Showing papers by "Vamsi K. Mootha published in 2016"

MitoCarta2.0: an updated inventory of mammalian mitochondrial proteins.

Abstract: Mitochondria are complex organelles that house essential pathways involved in energy metabolism, ion homeostasis, signalling and apoptosis. To understand mitochondrial pathways in health and disease, it is crucial to have an accurate inventory of the organelle's protein components. In 2008, we made substantial progress toward this goal by performing in-depth mass spectrometry of mitochondria from 14 organs, epitope tagging/microscopy and Bayesian integration to assemble MitoCarta (www.broadinstitute.org/pubs/MitoCarta): an inventory of genes encoding mitochondrial-localized proteins and their expression across 14 mouse tissues. Using the same strategy we have now reconstructed this inventory separately for human and for mouse based on (i) improved gene transcript models, (ii) updated literature curation, including results from proteomic analyses of mitochondrial sub-compartments, (iii) improved homology mapping and (iv) updated versions of all seven original data sets. The updated human MitoCarta2.0 consists of 1158 human genes, including 918 genes in the original inventory as well as 240 additional genes. The updated mouse MitoCarta2.0 consists of 1158 genes, including 967 genes in the original inventory plus 191 additional genes. The improved MitoCarta 2.0 inventory provides a molecular framework for system-level analysis of mammalian mitochondria.

Mitochondrial dysfunction remodels one-carbon metabolism in human cells

Abstract: Mitochondrial dysfunction is associated with a spectrum of human disorders, ranging from rare, inborn errors of metabolism to common, age-associated diseases such as neurodegeneration. How these lesions give rise to diverse pathology is not well understood, partly because their proximal consequences have not been well-studied in mammalian cells. Here we provide two lines of evidence that mitochondrial respiratory chain dysfunction leads to alterations in one-carbon metabolism pathways. First, using hypothesis-generating metabolic, proteomic, and transcriptional profiling, followed by confirmatory experiments, we report that mitochondrial DNA depletion leads to an ATF4-mediated increase in serine biosynthesis and transsulfuration. Second, we show that lesioning the respiratory chain impairs mitochondrial production of formate from serine, and that in some cells, respiratory chain inhibition leads to growth defects upon serine withdrawal that are rescuable with purine or formate supplementation. Our work underscores the connection between the respiratory chain and one-carbon metabolism with implications for understanding mitochondrial pathogenesis.

Hypoxia as a therapy for mitochondrial disease

Abstract: Defects in the mitochondrial respiratory chain (RC) underlie a spectrum of human conditions, ranging from devastating inborn errors of metabolism to aging. We performed a genome-wide Cas9-mediated screen to identify factors that are protective during RC inhibition. Our results highlight the hypoxia response, an endogenous program evolved to adapt to limited oxygen availability. Genetic or small-molecule activation of the hypoxia response is protective against mitochondrial toxicity in cultured cells and zebrafish models. Chronic hypoxia leads to a marked improvement in survival, body weight, body temperature, behavior, neuropathology, and disease biomarkers in a genetic mouse model of Leigh syndrome, the most common pediatric manifestation of mitochondrial disease. Further preclinical studies are required to assess whether hypoxic exposure can be developed into a safe and effective treatment for human diseases associated with mitochondrial dysfunction.

Complementation of mitochondrial electron transport chain by manipulation of the NAD+/NADH ratio.

Abstract: A decline in electron transport chain (ETC) activity is associated with many human diseases. Although diminished mitochondrial adenosine triphosphate production is recognized as a source of pathology, the contribution of the associated reduction in the ratio of the amount of oxidized nicotinamide adenine dinucleotide (NAD(+)) to that of its reduced form (NADH) is less clear. We used a water-forming NADH oxidase from Lactobacillus brevis (LbNOX) as a genetic tool for inducing a compartment-specific increase of the NAD(+)/NADH ratio in human cells. We used LbNOX to demonstrate the dependence of key metabolic fluxes, gluconeogenesis, and signaling on the cytosolic or mitochondrial NAD(+)/NADH ratios. Expression of LbNOX in the cytosol or mitochondria ameliorated proliferative and metabolic defects caused by an impaired ETC. The results underscore the role of reductive stress in mitochondrial pathogenesis and demonstrate the utility of targeted LbNOX for direct, compartment-specific manipulation of redox state.

