Crucial protein recruits help to protect itself while it forms

Proteins are often called the building blocks of cells, but even those building blocks need to be built. One of the most important steps in the process of building proteins is glycosylation, when sugar molecules (glycans) are attached to the maturing protein. These sugars can affect how the protein folds and functions, and mistakes during glycosylation can lead to disease.

A new study from Robert Keenan’s group at the University of Chicago, in collaboration with Rajat Rohatgi’s lab at Stanford University, sheds light on how this fundamental process can be regulated.

“It’s a complicated story that has many interesting layers, but it's yet another example where curiosity driven research reveals the underlying mechanism of a very basic cellular process that is linked to human disease,” said Keenan, who is a Professor of Biochemistry and Molecular Biology at UChicago. The paper was published this week in Nature. 

Capturing a protein being made 

Keenan has spent most of his career focusing on how proteins are made inside cells, especially the machinery involved in how ribosomes— machines that translate genetic information into proteins—dock to the membrane and help transport proteins inside. Of the roughly 20,000 proteins encoded by the human genome, about 7,000 are made on ribosomes docked at the endoplasmic reticulum (ER), an organelle that’s like a cellular transit hub to help molecules move inside or outside cells. After a ribosome docks to the ER membrane, the growing chain can be threaded into the ER, where it starts to fold or undergo modifications like glycosylation.

Last year, Mengxiao Ma, a postdoc in the Rohatgi lab at Stanford, published a study showing how a protein called GRP94, which helps fold and mature proteins in the ER, avoids becoming “hyperglycosylated,” meaning too many sugar molecules are attached to it. When GRP94 is hyperglycosylated, it gets flagged by the cell for destruction. This can have downstream effects on other proteins that rely on it, including cell surface signaling receptors involved in tissue development and immune responses.  

When GRP94 is being formed, it teams up with another protein called CCDC134 to block the ability of the oligosaccharyl transferase complex (OST), the cellular machine that facilitates glycosylation, from doing its job. Mutations that disrupt CCDC134 lead to GRP94 hyperglycosylation, causing a bone disorder called osteogenesis imperfecta.

Meanwhile, Keenan’s group had been studying how the OST works and saw that another protein called FKBP11 often binds to the ribosome machinery as proteins are being formed. Unexpectedly, GRP94 and CCDC134—the same proteins Rohatgi’s group was studying— were also present.

Mel Yamsek and Roshan Jha, postdocs in the Keenan lab, used cryogenic electron microscopy (cryo-EM) to try to capture images of how these proteins fit together during this process. The cryo-EM images showed a partially made form of GRP94 that looked different than the final protein. This version of GRP94 recruited CCDC134 and FKBP11 as “chaperones” to help shield it and block the ability of OST to glycosylate it while it was being formed. 

“We trapped GRP94 in the process of being made,” Keenan said. “There are very few examples of any protein being observed like this. So, this was serendipity, a bit of good fortune.” 

Recruiting chaperones for extra protection 

Because of its links to diabetes and cancer, there is great interest in trying to disrupt GRP94. This work provides a window into how future drug treatments could target the protein without disrupting other important cellular processes.  Such attempts have failed so far, however, often because potential drugs can also bind to other GRP94-like proteins in the cell, with unintended consequences. Targeting CCDC134 or FKBP11 could be a new route to selectively disrupt GRP94 by removing its built-in protection from hyperglycosylation. 

“Thinking about it in terms of evolution, maybe the early function of FKBP11 and CCDC134 was to shield any nascent protein chain as it enters the ER, to prevent any sort of inappropriate interactions with other stuff in the cell that could cause problems,” Keenan said.

"Later, GRP94 might have evolved the ability to bind much more tightly so it could inhibit its own glycosylation,” he continued. “It's the first example we’ve ever seen for directly regulating the activity of OST, which is fascinating because this is such a fundamental process in cells.” 

The study, “Structural basis of regulated N-glycosylation at the secretory translocon,” was supported by the National Institutes of Health, the American Cancer Society, the AP Giannini Foundation, and the National Science Foundation.

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