RESUMEN
Mfn2 is a mitochondrial outer membrane fusion protein with the additional role of tethering mitochondria to the ER. Here, we describe a novel connection between Mfn2 and calcium release from mitochondria. We show that Mfn2 controls the mitochondrial inner membrane sodium-calcium exchange protein NCLX, which is a major source for calcium release from mitochondria. This discovery was made with the fungal toxin Phomoxanthone (PXA), which induces calcium release from mitochondria. PXA-induced calcium release is blocked by a chemical inhibitor of NCLX, while NCLX and Mfn2 deletions both also prevent PXA-induced calcium release. CETSA experiments show that PXA directly targets Mfn2, which likely controls NCLX through physical interactions since co-immunoprecipitation and proximity ligation assays show increased association between Mfn2 and NCLX upon treatment with PXA. Interactions between Mfn2 and NCLX also increase when cells are treated with mitochondrial ROS-inducing conditions, such as oligomycin treatment of respiring cells, while the interactions do not increase in Oma1 -/- cells. It seems likely that opening of cristae by Oma1-mediated cleavage of Opa1 promotes translocation of NCLX from cristae to the rim where it can come into contact with Mfn2 thus promoting PXA-induced calcium release from mitochondria. These results therefore delineate a pathway that connects ROS produced inside mitochondria with calcium release and signaling in the cytosol.
RESUMEN
In addition to mitochondrial DNA, mitochondrial double-stranded RNA (mtdsRNA) is exported from mitochondria. However, specific channels for RNA transport have not been demonstrated. Here, we begin to characterize channel candidates for mtdsRNA export from the mitochondrial matrix to the cytosol. Down-regulation of SUV3 resulted in the accumulation of mtdsRNAs in the matrix, whereas down-regulation of PNPase resulted in the export of mtdsRNAs to the cytosol. Targeting experiments show that PNPase functions in both the intermembrane space and matrix. Strand-specific sequencing of the double-stranded RNA confirms the mitochondrial origin. Inhibiting or down-regulating outer membrane proteins VDAC1/2 and BAK/BAX or inner membrane proteins PHB1/2 strongly attenuated the export of mtdsRNAs to the cytosol. The cytosolic mtdsRNAs subsequently localized to large granules containing the stress protein TIA-1 and activated the type 1 interferon stress response pathway. Abundant mtdsRNAs were detected in a subset of non-small-cell lung cancer cell lines that were glycolytic, indicating relevance in cancer biology. Thus, we propose that mtdsRNA is a new damage-associated molecular pattern that is exported from mitochondria in a regulated manner.
Asunto(s)
Citosol , Mitocondrias , Prohibitinas , ARN Bicatenario , ARN Mitocondrial , Humanos , Citosol/metabolismo , Mitocondrias/metabolismo , ARN Bicatenario/metabolismo , ARN Mitocondrial/metabolismo , ARN Mitocondrial/genética , Línea Celular Tumoral , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Transporte de ARN , Exorribonucleasas/metabolismo , Exorribonucleasas/genética , Canal Aniónico 1 Dependiente del Voltaje/metabolismo , Canal Aniónico 1 Dependiente del Voltaje/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas MitocondrialesRESUMEN
Mitochondria are critical to the governance of metabolism and bioenergetics in cancer cells1. The mitochondria form highly organized networks, in which their outer and inner membrane structures define their bioenergetic capacity2,3. However, in vivo studies delineating the relationship between the structural organization of mitochondrial networks and their bioenergetic activity have been limited. Here we present an in vivo structural and functional analysis of mitochondrial networks and bioenergetic phenotypes in non-small cell lung cancer (NSCLC) using an integrated platform consisting of positron emission tomography imaging, respirometry and three-dimensional scanning block-face electron microscopy. The diverse bioenergetic phenotypes and metabolic dependencies we identified in NSCLC tumours align with distinct structural organization of mitochondrial networks present. Further, we discovered that mitochondrial networks are organized into distinct compartments within tumour cells. In tumours with high rates of oxidative phosphorylation (OXPHOSHI) and fatty acid oxidation, we identified peri-droplet mitochondrial networks wherein mitochondria contact and surround lipid droplets. By contrast, we discovered that in tumours with low rates of OXPHOS (OXPHOSLO), high glucose flux regulated perinuclear localization of mitochondria, structural remodelling of cristae and mitochondrial respiratory capacity. Our findings suggest that in NSCLC, mitochondrial networks are compartmentalized into distinct subpopulations that govern the bioenergetic capacity of tumours.