New Collaboration Uncovering a Mitochondrial Mystery


Mistakes happen. Inside every cell, the functions of life rely on the basic process of building proteins. But, about half the time, cells make errors when building proteins and have to recycle the pieces and start again. One important player in the cell’s recycling process, an enzyme called N-glycanase 1 (NGLY1), is at the center of a new, fundamental biological mystery that researchers at The Children’s Hospital of Philadelphia are setting out to solve.

Two young patients brought this mystery to the team’s attention. Both children arrived within a short time of each other with symptoms of suspected mitochondrial disease at CHOP’s Mitochondrial-Genetic Disease Clinic, which Marni Falk, MD, directs. Mitochondria are the organelles inside of cells that act as the cell’s energy generator, and diseases of mitochondria can have wide-ranging effects across every organ system and commonly include neurological and cardiac complications.

“There are a lot of areas of mitochondrial biology that are still not known at all,” said Dr. Falk, an attending physician at CHOP. “We’ve been so intrigued with this project because, every time we asked a question, three more questions followed.”

Dr. Falk and her team found that, instead of a primary mitochondrial disease, these two children had an extremely rare genetic disorder that was only recently identified, caused by an inherited deficiency in the protein-recycling enzyme, NGLY1. This was fundamentally weird. There was no evident logical reason for a disease of NGLY1 dysfunction to so closely resemble diseases of mitochondrial dysfunction because the proteins in mitochondria do not require NGLY1’s services, or so says conventional wisdom.

Evidence Builds for Mysterious Two-Way Connection

These proteins are believed to be unadorned with the modification (a carbohydrate addition called N-linked glycosyl groups) that NGLY1 removes when recycling proteins. Still, there appeared to be some connection. When the CHOP team looked closer at tissue samples biopsied from their two patients with NGLY1 deficiency, the mitochondria appeared abnormal both in quantity and appearance.

“We were intrigued with why something that’s involved in how you deglycosylate, and turn over your cellular proteins, should affect your mitochondria,” Dr. Falk said. “If the proteins in your mitochondria are not glycosylated, why should it matter at all?”

Preliminary evidence continued to mount that, for whatever reason, the action of NGLY1 did have some association with mitochondria, and past studies suggesting otherwise might be mistaken. When Dr. Falk collaborated with Zhe Zhang, PhD, in CHOP’s Center for Biomedical Informatics to evaluate data available about gene expression changes in nearly 100 mitochondrial disease patients from across eight different studies, they found that the NGLY1 gene was highly dysregulated in patients with mitochondrial disease. In fact, it was the second-most upregulated gene across all mitochondrial disease patients. They knew something had to be going on.

Multidisciplinary Approach to Understanding Mitochondrial Proteins

Now Dr. Falk and a multidisciplinary team of investigators from CHOP and the University of Pennsylvania intend to dissect the process further to find out more about this mysterious two-way connection between mitochondrial disease and protein deglycosylation by NGLY1. Under a newly awarded multi-PI NIH grant to Dr. Falk, Yair Argon, PhD, and Miao He, PhD, at CHOP, and Penn biochemist Eiko Nakamaru-Ogiso, PhD, the team will combine their complementary expertise in subjects including mitochondrial disease, clinical genetics, protein synthesis, glycosylation, and biochemistry. Their study will look in detail at the possible glycosylation of mitochondrial proteins that carry out the fundamental chain reaction of chemical processes within mitochondria that generates cellular energy, known as the electron transport chain.

For this study, they have access to cells from many dozens of mitochondrial disease patients who have enrolled in the CHOP mitochondrial disease research study, as well as cells from more than a dozen patients now identified with this rare NGLY1 deficiency, through collaboration with a natural history study of NGLY1 disease that is coordinated at the NIH by Lynne Wolfe, MS, CRNP. Thanks to a generous contribution by collaborator Tadashi Suzuki, DSci, of the Riken Advanced Science Institute in Japan, the team has embryonic fibroblast cells for study from a nonviable NGLY1 knockout mouse model.

“We’ve been working together to figure out how to isolate really pure mitochondria, so that we can apply sensitive methods to determine if the mitochondrial proteins, and specifically the proteins that work to directly make energy, are N-glycosylated,” Dr. Falk said. “Then, we can begin to understand how those protein modifications may be changed in either primary mitochondrial disease or NGLY1 disease.”

‘The Implications are Profound’

They suspect the answer has to do with cellular stress. To better understand the roles of NGLY1 and mitochondria in the cell’s stress response, and to translate that understanding into meaningful therapies, the team is combining its NIH-funded work with a project funded by the Grace Wilsey Foundation. They are working with cell lines and animal models of NGLY1 deficiency to characterize the nature of both cellular and mitochondrial stress responses that occur when NGLY1 is missing, and to develop possible NGLY1 disease therapies they can test in these models.

At the same time, the obscure-sounding scientific mystery of whether mitochondrial proteins are N-glycoslated after all, could add substantial pieces to the puzzle of some aspects of basic cell biology.

“The implications are profound,” Dr. Falk said. “If we’re right in our belief that mitochondrial proteins are glycosylated, there’s going to be a whole machinery to do this, potentially inside the mitochondria. That might open up understanding of new diseases or opportunities for new therapies. If you don’t even know a process is happening, you can’t possibly account for it, monitor it, and treat it.”

In addition to her titles at CHOP, Dr. Falk is an assistant professor of Pediatrics at the Perelman School of Medicine at the University of Pennsylvania. Dr. Argon is chief of the Cell Pathology Division in the Department of Pathology and Laboratory Medicine at CHOP and professor of Pathology and Laboratory Medicine at Penn. Dr. He is co-director of the Metabolic Disease Laboratory at CHOP and assistant professor of Pathology and Laboratory Medicine at Penn. Dr. Nakamuru-Ogiso, is a research assistant professor in the Department of Biochemistry and Biophysics at Penn.

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