RESUMO
The regular functioning of the nucleolus and nucleus-mitochondria crosstalk are considered unrelated processes, yet cytochrome c (Cc) migrates to the nucleus and even the nucleolus under stress conditions. Nucleolar liquid-liquid phase separation usually serves the cell as a fast, smart mechanism to control the spatial localization and trafficking of nuclear proteins. Actually, the alternative reading frame (ARF), a tumor suppressor protein sequestered by nucleophosmin (NPM) in the nucleoli, is shifted out from NPM upon DNA damage. DNA damage also triggers early translocation of respiratory Cc to nucleus before cytoplasmic caspase activation. Here, we show that Cc can bind to nucleolar NPM by triggering an extended-to-compact conformational change, driving ARF release. Such a NPM-Cc nucleolar interaction can be extended to a general mechanism for DNA damage in which the lysine-rich regions of Cc-rather than the canonical, arginine-rich stretches of membrane-less organelle components-controls the trafficking and availability of nucleolar proteins.
Assuntos
Citocromos c , Nucleofosmina , Arginina , Caspases , Lisina , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supressoras de TumorRESUMO
Repair of injured DNA relies on nucleosome dismantling by histone chaperones and de-phosphorylation events carried out by Protein Phosphatase 2A (PP2A). Typical histone chaperones are the Acidic leucine-rich Nuclear Phosphoprotein 32 family (ANP32) members, e.g. ANP32A, which is also a well-known PP2A inhibitor (a.k.a. I1PP2A). Here we report the novel interaction between the endogenous family member B-so-called ANP32B-and endogenous cytochrome c in cells undergoing camptothecin-induced DNA damage. Soon after DNA lesions but prior to caspase cascade activation, the hemeprotein translocates to the nucleus to target the Low Complexity Acidic Region (LCAR) of ANP32B; in a similar way, our group recently reported that the hemeprotein targets the acidic domain of SET/Template Activating Factor-Iß (SET/TAF-Iß), which is another histone chaperone and PP2A inhibitor (a.k.a. I2PP2A). The nucleosome assembly activity of ANP32B is indeed unaffected by cytochrome c binding. Like ANP32A, ANP32B inhibits PP2A activity and is thus herein referred to as I3PP2A. Our data demonstrates that ANP32B-dependent inhibition of PP2A is regulated by respiratory cytochrome c, which induces long-distance allosteric changes in the structured N-terminal domain of ANP32B upon binding to the C-terminal LCAR. In agreement with the reported role of PP2A in the DNA damage response, we propose a model wherein cytochrome c is translocated from the mitochondria into the nucleus upon DNA damage to modulate PP2A activity via its interaction with ANP32B.
Assuntos
Citocromos c , Chaperonas de Histonas , Núcleo Celular , Dano ao DNA , Proteína Fosfatase 2RESUMO
TRIM37 is an E3 ubiquitin ligase mutated in Mulibrey nanism, a disease with impaired organ growth and increased tumor formation. TRIM37 depletion from tissue culture cells results in supernumerary foci bearing the centriolar protein Centrin. Here, we characterize these centriolar protein assemblies (Cenpas) to uncover the mechanism of action of TRIM37. We find that an atypical de novo assembly pathway can generate Cenpas that act as microtubule-organizing centers (MTOCs), including in Mulibrey patient cells. Correlative light electron microscopy reveals that Cenpas are centriole-related or electron-dense structures with stripes. TRIM37 regulates the stability and solubility of Centrobin, which accumulates in elongated entities resembling the striped electron dense structures upon TRIM37 depletion. Furthermore, Cenpas formation upon TRIM37 depletion requires PLK4, as well as two parallel pathways relying respectively on Centrobin and PLK1. Overall, our work uncovers how TRIM37 prevents Cenpas formation, which would otherwise threaten genome integrity.
