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1.
Nat Commun ; 12(1): 720, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33526781

RESUMO

Cellular senescence is induced by stresses and results in a stable proliferation arrest accompanied by a pro-inflammatory secretome. Senescent cells accumulate during aging, promoting various age-related pathologies and limiting lifespan. The endoplasmic reticulum (ER) inositol 1,4,5-trisphosphate receptor, type 2 (ITPR2) calcium-release channel and calcium fluxes from the ER to the mitochondria are drivers of senescence in human cells. Here we show that Itpr2 knockout (KO) mice display improved aging such as increased lifespan, a better response to metabolic stress, less immunosenescence, as well as less liver steatosis and fibrosis. Cellular senescence, which is known to promote these alterations, is decreased in Itpr2 KO mice and Itpr2 KO embryo-derived cells. Interestingly, ablation of ITPR2 in vivo and in vitro decreases the number of contacts between the mitochondria and the ER and their forced contacts induce premature senescence. These findings shed light on the role of contacts and facilitated exchanges between the ER and the mitochondria through ITPR2 in regulating senescence and aging.


Assuntos
Senescência Celular/fisiologia , Retículo Endoplasmático/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Longevidade/fisiologia , Mitocôndrias/metabolismo , Animais , Cálcio/metabolismo , Retículo Endoplasmático/ultraestrutura , Feminino , Fibroblastos , Células HEK293 , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Masculino , Camundongos , Camundongos Knockout , Microscopia Confocal , Mitocôndrias/ultraestrutura , RNA Interferente Pequeno , Período Refratário Eletrofisiológico , Análise de Célula Única
2.
Nat Commun ; 12(1): 1049, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594064

RESUMO

Eukaryotic phytoplankton have a small global biomass but play major roles in primary production and climate. Despite improved understanding of phytoplankton diversity and evolution, we largely ignore the cellular bases of their environmental plasticity. By comparative 3D morphometric analysis across seven distant phytoplankton taxa, we observe constant volume occupancy by the main organelles and preserved volumetric ratios between plastids and mitochondria. We hypothesise that phytoplankton subcellular topology is modulated by energy-management constraints. Consistent with this, shifting the diatom Phaeodactylum from low to high light enhances photosynthesis and respiration, increases cell-volume occupancy by mitochondria and the plastid CO2-fixing pyrenoid, and boosts plastid-mitochondria contacts. Changes in organelle architectures and interactions also accompany Nannochloropsis acclimation to different trophic lifestyles, along with respiratory and photosynthetic responses. By revealing evolutionarily-conserved topologies of energy-managing organelles, and their role in phytoplankton acclimation, this work deciphers phytoplankton responses at subcellular scales.


Assuntos
Metabolismo Energético , Imageamento Tridimensional , Fitoplâncton/citologia , Fitoplâncton/fisiologia , Aclimatação/efeitos da radiação , Metabolismo Energético/efeitos da radiação , Luz , Microalgas/metabolismo , Microalgas/efeitos da radiação , Microalgas/ultraestrutura , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Mitocôndrias/ultraestrutura , Fitoplâncton/efeitos da radiação , Fitoplâncton/ultraestrutura , Plastídeos/metabolismo , Frações Subcelulares/metabolismo
3.
Nat Commun ; 12(1): 120, 2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33402698

RESUMO

Mitochondrial ATP synthase plays a key role in inducing membrane curvature to establish cristae. In Apicomplexa causing diseases such as malaria and toxoplasmosis, an unusual cristae morphology has been observed, but its structural basis is unknown. Here, we report that the apicomplexan ATP synthase assembles into cyclic hexamers, essential to shape their distinct cristae. Cryo-EM was used to determine the structure of the hexamer, which is held together by interactions between parasite-specific subunits in the lumenal region. Overall, we identified 17 apicomplexan-specific subunits, and a minimal and nuclear-encoded subunit-a. The hexamer consists of three dimers with an extensive dimer interface that includes bound cardiolipins and the inhibitor IF1. Cryo-ET and subtomogram averaging revealed that hexamers arrange into ~20-megadalton pentagonal pyramids in the curved apical membrane regions. Knockout of the linker protein ATPTG11 resulted in the loss of pentagonal pyramids with concomitant aberrantly shaped cristae. Together, this demonstrates that the unique macromolecular arrangement is critical for the maintenance of cristae morphology in Apicomplexa.


