How Proteases In Cell Membrane 'Cut Out' Proteins
Johns Hopkins scientists have discovered a new mode of action for enzymes immersed in a cell's membrane. Instead of recognizing proteins based on amino acid sequences, they recognize unstable proteins and cut them out based on shape
In a report published online Nov. 13 in the new journal eLife, the Johns Hopkins scientists say their study results are the first to shed light on how some enzymes use their native environment to function.
The particular “cellular scissors” studied,
known as rhomboid proteases, are unusual among
proteases because they cut target proteins
from inside the cell membrane.
And because these and other membrane proteases
have roles to play in everything from malaria to
Parkinson’s disease, uncovering their “inside”
work could have profound implications
for human health.
“The evolution of these proteases, which are found in all types of living organisms, gave cells a whole new set of tools for regulating biology,” says principal investigator Sinisa Urban, Ph.D., a Howard Hughes Medical Institute researcher and associate professor of molecular biology and genetics at the Institute for Basic Biomedical Sciences at Johns Hopkins.
Proteases cut proteins for many reasons. The stomach relies on them to indiscriminately break down and digest various proteins people eat. Other proteases are more specialized and help regulate the immune system. Each of these specialized proteases recognizes only specific protein “clients” and only cuts its clients at one specific site.
Urban: “Until we did this work, it was thought that specialized proteases decided which proteins to cut based on the presence or absence of a specific sequence of amino acids they recognized. But while most proteases work in watery environments, rhomboid proteases work in oily membranes. Their unique environment suggested to us that they may also have unique properties within the cell.”
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, 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 thoroughly assess the affect of the protease’s environment on its function, they developed ways to reassemble rhomboid proteases and their clients within real cell membranes.
This allowed them to use cutting-edge biophysical techniques to compare how the enzymes and clients behaved in real membranes versus substitute membranes. They report that rhomboid proteases allow more proteins through their gates and cut them at different places when they are in a gel rather than when they are in a 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, researchers found that specific
sequences were not the main determinant of 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 had previously been floppy and shapeless
outside of the membrane in a watery environment.
“Rhomboid proteases have watery inner chambers.
If the slinky shape falls apart inside, the protein gets cut.
If the slinky shape remains intact, it doesn’t get cut.”
Sinisa Urban, Ph.D.
Associate Professor, Molecular Biology
Johns Hopkins' Institute for Basic Biomedical Sciences
This insight, says Urban, opens possibilities for better understanding several diseases and ultimately for developing treatments. For example, he says, the protein that builds up in the brain of Alzheimer’s patients is a target for another type of membrane-resident protease that isn’t well understood either.
Co-authors of the report are Syed Moin and Sinisa Urban from the Johns Hopkins University School of Medicine and the Howard Hughes Medical Institute.
The research was supported by grants from the National Institute of Allergy and Infectious Diseases (AI066025) and the Howard Hughes Medical Institute.
Original article: http://www.hopkinsmedicine.org/news/media/releases/