Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 12.900
Filtrar
1.
Int J Nanomedicine ; 14: 7003-7016, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31564862

RESUMO

Background: Yttria-stabilized zirconia (Y2O3/ZrO2) nanoparticles are one of the important nanoparticles extensively used in manufacturing of plastics, textiles, catalyst, etc. Still, the cytotoxic and apoptotic effects of yttria-stabilized zirconia nanoparticles have not been well identified on human skin keratinocyte (HaCaT) cells. Therefore, in this study, we have designed to examine the cytotoxic potential of yttria-stabilized zirconia nanoparticles in HaCaT cells. Methods: Prior to treatment, the yttria-stabilized zirconia nanoparticles were characterized by using different advanced instruments viz. dynamic light scattering (DLS), scanning electron microscope (SEM) and transmission electron microscope (TEM). Cell viability of HaCaT cells was measured by using MTS and NRU assays and viability of cells was reduced in a dose- and time-dependent manner. Results: Reduction in the viability of cells was correlated with the rise of reactive oxygen species generation, increased caspase-3, mitochondria membrane potential and evidence of DNA strand breakage. These were consistent with the possibility that mitochondria damage can play a significant role in the cytotoxic response. Moreover, the activity of oxidative enzymes such as lipid peroxide (LPO) was increased and glutathione was reduced in HaCaT cells exposed with yttria-stabilized zirconia nanoparticles. It is also important to indicate that HaCaT cells appear to be more susceptible to yttria-stabilized zirconia nanoparticles exposure after 24 hrs. Conclusion: This result provides a dose- and time-dependent apoptosis and genotoxicity of yttria-stabilized zirconia nanoparticles in HaCaT cells.


Assuntos
Apoptose , Dano ao DNA , Células Epiteliais/citologia , Nanopartículas Metálicas/química , Pele/citologia , Ítrio/química , Zircônio/química , Acetilcisteína/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Células Epiteliais/metabolismo , Glutationa/metabolismo , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Nanopartículas Metálicas/ultraestrutura , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
2.
Mol Cell ; 76(2): 295-305, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31604601

RESUMO

Biomolecular condensation is emerging as an essential process for cellular compartmentalization. The formation of biomolecular condensates can be driven by liquid-liquid phase separation, which arises from weak, multivalent interactions among proteins and nucleic acids. A substantial body of recent work has revealed that diverse cellular processes rely on biomolecular condensation and that aberrant phase separation may cause disease. Many proteins display an intrinsic propensity to undergo phase separation. However, the mechanisms by which cells regulate phase separation to build functional condensates at the appropriate time and location are only beginning to be understood. Here, we review three key cellular mechanisms that enable the control of biomolecular phase separation: membrane surfaces, post-translational modifications, and active processes. We discuss how these mechanisms may function in concert to provide robust control over biomolecular condensates and suggest new research avenues that will elucidate how cells build and maintain these key centers of cellular compartmentalization.


Assuntos
Compartimento Celular , Membrana Celular/metabolismo , Ácidos Nucleicos/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas/metabolismo , Animais , Membrana Celular/química , Endocitose , Humanos , Membranas Intracelulares/metabolismo , Chaperonas Moleculares/metabolismo , Conformação de Ácido Nucleico , Ácidos Nucleicos/química , Conformação Proteica , Proteínas/química , Solubilidade , Relação Estrutura-Atividade
3.
PLoS Biol ; 17(10): e3000475, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31584943

RESUMO

The Toxoplasma gondii inner membrane complex (IMC) is an important organelle involved in parasite motility and replication. The IMC resides beneath the parasite's plasma membrane and is composed of both membrane and cytoskeletal components. Although the protein composition of the IMC is becoming better understood, the protein-protein associations that enable proper functioning of the organelle remain largely unknown. Determining protein interactions in the IMC cytoskeletal network is particularly challenging, as disrupting the cytoskeleton requires conditions that disrupt protein complexes. To circumvent this problem, we demonstrate the application of a photoreactive unnatural amino acid (UAA) crosslinking system to capture protein interactions in the native intracellular environment. In addition to identifying binding partners, the UAA approach maps the binding interface of the bait protein used for crosslinking, providing structural information of the interacting proteins. We apply this technology to the essential IMC protein ILP1 and demonstrate that distinct regions of its C-terminal coiled-coil domain crosslink to the alveolins IMC3 and IMC6, as well as IMC27. We also show that the IMC3 C-terminal domain and the IMC6 N-terminal domain are necessary for binding to ILP1, further mapping interactions between ILP1 and the cytoskeleton. Together, this study develops a new approach to study protein-protein interactions in Toxoplasma and provides the first insight into the architecture of the cytoskeletal network of the apicomplexan IMC.


