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1.
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
2.
Nat Commun ; 11(1): 3699, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709877

RESUMO

Mitochondria play a critical role in generating energy to support the entire lifecycle of biological cells, yet it is still unclear how their morphological structures evolve to regulate their functionality. Conventional fluorescence microscopy can only provide ~300 nm resolution, which is insufficient to visualize mitochondrial cristae. Here, we developed an enhanced squaraine variant dye (MitoESq-635) to study the dynamic structures of mitochondrial cristae in live cells with a superresolution technique. The low saturation intensity and high photostability of MitoESq-635 make it ideal for long-term, high-resolution (stimulated emission depletion) STED nanoscopy. We performed time-lapse imaging of the mitochondrial inner membrane over 50 min (3.9 s per frame, with 71.5 s dark recovery) in living HeLa cells with a resolution of 35.2 nm. The forms of the cristae during mitochondrial fusion and fission can be clearly observed. Our study demonstrates the emerging capability of optical STED nanoscopy to investigate intracellular physiological processes with nanoscale resolution for an extended period of time.


Assuntos
Ciclobutanos , Membranas Mitocondriais/ultraestrutura , Nanotecnologia/métodos , Fenóis , Linhagem Celular , Corantes Fluorescentes/química , Células HeLa , Humanos , Microscopia de Fluorescência/métodos , Mitocôndrias , Dinâmica Mitocondrial/fisiologia , Coloração e Rotulagem/métodos
3.
Nat Commun ; 11(1): 3301, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620849

RESUMO

Many cellular stresses are transduced into apoptotic signals through modification or up-regulation of the BH3-only subfamily of BCL2 proteins. Through direct or indirect mechanisms, these proteins activate BAK and BAX to permeabilize the mitochondrial outer membrane. While the BH3-only proteins BIM, PUMA, and tBID have been confirmed to directly activate BAK through its canonical BH3 binding groove, whether the BH3-only proteins BMF, HRK or BIK can directly activate BAK is less clear. Here we show that BMF and HRK bind and directly activate BAK. Through NMR studies, site-directed mutagenesis, and advanced molecular dynamics simulations, we also find that BAK activation by BMF and possibly HRK involves a previously unrecognized binding groove formed by BAK α4, α6, and α7 helices. Alterations in this groove decrease the ability of BMF and HRK to bind BAK, permeabilize membranes and induce apoptosis, suggesting a potential role for this BH3-binding site in BAK activation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Sítios de Ligação/genética , Células Cultivadas , Humanos , Células Jurkat , Espectroscopia de Ressonância Magnética , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Domínios Proteicos , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Homologia de Sequência de Aminoácidos , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/genética
4.
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
5.
Mol Cell ; 79(1): 68-83.e7, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32533918

RESUMO

BAX is a pro-apoptotic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane. How BAX monomers assemble into a higher-order conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a homogeneous BAX oligomer (BAXO) for analysis. Here, we report the production and characterization of a full-length BAXO that recapitulates physiologic BAX activation. Multidisciplinary studies revealed striking conformational consequences of oligomerization and insight into the macromolecular structure of oligomeric BAX. Importantly, BAXO enabled the assignment of specific roles to particular residues and α helices that mediate individual steps of the BAX activation pathway, including unexpected functionalities of BAX α6 and α9 in driving membrane disruption. Our results provide the first glimpse of a full-length and functional BAXO, revealing structural requirements for the elusive execution phase of mitochondrial apoptosis.


