Altering a worm’s diet gives insight into a rare genetic disorder affecting proteasome function

Recent research from the lab of Dr. Nicolas Lehrbach, an Assistant Professor in the Basic Sciences Division at Fred Hutch, discovered that altering nucleotide metabolism may bypass proteasome defects that occur in the rare genetic disorder, NGLY1 deficiency. While this work was recently published in Plos Biology, its unique backstory began over a decade ago and highlights the translational power of basic science research.

In May 2012, Matt Might published an article titled “Hunting Down My Son’s Killer.” This essay published to his personal website proceeded to go viral, becoming a top trending story on both Twitter and Reddit within 24 hours of its posting. To clarify, Matt’s son Bertrand is very much alive—their emotional article chronicles Matt and his wife’s three-year journey to identify their son’s unknown medical condition, which was finally identified as a new glycosylation disorder caused by mutations in the gene NGLY1. This gene encodes a deglycosylation enzyme that removes sugar chains (glycans) from proteins, which is an important step to break down abnormal proteins. NGLY1 deficiency results in the buildup of abnormal proteins and leads to symptoms including developmental delays, intellectual disability, movement disorders and seizures. At that time, Bertnard Might became the first clinical diagnosis of NGLY1 deficiency.

Simultaneously, Matt and Kristen Wilsey’s young daughter Grace was struggling with developmental delays and various neurological symptoms when the Wilsey’s came across Matt Might’s article. While Bertnard and Grace exhibited different symptoms, the pair did share some symptoms, including more unusual ones like an inability to produce tears. The Wilsey’s soon found out that Grace too had a mutation in the NGLY1 gene, and the parents connected online. The two families teamed up to form a foundation dedicated to researching their children’s condition. With both families being well connected and having the financial means to do so, they reached out to clinicians and researchers to collaborate and learn more about this newly identified rare disease. The Grace Science Foundation was created to enable this research, as at this time, funding for this rare disease was nonexistent.

Meanwhile, Dr. Nicolas ‘Nic’ Lehrbach was conducting his postdoctoral research in Dr. Gary Ruvkun’s lab at Harvard Medical School studying transcriptional control of the proteasome— a piece of cellular machinery that destroys damaged or misfolded proteins and regulates protein homeostasis. Dr. Lehrbach explains that “cells adjust levels of the proteasome to cope with stress” and that accumulation of proteins—particularly damaged ones—is a common feature of aging and neurodegenerative diseases. Since the proteasome is an essential cellular complex, it’s difficult to study because inhibiting or removing its components is often lethal to cells. To study proteasome regulation, Dr. Lehrbach utilized the genetically elegant roundworm C. elegans as a model system, which has the added benefit of being very hard to kill, enabling him to perturb the proteasome to study its regulation. Specifically, Dr. Lehrbach was interested in a transcription factor, SKN-1A (homologous to Nrf1 in mammalian cells) which regulates proteasome levels through activating proteasomal subunit genes. Interestingly, Dr. Lehrbach’s postdoctoral work uncovered that SKN-1A is an unusual glycoprotein that requires PNG-1 (the C. elegans homologue of NGLY1) to remove sugars from it which then transforms SKN-1A into an active form. In the case of NGLY1 deficiency, SKN-1A/Nrf1 cannot activate proteasomal genes, leading to proteasome deficiency and a buildup of damaged proteins, which could contribute symptoms exhibited by patients with this rare genetic disorder. It was around this time when the Grace Science Foundation reached out to him and his postdoctoral mentor Dr. Ruvkun, “wanting to learn more about what is known about this gene at the basic biology level” and to financially support their work, Dr. Lehrbach describes. The foundation understood the power of basic science in the quest to better understand NGLY1 deficiency.

