RESUMO
Signaling circuits crucial to systemic physiology are widespread, yet uncovering their molecular underpinnings remains a barrier to understanding the etiology of many metabolic disorders. Here, we identified a copper-linked signaling circuit activated by disruption of mitochondrial function in the murine liver or heart that resulted in atrophy of the spleen and thymus and caused a peripheral white blood cell deficiency. We demonstrated that the leukopenia was caused by α-fetoprotein, which required copper and the cell surface receptor CCR5 to promote white blood cell death. We further showed that α-fetoprotein expression was upregulated in several cell types upon inhibition of oxidative phosphorylation. Collectively, our data argue that α-fetoprotein may be secreted by bioenergetically stressed tissue to suppress the immune system, an effect that may explain the recurrent or chronic infections that are observed in a subset of mitochondrial diseases or in other disorders with secondary mitochondrial dysfunction.
Assuntos
Cobre , Doenças Mitocondriais , Camundongos , Animais , Cobre/metabolismo , alfa-Fetoproteínas/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Terapia de ImunossupressãoRESUMO
Two common γ-chain family cytokines IL-2 and IL-15 stimulate the same mammalian target of rapamycin complex-1 (mTORC1) signaling yet induce effector T (TE) and memory T (TM) cell differentiation via a poorly understood mechanism(s). Here, we prepared in vitro IL-2-stimulated TE (IL-2/TE) and IL-15-stimulated TM (IL-15/TM) cells for characterization by flow cytometry, Western blotting, confocal microscopy and Seahorse-assay analyses. We demonstrate that IL-2 and IL-15 stimulate strong and weak mTORC1 signals, respectively, which lead to the formation of CD62 ligand (CD62L)- killer cell lectin-like receptor subfamily G member-1 (KLRG)+ IL-2/TE and CD62L+KLRG- IL-15/TM cells with short- and long-term survival following their adoptive transfer into mice. The IL-15/mTORC1Weak signal activates the forkhead box-O-1 (FOXO1), T cell factor-1 (TCF1) and Eomes transcriptional network and the metabolic adenosine monophosphate-activated protein kinase-α-1 (AMPKα1), Unc-51-like autophagy-activating kinase-1 (ULK1) and autophagy-related gene-7 (ATG7) axis, increasing the expression of mitochondrial regulators aquaporin-9 (AQP9), mitochondrial transcription factor-A (TFAM), peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), carnitine palmitoyl transferase-1 (CPT1α), microtubule-associated protein light chain-3 II (LC3II), Complex I and ortic atrophy-1 (OPA1), leading to promoting mitochondrial biogenesis and fatty-acid oxidation (FAO). Interestingly, AMPKα1 deficiency abrogates these downstream responses to IL-15/mTORC1Weak signaling, leading to the upregulation of mTORC1 and hypoxia-inducible factor-1α (HIF-1α), a metabolic switch from FAO to glycolysis and reduced cell survival. Taken together, our data demonstrate that IL-15/mTORC1Weak signaling controls T-cell memory via activation of the transcriptional FOXO1-TCF1-Eomes and metabolic AMPKα1-ULK1-ATG7 pathways, a finding that may greatly impact the development of efficient vaccines and immunotherapies for the treatment of cancer and infectious diseases.