Assuntos
Apoptose , Mitocôndrias/patologia , Membranas Mitocondriais/metabolismo , Multimerização Proteica , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Animais , Transporte Biológico , Permeabilidade da Membrana Celular , Citosol/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Modelos Moleculares , Conformação Proteica , Proteínas Proto-Oncogênicas c-fos
6.
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
7.
Food Chem ; 328: 127174, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32492604

RESUMO

This study investigated the effect of lysosomal iron involvement in the mechanism of mitochondrial apoptosis on bovine muscle protein degradation during postmortem aging. Six crossbred cattle were studied to evaluate intracellular reactive oxygen species (ROS), antioxidant enzyme activity, lysosomal membrane stability, mitochondrial dysfunction-induced apoptosis, desmin and troponin-T degradation in both control and iron chelator desferrioxamine (DFO) groups. Results showed that lysosomal iron induced ROS accumulation and lysosomal membrane destabilization by decreasing the antioxidant enzyme activity (P < 0.05). Subsequently, lysosomal dysfunction mediated by iron increased mitochondrial membrane permeability and decreased mitochondrial membrane potential, thereby enhancing Bid and cytochrome c release and caspase-9/-3 activation (P < 0.05). Ultimately, lysosomal iron mediated lysosomal-mitochondrial apoptosis increased the postmortem bovine muscle desmin and troponin-T degradation (P < 0.05). The results indicated that lysosomal iron contributes to postmortem meat tenderization through the lysosomal-mitochondrial dysfunction-induced apoptosis pathway.


Assuntos
Ferro/metabolismo , Lisossomos/metabolismo , Proteínas de Carne/metabolismo , Mitocôndrias Musculares/patologia , Membranas Mitocondriais/patologia , Animais , Apoptose/efeitos dos fármacos , Autopsia , Bovinos , Permeabilidade da Membrana Celular , Citocromos c/metabolismo , Masculino , Mitocôndrias/metabolismo , Mitocôndrias Musculares/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Musculares/metabolismo , Proteólise , Espécies Reativas de Oxigênio/metabolismo
8.
Toxicol Appl Pharmacol ; 401: 115076, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32479918

RESUMO

Statin induced myopathy (SIM) is a main deleterious effect leading to the poor treatment compliance, while the preventive or therapeutic treatments are absent. Mounting evidences demonstrated that vitamin D plays a vital role in muscle as a direct modulator. The deficiency of vitamin D was considered as a cause of muscle dysfunction, whereas the supplementation resulted in a remission. However, there is no causal proof that vitamin D supplementation rescues SIM. Here, using the mice model of simvastatin-induced myopathy, we investigated the role of vitamin D supplementation and the mechanisms associated with mitochondria. Results indicated that simvastatin administration (80 mg/kg) impaired skeletal muscle with the increased serum creatine kinase (CK) level and the declined grip strength, which were alleviated by vitamin D supplementation. Moreover, vitamin D supplementation rescued the energy metabolism dysfunction in simvastatin-treated mice gastrocnemius by reducing the abnormal aggregation of muscular glycogen and lactic acid. Mitochondrial homeostasis plays a key role in the process of energy metabolism. Thus, the mitochondrial dysfunction is a mortal damage for the highly energy-requiring tissue. In our study, the mitochondrial cristae observed under transmission electron microscope (TEM) were lytic in simvastatin-treated gastrocnemius. Interestingly, vitamin D supplementation improved the mitochondrial cristae shape by regulating the expression of mitofusin-1/2 (MFN1/2), optic atrophy 1 (OPA1) and dynamin-related protein 1 (Drp1). As expected, the mitochondrial dysfunction and oxidative stress was mitigated by vitamin D supplementation. In conclusion, these findings suggested that moderate vitamin D supplementation rescued simvastatin induced myopathy via improving the mitochondrial cristae shape and function.