Assuntos
Azidas/química , Reagentes para Ligações Cruzadas/química , Proteínas do Citoesqueleto/química , Citoesqueleto/metabolismo , Membranas Intracelulares/metabolismo , Fenilalanina/análogos & derivados , Proteínas de Protozoários/química , Toxoplasma/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/genética , Citoesqueleto/ultraestrutura , Expressão Gênica , Membranas Intracelulares/ultraestrutura , Fenilalanina/química , Processos Fotoquímicos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas/métodos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/genética , Toxoplasma/ultraestrutura , Raios Ultravioleta
4.
J Biosci ; 44(4)2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31502569

RESUMO

Intracellular trafficking is a field that has been intensively studied for years and yet there remains much to be learned. Part of the reason that there is so much obscurity remaining in this field is due to all the pathways and the stages that define cellular trafficking. One of the major steps in cellular trafficking is fusion. Fusion is defined as the terminal step that occurs when a cargo-laden vesicle arrives at the proper destination. There are two types of fusion within a cell: homotypic and heterotypic fusion. Homotypic fusion occurs when the two membranes merging together are of the same type such as vacuole to vacuole fusion. Heterotypic fusion occurs when the two membranes at play are of different types such as when an endosomal membrane fuses with a Golgi membrane. In this review, we will focus on all the protein components - Rabs, Golgins, Multisubunit tethers, GTPases, protein phosphatases and SNAREs - that have been known to function in both of these types of fusion. We hope to develop a model of how all of these constituents function together to achieve membrane fusion. Membrane fusion is a biological process absolutely necessary for proper intracellular trafficking. Due to the degree of importance multiple proteins are required for it to be properly carried through. Whether we are talking about heterotypic or homotypic fusion, any defects in the fusion machinery can result in disease states such as Parkinson's and Alzheimer's disease. Although much research has significantly expanded our knowledge of fusion, there is still much more to be learned.


Assuntos
Citoplasma/genética , GTP Fosfo-Hidrolases/genética , Membranas Intracelulares/metabolismo , Fusão de Membrana/genética , Transporte Biológico/genética , Citoplasma/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Complexo de Golgi/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vacúolos/genética , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/genética
5.
Int J Mol Sci ; 20(19)2019 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-31561436

RESUMO

The Rab5 small GTPase is a regulator of endosomal trafficking and vesicle fusion. It possesses two adjacent cysteine residues for post-translational geranylgeranylation at its C-terminus for the protein to associate with the early endosome membrane. We compare the effect of mono-lipidification of only one cysteine residue with the doubly modified, fully functional Rab protein in both guanosine diphosphate (GDP)- and guanosine triphosphate (GTP)-bound states and in different membranes (one, three, and six-component membranes). Molecular simulations show that the mono-geranylgeranylated protein is less strongly associated with the membranes and diffuses faster than the doubly lipidated protein. The geranylgeranyl anchor membrane insertion depth is smaller and the protein-membrane distance distribution is broad and uncharacteristic for the membrane composition. The mono-geranylgeranylated protein reveals an unspecific association with the membrane and an orientation at the membrane that does not allow a nucleotide-specific recruitment of further effector proteins. This work shows that double-lipidification is critical for Rab5 to perform its physiological function and mono-geranylgeranylation renders it membrane-associated but non-functional.