Assuntos
Proteínas Quinases Ativadas por AMP , Autofagia , Diferenciação Celular , Interleucina-15 , Interleucina-2 , Respiração , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Autofagia/fisiologia , Interleucina-15/farmacologia , Mamíferos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Linfócitos TRESUMO
CD8+ memory T (TM) cells play a critical role in immune defense against infection. Two common γ-chain family cytokines, IL-2 and IL-7, although triggering the same mTORC1-S6K pathway, distinctly induce effector T (TE) cells and TM cells, respectively, but the underlying mechanism(s) remains elusive. In this study, we generated IL-7R-/and AMPKα1-knockout (KO)/OTI mice. By using genetic and pharmaceutical tools, we demonstrate that IL-7 deficiency represses expression of FOXO1, TCF1, p-AMPKα1 (T172), and p-ULK1 (S555) and abolishes T cell memory differentiation in IL-7R KO T cells after Listeria monocytogenesis rLmOVA infection. IL-2- and IL-7-stimulated strong and weak S6K (IL-2/S6Kstrong and IL-7/S6Kweak) signals control short-lived IL-7R-CD62L-KLRG1+ TE and long-term IL-7R+CD62L+KLRG1- TM cell formations, respectively. To assess underlying molecular pathway(s), we performed flow cytometry, Western blotting, confocal microscopy, and Seahorse assay analyses by using the IL-7/S6Kweak-stimulated TM (IL-7/TM) and the control IL-2/S6Kstrong-stimulated TE (IL-2/TE) cells. We determine that the IL-7/S6Kweak signal activates transcriptional FOXO1, TCF1, and Id3 and metabolic p-AMPKα1, p-ULK1, and ATG7 molecules in IL-7/TM cells. IL-7/TM cells upregulate IL-7R and CD62L, promote mitochondria biogenesis and fatty acid oxidation metabolism, and show long-term cell survival and functional recall responses. Interestingly, AMPKα1 deficiency abolishes the AMPKα1 but maintains the FOXO1 pathway and induces a metabolic switch from fatty acid oxidation to glycolysis in AMPKα1 KO IL-7/TM cells, leading to loss of cell survival and recall responses. Taken together, our data demonstrate that IL-7-stimulated weak strength of mTORC1-S6K signaling controls T cell memory via activation of transcriptional FOXO1-TCF1-Id3 and metabolic AMPKα1-ULK1-ATG7 pathways. This (to our knowledge) novel finding provides a new mechanism for a distinct IL-2/IL-7 stimulation model in T cell memory and greatly impacts vaccine development.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteína 7 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Linfócitos T CD8-Positivos/imunologia , Proteína Forkhead Box O1/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Proteínas Inibidoras de Diferenciação/metabolismo , Interleucina-7/metabolismo , Listeria monocytogenes/fisiologia , Listeriose/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Células T de Memória/imunologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Citotoxicidade Imunológica , Ácidos Graxos/metabolismo , Proteína Forkhead Box O1/genética , Regulação da Expressão Gênica , Glicólise , Fator 1-alfa Nuclear de Hepatócito/genética , Memória Imunológica , Proteínas Inibidoras de Diferenciação/genética , Interleucina-7/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-7/genética , Transdução de Sinais , Desenvolvimento de VacinasRESUMO
Histone variants are crucial regulators of chromatin structure and gene transcription, yet their functions within the brain remain largely unexplored. Here, we show that the H2A histone variant H2A.Z is essential for neuronal survival. Mice lacking H2A.Z in GABAergic neurons or Purkinje cells (PCs) present with a progressive cerebellar ataxia accompanied by widespread degeneration of PCs. Ablation of H2A.Z in other neuronal subtypes also triggers cell death. H2A.Z binds to the promoters of key nuclear-encoded mitochondrial genes to regulate their expression and promote organelle function. Bolstering mitochondrial activity genetically or by organelle transplant enhances the survival of H2A.Z-ablated neurons. Changes in bioenergetic status alter H2A.Z occupancy at the promoters of nuclear-encoded mitochondrial genes, an adaptive response essential for cell survival. Our results highlight that H2A.Z fulfills a key, conserved role in neuronal survival by acting as a transcriptional rheostat to regulate the expression of genes critical to mitochondrial function.
Assuntos
Núcleo Celular/metabolismo , Histonas/genética , Mitocôndrias/metabolismo , Transcriptoma , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo , Fibroblastos/citologia , Fibroblastos/metabolismo , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Histonas/deficiência , Histonas/metabolismo , Metformina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Proteínas Mitocondriais/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fosforilação Oxidativa , Células de Purkinje/citologia , Células de Purkinje/metabolismo , Transcriptoma/efeitos dos fármacos , Regulação para CimaRESUMO
The mitochondrial carrier family protein SLC25A3 transports both copper and phosphate in mammals, yet in Saccharomyces cerevisiae the transport of these substrates is partitioned across two paralogs: PIC2 and MIR1. To understand the ancestral state of copper and phosphate transport in mitochondria, we explored the evolutionary relationships of PIC2 and MIR1 orthologs across the eukaryotic tree of life. Phylogenetic analyses revealed that PIC2-like and MIR1-like orthologs are present in all major eukaryotic supergroups, indicating an ancient gene duplication created these paralogs. To link this phylogenetic signal to protein function, we used structural modeling and site-directed mutagenesis to identify residues involved in copper and phosphate transport. Based on these analyses, we generated an L175A variant of mouse SLC25A3 that retains the ability to transport copper but not phosphate. This work highlights the utility of using an evolutionary framework to uncover amino acids involved in substrate recognition by mitochondrial carrier family proteins.
