Showing papers by "Vamsi K. Mootha published in 2021"

MitoCarta3.0: an updated mitochondrial proteome now with sub-organelle localization and pathway annotations.

Abstract: The mammalian mitochondrial proteome is under dual genomic control, with 99% of proteins encoded by the nuclear genome and 13 originating from the mitochondrial DNA (mtDNA). We previously developed MitoCarta, a catalogue of over 1000 genes encoding the mammalian mitochondrial proteome. This catalogue was compiled using a Bayesian integration of multiple sequence features and experimental datasets, notably protein mass spectrometry of mitochondria isolated from fourteen murine tissues. Here, we introduce MitoCarta3.0. Beginning with the MitoCarta2.0 inventory, we performed manual review to remove 100 genes and introduce 78 additional genes, arriving at an updated inventory of 1136 human genes. We now include manually curated annotations of sub-mitochondrial localization (matrix, inner membrane, intermembrane space, outer membrane) as well as assignment to 149 hierarchical 'MitoPathways' spanning seven broad functional categories relevant to mitochondria. MitoCarta3.0, including sub-mitochondrial localization and MitoPathway annotations, is freely available at http://www.broadinstitute.org/mitocarta and should serve as a continued community resource for mitochondrial biology and medicine.

SARS-CoV-2 hijacks folate and one-carbon metabolism for viral replication.

Abstract: The recently identified Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. How this novel beta-coronavirus virus, and coronaviruses more generally, alter cellular metabolism to support massive production of ~30 kB viral genomes and subgenomic viral RNAs remains largely unknown. To gain insights, transcriptional and metabolomic analyses are performed 8 hours after SARS-CoV-2 infection, an early timepoint where the viral lifecycle is completed but prior to overt effects on host cell growth or survival. Here, we show that SARS-CoV-2 remodels host folate and one-carbon metabolism at the post-transcriptional level to support de novo purine synthesis, bypassing viral shutoff of host translation. Intracellular glucose and folate are depleted in SARS-CoV-2-infected cells, and viral replication is exquisitely sensitive to inhibitors of folate and one-carbon metabolism, notably methotrexate. Host metabolism targeted therapy could add to the armamentarium against future coronavirus outbreaks.

Circulating markers of NADH-reductive stress correlate with mitochondrial disease severity

Abstract: Mitochondrial disorders represent a large collection of rare syndromes that are difficult to manage both because we do not fully understand biochemical pathogenesis and because we currently lack facile markers of severity. The m.3243A>G variant is the most common heteroplasmic mitochondrial DNA mutation and underlies a spectrum of diseases, notably mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS). To identify robust circulating markers of m.3243A>G disease, we first performed discovery proteomics, targeted metabolomics, and untargeted metabolomics on plasma from a deeply phenotyped cohort (102 patients, 32 controls). In a validation phase, we measured concentrations of prioritized metabolites in an independent cohort using distinct methods. We validated 20 analytes (1 protein, 19 metabolites) that distinguish patients with MELAS from controls. The collection includes classic (lactate, alanine) and more recently identified (GDF-15, α-hydroxybutyrate) mitochondrial markers. By mining untargeted mass-spectra we uncovered 3 less well-studied metabolite families: N-lactoyl-amino acids, β-hydroxy acylcarnitines, and β-hydroxy fatty acids. Many of these 20 analytes correlate strongly with established measures of severity, including Karnofsky status, and mechanistically, nearly all markers are attributable to an elevated NADH/NAD+ ratio, or NADH-reductive stress. Our work defines a panel of organelle function tests related to NADH-reductive stress that should enable classification and monitoring of mitochondrial disease.

Airway stem cells sense hypoxia and differentiate into protective solitary neuroendocrine cells.

Abstract: Neuroendocrine (NE) cells are epithelial cells that possess many of the characteristics of neurons, including the presence of secretory vesicles and the ability to sense environmental stimuli. The normal physiologic functions of solitary airway NE cells remain a mystery. We show that mouse and human airway basal stem cells sense hypoxia. Hypoxia triggers the direct differentiation of these stem cells into solitary NE cells. Ablation of these solitary NE cells during hypoxia results in increased epithelial injury, whereas the administration of the NE cell peptide CGRP rescues this excess damage. Thus, we identify stem cells that directly sense hypoxia and respond by differentiating into solitary NE cells that secrete a protective peptide that mitigates hypoxic injury.

Fatal Perinatal Mitochondrial Cardiac Failure Caused by Recurrent De Novo Duplications in the ATAD3 Locus

Abstract: Summary Background In about half of all patients with a suspected monogenic disease, genomic investigations fail to identify the diagnosis. A contributing factor is the difficulty with repetitive regions of the genome, such as those generated by segmental duplications. The ATAD3 locus is one such region in which recessive deletions and dominant duplications have recently been reported to cause lethal perinatal mitochondrial diseases characterized by pontocerebellar hypoplasia or cardiomyopathy, respectively. Methods Whole-exome, whole-genome, and long-read DNA sequencing techniques combined with studies of RNA and quantitative proteomics were used to investigate 17 subjects from 16 unrelated families with suspected mitochondrial disease. Findings We report 6 different de novo duplications in the ATAD3 gene locus causing a distinctive presentation, including lethal perinatal cardiomyopathy, persistent hyperlactacidemia, and frequently, corneal clouding or cataracts and encephalopathy. The recurrent 68-kb ATAD3 duplications are identifiable from genome and exome sequencing but usually missed by microarrays. The ATAD3 duplications result in the formation of identical chimeric ATAD3A/ATAD3C proteins, altered ATAD3 complexes, and a striking reduction in mitochondrial oxidative phosphorylation complex I and its activity in heart tissue. Conclusions ATAD3 duplications appear to act in a dominant-negative manner and the de novo inheritance infers a low recurrence risk for families, unlike most pediatric mitochondrial diseases. More than 350 genes underlie mitochondrial diseases. In our experience, the ATAD3 locus is now one of the five most common causes of nuclear-encoded pediatric mitochondrial disease, but the repetitive nature of the locus means ATAD3 diagnoses may be frequently missed by current genomic strategies. Funding Australian NHMRC, US Department of Defense, US National Institutes of Health, Japanese AMED and JSPS agencies, Australian Genomics Health Alliance, and Australian Mito Foundation.

