Researchers at the La Jolla Institute of Immunology (LJI) have finally discovered the role of an enzyme called O-GlcNAc transferase (OGT) in maintaining cell health. Findings published in Proceedings of the National Academies of SciencesIt offers important information about cell biology and could pave the way for major medical breakthroughs.

The first author of the new study, Ph.D. “Many diseases are linked to OGT function,” says LJI instructor Xiang Li. “For example, many studies have demonstrated the abnormal function of OGT in cancer, diabetes, and cardiovascular disease.”

The new research, led by Li and co-led by LJI Professor Anjana Rao, PhD, and LJI Assistant Professor Samuel Myers, PhD, is the first to show that OGT controls cell survival by regulating a critical protein called mTOR. Cells rely on mTOR to keep their mitochondrial powerhouses running. Without functional mTOR, cells cannot perform nearly all of their essential functions, from protein synthesis to cell proliferation. Not surprisingly, mTOR dysfunction is also a hallmark of many diseases.

“OGT is important for every cell in the body,” Myers explains. “With this work, we now have a model of what each part of the OGT does that we can use for future research.”

Single OGT

OGT is an enzyme called transferase. This type of enzyme does a job called glycosylation, in which sugar molecules are added to newly synthesized proteins. OGT is unique among transferases in that it modifies proteins inside cells rather than cell surface proteins or secreted proteins. In fact, OGT’s glycosylation work is so important that embryonic cells die without it. But until now, scientists didn’t know why.

As Myers explains, the fundamental nature of OGT is what makes it so difficult to study. Scientists often study enzymes and other proteins by growing cells that do not have the genes for these proteins. They produce new dysfunctional cells and then investigate why things went wrong.

However, such an experiment with the OGT would be over before it even started. Because it’s only an OGT, scientists can’t remove it or reduce its function without killing the cells they need to work. “We knew OGT was important for cell survival, but for over 20 years we didn’t know why,” Lee says.

For the new study, Lee managed to circumvent this problem by using an inducible system to delete the OGT gene. He worked with mouse embryonic stem cells and then used an inducible version of a protein known as Cre to delete the OGT gene. This meant that the cells could grow normally until the scientists decided to activate the process, at which point the cells that had lost the OGT gene stopped proliferating and began to die. The team found that deletion of the OGT gene led to an abnormal increase in the function of a key enzyme called mTOR, which regulates cellular metabolism. Deletion of the OGT gene also triggered an important but potentially dangerous process in cells called mitochondrial oxidative phosphorylation.

Why is mitochondrial oxidative phosphorylation so dangerous? This process in cells is part of a delicate pathway that allows cells to produce ATP (the molecule that powers the cell). ATP can be produced by glycolysis as well as by mitochondrial oxidative phosphorylation, and disruption of this balance can have devastating consequences for cells.

Fortunately, OGT preserves mTOR activity and mitochondrial vitality by preserving protein synthesis and regulating amino acid levels in cells. More importantly, the researchers found the same protective role of OGT in CD8+ T cells, suggesting that the enzyme acts equally in all mammalian cell types, not just mouse embryonic stem cells.

Researchers to assist

Even dysfunctional cells devoid of OGT were not doomed forever. Using a new advanced gene-editing technology called CRISPR/Cas9, scientists were able to “save” dysfunctional cells. After asking whether a second gene in mouse embryonic stem cells could restore the growth of cells without OGT, Li found that mTOR and mitochondrial oxidative phosphorylation in cells without OGT were hyperactive and could be rescued by weakening the cells’ function.

This is good news for scientists hoping to learn more about OGT’s role in the body. “Now that we can delete the OGT gene while keeping cells alive, we can try restoring only the OGT fragments to learn more about how OGT works to keep cells alive,” Myers says.

Li says his new discovery may allow researchers to continue to examine the role of OGT and find therapeutic targets to potentially counteract the abnormal activity. “In the future, we hope our research will help shed light on issues related to OGT dysfunction in cancer and other diseases,” says Lee.