Assuntos
Evolução Biológica , Proteínas Mitocondriais/genética , Proteínas de Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas de Transporte de Cobre/genética , Proteínas de Transporte de Cobre/metabolismo , Eucariotos , Camundongos , Mitocôndrias , Proteínas Mitocondriais/metabolismo , Mutagênese Sítio-Dirigida , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismo , Filogenia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The pool of ß-Amyloid (Aß) length variants detected in preclinical and clinical Alzheimer disease (AD) samples suggests a diversity of roles for Aß peptides. We examined how a naturally occurring variant, e.g. Aß(1-38), interacts with the AD-related variant, Aß(1-42), and the predominant physiological variant, Aß(1-40). Atomic force microscopy, Thioflavin T fluorescence, circular dichroism, dynamic light scattering, and surface plasmon resonance reveal that Aß(1-38) interacts differently with Aß(1-40) and Aß(1-42) and, in general, Aß(1-38) interferes with the conversion of Aß(1-42) to a ß-sheet-rich aggregate. Functionally, Aß(1-38) reverses the negative impact of Aß(1-42) on long-term potentiation in acute hippocampal slices and on membrane conductance in primary neurons, and mitigates an Aß(1-42) phenotype in Caenorhabditis elegans. Aß(1-38) also reverses any loss of MTT conversion induced by Aß(1-40) and Aß(1-42) in HT-22 hippocampal neurons and APOE ε4-positive human fibroblasts, although the combination of Aß(1-38) and Aß(1-42) inhibits MTT conversion in APOE ε4-negative fibroblasts. A greater ratio of soluble Aß(1-42)/Aß(1-38) [and Aß(1-42)/Aß(1-40)] in autopsied brain extracts correlates with an earlier age-at-death in males (but not females) with a diagnosis of AD. These results suggest that Aß(1-38) is capable of physically counteracting, potentially in a sex-dependent manner, the neuropathological effects of the AD-relevant Aß(1-42).
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/efeitos adversos , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/farmacologia , Fragmentos de Peptídeos/efeitos adversos , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/epidemiologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Encéfalo/patologia , Caenorhabditis elegans , Células Cultivadas , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismoRESUMO
Energy sensors mTORC1 and AMPKα1 regulate T-cell metabolism and differentiation, while rapamycin (Rapa)-inhibition of mTORC1 (RIM) promotes T-cell memory. However, the underlying pathway and the role of AMPKα1 in Rapa-induced T-cell memory remain elusive. Using genetic and pharmaceutical tools, we demonstrate that Rapa promotes T-cell memory in mice in vivo post Listeria monocytogenesis rLmOVA infection and in vitro transition of effector T (TE) to memory T (TM) cells. IL-2- and IL-2+Rapa-stimulated T [IL-2/T and IL-2(Rapa+)/T] cells, when transferred into mice, differentiate into short-term IL-7R-CD62L-KLRG1+ TE and long-lived IL-7R+CD62L+KLRG1- TM cells, respectively. To assess the underlying pathways, we performed Western blotting, confocal microscopy and Seahorse-assay analyses using IL-2/T and IL-2(Rapa+)/T-cells. We determined that IL-2(Rapa+)/T-cells activate transcription FOXO1, TCF1 and Eomes and metabolic pAMPKα1(T172), pULK1(S555) and ATG7 molecules and promote mitochondrial biogenesis and fatty-acid oxidation (FAO). We found that rapamycin-treated AMPKα-deficient AMPKα1-KO IL-2(Rapa+)/TM cells up-regulate transcription factor HIF-1α and induce a metabolic switch from FAO to glycolysis. Interestingly, despite the rapamycin treatment, AMPKα-deficient TM cells lost their cell survival capacity. Taken together, our data indicate that rapamycin promotes T-cell memory via transcriptional FOXO1-TCF1-Eomes programs and AMPKα1-ULK1-ATG7 metabolic axis, and that AMPKα1 plays a critical role in RIM-induced T-cell memory.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Memória Imunológica/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Sirolimo/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Proteína Forkhead Box O1/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Interleucina-2/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas com Domínio T/metabolismoRESUMO
