Urban notes that rhomboid proteases are like barrels with a gate that only allows certain proteins inside. Once proteins get past the gate, they interact with the "scissors" inside the barrel and get clipped and released.
For their research, Urban and his team analyzed the activity of rhomboid proteases in microscopic gel-like droplets, which are traditionally used as substitutes for cell membranes, but which are incomplete imitations. To more thoroughly assess the role of the protease's environment in its function, they also developed ways to reassemble rhomboid proteases and their clients in real cell membranes. This allowed them to use cutting-edge biophysical techniques to compare how the enzymes and clients behaved in real membranes versus membrane substitutes.
They report that rhomboid proteases allow more proteins through their gates and cut them at different places when they are in the gel than when they are in the membrane. "That told us that these proteases are less accurate in recognizing which proteins to cut in the artificial environment than in their natural one," says Urban. "The membrane clearly helps to keep the gate from swinging open and letting unnatural sites to be cut."
The researchers then took a series of different proteins and changed their makeups in a variety of ways to see which ones the rhomboid proteases could cut in living cells. By analyzing dozens of individual changes to various proteins, they found that specific sequences were not the main thing that determined which proteins were cut. Instead, the key factor was whether the protein target was unstable in a watery environment.
Urban explains that when a protein contains a segment that crosses the viscous, oily cell membrane, that segment takes on a curly cue shape, like a slinky, even if it's floppy and shapeless outside the membrane in a watery environme
|Contact: Catherine Kolf|
Johns Hopkins Medicine