Assuntos
Proteínas de Ciclo Celular/genética , Centro Organizador dos Microtúbulos/metabolismo , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Centríolos/metabolismo , Células HeLa , Humanos , Nanismo de Mulibrey/genética , Nanismo de Mulibrey/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The T cell immune synapse is a site of intense vesicular trafficking. Here we show that the golgin GMAP210, known to capture vesicles and organize membrane traffic at the Golgi, is involved in the vesicular transport of LAT to the immune synapse. Upon activation, more GMAP210 interact with LAT-containing vesicles and go together with LAT to the immune synapse. Regulating LAT recruitment and LAT-dependent signaling, GMAP210 controls T cell activation. Using a rerouting and capture assay, we show that GMAP210 captures VAMP7-decorated vesicles. Overexpressing different domains of GMAP210, we also show that GMAP210 allows their specific delivery to the immune synapse by tethering LAT-vesicles to the Golgi. Finally, in a model of ectopic expression of LAT in ciliated cells, we show that GMAP210 tethering activity controls the delivery of LAT to the cilium. Hence, our results reveal a function for the golgin GMAP210 conveying specific vesicles to the immune synapse.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Complexo de Golgi/fisiologia , Leucócitos Mononucleares/fisiologia , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Vesículas Transportadoras/fisiologia , Linhagem Celular , Proteínas do Citoesqueleto , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Proteínas Nucleares/genética , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Transdução de Sinais , Linfócitos T/fisiologiaRESUMO
Objective: Noninvasive brain stimulation (NIBS) techniques, such as transcranial direct current stimulation (tDCS) and repetitive transcranial magnetic stimulation (rTMS), are increasingly being used to treat mental disorders, particularly major depression. The aim of this comprehensive review is to summarize the main advances, limitations, and perspectives of the field. Methods: We searched PubMed and other databases from inception to July 2017 for articles, particularly systematic reviews and meta-analyses, evaluating the use of NIBS in psychiatric disorders. Results: We reviewed the mechanisms of action, safety, tolerability, efficacy, and relevant clinical parameters of NIBS. Repetitive TMS is already an established technique for the treatment of depression, and there is theoretically room for further methodological development towards a high-end therapeutic intervention. In contrast, tDCS is a technically easier method and therefore potentially suitable for wider clinical use. However the evidence of its antidepressant efficacy is less sound, and a recent study found tDCS to be inferior to antidepressant pharmacotherapy. Clinical trials using rTMS for other mental disorders produced mixed findings, whereas tDCS use has not been sufficiently appraised. Conclusion: The most promising results of NIBS have been obtained for depression. These techniques excel in safety and tolerability, although their efficacy still warrants improvement.
Assuntos
Humanos , Estimulação Magnética Transcraniana/métodos , Estimulação Transcraniana por Corrente Contínua/métodos , Transtornos Mentais/terapia , Ensaios Clínicos como Assunto , Medicina Baseada em EvidênciasRESUMO
OBJECTIVE: Noninvasive brain stimulation (NIBS) techniques, such as transcranial direct current stimulation (tDCS) and repetitive transcranial magnetic stimulation (rTMS), are increasingly being used to treat mental disorders, particularly major depression. The aim of this comprehensive review is to summarize the main advances, limitations, and perspectives of the field. METHODS: We searched PubMed and other databases from inception to July 2017 for articles, particularly systematic reviews and meta-analyses, evaluating the use of NIBS in psychiatric disorders. RESULTS: We reviewed the mechanisms of action, safety, tolerability, efficacy, and relevant clinical parameters of NIBS. Repetitive TMS is already an established technique for the treatment of depression, and there is theoretically room for further methodological development towards a high-end therapeutic intervention. In contrast, tDCS is a technically easier method and therefore potentially suitable for wider clinical use. However the evidence of its antidepressant efficacy is less sound, and a recent study found tDCS to be inferior to antidepressant pharmacotherapy. Clinical trials using rTMS for other mental disorders produced mixed findings, whereas tDCS use has not been sufficiently appraised. CONCLUSION: The most promising results of NIBS have been obtained for depression. These techniques excel in safety and tolerability, although their efficacy still warrants improvement.