Mitochondria accumulate copper in their matrix for the eventual maturation of the cuproenzymes cytochrome c oxidase and superoxide dismutase. Transport into the matrix is achieved by mitochondrial carrier family (MCF) proteins. The major copper transporting MCF described to date in yeast is Pic2, which imports the metal ion into the matrix. Pic2 is one of ~30 MCFs that move numerous metabolites, nucleotides and co-factors across the inner membrane for use in the matrix. Genetic and biochemical experiments showed that Pic2 is required for cytochrome c oxidase activity under copper stress, and that it is capable of transporting ionic and complexed forms of copper. The Pic2 ortholog SLC25A3, one of 53 mammalian MCFs, functions as both a copper and a phosphate transporter. Depletion of SLC25A3 results in decreased accumulation of copper in the matrix, a cytochrome c oxidase defect and a modulation of cytosolic superoxide dismutase abundance. The regulatory roles for copper and cuproproteins resident to the mitochondrion continue to expand beyond the organelle. Mitochondrial copper chaperones have been linked to the modulation of cellular copper uptake and export and the facilitation of inter-organ communication. Recently, a role for matrix copper has also been proposed in a novel cell death pathway termed cuproptosis. This review will detail our understanding of the maturation of mitochondrial copper enzymes, the roles of mitochondrial signals in regulating cellular copper content, the proposed mechanisms of copper transport into the organelle and explore the evolutionary origins of copper homeostasis pathways.
Assuntos
Cobre/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas de Transporte de Fosfato/genética , Proteínas de Saccharomyces cerevisiae/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Mitocôndrias/genética , Chaperonas Moleculares/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
Synchrotron-based X-ray fluorescence microscopy (XFM) has become an important imaging technique to investigate elemental concentrations and distributions in biological specimens. Advances in technology now permit imaging at resolutions rivaling that of electron microscopy, and researchers can now visualize elemental concentrations in subcellular organelles when using appropriate correlative methods. XFM is an especially valuable tool to determine the distribution of endogenous trace metals that are involved in neurodegenerative diseases. Here, we discuss the latest research on the unusual copper (Cu) storage vesicles that were originally identified in mouse brains and the involvement of Cu in Alzheimer's disease. Finally, we provide an outlook of how future improvements to XFM will drive current trace element research forward.
Assuntos
Cobre/análise , Microscopia de Fluorescência/métodos , Doenças Neurodegenerativas/diagnóstico por imagem , Fatores Etários , Animais , Cobre/metabolismo , Humanos , Camundongos Knockout , Imagem Óptica , Organelas/metabolismo , Tecido Parenquimatoso/metabolismo , Espectrometria por Raios X , SíncrotronsRESUMO
In eukaryotes, cellular respiration is driven by mitochondrial cytochrome c oxidase (CcO), an enzyme complex that requires copper cofactors for its catalytic activity. Insertion of copper into its catalytically active subunits, including COX2, is a complex process that requires metallochaperones and redox proteins including SCO1, SCO2, and COA6, a recently discovered protein whose molecular function is unknown. To uncover the molecular mechanism by which COA6 and SCO proteins mediate copper delivery to COX2, we have solved the solution structure of COA6, which reveals a coiled-coil-helix-coiled-coil-helix domain typical of redox-active proteins found in the mitochondrial inter-membrane space. Accordingly, we demonstrate that COA6 can reduce the copper-coordinating disulfides of its client proteins, SCO1 and COX2, allowing for copper binding. Finally, our determination of the interaction surfaces and reduction potentials of COA6 and its client proteins provides a mechanism of how metallochaperone and disulfide reductase activities are coordinated to deliver copper to CcO.