Architecture of the mitochondrial calcium uniporter

Abstract: Mitochondria from many eukaryotic clades take up large amounts of calcium (Ca(2+)) via an inner membrane transporter called the uniporter. Transport by the uniporter is membrane potential dependent and sensitive to ruthenium red or its derivative Ru360 (ref. 1). Electrophysiological studies have shown that the uniporter is an ion channel with remarkably high conductance and selectivity. Ca(2+) entry into mitochondria is also known to activate the tricarboxylic acid cycle and seems to be crucial for matching the production of ATP in mitochondria with its cytosolic demand. Mitochondrial calcium uniporter (MCU) is the pore-forming and Ca(2+)-conducting subunit of the uniporter holocomplex, but its primary sequence does not resemble any calcium channel studied to date. Here we report the structure of the pore domain of MCU from Caenorhabditis elegans, determined using nuclear magnetic resonance (NMR) and electron microscopy (EM). MCU is a homo-oligomer in which the second transmembrane helix forms a hydrophilic pore across the membrane. The channel assembly represents a new solution of ion channel architecture, and is stabilized by a coiled-coil motif protruding into the mitochondrial matrix. The critical DXXE motif forms the pore entrance, which features two carboxylate rings; based on the ring dimensions and functional mutagenesis, these rings appear to form the selectivity filter. To our knowledge, this is one of the largest membrane protein structures characterized by NMR, and provides a structural blueprint for understanding the function of this channel.

Homozygous deletion in MICU1 presenting with fatigue and lethargy in childhood

Abstract: Objective: To define the mechanism responsible for fatigue, lethargy, and weakness in 2 cousins who had a normal muscle biopsy. Methods: Exome sequencing, long-range PCR, and Sanger sequencing to identify the pathogenic mutation. Functional analysis in the patient fibroblasts included oxygen consumption measurements, extracellular acidification studies, Western blotting, and calcium imaging, followed by overexpression of the wild-type protein. Results: Analysis of the exome sequencing depth revealed a homozygous deletion of exon 1 of MICU1 within a 2,755-base pair deletion. No MICU1 protein was detected in patient fibroblasts, which had impaired mitochondrial calcium uptake that was rescued through the overexpression of the wild-type allele. Conclusions: MICU1 mutations cause fatigue and lethargy in patients with normal mitochondrial enzyme activities in muscle. The fluctuating clinical course is likely mediated through the mitochondrial calcium uniporter, which is regulated by MICU1.

Natural underlying mtDNA heteroplasmy as a potential source of intra‐person hiPSC variability

Abstract: Functional variability among human clones of induced pluripotent stem cells (hiPSCs) remains a limitation in assembling high-quality biorepositories. Beyond inter-person variability, the root cause of intra-person variability remains unknown. Mitochondria guide the required transition from oxidative to glycolytic metabolism in nuclear reprogramming. Moreover, mitochondria have their own genome (mitochondrial DNA [mtDNA]). Herein, we performed mtDNA next-generation sequencing (NGS) on 84 hiPSC clones derived from a cohort of 19 individuals, including mitochondrial and non-mitochondrial patients. The analysis of mtDNA variants showed that low levels of potentially pathogenic mutations in the original fibroblasts are revealed through nuclear reprogramming, generating mutant hiPSCs with a detrimental effect in their differentiated progeny. Specifically, hiPSC-derived cardiomyocytes with expanded mtDNA mutations non-related with any described human disease, showed impaired mitochondrial respiration, being a potential cause of intra-person hiPSC variability. We propose mtDNA NGS as a new selection criterion to ensure hiPSC quality for drug discovery and regenerative medicine.

Associations between plasma branched-chain amino acids, β-aminoisobutyric acid and body composition.

