CMTR-1 RNA methyltransferase mutations activate widespread expression of a dopaminergic neuron-specific mitochondrial complex I gene.

The mitochondrial proteome is comprised of approximately 1,100 proteins,1 all but 12 of which are encoded by the nuclear genome in C. elegans. The expression of nuclear-encoded mitochondrial proteins varies widely across cell lineages and metabolic states,2,3,4 but the factors that specify these programs are not known. Here, we identify mutations in two nuclear-localized mRNA processing proteins, CMTR1/CMTR-1 and SRRT/ARS2/SRRT-1, which we show act via the same mechanism to rescue the mitochondrial complex I mutant NDUFS2/gas-1(fc21). CMTR-1 is an FtsJ-family RNA methyltransferase that, in mammals, 2'-O-methylates the first nucleotide 3' to the mRNA CAP to promote RNA stability and translation5,6,7,8. The mutations isolated in cmtr-1 are dominant and lie exclusively in the regulatory G-patch domain. SRRT-1 is an RNA binding partner of the nuclear cap-binding complex and determines mRNA transcript fate.9 We show that cmtr-1 and srrt-1 mutations activate embryonic expression of NDUFS2/nduf-2.2, a paralog of NDUFS2/gas-1 normally expressed only in dopaminergic neurons, and that nduf-2.2 is necessary for the complex I rescue by the cmtr-1 G-patch mutant. Additionally, we find that loss of the cmtr-1 G-patch domain cause ectopic localization of CMTR-1 protein to processing bodies (P bodies), phase-separated organelles involved in mRNA storage and decay.10 P-body localization of the G-patch mutant CMTR-1 contributes to the rescue of the hyperoxia sensitivity of the NDUFS2/gas-1 mutant. This study suggests that mRNA methylation at P bodies may control nduf-2.2 gene expression, with broader implications for how the mitochondrial proteome is translationally remodeled in the face of tissue-specific metabolic requirements and stress.

Mechanism of Action and Translational Potential of (S)-Meclizine in Preemptive Prophylaxis Against Stroke.

BACKGROUND: Mild chemical inhibition of mitochondrial respiration can confer resilience against a subsequent stroke or myocardial infarction, also known as preconditioning. However, the lack of chemicals that can safely inhibit mitochondrial respiration has impeded the clinical translation of the preconditioning concept. We previously showed that meclizine, an over-the-counter antivertigo drug, can toggle metabolism from mitochondrial respiration toward glycolysis and protect against ischemia-reperfusion injury in the brain, heart, and kidney. Here, we examine the mechanism of action of meclizine and report the efficacy and improved safety of the (S) enantiomer. METHODS: We determined the anoxic depolarization latency, tissue and neurological outcomes, and glucose uptake using micro-positron emission tomography after transient middle cerebral artery occlusion in mice pretreated (-17 and -3 hours) with either vehicle or meclizine. To exclude a direct effect on tissue excitability, we also examined spreading depression susceptibility. Furthermore, we accomplished the chiral synthesis of (R)- and (S)-meclizine and compared their effects on oxygen consumption and histamine H1 receptor binding along with their brain concentrations. RESULTS: Micro-positron emission tomography showed meclizine increases glucose uptake in the ischemic penumbra, providing the first in vivo evidence that the neuroprotective effect of meclizine indeed stems from its ability to toggle metabolism toward glycolysis. Consistent with reduced reliance on oxidative phosphorylation to sustain the metabolism, meclizine delayed anoxic depolarization onset after middle cerebral artery occlusion. Moreover, the (S) enantiomer showed reduced H1 receptor binding, a dose-limiting side effect for the racemate, but retained its effect on mitochondrial respiration. (S)-meclizine was at least as efficacious as the racemate in delaying anoxic depolarization onset and decreasing infarct volumes after middle cerebral artery occlusion. CONCLUSIONS: Our data identify (S)-meclizine as a promising new drug candidate with high translational potential as a chemical preconditioning agent for preemptive prophylaxis in patients with high imminent stroke or myocardial infarction risk.