Assuntos
Membranas Intracelulares/química , Lipídeos de Membrana/química , Proteínas rab5 de Ligação ao GTP/química , Sequência de Aminoácidos , Difusão , Membranas Intracelulares/metabolismo , Lipídeos de Membrana/metabolismo , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Proteínas rab5 de Ligação ao GTP/metabolismo
6.
Int J Mol Sci ; 20(18)2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31487853

RESUMO

The human cytochrome P450 (CYP) 2C9 and 2C19 enzymes are two highly similar isoforms with key roles in drug metabolism. They are anchored to the endoplasmic reticulum membrane by their N-terminal transmembrane helix and interactions of their cytoplasmic globular domain with the membrane. However, their crystal structures were determined after N-terminal truncation and mutating residues in the globular domain that contact the membrane. Therefore, the CYP-membrane interactions are not structurally well-characterized and their dynamics and the influence of membrane interactions on CYP function are not well understood. We describe herein the modeling and simulation of CYP 2C9 and CYP 2C19 in a phospholipid bilayer. The simulations revealed that, despite high sequence conservation, the small sequence and structural differences between the two isoforms altered the interactions and orientations of the CYPs in the membrane bilayer. We identified residues (including K72, P73, and I99 in CYP 2C9 and E72, R73, and H99 in CYP 2C19) at the protein-membrane interface that contribute not only to the differing orientations adopted by the two isoforms in the membrane, but also to their differing substrate specificities by affecting the substrate access tunnels. Our findings provide a mechanistic interpretation of experimentally observed effects of mutagenesis on substrate selectivity.


Assuntos
Citocromo P-450 CYP2C19/química , Citocromo P-450 CYP2C9/química , Fosfolipídeos/metabolismo , Sítios de Ligação , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica
7.
Biochem Soc Trans ; 47(4): 1173-1185, 2019 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-31383818

RESUMO

Lysosomes are the main degradative compartments of mammalian cells and serve as platforms for cellular nutrient signaling and sterol transport. The diverse functions of lysosomes and their adaptation to extracellular and intracellular cues are tightly linked to the spatiotemporally controlled synthesis, turnover and interconversion of lysosomal phosphoinositides, minor phospholipids that define membrane identity and couple membrane dynamics to cell signaling. How precisely lysosomal phosphoinositides act and which effector proteins within the lysosome membrane or at the lysosomal surface recognize them is only now beginning to emerge. Importantly, mutations in phosphoinositide metabolizing enzyme cause lysosomal dysfunction and are associated with numerous diseases ranging from neurodegeneration to cancer. Here, we discuss the phosphoinositides and phosphoinositide metabolizing enzymes implicated in lysosome function and homeostasis and outline perspectives for future research.


Assuntos
Homeostase , Lisossomos/metabolismo , Fosfatidilinositóis/metabolismo , Animais , Autofagossomos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Transporte Biológico , Humanos , Membranas Intracelulares/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Esteróis/metabolismo
8.
Results Probl Cell Differ ; 67: 49-79, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31435792

RESUMO

The intracellular transport is the most confusing issue in the field of cell biology. The Golgi complex (GC) is the central station along the secretory pathway. It contains Golgi glycosylation enzymes, which are responsible for protein and lipid glycosylation, and in many cells, it is organized into a ribbon. Position and structure of the GC depend on the position and function of the centriole. Here, we analyze published data related to the role of centriole and intracellular transport (ICT) for the formation of Golgi ribbon and specifically stress the importance of the delivery of membranes containing cargo and membrane proteins to the cell centre where centriole/centrosome is localized. Additionally, we re-examined the formation of Golgi ribbon from the point of view of different models of ICT.


Assuntos
Centríolos/metabolismo , Complexo de Golgi/química , Complexo de Golgi/metabolismo , Transporte Biológico , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo
9.
Results Probl Cell Differ ; 67: 441-485, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31435807

RESUMO

The Golgi apparatus is a central intracellular membrane-bound organelle with key functions in trafficking, processing, and sorting of newly synthesized membrane and secretory proteins and lipids. To best perform these functions, Golgi membranes form a unique stacked structure. The Golgi structure is dynamic but tightly regulated; it undergoes rapid disassembly and reassembly during the cell cycle of mammalian cells and is disrupted under certain stress and pathological conditions. In the past decade, significant amount of effort has been made to reveal the molecular mechanisms that regulate the Golgi membrane architecture and function. Here we review the major discoveries in the mechanisms of Golgi structure formation, regulation, and alteration in relation to its functions in physiological and pathological conditions to further our understanding of Golgi structure and function in health and diseases.