Abstract: Plasma branched-chain amino acids (BCAA) are elevated in obesity and associated with increased cardiometabolic risk. β-Aminoisobutyric acid (B-AIBA), a recently identified small molecule metabolite, is associated with decreased cardiometabolic risk. Therefore, we investigated the association of BCAA and B-AIBA with each other and with detailed body composition parameters, including abdominal visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT). A cross-sectional study was carried out with lean (n 15) and obese (n 33) men and women. Detailed metabolic evaluations, including measures of body composition, insulin sensitivity and plasma metabolomics were completed. Plasma BCAA were higher (1·6 (se 0·08) (×107) v. 1·3 (se 0·06) (×107) arbitrary units; P = 0·005) in obese v. lean subjects. BCAA were positively associated with VAT (R 0·49; P = 0·0006) and trended to an association with SAT (R 0·29; P = 0·052). The association between BCAA and VAT, but not SAT, remained significant after controlling for age, sex and race on multivariate modelling (P 0·05). Plasma B-AIBA was associated with parameters of insulin sensitivity (Matsuda index R 0·36, P = 0·01; glucose AUC: R −0·30, P = 0·04). Plasma BCAA levels were positively correlated with VAT and markers of insulin resistance. The results suggest a possible complex role of adipose tissue in BCAA homeostasis and insulin resistance.

Natural Product Screening Reveals Naphthoquinone Complex I Bypass Factors.

Abstract: Deficiency of mitochondrial complex I is encountered in both rare and common diseases, but we have limited therapeutic options to treat this lesion to the oxidative phosphorylation system (OXPHOS). Idebenone and menadione are redox-active molecules capable of rescuing OXPHOS activity by engaging complex I-independent pathways of entry, often referred to as “complex I bypass.” In the present study, we created a cellular model of complex I deficiency by using CRISPR genome editing to knock out Ndufa9 in mouse myoblasts, and utilized this cell line to develop a high-throughput screening platform for novel complex I bypass factors. We screened a library of ~40,000 natural product extracts and performed bioassay-guided fractionation on a subset of the top scoring hits. We isolated four plant-derived 1,4-naphthoquinone complex I bypass factors with structural similarity to menadione: chimaphilin and 3-chloro-chimaphilin from Chimaphila umbellata and dehydro-α-lapachone and dehydroiso-α-lapachone from Stereospermum euphoroides. We also tested a small number of structurally related naphthoquinones from commercial sources and identified two additional compounds with complex I bypass activity: 2-methoxy-1,4-naphthoquinone and 2-methoxy-3-methyl-1,4,-naphthoquinone. The six novel complex I bypass factors reported here expand this class of molecules and will be useful as tool compounds for investigating complex I disease biology.

Hypoxia as a therapy for mitochondrial disease

Abstract: Defects in the mitochondrial respiratory chain (RC) underlie a spectrum of human conditions, ranging from devastating inborn errors of metabolism to aging. We performed a genome-wide Cas9-mediated screen to identify factors that are protective during RC inhibition. Our results highlight the hypoxia response, an endogenous program evolved to adapt to limited oxygen availability. Genetic or small-molecule activation of the hypoxia response is protective against mitochondrial toxicity in cultured cells and zebrafish models. Chronic hypoxia leads to a marked improvement in survival, body weight, body temperature, behavior, neuropathology, and disease biomarkers in a genetic mouse model of Leigh syndrome, the most common pediatric manifestation of mitochondrial disease. Further preclinical studies are required to assess whether hypoxic exposure can be developed into a safe and effective treatment for human diseases associated with mitochondrial dysfunction.

Compositions and methods that promote the hypoxia response for treatment of mitochondrial dysfunction and oxidative stress disorders

Abstract: Methods of promoting hypoxia or the hypoxia response for the treatment or prevention of mitochondrial dysfunction and oxidative stress disorders are described. Methods for screening for targets of mitochondrial dysfunction and oxidative stress disorders are also described.

Protein prostheses for mitochondrial diseases or conditions

Abstract: The invention encompasses water-forming NADH and NADPH oxidases and the use of these enzymes to treat mammalian diseases or conditions associated with an elevated NADH/NAD+ ratio or NADPH/NADP+ ratio. Such pathologies include disorders caused by one or more defects in the mitochondrial respiratory chain, glucose metabolism disorders, cancers associated with reductive stress, and aging. The invention also provides a research tool for investigating the effect of exogenous water-forming NADH or NADPH oxidases on the metabolism of a mammalian cell, such as a human cell, and for elucidating the role of respiratory chain proteins in mitochondrial disorders.

Compositions and methods that promote hypoxia or the hypoxia response for treatment and prevention of mitochondrial dysfunction and oxidative stress disorders

Abstract: Methods of promoting hypoxia or the hypoxia response for the treatment or prevention of mitochondrial dysfunction and oxidative stress disorders are described. Methods for screening for targets of mitochondrial dysfunction and oxidative stress disorders are also described.