Assuntos
Suplementos Nutricionais , Mitocôndrias/efeitos dos fármacos , Doenças Musculares/induzido quimicamente , Doenças Musculares/tratamento farmacológico , Sinvastatina/toxicidade , Vitamina D/administração & dosagem , Animais , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Inibidores de Hidroximetilglutaril-CoA Redutases/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/patologia , Doenças Musculares/metabolismo , Distribuição Aleatória
9.
PLoS One ; 15(6): e0234653, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579577

RESUMO

We previously demonstrated that hexokinase II (HK2) dissociation from mitochondria during cardiac ischemia correlates with cytochrome c (cyt-c) loss, oxidative stress and subsequent reperfusion injury. However, whether HK2 release is the primary signal mediating this ischemia-induced mitochondrial dysfunction was not established. To investigate this, we studied the effects of dissociating HK2 from isolated heart mitochondria. Mitochondria isolated from Langendorff-perfused rat hearts before and after 30 min global ischemia ± ischemic preconditioning (IPC) were subject to in vitro dissociation of HK2 by incubation with glucose-6-phosphate at pH 6.3. Prior HK2 dissociation from pre- or end-ischemic heart mitochondria had no effect on their cyt-c release, respiration (± ADP) or mitochondrial permeability transition pore (mPTP) opening. Inner mitochondrial membrane morphology was assessed indirectly by monitoring changes in light scattering (LS) and confirmed by transmission electron microscopy. Although no major ultrastructure differences were detected between pre- and end-ischemia mitochondria, the amplitude of changes in LS was reduced in the latter. This was prevented by IPC but not mimicked in vitro by HK2 dissociation. We also observed more Drp1, a mitochondrial fission protein, in end-ischemia mitochondria. IPC failed to prevent this increase but did decrease mitochondrial-associated dynamin 2. In vitro HK2 dissociation alone cannot replicate ischemia-induced effects on mitochondrial function implying that in vivo dissociation of HK2 modulates end-ischemia mitochondrial function indirectly perhaps involving interaction with mitochondrial fission proteins. The resulting changes in mitochondrial morphology and cristae structure would destabilize outer / inner membrane interactions, increase cyt-c release and enhance mPTP sensitivity to [Ca2+].


Assuntos
Hexoquinase/metabolismo , Mitocôndrias Cardíacas/enzimologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Isquemia Miocárdica/enzimologia , Animais , Respiração Celular/efeitos dos fármacos , Dinaminas/metabolismo , Glucose-6-Fosfato/farmacologia , Hemodinâmica/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Precondicionamento Isquêmico , Ligantes , Masculino , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/ultraestrutura , Dinâmica Mitocondrial/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Isquemia Miocárdica/patologia , Ligação Proteica/efeitos dos fármacos , Ratos Wistar
10.
Proc Natl Acad Sci U S A ; 117(22): 12109-12120, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414919

RESUMO

The mitochondria-associated membrane (MAM) has emerged as a cellular signaling hub regulating various cellular processes. However, its molecular components remain unclear owing to lack of reliable methods to purify the intact MAM proteome in a physiological context. Here, we introduce Contact-ID, a split-pair system of BioID with strong activity, for identification of the MAM proteome in live cells. Contact-ID specifically labeled proteins proximal to the contact sites of the endoplasmic reticulum (ER) and mitochondria, and thereby identified 115 MAM-specific proteins. The identified MAM proteins were largely annotated with the outer mitochondrial membrane (OMM) and ER membrane proteins with MAM-related functions: e.g., FKBP8, an OMM protein, facilitated MAM formation and local calcium transport at the MAM. Furthermore, the definitive identification of biotinylation sites revealed membrane topologies of 85 integral membrane proteins. Contact-ID revealed regulatory proteins for MAM formation and could be reliably utilized to profile the proteome at any organelle-membrane contact sites in live cells.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteoma/análise , Proteínas de Ligação a Tacrolimo/metabolismo , Cálcio/metabolismo , Humanos , Biogênese de Organelas , Proteoma/metabolismo , Transdução de Sinais
11.
Proc Natl Acad Sci U S A ; 117(22): 12143-12154, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32424107

