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1.
Nat Neurosci ; 22(10): 1731-1742, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31501572

RESUMO

Mitochondria vary in morphology and function in different tissues; however, little is known about their molecular diversity among cell types. Here we engineered MitoTag mice, which express a Cre recombinase-dependent green fluorescent protein targeted to the outer mitochondrial membrane, and developed an isolation approach to profile tagged mitochondria from defined cell types. We determined the mitochondrial proteome of the three major cerebellar cell types (Purkinje cells, granule cells and astrocytes) and identified hundreds of mitochondrial proteins that are differentially regulated. Thus, we provide markers of cell-type-specific mitochondria for the healthy and diseased mouse and human central nervous systems, including in amyotrophic lateral sclerosis and Alzheimer's disease. Based on proteomic predictions, we demonstrate that astrocytic mitochondria metabolize long-chain fatty acids more efficiently than neuronal mitochondria. We also characterize cell-type differences in mitochondrial calcium buffering via the mitochondrial calcium uniporter (Mcu) and identify regulator of microtubule dynamics protein 3 (Rmdn3) as a determinant of endoplasmic reticulum-mitochondria proximity in Purkinje cells. Our approach enables exploring mitochondrial diversity in many in vivo contexts.


Assuntos
Encéfalo/citologia , Mitocôndrias/metabolismo , Neurônios/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Astrócitos/metabolismo , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Células Cultivadas , Cerebelo/citologia , Ácidos Graxos/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Membranas Mitocondriais/metabolismo , Proteômica , Células de Purkinje/metabolismo
2.
Dokl Biochem Biophys ; 486(1): 220-223, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31367826

RESUMO

Cardiolipin (CL) plays a central role in lipid peroxidation (LPO) of the mitochondrial inner membrane due to higher content of unsaturated fatty acids in CL in comparison with the other phospholipids. CL oxidation plays an important role in the regulation of various intracellular signaling pathways and its excessive oxidation contributes to the development of various pathologies and, possibly, participates in the aging process. Mitochondria-targeted antioxidants containing triphenylphosphonium (TPP+) effectively protect CL from oxidation. It is assumed that fluorescent probes on the basis of the C11-BODIPY fluorophore sensitive to LPO and containing TPP+ can selectively register CL oxidation. To test this possibility, we carried out a molecular dynamic simulation of such probes in a model mitochondrial membrane. It is shown that the probes are located in the membrane at the same depth as the unsaturated bonds in CL molecules sensitive to oxidation. Increasing the length of the linker that binds the fluorophore and TPP+ residue ha little effect on the position of the probe in the membrane. This indicates the possibility of modifying the linker to increase the selectivity of the probes to CL.


Assuntos
Corantes Fluorescentes/metabolismo , Peroxidação de Lipídeos , Membranas Mitocondriais/metabolismo , Simulação de Dinâmica Molecular , Compostos de Boro/metabolismo
3.
J Agric Food Chem ; 67(33): 9265-9276, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31361479

RESUMO

Fungal infections significantly alter the emissions of volatile organic compounds (VOCs) by plants, but the mechanisms for VOCs affecting fungal infections of plants remain largely unknown. Here, we found that infection by Botrytis cinerea upregulated linalool production by strawberries and fumigation with linalool was able to inhibit the infection of fruits by the fungus. Linalool treatment downregulated the expression of rate-limiting enzymes in the ergosterol biosynthesis pathway, and this reduced the ergosterol content in the fungi cell membrane and impaired membrane integrity. Linalool treatment also caused damage to mitochondrial membranes by collapsing mitochondrial membrane potential and also downregulated genes involved in adenosine triphosphate (ATP) production, resulting in a significant decrease in the ATP content. Linalool treatment increased the levels of reactive oxygen species (ROS), in response to which the treated fungal cells produced more of the ROS scavenger pyruvate. RNA-Seq and proteomic analysis data showed that linalool treatment slowed the rates of transcription and translation.