Assuntos
Doença , Complexo de Golgi/química , Complexo de Golgi/fisiologia , Saúde , Estresse Fisiológico , Animais , Transporte Biológico , Ciclo Celular , Humanos , Membranas Intracelulares/metabolismo
10.
Proc Jpn Acad Ser B Phys Biol Sci ; 95(7): 312-320, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31406056

RESUMO

Intracellular organelles were long viewed as isolated compartments floating in the cytosol. However, this view has been radically changed within the last decade by the discovery that most organelles communicate with the endoplasmic reticulum (ER) network via membrane contact sites (MCSs) that are essential for intracellular homeostasis. Protrudin is an ER resident protein that was originally shown to regulate neurite formation by promoting endosome trafficking. More recently, however, protrudin has been found to serve as a tethering factor at MCSs. The roles performed by protrudin at MCSs are mediated by its various domains, including inactivation of the small GTPase Rab11, bending of the ER membrane, and functional interactions with other molecules such as the motor protein KIF5 and the ER protein VAP. Mutations in the protrudin gene (ZFYVE27) are associated with hereditary spastic paraplegia, an axonopathy that results from defective ER structure. This review, examines the pleiotropic molecular functions of protrudin and its role in interorganellar communication.


Assuntos
Membranas Intracelulares/metabolismo , Organelas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Sequência de Bases , Humanos , Mutação , Domínios Proteicos , Transporte Proteico , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética
11.
Aquat Toxicol ; 215: 105266, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31401474

RESUMO

The persistent pollutants polybrominated diphenyl ethers (PBDEs) have been demonstrated to produce several negative effects on marine organisms. Although Mytilus galloprovincialis was extensively studied as model system, the effects of PBDEs on the innate immune system of mussels remains unclear. In this study, except for the control treatment, specimens of M. galloprovincialis were fed with microalgae treated with increasing concentrations of PBDEs (maximum level 100 ng L-1 of BDE-47 per day). BDE-47 treatment was maintained for 15 days and then the animals were fed with the same control diet, without contaminants, for 15 days. Samples of haemolymph (HL) were obtained at T0, T15 and T30 days of the experiment to evaluate different parameters related to immunity, such as neutral red retention time, and peroxidase, protease, antiprotease, lysozyme and bactericidal activities. BDE-47 exposure for 15 days affected both the stability of haemocytes and humoral parameters. In addition, the obtained results indicated that, at 30 days, after 15 days of culture without contaminant, the immune parameters were still affected, as some of them did not return to the basal levels, and others remained stimulated. Overall the results indicate that BDE-47 exposures at environmentally realistic levels may affect various aspects of immune function in M. galloprovincialis, acting as stressor that can compromise the general welfare.


Assuntos
Exposição Ambiental , Éteres Difenil Halogenados/toxicidade , Mytilus/imunologia , Animais , Antibacterianos/farmacologia , Comportamento Alimentar/efeitos dos fármacos , Hemócitos/efeitos dos fármacos , Hemócitos/metabolismo , Hemolinfa/efeitos dos fármacos , Hemolinfa/metabolismo , Hemolinfa/microbiologia , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Microalgas/fisiologia , Mytilus/efeitos dos fármacos , Mytilus/microbiologia , Peptídeo Hidrolases/metabolismo , Análise de Sobrevida , Poluentes Químicos da Água/toxicidade
12.
Cells ; 8(7)2019 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-31262095

