Prions are modular

January 28, 2000

In complimentary papers coming out in Science and Molecular Cell, researchers at the University of Chicago describe how prions--proteins that can exist in two different conformations and can pass their particular conformation from one generation to the next without any change in DNA--are modular. This discovery may convert these esoteric proteins into one of the most valuable tools in modern molecular biology.

A prion is a normal protein that has folded into an unusual shape. In its "prion state" the protein can entice healthy proteins of the same kind to adopt the misfolded prion form--a kind of protein misfolding chain reaction. Additionally, prions are both "infectious" and heritable—they are passed from generation to generation with no change in the nucleic acid sequence of the protein.

The researchers, led by Susan Lindquist, PhD, the Albert D. Lasker professor of molecular genetics and cell biology and Howard Hughes Investigator, found that the prion--determining region of prion proteins--the part of the prion protein that misfolds-can be transplanted onto other proteins, causing them to become prion-like.

"We've discovered a method to create novel prions which ultimately can have a lot of applications," said Lindquist.

Previous research has shown that prions are composed of two subunits--the prion-determining region and a functional domain which performs some function. "Once the prion part misfolds it entices other proteins of the same kind to fold incorrectly and they can clump together," Lindquist said. "The functional part may still be active. But if its job needs to be done in a particular place, it can't get there because it’s stuck."

In her January 28, 2000 Science paper, Lindquist and postdoctoral fellow Liming Li created a novel prion by taking the prion-determining part of Sup35, a known yeast prion, and linking it to a mammalian hormone response factor.

Lindquist and Li observed that the new prion existed in its functional state and also in the misfolded state. In cells containing the misfolded protein, the hormone response factor was unable to function properly. "We think this is because when the prions misfold and lump together, the functional subunit can't do its job," Lindquist stated.

Lindquist and Li were able to make the new protein switch between its functional state to the misfolded, prion state by tampering with levels of another protein in the cell-Hsp104. Although Hsp104 helps to ensure that normal proteins remain in their properly folded states, it helps to keep prions in their misfolded, aggregated state. Removing Hsp104 allows prion clumps to untangle and go back into solution in the cell.

"The new prion had the same characteristics as yeast prions," said Lindquist. "It could switch from its normal state to the prion state and responded to experimental manipulations in the same way a natural prion would," explained Lindquist.

In a complimentary paper coming out in Molecular Cell, Lindquist and graduate student Neal Sondheimer describe a newly discovered yeast prion. "We already know that two of the 6,200 proteins in yeast can be prions," said Sondheimer. "We wanted to know if there were more."

Sondheimer focused his search on a handful of suspect proteins that possessed regions that looked a lot like the prion-determining regions of known yeast prions Sup35 and Ure2. He used a fluorescent marker called GFP (Green Fluorescent Protein) to label the suspected prion-determining domains of these proteins and looked to see if they aggregated in cells-a sign that they may be misfolding and an indication of prion-like activity. Four of the proteins Sondheimer studied formed clumps in cells, which appeared as large green fluorescent dots.

Next, Sondheimer looked at the effect of Hsp104 on the proteins. One of the proteins, Rnq1 (pronounced "rink 1") behaved exactly like a prion in its response to different concentrations of Hsp104. When Hsp104 was removed from the cell, Rnq1 aggregates disassociated. When Hsp104 was added back, Rnq1 formed clumps. "Every comparison to Sup35 that checked out gave me more confidence that I was looking at a prion," Sondheimer said.

Sondheimer then looked to see if he could find Rnq1 in two distinct states and if the states could be induced to switch using the same tricks used to entice Sup35 to switch. "Again, this test checked out for Rnq1," Sondheimer said.

Finally, he looked to see whether the prion state of Rnq1 could entice Rnq1 proteins in the normal state to switch to the prion state. "I exposed a cell that had soluble, or non-aggregated Rnq1 to Rnq1 in its misfolded prion state, and the cell would become infected-all the Rnq1 in that cell would be aggregated," explained Sondheimer.

Once Sondheimer was convinced that Rnq1 was a prion, he took its prion determining domain and switched it with that of Sup35. The new protein behaved exactly like Sup35.

"The punchline of these two papers is that prions are modular," said Sondheimer. "They are composed of regions that can be swapped with each other like Legos."

Some of the applications of this research include using prion-determining domains to reversibly "knockout" single proteins in cells in order to better determine the protein's normal function. Because the change in function is produced by a change in protein conformation rather than a change in DNA, it can readily be reversed by experimental manipulation. "The new protein can be endlessly switched back and forth from the active to the inactive state," said Lindquist.

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