Assuntos
Botrytis/efeitos dos fármacos , Fragaria/metabolismo , Frutas/microbiologia , Monoterpenos/metabolismo , Doenças das Plantas/microbiologia , Compostos Orgânicos Voláteis/metabolismo , Trifosfato de Adenosina/metabolismo , Botrytis/crescimento & desenvolvimento , Fragaria/química , Fragaria/microbiologia , Frutas/química , Frutas/metabolismo , Interações Hospedeiro-Patógeno , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Monoterpenos/farmacologia , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteômica , Espécies Reativas de Oxigênio/metabolismo , Compostos Orgânicos Voláteis/farmacologia
4.
Nature ; 571(7765): 429-433, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31292547

RESUMO

Balanced fusion and fission are key for the proper function and physiology of mitochondria1,2. Remodelling of the mitochondrial inner membrane is mediated by the dynamin-like protein mitochondrial genome maintenance 1 (Mgm1) in fungi or the related protein optic atrophy 1 (OPA1) in animals3-5. Mgm1 is required for the preservation of mitochondrial DNA in yeast6, whereas mutations in the OPA1 gene in humans are a common cause of autosomal dominant optic atrophy-a genetic disorder that affects the optic nerve7,8. Mgm1 and OPA1 are present in mitochondria as a membrane-integral long form and a short form that is soluble in the intermembrane space. Yeast strains that express temperature-sensitive mutants of Mgm19,10 or mammalian cells that lack OPA1 display fragmented mitochondria11,12, which suggests that Mgm1 and OPA1 have an important role in inner-membrane fusion. Consistently, only the mitochondrial outer membrane-not the inner membrane-fuses in the absence of functional Mgm113. Mgm1 and OPA1 have also been shown to maintain proper cristae architecture10,14; for example, OPA1 prevents the release of pro-apoptotic factors by tightening crista junctions15. Finally, the short form of OPA1 localizes to mitochondrial constriction sites, where it presumably promotes mitochondrial fission16. How Mgm1 and OPA1 perform their diverse functions in membrane fusion, scission and cristae organization is at present unknown. Here we present crystal and electron cryo-tomography structures of Mgm1 from Chaetomium thermophilum. Mgm1 consists of a GTPase (G) domain, a bundle signalling element domain, a stalk, and a paddle domain that contains a membrane-binding site. Biochemical and cell-based experiments demonstrate that the Mgm1 stalk mediates the assembly of bent tetramers into helical filaments. Electron cryo-tomography studies of Mgm1-decorated lipid tubes and fluorescence microscopy experiments on reconstituted membrane tubes indicate how the tetramers assemble on positively or negatively curved membranes. Our findings convey how Mgm1 and OPA1 filaments dynamically remodel the mitochondrial inner membrane.


Assuntos
Chaetomium/química , Microscopia Crioeletrônica , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/química , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Cristalografia por Raios X , Proteínas Fúngicas/ultraestrutura , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/ultraestrutura , Galactosilceramidas/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/ultraestrutura , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica
5.
Life Sci ; 231: 116587, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31220526

RESUMO

The endoplasmic reticulum (ER) and mitochondria are two important organelles in cells. Mitochondria-associated membranes (MAMs) are lipid raft-like domains formed in the ER membranes that are in close apposition to mitochondria. They play an important role in signal transmission between these two essential organelles. When cells are exposed to internal or external stressful stimuli, the ER will activate an adaptive response called the ER stress response, which has a significant effect on mitochondrial function. Mitochondrial quality control is an important mechanism to ensure the functional integrity of mitochondria and the effect of ER stress on mitochondrial quality control through MAMs is of great significance. Therefore, in this review, we introduce ER stress and mitochondrial quality control, and discuss how ER stress signals are transmitted to mitochondria through MAMs. We then review the important roles of MAMs in mitochondrial quality control under ER stress.


Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Retículo Endoplasmático/metabolismo , Humanos , Microdomínios da Membrana , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/fisiologia
6.
Artigo em Inglês | MEDLINE | ID: mdl-31176865

RESUMO

Antarctic notothenioid fishes are highly stenothermal, yet their tolerance for warming is species-dependent. Because a body of literature points to the loss of cardiac function as underlying thermal limits in ectothermic animals, we investigated potential relationships among properties of ventricular mitochondrial membranes in notothenioids with known differences in both cardiac mitochondrial metabolism and organismal thermal tolerance. Fluidity of mitochondrial membranes was quantified by fluorescence depolarization for the white-blooded Chaenocephalus aceratus and the red-blooded Notothenia coriiceps. In these same membranes, lipid compositions and products of lipid peroxidation, the latter of which can disrupt membrane order, were analyzed in both species and in a second icefish, Pseudochaenichthys georgianus. Mitochondrial membranes from C. aceratus were significantly more fluid than those of the more thermotolerant species N. coriiceps (P < .0001). Consistent with this, ratios of total phosphatidylethanolamine (PE) to total phosphatidylcholine (PC) were lower in membranes from both species of icefishes, compared to those of N. coriiceps (P < .05). However, membranes of N. coriiceps displayed a greater unsaturation index (P < .0001). No differences among species were found in membrane products of lipid peroxidation. With rising temperatures, greater contents of PC in mitochondrial membranes from ventricles of icefishes are likely to promote membrane hyperfluidization at a lower temperature than for cardiac mitochondrial membranes from the red-blooded notothenioid. We propose that physical and chemical properties of the mitochondrial membranes may contribute to some of the observed differences in thermal sensitivity of physiological function among these species.


Assuntos
Lipídeos de Membrana/metabolismo , Miocárdio/metabolismo , Perciformes/metabolismo , Fosfolipídeos/metabolismo , Animais , Regiões Antárticas , Aquecimento Global , Peroxidação de Lipídeos , Fluidez de Membrana , Lipídeos de Membrana/química , Membranas Mitocondriais/metabolismo , Fosfolipídeos/química , Especificidade da Espécie , Termotolerância
7.
PLoS Genet ; 15(6): e1008085, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31170154

RESUMO

Mitochondrial dynamics is an essential physiological process controlling mitochondrial content mixing and mobility to ensure proper function and localization of mitochondria at intracellular sites of high-energy demand. Intriguingly, for yet unknown reasons, severe impairment of mitochondrial fusion drastically affects mtDNA copy number. To decipher the link between mitochondrial dynamics and mtDNA maintenance, we studied mouse embryonic fibroblasts (MEFs) and mouse cardiomyocytes with disruption of mitochondrial fusion. Super-resolution microscopy revealed that loss of outer mitochondrial membrane (OMM) fusion, but not inner mitochondrial membrane (IMM) fusion, leads to nucleoid clustering. Remarkably, fluorescence in situ hybridization (FISH), bromouridine labeling in MEFs and assessment of mitochondrial transcription in tissue homogenates revealed that abolished OMM fusion does not affect transcription. Furthermore, the profound mtDNA depletion in mouse hearts lacking OMM fusion is not caused by defective integrity or increased mutagenesis of mtDNA, but instead we show that mitochondrial fusion is necessary to maintain the stoichiometry of the protein components of the mtDNA replisome. OMM fusion is necessary for proliferating MEFs to recover from mtDNA depletion and for the marked increase of mtDNA copy number during postnatal heart development. Our findings thus link OMM fusion to replication and distribution of mtDNA.


