RESUMO
The oxidative phosphorylation system1 in mammalian mitochondria plays a key role in transducing energy from ingested nutrients2. Mitochondrial metabolism is dynamic and can be reprogrammed to support both catabolic and anabolic reactions, depending on physiological demands or disease states. Rewiring of mitochondrial metabolism is intricately linked to metabolic diseases and promotes tumour growth3-5. Here, we demonstrate that oral treatment with an inhibitor of mitochondrial transcription (IMT)6 shifts whole-animal metabolism towards fatty acid oxidation, which, in turn, leads to rapid normalization of body weight, reversal of hepatosteatosis and restoration of normal glucose tolerance in male mice on a high-fat diet. Paradoxically, the IMT treatment causes a severe reduction of oxidative phosphorylation capacity concomitant with marked upregulation of fatty acid oxidation in the liver, as determined by proteomics and metabolomics analyses. The IMT treatment leads to a marked reduction of complex I, the main dehydrogenase feeding electrons into the ubiquinone (Q) pool, whereas the levels of electron transfer flavoprotein dehydrogenase and other dehydrogenases connected to the Q pool are increased. This rewiring of metabolism caused by reduced mtDNA expression in the liver provides a principle for drug treatment of obesity and obesity-related pathology.
Assuntos
DNA Mitocondrial , Dieta Hiperlipídica , Obesidade , Transcrição Gênica , Animais , Obesidade/metabolismo , Obesidade/etiologia , Camundongos , DNA Mitocondrial/metabolismo , Masculino , Fígado Gorduroso/metabolismo , Fígado Gorduroso/etiologia , Fosforilação Oxidativa , Fígado/metabolismo , Ácidos Graxos/metabolismo , Camundongos Endogâmicos C57BL , OxirreduçãoRESUMO
Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system1-6. The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines. To verify the cellular target, we performed exome sequencing of mutagenized cells and identified a cluster of amino acid substitutions in POLRMT that cause resistance to IMTs. We obtained a cryo-electron microscopy (cryo-EM) structure of POLRMT bound to an IMT, which further defined the allosteric binding site near the active centre cleft of POLRMT. The growth of cancer cells and the persistence of therapy-resistant cancer stem cells has previously been reported to depend on OXPHOS7-17, and we therefore investigated whether IMTs have anti-tumour effects. Four weeks of oral treatment with an IMT is well-tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in xenografts of human cancer cells. In summary, IMTs provide a potent and specific chemical biology tool to study the role of mtDNA expression in physiology and disease.
Assuntos
Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Microscopia Crioeletrônica , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação para Baixo/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Mitocondriais/efeitos dos fármacos , Humanos , Masculino , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Especificidade por Substrato/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E. coli transposition-based recombination bacmid technology (e.g. Bac-to-Bac®) or homologous recombination transfection within insect cells (e.g. flashBAC™). Limited data is presented in the literature to benchmark the protocols used for these baculovirus vectors to facilitate the selection of a system for optimal production of target proteins. Taking advantage of the Protein Production and Purification Partnership in Europe (P4EU) scientific network, a benchmarking initiative was designed to compare the diverse protocols established in thirteen individual laboratories. This benchmarking initiative compared the expression of four selected intracellular proteins (mouse Dicer-2, 204â¯kDa; human ABL1 wildtype, 126â¯kDa; human FMRP, 68â¯kDa; viral vNS1-H1, 76â¯kDa). Here, we present the expression and purification results on these proteins and highlight the significant differences in expression yields obtained using different commercially-packaged baculovirus vectors. The highest expression level for difficult-to-express intracellular protein candidates were observed with the EmBacY baculovirus vector system.
Assuntos
Baculoviridae/genética , Vetores Genéticos/genética , Proteínas Recombinantes/metabolismo , Animais , Linhagem Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Recombinantes/genética , Ribonuclease III/genética , Ribonuclease III/metabolismo , Células Sf9RESUMO
Mitochondria harbor the oxidative phosphorylation (OXPHOS) system, which under aerobic conditions produces the bulk of cellular adenosine triphosphate (ATP). The mitochondrial genome encodes key components of the OXPHOS system, and it is transcribed by the mitochondrial RNA polymerase, POLRMT. The levels of mitochondrial transcription correlate with the respiratory activity of the cell. Therefore, compounds that can increase or decrease mitochondrial gene transcription may be useful for fine-tuning metabolism and could be used to treat metabolic diseases or certain forms of cancer. We here report the establishment of a novel high-throughput assay technology that has allowed us to screen a library of 430,000 diverse compounds for effects on mitochondrial transcription in vitro. Following secondary screens facilitated by the same assay principle, we identified 55 compounds that efficiently and selectively inhibit mitochondrial transcription and that are active also in cell culture. Our method is easily adaptable to other RNA or DNA polymerases and varying spectroscopic detection technologies.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Ensaios de Triagem em Larga Escala , Mitocôndrias/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/biossíntese , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Células HeLa , Humanos , Cinética , Metiltransferases/antagonistas & inibidores , Metiltransferases/genética , Metiltransferases/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas/química , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Sirtuin enzymes regulate metabolism and aging processes through deacetylation of acetyl-lysines in target proteins. More than 6,800 mammalian acetylation sites are known, but few targets have been assigned to most sirtuin isoforms, hampering our understanding of sirtuin function. Here we describe a peptide microarray system displaying 6,802 human acetylation sites for the parallel characterisation of their modification by deacetylases. Deacetylation data for all seven human sirtuins obtained with this system reveal isoform-specific substrate preferences and deacetylation substrate candidates for all sirtuin isoforms, including Sirt4. We confirm malate dehydrogenase protein as a Sirt3 substrate and show that peroxiredoxin 1 and high-mobility group B1 protein are deacetylated by Sirt5 and Sirt1, respectively, at the identified sites, rendering them likely new in vivo substrates. Our microarray platform enables parallel studies on physiological acetylation sites and the deacetylation data presented provide an exciting resource for the identification of novel substrates for all human sirtuins.