bacterial genomes for proteins that have domains that would be predicted to be able to enter into a prion conformation. Among the proteins they found was the Rho protein in Clostridium botulinum E3 strain Alaska E43. Like the authors, I’ll call this protein Cb-Rho from here on out.

As you might remember, Rho is that famous transcription termination protein found in many different bacteria including E. coli. The E. coli version, however, does not look particularly like a prion nor did the authors find that it acts like one either.

A hallmark of prions is that one of the conformations involves their ability to form amyloid aggregates. These authors used a bacterial assay to show that this happened with Cb-Rho.

They next checked whether the prion portion of the protein behaved as a prion in a yeast cell. They substituted the prion domain from Cb-Rho with that of the one from Sup35p, a yeast prion. This chimeric protein behaved similarly to wild type Sup35p.

pipe

If you have a broken pipe, you find a plumber. But if you want to find and characterize a prion, you turn to yeast. Image from flickr.

They next tested their protein in E. coli. Often the prion conformation of the prion protein results in decreased activity of the protein. This is true in the Sup35p case, for example.

Sup35 acts as a translation release factor – it is an important factor in stopping translation at a stop codon. It is less active in its prion form meaning that there is now more read through of stop codons.

Rho is a transcription termination factor and so it would make sense that the prion conformation of Rho would result in lower levels of transcription termination. This is just what the authors found when they tested Cb-Rho in E. coli.

They used a reporter in which a transcription termination site was placed upstream of the lacZ gene. The idea is that if transcription termination is compromised, more lacZ will get made making the colonies a darker blue. And since termination is not 100% efficient, if Rho is doing its job, the colonies will be light blue.

When they plated out E. coli containing an engineered form of Cb-Rho, they got two classes of colonies, light blue and dark blue. And more importantly, this colony color was inheritable. In other words, light blue colonies gave more light blue colonies and dark blue colonies gave more dark blue colonies.

This isn’t to say that the colony color was a permanent state. It wasn’t. A bit under 1% of the colonies would spontaneously revert to the other form, as you’d expect from a prion protein.

So with the invaluable help of yeast, these authors were able to find and validate a protein from bacteria that can act as a prion. If they find a function for this in the wild, then yeast will have helped us better understand the evolution of life on Earth. Again. #APOYG!

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

Membrane Snorkeling with Arginine

February 6, 2017


If this swimmer’s snorkel were a glutamic acid and he was in a membrane, he couldn’t stay there for very long. His snorkeling tube would be too short! Image from pixabay.

Snorkeling is a blast. With a small tube stuck out into the air you can explore the wonders of the sea for much longer than you would be able to otherwise.

It is obviously important that the snorkel be long enough to reach out of the water. If it isn’t, you’ll be sucking down a lungful of water in no time.

Something similar can happen with membrane spanning proteins except that in this case, a snorkel is not essential to the protein swimming in the greasy confines of a membrane. Instead, positively charged amino acids like lysine or arginine can be tolerated much more often than you might think because of their structure.

Since membranes are hydrophobic, the amino acids in the part of the protein that span the membrane tend to be hydrophobic as well. Charged amino acids are usually trouble.

A proposed exception is amino acids like arginine and lysine. Their positive charges are each at the end of a long aliphatic chain:

Lysine & Arginine

What is thought to happen in that the aliphatic chain of the lysine or arginine snuggles up to the greasy part of the phospholipid and the positive part of these amino acids sticks out of the membrane to the more aqueous environment on the other side. The aliphatic side chain is long enough for the charged group to get out of the hydrophobic part. These amino acids may also work well because they can interact with the net negative charge that some phospholipid head groups have.

This is all very cool but, to date, there is very little in vivo work that supports this idea. Which of course means we need to turn to that workhorse model organism Saccharomyces cerevisiae to get some evidence!

In a new study out in GENETICS, Keskin and coworkers use yeast to provide some in vivo evidence that these positively charged amino acids are well tolerated in the membrane-anchoring domain of the yeast protein Fis1p. While I won’t have the space to go into some of the other fascinating experiments in this study, please read it over to learn more about how proteins are inserted into the mitochondrial outer membrane and the structure of this particular carboxy-terminal anchor.

The authors investigated the 27 amino acid carboxy-terminal anchor of this protein by first individually changing every one of its amino acids into every other amino acid (deep mutational scanning). Well, they didn’t get every possible combination. But 98.9% of them is pretty good!

Next they used a very clever screen to find which of these Fis1p mutants that could not properly insert into the mitochondrial membrane. Basically, they fused transcription activator Gal4p to their mutant library. If a mutant protein cannot insert into the membrane, then it is free to enter the nucleus and activate transcription of either a HIS3 or URA3 reporter.

When they did this they were surprised to see that the positively charged amino acids arginine and lysine were well tolerated in the membrane spanning portion of the protein. As expected, mutants with the negatively charged amino acids aspartic acid or glutamic acid in this region of the protein were not.

Here are these four amino acids:

LysArgAspGlu

The key difference here (besides the different charges) is the length of the aliphatic chain, the “snorkel” part of the amino acid. Both of the negatively charged amino acids are too short to be able to stick the charged part of the amino acid out of the membrane layer. They are like a snorkeler trying to snorkel with a tube that’s too short. It won’t work well.

So membrane spanning regions are not as hydrophilic fearing as many scientists and protein prediction programs may think.

