RESUMO
Mitochondrial dysfunction is associated with a spectrum of human conditions, ranging from rare, inborn errors of metabolism to the aging process. To identify pathways that modify mitochondrial dysfunction, we performed genome-wide CRISPR screens in the presence of small-molecule mitochondrial inhibitors. We report a compendium of chemical-genetic interactions involving 191 distinct genetic modifiers, including 38 that are synthetic sick/lethal and 63 that are suppressors. Genes involved in glycolysis (PFKP), pentose phosphate pathway (G6PD), and defense against lipid peroxidation (GPX4) scored high as synthetic sick/lethal. A surprisingly large fraction of suppressors are pathway intrinsic and encode mitochondrial proteins. A striking example of such "intra-organelle" buffering is the alleviation of a chemical defect in complex V by simultaneous inhibition of complex I, which benefits cells by rebalancing redox cofactors, increasing reductive carboxylation, and promoting glycolysis. Perhaps paradoxically, certain forms of mitochondrial dysfunction may best be buffered with "second site" inhibitors to the organelle.
Assuntos
Genes Modificadores , Mitocôndrias/genética , Mitocôndrias/patologia , Autoantígenos/metabolismo , Morte Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Epistasia Genética/efeitos dos fármacos , Ferroptose/efeitos dos fármacos , Ferroptose/genética , Genoma , Glutationa Peroxidase/metabolismo , Glicólise/efeitos dos fármacos , Glicólise/genética , Humanos , Células K562 , Mitocôndrias/efeitos dos fármacos , Oligomicinas/toxicidade , Oxirredução , Fosforilação Oxidativa/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Via de Pentose Fosfato/genética , Espécies Reativas de Oxigênio/metabolismo , Ribonucleoproteínas/metabolismo , Antígeno SS-BRESUMO
BACKGROUND: VERVE-101 is an investigational in vivo CRISPR base-editing medicine designed to alter a single DNA base in the PCSK9 gene, permanently turn off hepatic protein production, and thereby durably lower low-density lipoprotein cholesterol. We test the efficacy, durability, tolerability, and potential for germline editing of VERVE-101 in studies of nonhuman primates and a murine F1 progeny study. METHODS: Cynomolgus monkeys were given a single intravenous infusion of a vehicle control (n=10) or VERVE-101 at a dose of 0.75 mg/kg (n=4) or 1.5 mg/kg (n=22) with subsequent follow-up up to 476 days. Two studies assessed the potential for germline editing, including sequencing sperm samples from sexually mature male nonhuman primates treated with VERVE-101 and genotyping offspring from female mice treated with the murine surrogate of VERVE-101 (VERVE-101mu). RESULTS: Liver biopsies 14 days after dosing noted mean PCSK9 editing of 46% and 70% in monkeys treated with VERVE-101 at 0.75 and 1.5 mg/kg, respectively. This translated into mean reductions in blood PCSK9 (proprotein convertase subtilisin/kexin type 9) of 67% and 83% and reductions of low-density lipoprotein cholesterol of 49% and 69% at the 0.75 and 1.5 mg/kg doses, respectively, assessed as time-weighted average change from baseline between day 28 and up to 476 days after dosing. Liver safety monitoring noted a transient rise in alanine aminotransferase and aspartate aminotransferase concentrations after infusion that fully resolved by day 14 with no accompanying change in total bilirubin. In a subset of monkeys necropsied 1 year after dosing, no findings related to VERVE-101 were identified on macroscopic and histopathologic assessment of the liver and other organs. In the study to assess potential germline editing of male nonhuman primates, sperm samples collected after VERVE-101 dosing showed no evidence of PCSK9 editing. Among 436 offspring of female mice treated with a saturating dose of VERVE-101mu, the PCSK9 edit was transmitted in 0 of 436 animals. CONCLUSIONS: VERVE-101 was well tolerated in nonhuman primates and led to 83% lower blood PCSK9 protein and 69% lower low-density lipoprotein cholesterol with durable effects up to 476 days after dosing. These results have supported the initiation of a first-in-human clinical trial in patients with heterozygous familial hypercholesterolemia and atherosclerotic cardiovascular disease.
