Human peroxisomal NAD plus /NADH homeostasis is regulated by two independent NAD(H) shuttle systems

Reduced (NADH) and oxidized (NAD+) nicotinamide adenine dinucleotides are ubiquitous hydride-donating/ accepting cofactors that are essential for cellular bioenergetics. Peroxisomes are single-membrane-bounded or-ganelles that are involved in multiple lipid metabolism pathways, including beta-oxidation of fatty acids, and which contain several NAD(H)-dependent enzymes. Although maintenance of NAD(H) homeostasis in peroxi-somes is considered essential for peroxisomal beta-oxidation, little is known about the regulation thereof. To resolve this issue, we have developed methods to specifically measure intraperoxisomal NADH levels in human cells using peroxisome-targeted NADH biosensors. By targeted CRISPR-Cas9-mediated genome editing of human cells, we showed with these sensors that the NAD+/NADH ratio in cytosol and peroxisomes are closely connected and that this crosstalk is mediated by intraperoxisomal lactate and malate dehydrogenases, generated via translational stop codon readthrough of the LDHB and MDH1 mRNAs. Our study provides evidence for the ex-istence of two independent redox shuttle systems in human peroxisomes that regulate peroxisomal NAD+/NADH homeostasis. This is the first study that shows a specific metabolic function of protein isoforms generated by translational stop codon readthrough in humans.

Automated summary

This study developed a method to measure intraperoxisomal NADH levels in human cells and found that the NAD+/NADH ratio in cytosol and peroxisomes are closely connected, mediated by intraperoxisomal lactate and malate dehydrogenases. This study provides evidence for the existence of two independent redox shuttle systems in human peroxisomes that regulate peroxisomal NAD+/NADH homeostasis. It also demonstrates the specific metabolic function of protein isoforms generated by translational stop codon readthrough in humans.