November 13, 2023
A novel genetically encoded tool manipulates redox metabolism with sub-cellular precision and can be used to interrogate the role of reductive stress in various pathologies
Scintillon Institute scientists validated a soluble transhydrogenase from E. coli (EcSTH) as a genetically encoded tool to induce NADH reductive stress in both mammalian cells and mouse liver. Characterization of the reductive stress using this tool revealed unique transcriptional and metabolic signatures. This new technology will be extremely useful for future studies aimed at identifying the root causes of multiple diseases or conditions linked to mitochondrial metabolism or an imbalance of redox cofactor NAD.
San Diego, CA – An evocative central question that continues to puzzle biologists is the role of mitochondrial (dys)function in various pathologies, ranging from cancer and neurodegeneration to the aging process itself. Do impaired mitochondria cause various metabolic changes leading to disease? Or is mitochondrial dysfunction a subsequent outcome of cascades of various disease etiologies?
Contrary to popular belief, answering these questions is not as simple as stating that mitochondria are “powerhouses of the cell” as these complex organelles support various cellular functions. However, the latter definition does highlight one of the most important functions of mitochondria, as they host complex cellular machinery that allows cells to effectively metabolize nutrients and produce energy in the form of adenosine triphosphate (ATP).
In supporting ATP production and oxidative phosphorylation (OXPHOS), mitochondria are crucial molecular rheostats in maintaining optimal cellular redox state. Indeed, the respiratory chain within the inner mitochondrial membrane metabolizes reduced nicotinamide adenine dinucleotide (NADH) and O2, ultimately producing water and oxidized NAD+. Diminished activity of the respiratory chain in multiple backgrounds, including genetic lesions, leads to an overabundance of NADH, termed NADH reductive stress. Until recently, there was no reliable technology to precisely model NADH reductive stress in mammalian cells.
“All previous methods to induce NADH reductive stress, such as genetic or pharmacologic oblation of the respiratory chain, were not as precise. Plus, we also wanted to study the role of NADH reductive stress in cells with intact mitochondria,” says senior author Valentin Cracan, PhD, an assistant professor at Scintillon Institute and adjunct assistant professor of Chemistry at Scripps Research. “Our technology allows us to target our novel tool to different cellular compartments and does not require inhibition of the respiratory chain or supplementation of cells with ethanol, which is another widely used (but less precise) method to generate an excess of reducing equivalents of NADH via the activity of cellular alcohol dehydrogenases.”
In their new paper published in Nature Chemical Biology, Xingxiu Pan, PhD, a postdoctoral fellow in the Cracan lab, and colleagues describe their discovery that enzymes from bacteria, called soluble transhydrogenases (STHs), can be used as a reagent to modulate NADH reductive stress when introduced into mammalian cells. First, members of the Cracan lab screened several bacterial STHs and identified an enzyme from E. coli (EcSTH) as the most promising candidate for modulating the cellular NADH/NAD+ ratio. Next, as a part of a collaboration with researchers Sara Violante, PhD and Justin Cross, PhD from the Memorial Sloan Kettering Cancer Center, New York City, extensive characterization of the NADH reductive stress was performed using a technology called liquid chromatography-mass spectrometry (LC-MS). This technology allows researchers to detect hundreds of cellular metabolites with extremely high sensitivity and precision. Using LC-MS, the authors identified novel accumulating metabolites in cells experiencing reductive stress that previously were not recognized as “regular” mammalian metabolites. These novel metabolites are likely the result of oversaturation of the pentose phosphate pathway (PPP), a fundamental component of cellular metabolism that takes place in the cytosol.
In addition to these extensive in vitro studies, Scintillon Institute researchers reached out to their colleagues at the Massachusetts General Hospital (MGH) to examine the utility of the new EcSTH tool in perturbing in vivo physiology. When Russell Goodman, MD, DPhil, assistant professor at Harvard Medical School and clinical hepatologist in the Division of Gastroenterology Medicine at MGH, expressed EcSTH in the liver of mice, he found that a protein previously known as a circulating biomarker of mitochondrial disease and associated NADH reductive stress, called Growth/differentiation factor 15 (GDF15), is extensively overproduced in the livers of these mice.
“Our new tool allowed us to identify GDF15 upregulation as a feature of the NADH reductive stress both in cell culture as well as in vivo. GDF15 was recently nominated as a biomarker of mitochondrial disease severity, yet our study is the first to show that the NADH reductive stress alone, even when the respiratory chain is intact, is sufficient to trigger GDF15 upregulation,” adds Dr. Cracan.
The researchers now plan to follow up directly on the multiple findings presented in their publication by exploring how redox dysfunction is impacting various pathologies, including aging-associated metabolic changes.
In addition to Cracan, Pan, Heacock, Abdulaziz, Zuckerman and Yao, authors of the study, “A genetically encoded tool to increase cellular NADH/NAD+ ratio in living cells,” include Sara Violante and Justin R. Cross of Memorial Sloan Kettering Cancer Center; and Nirajan Shrestha and Russell P. Goodman of the Massachusetts General Hospital.
The article can be accessed here:
Or here:
https://www.nature.com/articles/s41589-023-01460-w
DOI https://doi.org/10.1038/s41589-023-01460-w
This work was supported by funding from the National Institutes of Health (R00GM121856, R03AG067301, R35GM142495, R35GM142495-02S1, R01DK134675).