RESUMO

The yeast vacuole is a vital organelle, which is required for the degradation of aberrant intracellular or extracellular substrates and the recycling of the resulting nutrients as newly available building blocks for the cellular metabolism. Like the plant vacuole or the mammalian lysosome, the yeast vacuole is the destination of biosynthetic trafficking pathways that transport the vacuolar enzymes required for its functions. Moreover, substrates destined for degradation, like extracellular endocytosed cargoes that are transported by endosomes/multivesicular bodies as well as intracellular substrates that are transported via different forms of autophagosomes, have the vacuole as destination. We found that non-selective bulk autophagy of cytosolic proteins as well as the selective autophagic degradation of peroxisomes (pexophagy) and ribosomes (ribophagy) was dependent on the armadillo repeat protein Vac8 in Saccharomyces cerevisiae. Moreover, we showed that pexophagy and ribophagy depended on the palmitoylation of Vac8. In contrast, we described that Vac8 was not involved in the acidification of the vacuole nor in the targeting and maturation of certain biosynthetic cargoes, like the aspartyl-protease Pep4 (PrA) and the carboxy-peptidase Y (CPY), indicating a role of Vac8 in the uptake of selected cargoes. In addition, we found that the hallmark phenotype of the vac8 strain, namely the characteristic appearance of fragmented and clustered vacuoles, depended on the growth conditions. This fusion defect observed in standard glucose medium can be complemented by the replacement with oleic acid or glycerol medium. This complementation of vacuolar morphology also partially restores the degradation of peroxisomes. In summary, we found that Vac8 controlled vacuolar morphology and activity in a context- and cargo-dependent manner.


Assuntos
Autofagia , Membranas Intracelulares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Lipoilação , Peroxissomos/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência , Proteínas de Transporte Vesicular/genética
13.
FEBS Open Bio ; 9(7): 1184-1193, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31206247

RESUMO

Voltage-dependent anion channel isoform 2 of the yeast Saccharomyces cerevisiae (yVDAC2) was believed for many years to be devoid of channel activity. Recently, we isolated yVDAC2 and showed that it exhibits channel-forming activity in the planar lipid bilayer system when in its so-called native form. Here, we describe an alternative strategy for yVDAC2 isolation, through heterologous expression in bacteria and refolding in vitro. Recombinant yVDAC2, like its native form, is able to form voltage-dependent channels. However, some differences between native and recombinant yVDAC2 emerged in terms of voltage dependence and ion selectivity, suggesting that, in this specific case, the recombinant protein might be depleted of post-translational modification(s) that occur in eukaryotic cells.


Assuntos
Engenharia de Proteínas/métodos , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Canal de Ânion 2 Dependente de Voltagem/fisiologia , Sequência de Aminoácidos , Fenômenos Eletrofisiológicos , Membranas Intracelulares/metabolismo , Bicamadas Lipídicas/metabolismo , Mitocôndrias/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo
15.
Protein J ; 38(3): 351-362, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31054036

RESUMO

Blobel and coworkers discovered in 1978 that peroxisomal proteins are synthesized on free ribosomes in the cytosol and thus provided the grounds for the conception of peroxisomes as self-containing organelles. Peroxisomes are highly adaptive and versatile organelles carrying out a wide variety of metabolic functions. A striking feature of the peroxisomal import machinery is that proteins can traverse the peroxisomal membrane in a folded and even oligomeric state via cycling receptors. We outline essential steps of peroxisomal matrix protein import, from targeting of the proteins to the peroxisomal membrane, their translocation via transient pores and export of the corresponding cycling import receptors with emphasis on the situation in yeast. Peroxisomes can contribute to the adaptation of cells to different environmental conditions. This is realized by changes in metabolic functions and thus the enzyme composition of the organelles is adopted according to the cellular needs. In recent years, it turned out that this organellar diversity is based on an elaborate regulation of gene expression and peroxisomal protein import. The latter is in the focus of this review that summarizes our knowledge on the composition and function of the peroxisomal protein import machinery with emphasis on novel alternative protein import pathways.


Assuntos
Membranas Intracelulares/metabolismo , Peroxissomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico , Ubiquitinação
16.
J Cardiothorac Surg ; 14(1): 92, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088484