Single-cell analysis reveals context-dependent, cell-level selection of mtDNA.

Heteroplasmy occurs when wild-type and mutant mitochondrial DNA (mtDNA) molecules co-exist in single cells1. Heteroplasmy levels change dynamically in development, disease and ageing2,3, but it is unclear whether these shifts are caused by selection or drift, and whether they occur at the level of cells or intracellularly. Here we investigate heteroplasmy dynamics in dividing cells by combining precise mtDNA base editing (DdCBE)4 with a new method, SCI-LITE (single-cell combinatorial indexing leveraged to interrogate targeted expression), which tracks single-cell heteroplasmy with ultra-high throughput. We engineered cells to have synonymous or nonsynonymous complex I mtDNA mutations and found that cell populations in standard culture conditions purge nonsynonymous mtDNA variants, whereas synonymous variants are maintained. This suggests that selection dominates over simple drift in shaping population heteroplasmy. We simultaneously tracked single-cell mtDNA heteroplasmy and ancestry, and found that, although the population heteroplasmy shifts, the heteroplasmy of individual cell lineages remains stable, arguing that selection acts at the level of cell fitness in dividing cells. Using these insights, we show that we can force cells to accumulate high levels of truncating complex I mtDNA heteroplasmy by placing them in environments where loss of biochemical complex I activity has been reported to benefit cell fitness. We conclude that in dividing cells, a given nonsynonymous mtDNA heteroplasmy can be harmful, neutral or even beneficial to cell fitness, but that the 'sign' of the effect is wholly dependent on the environment.

Circulating N-lactoyl-amino acids and N-formyl-methionine reflect mitochondrial dysfunction and predict mortality in septic shock.

INTRODUCTION: Sepsis is a highly morbid condition characterized by multi-organ dysfunction resulting from dysregulated inflammation in response to acute infection. Mitochondrial dysfunction may contribute to sepsis pathogenesis, but quantifying mitochondrial dysfunction remains challenging. OBJECTIVE: To assess the extent to which circulating markers of mitochondrial dysfunction are increased in septic shock, and their relationship to severity and mortality. METHODS: We performed both full-scan and targeted (known markers of genetic mitochondrial disease) metabolomics on plasma to determine markers of mitochondrial dysfunction which distinguish subjects with septic shock (n = 42) from cardiogenic shock without infection (n = 19), bacteremia without sepsis (n = 18), and ambulatory controls (n = 19) - the latter three being conditions in which mitochondrial function, proxied by peripheral oxygen consumption, is presumed intact. RESULTS: Nine metabolites were significantly increased in septic shock compared to all three comparator groups. This list includes N-formyl-L-methionine (f-Met), a marker of dysregulated mitochondrial protein translation, and N-lactoyl-phenylalanine (lac-Phe), representative of the N-lactoyl-amino acids (lac-AAs), which are elevated in plasma of patients with monogenic mitochondrial disease. Compared to lactate, the clinical biomarker used to define septic shock, there was greater separation between survivors and non-survivors of septic shock for both f-Met and the lac-AAs measured within 24 h of ICU admission. Additionally, tryptophan was the one metabolite significantly decreased in septic shock compared to all other groups, while its breakdown product kynurenate was one of the 9 significantly increased. CONCLUSION: Future studies which validate the measurement of lac-AAs and f-Met in conjunction with lactate could define a sepsis subtype characterized by mitochondrial dysfunction.

T cell activation contributes to purifying selection against the MELAS-associated m.3243A>G pathogenic variant in blood.