Assuntos
DNA Mitocondrial/genética , Mitocôndrias Cardíacas/genética , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/genética , Animais , Variações do Número de Cópias de DNA/genética , Replicação do DNA/genética , Fibroblastos , Humanos , Hibridização in Situ Fluorescente , Fusão de Membrana/genética , Camundongos , Mitocôndrias Cardíacas/metabolismo , Membranas Mitocondriais/metabolismo , Mutagênese , Miócitos Cardíacos/metabolismo , Transcrição Genética
8.
Biochemistry (Mosc) ; 84(6): 593-607, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31238859

RESUMO

Mitochondria are among the most important cell organelles involved in the regulation of intracellular calcium homeostasis. During the last decade, a number of molecular structures responsible for the mitochondrial calcium transport have been identified including the mitochondrial Ca2+ uniporter (MCU), Na+/Ca2+ exchanger (NCLX), and Ca2+/H+ antiporter (Letm1). The review summarizes the data on the structure, regulation, and physiological role of such structures. The pathophysiological mechanism of Ca2+ transport through the cyclosporine A-sensitive mitochondrial permeability transition pore is discussed. An alternative mechanism for the mitochondrial pore opening, namely, formation of the lipid pore induced by saturated fatty acids, and its role in Ca2+ transport are described in detail.


Assuntos
Cálcio/metabolismo , Mitocôndrias/metabolismo , Animais , Canais de Cálcio/metabolismo , Citoplasma/metabolismo , Humanos , Transporte de Íons , Metabolismo dos Lipídeos , Membranas Mitocondriais/metabolismo , Estrutura Molecular , Trocador de Sódio e Cálcio/metabolismo
9.
Int J Mol Sci ; 20(9)2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31083380

RESUMO

Mitochondria-associated membranes (MAM) are a well-recognized contact link between the mitochondria and endoplasmic reticulum that affects mitochondrial biology and vascular smooth muscle cells (VSMCs) proliferation via the regulation of mitochondrial Ca2+(Ca2+m) influx. Nogo-B receptor (NgBR) plays a vital role in proliferation, epithelial-mesenchymal transition, and chemoresistance of some tumors. Recent studies have revealed that downregulation of NgBR, which stimulates the proliferation of VSMCs, but the underlying mechanism remains unclear. Here, we investigated the role of NgBR in MAM and VSMC proliferation. We analyzed the expression of NgBR in pulmonary arteries using a rat model of hypoxic pulmonary hypertension (HPH), in which rats were subjected to normoxic recovery after hypoxia. VSMCs exposed to hypoxia and renormoxia were used to assess the alterations in NgBR expression in vitro. The effect of NgBR downregulation and overexpression on VSMC proliferation was explored. The results revealed that NgBR expression was negatively related with VSMCs proliferation. Then, MAM formation and the phosphorylation of inositol 1,4,5-trisphosphate receptor type 3 (IP3R3) was detected. We found that knockdown of NgBR resulted in MAM disruption and augmented the phosphorylation of IP3R3 through pAkt, accompanied by mitochondrial dysfunction including decreased Ca2+m, respiration and mitochondrial superoxide, increased mitochondrial membrane potential and HIF-1α nuclear localization, which were determined by confocal microscopy and Seahorse XF-96 analyzer. By contrast, NgBR overexpression attenuated IP3R3 phosphorylation and HIF-1α nuclear localization under hypoxia. These results reveal that dysregulation of NgBR promotes VSMC proliferation via MAM disruption and increased IP3R3 phosphorylation, which contribute to the decrease of Ca2+m and mitochondrial impairment.


Assuntos
Membranas Mitocondriais/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Proteínas Nogo/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Proliferação de Células , Regulação para Baixo , Retículo Endoplasmático/metabolismo , Hipertensão Pulmonar , Hipóxia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Modelos Biológicos , Miócitos de Músculo Liso/ultraestrutura , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Artéria Pulmonar/metabolismo , Ratos , Transdução de Sinais
10.
Int J Mol Sci ; 20(9)2019 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-31085998