Assuntos
Transtornos Mentais/terapia , Estimulação Transcraniana por Corrente Contínua/métodos , Estimulação Magnética Transcraniana/métodos , Ensaios Clínicos como Assunto , Medicina Baseada em Evidências , HumanosRESUMO
Here, we address the regulation of microtubule nucleation during interphase by genetically ablating one, or two, of three major mammalian γ-TuRC-binding factors namely pericentrin, CDK5Rap2, and AKAP450. Unexpectedly, we find that while all of them participate in microtubule nucleation at the Golgi apparatus, they only modestly contribute at the centrosome where CEP192 has a more predominant function. We also show that inhibiting microtubule nucleation at the Golgi does not affect centrosomal activity, whereas manipulating the number of centrosomes with centrinone modifies microtubule nucleation activity of the Golgi apparatus. In centrosome-free cells, inhibition of Golgi-based microtubule nucleation triggers pericentrin-dependent formation of cytoplasmic-nucleating structures. Further depletion of pericentrin under these conditions leads to the generation of individual microtubules in a γ-tubulin-dependent manner. In all cases, a conspicuous MT network forms. Strikingly, centrosome loss increases microtubule number independently of where they were growing from. Our results lead to an unexpected view of the interphase centrosome that would control microtubule network organization not only by nucleating microtubules, but also by modulating the activity of alternative microtubule-organizing centers.
Assuntos
Centrossomo/metabolismo , Interfase/fisiologia , Microtúbulos/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Antígenos/genética , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular , Linhagem Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas do Citoesqueleto/genética , Técnicas de Inativação de Genes , Complexo de Golgi/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Tubulina (Proteína)/metabolismoRESUMO
Aryl hydrocarbon receptor (AhR) deficiency alters tissue homeostasis. However, how AhR regulates organ maturation and differentiation remains mostly unknown. Liver differentiation entails a polyploidization process fundamental for cell growth, metabolism, and stress responses. Here, we report that AhR regulates polyploidization during the preweaning-to-adult mouse liver maturation. Preweaning AhR-null (AhR-/-) livers had smaller hepatocytes, hypercellularity, altered cell cycle regulation, and enhanced proliferation. Those phenotypes persisted in adult AhR-/- mice and correlated with compromised polyploidy, predominance of diploid hepatocytes, and enlarged centrosomes. Phosphatidylinositol-3-phosphate kinase (PI3K), extracellular signal-regulated kinase (ERK), and Wnt/ß-catenin signaling remained upregulated from preweaning to adult AhR-null liver, likely increasing mammalian target of rapamycin (mTOR) activation. Metabolomics revealed the deregulation of mitochondrial oxidative phosphorylation intermediates succinate and fumarate in AhR-/- liver. Consistently, PI3K, ERK, and Wnt/ß-catenin inhibition partially rescued polyploidy in AhR-/- mice. Thus, AhR may integrate survival, proliferation, and metabolism for liver polyploidization. Since tumor cells tend to be polyploid, AhR modulation could have therapeutic value in the liver.
RESUMO
The CRISPR-Cas system is involved in bacterial immunity, virulence, gene regulation, biofilm formation and sporulation. In Salmonella enterica serovar Typhi, this system consists of five transcriptional units including antisense RNAs. It was determined that these genetic elements are expressed in minimal medium and are up-regulated by pH. In addition, a transcriptional characterization of cas3 and ascse2-1 is included herein.
Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , DNA Helicases/genética , Regulação Bacteriana da Expressão Gênica/genética , RNA Antissenso/genética , Salmonella typhi/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Transcrição Gênica/genética , Ativação Transcricional/genética , Regulação para Cima/genéticaRESUMO
Bipolar depression (BD) is a highly prevalent condition with limited therapeutic options. Deep (H1-coil) transcranial magnetic stimulation (dTMS) is a novel TMS modality with established efficacy for unipolar depression. We conducted a randomized sham-controlled trial to evaluate the efficacy and safety of dTMS in treatment-resistant BD patients. Patients received 20 sessions of active or sham dTMS over the left dorsolateral prefrontal cortex (H1-coil, 55 18 Hz 2 s 120% MT trains). The primary outcome was changes in the 17-item Hamilton Depression Rating Scale (HDRS-17) from baseline to endpoint (week 4). Secondary outcomes were changes from baseline to the end of the follow-up phase (week 8), and response and remission rates. Safety was assessed using a dTMS adverse effects questionnaire and the Young Mania Rating Scale to assess treatment-emergent mania switch (TEMS). Out of 50 patients, 43 finished the trial. There were 2 and 5 dropouts in the sham and active groups, respectively. Active dTMS was superior to sham at end point (difference favoring dTMS=4.88; 95% CI 0.43 to 9.32, p=0.03) but not at follow-up. There was also a trend for greater response rates in the active (48%) vs sham (24%) groups (OR=2.92; 95% CI=0.87 to 9.78, p=0.08). Remission rates were not statistically different. No TEMS episodes were observed. Deep TMS is a potentially effective and well-tolerated add-on therapy in resistant bipolar depressed patients receiving adequate pharmacotherapy.