Assuntos
Edição de Genes , Pró-Proteína Convertase 9 , Animais , Feminino , Humanos , Masculino , Camundongos , LDL-Colesterol/genética , LDL-Colesterol/metabolismo , Primatas/genética , Primatas/metabolismo , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/uso terapêutico , Sêmen/metabolismo , Edição de Genes/métodos , Sistemas CRISPR-Cas , Terapia Genética/métodos , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo II/terapia , Aterosclerose/genética , Aterosclerose/terapiaRESUMO
Mitochondria are complex organelles whose dysfunction underlies a broad spectrum of human diseases. Identifying all of the proteins resident in this organelle and understanding how they integrate into pathways represent major challenges in cell biology. Toward this goal, we performed mass spectrometry, GFP tagging, and machine learning to create a mitochondrial compendium of 1098 genes and their protein expression across 14 mouse tissues. We link poorly characterized proteins in this inventory to known mitochondrial pathways by virtue of shared evolutionary history. Using this approach, we predict 19 proteins to be important for the function of complex I (CI) of the electron transport chain. We validate a subset of these predictions using RNAi, including C8orf38, which we further show harbors an inherited mutation in a lethal, infantile CI deficiency. Our results have important implications for understanding CI function and pathogenesis and, more generally, illustrate how our compendium can serve as a foundation for systematic investigations of mitochondria.
Assuntos
Doença de Leigh/genética , Mitocôndrias/química , Proteínas Mitocondriais/análise , Proteoma , Animais , Bases de Dados de Proteínas , Complexo I de Transporte de Elétrons/metabolismo , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , Especificidade de ÓrgãosRESUMO
Much of our current knowledge about mitochondria has come from studying patients who have respiratory chain disorders. These disorders comprise a large collection of individually rare syndromes, each presenting in a unique and often devastating way. In recent years, there has been great progress in defining their genetic basis, but we still know little about the cascade of events that gives rise to such diverse pathology. Here, we review these disorders and explore them in the context of a contemporary understanding of mitochondrial evolution, biochemistry and genetics. Fully deciphering their pathogenesis is a challenging next step that will inspire the development of drug treatments for rare and common diseases.
Assuntos
Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Evolução Biológica , Transporte de Elétrons/fisiologia , Humanos , Mitocôndrias/patologiaRESUMO
Sodium benzoate is a widely used preservative found in many foods and soft drinks. It is metabolized within mitochondria to produce hippurate, which is then cleared by the kidneys. We previously reported that ingestion of sodium benzoate at the generally regarded as safe (GRAS) dose leads to a robust excursion in the plasma hippurate level [1]. Since previous reports demonstrated adverse effects of benzoate and hippurate on glucose homeostasis in cells and in animal models, we hypothesized that benzoate might represent a widespread and underappreciated diabetogenic dietary exposure in humans. Here, we evaluated whether acute exposure to GRAS levels of sodium benzoate alters insulin and glucose homeostasis through a randomized, controlled, cross-over study of 14 overweight subjects. Serial blood samples were collected following an oral glucose challenge, in the presence or absence of sodium benzoate. Outcome measurements included glucose, insulin, glucagon, as well as temporal mass spectrometry-based metabolic profiles. We did not find a statistically significant effect of an acute oral exposure to sodium benzoate on glucose homeostasis. Of the 146 metabolites targeted, four changed significantly in response to benzoate, including the expected rise in benzoate and hippurate. In addition, anthranilic acid, a tryptophan metabolite, exhibited a robust rise, while acetylglycine dropped. Although our study shows that GRAS doses of benzoate do not have an acute, adverse effect on glucose homeostasis, future studies will be necessary to explore the metabolic impact of chronic benzoate exposure.
Assuntos
Glucose/metabolismo , Metaboloma , Benzoato de Sódio/administração & dosagem , Benzoato de Sódio/metabolismo , Adolescente , Adulto , Anticonvulsivantes/sangue , Glicemia/metabolismo , Estudos Cross-Over , Dieta , Feminino , Conservantes de Alimentos/administração & dosagem , Conservantes de Alimentos/metabolismo , Glucagon/sangue , Glicina/análogos & derivados , Glicina/sangue , Hipuratos/sangue , Homeostase , Humanos , Insulina/sangue , Masculino , Sobrepeso , Adulto Jovem , ortoaminobenzoatos/sangueRESUMO
PPARgamma is a member of the nuclear receptor family for which agonist ligands have antigrowth effects. However, clinical studies using PPARgamma ligands as a monotherapy failed to show a beneficial effect. Here we have studied the effects of PPARgamma activation with chemotherapeutic agents in current use for specific cancers. We observed a striking synergy between rosiglitazone and platinum-based drugs in several different cancers both in vitro and using transplantable and chemically induced "spontaneous" tumor models. The effect appears to be due in part to PPARgamma-mediated downregulation of metallothioneins, proteins that have been shown to be involved in resistance to platinum-based therapy. These data strongly suggest combining PPARgamma agonists and platinum-based drugs for the treatment of certain human cancers.