RESUMO

Proximity labeling catalyzed by promiscuous enzymes, such as TurboID, have enabled the proteomic analysis of subcellular regions difficult or impossible to access by conventional fractionation-based approaches. Yet some cellular regions, such as organelle contact sites, remain out of reach for current PL methods. To address this limitation, we split the enzyme TurboID into two inactive fragments that recombine when driven together by a protein-protein interaction or membrane-membrane apposition. At endoplasmic reticulum-mitochondria contact sites, reconstituted TurboID catalyzed spatially restricted biotinylation, enabling the enrichment and identification of >100 endogenous proteins, including many not previously linked to endoplasmic reticulum-mitochondria contacts. We validated eight candidates by biochemical fractionation and overexpression imaging. Overall, split-TurboID is a versatile tool for conditional and spatially specific proximity labeling in cells.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteoma/análise , Biotinilação , Células HEK293 , Humanos , Proteoma/metabolismo , Coloração e Rotulagem
12.
Adv Exp Med Biol ; 1239: 439-451, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32451871

RESUMO

The birth of widely available genomic databases at the turn of the millennium led to the identification of many previously unknown myosin genes and identification of novel classes of myosin, including MYO19. Further sequence analysis has revealed the unique evolutionary history of class XIX myosins. MYO19 is found in species ranging from vertebrates to some unicellular organisms, while it has been lost from some lineages containing traditional experimental model organisms. Unique sequences in the motor domain suggest class-specific mechanochemistry that may relate to its cellular function as a mitochondria-associated motor. Work over the past 10 years has demonstrated that MYO19 is an actin-activated ATPase capable of actin-based transport, and investigation of some of the conserved differences within the motor domain indicate their importance in MYO19 motor activity. The cargo-binding MyMOMA tail domain contains two distinct mechanisms of interaction with mitochondrial outer membrane components, and perturbation of MYO19 expression leads to alterations in mitochondrial movement and dynamics that impact cell function. This chapter summarizes the current state of the field and highlights potential new directions of inquiry.


Assuntos
Miosinas , Actinas , Animais , Humanos , Mitocôndrias , Membranas Mitocondriais
13.
Nat Commun ; 11(1): 2355, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398688

RESUMO

Correct intracellular distribution of proteins is critical for the function of eukaryotic cells. Certain proteins are targeted to more than one cellular compartment, e.g. to mitochondria and peroxisomes. The protein phosphatase Ptc5 from Saccharomyces cerevisiae contains an N-terminal mitochondrial presequence followed by a transmembrane domain, and has been detected in the mitochondrial intermembrane space. Here we show mitochondrial transit of Ptc5 to peroxisomes. Translocation of Ptc5 to peroxisomes depended both on the C-terminal peroxisomal targeting signal (PTS1) and N-terminal cleavage by the mitochondrial inner membrane peptidase complex. Indirect targeting of Ptc5 to peroxisomes prevented deleterious effects of its phosphatase activity in the cytosol. Sorting of Ptc5 involves simultaneous interaction with import machineries of both organelles. We identify additional mitochondrial proteins with PTS1, which localize in both organelles and can increase their physical association. Thus, a tug-of-war-like mechanism can influence the interaction and communication of two cellular compartments.


Assuntos
Mitocôndrias/metabolismo , Peroxissomos/metabolismo , Proteína Fosfatase 2C/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Sinais Direcionadores de Proteínas , Saccharomyces cerevisiae/citologia
14.
Sci Total Environ ; 734: 139229, 2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32450398