RESUMO

In this study, we provide experimental evidence that a maternally inherited polymorphism in the mitochondrial cytochrome b gene (mt-Cytb; m.15124A>G, Ile-Val) in mitochondrial complex III resulted in middle-aged obesity and higher susceptibility to diet-induced obesity, as well as age-related inflammatory disease, e.g., ulcerative dermatitis, in mice. As a consequence of the gene variation, we observed alterations in body composition, metabolism and mitochondrial functions, i.e., increased mitochondrial oxygen consumption rate and higher levels of reactive oxygen species, as well as in the commensal bacterial composition in the gut, with higher abundance of Proteobacteria in mice carrying the variant. These observations are in line with the previously described links of the mitochondrial complex III gene with obesity and metabolic diseases in humans. Given that these functional changes by the G variant at m.15124 in the mt-Cytb are already present in young mice that were kept under normal condition, it is plausible that the m.15124A>G variant is a disease susceptibility modifier to the diseases induced by additional stressors, i.e., dietary and/or aging stress, and that the variant results in the higher incidence of clinical diseases presentation in C57BL/6J-mt129S1/SvlmJ than C57BL/6J mice. Thus, mtDNA variants could be potential biomarkers to evaluate the healthspan.


Assuntos
DNA Mitocondrial/genética , Genes Mitocondriais/genética , Animais , Bacteroidetes/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Membranas Mitocondriais/metabolismo , Mutação/genética , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Polimorfismo de Nucleotídeo Único/genética , Espécies Reativas de Oxigênio/metabolismo
11.
Microb Pathog ; 133: 103542, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31125686

RESUMO

The mitochondrion of kinetoplastida has unique characteristics both in structure and function. To better understand the mitochondrial proteome of the Leishmania tropica promastigote stage, liquid chromatography coupled with mass spectrometry (LC/MS/MS) approach was used. In the wake of mitochondria isolation and purity validation, 1212 proteins were identified, among which approximately 44% of proteins belonged to the mitochondrial proteome. Several functions were enriched in mitochondrial proteome including tricarboxylic acid cycle and respiratory chain, protein folding, signalling, transport, lipid metabolism, amino acid, and nucleotide metabolism. Furthermore, the result of the present research was compared with the previous related studies. Gaining more information about vital metabolism of the cell and molecules can be used for therapeutic purposes.


Assuntos
Leishmania tropica/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Dobramento de Proteína , Proteoma/metabolismo , Proteínas de Transporte , Chaperoninas , Cromatografia Líquida , Ciclo do Ácido Cítrico , Transporte de Elétrons , Proteínas de Choque Térmico , Leishmania/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/isolamento & purificação , Espectrometria de Massas em Tandem
12.
Nat Commun ; 10(1): 1689, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30976076

RESUMO

Gasdermin E (GSDME/DFNA5) cleavage by caspase-3 liberates the GSDME-N domain, which mediates pyroptosis by forming pores in the plasma membrane. Here we show that GSDME-N also permeabilizes the mitochondrial membrane, releasing cytochrome c and activating the apoptosome. Cytochrome c release and caspase-3 activation in response to intrinsic and extrinsic apoptotic stimuli are significantly reduced in GSDME-deficient cells comparing with wild type cells. GSDME deficiency also accelerates cell growth in culture and in a mouse model of melanoma. Phosphomimetic mutation of the highly conserved phosphorylatable Thr6 residue of GSDME, inhibits its pore-forming activity, thus uncovering a potential mechanism by which GSDME might be regulated. Like GSDME-N, inflammasome-generated gasdermin D-N (GSDMD-N), can also permeabilize the mitochondria linking inflammasome activation to downstream activation of the apoptosome. Collectively, our results point to a role of gasdermin proteins in targeting the mitochondria to promote cytochrome c release to augment the mitochondrial apoptotic pathway.