Assuntos
Mitocôndrias/ultraestrutura , Membranas Mitocondriais/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/química , Subunidades Proteicas/química , Proteínas de Protozoários/química , Toxoplasma/ultraestrutura , Sítios de Ligação , Cardiolipinas/química , Cardiolipinas/metabolismo , Microscopia Crioeletrônica , Expressão Gênica , Mitocôndrias/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Especificidade por Substrato , Termodinâmica , Toxoplasma/genética , Toxoplasma/metabolismo
4.
Int J Nanomedicine ; 16: 283-296, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33469287

RESUMO

Methods: In this study, we used MTT assays to demonstrate that a combination of SPIO-Serum and wild-type p53 overexpression can reduce ovarian cancer cell viability in vitro. Prussian blue staining and iron assays were used to determine changes in intracellular iron concentration following SPIO-Serum treatment. TEM was used to evaluate any mitochondrial damage induced by SPIO-Serum treatment, and Western blot was used to evaluate the expression of the iron transporter and lipid peroxidation regulator proteins. JC-1 was used to measure mitochondrial membrane potential, and ROS levels were estimated by flow cytometry. Finally, xCT protein expression and mitochondrial ROS levels were confirmed using fluorescence microscopy. Results: SPIO-Serum effectively induced lipid peroxidation and generated abundant toxic ROS. It also facilitated the downregulation of GPX4 and xCT, ultimately resulting in iron-dependent oxidative death. These effects could be reversed by iron chelator DFO and lipid peroxidation inhibitor Fer-1. SPIO-Serum treatment disrupted intracellular iron homeostasis by regulating iron uptake and the cells presented with missing mitochondrial cristae and ruptured outer mitochondrial membranes. Moreover, we were able to show that p53 contributed to SPIO-Serum-induced ferroptosis in ovarian cancer cells. Conclusion: SPIO-Serum induced ferroptosis and overexpressed p53 contributed to ferroptosis in ovarian cancer cells. Our data provide a theoretical basis for ferroptosis as a novel cell death phenotype induced by nanomaterials.


Assuntos
Ferroptose , Neoplasias Ovarianas/patologia , Soro/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Feminino , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Modelos Biológicos , Neoplasias Ovarianas/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Receptores da Transferrina/metabolismo , Transferrina/metabolismo
5.
Cell Prolif ; 54(2): e12964, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33314534

RESUMO

OBJECTIVES: 20-hydroxyeicosatetraenoic acid (20-HETE) is a metabolite of arachidonic acid catalysed by cytochrome P450 enzymes and plays an important role in cell death and proliferation. We hypothesized that 20-HETE synthesis inhibition may have protective effects in traumatic brain injury (TBI) and investigated possible underlying molecular mechanisms. MATERIALS AND METHODS: Neurologic deficits, and lesion volume, reactive oxygen species (ROS) levels and cell death as assessed using immunofluorescence staining, transmission electron microscopy and Western blotting were used to determine post-TBI effects of HET0016, an inhibitor of 20-HETE synthesis, and their underlying mechanisms. RESULTS: The level of 20-HETE was found to be increased significantly after TBI in mice. 20-HETE synthesis inhibition reduced neuronal apoptosis, ROS production and damage to mitochondrial structures after TBI. Mechanistically, HET0016 decreased the Drp1 level and increased the expression of Mfn1 and Mfn2 after TBI, indicating a reversal of the abnormal post-TBI mitochondrial dynamics. HET0016 also promoted the restoration of SIRT1 and PGC-1α in vivo, and a SIRT1 activator (SRT1720) reversed the downregulation of SIRT1 and PGC-1α and the abnormal mitochondrial dynamics induced by 20-HETE in vitro. Furthermore, plasma 20-HETE levels were found to be higher in TBI patients with unfavourable neurological outcomes and were correlated with the GOS score. CONCLUSIONS: The inhibition of 20-HETE synthesis represents a novel strategy to mitigate TBI-induced mitochondrial dysfunction and neuronal apoptosis by regulating the SIRT1/PGC-1α pathway.