RESUMO

The induced membrane damage is a key mechanism for the cytotoxicity of graphene nanosheets (GNSs). In this research, the physical interaction of GNSs on model membranes was investigated using artificial membranes and plasma membrane vesicles. The effects of the GNSs on plasma membrane, lysosomal and mitochondrial membranes were investigated using rat basophilic leukemia (RBL2H3) cells via lactate dehydrogenase (LDH) assay, acridine orange staining and JC-1 probe, respectively. The physical interaction with model membranes was dominated by electrostatic forces, and the adhered GNSs disrupted the membrane. The degree of physical membrane disruption was quantified by the quartz crystal microbalance with dissipation (QCM-D), confirming the serious membrane disruption. The internalized GNSs were mainly distributed in the lysosomes. They caused plasma membrane leakage, increased the lysosomal membrane permeability (LMP), and depolarized the mitochondrial membrane potential (MMP). The increased cellular levels of reactive oxygen species (ROS) were also detected after GNS exposure. The combination of physical interaction and the excess ROS production damaged the plasma and organelle membranes in living RBL-2H3 cells. The lysosomal and mitochondrial dysfunction, and the oxidative stress further induced cell apoptosis. Specially, the exposure to 25 mg/L GNSs caused severest cell mortality, plasma membrane damage, ROS generation, MMP depolarization and apoptosis. The research findings provide more comprehensive information on the graphene-induced plasma and organelle membrane damage, which is important to understand and predict the cytotoxicity of carbon-based nanomaterials.


Assuntos
Membranas Mitocondriais , Animais , Apoptose , Linhagem Celular Tumoral , Grafite , Lisossomos , Potencial da Membrana Mitocondrial , Ratos , Espécies Reativas de Oxigênio
15.
Gene ; 748: 144705, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32339625

RESUMO

Trypanosomatids are unicellular parasitic protozoa. Many of the species of this genera cause severe diseases in human, such as Leishmaniasis, African trypanosomiasis and Chagas disease. These parasites possess a single reticular mitochondrion with a concatenated structure of mitochondrial DNA known as kinetoplast or kDNA. kDNA encodes few essential mitochondrial proteins but no tRNAs. Therefore, trypanosomatid mitochondrion import a full set of nucleus-encoded tRNAs for mitochondrial translation. Recent advances indicated that mitochondrial protein translocases, particularly the subunits of the ATOM complex, are involved in the import of a tRNA in Trypanosoma brucei. However, the global picture and the role of the translocase components of the mitochondrial inner membrane (TbTims) are not well understood. Here we investigated the relative abundance of 16 different tRNAs in the cytosolic and mitochondrial fractions isolated from the six TbTims knockdown cell lines. We found that knockdown of TbTim17, one of the primary components of the TbTIM complex, reduced the abundance of all of these tRNAs into mitochondria and increased their abundance in the cytosol. Depletion of TbTim62, a TbTim17 associated proteins, also reduced the relative abundance of most of these tRNAs into mitochondria except for tRNAleu, tRNAmet, and tRNAglu. Whereas, knockdown of other TbTims, like TbTim50 and two small TbTims, TbTim10 and TbTim8/13, didn't have any effect on tRNA abundance either in the cytosol or mitochondria. Depletion of any of these TbTims showed minimal effect on the levels of total tRNAs in T. brucei. Absolute quantification of tRNA levels revealed that TbTim17 knockdown reduced the levels of different tRNAs in mitochondria from 3-6% to 0.8-1.4%, which is equivalent to ~70% reduction in average. Whereas, TbTim62 depletion showed somewhat selective effect. Overall, our results suggest that TbTim17 and TbTim62 are essential for tRNA import that further makes a connection between the tRNA and protein import into mitochondria in T. brucei.


Assuntos
Proteínas de Membrana Transportadoras/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/enzimologia , RNA de Transferência/genética , RNA de Transferência/metabolismo , Trypanosoma brucei brucei/enzimologia , Animais , Transporte Biológico
16.
Proc Natl Acad Sci U S A ; 117(17): 9329-9337, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32291341