Assuntos
Inflamassomos/metabolismo , Melanoma Experimental/patologia , Mitocôndrias/fisiologia , Piroptose/fisiologia , Receptores Estrogênicos/metabolismo , Neoplasias Cutâneas/patologia , Animais , Caspase 3/metabolismo , Citocromos c/metabolismo , Fibroblastos , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Mutação , Fosforilação/fisiologia , Cultura Primária de Células , Domínios Proteicos/genética , Receptores Estrogênicos/genética , Treonina/metabolismo
13.
Mol Biol Cell ; 30(10): 1138-1146, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30840537

RESUMO

The anti-apoptotic Bcl-2 family protein Bcl-xL plays a critical role in cell survival by protecting the integrity of the mitochondrial outer membrane (MOM). The mechanism through which Bcl-xL is recruited to the MOM has not been fully discerned. The retromer is a conserved endosomal scaffold complex involved in membrane trafficking. Here we identify VPS35 and VPS26, two core components of the retromer, as novel regulators of Bcl-xL. We observed interactions and colocalization between Bcl-xL, VPS35, VPS26, and MICAL-L1, a protein involved in recycling endosome biogenesis that also interacts with the retromer. We also found that upon VPS35 depletion, levels of nonmitochondrial Bcl-xL were increased. In addition, retromer-depleted cells displayed more rapid Bax activation and apoptosis. These results suggest that the retromer regulates apoptosis by facilitating Bcl-xL's transport to the MOM. Importantly, our studies suggest a previously uncharacterized relationship between the machineries of cell death/survival and endosomal trafficking.


Assuntos
Membranas Mitocondriais/metabolismo , Proteína bcl-X/metabolismo , Apoptose/fisiologia , Endossomos/metabolismo , Células HeLa , Humanos , Mitocôndrias/metabolismo , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas de Transporte Vesicular/metabolismo
14.
Nat Chem ; 11(4): 342-350, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30903037

RESUMO

The architecture and bioactivity of natural products frequently serve as embarkation points for the exploration of biologically relevant chemical space. Total synthesis followed by derivative synthesis has historically enabled a deeper understanding of structure-activity relationships. However, synthetic strategies towards a natural product are not always guided by hypotheses regarding the structural features required for bioactivity. Here, we report an approach to natural product total synthesis that we term 'pharmacophore-directed retrosynthesis'. A hypothesized, pharmacophore of a natural product is selected as an early synthetic target and this dictates the retrosynthetic analysis. In an ideal application, sequential increases in the structural complexity of this minimal structure enable development of a structure-activity relationship profile throughout the course of the total synthesis effort. This approach enables the identification of simpler congeners retaining bioactivity at a much earlier stage of a synthetic effort, as demonstrated here for the spongiane diterpenoid, gracilin A, leading to simplified derivatives with potent neuroprotective and immunosuppressive activity.


Assuntos
Acetatos/química , Diterpenos/química , Furanos/química , Imunossupressores/química , Fármacos Neuroprotetores/química , Acetatos/síntese química , Acetatos/farmacologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cristalografia por Raios X , Reação de Cicloadição , Diterpenos/síntese química , Diterpenos/farmacologia , Desenho de Drogas , Furanos/síntese química , Furanos/farmacologia , Humanos , Imunossupressores/síntese química , Imunossupressores/farmacologia , Membranas Mitocondriais/metabolismo , Conformação Molecular , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade
15.
Mol Biol Cell ; 30(10): 1198-1213, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30865555

RESUMO

Mitochondria are essential and dynamic organelles undergoing constant fission and fusion. The primary players in mitochondrial morphology (MFN1/2, OPA1, DRP1) have been identified, but their mechanism(s) of regulation are still being elucidated. ARL2 is a regulatory GTPase that has previously been shown to play a role in the regulation of mitochondrial morphology. Here we demonstrate that ELMOD2, an ARL2 GTPase-activating protein (GAP), is necessary for ARL2 to promote mitochondrial elongation. We show that loss of ELMOD2 causes mitochondrial fragmentation and a lower rate of mitochondrial fusion, while ELMOD2 overexpression promotes mitochondrial tubulation and increases the rate of fusion in a mitofusin-dependent manner. We also show that a mutant of ELMOD2 lacking GAP activity is capable of promoting fusion, suggesting that ELMOD2 does not need GAP activity to influence mitochondrial morphology. Finally, we show that ELMOD2, ARL2, Mitofusins 1 and 2, Miros 1 and 2, and mitochondrial phospholipase D (mitoPLD) all localize to discrete, regularly spaced puncta along mitochondria. These results suggest that ELMOD2 is functioning as an effector downstream of ARL2 and upstream of the mitofusins to promote mitochondrial fusion. Our data provide insights into the pathway by which mitochondrial fusion is regulated in the cell.


