LA JOLLA, Calif.—A study led by scientists at The Scripps Research Institute (TSRI) and the University of California, Berkeley, shows how a crucial molecular enzyme starts in a tucked-in position, and flips out when it encounters the correct target.
The new findings, published in eLife, shed light on the proteasome, a molecular machine that serves as a recycling center in cells. Proteasomes break down spent or damaged proteins and can even eliminate harmful misfolded proteins observed in many diseases. This research is the first study in almost 20 years to solve a large component of the proteasome at near-atomic resolution.
TSRI biologist Gabriel Lander, who was co-senior author of the study with Andreas Martin of UC Berkeley, said the breakthrough was possible with recent advances in cryo-electron microscopy (cryo-EM), noting: “Having an atomic-resolution structure and a better understanding of this mechanism gives us the ability to someday design therapeutics to combat cancer and neurodegeneration.”
Using cryo-EM, scientists investigated part of the proteasome that contains a deubiquitinase enzyme called Rpn11. This enzyme performs a crucial function called deubiquitination, during which it cleaves molecular tags from proteins scheduled for recycling in the proteasome. This is a key step in proteasomal processing—without Rpn11, the protein tags would clog the proteasome and the cell would die.
From previous studies, scientists knew Rpn11 and its surrounding proteins latch onto the proteasome to form a sort of lid. “The lid consists of nine different proteins, eight of which are necessary to assemble the lid complex; the remaining protein only appears to help with integration into the 26S proteasome, but it isn’t required,” says Lander. “The Rpn11 protein interacts in some way with every single one of the other required proteins, although the enzymatic portion of the Rpn11 protein is primarily blocked from function through interactions with one of these proteins, Rpn5.” The lid complex can also exist separately from the proteasome, which poses a potential problem: If Rpn11 cleaves tags from proteins that haven’t gotten to the proteasome yet, those proteins could skip the recycling stage and cause disease.
The study shows the lid in its free-floating conformation, in which Rpn11 is carefully nestled in the crook of surrounding proteins, stabilized and inactive.
“The lid only appears to have functionality in the context of the assembled proteasome. It’s intriguing that nature would evolve such a complex assembly pathway to house the deubiquitinating enzyme Rpn11, without endowing the resulting complex with some secondary function,” Lander tells DDNews.
“There’s a sophisticated network of interactions that pin the Rpn11 deubiquitinase against neighboring subunits to keep it inhibited in the isolated proteasome lid,” explained Corey M. Dambacher, a researcher at TSRI at the time of the study and now a senior scientist at Omniome Inc., who was first author of the study along with TSRI research associate Mark Herzik Jr. and Evan J. Worden of UC Berkeley.
“In order for Rpn11 to perform its job, it has to flip out of this inhibited conformation,” said Herzik. The new study also shows that, to flip out of the conformation at the proteasome, the proteins surrounding deubiquitinase pivot and rotate, binding to the proteasome and releasing the deubiquitinase active site from its nook.
“Based on the study data, the Rpn11 enzyme appears to be held in position through a network of hair-trigger interactions. Even a slight perturbation of the configuration results in an exposed Rpn11 enzyme,” Lander explains. “The subunits that surround the Rpn11 enzyme all have ‘arms’ that are able to interact with the proteasome. When these arms bind to the proteasome, it introduces a rearrangement of the lid complex, which is likely the trigger that causes the Rpn11 enzyme to be released for incorporation and activation within the proteasome.”
Lander called the system “finely tuned,” but said there may be ways to manipulate it. The study collaborators at UC Berkeley made small mutations to the proteins holding Rpn11 in position, and found that any small change will release the deubiquitinase, even when the lid is floating freely. The new understanding of the mechanism that activates Rpn11 could guide future therapies that remove damaged or misfolded proteins, according to Lander.
Going forward, the researchers hope to use the same cryo-EM techniques to investigate other components of the proteasome and figure out exactly how it recognizes and destroys proteins. “Despite decades of work into the proteasome, the specific mechanisms by which substrates are targeted, unfolded and translocated during degradation by the proteasome are still not very well understood. Even aspects of the deubiquitination mechanism are still in question,” Lander informs DDNews. “There is much to be learned. The proteasome can also be heavily influenced by a variety of different protein cofactors and additional ubiquitinating and deubiquitinating enzymes, and the role that all of these different cofactors have on protein degradation is something that we will be investigating through structure-function studies for many years to come.”
Cryo-EM has also recently enabled researchers from TSRI and the Duke University Medical Center to reveal the three-dimensional structure of a crucial ion channel. Their new findings depict this channel in more detail than ever before, shedding light on the channel’s possible role in immune functions such as detecting infection and inflammation.
“Our ability to perceive our environment—which includes sensing temperature and pain—is heavily reliant on these channels. Understanding their 3D structure paves the way for the development of a wide variety of new therapies,” said Lander, who was co-senior author of the study with biochemist Seok-Yong Lee of the Duke University Medical Center. The new study was recently published in the journal Nature Structural and Molecular Biology.
“The fact that the field of cryo-electron microscopy has advanced to where we can now solve the structures of these small membrane-embedded complexes to such high resolution is exciting,” said Herzik, who was co-first author of the study with Lejla Zubcevic of Duke University. Through the use of new sample preparation techniques, computer programs and a new generation of cameras, researchers at TSRI have improved the potential resolution of cryo-EM images to the point that TRPV2 could be imaged with near-atomic precision.
Lee and his colleagues focused on an ion channel called the transient receptor potential vanilloid-2 (TRPV2), which resides within the membranes of cells throughout the body. Previous research suggested TRPV2 was involved in sensing physical stresses such as changes in pressure and temperature, as well as in detecting immune challenges and activating the immune system’s T cells.
When researchers compared the structure of TRPV2 with TRPV1, a genetically similar ion channel found only in the nervous system, they noticed some significant differences. TRPV2’s architectural components near the central gate and the peripheral domains were in a previously unobserved configuration. Together, this led the authors to propose that this configuration represents a “desensitized” state, providing a new molecular snapshot of these ion channels at work.
“Since this is the first structure of the TRPV2 channel to be solved at high resolution, the previously solved structures of TRPV1 in different conformations played an important role in characterizing our data. We were surprised to see that the conformation that we observed in our structure didn’t precisely match any of the previously described states of TRPV1,” Landers says. “These comparisons led us to conclude that our TRPV2 is in a 'desensitized’ conformation. However, it is also entirely possible that the rules which dictate TRPV1’s conformational rearrangements may not apply to TRPV2, as these channels are not 100-percent identical. Future structural studies on TRPV2 will shed more light on whether this is truly a desensitized conformation, or if TRPV2 has a slightly different mechanism of activation relative to TRPV1.”
Lander said the next step is to find the structures of TRPV2 at different stages of opening and closing its gate. With the entire cycle imaged, researchers will have a better idea of how the ion channel works and how it might be manipulated therapeutically to treat autoimmune diseases.