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Metabolismo Energético , Neoplasias Pulmonares , Mitocondrias , Humanos , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/ultraestructura , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Gotas Lipídicas/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/ultraestructura , Microscopía Electrónica , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Fosforilación Oxidativa , Fenotipo , Tomografía de Emisión de PositronesRESUMEN
Presequence protease (PreP), a 117 kDa mitochondrial M16C metalloprotease vital for mitochondrial proteostasis, degrades presequence peptides cleaved off from nuclear-encoded proteins and other aggregation-prone peptides, such as amyloid ß (Aß). PreP structures have only been determined in a closed conformation; thus, the mechanisms of substrate binding and selectivity remain elusive. Here, we leverage advanced vitrification techniques to overcome the preferential denaturation of one of two ~55 kDa homologous domains of PreP caused by air-water interface adsorption. Thereby, we elucidate cryoEM structures of three apo-PreP open states along with Aß- and citrate synthase presequence-bound PreP at 3.3-4.6 Å resolution. Together with integrative biophysical and pharmacological approaches, these structures reveal the key stages of the PreP catalytic cycle and how the binding of substrates or PreP inhibitor drives a rigid body motion of the protein for substrate binding and catalysis. Together, our studies provide key mechanistic insights into M16C metalloproteases for future therapeutic innovations.
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Péptidos beta-Amiloides , Mitocondrias , Péptidos beta-Amiloides/metabolismo , Microscopía por Crioelectrón , Humanos , Metaloproteasas/metabolismo , Mitocondrias/metabolismo , Conformación Molecular , Conformación Proteica , Especificidad por SustratoRESUMEN
Yeast is a facultative anaerobe and uses diverse electron acceptors to maintain redox-regulated import of cysteine-rich precursors via the mitochondrial intermembrane space assembly (MIA) pathway. With the growing diversity of substrates utilizing the MIA pathway, understanding the capacity of the intermembrane space (IMS) to handle different types of stress is crucial. We used MS to identify additional proteins that interacted with the sulfhydryl oxidase Erv1 of the MIA pathway. Altered inheritance of mitochondria 32 (Aim32), a thioredoxin-like [2Fe-2S] ferredoxin protein, was identified as an Erv1-binding protein. Detailed localization studies showed that Aim32 resided in both the mitochondrial matrix and IMS. Aim32 interacted with additional proteins including redox protein Osm1 and protein import components Tim17, Tim23, and Tim22. Deletion of Aim32 or mutation of conserved cysteine residues that coordinate the Fe-S center in Aim32 resulted in an increased accumulation of proteins with aberrant disulfide linkages. In addition, the steady-state level of assembled TIM22, TIM23, and Oxa1 protein import complexes was decreased. Aim32 also bound to several mitochondrial proteins under nonreducing conditions, suggesting a function in maintaining the redox status of proteins by potentially targeting cysteine residues that may be sensitive to oxidation. Finally, Aim32 was essential for growth in conditions of stress such as elevated temperature and hydroxyurea, and under anaerobic conditions. These studies suggest that the Fe-S protein Aim32 has a potential role in general redox homeostasis in the matrix and IMS. Thus, Aim32 may be poised as a sensor or regulator in quality control for a broad range of mitochondrial proteins.