Assuntos
Proteínas de Transporte/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Mitocondriais/metabolismo , Proteína Dissulfeto Redutase (Glutationa)/metabolismo , Proteínas de Transporte/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Humanos , Espectroscopia de Ressonância Magnética , Proteínas Mitocondriais/genética , Chaperonas Moleculares/metabolismo , Mutação/genética , Ligação Proteica , Proteína Dissulfeto Redutase (Glutationa)/genéticaRESUMO
Gemini surfactants are promising molecules utilized as non-viral gene delivery vectors. However, little is known about their cellular uptake and distribution after they release their therapeutic cargo. Therefore, we quantitatively evaluated the cellular uptake and distribution of three gemini surfactants: unsubstituted (16-3-16), with pyridinium head groups (16(Py)-S-2-S-16(Py)) and substituted with a glycyl-lysine di-peptide (16-7N(GK)-16). We also assessed the relationship between cellular uptake and distribution of each gemini surfactant and its overall efficiency and toxicity. Epidermal keratinocytes PAM 212 were treated with gemini surfactant nanoparticles formulated with plasmid DNA and harvested at various time points to collect the enriched nuclear, mitochondrial, plasma membrane, and cytosolic fractions. Gemini surfactants were then extracted from each subcellular fraction and quantified using a validated flow injection analysis-tandem mass spectrometry (FIA-MS/MS) method. Mass spectrometry is superior to the use of fluorescent tags that alter the physicochemical properties and pharmacokinetics of the nanoparticles and can be cleaved from the gemini surfactant molecules within biological systems. Overall, a significantly higher cellular uptake was observed for 16-7N(GK)-16 (17.0%) compared with 16-3-6 (3.6%) and 16(Py)-S-2-S-16(Py) (1.4%), which explained the relatively higher transfection efficiency of 16-7N(GK)-16. Gemini surfactants 16-3-16 and 16(Py)-S-2-S-16(Py) displayed similar subcellular distribution patterns, with major accumulation in the nucleus, followed by the mitochondrion, cytosol, and plasma membrane. In contrast, 16-7N(GK)-16 was relatively evenly distributed across all four subcellular fractions. However, accumulation within the nucleus after 5 h of treatment was the highest for 16(Py)-S-2-S-16(Py) (50.3%), followed by 16-3-16 (41.8%) and then 16-7N(GK)-16 (33.4%), possibly leading to its relatively higher toxicity. Graphical Abstract.
Assuntos
Técnicas de Transferência de Genes , Queratinócitos/metabolismo , Nanopartículas , Tensoativos/química , Animais , Linhagem Celular , Análise de Injeção de Fluxo , Queratinócitos/citologia , Camundongos , Espectrometria de Massas em Tandem , Fatores de Tempo , TransfecçãoRESUMO
A simple, reliable flow injection analysis (FIA)-tandem mass spectrometric (MS/MS) method was developed for the determination of gemini surfactants, designated as 16-3-16, 16(Py)-S-2-S-(Py)16 and 16-7N(GK)-16, as gene delivery agents in cellular matrix. 16-3-16 is a conventional gemini surfactant bearing two quaternary amines, linked by a 3-carbon spacer region, 16(Py)-S-2-S-(Py)16 contains two pyridinium head groups, while 16-7N(GK)-16 bears a glycine-lysine di-peptide in the space region. The method was fully validated according to USFDA guidelines. It is the first time that FIA-MS/MS method was developed for the quantification of gemini surfactants, belonging to different structural families. The method was superior to existing liquid chromatographic (LC)-MS/MS methods in terms of sensitivity and time of analysis. Positive electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode were used on a triple quadrupole-linear ion trap (4000 QTRAP®) instrument. Deuterated internal standards were used to correct for matrix effects and variations in ionization within the ESI source. Isotope dilution standard curves were established in cellular matrix, with a linear range of 10 nM-1000 nM for 16-3-16 and 16(Py)-S-2-S-(Py)16, and 20 nM-2000 nM for 16-7N(GK)-16. The precision, accuracy, recovery and stability were all within the acceptable ranges as per the USFDA guidelines. The method was successfully applied for the quantification of target gemini surfactants in the nuclear fraction of PAM 212 keratinocyte cells treated with nanoparticles, which varied significantly and may explain differences in the observed efficiency and/or toxicity of these gemini surfactants in gene delivery.