T cells have been shown to maintain a lower percentage (heteroplasmy) of the pathogenic m.3243A>G variant (MT-TL1, associated with maternally inherited diabetes and deafness [MIDD] and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes [MELAS]). The mechanism(s) underlying this purifying selection, however, remain unknown. Here we report that purified patient memory CD4+ T cells have lower bulk m.3243A>G heteroplasmy compared to naïve CD4+ T cells. In vitro activation of naïve CD4+ m.3243A>G patient T cells results in lower bulk m.3243A>G heteroplasmy after proliferation. Finally, m.3243A>G patient T cell receptor repertoire sequencing reveals relative oligoclonality compared to controls. These data support a role for T cell activation in peripheral, purifying selection against high m.3243A>G heteroplasmy T cells at the level of the cell, in a likely cell-autonomous fashion.

PMF-seq: a highly scalable screening strategy for linking genetics to mitochondrial bioenergetics.

Our current understanding of mitochondrial organelle physiology has benefited from two broad approaches: classically, cuvette-based measurements with suspensions of isolated mitochondria, in which bioenergetic parameters are monitored acutely in response to respiratory chain substrates and inhibitors1-4, and more recently, highly scalable genetic screens for fitness phenotypes associated with coarse-grained properties of the mitochondrial state5-10. Here we introduce permeabilized-cell mitochondrial function sequencing (PMF-seq) to combine strengths of these two approaches to connect genes to detailed bioenergetic phenotypes. In PMF-seq, the plasma membranes within a pool of CRISPR mutagenized cells are gently permeabilized under conditions that preserve mitochondrial physiology, where detailed bioenergetics can be probed in the same way as with isolated organelles. Cells with desired bioenergetic parameters are selected optically using flow cytometry and subjected to next-generation sequencing. Using PMF-seq, we recover genes differentially required for mitochondrial respiratory chain branching and reversibility. We demonstrate that human D-lactate dehydrogenase specifically conveys electrons from D-lactate into cytochrome c to support mitochondrial membrane polarization. Finally, we screen for genetic modifiers of tBID, a pro-apoptotic protein that acts directly and acutely on mitochondria. We find the loss of the complex V assembly factor ATPAF2 acts as a genetic sensitizer of tBID's acute action. We anticipate that PMF-seq will be valuable for defining genes critical to the physiology of mitochondria and other organelles.

METTL17 is an Fe-S cluster checkpoint for mitochondrial translation.

Friedreich's ataxia (FA) is a debilitating, multisystemic disease caused by the depletion of frataxin (FXN), a mitochondrial iron-sulfur (Fe-S) cluster biogenesis factor. To understand the cellular pathogenesis of FA, we performed quantitative proteomics in FXN-deficient human cells. Nearly every annotated Fe-S cluster-containing protein was depleted, indicating that as a rule, cluster binding confers stability to Fe-S proteins. We also observed depletion of a small mitoribosomal assembly factor METTL17 and evidence of impaired mitochondrial translation. Using comparative sequence analysis, mutagenesis, biochemistry, and cryoelectron microscopy, we show that METTL17 binds to the mitoribosomal small subunit during late assembly and harbors a previously unrecognized [Fe4S4]2+ cluster required for its stability. METTL17 overexpression rescued the mitochondrial translation and bioenergetic defects, but not the cellular growth, of FXN-depleted cells. These findings suggest that METTL17 acts as an Fe-S cluster checkpoint, promoting translation of Fe-S cluster-rich oxidative phosphorylation (OXPHOS) proteins only when Fe-S cofactors are replete.

Hypoxia and intra-complex genetic suppressors rescue complex I mutants by a shared mechanism.

The electron transport chain (ETC) of mitochondria, bacteria, and archaea couples electron flow to proton pumping and is adapted to diverse oxygen environments. Remarkably, in mice, neurological disease due to ETC complex I dysfunction is rescued by hypoxia through unknown mechanisms. Here, we show that hypoxia rescue and hyperoxia sensitivity of complex I deficiency are evolutionarily conserved to C. elegans and are specific to mutants that compromise the electron-conducting matrix arm. We show that hypoxia rescue does not involve the hypoxia-inducible factor pathway or attenuation of reactive oxygen species. To discover the mechanism, we use C. elegans genetic screens to identify suppressor mutations in the complex I accessory subunit NDUFA6/nuo-3 that phenocopy hypoxia rescue. We show that NDUFA6/nuo-3(G60D) or hypoxia directly restores complex I forward activity, with downstream rescue of ETC flux and, in some cases, complex I levels. Additional screens identify residues within the ubiquinone binding pocket as being required for the rescue by NDUFA6/nuo-3(G60D) or hypoxia. This reveals oxygen-sensitive coupling between an accessory subunit and the quinone binding pocket of complex I that can restore forward activity in the same manner as hypoxia.