Assuntos
Antineoplásicos/farmacologia , Carboplatina/farmacologia , Divisão Celular/efeitos dos fármacos , PPAR gama/agonistas , Tiazolidinedionas/farmacologia , Sinergismo Farmacológico , Ligantes , RosiglitazonaRESUMO
BACKGROUND: Mitochondrial diseases comprise a diverse set of clinical disorders that affect multiple organ systems with varying severity and age of onset. Due to their clinical and genetic heterogeneity, these diseases are difficult to diagnose. We have developed a targeted exome sequencing approach to improve our ability to properly diagnose mitochondrial diseases and apply it here to an individual patient. Our method targets mitochondrial DNA (mtDNA) and the exons of 1,600 nuclear genes involved in mitochondrial biology or Mendelian disorders with multi-system phenotypes, thereby allowing for simultaneous evaluation of multiple disease loci. CASE PRESENTATION: Targeted exome sequencing was performed on a patient initially suspected to have a mitochondrial disorder. The patient presented with diabetes mellitus, diffuse brain atrophy, autonomic neuropathy, optic nerve atrophy, and a severe amnestic syndrome. Further work-up revealed multiple heteroplasmic mtDNA deletions as well as profound thiamine deficiency without a clear nutritional cause. Targeted exome sequencing revealed a homozygous c.1672C > T (p.R558C) missense mutation in exon 8 of WFS1 that has previously been reported in a patient with Wolfram syndrome. CONCLUSION: This case demonstrates how clinical application of next-generation sequencing technology can enhance the diagnosis of patients suspected to have rare genetic disorders. Furthermore, the finding of unexplained thiamine deficiency in a patient with Wolfram syndrome suggests a potential link between WFS1 biology and thiamine metabolism that has implications for the clinical management of Wolfram syndrome patients.
Assuntos
Análise Mutacional de DNA , Exoma , Sequenciamento de Nucleotídeos em Larga Escala , Doenças Mitocondriais/diagnóstico , Síndrome de Wolfram/diagnóstico , Atrofia , Encéfalo/patologia , DNA Mitocondrial/química , Diagnóstico Diferencial , Éxons , Homozigoto , Humanos , Imageamento por Ressonância Magnética , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Doenças Mitocondriais/genética , Mutação de Sentido Incorreto , Síndrome de Wolfram/genéticaRESUMO
PURPOSE: Current therapy for lung cancer involves multimodality therapies. However, many patients are either refractory to therapy or develop drug resistance. KRAS and epidermal growth factor receptor (EGFR) mutations represent some of the most common mutations in lung cancer, and many studies have shown the importance of these mutations in both carcinogenesis and chemoresistance. Genetically engineered murine models of mutant EGFR and KRAS have been developed that more accurately recapitulate human lung cancer. Recently, using cell-based experiments, we showed that platinum-based drugs and the antidiabetic drug rosiglitazone (PPARgamma ligand) interact synergistically to reduce cancer cell and tumor growth. Here, we directly determined the efficacy of the PPARgamma/carboplatin combination in these more relevant models of drug resistant non-small cell lung cancer. EXPERIMENTAL DESIGN: Tumorigenesis was induced by activation of either mutant KRAS or EGFR. Mice then received either rosiglitazone or carboplatin monotherapy, or a combination of both drugs. Change in tumor burden, pathology, and evidence of apoptosis and cell growth were assessed. RESULTS: Tumor burden remained unchanged or increased in the mice after monotherapy with either rosiglitazone or carboplatin. In striking contrast, we observed significant tumor shrinkage in mice treated with these drugs in combination. Immunohistochemical analyses showed that this synergy was mediated via both increased apoptosis and decreased proliferation. Importantly, this synergy between carboplatin and rosiglitazone did not increase systemic toxicity. CONCLUSIONS: These data show that the PPARgamma ligand/carboplatin combination is a new therapy worthy of clinical investigation in lung cancers, including those cancers that show primary resistance to platinum therapy or acquired resistance to targeted therapy.
Assuntos
Antineoplásicos/uso terapêutico , Carboplatina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Hipoglicemiantes/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Tiazolidinedionas/uso terapêutico , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quimioterapia Combinada , Receptores ErbB/fisiologia , Genes ras/fisiologia , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Imageamento por Ressonância Magnética , Camundongos , Camundongos Transgênicos , PPAR gama/agonistas , Inibidores de Proteínas Quinases/efeitos adversos , Rosiglitazona , Uteroglobina/fisiologiaRESUMO
This study assessed the effectiveness of surgical sympathetic denervation of the common hepatic artery (CHADN) in improving glucose tolerance. CHADN eliminated norepinephrine content in the liver and partially decreased it in the pancreas and the upper gut. We assessed oral glucose tolerance at baseline and after 4 weeks of high-fat high-fructose (HFHF) feeding. Dogs were then randomized to sham surgery (SHAM) (n = 9) or CHADN surgery (n = 11) and retested 2.5 or 3.5 weeks later while still on the HFHF diet. CHADN improved glucose tolerance by â¼60% in part because of enhanced insulin secretion, as indicated by an increase in the insulinogenic index. In a subset of dogs (SHAM, n = 5; CHADN, n = 6), a hyperinsulinemic-hyperglycemic clamp was used to assess whether CHADN could improve hepatic glucose metabolism independent of a change in insulin release. CHADN reduced the diet-induced defect in net hepatic glucose balance by 37%. In another subset of dogs (SHAM, n = 4; CHADN, n = 5) the HFHF diet was continued for 3 months postsurgery and the improvement in glucose tolerance caused by CHADN continued. In conclusion, CHADN has the potential to enhance postprandial glucose clearance in states of diet-induced glucose intolerance.