Assuntos
Análise de Injeção de Fluxo/métodos , Técnicas de Transferência de Genes , Tensoativos/análise , Espectrometria de Massas em Tandem/métodos , Animais , Linhagem Celular Tumoral , Núcleo Celular/química , Análise de Injeção de Fluxo/instrumentação , Queratinócitos , Camundongos , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/instrumentaçãoRESUMO
Copper is an essential cofactor of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain. Inherited loss-of-function mutations in several genes encoding proteins required for copper delivery to CcO result in diminished CcO activity and severe pathologic conditions in affected infants. Copper supplementation restores CcO function in patient cells with mutations in two of these genes, COA6 and SCO2, suggesting a potential therapeutic approach. However, direct copper supplementation has not been therapeutically effective in human patients, underscoring the need to identify highly efficient copper transporting pharmacological agents. By using a candidate-based approach, we identified an investigational anticancer drug, elesclomol (ES), that rescues respiratory defects of COA6-deficient yeast cells by increasing mitochondrial copper content and restoring CcO activity. ES also rescues respiratory defects in other yeast mutants of copper metabolism, suggesting a broader applicability. Low nanomolar concentrations of ES reinstate copper-containing subunits of CcO in a zebrafish model of copper deficiency and in a series of copper-deficient mammalian cells, including those derived from a patient with SCO2 mutations. These findings reveal that ES can restore intracellular copper homeostasis by mimicking the function of missing transporters and chaperones of copper, and may have potential in treating human disorders of copper metabolism.
Assuntos
Antineoplásicos/farmacologia , Cobre/deficiência , Drogas em Investigação/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Hidrazinas/farmacologia , Mitocôndrias/efeitos dos fármacos , Animais , Antineoplásicos/uso terapêutico , Transporte Biológico/genética , Proteínas de Transporte/genética , Linhagem Celular , Coenzimas/deficiência , Cobre/uso terapêutico , Transportador de Cobre 1 , Suplementos Nutricionais , Modelos Animais de Doenças , Reposicionamento de Medicamentos , Drogas em Investigação/uso terapêutico , Fibroblastos , Humanos , Hidrazinas/uso terapêutico , Proteínas de Membrana Transportadoras/genética , Erros Inatos do Metabolismo/tratamento farmacológico , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Chaperonas Moleculares , Mutagênese Sítio-Dirigida , Mutação , Ratos , Saccharomyces cerevisiae , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Cytochrome c oxidase (COX) was initially purified more than 70 years ago. A tremendous amount of insight into its structure and function has since been gleaned from biochemical, biophysical, genetic, and molecular studies. As a result, we now appreciate that COX relies on its redox-active metal centers (heme a and a3, CuA and CuB) to reduce oxygen and pump protons in a reaction essential for most eukaryotic life. Questions persist, however, about how individual structural subunits are assembled into a functional holoenzyme. Here, we focus on what is known and what remains to be learned about the accessory proteins that facilitate CuA site maturation.
Assuntos
Cobre , Complexo IV da Cadeia de Transporte de Elétrons , Heme/análogos & derivados , Subunidades Proteicas , Domínio Catalítico , Cobre/química , Cobre/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Complexo IV da Cadeia de Transporte de Elétrons/química , Heme/química , Heme/metabolismo , Transporte de Íons/fisiologia , Oxigênio/química , Oxigênio/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , PrótonsRESUMO
Copper is required for the activity of cytochrome c oxidase (COX), the terminal electron-accepting complex of the mitochondrial respiratory chain. The likely source of copper used for COX biogenesis is a labile pool found in the mitochondrial matrix. In mammals, the proteins that transport copper across the inner mitochondrial membrane remain unknown. We previously reported that the mitochondrial carrier family protein Pic2 in budding yeast is a copper importer. The closest Pic2 ortholog in mammalian cells is the mitochondrial phosphate carrier SLC25A3. Here, to investigate whether SLC25A3 also transports copper, we manipulated its expression in several murine and human cell lines. SLC25A3 knockdown or deletion consistently resulted in an isolated COX deficiency in these cells, and copper addition to the culture medium suppressed these biochemical defects. Consistent with a conserved role for SLC25A3 in copper transport, its heterologous expression in yeast complemented copper-specific defects observed upon deletion of PIC2 Additionally, assays in Lactococcus lactis and in reconstituted liposomes directly demonstrated that SLC25A3 functions as a copper transporter. Taken together, these data indicate that SLC25A3 can transport copper both in vitro and in vivo.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Proteínas Carreadoras de Solutos/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte de Cátions/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Humanos , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas de Transporte de Fosfato/genética , Proteínas Carreadoras de Solutos/genéticaRESUMO