Assuntos
Amidinas/farmacologia , Apoptose/efeitos dos fármacos , Lesões Encefálicas Traumáticas/patologia , Ácidos Hidroxieicosatetraenoicos/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Lesões Encefálicas Traumáticas/veterinária , Dinaminas/metabolismo , Feminino , Humanos , Ácidos Hidroxieicosatetraenoicos/sangue , Ácidos Hidroxieicosatetraenoicos/farmacologia , Modelos Logísticos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Neurônios/citologia , Neurônios/metabolismo , Neurônios/ultraestrutura , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 1/química , Sirtuína 1/metabolismo
6.
Nat Commun ; 11(1): 5187, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-33056988

RESUMO

Mitoribosomes are specialized protein synthesis machineries in mitochondria. However, how mRNA binds to its dedicated channel, and tRNA moves as the mitoribosomal subunit rotate with respect to each other is not understood. We report models of the translating fungal mitoribosome with mRNA, tRNA and nascent polypeptide, as well as an assembly intermediate. Nicotinamide adenine dinucleotide (NAD) is found in the central protuberance of the large subunit, and the ATPase inhibitory factor 1 (IF1) in the small subunit. The models of the active mitoribosome explain how mRNA binds through a dedicated protein platform on the small subunit, tRNA is translocated with the help of the protein mL108, bridging it with L1 stalk on the large subunit, and nascent polypeptide paths through a newly shaped exit tunnel involving a series of structural rearrangements. An assembly intermediate is modeled with the maturation factor Atp25, providing insight into the biogenesis of the mitoribosomal large subunit and translation regulation.


Assuntos
Mitocôndrias/metabolismo , Ribossomos Mitocondriais/metabolismo , Neurospora crassa/fisiologia , Biossíntese de Proteínas , Fracionamento Celular , Microscopia Crioeletrônica , Proteínas Fúngicas/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/ultraestrutura , Modelos Moleculares , NAD/metabolismo , Proteínas/metabolismo , RNA Mensageiro/metabolismo , RNA Ribossômico/metabolismo , RNA de Transferência/metabolismo , Proteínas Ribossômicas/metabolismo
7.
Mol Cell ; 80(1): 43-58.e7, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32937100

RESUMO

Immune cell function depends on specific metabolic programs dictated by mitochondria, including nutrient oxidation, macromolecule synthesis, and post-translational modifications. Mitochondrial adaptations have been linked to acute and chronic inflammation, but the metabolic cues and precise mechanisms remain unclear. Here we reveal that histone deacetylase 3 (HDAC3) is essential for shaping mitochondrial adaptations for IL-1ß production in macrophages through non-histone deacetylation. In vivo, HDAC3 promoted lipopolysaccharide-induced acute inflammation and high-fat diet-induced chronic inflammation by enhancing NLRP3-dependent caspase-1 activation. HDAC3 configured the lipid profile in stimulated macrophages and restricted fatty acid oxidation (FAO) supported by exogenous fatty acids for mitochondria to acquire their adaptations and depolarization. Rather than affecting nuclear gene expression, HDAC3 translocated to mitochondria to deacetylate and inactivate an FAO enzyme, mitochondrial trifunctional enzyme subunit α. HDAC3 may serve as a controlling node that balances between acquiring mitochondrial adaptations and sustaining their fitness for IL-1ß-dependent inflammation.


Assuntos
Ácidos Graxos/metabolismo , Histona Desacetilases/metabolismo , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Mitocôndrias/metabolismo , Adulto , Animais , Caspase 1/metabolismo , Feminino , Humanos , Inflamação/patologia , Metabolismo dos Lipídeos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Mitocôndrias/ultraestrutura , Subunidade alfa da Proteína Mitocondrial Trifuncional/metabolismo , Células Mieloides/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Oxirredução , Fosforilação Oxidativa , Adulto Jovem
8.
Nat Commun ; 11(1): 3830, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737313

RESUMO

The mammalian mitochondrial ribosome (mitoribosome) and its associated translational factors have evolved to accommodate greater participation of proteins in mitochondrial translation. Here we present the 2.68-3.96 Å cryo-EM structures of the human 55S mitoribosome in complex with the human mitochondrial elongation factor G1 (EF-G1mt) in three distinct conformational states, including an intermediate state and a post-translocational state. These structures reveal the role of several mitochondria-specific (mito-specific) mitoribosomal proteins (MRPs) and a mito-specific segment of EF-G1mt in mitochondrial tRNA (tRNAmt) translocation. In particular, the mito-specific C-terminal extension in EF-G1mt is directly involved in translocation of the acceptor arm of the A-site tRNAmt. In addition to the ratchet-like and independent head-swiveling motions exhibited by the small mitoribosomal subunit, we discover significant conformational changes in MRP mL45 at the nascent polypeptide-exit site within the large mitoribosomal subunit that could be critical for tethering of the elongating mitoribosome onto the inner-mitochondrial membrane.


Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Elongação Traducional da Cadeia Peptídica , Fator G para Elongação de Peptídeos/química , RNA Mitocondrial/química , RNA de Transferência/química , Proteínas Ribossômicas/química , Ribossomos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Microscopia Crioeletrônica , Células HEK293 , Humanos , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
9.
PLoS One ; 15(8): e0237930, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841274

RESUMO

Chinese hamster ovary cells have been the workhorse for the production of recombinant proteins in mammalian cells. Since biochemical, cellular and omics studies are usually affected by the lack of suitable fractionation procedures to isolate compartments from these cells, differential and isopycnic centrifugation based techniques were characterized and developed specially for them. Enriched fractions in intact nuclei, mitochondria, peroxisomes, cis-Golgi, trans-Golgi and endoplasmic reticulum (ER) were obtained in differential centrifugation steps and subsequently separated in discontinuous sucrose gradients. Nuclei, mitochondria, cis-Golgi, peroxisomes and smooth ER fractions were obtained as defined bands in 30-60% gradients. Despite the low percentage represented by the microsomes of the total cell homogenate (1.7%), their separation in a novel sucrose gradient (10-60%) showed enough resolution and efficiency to quantitatively separate their components into enriched fractions in trans-Golgi, cis-Golgi and ER. The identity of these organelles belonging to the classical secretion pathway that came from 10-60% gradients was confirmed by proteomics. Data are available via ProteomeXchange with identifier PXD019778. Components from ER and plasma membrane were the most frequent contaminants in almost all obtained fractions. The improved sucrose gradient for microsomal samples proved being successful in obtaining enriched fractions of low abundance organelles, such as Golgi apparatus and ER components, for biochemical and molecular studies, and suitable for proteomic research, which makes it a useful tool for future studies of this and other mammalian cell lines.


Assuntos
Microssomos/metabolismo , Proteômica , Animais , Células CHO , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Centrifugação , Cricetinae , Cricetulus , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Ontologia Genética , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Microssomos/ultraestrutura , Mitocôndrias/ultraestrutura , Proteoma/metabolismo , Software , Frações Subcelulares/metabolismo
10.
Nat Commun ; 11(1): 3290, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620929

RESUMO

In mitochondria, ß-barrel outer membrane proteins mediate protein import, metabolite transport, lipid transport, and biogenesis. The Sorting and Assembly Machinery (SAM) complex consists of three proteins that assemble as a 1:1:1 complex to fold ß-barrel proteins and insert them into the mitochondrial outer membrane. We report cryoEM structures of the SAM complex from Myceliophthora thermophila, which show that Sam50 forms a 16-stranded transmembrane ß-barrel with a single polypeptide-transport-associated (POTRA) domain extending into the intermembrane space. Sam35 and Sam37 are located on the cytosolic side of the outer membrane, with Sam35 capping Sam50, and Sam37 interacting extensively with Sam35. Sam35 and Sam37 each adopt a GST-like fold, with no functional, structural, or sequence similarity to their bacterial counterparts. Structural analysis shows how the Sam50 ß-barrel opens a lateral gate to accommodate its substrates.


Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Biossíntese de Proteínas , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Microscopia Crioeletrônica , Detergentes/química , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Conformação Proteica , Dobramento de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Sordariales/genética , Sordariales/metabolismo
11.
Nat Commun ; 11(1): 2932, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32522994

RESUMO

Translation initiation in human mitochondria relies upon specialized mitoribosomes and initiation factors, mtIF2 and mtIF3, which have diverged from their bacterial counterparts. Here we report two distinct mitochondrial pre-initiation assembly steps involving those factors. Single-particle cryo-EM revealed that in the first step, interactions between mitochondria-specific protein mS37 and mtIF3 keep the small mitoribosomal subunit in a conformation favorable for a subsequent accommodation of mtIF2 in the second step. Combination with fluorescence cross-correlation spectroscopy analyses suggests that mtIF3 promotes complex assembly without mRNA or initiator tRNA binding, where exclusion is achieved by the N-terminal and C-terminal domains of mtIF3. Finally, the association of large mitoribosomal subunit is required for initiator tRNA and leaderless mRNA recruitment to form a stable initiation complex. These data reveal fundamental aspects of mammalian protein synthesis that are specific to mitochondria.