Assuntos
Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Dinâmica Mitocondrial/fisiologia , Animais , Células COS , Linhagem Celular , Cercopithecus aethiops , GTP Fosfo-Hidrolases/metabolismo , Técnicas de Inativação de Genes/métodos , Humanos , Fusão de Membrana/fisiologia , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fosfolipase D/genética , Fosfolipase D/metabolismo
16.
Nature ; 567(7748): 341-346, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30842654

RESUMO

Cancer-specific inhibitors that reflect the unique metabolic needs of cancer cells are rare. Here we describe Gboxin, a small molecule that specifically inhibits the growth of primary mouse and human glioblastoma cells but not that of mouse embryonic fibroblasts or neonatal astrocytes. Gboxin rapidly and irreversibly compromises oxygen consumption in glioblastoma cells. Gboxin relies on its positive charge to associate with mitochondrial oxidative phosphorylation complexes in a manner that is dependent on the proton gradient of the inner mitochondrial membrane, and it inhibits the activity of F0F1 ATP synthase. Gboxin-resistant cells require a functional mitochondrial permeability transition pore that regulates pH and thus impedes the accumulation of Gboxin in the mitochondrial matrix. Administration of a metabolically stable Gboxin analogue inhibits glioblastoma allografts and patient-derived xenografts. Gboxin toxicity extends to established human cancer cell lines of diverse organ origin, and shows that the increased proton gradient and pH in cancer cell mitochondria is a mode of action that can be targeted in the development of antitumour reagents.


Assuntos
Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Aloenxertos , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Linhagem Celular Tumoral , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/metabolismo , Transplante de Neoplasias , Especificidade de Órgãos , Força Próton-Motriz/efeitos dos fármacos , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Nat Commun ; 10(1): 1432, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926815

RESUMO

Of the four separate PE biosynthetic pathways in eukaryotes, one occurs in the mitochondrial inner membrane (IM) and is executed by phosphatidylserine decarboxylase (Psd1). Deletion of Psd1 is lethal in mice and compromises mitochondrial function. We hypothesize that this reflects inefficient import of non-mitochondrial PE into the IM. Here, we test this by re-wiring PE metabolism in yeast by re-directing Psd1 to the outer mitochondrial membrane or the endomembrane system and show that PE can cross the IMS in both directions. Nonetheless, PE synthesis in the IM is critical for cytochrome bc1 complex (III) function and mutations predicted to disrupt a conserved PE-binding site in the complex III subunit, Qcr7, impair complex III activity similar to PSD1 deletion. Collectively, these data challenge the current dogma of PE trafficking and demonstrate that PE made in the IM by Psd1 support the intrinsic functionality of complex III.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Membranas Mitocondriais/metabolismo , Fosfatidiletanolaminas/metabolismo , Saccharomyces cerevisiae/metabolismo , Aerobiose , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Membranas Mitocondriais/ultraestrutura , Mutação/genética , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
Biomed Pharmacother ; 111: 859-872, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30841465

