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. An important step 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 with many interesting layers, and 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. 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 components that work with ribosomes— machines that translate genetic information into proteins—to make proteins at the endoplasmic reticulum (ER) membrane. Of the roughly 20,000 proteins encoded by the human genome, about 7,000 are made on ribosomes docked at the ER membrane. Here, the growing chain can be threaded into and across the ER, where it can begin to fold and undergo modifications like glycosylation, before being transported to its ultimate destination inside or outside the cell.

Last year Rohatgi and a postdoc in the lab, Mengxiao Ma, published a study showing how a protein called GRP94, which helps fold and mature proteins in the ER, avoids becoming “hyperglycosylated.” When GRP94 is hyperglycosylated, meaning too many sugar molecules are attached to it, it gets flagged by the cell for destruction. This can have downstream consequences for other proteins that rely on it, including cell surface signaling receptors involved in tissue development, inflammation and cancer. To avoid this fate, the growing GRP94 chainteams up with another protein called CCDC134 to block the oligosaccharyltransferase complex (OST), the cellular machine that facilitates glycosylation, from doing its job. Mutations that disrupt CCDC134 lead to GRP94 hyperglycosylation, causing a bone disorder calledosteogenesis 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 capture images of how these proteins work together during this process. The cryo-EM images showed a partially made form of GRP94 that looked different than the fully made protein. This version recruited CCDC134 and FKBP11 as “chaperones” to help GRP94 block the ability of OST to glycosylate it while it was being formed.

“We visualized 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

Keenan said that the original function of FKBP11 and CCDC134 could have evolved to shield any nascent protein chain as it enters the ER and prevent inappropriate interactions with other things in the cell that could cause problems. Later, GRP94 might have evolved the ability to bind much more tightly so it could also inhibit its own glycosylation. “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,” he said. 

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

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.