ChREBP is activated by reductive stress and mediates GCKR-associated metabolic traits.

Common genetic variants in glucokinase regulator (GCKR), which encodes GKRP, a regulator of hepatic glucokinase (GCK), influence multiple metabolic traits in genome-wide association studies (GWASs), making GCKR one of the most pleiotropic GWAS loci in the genome. It is unclear why. Prior work has demonstrated that GCKR influences the hepatic cytosolic NADH/NAD+ ratio, also referred to as reductive stress. Here, we demonstrate that reductive stress is sufficient to activate the transcription factor ChREBP and necessary for its activation by the GKRP-GCK interaction, glucose, and ethanol. We show that hepatic reductive stress induces GCKR GWAS traits such as increased hepatic fat, circulating FGF21, and circulating acylglycerol species, which are also influenced by ChREBP. We define the transcriptional signature of hepatic reductive stress and show its upregulation in fatty liver disease and downregulation after bariatric surgery in humans. These findings highlight how a GCKR-reductive stress-ChREBP axis influences multiple human metabolic traits.

Nuclear genetic control of mtDNA copy number and heteroplasmy in humans.

Mitochondrial DNA (mtDNA) is a maternally inherited, high-copy-number genome required for oxidative phosphorylation1. Heteroplasmy refers to the presence of a mixture of mtDNA alleles in an individual and has been associated with disease and ageing. Mechanisms underlying common variation in human heteroplasmy, and the influence of the nuclear genome on this variation, remain insufficiently explored. Here we quantify mtDNA copy number (mtCN) and heteroplasmy using blood-derived whole-genome sequences from 274,832 individuals and perform genome-wide association studies to identify associated nuclear loci. Following blood cell composition correction, we find that mtCN declines linearly with age and is associated with variants at 92 nuclear loci. We observe that nearly everyone harbours heteroplasmic mtDNA variants obeying two principles: (1) heteroplasmic single nucleotide variants tend to arise somatically and accumulate sharply after the age of 70 years, whereas (2) heteroplasmic indels are maternally inherited as mixtures with relative levels associated with 42 nuclear loci involved in mtDNA replication, maintenance and novel pathways. These loci may act by conferring a replicative advantage to certain mtDNA alleles. As an illustrative example, we identify a length variant carried by more than 50% of humans at position chrM:302 within a G-quadruplex previously proposed to mediate mtDNA transcription/replication switching2,3. We find that this variant exerts cis-acting genetic control over mtDNA abundance and is itself associated in-trans with nuclear loci encoding machinery for this regulatory switch. Our study suggests that common variation in the nuclear genome can shape variation in mtCN and heteroplasmy dynamics across the human population.

Lipoylation is dependent on the ferredoxin FDX1 and dispensable under hypoxia in human cells.

Iron-sulfur clusters (ISC) are essential cofactors that participate in electron transfer, environmental sensing, and catalysis. Amongst the most ancient ISC-containing proteins are the ferredoxin (FDX) family of electron carriers. Humans have two FDXs- FDX1 and FDX2, both of which are localized to mitochondria, and the latter of which is itself important for ISC synthesis. We have previously shown that hypoxia can eliminate the requirement for some components of the ISC biosynthetic pathway, but FDXs were not included in that study. Here, we report that FDX1, but not FDX2, is dispensable under 1% O2 in cultured human cells. We find that FDX1 is essential for production of the lipoic acid cofactor, which is synthesized by the ISC-containing enzyme lipoyl synthase. While hypoxia can rescue the growth phenotype of either FDX1 or lipoyl synthase KO cells, lipoylation in these same cells is not rescued, arguing against an alternative biosynthetic route or salvage pathway for lipoate in hypoxia. Our work reveals the divergent roles of FDX1 and FDX2 in mitochondria, identifies a role for FDX1 in lipoate synthesis, and suggests that loss of lipoic acid can be tolerated under low oxygen tensions in cell culture.