RESUMO

BACKGROUND: Mitochondrial impairment can result from myocardial ischemia reperfusion injury (IR). Despite cardioplegic arrest, IR-associated cardiodepression is a major problem in heart surgery. We determined the effect of increasing ischemia time on the respiratory chain (RC) function, the inner membrane polarization and Ca2+ homeostasis of rat cardiac subsarcolemmal mitochondria (SSM). METHODS: Wistar rat hearts were divided into 4 groups of stop-flow induced warm global IR using a pressure-controlled Langendorff system: 0, 15, 30 and 40 min of ischemia with 30 min of reperfusion, respectively. Myocardial contractility was determined from left ventricular pressure records (dP/dt, dPmax) with an intraventricular balloon. Following reperfusion, SSM were isolated and analyzed regarding electron transport chain (ETC) coupling by polarography (Clark-Type electrode), membrane polarization (JC1 fluorescence) and Ca2+-handling in terms of Ca2+-induced swelling and Ca2+-uptake/release (Calcium Green-5 N® fluorescence). RESULTS: LV contractility and systolic pressure during reperfusion were impaired by increasing ischemic times. Ischemia reduced ETC oxygen consumption in IR40/30 compared to IR0/30 at complex I-V (8.1 ± 1.2 vs. 18.2 ± 2.0 nmol/min) and II-IV/V (16.4 ± 2.6/14.8 ± 2.3 vs. 2.3 ± 0.6 nmol/min) in state 3 respiration (p < 0.01). Relative membrane potential revealed a distinct hyperpolarization in IR30/30 and IR40/30 (171.5 ± 17.4% and 170.9 ± 13.5%) compared to IR0/30 (p < 0.01), wearing off swiftly after CCCP-induced uncoupling. Excess mitochondrial permeability transition pore (mPTP)-gated Ca2+-induced swelling was recorded in all groups and was most pronounced in IR40/30. Pyruvate addition for mPTP blocking strongly reduced SSM swelling in IR40/30 (relative AUC, ± pyruvate; IR0/30: 1.00 vs. 0.61, IR15/30: 1.68 vs. 1.00, IR30/30: 1.42 vs. 0.75, IR40/30: 1.97 vs. 0.85; p < 0.01). Ca2+-uptake remained unaffected by previous IR. Though Ca2+-release was delayed for ≥30 min of ischemia (p < 0.01), Ca2+ retention was highest in IR15/30 (RFU; IR0/30: 6.3 ± 3.6, IR 15/30 42.9 ± 5.0, IR30/30 15.9 ± 3.8, IR40/30 11.5 ± 6.6; p ≤ 0.01 for IR15/30 against all other groups). CONCLUSIONS: Ischemia prolongation in IR injury gradually impaired SSM in terms of respiratory chain function and Ca2+-homeostasis. Membrane hyperpolarization appears to be responsible for impaired Ca2+-cycling and ETC function. Ischemia time should be considered an important factor influencing IR experimental data on subsarcolemmal mitochondria. Periods of warm global ischemia should be minimized during cardiac surgery to avoid excessive damage to SSMs.


Assuntos
Cálcio/metabolismo , Transporte de Elétrons/fisiologia , Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Sarcolema/metabolismo , Isquemia Quente/efeitos adversos , Animais , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Cátions/metabolismo , Modelos Animais de Doenças , Parada Cardíaca Induzida , Membranas Intracelulares/metabolismo , Preparação de Coração Isolado , Masculino , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Consumo de Oxigênio/fisiologia , Ratos , Ratos Wistar , Recuperação de Função Fisiológica , Fatores de Tempo
17.
EBioMedicine ; 43: 607-619, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31060900

RESUMO

BACKGROUND: The mitochondrial associated endoplasmic reticulum (ER) membrane (MAM) provides a platform for communication between the mitochondria and ER, and it plays a vital role in many biological functions. Disulphide-bond A oxidoreductase-like protein (DsbA-L), expressed in the MAM, serves as an antioxidant and reduces ER stress. However, the role of DsbA-L and MAM in kidney pathobiology remains unclear. METHODS: Molecular biology techniques, transmission electron microscopy (TEM), in situ proximity ligation assays (PLAs), confocal microscopy, TUNEL staining and flow cytometry were utilized to analyse apoptosis and status of MAM in DsbA-L mutant mice. FINDINGS: We showed that MAM was significantly reduced in the kidneys of streptozotocin-induced diabetic mice, which correlated with the extent of renal injury. We also observed a correlation between the loss of MAM integrity and increased apoptosis and renal injury in diabetic nephropathy (DN). These alterations were further exacerbated in diabetic DsbA-L gene-deficient mice (DsbA-L-/-). In vitro, overexpression of DsbA-L in HK-2 cells restored MAM integrity and reduced apoptosis induced by high-glucose ambience. These beneficial effects were partially blocked by overexpression of FATE-1, a MAM uncoupling protein. Finally, the expression of DsbA-L was positively correlated with MAM integrity in the kidneys of DN patients but negatively correlated with apoptosis and renal injury. INTERPRETATION: Our results indicate that DsbA-L exerts an antiapoptotic effect by maintaining MAM integrity, which is apparently disrupted in DN. FUND: This work was supported by the National Natural Science Foundation of China (81730018), the National Key R&D Program of China (2016YFC1305501) and NIH (DK60635).