Assuntos
Dieta Hiperlipídica , Açúcares da Dieta , Intolerância à Glucose/metabolismo , Artéria Hepática/inervação , Fígado/metabolismo , Norepinefrina/metabolismo , Simpatectomia , Animais , Cães , Frutose , Técnica Clamp de Glucose , Teste de Tolerância a Glucose , Masculino , Pâncreas/metabolismo , Distribuição Aleatória , Trato Gastrointestinal Superior/metabolismoRESUMO
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.
Assuntos
Produtos Biológicos/farmacologia , Complexo I de Transporte de Elétrons/deficiência , Complexo I de Transporte de Elétrons/metabolismo , Naftoquinonas/farmacologia , Animais , Bignoniaceae/química , Produtos Biológicos/química , Sistemas CRISPR-Cas , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Complexo I de Transporte de Elétrons/genética , Ericaceae/química , Edição de Genes , Técnicas de Inativação de Genes , Ensaios de Triagem em Larga Escala , Camundongos , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Naftoquinonas/química , Fosforilação Oxidativa , Smegmamorpha/metabolismoRESUMO
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.
Assuntos
Carbono/metabolismo , Fosforilação Oxidativa , Linhagem Celular , Perfilação da Expressão Gênica , Humanos , Metaboloma , Proteoma/análiseRESUMO
Tau hyperphosphorylation is a central event in the development of Alzheimer's Disease (AD). Protein phosphatase 2A (PP2A) heterotrimer formation is necessary for efficient dephosphorylation of the tau protein. S-Adenosylmethionine-dependent carboxyl methylation is essential for the assembly of PP2A heterotrimers. Epidemiological evidence indicates that elevated plasma homocysteine is an independent risk factor for AD. Homocysteine is a key intermediate in the methyl cycle and elevated plasma homocysteine results in a global decrease in cellular methylation. We propose that the PP2A methylation system is the link relating elevated plasma homocysteine to AD.
Assuntos
Doença de Alzheimer/etiologia , Homocisteína/sangue , Modelos Biológicos , Fosfoproteínas Fosfatases/metabolismo , Doença de Alzheimer/sangue , Doença de Alzheimer/enzimologia , Humanos , Metilação , Fosforilação , Proteína Fosfatase 2 , Fatores de Risco , Proteínas tau/metabolismoRESUMO
Despite the role of aerobic glycolysis in cancer, recent studies highlight the importance of the mitochondria and biosynthetic pathways as well. PPARγ coactivator 1α (PGC1α) is a key transcriptional regulator of several metabolic pathways including oxidative metabolism and lipogenesis. Initial studies suggested that PGC1α expression is reduced in tumors compared with adjacent normal tissue. Paradoxically, other studies show that PGC1α is associated with cancer cell proliferation. Therefore, the role of PGC1α in cancer and especially carcinogenesis is unclear. Using Pgc1α(-/-) and Pgc1α(+/+) mice, we show that loss of PGC1α protects mice from azoxymethane-induced colon carcinogenesis. Similarly, diethylnitrosamine-induced liver carcinogenesis is reduced in Pgc1α(-/-) mice as compared with Pgc1α(+/+) mice. Xenograft studies using gain and loss of PGC1α expression showed that PGC1α also promotes tumor growth. Interestingly, while PGC1α induced oxidative phosphorylation and tricarboxylic acid cycle gene expression, we also observed an increase in the expression of two genes required for de novo fatty acid synthesis, ACC and FASN. In addition, SLC25A1 and ACLY, which are required for the conversion of glucose into acetyl-CoA for fatty acid synthesis, were also increased by PGC1α, thus linking the oxidative and lipogenic functions of PGC1α. Indeed, using stable (13)C isotope tracer analysis, we show that PGC1α increased de novo lipogenesis. Importantly, inhibition of fatty acid synthesis blunted these progrowth effects of PGC1α. In conclusion, these studies show for the first time that loss of PGC1α protects against carcinogenesis and that PGC1α coordinately regulates mitochondrial and fatty acid metabolism to promote tumor growth.