Assuntos
Mitocôndrias/metabolismo , Microscopia Crioeletrônica , Humanos , Mitocôndrias/ultraestrutura , RNA Mensageiro/metabolismo , Ribossomos/metabolismo
12.
Arch Biochem Biophys ; 690: 108416, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32502471

RESUMO

Alkaptonuria (AKU) is a rare metabolic disease correlated with the deficiency of homogentisate 1,2-dioxygenase and leading to an accumulation of the metabolite homogentisic acid (HGA) which can be subjected to oxidation and polymerization reactions. These events are considered a trigger for the induction of oxidative stress in AKU but, despite the large description of an altered redox status, the underlying pathogenetic processes are still unstudied. In the present study, we investigated the molecular mechanisms responsible for the oxidative damage present in an osteoblast-based cellular model of AKU. Bone, in fact, is largely affected in AKU patients: severe osteoclastic resorption, osteoporosis, even for pediatric cases, and an altered rate of remodeling biomarkers have been reported. In our AKU osteoblast cell model, we found a clear altered redox homeostasis, determined by elevated hydrogen peroxide (H2O2) levels and 4HNE protein adducts formation. These findings were correlated with increased NADPH oxidase (NOX) activity and altered mitochondrial respiration. In addition, we observed a decreased activity of superoxide dismutase (SOD) and reduced levels of thioredoxin (TRX) that parallel the decreased Nrf2-DNA binding. Overall, our results reveal that HGA is able to alter the cellular redox homeostasis by modulating the endogenous ROS production via NOX activation and mitochondrial dysfunctions and impair the cellular response mechanism. These findings can be useful for understanding the pathophysiology of AKU, not yet well studied in bones, but which is an important source of comorbidities that affect the life quality of the patients.


Assuntos
Alcaptonúria/metabolismo , Homeostase/fisiologia , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Ácido Homogentísico/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , NADPH Oxidases/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Osteoblastos/citologia , Oxirredução , Estresse Oxidativo/fisiologia , Transdução de Sinais , Superóxido Dismutase/metabolismo , Tiorredoxinas/metabolismo
13.
PLoS One ; 15(6): e0233856, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32492073

RESUMO

The enzyme ß-glucosidase 2 (GBA2) is clinically relevant because it is targeted by the drug miglustat (Zavesca®) and because it is involved in inherited diseases. Mutations in the GBA2 gene are associated with two neurological diseases on the ataxia-spasticity spectrum, hereditary spastic paraplegia 46 (SPG46) and Marinesco-Sjögren-like syndrome (MSS). To establish how GBA2 mutations give rise to neurological pathology, we have begun to investigate mutant forms of GBA2 encoded by disease-associated GBA2 alleles. Previously, we found that five GBA2 missense mutants and five C-terminally truncated mutants lacked enzyme activity. Here we have examined the cellular locations of wild-type (WT) and mutant forms of GBA2 by confocal and electron microscopy, using transfected cells. Similar to GBA2-WT, the D594H and M510Vfs*17 GBA2 mutants were located at the plasma membrane, whereas the C-terminally truncated mutants terminating after amino acids 233 and 339 (GBA2-233 and -339) were present in the mitochondrial matrix, induced mitochondrial fragmentation and loss of mitochondrial transmembrane potential. Deletional mutagenesis indicated that residues 161-200 are critical for the mitochondrial fragmentation of GBA2-233 and -339. Considering that the mitochondrial fragmentation induced by GBA2-233 and -339 is consistently accompanied by their localization to the mitochondrial matrix, our deletional analysis raises the possibility that that GBA2 residues 161-200 harbor an internal targeting sequence for transport to the mitochondrial matrix. Altogether, our work provides new insights into the behaviour of GBA2-WT and disease-associated forms of GBA2.


