January 20, 2016
Eggs start out as slimy and awful, but can end up warm, firm and wonderful. All it takes is some heat to denature the egg proteins and voilà, a tasty breakfast.
Not that anyone would want to do it, but of course it is impossible to do the reverse. You can’t take a fried egg and turn it back into a raw one. The denaturation is pretty much permanent.
When a cell is hit with high temperatures, its proteins start to denature as well. And scientists thought that most of the denaturation of many of these proteins was as irreversible as the eggs. The thought was that many or most of these denatured proteins were “eaten” through proteolytic degradation. Although cellular chaperones are capable of disaggregating and refolding some heat-denatured proteins, it wasn’t known which aggregated proteins met which fate in a living cell.
A new study out in Cell by Wallace and colleagues shows that at least in yeast, most eggs get unfried. After a heat shock, aggregated proteins in the cell return to their unaggregated form and get back to work.
Now those earlier scientists weren’t crazy or anything. The proteins they looked at did indeed clump up and get broken down by the cell after a heat shock. But these were proteins introduced to the cell.
In the current study, Wallace and colleagues looked at normal yeast proteins being made at their normal levels. And now what happens after a brief heat shock is an entirely different story.
The first experiment they did looked at which endogenous yeast proteins aggregated after they were shifted from their normal 30 to 46 degrees Celsius for 2, 4, or 8 minutes. The researchers detected aggregation using ultracentrifugation—those proteins that shifted from the supernatant to the pellet after a spin in the centrifuge were said to have aggregated.
Using stable isotope labeling and liquid chromatography coupled to tandem mass spectroscopy (LC-MS/MS), they were able to detect 982 yeast proteins easily. Of these, 177 went from the supernatant to the pellet after the temperature shift. (And 4 did the reverse and went from the pellet to the supernatant!)
After doing some important work investigating these aggregated proteins, the researchers next set out to see what happened to them when the cells are returned to 30 degrees Celsius. Are they chewed up and recycled, or nursed back to health and returned to the wild?
To figure this out they did an experiment where proteins are labeled at two different times using two different labels. The researchers first grew the yeast cells at 30 degrees Celsius in the presence of arginine and lysine with a “light” label. This labels all of the proteins in the cell that have an arginine and/or lysine.
Then the cells are washed and a new media is added that contains “heavy” labeled arginine and lysine. The cells are shifted to 42 degrees Celsius for 10 minutes and then allowed to recover for 0, 20, or 60 minutes.
After 60 minutes of recovery, the ratio of light to heavy aggregated proteins looked the same as proteins that hadn’t aggregated. In other words, aggregation did not cause proteins to turn over more quickly.
It looks as if aggregated proteins are untangled and allowed to go about their business. So after a heat shock the cell doesn’t throw its hands in the air and simply start things over.
Other experiments done by Wallace and coworkers in this study, that we do not have the space to tackle here, suggest that the cell has an orderly process for dealing with heat stress. After a heat shock, certain proteins aggregate with chaperones in specific areas of the cell. Once the temperature returns to normal, these stress granules disassemble and the aggregated proteins are released intact.
None of this will help us unfry an egg — a denatured egg protein is obviously significantly different than an aggregated protein protected by chaperones in a stress granule. But this study does help us better understand how our cells work. And that’s a good thing.
Unlike Mr. Bill’s dog, most aggregated yeast proteins can return from a heat shock.
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight
August 06, 2015
Some kids are born troublemakers, wreaking havoc and destruction everywhere they go. They can’t help themselves; it’s in their nature to be that way. But if they have concerned and protective adults in their lives, children can overcome this tendency and grow up to become productive members of society.
Within the cell, ribosomal proteins are problem children. Although they grow up to have essential and productive roles, as newborns they can cause big trouble.
Many of them have highly charged, unstructured regions that give them a tendency to aggregate with other proteins. And they have a complicated journey to adulthood, since ribosome assembly happens in multiple cellular compartments. With an estimated 160,000 ribosomal proteins synthesized every minute in rapidly growing S. cerevisiae cells, these troublemakers could cause major problems if left to their own devices.