RESUMO

This study investigated the effects of post-treatment with kolaviron on a 2-Vessel Occlusion (2-VO) model of cerebral ischemia/reperfusion (I/R) injury in rats to ascertain its level of efficacy as a potential therapeutic agent for stroke. Male Wistar rats submitted to 30 min of bilateral common carotid artery occlusion and 24 h of reperfusion were treated with kolaviron (25-100 mg/kg) or 20 mg/kg quercetin immediately after reperfusion and 2 h post reperfusion. At the end of the period of reperfusion, animals were scored for motor and cognitive deficits. Brain relative weight and water content were determined. Cortices, striata and hippocampi were dissected and processed for estimation of markers of oxidative stress, inflammation, neurotransmitter dysregulation and excitotoxicity. In addition, assessment of hippocampal mitochondrial integrity and histopathological examination of the cortical, striatal and hippocampal regions were carried out. There was reversal of 2-VO ischemia/reperfusion (I/R) induced motor and cognitive deficits by kolaviron post-treatment. Post-treatment with kolaviron also attenuated I/R-induced oxidative stress, neuroinflammatory events, excitotoxicity as well as mitochondrial dysfunction in brain tissues. Histopathological findings showed amelioration of I/R-induced neuronal cell damage by kolaviron post-treatment. The results revealed the multi-target neurotherapeutic activity of kolaviron and suggest that it is a promising candidate for drug development against stroke.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Transporte de Elétrons/efeitos dos fármacos , Flavonoides/farmacologia , Mitocôndrias/efeitos dos fármacos , Neurotransmissores/metabolismo , Oxirredução/efeitos dos fármacos , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Isquemia Encefálica/metabolismo , Modelos Animais de Doenças , Masculino , Mitocôndrias/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/metabolismo
19.
Methods Mol Biol ; 1949: 69-93, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30790250

RESUMO

Membrane biogenesis requires an extensive traffic of lipids between different cell compartments. Two main pathways, the vesicular and non-vesicular pathways, are involved in such a process. Whereas the mechanisms involved in vesicular trafficking are well understood, fewer is known about non-vesicular lipid trafficking, particularly in plants. This pathway involves the direct exchange of lipids at membrane contact sites (MCSs) between organelles. In plants, an extensive traffic of the chloroplast-synthesized digalactosyldiacylglycerol (DGDG) to mitochondria occurs during phosphate starvation. This lipid exchange occurs by non-vesicular trafficking pathways at MCSs between mitochondria and plastids. By a biochemical approach, a mitochondrial lipoprotein super-complex called MTL (Mitochondrial Transmembrane Lipoprotein complex) involved in mitochondria lipid trafficking has been identified in Arabidopsis thaliana. This protocol describes the method to isolate the MTL complex and to study the implication of a component of this complex (AtMic60) in mitochondria lipid trafficking.


Assuntos
Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Complexos Multiproteicos/metabolismo , Transporte Biológico , Cromatografia Líquida , Lipoproteínas/isolamento & purificação , Espectrometria de Massas , Proteínas de Membrana/isolamento & purificação , Solubilidade
20.
Biochim Biophys Acta Mol Cell Res ; 1866(5): 930-944, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30802482

RESUMO

Mitochondria are pivotal organelles for cellular signaling and metabolism, and their dysfunction leads to severe cellular stress. About 60-70% of the mitochondrial proteome consists of preproteins synthesized in the cytosol with an amino-terminal cleavable presequence targeting signal. The TIM23 complex transports presequence signals towards the mitochondrial matrix. Ultimately, the mature protein segments are either transported into the matrix or sorted to the inner membrane. To ensure accurate preprotein import into distinct mitochondrial sub-compartments, the TIM23 machinery adopts specific functional conformations and interacts with different partner complexes. Regulatory subunits modulate the translocase dynamics, tailoring the import reaction to the incoming preprotein. The mitochondrial membrane potential and the ATP generated via oxidative phosphorylation are key energy sources in driving the presequence import pathway. Thus, mitochondrial dysfunctions have rapid repercussions on biogenesis. Cellular mechanisms exploit the presequence import pathway to monitor mitochondrial dysfunctions and mount transcriptional and proteostatic responses to restore functionality.


Assuntos
Homeostase/fisiologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Biogênese de Organelas , Animais , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Transporte Proteico/fisiologia
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