Assuntos
Glucosilceramidase/metabolismo , Mitocôndrias/patologia , Alelos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Glucosilceramidase/genética , Humanos , Potencial da Membrana Mitocondrial , Microscopia Eletrônica , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Mutação , Paraplegia Espástica Hereditária/genética , Degenerações Espinocerebelares/genética
14.
Proc Natl Acad Sci U S A ; 117(27): 15684-15693, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571921

RESUMO

Mitochondria are known to play an essential role in photoreceptor function and survival that enables normal vision. Within photoreceptors, mitochondria are elongated and extend most of the inner-segment length, where they supply energy for protein synthesis and the phototransduction machinery in the outer segment, as well as acting as a calcium store. Here, we examined the arrangement of the mitochondria within the inner segment in detail using three-dimensional (3D) electron microscopy techniques and show they are tethered to the plasma membrane in a highly specialized arrangement. Remarkably, mitochondria and their cristae openings align with those of neighboring inner segments. The pathway by which photoreceptors meet their high energy demands is not fully understood. We propose this to be a mechanism to share metabolites and assist in maintaining homeostasis across the photoreceptor cell layer. In the extracellular space between photoreceptors, Müller glial processes were identified. Due to the often close proximity to the inner-segment mitochondria, they may, too, play a role in the inner-segment mitochondrial arrangement as well as metabolite shuttling. OPA1 is an important factor in mitochondrial homeostasis, including cristae remodeling; therefore, we examined the photoreceptors of a heterozygous Opa1 knockout mouse model. The cristae structure in the Opa1 +/- photoreceptors was not greatly affected, but the mitochondria were enlarged and had reduced alignment to neighboring inner-segment mitochondria. This indicates the importance of key regulators in maintaining this specialized photoreceptor mitochondrial arrangement.


Assuntos
GTP Fosfo-Hidrolases/genética , Mitocôndrias/genética , Membranas Mitocondriais/ultraestrutura , Visão Ocular/genética , Animais , Membrana Celular/genética , Membrana Celular/ultraestrutura , Células Ependimogliais/metabolismo , Células Ependimogliais/ultraestrutura , Humanos , Camundongos , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Células Fotorreceptoras/ultraestrutura , Visão Ocular/fisiologia
15.
Nat Commun ; 11(1): 2857, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32504069

RESUMO

Virtual memory T (TVM) cells are antigen-naïve CD8+ T cells that exist in a semi-differentiated state and exhibit marked proliferative dysfunction in advanced age. High spare respiratory capacity (SRC) has been proposed as a defining metabolic characteristic of antigen-experienced memory T (TMEM) cells, facilitating rapid functionality and survival. Given the semi-differentiated state of TVM cells and their altered functionality with age, here we investigate TVM cell metabolism and its association with longevity and functionality. Elevated SRC is a feature of TVM, but not TMEM, cells and it increases with age in both subsets. The elevated SRC observed in aged mouse TVM cells and human CD8+ T cells from older individuals is associated with a heightened sensitivity to IL-15. We conclude that elevated SRC is a feature of TVM, but not TMEM, cells, is driven by physiological levels of IL-15, and is not indicative of enhanced functionality in CD8+ T cells.


Assuntos
Envelhecimento/imunologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Subpopulações de Linfócitos T/imunologia , Adulto , Idoso , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/ultraestrutura , Diferenciação Celular/imunologia , Proliferação de Células , Modelos Animais de Doenças , Feminino , Humanos , Vírus da Influenza A/imunologia , Influenza Humana/sangue , Influenza Humana/imunologia , Influenza Humana/virologia , Masculino , Camundongos , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/ultraestrutura , Adulto Jovem
16.
Life Sci ; 256: 117971, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32553925

RESUMO

AIMS: Multiple myeloma (MM) was recently reported to rely on increased oxidative phosphorylation (OXPHOS) for survival, providing a potential opportunity for MM therapy. Herein, we aimed to propose a novel targeted drug for MM treatment, followed by the exploration of reason for OXPHOS enhancement in MM cells. MATERIALS AND METHODS: The expression of OXPHOS genes and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) was analyzed using bioinformatics analyses, followed by verification in MM cell lines. The effects of SR18292 on OXPHOS were measured by qRT-PCR, Western blot, transmission electron microscopy, oxygen consumption rate and so on. The proliferation and apoptosis were evaluated by CCK-8, flow cytometry and Western blot. The efficiency and safety of SR18292 were assessed in a mouse model of MM. KEY FINDINGS: The OXPHOS genes were generally overexpressed in MM cells, which was associated with poorer prognosis of MM patients. PGC-1α, a transcriptional coactivator, was upregulated in MM cells, and MM patients with higher PGC-1α expression exhibited increased enrichment of the OXPHOS gene set. Treatment with SR18292 (an inhibitor of PGC-1α) significantly impaired the proliferation and survival of MM cells due to OXPHOS metabolism dysfunction, which leads to energy exhaustion and oxidative damage. Besides, SR18292 potently inhibited tumor growth at a well-tolerated dose in MM model mice. SIGNIFICANCE: The overexpression of OXPHOS gene set mediated by upregulated PGC-1α provides a structural basis for enhanced OXPHOS in MM cells, and SR18292 (a PGC-1α inhibitor) exerts potent antimyeloma effects, offering a potential tangible avenue for MM therapy.