Continuous, but not intermittent, regimens of hypoxia prevent and reverse ataxia in a murine model of Friedreich's ataxia.

Friedreich's ataxia (FA) is a devastating, multi-systemic neurodegenerative disease affecting thousands of people worldwide. We previously reported that oxygen is a key environmental variable that can modify FA pathogenesis. In particular, we showed that chronic, continuous normobaric hypoxia (11% FIO2) prevents ataxia and neurological disease in a murine model of FA, although it did not improve cardiovascular pathology or lifespan. Here, we report the pre-clinical evaluation of seven 'hypoxia-inspired' regimens in the shFxn mouse model of FA, with the long-term goal of designing a safe, practical and effective regimen for clinical translation. We report three chief results. First, a daily, intermittent hypoxia regimen (16 h 11% O2/8 h 21% O2) conferred no benefit and was in fact harmful, resulting in elevated cardiac stress and accelerated mortality. The detrimental effect of this regimen is likely owing to transient tissue hyperoxia that results when daily exposure to 21% O2 combines with chronic polycythemia, as we could blunt this toxicity by pharmacologically inhibiting polycythemia. Second, we report that more mild regimens of chronic hypoxia (17% O2) confer a modest benefit by delaying the onset of ataxia. Third, excitingly, we show that initiating chronic, continuous 11% O2 breathing once advanced neurological disease has already started can rapidly reverse ataxia. Our studies showcase both the promise and limitations of candidate hypoxia-inspired regimens for FA and underscore the need for additional pre-clinical optimization before future translation into humans.

Effectors Enabling Adaptation to Mitochondrial Complex I Loss in Hürthle Cell Carcinoma.

Oncocytic (Hürthle cell) carcinoma of the thyroid (HCC) is genetically characterized by complex I mitochondrial DNA mutations and widespread chromosomal losses. Here, we utilize RNA sequencing and metabolomics to identify candidate molecular effectors activated by these genetic drivers. We find glutathione biosynthesis, amino acid metabolism, mitochondrial unfolded protein response, and lipid peroxide scavenging to be increased in HCC. A CRISPR-Cas9 knockout screen in a new HCC model reveals which pathways are key for fitness, and highlights loss of GPX4, a defense against lipid peroxides and ferroptosis, as a strong liability. Rescuing complex I redox activity with the yeast NADH dehydrogenase (NDI1) in HCC cells diminishes ferroptosis sensitivity, while inhibiting complex I in normal thyroid cells augments ferroptosis induction. Our work demonstrates unmitigated lipid peroxide stress to be an HCC vulnerability that is mechanistically coupled to the genetic loss of mitochondrial complex I activity. SIGNIFICANCE: HCC harbors abundant mitochondria, mitochondrial DNA mutations, and chromosomal losses. Using a CRISPR-Cas9 screen inspired by transcriptomic and metabolomic profiling, we identify molecular effectors essential for cell fitness. We uncover lipid peroxide stress as a vulnerability coupled to mitochondrial complex I loss in HCC. See related article by Frank et al., p. 1884. This article is highlighted in the In This Issue feature, p. 1749.

Salvage of ribose from uridine or RNA supports glycolysis in nutrient-limited conditions.