To help control this unruly mob, certain proteins in the cell act as designated chaperones for ribosomal proteins. In a new paper in Nature Communications, Pausch and colleagues found that, surprisingly, these specialized nurses catch their client proteins even as they’re being born. They swaddle them from the first moment they start to emerge as nascent proteins, and keep them from causing any harm until they can be delivered safely to their final destination.
The researchers first looked at the proteins Rrb1 and Sqt1. Previous work had suggested they might act as specific chaperones for the ribosomal proteins Rpl3 and Rpl10, respectively. And Pausch and colleagues confirmed these results, showing that TAP-tagged Rrb1 pulled down only Rpl3 and Sqt1 only pulled down Rpl10. Each of these troublesome tots had its own personal chaperone!
But surprisingly, very little of the protein was needed for these chaperones to keep ahold of their respective charges. When the authors trimmed down the ribosomal proteins to shorter and shorter lengths, they saw that just the N-terminal 15-20 amino acids of each ribosomal protein were necessary and sufficient for interaction with its chaperone.
They decided to use X-ray crystallography to look in detail at the Sqt1-Rpl10 interaction. First they determined the crystal structure of Sqt1 on its own, and found that it forms an eight-bladed WD-repeat beta-propeller, looking much like a round electric fan. The amino acids positioned on the surface of the blades are negatively charged.
Next, the authors co-crystallized Sqt1 with a peptide corresponding to amino acids 2-15 of Rpl10. The structure showed that the positively charged peptide was cradled in the negatively charged surface.
To test whether these charged residues were important for the interaction, they mutated the charged residues of Sqt1 and of the peptide and combined them in various ways. Sure enough, changing the charged residues of either partner disrupted or diminished the interaction.
Pausch and colleagues went on to test whether those same charged residues are important in vivo. An sqt1 mutation changing glutamate residue 315 to lysine (E315K), that abolished the Sqt1-Rpl10 interaction in vitro, was lethal for yeast cells, confirming the importance of the interaction.
The researchers also detected many allele-specific genetic interactions between the charged residues of the two proteins, and even found that switching the charges in an interacting pair of amino acids (changing an Sqt1 residue to a positive charge and its Rpl10 binding partner to a negative charge) would improve growth compared to either single mutant.
The lethality of that sqt1-E315K mutation, and even the lethality of an sqt1 null mutation, were weakly suppressed by overproduction of Rpl10. So yeast cells can get by (just barely) with an un-chaperoned Rpl10, as long as there’s enough of it around. This result also confirmed that Rpl10 is the only client of Sqt1.
As yet another verification that Sqt1 acts as a chaperone, the authors looked to see what happens to the Rpl10 protein in sqt1 mutants. If cells carrying wild-type SQT1 are lysed and separated into a pellet and supernatant, most Rpl10 spins down in the pellet but a significant amount is soluble in the supernatant. However, if the cells carry any of several sqt1 mutant alleles that alter the charged residues and diminish the interaction with Rpl10, all of the Rpl10 is found glommed together in the pellet.
The two chaperone-ribosomal protein interactions that Pausch and colleagues investigated, Sqt1-Rpl10 and Rrb1-Rpl3, both involved the extreme N termini of the ribosomal proteins. Previous studies had also shown that two other chaperones for ribosomal proteins, Yar1 and Syo1, also interact with the N termini of their clients. So the authors wondered whether interactions between ribosomal proteins and their chaperones might even start during translation of the ribosomal proteins.
In a final experiment, the researchers treated yeast with cycloheximide to freeze translation and then pulled down each of the four chaperones via affinity tags. Each chaperone specifically pulled down the mRNA encoding its client protein, showing that it was binding to the nascent protein as it first started to emerge from the translating ribosome.
So this study has defined a new step in ribosomal biogenesis. Certain specific ribosomal proteins are such troublemakers that it’s too dangerous for the cell to just release them into the cytoplasm after they’re translated.