SCO1 is a ubiquitously expressed, mitochondrial protein with essential roles in cytochrome c oxidase (COX) assembly and the regulation of copper homeostasis. SCO1 patients present with severe forms of early onset disease, and ultimately succumb from liver, heart or brain failure. However, the inherent susceptibility of these tissues to SCO1 mutations and the clinical heterogeneity observed across SCO1 pedigrees remain poorly understood phenomena. To further address this issue, we generated Sco1hrt/hrt and Sco1stm/stm mice in which Sco1 was specifically deleted in heart and striated muscle, respectively. Lethality was observed in both models due to a combined COX and copper deficiency that resulted in a dilated cardiomyopathy. Left ventricular dilation and loss of heart function was preceded by a temporal decrease in COX activity and copper levels in the longer-lived Sco1stm/stm mice. Interestingly, the reduction in copper content of Sco1stm/stm cardiomyocytes was due to the mislocalisation of CTR1, the high affinity transporter that imports copper into the cell. CTR1 was similarly mislocalized to the cytosol in the heart of knockin mice carrying a homozygous G115S substitution in Sco1, which in humans causes a hypertrophic cardiomyopathy. Our current findings in the heart are in marked contrast to our prior observations in the liver, where Sco1 deletion results in a near complete absence of CTR1 protein. These data collectively argue that mutations perturbing SCO1 function have tissue-specific consequences for the machinery that ultimately governs copper homeostasis, and further establish the importance of aberrant mitochondrial signaling to the etiology of copper handling disorders.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miocárdio/metabolismo , Animais , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Membrana Celular/metabolismo , Cobre/deficiência , Transportador de Cobre 1 , Modelos Animais de Doenças , Complexo IV da Cadeia de Transporte de Elétrons/genética , Homeostase , Transporte de Íons , Metalochaperonas/genética , Metalochaperonas/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares , Miócitos Cardíacos/metabolismo , Oxirredução , Transdução de SinaisRESUMO
All known eukaryotes require copper for their development and survival. The essentiality of copper reflects its widespread use as a co-factor in conserved enzymes that catalyze biochemical reactions critical to energy production, free radical detoxification, collagen deposition, neurotransmitter biosynthesis and iron homeostasis. However, the prioritized use of copper poses an organism with a considerable challenge because, in its unbound form, copper can potentiate free radical production and displace iron-sulphur clusters to disrupt protein function. Protective mechanisms therefore evolved to mitigate this challenge and tightly regulate the acquisition, trafficking and storage of copper such that the metal ion is rarely found in its free form in the cell. Findings by a number of groups over the last ten years emphasize that this regulatory framework forms the foundation of a system that is capable of monitoring copper status and reprioritizing copper usage at both the cellular and systemic levels of organization. While the identification of relevant molecular mechanisms and signaling pathways has proven to be difficult and remains a barrier to our full understanding of the regulation of copper homeostasis, mounting evidence points to the mitochondrion as a pivotal hub in this regard in both healthy and diseased states. Here, we review our current understanding of copper handling pathways contained within the organelle and consider plausible mechanisms that may serve to functionally couple their activity to that of other cellular copper handling machinery to maintain copper homeostasis.
Assuntos
Cobre/metabolismo , Homeostase , Mitocôndrias/metabolismo , Modelos Biológicos , Animais , Transporte Biológico , Proteínas de Transporte de Cátions/metabolismo , Transportador de Cobre 1 , Glutationa/metabolismo , HumanosRESUMO
Death Receptor 5 (DR5) is a promising target for cancer therapy due to its ability to selectively induce apoptosis in cancer cells. However, the therapeutic usefulness of DR5 agonists is currently limited by the frequent resistance of malignant tumours to its activation. The identification of molecular mechanisms that determine outcomes of DR5 action is therefore crucial for improving the efficiency of DR5-activating reagents in cancer treatment. Here, we provide evidence that an intrinsically kinase-inactive member of the Eph group of receptor tyrosine kinases, EPHB6, induces marked fragmentation of the mitochondrial network in breast cancer cells of triple-negative origin, lacking expression of the estrogen, progesterone and HER2 receptors. Remarkably, this response renders cancer cells more susceptible to DR5-mediated apoptosis. EPHB6 action in mitochondrial fragmentation proved to depend on its ability to activate the ERK-DRP1 pathway, which increases the frequency of organelle fission. Moreover, DRP1 activity is also essential to the EPHB6-mediated pro-apoptotic response that we observe in the context of DR5 activation. These findings provide the first description of a member of the receptor tyrosine kinase family capable of producing a pro-apoptotic effect through the activation of ERK-DRP1 signaling and subsequent mitochondrial fragmentation. Our observations are of potential practical importance, as they imply that DR5-activating therapeutic approaches should be applied in a more personalized manner to primarily treat EPHB6-expressing tumours. Finally, our findings also suggest that the EPHB6 receptor itself may represent a promising target for cancer therapy, since EPHB6 and DR5 co-activation should support more efficient elimination of cancer cells.