RESUMO

The organization of the mitochondrial electron transport chain proteins into supercomplexes (SCs) is now undisputed; however, their assembly process, or the role of differential expression isoforms, remain to be determined. In Saccharomyces cerevisiae, cytochrome c oxidase (CIV) forms SCs of varying stoichiometry with cytochrome bc 1 (CIII). Recent studies have revealed, in normoxic growth conditions, an interface made exclusively by Cox5A, the only yeast respiratory protein that exists as one of two isoforms depending on oxygen levels. Here we present the cryo-EM structures of the III2-IV1 and III2-IV2 SCs containing the hypoxic isoform Cox5B solved at 3.4 and 2.8 Å, respectively. We show that the change of isoform does not affect SC formation or activity, and that SC stoichiometry is dictated by the level of CIII/CIV biosynthesis. Comparison of the CIV5B- and CIV5A-containing SC structures highlighted few differences, found mainly in the region of Cox5. Additional density was revealed in all SCs, independent of the CIV isoform, in a pocket formed by Cox1, Cox3, Cox12, and Cox13, away from the CIII-CIV interface. In the CIV5B-containing hypoxic SCs, this could be confidently assigned to the hypoxia-induced gene 1 (Hig1) type 2 protein Rcf2. With conserved residues in mammalian Hig1 proteins and Cox3/Cox12/Cox13 orthologs, we propose that Hig1 type 2 proteins are stoichiometric subunits of CIV, at least when within a III-IV SC.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Microscopia Crioeletrônica/métodos , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/fisiologia , Hipóxia/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Isoformas de Proteínas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia
17.
Proc Natl Acad Sci U S A ; 117(17): 9349-9355, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32291342

RESUMO

Mitochondria metabolize almost all the oxygen that we consume, reducing it to water by cytochrome c oxidase (CcO). CcO maximizes energy capture into the protonmotive force by pumping protons across the mitochondrial inner membrane. Forty years after the H+/e- stoichiometry was established, a consensus has yet to be reached on the route taken by pumped protons to traverse CcO's hydrophobic core and on whether bacterial and mitochondrial CcOs operate via the same coupling mechanism. To resolve this, we exploited the unique amenability to mitochondrial DNA mutagenesis of the yeast Saccharomyces cerevisiae to introduce single point mutations in the hydrophilic pathways of CcO to test function. From adenosine diphosphate to oxygen ratio measurements on preparations of intact mitochondria, we definitely established that the D-channel, and not the H-channel, is the proton pump of the yeast mitochondrial enzyme, supporting an identical coupling mechanism in all forms of the enzyme.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/química , Heme/química , Oxirredutases/química , Bactérias/metabolismo , Cobre/química , Cobre/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Transporte de Íons , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Oxirredução , Oxirredutases/metabolismo , Oxigênio/metabolismo , Bombas de Próton/metabolismo , Prótons , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
Sheng Li Xue Bao ; 72(2): 205-219, 2020 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-32328614

RESUMO

The mitochondrial respiratory chain supercomplex (mitoSC) is a complex super-assembly formed by free complexes on the mitochondrial inner membrane respiratory chain through the interaction between their subunits, mainly including mitoSCI1+III2+IV1-4, mitoSCI1+III2, mitoSCIII2+IV1-2, high molecular weight mitoSC (HMW mitoSC) and mitochondrial metacomplex (mitoMC). mitoSC has been shown to improve the efficiency of electron transport in the respiratory chain and reduce the production of reactive oxygen species. The species and content of mitoSC change in different tissues in aging and many mitochondria-related diseases. By summarizing the structure and function of mitoSC in different tissues of human and mammals, and the changes of mitoSC under conditions of aging, heart disease, type 2 diabetes, cancer and genetic defects, this review focuses on the effects of exercise on mitoSC and its related regulation mechanisms in order to offer an insight for exercise interventions in mitochondria-related diseases.