Mitochondrial Clearance of Calcium Facilitated by MICU2 Controls Insulin Secretion

Abstract: Objective Transport of Ca2+ into pancreatic β-cell mitochondria facilitates nutrient-mediated insulin secretion. The underlying mechanism, however, is unclear. Recent establishment of the molecular identity of the mitochondrial Ca2+ uniporter (MCU) and associated proteins allows modification of mitochondrial Ca2+ transport in intact cells. We examined the consequences of deficiency of the accessory protein, MICU2, in rat and human insulin-secreting cells and mouse islets. Methods siRNA-silencing of Micu2 in INS1-832/13 and EndoC-βH1 cell lines was performed; Micu2-/- mice were also studied. Insulin secretion and mechanistic analyses, utilizing live confocal imaging to assess mitochondrial function and intracellular Ca2+ dynamics, were performed. Results Silencing of Micu2 abrogated GSIS in INS1 832/13 and EndoC-βH1 cells. Micu2-/- mice also displayed attenuated GSIS. Mitochondrial Ca2+ uptake declined in MICU2-deficient INS1 832/13 and EndoC-βH1 cells in response to high glucose and high K+. Furthermore, MICU2 silencing in INS1 832/13 cells, presumably through its effects on mitochondrial Ca2+ uptake, perturbed mitochondrial function illustrated by absent mitochondrial membrane hyperpolarization and lowering of the ATP/ADP ratio in response to an elevation of glucose. Despite the loss of mitochondrial Ca2+ uptake, cytosolic Ca2+ was lower in siMICU2-treated INS1 832/13 cells in response to high K+. It was hypothesized that Ca2+ was accumulating in the submembrane compartment in MICU2-deficient cells, resulting in desensitization of voltage-dependent Ca2+ channels, thereby lowering total cytosolic Ca2+. Indeed, upon high K+ stimulation, MICU2-silenced cells showed higher and prolongated rises in submembrane Ca2+ levels. Conclusions MICU2 plays a critical role in β-cell mitochondrial Ca2+ uptake. β-cell mitochondria sequester Ca2+ from the submembrane compartment preventing desensitization of voltage-dependent Ca2+ channels, thereby facilitating GSIS.

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

Abstract: Getting older increases our risk of experiencing a wide range of diseases, such as diabetes, heart disease and neurodegenerative disease. The genetic variations that we inherit from our parents play a major role in predicting this risk. However, the biological networks involved in this process are extremely complex and remain challenging to decipher. Prior studies have suggested that specialised structures inside our body’s cells, called organelles, may have an important role to play in aging. Organelles represent self-contained biological factories inside each cell, designed to perform specific tasks. Examples include the nucleus, which harbours most of the cell’s genetic material, and mitochondria, which help provide cells with energy. Organelles tend to deteriorate and become dysfunctional with age, and mitochondria in particular are badly affected by the ageing process. A decline in organelle activity has been thought to explain ageing and the development of age-related diseases. However, this has never been systematically tested on a large scale at the inherited genetic level. Gupta et al. assessed whether common inherited genetic variation in genes associated with ten different organelles could affect the risk of age-related disease, using a database of DNA samples from more than 300,000 individuals. They considered 24 diseases and traits that become more common with advanced age. Gupta et al. discovered that inherited variants in or near genes associated with the nucleus were consistently linked to age-related disease risks. Most of this signal arose from genes encoding the nuclear transcription factors, proteins that help to control the rate at which genes are expressed. However, variants in genes associated with other organelles, including mitochondria, did not appear to be linked to age-related diseases. This research suggests that inherited variation in transcription factors in the nucleus could act as genetic levers that increase the risk of common, age-related diseases. It also suggests that common genetic variation in other cellular organelles may not be as heavily involved in the development of such diseases. Such insights into the cellular structures and biological pathways involved in ageing and age-related disease also establish new targets for drugs to prevent or treat disease.

Mitochondrial DNA variation across 56,434 individuals in gnomAD

Abstract: 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.

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

Abstract: Aging is associated with defects in many organelles, but an open question is whether the inherited risk for age-related disease is enriched within loci relevant to each organelle. Here, we begin with a focus on mitochondria, as mitochondrial dysfunction is a hallmark of age-related disease. We report a striking lack of enrichment of mitochondria-relevant loci across GWAS for 24 age-related traits. Analyses of nine additional organelles reveal enrichment only for the nucleus, particularly nuclear transcription factors. Consistent with these results, natural selection appears to exert stronger purifying selection against protein-truncating variants for transcription factors compared to mitochondrial pathways, underscoring the importance of inherited variation in gene-regulation in age-related traits.

Human genetic analyses of organelles highlight the nucleus, but not the mitochondrion, in age-related trait heritability

Abstract: 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 impacting each organelle contributes to age-related disease pathogenesis. Here, we evaluate if organelle-relevant loci 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 relevant to mitochondria 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 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 variation contributes to age-related organelle deterioration.

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

Abstract: Summary paragraph Glucose 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.

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

Abstract: 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.