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
Tags: chaperones, heat shock, protein aggregates, protein aggregation
June 05, 2014
In A World Out of Time by Larry Niven, people live forever by teleporting the unfolded protein aggregates associated with aging out of their cells. Turns out that our very clever yeast Saccharomyces cerevisiae can do the same thing and it doesn’t need a machine.
Instead of teleporting the aggregates away, yeast saddles the mother cell with them when it buds. The daughter has now regained her youth and the mother is left to struggle with old age.
In a new study in Science, Hill and coworkers show that the yeast metacaspase gene MCA1 is critical in this process. But it doesn’t look like it is involved in segregating these bundles to the mother cell. Instead, it appears to help clear away many of the bundles left in the daughter. If it were in Niven’s original story, Mca1p might be a little nanobot that chewed up any aggregates the teleporter missed.
This all makes sense given caspases’ role in multicellular beasts. There, these executioner proteases chew up cellular proteins during apoptosis, the process of programmed cell death that is a critically important part of development and growth.
Although apoptosis has been observed in yeast and Mca1p is involved in the process, it has always been a bit of a mystery why a single-celled organism needs a mechanism for suicide. This study now suggests that yeast’s only caspase, Mca1p, has a role as a healer as well as an executioner. It saves the daughter by degrading and proteolytically clearing away the aggregated bundles clogging up her cell.
Scientists already knew that HSP104 was a key player in making sure that aggregates stayed with mom. Hill and coworkers used this fact and performed a genetic interaction screen using HSP104 to identify MCA1 as required to keep protein aggregates out of the daughter in response to a heat shock. Follow up work confirmed this result by showing that overexpressing MCA1 led to more efficient segregation of aggregates and that deleting it led to poor segregation of aggregates.
Digging deeper, these authors found that this poor segregation was because Mca1p was not eliminating aggregates in the daughter, as opposed to affecting the segregation itself. They also showed that the protease activity of Mca1p was needed for this effect.
In the final set of experiments we’ll discuss, the authors looked to see what effect MCA1 has on the life span of a yeast cell. They saw little effect of deleting MCA1 unless a second gene was also deleted: YDJ1, which encodes an HSP40 co-chaperone. The double deletion mutant yeast were able to divide fewer times before petering out. Consistent with this, overexpressing MCA1 led to increased life span and this effect was enhanced in the absence of YDJ1.
Finally, cells lived for a shorter time if just the active site of the Mca1p protease was compromised in a ydj1 deletion background. This again confirms that proteolysis is key to MCA1’s effects on aging.
So yeast attains eternal youth by both dumping its age-related aggregates on its mother and by using Mca1p to destroy any aggregates that managed to get into the daughter. The daughter gets a reset until she builds up too many aggregates, in which case she gets saddled with them.
Yeast may be showing us another way to live a longer life. If we can specifically degrade our aggregates without causing our cells to commit mass suicide, maybe we can extend our lives. And we don’t even need fancy teleporting machinery; we just need to adapt the molecular machinery yeast is born with. Feel free to use this idea for a new science fiction story!
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight
Tags: ageing, caspase, protein aggregates, Saccharomyces cerevisiae