Study Reveals the "Black Box" of Protein Quality Control in the ER

Over the past few years, researchers in the Laboratory of Metabolic Regulation and Genetics at Rockefeller University have uncovered significant insights into the antioxidant glutathione, which has numerous vital functions in the body, including eliminating free radicals and repairing cellular damage.

Researchers have uncovered, among other findings, the metabolite’s complex relationship with mitochondria, the cell’s energy center, where it both sustains energy production and can promote breast cancer metastasis, as well as how glutathione regulates iron levels. They have now also shown that glutathione plays a crucial role in keeping the endoplasmic reticulum (ER), a region in the cell that produces proteins, operating smoothly. Their observations were presented in a study that was published in Nature Cell Biology.

Rockefeller has an incredibly rich history of research on the endoplasmic reticulum, so we know that when things go wrong in this organelle, many diseases ranging from neurodegeneration to cancer can result. We discovered a glutathione regulator in the ER that likely plays a key role in these conditions.

Kivanç Birsoy, Joseph L. Goldstein Professor, Rockefeller University

They discovered that this regulator serves as an essential proofreader, guaranteeing the proper folding of proteins in the ER.

Striking the Right Balance

A few years ago, the team found that all systems collapse if glutathione levels in mitochondria are not exactly regulated. Following their initial discoveries, the team began to consider the role of glutathione in the ER, which collaborates with mitochondria to maintain cell homeostasis.

The scientists knew from earlier research that glutathione plays a role in preserving the tightly controlled, Goldilocks-like environment of the ER, where ribosome-produced secretory and membrane proteins are folded for export.

After being exported into the cytosol, the jelly-like fluid that fills the cell, these proteins proceed to carry out their designated functions. The ER prefers an oxidized environment, whereas the mitochondria favor the unoxidized form of glutathione. Working with Hite's lab, the team went out to determine the mechanisms that calibrate the ideal ratio and why.

Quality Control

Liu started to directly study organelle functioning after creating a novel technique to quickly profile the chemical landscape inside the ER. She found that the ER imports an oxidized form of glutathione called GSSG from the cytosol and exports a reduced version called GSH in order to maintain its oxidized balance.

By maintaining a high GSSG-to-GSH ratio, the ER maintains its equilibrium. This process is regulated by the transporter SLC33A1, according to genetic screening. In addition to confirming that the SLC33A1 protein does, in fact, transport GSSG, structural investigations also provided insights into the biochemical mechanisms underlying this transport.

Before this work, we knew the ER needed to stay oxidized to fold proteins correctly, but the machinery responsible for maintaining that balance was essentially a black box.

Mark Gad, Memorial Sloan Kettering Cancer Center

“We discovered that the correct glutathione ratio is essential to a proofreading step in protein folding. It may even be its primary job,” Liu says. “So, if something goes wrong and the GSSG accumulates, it inhibits an enzyme that relies on the correct oxidation of the ER environment to operate a protein quality control system.”

Additionally, they found that misfolded proteins accumulate in the ER because they cannot be exported if they fail quality control. Cell death may eventually result from this extra debris.

“Identifying SLC33A1 as the key exporter – and being able to visualize exactly how it binds its cargo – gives us a handle on a process that, when it goes wrong, is linked to neurodegeneration and cancer,” notes Gad.

Neurodevelopmental Disorders and Cancer

To date, the researchers have uncovered glutathione-linked molecular processes that may contribute to a variety of disorders. The first is Huppke-Brendel Syndrome, a severe neurodevelopmental condition marked by increasing neurodegeneration, motor impairments, and severe intellectual disability. Up until today, scientists only knew that it was connected to mutations in the gene that makes the SLC33A1 transporter.

Our findings raise the possibility that the dysfunction of this gene alters the delicate glutathione balance in the ER and leads to protein misfolding during brain development. We think this could lead to new interventions, such as reducing the glutathione overload through synthesis inhibitors or compounds that can dissipate it.

Shanshan Liu, Rockefeller University

The results also have implications for possible treatments for lung malignancies caused by KEAP1 gene alterations.

“These cancer cells rely on a high level of glutathione synthesis,” Liu adds. “So, if we were to inhibit the SLC33A1 transporter, the GSSG would accumulate, and the cancer cells would die.”

“Our work demonstrates that defining how nutrients and metabolites are transported across cellular and organelle membranes reveals fundamental principles of cell biology while uncovering a major class of disease-relevant and therapeutically tractable proteins,” Birsoy says. “We will continue to illuminate this largely uncharted area in future work.”