Assuntos
Transporte de Elétrons , Exercício Físico , Membranas Mitocondriais/enzimologia , Animais , Humanos , Mitocôndrias , Doenças Mitocondriais
19.
Metabolism ; 107: 154227, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32275974

RESUMO

OBJECTIVE: L5, a highly electronegative subtype of low-density lipoprotein (LDL), is likely associated with the development of atherosclerosis and cardiovascular diseases. Normal LDL is composed mainly of apolipoprotein (Apo) B, but L5 has additional proteins such as ApoE. We previously demonstrated that L5 induces endothelial cell senescence by increasing mitochondrial reactive oxygen species. In the present study, we examined the effect of L5 on mitochondrial function in cardiomyocytes. METHODS: We used the Seahorse XF24 extracellular flux analyzer to examine the effect of L5 and its components on mitochondrial energy production. The effects of L5 on mitochondrial morphology were examined by immunofluorescence using MitoTracker Green FM and the corresponding probes in H9c2 cardiomyoblasts. Mitochondrial permeability was assessed by using a calcium-induced swelling assay with a voltage-dependent anion-selective channel (VDAC) inhibitor to determine VDAC-dependence both in vitro and in vivo. L5 without ApoE, referred to as △L5, was used to clarify the role of ApoE in L5-induced mitochondrial dysfunction. RESULTS: L5 not only significantly decreased basal (P < 0.05) and maximal respiration (P < 0.01) but also reduced spare respiratory capacity (P < 0.01) in H9c2 cells. Additionally, L5 caused phosphorylation of Drp1 and mitochondrial fission. Recombinant ApoE mimicked the mitochondrial effects of L5, but △L5 did not cause similar effects. After entering cells, ApoE on L5 colocalized with mitochondrial VDAC and caused mitochondria swelling both in vitro and in vivo. This effect was also seen with recombinant ApoE but not △L5. CONCLUSIONS: ApoE may play an important role in electronegative LDL-induced mitochondrial dysfunction through the opening of the mitochondrial permeability transition pore via the interaction of ApoE and VDAC.


Assuntos
Apolipoproteínas E/metabolismo , Lipoproteínas LDL/farmacologia , Doenças Mitocondriais/induzido quimicamente , Doenças Mitocondriais/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Linhagem Celular , Metabolismo Energético/efeitos dos fármacos , Humanos , Técnicas In Vitro , Lipoproteínas LDL/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Membranas Mitocondriais/metabolismo , Dilatação Mitocondrial/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Espécies Reativas de Oxigênio
20.
Life Sci ; 253: 117626, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32247002

RESUMO

AIMS: Postoperative cognitive dysfunction (POCD) is a common postoperative complication that is associated with increased morbidity and mortality. However, the mechanism of pathogenesis of POCD still remains largely unknown. The aim of the study was to investigate the function and mechanism of lncRNA PCAI in POCD. MATERIALS AND METHODS: Knockdown and overexpression studies were performed to analyze the function of lncRNA PCAI in cultured BV-2 cell lines treated with LPS to mimic the neuroinflammation. Real-time PCR, western blot, ELISA were used to determine the expression level of inflammation markers. Rescue experiment was performed to prove the relationship between PCAI and SUZ12. RESULTS: We found that the expression of lncRNA PCAI was decreased with the increasing concentrations of LPS. Knockdown of lncRNA PCAI inhibited the cell death rates and attenuated the cell inflammation via ELISA and real-time PCR. Besides, downregulated of lncRNA PCAI can protect the mitochondrial function via membrane potential assay. Overexpression of lncRNA PCAI can promote the cell death and inflammation response induced by LPS. We also provided mechanism study about lncRNA PCAI that negatively regulating SUZ12. Rescue experiment also verified the results. CONCLUSION: We performed comprehensive study of functional analysis of lncRNA PCAI in POCD and proved its mechanism, which negatively regulate SUZ12. Our study provided new clues for the clinical intervention and targets for POCD.


Assuntos
Disfunção Cognitiva/etiologia , Hipocampo/metabolismo , Inflamação/metabolismo , Complicações Cognitivas Pós-Operatórias/prevenção & controle , Complicações Pós-Operatórias/prevenção & controle , RNA Longo não Codificante/genética , Animais , Linhagem Celular , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Inflamação/patologia , Camundongos , Membranas Mitocondriais/metabolismo , Complexo Repressor Polycomb 2/genética , Substâncias Protetoras/metabolismo , Substâncias Protetoras/farmacologia
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