Glucose is vital for life, serving as both a source of energy and carbon building block for growth. When glucose is limiting, alternative nutrients must be harnessed. To identify mechanisms by which cells can tolerate complete loss of glucose, we performed nutrient-sensitized genome-wide genetic screens and a PRISM growth assay across 482 cancer cell lines. We report that catabolism of uridine from the medium enables the growth of cells in the complete absence of glucose. While previous studies have shown that uridine can be salvaged to support pyrimidine synthesis in the setting of mitochondrial oxidative phosphorylation deficiency1, our work demonstrates that the ribose moiety of uridine or RNA can be salvaged to fulfil energy requirements via a pathway based on: (1) the phosphorylytic cleavage of uridine by uridine phosphorylase UPP1/UPP2 into uracil and ribose-1-phosphate (R1P), (2) the conversion of uridine-derived R1P into fructose-6-P and glyceraldehyde-3-P by the non-oxidative branch of the pentose phosphate pathway and (3) their glycolytic utilization to fuel ATP production, biosynthesis and gluconeogenesis. Capacity for glycolysis from uridine-derived ribose appears widespread, and we confirm its activity in cancer lineages, primary macrophages and mice in vivo. An interesting property of this pathway is that R1P enters downstream of the initial, highly regulated steps of glucose transport and upper glycolysis. We anticipate that 'uridine bypass' of upper glycolysis could be important in the context of disease and even exploited for therapeutic purposes.

Multi-omics identifies large mitoribosomal subunit instability caused by pathogenic MRPL39 variants as a cause of pediatric onset mitochondrial disease.

MRPL39 encodes one of 52 proteins comprising the large subunit of the mitochondrial ribosome (mitoribosome). In conjunction with 30 proteins in the small subunit, the mitoribosome synthesizes the 13 subunits of the mitochondrial oxidative phosphorylation (OXPHOS) system encoded by mitochondrial Deoxyribonucleic acid (DNA). We used multi-omics and gene matching to identify three unrelated individuals with biallelic variants in MRPL39 presenting with multisystem diseases with severity ranging from lethal, infantile-onset (Leigh syndrome spectrum) to milder with survival into adulthood. Clinical exome sequencing of known disease genes failed to diagnose these patients; however quantitative proteomics identified a specific decrease in the abundance of large but not small mitoribosomal subunits in fibroblasts from the two patients with severe phenotype. Re-analysis of exome sequencing led to the identification of candidate single heterozygous variants in mitoribosomal genes MRPL39 (both patients) and MRPL15. Genome sequencing identified a shared deep intronic MRPL39 variant predicted to generate a cryptic exon, with transcriptomics and targeted studies providing further functional evidence for causation. The patient with the milder disease was homozygous for a missense variant identified through trio exome sequencing. Our study highlights the utility of quantitative proteomics in detecting protein signatures and in characterizing gene-disease associations in exome-unsolved patients. We describe Relative Complex Abundance analysis of proteomics data, a sensitive method that can identify defects in OXPHOS disorders to a similar or greater sensitivity to the traditional enzymology. Relative Complex Abundance has potential utility for functional validation or prioritization in many hundreds of inherited rare diseases where protein complex assembly is disrupted.

Hypoxia extends lifespan and neurological function in a mouse model of aging.

There is widespread interest in identifying interventions that extend healthy lifespan. Chronic continuous hypoxia delays the onset of replicative senescence in cultured cells and extends lifespan in yeast, nematodes, and fruit flies. Here, we asked whether chronic continuous hypoxia is beneficial in mammalian aging. We utilized the Ercc1 Δ/- mouse model of accelerated aging given that these mice are born developmentally normal but exhibit anatomic, physiological, and biochemical features of aging across multiple organs. Importantly, they exhibit a shortened lifespan that is extended by dietary restriction, the most potent aging intervention across many organisms. We report that chronic continuous 11% oxygen commenced at 4 weeks of age extends lifespan by 50% and delays the onset of neurological debility in Ercc1 Δ/- mice. Chronic continuous hypoxia did not impact food intake and did not significantly affect markers of DNA damage or senescence, suggesting that hypoxia did not simply alleviate the proximal effects of the Ercc1 mutation, but rather acted downstream via unknown mechanisms. To the best of our knowledge, this is the first study to demonstrate that "oxygen restriction" can extend lifespan in a mammalian model of aging.

Nuclear genetic control of mtDNA copy number and heteroplasmy in humans.

Human mitochondria contain a high copy number, maternally transmitted genome (mtDNA) that encodes 13 proteins required for oxidative phosphorylation. Heteroplasmy arises when multiple mtDNA variants co-exist in an individual and can exhibit complex dynamics in disease and in aging. As all proteins involved in mtDNA replication and maintenance are nuclear-encoded, heteroplasmy levels can, in principle, be under nuclear genetic control, however this has never been shown in humans. Here, we develop algorithms to quantify mtDNA copy number (mtCN) and heteroplasmy levels using blood-derived whole genome sequences from 274,832 individuals of diverse ancestry and perform GWAS to identify nuclear loci controlling these traits. After careful correction for blood cell composition, we observe that mtCN declines linearly with age and is associated with 92 independent nuclear genetic loci. We find that nearly every individual carries heteroplasmic variants that obey two key patterns: (1) heteroplasmic single nucleotide variants are somatic mutations that accumulate sharply after age 70, while (2) heteroplasmic indels are maternally transmitted as mtDNA mixtures with resulting levels influenced by 42 independent nuclear loci involved in mtDNA replication, maintenance, and novel pathways. These nuclear loci do not appear to act by mtDNA mutagenesis, but rather, likely act by conferring a replicative advantage to specific mtDNA molecules. As an illustrative example, the most common heteroplasmy we identify is a length variant carried by >50% of humans at position m.302 within a G-quadruplex known to serve as a replication switch. We find that this heteroplasmic variant exerts cis -acting genetic control over mtDNA abundance and is itself under trans -acting genetic control of nuclear loci encoding protein components of this regulatory switch. Our study showcases how nuclear haplotype can privilege the replication of specific mtDNA molecules to shape mtCN and heteroplasmy dynamics in the human population.

A genetically encoded system for oxygen generation in living cells.

Oxygen plays a key role in supporting life on our planet. It is particularly important in higher eukaryotes where it boosts bioenergetics as a thermodynamically favorable terminal electron acceptor and has important roles in cell signaling and development. Many human diseases stem from either insufficient or excessive oxygen. Despite its fundamental importance, we lack methods with which to manipulate the supply of oxygen with high spatiotemporal resolution in cells and in organisms. Here, we introduce a genetic system, SupplemeNtal Oxygen Released from ChLorite (SNORCL), for on-demand local generation of molecular oxygen in living cells, by harnessing prokaryotic chlorite O2-lyase (Cld) enzymes that convert chlorite (ClO2-) into molecular oxygen (O2) and chloride (Cl-). We show that active Cld enzymes can be targeted to either the cytosol or mitochondria of human cells, and that coexpressing a chlorite transporter results in molecular oxygen production inside cells in response to externally added chlorite. This first-generation system allows fine temporal and spatial control of oxygen production, with immediate research applications. In the future, we anticipate that technologies based on SNORCL will have additional widespread applications in research, biotechnology, and medicine.

Congenital Hypermetabolism and Uncoupled Oxidative Phosphorylation.

We describe the case of identical twin boys who presented with low body weight despite excessive caloric intake. An evaluation of their fibroblasts showed elevated oxygen consumption and decreased mitochondrial membrane potential. Exome analysis revealed a de novo heterozygous variant in ATP5F1B, which encodes the ß subunit of mitochondrial ATP synthase (also called complex V). In yeast, mutations affecting the same region loosen coupling between the proton motive force and ATP synthesis, resulting in high rates of mitochondrial respiration. Expression of the mutant allele in human cell lines recapitulates this phenotype. These data support an autosomal dominant mitochondrial uncoupling syndrome with hypermetabolism. (Funded by the National Institutes of Health.).