Asunto(s)
Ferredoxinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Ferredoxinas/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Mitochondrial Ca2 + uptake influences energy production, cell survival, and Ca2 + signaling. The mitochondrial calcium uniporter, MCU, is the primary route for uptake of Ca2 + into the mitochondrial matrix. We have generated a zebrafish MCU mutant that survives to adulthood and exhibits dramatic cardiac phenotypes resembling cardiomyopathy and sinus arrest. MCU hearts contract weakly and have a smaller ventricle with a thin compact layer and reduced trabecular density. Damaged myofibrils and swollen mitochondria were present in the ventricles of MCU mutants, along with gene expression changes indicative of cell stress and altered cardiac structure and function. Using electrocardiography, we found that MCU hearts display conduction system defects and abnormal rhythm, with extended pauses resembling episodes of sinus arrest. Together, our findings suggest that proper mitochondrial Ca2 + homeostasis is crucial for maintaining a healthy adult heart, and establish the MCU mutant as a useful model for understanding the role of mitochondrial Ca2 + handling in adult cardiac biology.
RESUMEN
Mitochondria are essential regulators of cellular energy and metabolism, and have a crucial role in sustaining the growth and survival of cancer cells. A central function of mitochondria is the synthesis of ATP by oxidative phosphorylation, known as mitochondrial bioenergetics. Mitochondria maintain oxidative phosphorylation by creating a membrane potential gradient that is generated by the electron transport chain to drive the synthesis of ATP1. Mitochondria are essential for tumour initiation and maintaining tumour cell growth in cell culture and xenografts2,3. However, our understanding of oxidative mitochondrial metabolism in cancer is limited because most studies have been performed in vitro in cell culture models. This highlights a need for in vivo studies to better understand how oxidative metabolism supports tumour growth. Here we measure mitochondrial membrane potential in non-small-cell lung cancer in vivo using a voltage-sensitive, positron emission tomography (PET) radiotracer known as 4-[18F]fluorobenzyl-triphenylphosphonium (18F-BnTP)4. By using PET imaging of 18F-BnTP, we profile mitochondrial membrane potential in autochthonous mouse models of lung cancer, and find distinct functional mitochondrial heterogeneity within subtypes of lung tumours. The use of 18F-BnTP PET imaging enabled us to functionally profile mitochondrial membrane potential in live tumours.
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Carcinoma de Pulmón de Células no Pequeñas/fisiopatología , Neoplasias Pulmonares/fisiopatología , Potencial de la Membrana Mitocondrial , Células A549 , Animales , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico por imagen , Humanos , Neoplasias Pulmonares/diagnóstico por imagen , Ratones , Ratones Transgénicos , Compuestos Organofosforados , Tomografía de Emisión de PositronesRESUMEN
Erythropoietin (EPO) signaling is critical to many processes essential to terminal erythropoiesis. Despite the centrality of iron metabolism to erythropoiesis, the mechanisms by which EPO regulates iron status are not well-understood. To this end, here we profiled gene expression in EPO-treated 32D pro-B cells and developing fetal liver erythroid cells to identify additional iron regulatory genes. We determined that FAM210B, a mitochondrial inner-membrane protein, is essential for hemoglobinization, proliferation, and enucleation during terminal erythroid maturation. Fam210b deficiency led to defects in mitochondrial iron uptake, heme synthesis, and iron-sulfur cluster formation. These defects were corrected with a lipid-soluble, small-molecule iron transporter, hinokitiol, in Fam210b-deficient murine erythroid cells and zebrafish morphants. Genetic complementation experiments revealed that FAM210B is not a mitochondrial iron transporter but is required for adequate mitochondrial iron import to sustain heme synthesis and iron-sulfur cluster formation during erythroid differentiation. FAM210B was also required for maximal ferrochelatase activity in differentiating erythroid cells. We propose that FAM210B functions as an adaptor protein that facilitates the formation of an oligomeric mitochondrial iron transport complex, required for the increase in iron acquisition for heme synthesis during terminal erythropoiesis. Collectively, our results reveal a critical mechanism by which EPO signaling regulates terminal erythropoiesis and iron metabolism.