Assuntos
Retículo Endoplasmático/metabolismo , Glucose/metabolismo , Glutationa Transferase/metabolismo , Membranas Intracelulares/metabolismo , Túbulos Renais/metabolismo , Animais , Biomarcadores , Glicemia , Linhagem Celular , Diabetes Mellitus Experimental , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Feminino , Túbulos Renais/patologia , Túbulos Renais/ultraestrutura , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo
18.
Dev Cell ; 49(5): 786-801.e6, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31056345

RESUMO

How can anterograde membrane trafficking be modulated by physiological cues? A screen of Golgi-associated proteins revealed that the ARF-GEF GBF1 can selectively modulate the ER-Golgi trafficking of prohaemostatic von Willebrand factor (VWF) and extracellular matrix (ECM) proteins in human endothelial cells and in mouse fibroblasts. The relationship between levels of GBF1 and the trafficking of VWF into forming secretory granules confirmed GBF1 is a limiting factor in this process. Further, GBF1 activation by AMPK couples its control of anterograde trafficking to physiological cues; levels of glucose control GBF1 activation in turn modulating VWF trafficking into secretory granules. GBF1 modulates both ER and TGN exit, the latter dramatically affecting the size of the VWF storage organelles, thereby influencing the hemostatic capacity of the endothelium. The role of AMPK as a central integrating element of cellular pathways with intra- and extra-cellular cues can now be extended to modulation of the anterograde secretory pathway.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fator de von Willebrand/metabolismo , Fator 1 de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/genética , Proteínas Quinases Ativadas por AMP/genética , Animais , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Membranas Intracelulares/metabolismo , Camundongos , Fosforilação , Transporte Proteico , Fator de von Willebrand/genética
19.
Mol Cell Proteomics ; 18(7): 1285-1306, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30962257

RESUMO

The chloroplast is a major plant cell organelle that fulfills essential metabolic and biosynthetic functions. Located at the interface between the chloroplast and other cell compartments, the chloroplast envelope system is a strategic barrier controlling the exchange of ions, metabolites and proteins, thus regulating essential metabolic functions (synthesis of hormones precursors, amino acids, pigments, sugars, vitamins, lipids, nucleotides etc.) of the plant cell. However, unraveling the contents of the chloroplast envelope proteome remains a difficult challenge; many proteins constituting this functional double membrane system remain to be identified. Indeed, the envelope contains only 1% of the chloroplast proteins (i.e. 0.4% of the whole cell proteome). In other words, most envelope proteins are so rare at the cell, chloroplast, or even envelope level, that they remained undetectable using targeted MS studies. Cross-contamination of chloroplast subcompartments by each other and by other cell compartments during cell fractionation, impedes accurate localization of many envelope proteins. The aim of the present study was to take advantage of technologically improved MS sensitivity to better define the proteome of the chloroplast envelope (differentiate genuine envelope proteins from contaminants). This MS-based analysis relied on an enrichment factor that was calculated for each protein identified in purified envelope fractions as compared with the value obtained for the same protein in crude cell extracts. Using this approach, a total of 1269 proteins were detected in purified envelope fractions, of which, 462 could be assigned an envelope localization by combining MS-based spectral count analyses with manual annotation using data from the literature and prediction tools. Many of such proteins being previously unknown envelope components, these data constitute a new resource of significant value to the broader plant science community aiming to define principles and molecular mechanisms controlling fundamental aspects of plastid biogenesis and functions.


Assuntos
Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Membranas Intracelulares/metabolismo , Espectrometria de Massas/métodos , Proteoma/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Extratos Celulares , Bases de Dados de Proteínas , Proteínas de Membrana/metabolismo , Frações Subcelulares/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA