Biology subjects

Mootha, V. K.

Publications and source records attributed to Mootha, V. K..

15 recordsLinked to original sources

bioRxiv2026

A genetically encoded redox bottleneck constrains human developmental rate

The intrinsically slow pace of human development poses challenges for regenerative medicine and disease modeling. This trait is attributed to low metabolic rates, yet the endogenous mechanisms determining species-specific metabolic flux remain unknown. Here, we identify coupling between glycolytic NADH production and mitochondrial oxidation through the glycerol-3-phosphate (G3P) shuttle as a genetic bottleneck constraining human developmental tempo. Using stem cell-derived models of the segmentation clock, an oscillator whose period reflects developmental rate, we show that low expression of the G3P shuttle enzyme GPD1L limits NADH oxidation in human progenitors compared to mouse. Overexpressing GPD1L boosts metabolic flux, accelerating the segmentation clock, cell cycle, and differentiation across germ layers. G3P-mediated redox coupling is thus a genetically encoded, rate-limiting mechanism that sets the tempo of human development.

developmental biology↗

bioRxiv2026

An optimized "hypoxia in a pill" regimen reverses neurodegenerative disease phenotypes in multiple preclinical models

A growing body of pre-clinical research is demonstrating the therapeutic potential of chronic, continuous hypoxia (11% FIO2) for rare and common diseases (1). However, the chronic delivery of hypoxic gas poses both practical challenges and long-term safety concerns. We previously introduced a small-molecule, ''hypoxia-in-a-pill'' combining a hemoglobin affinity enhancer (GBT440) to limit oxygen delivery with a HIF-2 inhibitor (PT2399) to prevent detrimental compensatory erythropoiesis (2). Although this small-molecule regimen extended the lifespan of the Ndufs4 KO mouse model of mitochondrial complex I deficiency and Leigh syndrome, its efficacy did not match chronic 11% FIO2. Here we optimize this regimen using GBT601, a second-generation hemoglobin affinity enhancer with longer half-life and greater hemoglobin occupancy, and show it rescues key neurodegenerative phenotypes in multiple models. When we initiate therapy in 50 day old Ndufs4 KO mice with advanced disease, GBT601 monotherapy alleviated neurological disease phenotypes and extended median lifespan from 62 days to 105 days, while dual therapy with the GBT601/PT2399 combination extended median lifespan to 158 days. When initiated after onset of advanced disease in a mouse model of Friedreich's ataxia, the combination halted further progression of motor phenotypes. In a mouse model of Parkinson's disease due to a-synuclein toxicity, initiating the GBT601/PT2399 combination after onset of neurodegeneration attenuates brain hyperoxia and lipid peroxidation and reverses motor phenotypes. Importantly, the combination maintained body weight without inducing any signs of pulmonary hypertension. Our findings motivate further pre-clinical and clinical evaluation of our ''hypoxia in a pill'' approach for diseases with high unmet need.

physiology↗

bioRxiv2026

4,5-dihydroxyhexanoic acid is a robust circulating and urine marker of mitochondrial disease and its severity

Management of patients with mitochondrial respiratory chain diseases is challenging, in part because of our incomplete understanding of pathogenesis and a lack of biomarkers. Unknown metabolites account for >90% of detected features in modern metabolomics experiments and hold immense untapped promise for new basic and biomedical research. We recently used mass spectrometry-based metabolomics to identify and validate 19 circulating blood-based biomarkers for patients with the mitochondrial DNA (mtDNA) m.3243A>G pathogenic variant, which is the most frequent cause of the mitochondrial disorder MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). However, the most significantly changing biomarker corresponded to an "unknown" metabolite. Here, we combine cheminformatics with analytical chemistry and identify that feature as 4,5-dihydroxyhexanoic acid (4,5-DHHA), a metabolite previously associated with inherited defects of gamma-aminobutyric acid (GABA) catabolism, but with no prior links to mitochondrial respiratory chain disorders. We validate this finding in an independent MELAS cohort and further show that 4,5-DHHA levels correlate with disease severity and are elevated in patients with other forms of mitochondrial disease and sepsis. Furthermore, brain 4,5-DHHA levels were elevated in two genetic mouse models of mitochondrial disease. In vitro and tissue culture experiments indicate that 4,5-DHHA is generated when the GABA catabolite succinic semialdehyde reacts with an intermediate of the pyruvate dehydrogenase reaction and is sensitive to mitochondrial complex I function. Our work identifies 4,5-DHHA as a robust plasma and urine marker of mitochondrial dysfunction in humans and reveals new connections between the respiratory chain and GABA metabolism. Significance StatementInborn errors of the mitochondrial respiratory chain cause severe, progressive diseases, yet effective treatments and biomarkers remain limited. Modern metabolomics detects thousands of molecules in biofluids, but the vast majority are unidentified. In this study, we investigate the most significantly altered blood metabolite in patients with the most common mitochondrial disease - MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) - and identify it as an 4,5-dihydroxyhexanoic acid (4,5-DHHA). We show that 4,5-DHHA is reproducibly elevated and correlates with severity. Levels are increased across multiple mitochondrial disorders as well as in sepsis and rise when respiratory chain function is impaired. These findings establish 4,5-DHHA as a promising biomarker of mitochondrial dysfunction and reveal a link to dysregulated GABA metabolism.

biochemistry↗

bioRxiv2025

Mitochondrial depolarization stabilizes the vitamin B12 chaperone MMADHC in the cytosol to increase MTR activity

Of the [~]1100 mitochondrial proteins, only a handful like PINK1 and ATFS-1 are known to stabilize and relocalize upon collapse of the proton motive force (PMF) to execute signaling roles. To systematically identify genes that increase exclusively at the protein level upon PMF collapse, we performed a joint proteomic and RNA-seq screen. The screen revealed 10 candidates (six mitochondrial), including the vitamin B12 chaperone MMADHC and cytosolic B12-dependent methionine synthase (MTR). MMADHC is short-lived across cell types and we show that its levels increase with PMF collapse. MMADHC stabilization precedes PINK1 activation in a time course of increasing mtDNA depletion, suggesting greater sensitivity to PMF collapse. MMADHC accumulates in mitochondria with LONP1 inhibition but in the cytosol upon PMF collapse, likely due to mitochondrial import failure. Cytosol-stabilized MMADHC increases MTR levels and activity. Altogether, the mitochondrial PMF regulates the cytosolic B12-dependent MTR, integral to one-carbon metabolism, by controlling the stability and compartmentalization of the B12 chaperone MMADHC. Significance StatementHumans have only two vitamin B12-dependent enzymes - mitochondrial MMUT and cytosolic MTR - and both require a common B12 chaperone MMADHC. We discover that MMADHC is a low abundant, short-lived protein that is continuously imported and degraded by energized mitochondria. Upon collapse of the mitochondrial proton motive force, MMADHC accumulates in the cytosol and increases the levels and activity of MTR, critical for one-carbon metabolism. This PMF-dependent regulation of MMADHC stability and localization is important for understanding cofactor rationing and spatiotemporal compartmentalization of B12 metabolism.

biochemistry↗

bioRxiv2025

The proton motive force maintains mtDNA euploidy by balancing mtDNA replication with cell proliferation

Human mitochondrial DNA (mtDNA) encodes 13 essential components of the electron transport chain (ETC)1. A typical cell contains [~]1000s of copies of mtDNA, but how this copy number is stably maintained is unclear. Here, we track mtDNA copy number (mtCN) recovery in K562 cells following transient, chemically induced depletion to uncover principles of mtCN stability. Below a critical mtCN, ETC activity fails to sustain the proton motive force (PMF) and de novo pyrimidine synthesis--both required for mtDNA replication. PMF-dependent processes like Fe-S cluster biogenesis are also disrupted and stress responses are activated that impair cell proliferation and limit further mtCN dilution by cell division. Nonetheless, mtDNA replication and recovery remain possible via mtDNA-independent PMF, generated by complex V reversal, and uridine salvage. Once mtCN is restored, the ETC and forward complex V activity re-engage, stress responses subside, and proliferation recommences. Each cell division then dilutes mtDNA, serving as a built- in brake on mtCN. Our findings suggest that mtCN homeostasis emerges from the balance of two opposing PMF-driven processes -- mtDNA replication and cell proliferation -- revealing a bioenergetic logic that preserves mtDNA euploidy through repeated cell divisions.

cell biology↗

bioRxiv2025

Mechanism of age-related accumulation of mitochondrial DNA mutations in human blood

One of the strongest signatures of aging is an accumulation of mutant mitochondrial DNA (mtDNA) heteroplasmy. Here we investigate the mechanism underlying this phenomenon by calling mtDNA sequence, abundance, and heteroplasmic variation in human blood using whole genome sequences from [~]750,000 individuals. Our analyses reveal a simple, two-step mechanism: first, individual cells randomly accumulate low levels of "cryptic" mtDNA mutations; then, when a cell clone proliferates, the cryptic mtDNA variants are carried as passenger mutations and become detectable in whole blood. Four lines of evidence support this model: (1) the mutational spectrum of age-accumulating mtDNA variants is consistent with a well-established model of mtDNA replication errors, (2) these mutations are found primarily at low levels of heteroplasmy and do not show evidence of positive selection, (3) high mtDNA mutation burden tends to co-occur in samples harboring somatic driver mutations for clonal hematopoiesis (CH), and (4) nuclear GWAS reveals that germline variants predisposing to CH (such as those near TERT, TCL1A, and SMC4) also increase mtDNA mutation burden. We propose that the high copy number and high mutation rate of mtDNA make it a particularly sensitive blood-based marker of CH. Importantly, our work helps to mechanistically unify three prominent signatures of aging: common germline variants in TERT, clonal hematopoiesis, and observed mtDNA mutation accrual.

genetics↗

bioRxiv2024

Cytosolic N6AMT1-dependent translation supports mitochondrial RNA processing

Mitochondrial biogenesis relies on both the nuclear and mitochondrial genomes, and imbalance in their expression can lead to inborn error of metabolism, inflammation, and aging. Here, we investigate N6AMT1, a nucleo-cytosolic methyltransferase that exhibits genetic co-dependency with mitochondria. We determine transcriptional and translational profiles of N6AMT1 and report that it is required for the cytosolic translation of TRMT10C (MRPP1) and PRORP (MRPP3), two subunits of the mitochondrial RNAse P enzyme. In the absence of N6AMT1, or when its catalytic activity is abolished, RNA processing within mitochondria is impaired, leading to the accumulation of unprocessed and double-stranded RNA, thus preventing mitochondrial protein synthesis and oxidative phosphorylation. Our work sheds light on the function of N6AMT1 in protein synthesis and highlights a cytosolic program required for proper mitochondrial biogenesis.

molecular biology↗

bioRxiv2022

METTL17 is an Fe-S cluster checkpoint for mitochondrial translation

AO_SCPLOWBSTRACTC_SCPLOWFriedreichs ataxia (FA) is the most common monogenic mitochondrial disease. FA is caused by a depletion of the mitochondrial protein frataxin (FXN), an iron-sulfur (Fe-S) cluster biogenesis factor. To better understand the cellular consequences of FA, we performed quantitative proteome profiling of human cells depleted for FXN. Nearly every known Fe-S cluster-containing protein was depleted in the absence of FXN, indicating that as a rule, cluster binding confers stability to Fe-S proteins. Proteomic and genetic interaction mapping identified impaired mitochondrial translation downstream of FXN loss, and specifically highlighted the methyltransferase-like protein METTL17 as a candidate effector. Using comparative sequence analysis, mutagenesis, biochemistry and cryogenic electron 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 on the mitoribosome. Notably, METTL17 overexpression rescued the mitochondrial translation and bioenergetic defects, but not the cellular growth, of FXN null cells. Our data suggest that METTL17 serves as an Fe-S cluster checkpoint: promoting the translation and assembly of Fe-S cluster rich OXPHOS proteins only when Fe-S cluster levels are replete.

cell biology↗

bioRxiv2022

A natural fusion of flavodiiron, rubredoxin, and NADH:rubredoxin oxidoreductase domains is the highly efficient water-forming oxidase of T. vaginalis

Microaerophilic pathogens such as Giardia lamblia and Trichomonas vaginalis have robust oxygen consumption systems to detoxify oxygen and maintain the intracellular redox balance. This oxygen consumption is a result of the H2O-forming NADH oxidase activity of two distinct flavin-containing systems: H2O-forming NADH oxidases (NOXes) and multicomponent flavodiiron proteins (FDPs). Both systems are not membrane-bound and recycle NADH into oxidized NAD+ while simultaneously removing O2 from the local environment, making them crucial for the survival of human microaerophilic pathogens. In this study, using bioinformatic and biochemical analysis, we show that T. vaginalis lacks a NOX-like enzyme, and instead harbors three proteins that are very close in their amino acid sequence and represent a natural fusion between N-terminal FDP, central rubredoxin and C-terminal NADH:rubredoxin oxidoreductase domains. We demonstrate that this natural fusion protein with fully populated flavin redox centers unlike a "stand-alone" FDP (also present in T. vaginalis), directly accepts reducing equivalents of NADH to catalyze the four-electron reduction of O2 to water within a single polypeptide and with an extremely high turnover. Using single particle electron cryo-microscopy (cryo-EM) we present structural insight into the spatial organization of the FDP core within this multidomain fusion protein. Our studies represent an important addition to our understanding of systems that allow human protozoan parasites to maintain their optimal redox balance and survive transient exposure to oxic conditions.

biochemistry↗

bioRxiv2021

Combinatorial G x G x E CRISPR screening and functional analysis highlights SLC25A39 in mitochondrial GSH transport

The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes1-3. The family is highly redundant and their transport activities coupled to metabolic state. Here, we introduce a pooled, dual CRISPR screening strategy that knocks out pairs of transporters in four metabolic states -- glucose, galactose, OXPHOS inhibition, and absence of pyruvate -- designed to unmask the inter-dependence of these genes. In total, we screened 63 genes in four metabolic states, corresponding to 2016 single and pair-wise genetic perturbations. We recovered 19 gene-by-environment (GxE) interactions and 9 gene-by-gene (GxG) interactions. One GxE interaction hit illustrated that the fitness defect in the mitochondrial folate carrier (SLC25A32) KO cells was genetically buffered in galactose due to a lack of substrate in de novo purine biosynthesis. Another GxE interaction hit revealed non-equivalence of the paralogous ATP/ADP exchangers (ANTs) with ANT2 specifically required during OXPHOS inhibition. GxG analysis highlighted a buffering interaction between the iron transporter SLC25A37 and the poorly characterized SLC25A39. Mitochondrial metabolite profiling, organelle transport assays, and structure-guided mutagenesis suggests SLC25A39 is critical for mitochondrial glutathione (GSH) transport. Our work underscores the importance of systemetically investigating family-wide genetic interactions between mitochondrial transporters across many metabolic environments.

cell biology↗

bioRxiv2021

Mitochondrial DNA variation across 56,434 individuals in gnomAD

Databases of allele frequency are extremely helpful for evaluating clinical variants of unknown significance; however, until now, genetic databases such as the Genome Aggregation Database (gnomAD) have ignored the mitochondrial genome (mtDNA). Here we present a pipeline to call mtDNA variants that addresses three technical challenges: (i) detecting homoplasmic and heteroplasmic variants, present respectively in all or a fraction of mtDNA molecules, (ii) circular mtDNA genome, and (iii) misalignment of nuclear sequences of mitochondrial origin (NUMTs). We observed that mtDNA copy number per cell varied across gnomAD cohorts and influenced the fraction of NUMT-derived false-positive variant calls, which can account for the majority of putative heteroplasmies. To avoid false positives, we excluded samples prone to NUMT misalignment (few mtDNA copies per cell), cell line artifacts (many mtDNA copies per cell), or with contamination and we reported variants with heteroplasmy greater than 10%. We applied this pipeline to 56,434 whole genome sequences in the gnomAD v3.1 database that includes individuals of European (58%), African (25%), Latino (10%), and Asian (5%) ancestry. Our gnomAD v3.1 release contains population frequencies for 10,850 unique mtDNA variants at more than half of all mtDNA bases. Importantly, we report frequencies within each nuclear ancestral population and mitochondrial haplogroup. Homoplasmic variants account for most variant calls (98%) and unique variants (85%). We observed that 1/250 individuals carry a pathogenic mtDNA variant with heteroplasmy above 10%. These mitochondrial population allele frequencies are publicly available at gnomad.broadinstitute.org and will aid in diagnostic interpretation and research studies.

genomics↗

bioRxiv2021

Salvage of Ribose from Uridine or RNA Supports Glycolysis when Glucose is Limiting

Summary paragraphGlucose is vital for life, serving both as a source of energy and as a carbon building block for growth. When glucose availability 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 screening and growth assays of 482 pooled PRISM cancer cell lines. We report that catabolism of uridine enables the growth of cells in the complete absence of glucose. While previous studies have shown that the uracil base of uridine can be salvaged to support growth in the setting of mitochondrial electron transport chain deficiency (1), our work shows that the ribose moiety of uridine can be salvaged via a pathway we call "uridinolysis" defined as: [1] the phosphorylytic cleavage of uridine by UPP1/2 into uracil and ribose-1-phosphate (R1P), [2] the conversion of R1P into fructose-6-P and glyceraldehyde-3-P by PGM2 and the non-oxidative branch of the pentose phosphate pathway (non-oxPPP), and [3] their glycolytic utilization to fuel ATP production, biosynthesis and gluconeogenesis. Intriguingly, we report that uridine nucleosides derived from RNA are also a substrate for uridinolysis and that RNA can support growth in glucose-limited conditions. Our results underscore the malleability of central carbon metabolism and raise the provocative hypothesis that RNA can also serve as a potential storage for energy.

biochemistry↗

bioRxiv2021

Human genetic analyses of organelles highlight the nucleus in age-related trait heritability

Most age-related human diseases are accompanied by a decline in cellular organelle integrity, including impaired lysosomal proteostasis and defective mitochondrial oxidative phosphorylation. An open question, however, is the degree to which inherited variation in or near genes encoding each organelle contributes to age-related disease pathogenesis. Here, we evaluate if genetic loci encoding organelle proteomes confer greater-than-expected age-related disease risk. As mitochondrial dysfunction is a "hallmark" of aging, we begin by assessing nuclear and mitochondrial DNA loci near genes encoding the mitochondrial proteome and surprisingly observe a lack of enrichment across 24 age-related traits. Within nine other organelles, we find no enrichment with one exception: the nucleus, where enrichment emanates from nuclear transcription factors. In agreement, we find that genes encoding several organelles tend to be "haplosufficient," while we observe strong purifying selection against heterozygous protein-truncating variants impacting the nucleus. Our work identifies common variation near transcription factors as having outsize influence on age-related trait risk, motivating future efforts to determine if and how this inherited variation then contributes to observed age-related organelle deterioration.

genomics↗

bioRxiv2020

Structural insights into the Ca 2+-dependent gating of the human mitochondrial calcium uniporter

Mitochondrial Ca2+ uptake plays an important role in cellular physiology such as modulating ATP production, regulating cytoplasmic Ca2+ dynamics, and triggering cell death, and is mediated by the mitochondrial calcium uniporter, a highly selective calcium channel localized to the inner mitochondrial membrane. In humans, the uniporter functions as a holocomplex consisting of MCU, EMRE, MICU1 and MICU2, among which MCU and EMRE form a subcomplex and function as the conductive channel while MICU1 and MICU2 are EF-hand proteins that regulate the channel activity in a Ca2+ dependent manner. Here we present the EM structures of the human mitochondrial calcium uniporter holocomplex (uniplex) in the presence and absence of Ca2+, revealing distinct Ca2+ dependent assembly of the uniplex. In the presence of Ca2+, MICU1 and MICU2 form a heterotetramer of MICU1-(MICU2)2-MICU1 and bridge the dimeric form of the MCU-EMRE subcomplex through electrostatic interactions between MICU1 and EMRE, leaving the MCU channel pore unblocked. In the absence of Ca2+, multiple uniplex assemblies are observed but is predominantly occupied by the MICU1 subunit from a MICU1-MICU2 heterodimer blocking the MCU channel pore. Our structural observations suggest that Ca2+ changes the dimerization interaction between MICU1 and MICU2, which in turn determines how the MICU1-MICU2 subcomplex interacts with the MCU-EMRE channel and, consequently, changes the distribution of the uniplex assemblies between the blocked and unblocked states.View Full Text

biochemistry↗

bioRxiv2019

Molecular basis of EMRE-dependence of the human mitochondrial calcium uniporter

The mitochondrial uniporter is calcium-activated calcium channel complex critical for cellular signaling and bioenergetics. MCU, the pore-forming subunit of the uniporter, contains two transmembrane domains and is found in all major eukaryotic taxa. In amoeba and fungi, MCU homologs are sufficient to form a functional calcium channel, whereas human MCU exhibits a strict requirement for the metazoan-specific, single-pass transmembrane protein EMRE for conductance. Here, we exploit this evolutionary divergence to decipher the molecular basis of the human MCUs dependence on EMRE. By systematically generating chimeric proteins that consist of EMRE-independent D. discoideum MCU (DdMCU) and H. sapiens MCU (HsMCU), we converged on a stretch of 10 amino acids in DdMCU that can be transplanted to HsMCU to render it EMRE-dependent. We call this region in human MCU the EMRE-dependence domain (EDD). Crosslinking experiments show that HsEMRE directly interacts with MCU at both of its transmembrane domains as well as the EDD. Based on previously published structures of fungal MCU homologs, the EDD segment is located distal to the calcium pores selectivity filter and appears flexible. We propose that EMRE stabilizes EDD of MCU, permitting both channel opening and calcium conductance

biochemistry↗