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Células Eritroides/metabolismo , Eritropoyetina/metabolismo , Ferroquelatasa/metabolismo , Hemo/biosíntesis , Hierro/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , Células Eritroides/citología , Eritropoyesis , Células HEK293 , Humanos , Proteínas de la Membrana/química , Ratones , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/química , Transporte de ProteínasRESUMEN
Mutations of EXOSC3 have been linked to the rare neurological disorder known as Pontocerebellar Hypoplasia type 1B (PCH1B). EXOSC3 is one of three putative RNA-binding structural cap proteins that guide RNA into the RNA exosome, the cellular machinery that degrades RNA. Using RNAcompete, we identified a G-rich RNA motif binding to EXOSC3. Surface plasmon resonance (SPR) and microscale thermophoresis (MST) indicated an affinity in the low micromolar range of EXOSC3 for long and short G-rich RNA sequences. Although several PCH1B-causing mutations in EXOSC3 did not engage a specific RNA motif as shown by RNAcompete, they exhibited lower binding affinity to G-rich RNA as demonstrated by MST. To test the hypothesis that modification of the RNA-protein interface in EXOSC3 mutants may be phenocopied by small molecules, we performed an in-silico screen of 50â¯000 small molecules and used enzyme-linked immunosorbant assays (ELISAs) and MST to assess the ability of the molecules to inhibit RNA-binding by EXOSC3. We identified a small molecule, EXOSC3-RNA disrupting (ERD) compound 3 (ERD03), which ( i) bound specifically to EXOSC3 in saturation transfer difference nuclear magnetic resonance (STD-NMR), ( ii) disrupted the EXOSC3-RNA interaction in a concentration-dependent manner, and ( iii) produced a PCH1B-like phenotype with a 50% reduction in the cerebellum and an abnormally curved spine in zebrafish embryos. This compound also induced modification of zebrafish RNA expression levels similar to that observed with a morpholino against EXOSC3. To our knowledge, this is the first example of a small molecule obtained by rational design that models the abnormal developmental effects of a neurodegenerative disease in a whole organism.
Asunto(s)
Modelos Animales de Enfermedad , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Isoquinolinas/farmacología , Isoquinolinas/toxicidad , Atrofias Olivopontocerebelosas/genética , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Pez Cebra/anomalías , Animales , Atrofia , Cerebelo/patología , Regulación hacia Abajo , Complejo Multienzimático de Ribonucleasas del Exosoma/química , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Técnicas de Silenciamiento del Gen , Humanos , Isoquinolinas/metabolismo , Simulación del Acoplamiento Molecular , Mutación , Atrofias Olivopontocerebelosas/inducido químicamente , Atrofias Olivopontocerebelosas/patología , Fenotipo , Unión Proteica , Dominios Proteicos , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Curvaturas de la Columna Vertebral/inducido químicamente , Transcriptoma/efectos de los fármacos , Regulación hacia ArribaRESUMEN
Polynucleotide phosphorylase (PNPase) is an essential mitochondria-localized exoribonuclease implicated in multiple biological processes and human disorders. To reveal role(s) for PNPase in mitochondria, we established PNPase knockout (PKO) systems by first shifting culture conditions to enable cell growth with defective respiration. Interestingly, PKO established in mouse embryonic fibroblasts (MEFs) resulted in the loss of mitochondrial DNA (mtDNA). The transcriptional profile of PKO cells was similar to rho0 mtDNA deleted cells, with perturbations in cholesterol (FDR = 6.35 x 10-13), lipid (FDR = 3.21 x 10-11), and secondary alcohol (FDR = 1.04x10-12) metabolic pathway gene expression compared to wild type parental (TM6) MEFs. Transcriptome analysis indicates processes related to axonogenesis (FDR = 4.49 x 10-3), axon development (FDR = 4.74 x 10-3), and axonal guidance (FDR = 4.74 x 10-3) were overrepresented in PKO cells, consistent with previous studies detailing causative PNPase mutations in delayed myelination, hearing loss, encephalomyopathy, and chorioretinal defects in humans. Overrepresentation analysis revealed alterations in metabolic pathways in both PKO and rho0 cells. Therefore, we assessed the correlation of genes implicated in cell cycle