It may be that the algorithms used to predict membrane spanning regions of proteins are missing some of them because they are too strict about the presence of charged amino acids like lysine and arginine. Perhaps these programs need to be modified to better allow for the presence of an errant arginine or lysine lurking in a long stretch of hydrophobic amino acids.

Of course we turned to yeast to help us better understand reality so we can come up with better algorithms. We may need to listen to yeast so that the programs can be adjusted to think about more than charge, and focus on the length of the snorkel too. #APOYG!

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

Don’t miss Fungal Pathogen Genomics!

January 30, 2017


The application deadline for the Fungal Pathogen Genomics workshop to be held May 11-17, 2017 at the Wellcome Genome Campus in Hinxton, Cambridge, UK is fast approaching! Be sure to apply by this Friday, February 3!

This exciting new week-long course aims to provide experimental biologists working on fungal organisms with hands-on experience in genomic-scale data analysis; including genome browsers and comparison tools, data mining using resources such as FungiDB, Ensembl/PhytoPathDB, PomBase, SGD/CGD, MycoCosm, analysis of genome annotation, and next generation sequence analysis and visualization (including RNA sequence analysis and variant calling). An important aim is that the participants should understand the origin of data available in public resources and how to analyse it in conjunction with their own.

The course is taught as a collaborative effort between available fungal informatics resources. The majority of this intensive course will be based on hands-on exercises, supplemented by lectures on genomics and bioinformatics techniques and keynote presentations by distinguished guest speakers.

Don’t miss out – apply now!

Not Recycling (RNA) Can be Bad for your Health

January 12, 2017


Improper disposal is bad for the environment and bad for cells. (Image from Wikimedia Commons)

Not too long ago, it was common to see people pouring used motor oil into street drains. Or to have people dumping old prescription drugs down their sinks.

Practices like these were (and are) terrible for the environment. Nature simply can’t deal with a buildup of this stuff (click here for some examples of the effects of pharmaceuticals on the environment).

Which is why it is so great that there are now ways to deal with waste like this. We can recycle it or at the very least dispose of it more carefully.

Turns out that things are similar in a cell. When its trash isn’t disposed of properly and/or recycled, the cell can suffer. And if cells suffer, so can the person made up of those cells.

One case where something like this is probably happening is in patients with the neurological disorder Pontocerebellar hypoplasia type 1B (PCH1B). These people have a mutated EXOSC3, an important gene for a cell’s RNA exosome. Presumably, this terrible disease is the result of certain cells not being able to properly clear some of their old RNAs.

In a new study out in GENETICS, Fasken and coworkers use good old Saccharomyces cerevisiae to begin to figure out what might be going on in the cells of these patients. They found that the most severe mutation seems to make it harder for the mutated protein to be part of the RNA exosome. As a result of being left out, the mutant protein is degraded more quickly leading to a buildup of some RNAs.

These sets of experiments were made a bit more complicated by the fact that human EXOSC3 cannot substitute for RRP40, the equivalent gene in yeast. This meant the researchers needed to focus on only those disease-causing mutations that hit the most highly conserved residues: EXOSC3-G31A, D132A and W238R.

Of these three, only the W238R yeast equivalent, rrp40-195R had much of an effect on the yeast. Fasken and coworkers propose that this is because this is the most deleterious of the three mutants.

Yeast harboring rrp40-195R grew more slowly at both 30 and 37 degrees C with the more pronounced effect at the higher temperature. At 37 degrees C, this mutant had higher levels of certain RNAs but not others. The RNA exosome was compromised for some but not all yeast RNAs.

And it wasn’t compromised everywhere. Although the RNA exosome works both in the nucleus and the cytoplasm, this mutant appeared to only be compromised in the nucleus. (Check the paper out for the cool way they figured this out.)

Next, the authors wanted to work out what this mutation did to the protein and the exosome. They were able to show that the mutant protein was more unstable than the wild type version and, interestingly, was even less stable when co-expressed with the wild type protein. They also showed that the mutant protein associated less well with the exosome complex and, again, this was exacerbated if the wild type protein was also present.

A reasonable model here is that RRP40 is more prone to degradation when it is not part of the RNA exosome. If true, then the mutant version of the protein is less stable because it is less often a part of the RNA exosome. And wild type RRP40 outcompetes the mutant protein making the mutant stay out of the complex for even more time.

Bad things can happen when you don’t recycle. (Image from Pixabay)

OK, so they have done some good work showing why this mutant of RRP40 affects the growth of a yeast cell. But what we really want to know is if these results explain what is going on in the cells of people with the disease.

Fasken and coworkers tackled this by looking at the effect of expressing the equivalent mouse Exosc3 mutant in the presence of wild type endogenous Exosc3 in mouse neuronal cells (the types of cells affected in PCH1B patients). They found that just like in yeast cells, the mutant was less stable in mouse neuronal cells.

So it looks like the recycling machinery for RNA is broken in these cells because of an unstable component and that this leads to a buildup of toxic RNAs. But if the yeast experiments hold up, not all RNAs are affected.

It is more like people still being able to recycle their cans and bottles but not their motor oil. Certain parts of the environment like waterways take a hit but other parts are left relatively unscathed.

This makes sense when you think about PCH1B. Only a few cell types are affected by the mutation in the EXOSC3 gene. In other words, most cells can deal with a slightly wonky RNA exosome.

Yeast has again helped researchers better understand a genetic disease. Awesome indeed. #APOYG

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

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