Assuntos
Antineoplásicos/uso terapêutico , Indóis/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Fosforilação Oxidativa , Propanóis/uso terapêutico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Metabolismo Energético/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Indóis/farmacologia , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Mieloma Múltiplo/ultraestrutura , Fosforilação Oxidativa/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Prognóstico , Propanóis/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Nat Commun ; 11(1): 2475, 2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32424134

RESUMO

Autoimmune ß-cell destruction leads to type 1 diabetes, but the pathophysiological mechanisms remain unclear. To help address this void, we created an open-access online repository, unprecedented in its size, composed of large-scale electron microscopy images ('nanotomy') of human pancreas tissue obtained from the Network for Pancreatic Organ donors with Diabetes (nPOD; www.nanotomy.org). Nanotomy allows analyses of complete donor islets with up to macromolecular resolution. Anomalies we found in type 1 diabetes included (i) an increase of 'intermediate cells' containing granules resembling those of exocrine zymogen and endocrine hormone secreting cells; and (ii) elevated presence of innate immune cells. These are our first results of mining the database and support recent findings that suggest that type 1 diabetes includes abnormalities in the exocrine pancreas that may induce endocrine cellular stress as a trigger for autoimmunity.


Assuntos
Bases de Dados como Assunto , Diabetes Mellitus Tipo 1/patologia , Ilhotas Pancreáticas/ultraestrutura , Microscopia Eletrônica , Autoanticorpos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Granulócitos/imunologia , Humanos , Imunidade Inata , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/patologia , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Doadores de Tecidos
18.
Am J Physiol Endocrinol Metab ; 318(6): E848-E855, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32369416

RESUMO

Disturbances in skeletal muscle lipid oxidation might induce ectopic fat deposition and lipotoxicity. Nevertheless, the cellular mechanisms that regulate skeletal muscle lipid oxidation have not been fully determined. We aimed to determine whether there was an association between relative whole body lipid oxidation and mitochondrial size or mitochondria-sarcoplasmic reticulum interactions in the skeletal muscle. Twelve healthy men were included [mean (standard deviation), 24.7 (1.5) yr old, 24.4 (2.6) kg/m2]. The respiratory quotient (RQ) was used to estimate relative lipid oxidation at rest and during exercise (50% maximal oxygen consumption, 600 kcal expended). A skeletal muscle biopsy was obtained from the vastus lateralis at rest. Transmission electron microscopy was used to determine mitochondrial size and mitochondria-sarcoplasmic reticulum interactions (≤50 nm of distance between organelles). Protein levels of fusion/fission regulators were measured in skeletal muscle by Western blot. Resting RQ and exercise RQ associated inversely with intermyofibrillar mitochondrial size (r = -0.66 and r = -0.60, respectively, P < 0.05). Resting RQ also associated inversely with the percentage of intermyofibrillar mitochondria-sarcoplasmic reticulum interactions (r = -0.62, P = 0.03). Finally, intermyofibrillar mitochondrial size associated inversely with lipid droplet density (r = -0.66, P = 0.01) but directly with mitochondria fusion-to-fission ratio (r = 0.61, P = 0.03). Our results show that whole body lipid oxidation is associated with skeletal muscle intermyofibrillar mitochondrial size, fusion phenotype, and mitochondria-sarcoplasmic-reticulum interactions in nondiabetic humans.