Assuntos
Mitocôndrias/metabolismo , Receptores da Família Eph/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Apoptose/fisiologia , Linhagem Celular Tumoral , Dinaminas , GTP Fosfo-Hidrolases/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais/metabolismo , Neoplasias de Mama Triplo Negativas/enzimologia , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
BACKGROUND: Lymphopenia promotes naïve T-cell homeostatic proliferation and adoptive effector T-cell survival and memory formation. IL-7 plays a critical role in homeostatic proliferation, survival and memory formation of naïve T-cells in lymphopenia, and its underlying molecular mechanism has also been well studied. However, the mechanism for adoptively transferred effector T-cell survival and memory formation is not fully understood. Here, we transferred in vitro-activated transgenic OT-I CD8(+) effector T-cells into irradiation (600 rads)-induced lymphopenic C57BL/6, IL-7 knockout (KO) and IL-15 KO mice, and investigated the survival and memory formation of transferred T-cells in lymphopenia. RESULTS: We demonstrate that transferred T-cells prolong their survival and enhance their memory in lymphopenic mice, in a manner that depends on IL-15 signaling, but not IL-7. We determine that in vitro stimulation of naïve or effector T-cells with IL-7 and IL-15 reduces IL-7Rα, and increases and/or maintains IL-15Rß expression, respectively. Consistent with these findings, the expression of IL-7Rα and IL-15Rß is down- and up-regulated, respectively, in vivo on transferred T-cells in an early phase post T-cell transfer in lymphopenia. We further show that in vitro IL-15 restimulation-induced memory T-cells (compared to IL-2 restimulation-induced effector T-cells) and in vivo transferred T-cells in irradiated IL-15-sufficient C57BL/6 mice (compared to IL-15-deficient IL-15 KO mice) have increased mitochondrial content, but less NADH and lower mitochondrial potential (ΔΨm), and demonstrate greater phosphorylation of signal transducers and activators of transcription-5 (STAT5) and Unc-51-like kinase-1 (ULK1), and higher expression of B-cell leukemia/lymphoma-2 (Bcl2) and memory-, autophagy- and mitochondrial biogenesis-related molecules. CONCLUSION: Irradiation-induced lymphopenia promotes effector T-cell survival via IL-15 signaling the STAT5/Bcl2 pathway, enhances T-cell memory formation via IL-15 activation of the forkhead-box family of transcription factor (FOXO)/eomesodermin (Eomes) memory and ULK1/autophagy-related gene-7 (ATG7) autophagy pathways, and via IL-15 activation of the mitochondrial remodeling. Our data thus identify some important targets to consider when designing potent adoptive T-cell immunotherapies of cancer.
RESUMO
Three mitochondria-encoded subunits form the catalytic core of cytochrome c oxidase, the terminal enzyme of the respiratory chain. COX1 and COX2 contain heme and copper redox centers, which are integrated during assembly of the enzyme. Defects in this process lead to an enzyme deficiency and manifest as mitochondrial disorders in humans. Here we demonstrate that COA6 is specifically required for COX2 biogenesis. Absence of COA6 leads to fast turnover of newly synthesized COX2 and a concomitant reduction in cytochrome c oxidase levels. COA6 interacts transiently with the copper-containing catalytic domain of newly synthesized COX2. Interestingly, similar to the copper metallochaperone SCO2, loss of COA6 causes cardiomyopathy in humans. We show that COA6 and SCO2 interact and that corresponding pathogenic mutations in each protein affect complex formation. Our analyses define COA6 as a constituent of the mitochondrial copper relay system, linking defects in COX2 metallation to cardiac cytochrome c oxidase deficiency.