Assuntos
Transtorno Bipolar/terapia , Estimulação Magnética Transcraniana , Adulto , Antidepressivos/uso terapêutico , Método Duplo-Cego , Feminino , Seguimentos , Humanos , Masculino , Pacientes Desistentes do Tratamento , Córtex Pré-Frontal , Escalas de Graduação Psiquiátrica , Indução de Remissão , Fatores de Tempo , Estimulação Magnética Transcraniana/efeitos adversos , Estimulação Magnética Transcraniana/métodos , Resultado do TratamentoRESUMO
Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis.
Assuntos
Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas dos Microtúbulos/genética , Morfogênese/genética , Proteínas Nucleares/genética , alfa Catenina/genética , Junções Aderentes/metabolismo , Junções Aderentes/ultraestrutura , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Adesão Celular , Linhagem Celular , Polaridade Celular , Forma Celular , Cães , Embrião não Mamífero , Células Epiteliais/citologia , Vetores Genéticos , Humanos , Lentivirus/genética , Células Madin Darby de Rim Canino , Proteínas dos Microtúbulos/antagonistas & inibidores , Proteínas dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Oryzias , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Técnicas do Sistema de Duplo-Híbrido , alfa Catenina/metabolismoRESUMO
A shared feature among all microtubule (MT)-dependent processes is the requirement for MTs to be organized in arrays of defined geometry. At a fundamental level, this is achieved by precisely controlling the timing and localization of the nucleation events that give rise to new MTs. To this end, MT nucleation is restricted to specific subcellular sites called MT-organizing centres. The primary MT-organizing centre in proliferating animal cells is the centrosome. However, the discovery of MT nucleation capacity of the Golgi apparatus (GA) has substantially changed our understanding of MT network organization in interphase cells. Interestingly, MT nucleation at the Golgi apparently relies on multiprotein complexes, similar to those present at the centrosome, that assemble at the cis-face of the organelle. In this process, AKAP450 plays a central role, acting as a scaffold to recruit other centrosomal proteins important for MT generation. MT arrays derived from either the centrosome or the GA differ in their geometry, probably reflecting their different, yet complementary, functions. Here, I review our current understanding of the molecular mechanisms involved in MT nucleation at the GA and how Golgi- and centrosome-based MT arrays work in concert to ensure the formation of a pericentrosomal polarized continuous Golgi ribbon structure, a critical feature for cell polarity in mammalian cells. In addition, I comment on the important role of the Golgi-nucleated MTs in organizing specialized MT arrays that serve specific functions in terminally differentiated cells.
Assuntos
Centrossomo/fisiologia , Complexo de Golgi/fisiologia , Microtúbulos/fisiologia , Mitose/fisiologia , Modelos Biológicos , Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas do Citoesqueleto/metabolismoRESUMO
Recent evidence suggests that most influenza A virus gene segments can contribute to the pathogenicity of the virus. In this regard, the hemagglutinin (HA) subtype of the circulating strains has been closely surveyed, but the reassortment of internal gene segments is usually not monitored as a potential source of an increased pathogenicity. In this work, an oligonucleotide DNA microarray (PhyloFlu) designed to determine the phylogenetic origins of the eight segments of the influenza virus genome was constructed and validated. Clades were defined for each segment and also for the 16 HA and 9 neuraminidase (NA) subtypes. Viral genetic material was amplified by reverse transcription-PCR (RT-PCR) with primers specific to the conserved 5' and 3' ends of the influenza A virus genes, followed by PCR amplification with random primers and Cy3 labeling. The microarray unambiguously determined the clades for all eight influenza virus genes in 74% (28/38) of the samples. The microarray was validated with reference strains from different animal origins, as well as from human, swine, and avian viruses from field or clinical samples. In most cases, the phylogenetic clade of each segment defined its animal host of origin. The genomic fingerprint deduced by the combined information of the individual clades allowed for the determination of the time and place that strains with the same genomic pattern were previously reported. PhyloFlu is useful for characterizing and surveying the genetic diversity and variation of animal viruses circulating in different environmental niches and for obtaining a more detailed surveillance and follow up of reassortant events that can potentially modify virus pathogenicity.
Assuntos
Impressões Digitais de DNA/métodos , Genoma Viral , Técnicas de Genotipagem/métodos , Vírus da Influenza A/classificação , Influenza Humana/virologia , Análise em Microsséries/métodos , Infecções por Paramyxoviridae/veterinária , Animais , Análise por Conglomerados , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Infecções por Paramyxoviridae/virologia , Filogenia , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Virologia/métodosRESUMO
Polarized trafficking of adhesion receptors plays a pivotal role in controlling cellular behavior during morphogenesis. Particularly, clathrin-dependent endocytosis of integrins has long been acknowledged as essential for cell migration. However, little is known about the contribution of integrin trafficking to epithelial tissue morphogenesis. Here we show how the transmembrane protein Opo, previously described for its essential role during optic cup folding, plays a fundamental role in this process. Through interaction with the PTB domain of the clathrin adaptors Numb and Numbl via an integrin-like NPxF motif, Opo antagonizes Numb/Numbl function and acts as a negative regulator of integrin endocytosis in vivo. Accordingly, numb/numbl gain-of-function experiments in teleost embryos mimic the retinal malformations observed in opo mutants. We propose that developmental regulator Opo enables polarized integrin localization by modulating Numb/Numbl, thus directing the basal constriction that shapes the vertebrate retina epithelium.
Assuntos
Endocitose , Integrinas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Epitélio Pigmentado da Retina/embriologia , Epitélio Pigmentado da Retina/metabolismo , Animais , Células Cultivadas , Proteínas de Peixes/antagonistas & inibidores , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Oryzias , Epitélio Pigmentado da Retina/citologiaRESUMO
Regulation of proteasome abundance to meet cell needs under stress conditions is critical for maintaining cellular homeostasis. However, the effects of aging on this homeostatic response remain unknown. In this report, we analyzed in young and aged rat hippocampus, the dynamics of proteasome recovery induced by proteasome stress. Proteasome inhibition in young rats leads to an early and coordinate transcriptional and translational up-regulation of both the catalytic subunits of constitutive proteasome and the proteasome maturation protein. By contrast, aged rats up-regulated the inducible catalytic subunits and showed a lower and shorter expression of proteasome maturation protein. This resulted in a faster recovery of proteasome activity in young rats. Importantly, proteasome inhibition highly affected pyramidal cells, leading to the accumulation of ubiquitinated proteins in perinuclear regions of aged, but not young pyramidal neurons. These data strongly suggest that age-dependent differences in proteasome level and composition could contribute to neurodegeneration induced by proteasome dysfunction in normal and pathological aging.
Assuntos
Envelhecimento , Hipocampo/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Regulação para Cima/fisiologia , Fatores Etários , Animais , Domínio Catalítico/fisiologia , Nucléolo Celular/metabolismo , Hipocampo/citologia , Imunoproteínas/metabolismo , Masculino , Neurônios/citologia , Neurônios/metabolismo , Ratos , Ratos WistarRESUMO
BACKGROUND: Neuroinflammation and protein accumulation are characteristic hallmarks of both normal aging and age-related neurodegenerative diseases. However, the relationship between these factors in neurodegenerative processes is poorly understood. We have previously shown that proteasome inhibition produced higher neurodegeneration in aged than in young rats, suggesting that other additional age-related events could be involved in neurodegeneration. We evaluated the role of lipopolysaccharide (LPS)-induced neuroinflammation as a potential synergic risk factor for hippocampal neurodegeneration induced by proteasome inhibition. METHODS: Young male Wistar rats were injected with 1 µL of saline or LPS (5 mg/mL) into the hippocampus to evaluate the effect of LPS-induced neuroinflammation on protein homeostasis. The synergic effect of LPS and proteasome inhibition was analyzed in young rats that first received 1 µL of LPS and 24 h later 1 µL (5 mg/mL) of the proteasome inhibitor lactacystin. Animals were sacrificed at different times post-injection and hippocampi isolated and processed for gene expression analysis by real-time polymerase chain reaction; protein expression analysis by western blots; proteasome activity by fluorescence spectroscopy; immunofluorescence analysis by confocal microscopy; and degeneration assay by Fluoro-Jade B staining. RESULTS: LPS injection produced the accumulation of ubiquitinated proteins in hippocampal neurons, increased expression of the E2 ubiquitin-conjugating enzyme UB2L6, decreased proteasome activity and increased immunoproteasome content. However, LPS injection was not sufficient to produce neurodegeneration. The combination of neuroinflammation and proteasome inhibition leads to higher neuronal accumulation of ubiquitinated proteins, predominant expression of pro-apoptotic markers and increased neurodegeneration, when compared with LPS or lactacystin (LT) injection alone. CONCLUSIONS: Our results identify neuroinflammation as a risk factor that increases susceptibility to neurodegeneration induced by proteasome inhibition. These results highlight the modulation of neuroinflammation as a mechanism for neuronal protection that could be relevant in situations where both factors are present, such as aging and neurodegenerative diseases.
Assuntos
Hipocampo/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Degeneração Neural/induzido quimicamente , Inibidores de Proteassoma/toxicidade , Acetilcisteína/análogos & derivados , Acetilcisteína/toxicidade , Envelhecimento/efeitos dos fármacos , Envelhecimento/fisiologia , Animais , Sinergismo Farmacológico , Hipocampo/enzimologia , Hipocampo/patologia , Inflamação/induzido quimicamente , Inflamação/epidemiologia , Inflamação/patologia , Masculino , Degeneração Neural/epidemiologia , Degeneração Neural/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos , Ratos Wistar , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/fisiologiaRESUMO
Lewy bodies containing the centrosomal protein γ-tubulin and fragmentation of Golgi apparatus (GA) have been described in nigral neurons of Parkinson's disease (PD) patients. However, the relevance of these features in PD pathophysiology remains unknown. We analyzed the impact of proteasome inhibition in the formation of γ-tubulin-containing aggregates as well as on GA structure. SH-SY5Y cells were treated with the proteasome inhibitor Z-Leu-Leu-Leu-al (MG132) to induce centrosomal-protein aggregates. Then, microtubules (MTs) and Golgi dynamics, as well as the vesicular transport of dopamine transporter (DAT) were evaluated both in vitro and in living cells. MG132 treatment induced γ-tubulin aggregates which altered microtubule nucleation. MG132-treated cells containing γ-tubulin aggregates showed fragmentation of GA and perturbation of the trans-Golgi network. Under these conditions, the DAT accumulated at the centrosomal-Golgi region indicating that the vesicular transport of DAT was disrupted. Thus, centrosomal aggregates and fragmentation of GA are 2 closely related processes that could result in the disruption of the vesicular transport of DAT toward the plasma membrane in a model of dopaminergic neuronal degeneration.
Assuntos
Centrossomo/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Complexo de Golgi/metabolismo , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Linhagem Celular , Centrossomo/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Humanos , Leupeptinas/farmacologia , Microtúbulos/metabolismo , Inibidores de Proteassoma/farmacologia , Tubulina (Proteína)/metabolismoRESUMO
Pituitary tumor transforming gene 1 (PTTG1), also known as securin, has been implicated in many biological functions, including inhibition of sister chromatid separation, DNA repair, organ development, and regulation of the expression and secretion of angiogenic and metastatic factors. Although most of these functions of securin seem to depend on the localization of PTTG1 in the nucleus of the cell, a fraction of the protein has been also detected in the cytoplasm. Here we demonstrate that, in different cell types, a portion of cytoplasmic PTTG1 is associated with the cis face of the Golgi apparatus and that this localization depends on PTTG1 phosphorylation status. In this organelle, PTTG1 forms a complex with proteins involved in microtubule nucleation, including GM130, AKAP450, and γ-tubulin. RNA interference-mediated depletion of PTTG1 produces a delay in centrosomal and noncentrosomal microtubule nucleation. Cells lacking PTTG1 show severe defects in both cell polarization and migration in wound-healing assays. To our knowledge, this is the first study reporting the role of PTTG1 in microtubule nucleation and cell polarization, two processes directly involved in cell migration. We believe that these findings will contribute to understanding the mechanisms underlying PTTG1-mediated biological functions.
Assuntos
Movimento Celular , Complexo de Golgi/metabolismo , Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Ancoragem à Quinase A/metabolismo , Anticorpos Monoclonais/imunologia , Autoantígenos/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Polaridade Celular , Centrossomo , Proteínas do Citoesqueleto/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Fosforilação , Interferência de RNA , RNA Interferente Pequeno , Securina , Tubulina (Proteína)/metabolismo , CicatrizaçãoRESUMO
Mammalian cells exhibit a frequent pericentrosomal Golgi ribbon organization. In this paper, we show that two AKAP450 N-terminal fragments, both containing the Golgi-binding GM130-interacting domain of AKAP450, dissociated endogenous AKAP450 from the Golgi and inhibited microtubule (MT) nucleation at the Golgi without interfering with centrosomal activity. These two fragments had, however, strikingly different effects on both Golgi apparatus (GA) integrity and positioning, whereas the short fragment induced GA circularization and ribbon fragmentation, the large construct that encompasses an additional p150glued/MT-binding domain induced separation of the Golgi ribbon from the centrosome. These distinct phenotypes arose by specific interference of each fragment with either Golgi-dependent or centrosome-dependent stages of Golgi assembly. We could thus demonstrate that breaking the polarity axis by perturbing GA positioning has a more dramatic effect on directional cell migration than disrupting the Golgi ribbon. Both features, however, were required for ciliogenesis. We thus identified AKAP450 as a key determinant of pericentrosomal Golgi ribbon integrity, positioning, and function in mammalian cells.
Assuntos
Movimento Celular , Centrossomo/metabolismo , Cílios/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Ancoragem à Quinase A/metabolismo , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Humanos , Microtúbulos/metabolismoRESUMO
We describe the localization of the golgin GMAP210 and the intraflagellar protein IFT88 in the Golgi of spermatids and the participation of these two proteins in the development of the acrosome-acroplaxome complex, the head-tail coupling apparatus (HTCA) and the spermatid tail. Immunocytochemical experiments show that GMAP210 predominates in the cis-Golgi, whereas IFT88 prevails in the trans-Golgi network. Both proteins colocalize in proacrosomal vesicles, along acrosome membranes, the HTCA and the developing tail. IFT88 persists in the acrosome-acroplaxome region of the sperm head, whereas GMAP210 is no longer seen there. Spermatids of the Ift88 mouse mutant display abnormal head shaping and are tail-less. GMAP210 is visualized in the Ift88 mutant during acrosome-acroplaxome biogenesis. However, GMAP210-stained vesicles, mitochondria and outer dense fiber material build up in the manchette region and fail to reach the abortive tail stump in the mutant. In vitro disruption of the spermatid Golgi and microtubules with Brefeldin-A and nocodazole blocks the progression of GMAP210- and IFT88-stained proacrosomal vesicles to the acrosome-acroplaxome complex but F-actin distribution in the acroplaxome is not affected. We provide the first evidence that IFT88 is present in the Golgi of spermatids, that the microtubule-associated golgin GMAP210 and IFT88 participate in acrosome, HTCA, and tail biogenesis, and that defective intramanchette transport of cargos disrupts spermatid tail development.