Assuntos
Exercício Físico/fisiologia , Metabolismo dos Lipídeos , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Quadríceps/ultraestrutura , Retículo Sarcoplasmático/ultraestrutura , Adolescente , Adulto , Humanos , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/ultraestrutura , Masculino , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Tamanho Mitocondrial , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Oxirredução , Consumo de Oxigênio , Músculo Quadríceps/metabolismo , Adulto Jovem
19.
PLoS One ; 15(5): e0232759, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32453737

RESUMO

SUMMARY: Reprogramming autologous adult cells to pluripotent cells allows for relatively safe cell replacement therapy. This can be achieved by nuclear transfer, cell fusion, or induced pluripotent stem cell technology However, the epigenetic memory of the cell is considered as a great challenge facing the complete reprograming of cells by these methods. Introducing oocyte-specific factors into differentiated cells may present a promising approach by mimicking cellular reprogramming during fertilization. METHODS: Human bone marrow mesenchymal stromal cells (hBM-MSCs) were cultured with different concentrations of human metaphase II (M II) oocyte extract (0.1, 1, 5, 10, 30 ng/µl). Reprogramming was assessed at various exposure times (1, 4, 7 days). Cells were tested for their proliferation rate, morphological changes, expression of pluripotency markers, expression of mesenchymal to epithelial transition markers, and mitochondrial rejuvenation. (mitochondrial localization, morphological changes, bioenergetics, transmembrane potential, and levels of reactive oxygen species, ROS). RESULTS: Treatment of human BM-MSCs with 10 ng/µl oocyte extract resulted in increased cell proliferation, which was associated with the upregulation of the pluripotency genes OCT-4, NANOG, and SOX-2 and a concomitant downregulation of mesenchymal-specific genes. MSCs exhibited small, immature round mitochondria with few swollen cristae localized proximal to the cell nucleus. This was accompanied by morphological cell changes, a metabolic shift towards oxidative phosphorylation, a high mitochondrial membrane potential, and increased ROS production. CONCLUSION: These data show that treatment with 10 ng/µl human MII-phase oocyte extract induced genetic and mitochondrial reprogramming of human BM-MSCs to a more embryonic phenotype.


Assuntos
Extratos Celulares/farmacologia , Reprogramação Celular/genética , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Oócitos/metabolismo , Biomarcadores/metabolismo , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Reprogramação Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Potenciais da Membrana/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Oócitos/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Fatores de Tempo
20.
Neurology ; 94(23): e2404-e2411, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32457210

RESUMO

OBJECTIVE: To test the hypothesis that ApoE isoforms affect mitochondrial structure and function that are related to cognitive impairment in Alzheimer disease (AD), we systematically investigated the effects of ApoE isoforms on mitochondrial biogenesis and dynamics, oxidative stress, synapses, and cognitive performance in AD. METHODS: We obtained postmortem human brain tissues and measured proteins that are responsible for mitochondrial biogenesis (peroxisome proliferator-activated receptor-gamma coactivator-1α [PGC-1α] and sirtuin 3 [SIRT3]), for mitochondrial dynamics (mitofusin 1 [MFN1], mitofusin 2 [MFN2], and dynamin-like protein 1 [DLP1]), for oxidative stress (superoxide dismutase 2 [SOD2] and forkhead-box protein O3a [Foxo3a]), and for synapses (postsynaptic density protein 95 [PSD95] and synapsin1 [Syn1]). A total of 46 cases were enrolled, including ApoE-ɛ4 carriers (n = 21) and noncarriers (n = 25). RESULTS: Levels of these proteins were compared between ApoE-ɛ4 carriers and noncarriers. ApoE-ɛ4 was associated with impaired mitochondrial structure and function, oxidative stress, and synaptic integrity in the human brain. Correlation analysis revealed that mitochondrial proteins and the synaptic protein were strongly associated with cognitive performance. CONCLUSION: ApoE isoforms influence mitochondrial structure and function, which likely leads to alteration in oxidative stress, synapses, and cognitive function. These mitochondria-related proteins may be a harbinger of cognitive decline in ApoE-ɛ4 carriers and provide novel therapeutic targets for prevention and treatment of AD.


Assuntos
Doença de Alzheimer/metabolismo , Apolipoproteínas E/fisiologia , Mitocôndrias/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/psicologia , Apolipoproteína E4/genética , Apolipoproteína E4/fisiologia , Química Encefálica , Feminino , Humanos , Masculino , Testes de Estado Mental e Demência , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial , Proteínas Mitocondriais/análise , Proteínas do Tecido Nervoso/análise , Plasticidade Neuronal/genética , Biogênese de Organelas , Estresse Oxidativo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/análise , Isoformas de Proteínas/fisiologia , Sirtuína 3/análise , Aprendizagem Verbal
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