As part of this postdoctoral work, Dr. Lehrbach wondered if he could “use a worm genetic system to give clues as to how to bypass the defect in the proteasome or help boost proteasome levels.” This approach could identify genes that make cells less sensitive to proteasomal inhibition and offer new treatment avenues for NGLY1 deficiency. Using a large scale forward genetic screen (EMS mutagenesis) in C. elegans, Dr. Lehrbach inhibited the proteasome in png-1 mutant animals to model NGLY1 deficiency. Proteasome inhibition in these mutants resulted in lethal growth arrest. The goal was to find genes that when mutated would mitigate this phenotype. Dr. Lehrbach identified loss of function mutations in three genes—ent-4, tald-1 and rsks-1— which improved the ability of png-1 mutant animals to survive with a compromised proteasome. The proteins encoding these genes had unexpected functions, including nucleotide cellular transport and metabolic roles, but it was unclear exactly how they were connected to the proteasome or the accumulation of damaged proteins. In fact, the results were so surprising that Dr. Lehrbach “questioned whether the screen was set up properly since it wasn’t hitting anything obviously related to the proteasome.” And so this story sat while Dr. Lehrbach worked on other proteasome research and started his lab at Fred Hutch.

Enter Dr. Katie Yanagi, a postdoctoral researcher in the Lehrbach lab. While Dr. Yanagi pursued her own independent research in the Lehrbach lab, she began working on the proteasomal inhibition in png-1 mutants story as a side project to see if she could piece together what those three genes that had been identified as png-1 suppressors were doing. Unexpectedly, the researchers found that the suppressor mutations were able to elicit resistance to proteasome inhibition independent of SKN-1A; instead, they appeared to provide a general resistance to proteasomal inhibition by improving proteasomal function. One of these three genes, ent-4, encodes a nucleotide transporter and is specifically expressed in intestine cells, while another gene, tald-1, plays an important role in nucleotide metabolism. Getting creative, Dr. Yanagi began testing whether ENT-4 may mediate uptake of nucleotides from the diet and how altering nucleotide metabolism could influence proteasome function. Dr. Yanagi began inhibiting nucleotide biosynthesis and then altering nucleotide availability in the worms’ diet. To her surprise she “found that animals that do not have a functional copy of PNG-1/NGLY1 do better with lower levels of dietary nucleotides,” Dr. Yanagi stated, whereas increasing dietary nucleotide availability makes proteasome dysfunction worse in PNG-1/NGLY1 mutant animals.

“I think it’s great how much we can learn from work in C. elegans especially when it comes to diseases. We can model mutations found in patients or knock out the whole gene to better understand its function. The work we do with worms can provide a foundation for new therapies for hard to treat and rare diseases,” exclaims Dr. Yanagi. Dr. Lehrbach echoes this statement but emphasizes that this story is still a bit of “a black box, as we don’t exactly know how nucleotide metabolism connects to the proteasome or how this would translate to patients.” Now, his lab is “shifting this work towards understanding the intricacies of the proteasome. There’s so much to learn about complexity of it and we’re working to find ways to boost the proteasome,” which is relevant for various disease and cancer therapeutic approaches. Furthermore, Dr. Lehrbach continues to be part of the Grace Science Foundation community, sharing that “the foundation supports a couple dozen labs and brings together researchers working in diverse systems— worms, flies, mice— with biochemists and clinicians. Everyone is interested in the same problem and approaching it through different lenses.” This unique group meets regularly online and in person at an annual scientific conference to share their work and ideas. In the case of NGLY1 deficiency and through the Grace Science Foundation, “rare disease research could be the steppingstone to developing therapies for much more common diseases,” Dr. Lehrbach adds, as the foundation is committed to finding treatments for this rare genetic condition in addition to others where modulating proteasome function could be beneficial including cancers, diabetes and neurological disorders.

This work was supported by the Grace Science Foundation and the National Institutes of Health.

Fred Hutch/UW/Seattle Children’s Cancer Consortium member Dr. Nicolas Lehrbach contributed to this work.

Yanagi KS, Jochim B, Kunjo SO, Breen P, Ruvkun G, Lehrbach N. Mutations in nucleotide metabolism genes bypass proteasome defects in png-1/NGLY1-deficient Caenorhabditis elegans. 2024. PLoS Biology.