Instead, these bouncing baby proteins are caught by their individual nurses before they’re even fully born, and wrapped up to protect both the ribosomal proteins themselves and the rest of the cell. Since ribosomal biogenesis is highly conserved across species and since defects in it are associated with many different diseases, further study of these cellular midwives could have important implications for human health. Perhaps some gentle guidance could help put wayward ribosomes on the right track.
by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD
Categories: Research Spotlight
April 01, 2013
When you think of a chaperone, you probably think of a strict adult at the prom who keeps a tight rein on the kids’ behavior. Well, in nature, a chaperone sometimes has to do the opposite to help new genes form more quickly. Sometimes the chaperone has to give the gene a longer leash to explore lots of different possibilities.
See, in theory, it is pretty easy to make a new gene. A cell accidentally makes an extra copy of an existing gene and this gene is then free to mutate into something new. A few mutations later and you have a new gene.
Turns out this is probably trickier than it sounds. First off, having an extra copy of a gene can cause problems. And second, getting to a new function is no walk in the park either. It usually takes a few sequential mutations to get there and, with proteins being such persnickety things, many of the intermediates along the way end up being unstable.
One way a cell might deal with these issues is to bring in a chaperone that lets the gene tolerate more mutations. Chaperones are proteins that help stabilize other proteins, often under trying conditions like high temperature. They coddle the protein and keep it stable so that it can still do its job. In addition, chaperones can also cause a protein to relocate to different parts of the cell.
So the idea is that if a duplicated gene gains a mutation that lets its protein interact with a chaperone, the protein may get more stability from that interaction or may be rerouted to where it won’t do any harm. Because the chaperone buffers the possible harmful effects for the cell, the gene is free to explore more different intermediates on the way to its new function.
A new study out in GENETICS by Lachowiec and coworkers lends support to this “capacitor hypothesis.” The authors used both Arabidopsis and Saccharomyces cerevisiae to show that genes whose proteins interact with the chaperone Hsp90 evolved more quickly than closely related genes that did not. This strongly supports the idea that chaperones can encourage new functions in duplicated genes.
The authors first looked at a couple of closely related transcription factors from Arabidopsis, BES1 and BZR1. Using a specific inhibitor of HSP90 called geldanamycin (GdA), they were able to show that BES1 was a client of HSP90 but BZR1 was not. They then created a phylogenetic tree of Arabidopsis BZR/BEH gene family and, by determining the ratio of non-synonymous to synonymous changes, found that BES1 had a higher rate of mutation. One explanation is that the stabilizing/relocalizing influence of HSP90 allowed BES1 to tolerate more mutations.
This result was an excellent first step in showing that the capacitor hypothesis may be true in some cases, but it is limited by being based on a single pair of proteins. To broaden their findings, Lachowiec and coworkers took advantage of the vast knowledge about Hsp90 interactions in Saccharomyces cerevisiae to look at many more genes.
At first this didn’t work out that well. The authors looked at a data set of yeast proteins that interacted with Hsp90 (encoded in yeast by the HSP82 and HSC82 genes) and, after removing any co-chaperones from the set, found no difference in the rate of evolution between those proteins that interacted with Hsp90 and those that did not. But as the authors note, this isn’t surprising as so many other factors play a role in the rate of evolution too.
To refine their analysis, they mimicked their BES1/BZR1 study and focused on pairs of closely related proteins where one interacted with Hsp90 and the other did not. They found that proteins that interacted with Hsp90 had a “longer branch length” than did their close relatives that did not interact. In other words, Hsp90 appeared to help along the formation of a new gene.
The authors then went back to Arabidopsis and showed that BZR and BES1 were found in distinct but overlapping parts of the cell. This lends credence to the idea that chaperones cause proteins to localize to different parts of the cell.
So it looks like an important function of chaperones may be to shepherd new gene formation. They are more like a 1960’s version of a chaperone…they let duplicated genes make lots of mistakes on their way to discovering who they really are.
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight