RESUMO
Macromolecules of various sizes induce crowding of the cellular environment. This crowding impacts on biochemical reactions by increasing solvent viscosity, decreasing the water-accessible volume and altering protein shape, function, and interactions. Although mitochondria represent highly protein-rich organelles, most of these proteins are somehow immobilized. Therefore, whether the mitochondrial matrix solvent exhibits macromolecular crowding is still unclear. Here, we demonstrate that fluorescent protein fusion peptides (AcGFP1 concatemers) in the mitochondrial matrix of HeLa cells display an elongated molecular structure and that their diffusion constant decreases with increasing molecular weight in a manner typical of macromolecular crowding. Chloramphenicol (CAP) treatment impaired mitochondrial function and reduced the number of cristae without triggering mitochondrial orthodox-to-condensed transition or a mitochondrial unfolded protein response. CAP-treated cells displayed progressive concatemer immobilization with increasing molecular weight and an eightfold matrix viscosity increase, compatible with increased macromolecular crowding. These results establish that the matrix solvent exhibits macromolecular crowding in functional and dysfunctional mitochondria. Therefore, changes in matrix crowding likely affect matrix biochemical reactions in a manner depending on the molecular weight of the involved crowders and reactants.
Assuntos
Mitocôndrias , Proteínas , Humanos , Células HeLa , Substâncias Macromoleculares/metabolismo , Proteínas/metabolismo , Solventes/metabolismo , Mitocôndrias/metabolismoRESUMO
Dengue is an important arboviral infectious disease for which there is currently no specific cure. We report gemini-like (geminoid) alkylated amphiphilic peptides containing lysines in combination with glycines or alanines (C15H31C(O)-Lys-(Gly or Ala)nLys-NHC16H33, shorthand notation C16-KXnK-C16 with X = A or G, and n = 0-2). The representatives with 1 or 2 Ala inhibit dengue protease and human furin, two serine proteases involved in dengue virus infection that have peptides with cationic amino acids as their preferred substrates, with IC50 values in the lower µM range. The geminoid C16-KAK-C16 combined inhibition of DENV2 protease (IC50 2.3 µM) with efficacy against replication of wildtype DENV2 in LLC-MK2 cells (EC50 4.1 µM) and an absence of toxicity. We conclude that the lysine-based geminoids have activity against dengue virus infection, which is based on their inhibition of the proteases involved in viral replication and are therefore promising leads to further developing antiviral therapeutics, not limited to dengue.
Assuntos
Antivirais , Vírus da Dengue , Furina , Inibidores de Proteases , Replicação Viral , Antivirais/farmacologia , Dengue/tratamento farmacológico , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/fisiologia , Furina/antagonistas & inibidores , Humanos , Peptídeo Hidrolases , Peptídeos/farmacologia , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
We introduce Frapbot, a free-of-charge open source software web application written in R, which provides manual and automated analyses of fluorescence recovery after photobleaching (FRAP) datasets. For automated operation, starting from data tables containing columns of time-dependent intensity values for various regions of interests within the images, a pattern recognition algorithm recognizes the relevant columns and identifies the presence or absence of prebleach values and the time point of photobleaching. Raw data, residuals, normalization, and boxplots indicating the distribution of half times of recovery (t1/2 ) of all uploaded files are visualized instantly in a batch-wise manner using a variety of user-definable fitting options. The fitted results are provided as .zip file, which contains .csv formatted output tables. Alternatively, the user can manually control any of the options described earlier. © 2017 International Society for Advancement of Cytometry.
Assuntos
Recuperação de Fluorescência Após Fotodegradação/métodos , Algoritmos , Fotodegradação , SoftwareRESUMO
Intracellular chemical reactions generally constitute reaction-diffusion systems located inside nanostructured compartments like the cytosol, nucleus, endoplasmic reticulum, Golgi, and mitochondrion. Understanding the properties of such systems requires quantitative information about solute diffusion. Here we present a novel approach that allows determination of the solvent-dependent solute diffusion constant (D(solvent)) inside cell compartments with an experimentally quantifiable nanostructure. In essence, our method consists of the matching of synthetic fluorescence recovery after photobleaching (FRAP) curves, generated by a mathematical model with a realistic nanostructure, and experimental FRAP data. As a proof of principle, we assessed D(solvent) of a monomeric fluorescent protein (AcGFP1) and its tandem fusion (AcGFP1(2)) in the mitochondrial matrix of HEK293 cells. Our results demonstrate that diffusion of both proteins is substantially slowed by barriers in the mitochondrial matrix (cristae), suggesting that cells can control the dynamics of biochemical reactions in this compartment by modifying its nanostructure.
Assuntos
Mitocôndrias/ultraestrutura , Proteínas/metabolismo , Compartimento Celular , Difusão , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Cinética , Mitocôndrias/metabolismo , Nanoestruturas/ultraestrutura , SoluçõesRESUMO
Studies employing native PAGE suggest that most nDNA-encoded CI subunits form subassemblies before assembling into holo-CI. In addition, in vitro evidence suggests that some subunits can directly exchange in holo-CI. Presently, data on the kinetics of these two incorporation modes for individual CI subunits during CI maintenance are sparse. Here, we used inducible HEK293 cell lines stably expressing AcGFP1-tagged CI subunits and quantified the amount of tagged subunit in mitoplasts and holo-CI by non-native and native PAGE, respectively, to determine their CI incorporation efficiency. Analysis of time courses of induction revealed three subunit-specific patterns. A first pattern, represented by NDUFS1, showed overlapping time courses, indicating that imported subunits predominantly incorporate into holo-CI. A second pattern, represented by NDUFV1, consisted of parallel time courses, which were, however, not quantitatively overlapping, suggesting that imported subunits incorporate at similar rates into holo-CI and CI assembly intermediates. The third pattern, represented by NDUFS3 and NDUFA2, revealed a delayed incorporation into holo-CI, suggesting their prior appearance in CI assembly intermediates and/or as free monomers. Our analysis showed the same maximum incorporation into holo-CI for NDUFV1, NDUFV2, NDUFS1, NDUFS3, NDUFS4, NDUFA2, and NDUFA12 with nearly complete loss of endogenous subunit at 24 h of induction, indicative of an equimolar stoichiometry and unexpectedly rapid turnover. In conclusion, the results presented demonstrate that newly formed nDNA-encoded CI subunits rapidly incorporate into holo-CI in a subunit-specific manner.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Homeostase/fisiologia , Proteínas Mitocondriais/metabolismo , Subunidades Proteicas/metabolismo , Animais , Cricetinae , Cricetulus , Complexo I de Transporte de Elétrons/genética , Células HEK293 , Humanos , Cinética , Proteínas Mitocondriais/genética , Subunidades Proteicas/genéticaRESUMO
Complex I (CI) of the oxidative phosphorylation system is assembled from 45 subunits encoded by both the mitochondrial and nuclear DNA. Defective mitochondrial translation is a major cause of mitochondrial disorders and proper understanding of its mechanisms and consequences is fundamental to rational treatment design. Here, we used a live cell approach to assess its consequences on CI assembly. The approach consisted of fluorescence recovery after photobleaching (FRAP) imaging of the effect of mitochondrial translation inhibition by chloramphenicol (CAP) on the dynamics of AcGFP1-tagged CI subunits NDUFV1, NDUFS3, NDUFA2 and NDUFB6 and assembly factor NDUFAF4. CAP increased the mobile fraction of the subunits, but not NDUFAF4, and decreased the amount of CI, demonstrating that CI is relatively immobile and does not associate with NDUFAF4. CAP increased the recovery kinetics of NDUFV1-AcGFP1 to the same value as obtained with AcGFP1 alone, indicative of the removal of unbound NDUFV1 from the mitochondrial matrix. Conversely, CAP decreased the mobility of NDUFS3-AcGFP1 and, to a lesser extent, NDUFB6-AcGFP1, suggestive of their enrichment in less mobile subassemblies. Little, if any, change in mobility of NDUFA2-AcGFP1 could be detected, suggesting that the dynamics of this accessory subunit of the matrix arm remains unaltered. Finally, CAP increased the mobility of NDUFAF4-AcGFP1, indicative of interaction with a more mobile membrane-bound subassembly. Our results show that the protein interactions of CI subunits and assembly factors are differently altered when mitochondrial translation is defective.
Assuntos
Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Mitocôndrias/fisiologia , Biossíntese de Proteínas , Subunidades Proteicas/metabolismo , Linhagem Celular , Complexo I de Transporte de Elétrons/genética , Recuperação de Fluorescência Após Fotodegradação , Humanos , Mitocôndrias/genética , Subunidades Proteicas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Mitochondrial isolated complex I deficiency is the most frequently encountered OXPHOS defect. We report a patient with an isolated complex I deficiency expressed in skin fibroblasts as well as muscle tissue. Because the parents were consanguineous, we performed homozygosity mapping to identify homozygous regions containing candidate genes such as NDUFA2 on chromosome 5. Screening of this gene on genomic DNA revealed a mutation that interferes with correct splicing and results in the skipping of exon 2. Exon skipping was confirmed on the mRNA level. The mutation in this accessory subunit causes reduced activity and disturbed assembly of complex I. Furthermore, the mutation is associated with a mitochondrial depolarization. The expression and activity of complex I and the depolarization was (partially) rescued with a baculovirus system expressing the NDUFA2 gene.
Assuntos
Complexo I de Transporte de Elétrons/genética , Doença de Leigh/enzimologia , Doença de Leigh/genética , Mutação , Consanguinidade , Primers do DNA/genética , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Éxons , Fibroblastos/enzimologia , Teste de Complementação Genética , Homozigoto , Humanos , Lactente , Masculino , Mitocôndrias/enzimologia , Músculos/enzimologia , RNA Mensageiro/genéticaRESUMO
Diffuse brain infiltration by glioma cells causes detrimental disease progression, but its multicellular coordination is poorly understood. We show here that glioma cells infiltrate the brain collectively as multicellular networks. Contacts between moving glioma cells are adaptive epithelial-like or filamentous junctions stabilized by N-cadherin, ß-catenin and p120-catenin, which undergo kinetic turnover, transmit intercellular calcium transients and mediate directional persistence. Downregulation of p120-catenin compromises cell-cell interaction and communication, disrupts collective networks, and both the cadherin and RhoA binding domains of p120-catenin are required for network formation and migration. Deregulating p120-catenin further prevents diffuse glioma cell infiltration of the mouse brain with marginalized microlesions as the outcome. Transcriptomics analysis has identified p120-catenin as an upstream regulator of neurogenesis and cell cycle pathways and a predictor of poor clinical outcome in glioma patients. Collective glioma networks infiltrating the brain thus depend on adherens junctions dynamics, the targeting of which may offer an unanticipated strategy to halt glioma progression.
Assuntos
Junções Aderentes/metabolismo , Cateninas/metabolismo , Adesão Celular/fisiologia , Glioma/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Caderinas/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/fisiologia , Glioma/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , delta CateninaRESUMO
Mitochondrial complex I deficiency is the most common defect of the OXPHOS system. We report a patient from consanguineous parents with a complex I deficiency expressed in skin fibroblasts. Homozygosity mapping revealed several homozygous regions with candidate genes, including the gene encoding an assembly factor for complex I, NDUFAF2. Screening of this gene on genomic DNA revealed a homozygous stop-codon resulting in a truncation of the protein at position 38. The mutation causes a severely reduced activity and a disturbed assembly of complex I. A baculovirus containing the GFP-tagged wild-type NDUFAF2 gene was used to prove the functional consequences of the mutation. The expression and activity of complex I was almost completely rescued by complementation of the patient fibroblasts with the baculovirus. Therefore, the homozygous substitution in NDUFAF2 is the disease-causing mutation, which results in a complex I deficiency in the fibroblasts of the patient.
Assuntos
Códon sem Sentido , DNA Mitocondrial/genética , Complexo I de Transporte de Elétrons/genética , Proteínas Mitocondriais/genética , Chaperonas Moleculares/genética , Baculoviridae/genética , Complexo I de Transporte de Elétrons/deficiência , Evolução Fatal , Feminino , Fibroblastos/patologia , Teste de Complementação Genética , Homozigoto , Humanos , Lactente , Pele/patologiaRESUMO
Within living cells, mitochondria are considered relevant sources of reactive oxygen species (ROS) and are exposed to reactive nitrogen species (RNS). During the last decade, accumulating evidence suggests that mitochondrial (dys)function, ROS/RNS levels, and aberrations in mitochondrial morphology are interconnected, albeit in a cell- and context-dependent manner. Here it is hypothesized that ROS and RNS are involved in the short-term regulation of mitochondrial morphology and function via non-transcriptional pathways. We review the evidence for such a mechanism and propose that it allows homeostatic control of mitochondrial function and morphology by redox signaling.
Assuntos
Mitocôndrias/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Animais , Humanos , Mitocôndrias/patologia , OxirreduçãoRESUMO
Virtually every mammalian cell contains mitochondria. These double-membrane organelles continuously change shape and position and contain the complete metabolic machinery for the oxidative conversion of pyruvate, fatty acids, and amino acids into ATP. Mitochondria are crucially involved in cellular Ca2+ and redox homeostasis and apoptosis induction. Maintenance of mitochondrial function and integrity requires an inside-negative potential difference across the mitochondrial inner membrane. This potential is sustained by the electron-transport chain (ETC). NADH:ubiquinone oxidoreductase or complex I (CI), the first and largest protein complex of the ETC, couples the oxidation of NADH to the reduction of ubiquinone. During this process, electrons can escape from CI and react with ambient oxygen to produce superoxide and derived reactive oxygen species (ROS). Depending on the balance between their production and removal by antioxidant systems, ROS may function as signaling molecules or induce damage to a variety of biomolecules or both. The latter ultimately leads to a loss of mitochondrial and cellular function and integrity. In this review, we discuss (a) the role of CI in mitochondrial functioning; (b) the composition, structure, and biogenesis of CI; (c) regulation of CI function; (d) the role of CI in ROS generation; and (e) adaptive responses to CI deficiency.
Assuntos
Complexo I de Transporte de Elétrons/fisiologia , Mamíferos/metabolismo , Mitocôndrias/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Transporte Biológico , Bovinos , Cromanos/farmacologia , Transporte de Elétrons/fisiologia , Complexo I de Transporte de Elétrons/deficiência , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Fibroblastos/efeitos dos fármacos , Fibroblastos/ultraestrutura , Humanos , Lipídeos de Membrana/fisiologia , Mitocôndrias/ultraestrutura , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/fisiologia , NADH Desidrogenase/fisiologia , Especificidade de Órgãos , Fosforilação Oxidativa , Rotenona/farmacologia , Transdução de Sinais/fisiologiaRESUMO
Disturbances in the assembly of mitochondrial complex I (CI) are a frequent cause of mitochondrial disorders. Several lines of evidence hint at a semi-sequential assembly pathway, in which the 45 individual subunits that form the holoenzyme are pieced together by means of smaller intermediates. To understand this process, it is necessary to explain the exact order, the rate-limiting steps, and the dynamics of subunit incorporation. In this chapter, we describe an approach to regulate the expression levels of an AcGFP(1)-tagged subunit (NDUFS3) in mammalian cells by means of a tetracycline-inducible promoter. This strategy allows the study of the dynamics of CI assembly intermediates in living cells on native gels. After establishing that the AcGFP(1) tag does not interfere with the activity and assembly of the enzyme, we show how this system can be used to trace the labeled subunit in an induction pulse-chase experiment or to study its accumulation in specific assembly intermediates after inhibition of mitochondrial translation.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Proteínas de Fluorescência Verde/genética , Complexo I de Transporte de Elétrons/genética , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos , HumanosRESUMO
Mitochondrial complex I (CI) is a large assembly of 45 different subunits, and defects in its biogenesis are the most frequent cause of mitochondrial disorders. In vitro evidence suggests a stepwise assembly process involving pre-assembled modules. However, whether these modules also exist in vivo is as yet unresolved. To answer this question, we here applied submitochondrial fluorescence recovery after photobleaching to HEK293 cells expressing 6 GFP-tagged subunits selected on the basis of current CI assembly models. We established that each subunit was partially present in a virtually immobile fraction, possibly representing the holo-enzyme. Four subunits (NDUFV1, NDUFV2, NDUFA2, and NDUFA12) were also present as highly mobile matrix-soluble monomers, whereas, in sharp contrast, the other two subunits (NDUFB6 and NDUFS3) were additionally present in a slowly mobile fraction. In the case of the integral membrane protein NDUFB6, this fraction most likely represented one or more membrane-bound subassemblies, whereas biochemical evidence suggested that for the NDUFS3 protein this fraction most probably corresponded to a matrix-soluble subassembly. Our results provide first time evidence for the existence of CI subassemblies in mitochondria of living cells.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , NADH NADPH Oxirredutases/química , Linhagem Celular , Membrana Celular/metabolismo , DNA Complementar/metabolismo , Complexo I de Transporte de Elétrons/química , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/química , Humanos , Cinética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , NADH Desidrogenase/química , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Fatores de TempoRESUMO
Biogenesis of human mitochondrial complex I (CI) requires the coordinated assembly of 45 subunits derived from both the mitochondrial and nuclear genome. The presence of CI subcomplexes in CI-deficient cells suggests that assembly occurs in distinct steps. However, discriminating between products of assembly or instability is problematic. Using an inducible NDUFS3-green fluorescent protein (GFP) expression system in HEK293 cells, we here provide direct evidence for the stepwise assembly of CI. Upon induction, six distinct NDUFS3-GFP-containing subcomplexes gradually appeared on a blue native Western blot also observed in wild type HEK293 mitochondria. Their stability was demonstrated by differential solubilization and heat incubation, which additionally allowed their distinction from specific products of CI instability and breakdown. Inhibition of mitochondrial translation under conditions of steady state labeling resulted in an accumulation of two of the NDUFS3-GFP-containing subcomplexes (100 and 150 kDa) and concomitant disappearance of the fully assembled complex. Lifting inhibition reversed this effect, demonstrating that these two subcomplexes are true assembly intermediates. Composition analysis showed that this event was accompanied by the incorporation of at least one mitochondrial DNA-encoded subunit, thereby revealing the first entry point of these subunits.
Assuntos
Complexo I de Transporte de Elétrons/química , Mitocôndrias/química , NADH Desidrogenase/química , Western Blotting , Células Cultivadas , DNA Mitocondrial/genética , Proteínas de Fluorescência Verde/química , Humanos , Subunidades ProteicasRESUMO
Ecsit is a cytosolic adaptor protein essential for inflammatory response and embryonic development via the Toll-like and BMP (bone morphogenetic protein) signal transduction pathways, respectively. Here, we demonstrate a mitochondrial function for Ecsit (an evolutionary conserved signaling intermediate in Toll pathways) in the assembly of mitochondrial complex I (NADH:ubiquinone oxidoreductase). An N-terminal targeting signal directs Ecsit to mitochondria, where it interacts with assembly chaperone NDUFAF1 in 500- to 850-kDa complexes as demonstrated by affinity purification and vice versa RNA interference (RNAi) knockdowns. In addition, Ecsit knockdown results in severely impaired complex I assembly and disturbed mitochondrial function. These findings support a function for Ecsit in the assembly or stability of mitochondrial complex I, possibly linking assembly of oxidative phosphorylation complexes to inflammatory response and embryonic development.