# Characterizing the biochemical regulation of mitochondrial one-carbon metabolism

> **NIH NIH F32** · MASSACHUSETTS GENERAL HOSPITAL · 2021 · $23,486

## Abstract

Project Summary/Abstract
Dysfunctions in one carbon (1C), or folate, metabolism are well-known for their deleterious effects on human
development, causing neural tube defects. However, the pathway is also implicated in both mitochondrial
dysfunction (found in aging as well as rare, genetic disorders), and many cancers. Because of the centrality of
1C metabolism across these diverse human diseases, it is already the target of drugs such as methotrexate.
Nevertheless, the underlying biochemical logic of the pathway remains incredibly complex, rendering its
fundamental functions difficult to understand, and therefore limiting its ability to be targeted by further drugs.
Contributing to the complexity of 1C metabolism are its subcellular compartmentalization and redox dependency.
It is not well appreciated that there are two parallel branches of 1C metabolism, one in the cytosol and one in
the mitochondria, which are controlled by the subcellular redox state of NADH and NADPH cofactors. Therefore,
to fully characterize the biochemical logic driving 1C metabolism, it is necessary to have tools to precisely perturb
the subcellular redox state of these cofactors. Recent work by the Mootha laboratory has provided tools to do
exactly that: a collection of four water-forming oxidases (NOXes) that can selectively oxidize the NADH or
NADPH pool in the cytoplasm or the mitochondria.
This proposal aims to use these powerful genetic tools to decipher, for the first time, the biochemical logic
underlying 1C metabolism in two states of cellular stress: mitochondrial dysfunction and hypoxia. These
perturbations are good models to probe the activity of 1C metabolism. Mitochondrial dysfunction upregulates the
pathway, and simultaneously reduces both the mitochondrial and cytosolic NADH pools. Hypoxia has also been
shown to significantly remodel the mitochondrial 1C branch and additionally produces cytotoxic reactive oxygen
species (ROS). To better characterize the complex interactions between 1C metabolism, subcellular redox state,
and ROS, this proposal will leverage high-resolution mass spectrometry to measure whole-metabolome
perturbations. Finally, this proposal will couple the metabolomics dataset with measurements of cytotoxic
reactive oxygen species and use an already-established cell line lacking a critical mitochondrial 1C enzyme to
isolate the contributions of 1C on ROS.
1C metabolism plays a critical role in human development, cancer, and mitochondrial dysfunction. However, its
underlying biochemical regulation remains poorly understood. Leveraging recently developed genetic tools to
modulate subcellular redox homeostasis with high-resolution metabolomics, this proposal aims to decipher the
biochemical logic of the 1C metabolic pathway with implications for current and pressing problems in human
health and disease.

## Key facts

- **NIH application ID:** 10410227
- **Project number:** 3F32GM133047-02S1
- **Recipient organization:** MASSACHUSETTS GENERAL HOSPITAL
- **Principal Investigator:** Owen Samuel Skinner
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $23,486
- **Award type:** 3
- **Project period:** 2019-07-01 → 2021-10-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10410227

## Citation

> US National Institutes of Health, RePORTER application 10410227, Characterizing the biochemical regulation of mitochondrial one-carbon metabolism (3F32GM133047-02S1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10410227. Licensed CC0.

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