While most enzymatic activity is conducted through chemical binding interactions between molecules, in some cases, the mere presence of an enzyme can affect intracellular functions.
While exploring the role of MTHFD1 in folate metabolism, a team led by Stefan Kubicek, PhD, at Research Center for Molecular Medicine of the Austrian Academy of Sciences, CeMM, uncovered a previously unknown role for NUDT5. This enzyme typically hydrolyzes metabolites, but as Kubicek explains, “our work reveals a completely different role—it acts as a structural regulator that determines whether the cell keeps producing purines or not.”
The team used a variety of approaches to analyze the role of NUDT5 and its interaction with other proteins in the folate pathway and in regulating purine synthesis.
“Because the folate and purine pathways are tightly linked, understanding this regulatory network could eventually inform new therapeutic approaches,” shared co-first author Jung-Ming George Lin, PhD.
“Our findings highlight that enzymes not only can act via the chemical reactions they catalyze, but also through their structure,” said Kubicek. “Sometimes, it’s the physical presence of a protein that makes the crucial difference.”
GEN spoke with Kubicek about his team’s research to gain better understanding of the drive behind the project and to expand on the collaborative work behind their study published in Science. Their paper is entitled, “A non-enzymatic role of Nudix hydrolase 5 in repressing purine de novo synthesis.”
This interview has been edited for length and clarity.
GEN: What was the drive behind beginning this project?
Kubicek: This paper originates from an earlier project in which we discovered an unexpected role of folate metabolic enzyme MTHFD1 in transcriptional regulation. We then wanted to understand the different enzymatic activities of the enzyme.
Surprisingly, we found that mutating one of its activities made cells dependent on adenosine, whereas mutating the other activity caused adenosine toxicity at very low micromolar concentrations. This enabled us to set up a genetic screen for modulators of this adenosine toxicity, which resulted in the discovery of a role for NUDT5 in regulating purine de novo synthesis.
GEN: For readers who may not be familiar with the technical details, could you describe the main methods you used in your study?
Kubicek: This is a highly interdisciplinary manuscript, that uses a multitude of methods in molecular biology, biochemistry, cell biology, and chemical biology. We genetically engineered multiple cell models for introducing point mutations in key proteins and performed genome-wide genetic loss of function screens as well as chemical screens for modulators of adenosine-mediated phenotypes.
To analyze the impact of mutations on nucleotide salvage and biosynthesis, we employed isotope tracing metabolomics. This was a joint project with Kilian Huber’s laboratory who developed a chemical PROTAC degrader dNUDT5 that contributed important data to showing that NUDT5 presence rather than enzymatic activity modulates purine de novo synthesis.
Interaction proteomics showed that NUDT5 physically binds to the rate limiting enzyme of purine de novo synthesis, and AlphaFold modeling allowed us to molecularly understand this interaction and predict mutations that impair the binding event.
Our collaborators in Kathrin Lang’s laboratory could biochemically show the repression of PPAT activity by NUDT5, the Rosenblatt and Froese laboratories contributed their expertise for experiments in cells from MTHFD1-deficiency patients, and the Casanova laboratory performed xenograft studies.
GEN: NUDT5 functioning as a mechanical block to purine formation was unexpected. Do you think that there are other related cases where metabolite enzymes, or other enzymes/proteins, may be identified to have alternate functions?
Kubicek: It is known that several metabolic enzymes can exert so called ‘moonlighting’ functions. In some instances, that means that they exert their normal metabolic function at an unexpected subcellular localization or on non-canonical substrates. But sometimes they also act through structural roles as binders to other proteins or nucleic acids.
I expect that through the recent breakthroughs in computational methods for systematic modeling and predicting these interactions, we will see more such functions characterized in the near future.
GEN: Your team has made connections between folate metabolism, purine synthesis, and MTHFD1 deficiency diseases. Can you expand on this connection? Will you be continuing this work towards clinical applications? If so, what are your next steps?
Kubicek: We assume that the adenosine toxicity in MTHFD1 cyclohydrolase mutantation is caused by a folate trap, whereby all folates accumulate as formyl-tetrahydrofolate.
Normally, this metabolite can be converted back to tetrahydrofolate, either through the activity of MTHFD1 or through purine de novo synthesis. In the MTHFD1 cyclohydrolase mutants, one of these routes is blocked through the mutation—which is also seen in MTHFD1 deficiency patients. All cells repress the other route, purine de novo synthesis, when external adenosine is available.
We found that this repression occurs via the interaction of NUDT5 with the rate-limiting enzyme of purine de novo synthesis, PPAT. This has important implications, as loss of NUDT5 not only reduces adenosine toxicity in MTHFD1 mutant cells, but also modulates the response to cancer therapeutics. When cells do not repress their own purine synthesis when they are exposed to purine antimetabolite drugs, this causes resistance to these agents.
Beyond potential diagnostic opportunities based on this finding, Kilian Huber’s laboratory in Oxford also has developed dNUDT5, a highly potent chemical degrader of NUDT5. In addition to using this compound for analyzing the basic biology of the protein, we are also considering translational possibilities, e.g. for protecting normal tissues for toxic effects of antimetabolite therapy.
GEN: What other next steps/follow up projects are you looking forward to?
Kubicek: Our project has opened up multiple new directions and research questions. Certainly, it will be interesting to develop chemical tools to directly modulate the interaction between NUDT5 and PPAT and thereby control the balance between nucleotide salvage and de novo synthesis.
I also believe that other modulators of the fundamental nucleotide synthesis pathways remain to be discovered. Finally, we are also working towards linking back our finding to the core interest of my laboratory in epigenetic control and chromatin structure that formed the basis of studying MTHFD1.
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