progression and total metabolism and observed a strong positive correlation between PKO cells and rho0 MEFs compared to TM6 MEFs. We quantified the normalized biomass accumulation rate of PKO clones at 1.7% (SD ± 2.0%) and 2.4% (SD ± 1.6%) per hour, which was lower than TM6 cells at 3.3% (SD ± 3.5%) per hour. Furthermore, PKO in mouse inner ear hair cells caused progressive hearing loss that parallels human familial hearing loss previously linked to mutations in PNPase. Combined, our study reports that knockout of a mitochondrial nuclease results in mtDNA loss and suggests that mtDNA maintenance could provide a unifying connection for the large number of biological activities reported for PNPase.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , ADN Mitocondrial/metabolismo , Regulación de la Expresión Génica , Pérdida Auditiva/fisiopatología , Mitocondrias/metabolismo , Polirribonucleótido Nucleotidiltransferasa/metabolismo , Animales , Ciclo Celular , ADN Mitocondrial/genética , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/genética , Mutación , Polirribonucleótido Nucleotidiltransferasa/genéticaRESUMEN
The formin-like protein INF2 is an important player in the polymerization of actin filaments. In this issue, Chakrabarti et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201709111) demonstrate that INF2 mediates actin polymerization at the endoplasmic reticulum (ER), resulting in increased ER-mitochondria contacts, calcium uptake by mitochondria, and mitochondrial division.
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Actinas/metabolismo , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Microfilamentos/metabolismo , Mitocondrias/metabolismo , Dinámicas Mitocondriales/fisiología , Señalización del Calcio/fisiología , División Celular/fisiología , Forminas , GTP Fosfohidrolasas/metabolismo , Humanos , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismoRESUMEN
Tim17 and Tim23 are the main subunits of the TIM23 complex, one of the two major essential mitochondrial inner-membrane protein translocon machineries (TIMs). No chemical probes that specifically inhibit TIM23-dependent protein import were known to exist. Here we show that the natural product stendomycin, produced by Streptomyces hygroscopicus, is a potent and specific inhibitor of the TIM23 complex in yeast and mammalian cells. Furthermore, stendomycin-mediated blockage of the TIM23 complex does not alter normal processing of the major regulatory mitophagy kinase PINK1, but TIM23 is required to stabilize PINK1 on the outside of mitochondria to initiate mitophagy upon membrane depolarization.
Asunto(s)
Proteínas Mitocondriales/metabolismo , Péptidos/farmacología , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Péptidos Catiónicos Antimicrobianos , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Estructura Molecular , Péptidos/química , Transporte de Proteínas/efectos de los fármacos , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Prokaryotes have aerobic and anaerobic electron acceptors for oxidative folding of periplasmic proteins. The mitochondrial intermembrane space has an analogous pathway with the oxidoreductase Mia40 and sulfhydryl oxidase Erv1, termed the mitochondrial intermembrane space assembly (MIA) pathway. The aerobic electron acceptors include oxygen and cytochrome c, but an acceptor that can function under anaerobic conditions has not been identified. Here we show that the fumarate reductase Osm1, which facilitates electron transfer from fumarate to succinate, fills this gap as a new electron acceptor. In addition to microsomes, Osm1 localizes to the mitochondrial intermembrane space and assembles with Erv1 in a complex. In reconstitution studies with reduced Tim13, Mia40, and Erv1, the addition of Osm1 and fumarate completes the disulfide exchange pathway that results in Tim13 oxidation. From in vitro import assays, mitochondria lacking Osm1 display decreased import of MIA substrates, Cmc1 and Tim10. Comparative reconstitution assays support that the Osm1/fumarate couple accepts electrons with similar efficiency to cytochrome c and that the cell has strategies to coordinate expression of the terminal electron acceptors. Thus Osm1/fumarate is a new electron acceptor couple in the mitochondrial intermembrane space that seems to function in both aerobic and anaerobic conditions.
