Showing papers by "Vamsi K. Mootha published in 2019"

Oxygen and mammalian cell culture: are we repeating the experiment of Dr. Ox?

Abstract: Mammalian cell culture represents a cornerstone of modern biomedical research. There is growing appreciation that the media conditions in which cells are cultured can profoundly influence the observed biology and reproducibility. Here, we consider a key but often ignored variable, oxygen, and review why being mindful of this environmental parameter is so important in the design and interpretation of cell culture studies.

Itaconyl-CoA forms a stable biradical in methylmalonyl-CoA mutase and derails its activity and repair

Abstract: Itaconate is an immunometabolite with both anti-inflammatory and bactericidal effects. Its coenzyme A (CoA) derivative, itaconyl-CoA, inhibits B12-dependent methylmalonyl-CoA mutase (MCM) by an unknown mechanism. We demonstrate that itaconyl-CoA is a suicide inactivator of human and Mycobacterium tuberculosis MCM, which forms a markedly air-stable biradical adduct with the 5′-deoxyadenosyl moiety of the B12 coenzyme. Termination of the catalytic cycle in this way impairs communication between MCM and its auxiliary repair proteins. Crystallography and spectroscopy of the inhibited enzyme are consistent with a metal-centered cobalt radical ~6 angstroms away from the tertiary carbon-centered radical and suggest a means of controlling radical trajectories during MCM catalysis. Mycobacterial MCM thus joins enzymes in the glyoxylate shunt and the methylcitrate cycle as targets of itaconate in pathogen propionate metabolism.

Crystal structure of MICU2 and comparison with MICU1 reveal insights into the uniporter gating mechanism

Abstract: The mitochondrial uniporter is a Ca2+-channel complex resident within the organelle’s inner membrane. In mammalian cells the uniporter’s activity is regulated by Ca2+ due to concerted action of MICU1 and MICU2, two paralogous, but functionally distinct, EF-hand Ca2+-binding proteins. Here we present the X-ray structure of the apo form of Mus musculus MICU2 at 2.5-A resolution. The core structure of MICU2 is very similar to that of MICU1. It consists of two lobes, each containing one canonical Ca2+-binding EF-hand (EF1, EF4) and one structural EF-hand (EF2, EF3). Two molecules of MICU2 form a symmetrical dimer stabilized by highly conserved hydrophobic contacts between exposed residues of EF1 of one monomer and EF3 of another. Similar interactions stabilize MICU1 dimers, allowing exchange between homo- and heterodimers. The tight EF1–EF3 interface likely accounts for the structural and functional coupling between the Ca2+-binding sites in MICU1, MICU2, and their complex that leads to the previously reported Ca2+-binding cooperativity and dominant negative effect of mutation of the Ca2+-binding sites in either protein. The N- and C-terminal segments of the two proteins are distinctly different. In MICU2 the C-terminal helix is significantly longer than in MICU1, and it adopts a more rigid structure. MICU2’s C-terminal helix is dispensable in vitro for its interaction with MICU1 but required for MICU2’s function in cells. We propose that in the MICU1–MICU2 oligomeric complex the C-terminal helices of both proteins form a central semiautonomous assembly which contributes to the gating mechanism of the uniporter.

A patient with homozygous nonsense variants in two Leigh syndrome disease genes: Distinguishing a dual diagnosis from a hypomorphic protein-truncating variant

Abstract: Leigh syndrome is a mitochondrial disease caused by pathogenic variants in over 85 genes. Whole exome sequencing of a patient with Leigh-like syndrome identified homozygous protein-truncating variants in two genes associated with Leigh syndrome; a reported pathogenic variant in PDHX (NP_003468.2:p.(Arg446*)), and an uncharacterized variant in complex I (CI) assembly factor TIMMDC1 (NP_057673.2:p.(Arg225*)). The TIMMDC1 variant was predicted to truncate 61 amino acids at the C-terminus and functional studies demonstrated a hypomorphic impact of the variant on CI assembly. However, the mutant protein could still rescue CI assembly in TIMMDC1 knockout cells and the patient's clinical phenotype was not clearly distinct from that of other patients with the same PDHX defect. Our data suggest that the hypomorphic effect of the TIMMDC1 protein-truncating variant does not constitute a dual diagnosis in this individual. We recommend cautious assessment of variants in the C-terminus of TIMMDC1 and emphasize the need to consider the caveats detailed within the American College of Medical Genetics and Genomics (ACMG) criteria when assessing variants.

Bayesian hidden Markov tree models for clustering genes with shared evolutionary history

Abstract: Determination of functions for poorly characterized genes is crucial for understanding biological processes and studying human diseases. Functionally associated genes are often gained and lost together through evolution. Therefore identifying co-evolution of genes can predict functional gene-gene associations. We describe here the full statistical model and computational strategies underlying the original algorithm CLustering by Inferred Models of Evolution (CLIME 1.0) recently reported by us (Cell 158 (2014) 213–225). CLIME 1.0 employs a mixture of tree-structured hidden Markov models for gene evolution process, and a Bayesian model-based clustering algorithm to detect gene modules with shared evolutionary histories (termed evolutionary conserved modules, or ECMs). A Dirichlet process prior was adopted for estimating the number of gene clusters and a Gibbs sampler was developed for posterior sampling. We further developed an extended version, CLIME 1.1, to incorporate the uncertainty on the evolutionary tree structure. By simulation studies and benchmarks on real data sets, we show that CLIME 1.0 and CLIME 1.1 outperform traditional methods that use simple metrics (e.g., the Hamming distance or Pearson correlation) to measure co-evolution between pairs of genes.

Impaired hypoxic pulmonary vasoconstriction in a mouse model of Leigh syndrome

Abstract: Hypoxic pulmonary vasoconstriction (HPV) is a physiological vasomotor response that maintains systemic oxygenation by matching perfusion to ventilation during alveolar hypoxia. Although mitochondri...

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

Abstract: 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 MCU’s 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 pore’s selectivity filter and appears flexible. We propose that EMRE stabilizes EDD of MCU, permitting both channel opening and calcium conductance

Biallelic mutations in MRPS34 lead to instability of the small mitoribosomal subunit and Leigh syndrome

Abstract: The synthesis of all 13 mitochondrial DNA (mtDNA)-encoded protein subunits of the human oxidative phosphorylation (OXPHOS) system is carried out by mitochondrial ribosomes (mitoribosomes). Defects in the stability of mitoribosomal proteins or mitoribosome assembly impair mitochondrial protein translation, causing combined OXPHOS enzyme deficiency and clinical disease. Here we report four autosomal-recessive pathogenic mutations in the gene encoding the small mitoribosomal subunit protein, MRPS34, in six subjects from four unrelated families with Leigh syndrome and combined OXPHOS defects. Whole-exome sequencing was used to independently identify all variants. Two splice-site mutations were identified, including homozygous c.321+1G>T in a subject of Italian ancestry and homozygous c.322-10G>A in affected sibling pairs from two unrelated families of Puerto Rican descent. In addition, compound heterozygous MRPS34 mutations were identified in a proband of French ancestry; a missense (c.37G>A [p.Glu13Lys]) and a nonsense (c.94C>T [p.Gln32∗]) variant. We demonstrated that these mutations reduce MRPS34 protein levels and the synthesis of OXPHOS subunits encoded by mtDNA. Examination of the mitoribosome profile and quantitative proteomics showed that the mitochondrial translation defect was caused by destabilization of the small mitoribosomal subunit and impaired monosome assembly. Lentiviral-mediated expression of wild-type MRPS34 rescued the defect in mitochondrial translation observed in skin fibroblasts from affected subjects, confirming the pathogenicity of MRPS34 mutations. Our data establish that MRPS34 is required for normal function of the mitoribosome in humans and furthermore demonstrate the power of quantitative proteomic analysis to identify signatures of defects in specific cellular pathways in fibroblasts from subjects with inherited disease.

Mitochondrial clearance of Ca2+ controls insulin secretion

Abstract: SUMMARY Transport of Ca2+ from the cytosol to the mitochondrial matrix of insulin-secreting pancreatic β-cells facilitates nutrient-mediated insulin secretion. However, the underlying mechanism is unclear. The establishment of the molecular identity of the mitochondrial Ca2+ uniporter (MCU) and associated proteins has allowed mitochondrial Ca2+ transport to be modified in intact cells. We examined the consequences of deficiency of the accessory protein, MICU2, in rat and human insulin-secreting cell lines as well as in mouse islets. Glucose-induced mitochondrial Ca2+ elevation and inner membrane hyperpolarization were reduced, together with cytosolic ATP/ADP-ratios and insulin secretion. Insulin secretion in Micu2 knock out mice was attenuated in vitro as well as in vivo. While KCl-evoked sub-plasmalemmal Ca2+ increases were more pronounced, the global cytosolic Ca2+ response was, surprisingly, diminished in MICU2-deficient cells. These findings were supported by selective inhibition of mitochondrial Ca2+ uptake by mitochondrial depolarization. It is concluded that mitochondrial Ca2+ transport plays an additional and hitherto unrecognized role in stimulated β-cells by regulating net Ca2+ entry across the plasma membrane. This is likely accounted for by clearing of sub-plasmalemmal Ca2+ levels by mitochondria located near the plasma membrane.