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Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Succinato Deshidrogenasa/metabolismo , Citocromos c/metabolismo , Disulfuros/metabolismo , Transporte de Electrón , Electrones , Fumaratos/metabolismo , Membranas Intracelulares/enzimología , Membranas Intracelulares/metabolismo , Microsomas/enzimología , Microsomas/metabolismo , Mitocondrias/enzimología , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Mitocondriales/genética , Oxidación-Reducción , Oxidorreductasas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Pliegue de Proteína , Transporte de Proteínas , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Succinato Deshidrogenasa/genéticaRESUMEN
Fibrils and oligomers are the aggregated protein agents of neuronal dysfunction in ALS diseases. Whereas we now know much about fibril architecture, atomic structures of disease-related oligomers have eluded determination. Here, we determine the corkscrew-like structure of a cytotoxic segment of superoxide dismutase 1 (SOD1) in its oligomeric state. Mutations that prevent formation of this structure eliminate cytotoxicity of the segment in isolation as well as cytotoxicity of the ALS-linked mutants of SOD1 in primary motor neurons and in a Danio rerio (zebrafish) model of ALS. Cytotoxicity assays suggest that toxicity is a property of soluble oligomers, and not large insoluble aggregates. Our work adds to evidence that the toxic oligomeric entities in protein aggregation diseases contain antiparallel, out-of-register ß-sheet structures and identifies a target for structure-based therapeutics in ALS.
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Esclerosis Amiotrófica Lateral/metabolismo , Superóxido Dismutasa-1/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Cristalografía por Rayos X/métodos , Ratones , Neuronas Motoras/metabolismo , Mutación/genética , Conformación Proteica en Lámina beta , Superóxido Dismutasa-1/genéticaRESUMEN
Current antifungal therapies have limited effectiveness in treating invasive fungal infections. Furthermore, the development of new antifungal is currently unable to keep pace with the urgent demand for safe and effective new drugs. Auranofin, an FDA-approved drug for the treatment of rheumatoid arthritis, inhibits growth of a diverse array of clinical isolates of fungi and represents a new antifungal agent with a previously unexploited mechanism of action. In addition to auranofin's potent antifungal activity against planktonic fungi, this drug significantly reduces the metabolic activity of Candida cells encased in a biofilm. Unbiased chemogenomic profiling, using heterozygous S. cerevisiae deletion strains, combined with growth assays revealed three probable targets for auranofin's antifungal activity-mia40, acn9, and coa4. Mia40 is of particular interest given its essential role in oxidation of cysteine rich proteins imported into the mitochondria. Biochemical analysis confirmed auranofin targets the Mia40-Erv1 pathway as the drug inhibited Mia40 from interacting with its substrate, Cmc1, in a dose-dependent manner similar to the control, MB-7. Furthermore, yeast mitochondria overexpressing Erv1 were shown to exhibit resistance to auranofin as an increase in Cmc1 import was observed compared to wild-type yeast. Further in vivo antifungal activity of auranofin was examined in a Caenorhabditis elegans animal model of Cryptococcus neoformans infection. Auranofin significantly reduced the fungal load in infected C. elegans. Collectively, the present study provides valuable evidence that auranofin has significant promise to be repurposed as a novel antifungal agent and may offer a safe, effective, and quick supplement to current approaches for treating fungal infections.
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Antifúngicos/farmacología , Auranofina/farmacología , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Mitocondriales/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/efectos de los fármacos , Biopelículas/efectos de los fármacos , Reposicionamiento de Medicamentos , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Haploinsuficiencia , Humanos , Potenciales de la Membrana , Pruebas de Sensibilidad Microbiana , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Consumo de Oxígeno , Especies Reactivas de Oxígeno/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Diverse protein import pathways into mitochondria use translocons on the outer membrane (TOM) and inner membrane (TIM). We adapted a genetic screen, based on Ura3 mistargeting from mitochondria to the cytosol, to identify small molecules that attenuated protein import. Small molecule mitochondrial import blockers of the Carla Koehler laboratory (MB)-10 inhibited import of substrates that require the TIM23 translocon. Mutational analysis coupled with molecular docking and molecular dynamics modeling revealed that MB-10 binds to a specific pocket in the C-terminal domain of Tim44 of the protein-associated motor (PAM) complex. This region was proposed to anchor Tim44 to the membrane, but biochemical studies with MB-10 show that this region is required for binding to the translocating precursor and binding to mtHsp70 in low ATP conditions. This study also supports a direct role for the PAM complex in the import of substrates that are laterally sorted to the inner membrane, as well as the mitochondrial matrix. Thus, MB-10 is the first small molecule modulator to attenuate PAM complex activity, likely through binding to the C-terminal region of Tim44.
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Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Saccharomyces cerevisiae/efectos de los fármacos , Animales , Sitios de Unión , Pruebas Genéticas , Células HeLa , Humanos , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Neurospora crassa , Transporte de Proteínas/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Pez CebraRESUMEN
SCL25A46 is a mitochondrial carrier protein that surprisingly localizes to the outer membrane and is distantly related to Ugo1. Here we show that a subset of SLC25A46 interacts with mitochondrial dynamics components and the MICOS complex. Decreased expression of SLC25A46 results in increased stability and oligomerization of MFN1 and MFN2 on mitochondria, promoting mitochondrial hyperfusion. A mutation at L341P causes rapid degradation of SLC25A46, which manifests as a rare disease, pontocerebellar hypoplasia. The E3 ubiquitin ligases MULAN and MARCH5 coordinate ubiquitylation of SLC25A46 L341P, leading to degradation by organized activities of P97 and the proteasome. Whereas outer mitochondrial membrane-associated degradation is typically associated with apoptosis or a specialized type of autophagy termed mitophagy, SLC25A46 degradation operates independently of activation of outer membrane stress pathways. Thus SLC25A46 is a new component in mitochondrial dynamics that serves as a regulator for MFN1/2 oligomerization. Moreover, SLC25A46 is selectively degraded from the outer membrane independently of mitophagy and apoptosis, providing a framework for mechanistic studies in the proteolysis of outer membrane proteins.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Dinámicas Mitocondriales/fisiología , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Apoptosis/fisiología , Autofagia/fisiología , Células HEK293 , Células HeLa , Humanos , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Disturbed mitochondrial fusion and fission have been linked to various neurodegenerative disorders. In siblings from two unrelated families who died soon after birth with a profound neurodevelopmental disorder characterized by pontocerebellar hypoplasia and apnoea, we discovered a missense mutation and an exonic deletion in the SLC25A46 gene encoding a mitochondrial protein recently implicated in optic atrophy spectrum disorder. We performed functional studies that confirmed the mitochondrial localization and pro-fission properties of SLC25A46. Knockdown of slc24a46 expression in zebrafish embryos caused brain malformation, spinal motor neuron loss, and poor motility. At the cellular level, we observed abnormally elongated mitochondria, which was rescued by co-injection of the wild-type but not the mutant slc25a46 mRNA. Conversely, overexpression of the wild-type protein led to mitochondrial fragmentation and disruption of the mitochondrial network. In contrast to mutations causing non-lethal optic atrophy, missense mutations causing lethal congenital pontocerebellar hypoplasia markedly destabilize the protein. Indeed, the clinical severity appears inversely correlated with the relative stability of the mutant protein. This genotype-phenotype correlation underscores the importance of SLC25A46 and fine tuning of mitochondrial fission and fusion in pontocerebellar hypoplasia and central neurodevelopment in addition to optic and peripheral neuropathy across the life span.